INTRODUCTION
R
espiratory diseases represent one of the largest health problems word wide. Diseases such as asthma and
the smoking related diseases are already common and increasing so we urgently need better approaches
to treat or cure these diseases. At the same time, new respiratory diseases such as those associated with viruses
threaten pandemics that challenge our national health systems.
With these continued challenges for new treatment with better patient care, clinical and respiratory researchers
have sought better approaches to all aspects of patient care from improved diagnoses to superior therapies.
This has lead to an explosion of new research with an increasingly better understanding of how to diagnose
diseases and then develop new therapies. Thus, for example ever improving technologies for imaging lung
disease have lead to increasingly better diagnoses, although challenges remain as we seek to further improve
resolution. At the same time, the revolution in molecular biology, culminating with the publication of the
complete human genome, has lead to hopes for finding more precise clues to disease susceptibility pathogenesis
in genetic analysis. This is leading to new concepts in pharmacogenomics as we start to use new drugs,
including those used for lung cancers, being directed at mutations associated with disease.
This is the first Encyclopaedia of Respiratory Medicine. It is our hope that it is comprehensive and captures
the key aspects of current patient care, as well as the exciting developments in respiratory science that we all
believe will eventually lead to better patient care in the twenty first century.
This encyclopaedia is comprehensive in scope and provides clinician and researcher with a snap shot of the
current state of knowledge in respiratory medicine. All entries have adhered to a structured layout, starting
with an abstract crystallizing the key facts and finishing with reading lists for those who want to delve further
into the subject. In addition, most entries have a colour diagram designed to help understanding and provide a
valuable aid for undergraduate and post-graduate teaching.
These are exciting times for respiratory medicine. We hope this encyclopedia will become a valuble tool for
clinicians and researches at all stages of their careers from those beginning their carreers to those established
but wanting to update themselves on the new developments.
Finally, we would like to thank our Advisory Editorial Board who helped so much in shaping the contents of
this works, as well as the authors who wrote the articles and faced the challenge of condensing areas of
respiratory medicine, often the subject of entire textbooks, into a short article of 4000 words or less.
GEOFFREY J. LAURENT
STEVEN D. SHAPIRO
FOREWORD
Animals live by two principal things, food and breath. Of these, by far the most important is the respiration, for if it is stopped, the man
will not endure long, but immediately dies. – Aretaeus the Cappocian (150–200 AD)
O
f course, not all medical specialists would agree with this statement, and those who disagree would be
quick to posit that it is the failure of ‘‘their’’ particular organ that tends to cause immediate death.
However, that is not the issue. The point of this quotation is to illustrate that the proper functioning of the lung
has been a subject of great interest for centuries. The Greek physician Aretaeus devoted many of his
observations to diabetes, but his manuscript ‘‘On the Causes and Indications of Acute and Chronic Diseases’’
also discussed lung diseases, such as pneumonia. Since his time, great numbers of physicians from all continents and cultures have contributed to our knowledge of respiratory diseases.
While acknowledging our rich history of discoveries about pulmonary and respiratory medicine— discoveries that were made by men and women whose names symbolize the great journey of this specialty—one
must concede that the field experienced an extraordinary growth spurt beginning in the 1940s. Knowledge of
respiratory physiology, which developed very fast during World War II, created a tidal wave of interest that
continued for years afterward. The ability to measure and understand respiratory physiology and its alterations became a diagnostic tool, and it opened the door to therapeutic or respiratory support procedures.
But, then, in the 1950s and 1960s cell biology and subcellular research entered the scene. The potential of
molecular biology and genetics was quickly recognized, and respiratory medicine appreciated that a better understanding of normal and disordered biological respiratory processes hinged on use of these new approaches.
Lung and respiratory researchers, impelled in part by the ever-increasing public health burdens of respiratory
diseases, seized the opportunity. The stage was set for progress to occur. The architects of this ‘‘revolution’’ in
respiratory medicine are well known; it is our good fortune that many have contributed to these four volumes.
Four volumes! y Encyclopedia! y Indeed, these four volumes truly constitute an encyclopedia of pulmonary biology and respiratory medicine!
Respiratory medicine is still growing. Because it is such a dynamic and exciting field, new investigators will
almost surely want to be part of it. However, to do so they will need to know about the established state of
knowledge that will be the basis of their work. New investigators in the science of respiratory medicine,
whether interested in fundamental research or clinical research or application, will find ideas and inspiration in
these volumes. All of the tools of the trade are assembled therein.
As noted, respiratory medicine has been a progressive and expanding field but, as is the case with many fields
of medicine, the transfer of what we know to the general practice of medicine has been slow and limited.
Translation, as it is called, is an emerging discipline in need of assistance; fortunately, the breadth of the
knowledge presented in these volumes provides tools to facilitate this translation process.
This four-volume encyclopedia is, at once, both a tribute to the centuries of pioneering investigations in the
field of respiratory medicine and a foundation for even greater accomplishments in the future. The presentation
of all this knowledge in these excellent and comprehensive volumes can only serve to stimulate further work of
equal or surpassing significance. The editors and the authors are to be commended for their contributions to
this singular effort. Because of their work, respiratory science and medicine will advance faster and patients
worldwide will be the beneficiaries.
Claude Lenfant, MD
Gaithersburg, Maryland
Notes on the Subject Index
To save space in the index, the following abbreviations have been used:
ALI
acute lung injury
ARDS
acute respiratory distress syndrome
BAL
bronchoalveolar lavage
BPD
bronchopulmonary dysplasia
CAP
community-acquired pneumonia
CFTR
cystic fibrosis transporter regulation
COP
cryptogenic organizing pneumonia
COPD
chronic obstructive pulmonary disease
CWP
coal workers’ pneumoconiosis
G-CSF
granulocyte colony-stimulating factor
GERD
gastroesophageal reflux disease
GM-CSF
granulocyte-macrophage colony-stimulating factor
HUVS
hypocomplementemic urticarial vasculitis syndrome
IL
interleukin
IPF
idiopathic pulmonary fibrosis
IPH
idiopathic pulmonary hemosiderosis
MCP
monocyte chemoattractant protein
M-CSF
macrophage colony-stimulating factor
MIP
macrophage inflammatory protein
MMP
matrix metalloproteinase
NSCLC
non-small cell lung carcinoma
PPAR
peroxisome proliferator-activated receptor
SCLC
small-cell lung carcinoma
SP
surfactant protein
TGF
transforming growth factor
TIMP
tissue inhibitor of metalloproteinases
TNF
tumor necrosis factor
VEGF
vascular endothelial growth factor
Editorial Advisory Board
Kenneth B. Adler, North Carolina State University, Raleigh, NC, USA
Peter J. Barnes, Imperial College London, UK
Paul Borm, Zuyd University, Heerlen, The Netherlands
Arnold R. Brody, Tulane Medical School, New Orleans, LA, USA
Rachel C. Chambers, University College London, UK
Augustine M. K. Choi, University of Pittsburgh, PA, USA
Jack A. Elias, Yale University School of Medicine, New Haven, CT, USA
Patricia W. Finn, University of California San Diego, La Jolla, CA, USA
Stephen T. Holgate, University of Southampton, Southampton, UK
Steven Idell, The University of Texas Health Center at Tyler, TX, USA
Sebastian L. Johnston, National Heart and Lung Institute, Imperial college London, UK
Talmadge E. King, Jr, University of California, San Francisco, CA, USA
Stella Kourembanas, Children’s Hospital Boston, Harvard Medical School, Boston, MA, USA
Y. C. Gary Lee, University College London, UK
Richard Marshall, University College London, UK
Sadis Matalon, University of Alabama, Birmingham, AL, USA
Joel Moss, National Institutes of Health, Bethesda, MD, USA
William C. Parks, University of Washington, Seattle, WA, USA
Charles G. Plopper, University of California, Davis, CA, USA
Bruce W. S. Robinson, The University of Western Australia, Nedlands, Australia
Neil Schluger, Columbia University College of Physicians and Surgeons, New York, NY, USA
Edwin K. Silverman, Brigham and Women’s Hospital Boston, MA, USA
Eric S. Silverman, Brigham and Women’s Hospital, Boston, MA, USA
Peter Sly, Institute for Child Health Research, West Perth, Australia
Kingman Strohl, Case Western Reserve University, Cleveland, OH, USA
Teresa D. Tetley, Imperial College London, UK
John B. West, University of California, San Diego, CA, USA
Editors
Geoffrey J Laurent, Royal Free and University College Medical School, London, UK
Steven D Shapiro, Brigham and Woman’s Hospital, Boston, USA
Dedication
To my family, Lal, Guy, David and Gabrielle (GJL).
My contribution to this work would not have been possible without the love and support from my wife Nicole
and my daughters Calli, Tess, Skylar, and Ellery. I also thank my mentors and trainees for my continual
education and the Division of Pulmonary and Critical Care Medicine at Brigham and Women’s Hospital who
took care of our patients allowing me the time to undertake this project (SDS)
Permission Acknowledgments
The following material is reproduced with kind permission of Lippincott Williams and Wilkins
Figure 4 and 8 of ARTERIAL BLOOD GASES
Table 1 of ARTERIES AND VEINS
Figure 2 and 3 of BREATHING | Breathing in the Newborn
Figure 2a, 2b, 3, 4 and 5 of DRUG-INDUCED PULMONARY DISEASE
Table 1, 2 and 3 of DRUG-INDUCED PULMONARY DISEASE
Figure 2 of ENVIRONMENTAL POLLUTANTS | Diesel exhaust particles
Figure 4 of EXERCISE PHYSIOLOGY
Figure 2 of FLUID BALANCE IN THE LUNG
Figure 2 of GASTROESOPHAGEAL REFLUX
Figure 2 of GENE REGULATION
Figure 2 of HIGH ALTITUDE, PHYSIOLOGY AND DISEASES
Figure 2 and 3 of IDIOPATHIC PULMONARY HEMOSIDEROSIS
Table 1 of IDIOPATHIC PULMONARY HEMOSIDEROSIS
Figure 1, 2 and 3 of OXYGEN-HEMOGLOBIN DISSOCIATION CURVE
Figure 10a, 10b and 11a of SYSTEMIC DISEASE | Eosinophilic Lung Diseases
http://www.lww.com
The following material is reproduced with kind permission of Nature Publishing Group
Figure 2 of COAGULATION CASCADE | iuPA, tPA, uPAR
Figure 1 of COAGULATION CASCADE | Tissue Factor
Figure 1a of MATRIX METALLOPROTEINASES
Figure 1 of MYOFIBROBLASTS
2
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http://www.nature.com/nature and http://www.nature.com/reviews
The following material is reproduced with kind permission of Taylor & Francis Ltd
Figure 2 of AUTOANTIBODIES
Table 1 of BASAL CELLS
Figure 1 of NEUROPHYSIOLOGY | Neuroendocrine Cells
Table 1 of NEUROPHYSIOLOGY | Neuroendocrine Cells
Figure 1 and 2 of SURFACANT | Overview
Tables 1, 2, 3 and 4 of SURFACANT | Overview
http://www.tandf.co.uk/journals
A
ACETYLCHOLINE
J Zaagsma and H Meurs, University of Groningen,
Groningen, The Netherlands
& 2006 Elsevier Ltd. All rights reserved.
Abstract
In the airways, acetylcholine is a neurotransmitter in parasympathetic ganglia and in postganglionic parasympathetic nerves,
as well as a nonneural paracrine mediator in various cells in the
airway wall. Ganglionic transmission by acetylcholine is mediated by nicotinic receptors, which are ligand-gated in channels,
whereas postganglionic transmission is through G-protein-coupled muscarinic receptors. Of the five mammalian muscarinic
receptor subtypes, mainly M1, M2, and M3 receptors are involved in airway functions. Gq-coupled M1 receptors facilitate
ganglionic transmission mediated by nicotinic receptors and
modulate surfactant production and fluid resorption in the alveoli. Prejunctional Gi/o-coupled M2 receptors in parasympathetic nerve terminals attenuate acetylcholine release upon nerve
stimulation. M2 receptors are also abundantly present in airway
smooth muscle; however, the major function of these postjunctional M2 receptors is unknown. Postjunctional Gq-coupled M3
receptors mediate airway smooth muscle contraction and mucus
secretion. Dysfunction of the prejunctional M2 autoreceptor induced by allergic airway inflammation has been implied in exaggerated vagal reflex activity and airway hyperresponsiveness
in asthma. Inflammation-induced increased M3 receptor stimulation may be involved in airway remodeling in chronic
asthma. Possible mechanisms include potentiation of growth
factor-induced proliferation of airway smooth muscle cells and
induction of a contractile phenotype of these cells. Exaggerated
M3 receptor stimulation may also cause reduced responsiveness to b2-adrenoceptor agonists by transductional cross-talk
between phosphoinositide metabolism and adenylyl cyclase,
which involves protein kinase C-induced uncoupling of the b2adrenoceptor from the effector system. Muscarinic receptor antagonists have been shown to be effective in airway diseases like
asthma and, especially, chronic obstructive pulmonary disease.
Of these, tiotropium bromide is particularly useful, due to its
long duration of action as well as its kinetic selectivity for the
M3 receptor.
Introduction
Acetylcholine is a neurotransmitter in the central and
peripheral nervous system where it plays a major role
in the afferent neurons of both the autonomic
and somatic (voluntary) branches. As a chemical
transmitter, it has been identified as ‘Vagusstoff’ in
1921 by Otto Loewi showing its release from
an isolated frog heart following stimulation of the
vagosympathetic trunk; when applied to a second,
unstimulated heart, the perfusate slowed its rate,
resembling the effect of vagus stimulation. In
1926 Loewi provided evidence for identification of
Vagusstoff as acetylcholine. Acetylcholine is the
neurotransmitter of all sympathetic and parasympathetic autonomic ganglia and of the postganglionic
parasympathetic nerves. In the airways, the parasympathetic ganglia are located near or within the airway
wall. Ganglionic transmission mediated by acetylcholine is through nicotinic receptors which belong to
the family of ligand-gated ion channels. Postganglionic transmission by acetylcholine, released from
parasympathetic nerve terminals, is through muscarinic receptors of which five different subtypes
have been identified, all being G-protein-coupled
receptors. During periods of airway inflammation
vagal release of acetylcholine may be increased by
various mechanisms. Hence, both in asthma and
(particularly) in chronic obstructive pulmonary disease (COPD) blockade of postjunctional muscarinic
receptors is the key to reversing airway obstructions.
Synthesis, Storage, and Release
Acetylcholine is synthesized from choline and acetylcoenzyme A (acetyl-CoA) in the cytoplasm of the
nerve terminal through the enzyme choline acetyltransferase (ChAT). Choline is taken up by the nerve
terminal from the extracellular fluid through a sodium-dependent carrier; this transport is the ratelimiting process in acetylcholine synthesis. Acetyl-CoA
is synthesized in mitochondria which are abundantly
present in the nerve endings. Most of the synthesized
acetylcholine is actively transported from the cytosol
into synaptic vesicles by a specific transporter; this
vesicular (‘quantal’) package of acetylcholine reaches
up to 50 000 molecules per vesicle.
Release of acetylcholine is initiated by influx of
Ca2 þ ions through voltage-operated N- or P-type calcium channels. The increased intracellular Ca2 þ ions
bind to a vesicle-associated protein (synaptotagmin)
which favors association of a second vesicle protein
(synaptobrevin) with one or more proteins in the
plasma membrane of the nerve terminal. Following
2 ACETYLCHOLINE
this vesicle-docking process, fusion between vesicle
membrane and plasma membrane occurs, followed by
exocytosis. After the expulsion of acetylcholine the
empty vesicle is recaptured by endocytosis and can be
reused. In the synaptic cleft, the released acetylcholine
will associate with post- and prejunctional receptors
and is also subject to rapid hydrolysis by the enzyme
acetylcholinesterase into choline and acetate. Over
50% of the choline formed will be taken up again by
the nerve terminal and reused for neurotransmitter
synthesis.
Acetylcholine is also present in nonneuronal cells.
In recent years it has become clear that in the airways
the majority of cells express ChAT and contain
acetylcholine, including epithelial cells, smooth muscle cells, mast cells, and migrated immune cells such
as alveolar macrophages, granulocytes, and lymphocytes. However, the regulatory role of this nonneuronal acetylcholine in inflammatory airways
diseases has yet to be established.
Regulation of Synaptic Transmission
and Activity
Ganglionic
Preganglionic nerves innervating the parasympathetic
ganglia in the airways evoke action potentials during
normal breathing with relatively high frequencies, in
the range of 1–20 Hz. As a result, basal airway
smooth muscle tone in vivo is mediated to a significant extent by cholinergic nerve activity. The pattern
of ganglionic action potential bursts coincides with
respiration, suggesting that the respiratory centers in
the brainstem govern preganglionic nerve activity.
However, in addition to this central drive, reflex
stimulation through mechanically sensitive afferent
nerve terminals in the lungs during respiration is
importantly involved as well. The fidelity by which
preganglionic impulses are translated into action
potentials in the postganglionic neurons is relatively
low in parasympathetic airway ganglia, implying a
filtering function of these ganglia. This filtering
function can be diminished by various inflammatory mediators. Thus, histamine, prostaglandin D2
(PGD2), and bradykinin are able to enhance ganglionic cholinergic transmission and the same is true for
tachykinins (substance P, neurokinin A) released by
nonmyelinated sensory C-fibers in the airways.
Postganglionic
The release of acetylcholine from parasympathetic
nerve terminals is regulated by a variety of prejunctional receptors, which may inhibit or facilitate
transmitter outflow. In the airways, autoinhibitory
muscarinic M2 receptors, activated by acetylcholine
itself, represent an important negative feedback,
limiting further release, at higher firing rates in
particular. In animal models of allergic airway inflammation and asthma as well as in human asthma,
dysfunction of these M2 autoreceptors has been
found to contribute to exaggerated acetylcholine release from vagal nerve endings, to increased cholinergic reflex activity in response to inhaled stimuli,
and to contribute to airway hyperresponsiveness.
Most of this receptor dysfunction is thought to be
caused by activated eosinophils that migrate to
cholinergic nerves and release major basic protein
(MBP) which acts as an allosteric antagonist of muscarinic M2 receptors. Since eosinophilic inflammation is far less prominent in COPD and since M2
autoreceptors are more prominent in larger airways,
it is no surprise that these receptors are still functional in patients with stable COPD; however,
this does not exclude a dysfunction during acute
exacerbations.
In addition to M2 autoreceptors, a variety of
heteroreceptors modulating acetylcholine release
have been identified on cholinergic nerve endings.
Catecholamines may inhibit or facilitate acetylcholine overflow through prejunctional a2- and b2-adrenoceptors, respectively. Neurokinins like substance
P may enhance cholinergic transmission through
facilitatory neurokinin 1 (NK1) and/or 2 (NK2) receptors. Interestingly, substance P may also induce
MBP release from eosinophils, causing M2 receptor
dysfunction, which could act synergistically to direct
facilitation. Allergic inflammation-derived prostanoids, including PGD2, PGF2a, and thromboxane
A2, as well as histamine, can also augment acetylcholine release through prejunctional receptors.
Taken together, the above observations indicate
that parasympathetic acetylcholine release is governed by various regulatory systems, the set-point of
which is subject to environmental modulations. During periods of airway inflammation these modulations
often result in enhanced cholinergic transmission.
Receptors and Biological Function
In the ganglia, acetylcholine interacts with nicotinic
receptors. These receptors consist of five polypeptide
subunits, together forming a cylindric structure of
about 8 nm diameter, which acts as an ion channel.
Each subunit passes the membrane four times, so
the central pore is surrounded by 20 membranespanning helices. The subunits have been subdivided
into five classes, designated a, b, g, d, and e. Of the a
and b subunits, 10 and 4 different subtypes have
been identified, respectively. Peripheral ganglionic
ACETYLCHOLINE 3
receptors consist of only a and b subunits, the main
subtype being (a3)2(b4)3. Each a subunit possesses a
binding site for acetylcholine; they need to be occupied both to induce channel opening, which will
enhance Na þ and K þ permeability. This results in
an inward flux of mainly Na þ ions, causing depolarization and action potential generation in the
postganglionic cell (provided the acetylcholine concentration is high enough).
Acetylcholine released by postganglionic parasympathetic nerves may choose between five muscarinic
receptor subtypes, designated M1 to M5, to interact
with. Most organs and tissues express more than one
subtype and this is true for many individual cells as
well. The five subtypes can be subdivided into two
main classes, the odd-numbered receptors (M1, M3,
M5), which couple preferentially to heterotrimeric Gqproteins, and the even-numbered (M2, M4) receptors
which show selectivity for Gi/o type of G-proteins.
The principal signaling route of Gq-coupled receptors is the activation of phospholipase C, mediating
hydrolysis of phosphatidylinositol 4,5-bisphosphate
into inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG) (Figure 1, left). IP3 mobilizes Ca2 þ
ions from intracellular stores, which generates a rapid
and transient rise of the free Ca2 þ concentration in
the cytosol. DAG triggers the translocation and activation of protein kinase C (PKC) which is able to
phosphorylate a variety of different protein substrates.
The main signal transduction of Gi/o-coupled
receptors is to inhibit adenylyl cyclase activity, which
reduces the intracellular cyclic AMP concentration.
In airway smooth muscle, activation of Gi/o by M2
receptors may also diminish opening of calcium-activated potassium channels (KCa or maxi-K channels)
induced by (Gs-coupled) b-adrenoceptors. Thus, K þ
efflux, membrane hyperpolarization, and subsequent
smooth muscle relaxation, initiated by b-agonist
administration, is under restraint of acetylcholine
through this mechanism.
In the airways of most mammalian species, including human, M1, M2, and M3 receptors are the
most important ones. So far, M4 receptors have only
been detected convincingly in bronchiolar smooth
muscle and alveolar walls of the rabbit, whereas
muscarinic M5 receptors, now known to mediate
dilatation of cerebral arteries and arterioles, are
absent in the lungs.
M1 receptors have been found in alveolar walls,
parasympathetic ganglia, and submucosal glands.
Rat and guinea pig lung studies have indicated their
presence in type II alveolar cells, mediating surfactant production and fluid reabsorption, respectively.
In parasympathetic airway ganglia of several species,
including human, M1 receptor stimulation is able to
facilitate ganglionic transmission mediated by nicotinic receptors. Thus, vagal bronchoconstriction,
induced by inhalation of SO2, has been found especially sensitive to inhaled pirenzepine, a M1-selective
antagonist. In submucosal glands, M1 receptors are
not involved in mucus secretion, which appears to be
mediated solely by M3 receptors.
M2 and M3 receptors represent the major receptor populations, both in intra- and extrapulmonary
Epinephrine,
2-agonists
Acetylcholine
PIP2
M3
Gq
PLC
DAG
2
PKC
−
IP3
Ca2+
Contraction
AC
Gs
+
P
P
−
P
−
ATP
P
ARK
cAMP
Relaxation
Figure 1 Cross-talk between M3 muscarinic receptors and b2-adrenoceptors in airway smooth muscle. Generation of 1,2-diacylglycerol (DAG) by M3 receptor-induced phosphoinositide (PIP2) metabolism causes activation of protein kinase C (PKC). PKC may phosphorylate the b2-adrenoceptor as well as Gs, causing uncoupling of the receptor from the effector system. Moreover, PKC may
phosphorylate b-adrenoceptor kinase(s) (bARK), which amplifies b-agonist-induced desensitization mediated by bARK-induced phosphorylation of the receptor. AC, adenylyl cyclase; cAMP, cyclic adenosine 30 , 50 -monophosphate; IP3, inositol 1,4,5-trisphosphate; PLC,
phospholipase C.
4 ACETYLCHOLINE
airways. As already discussed, inhibitory M2 autoreceptors located at parasympathetic nerve terminals
have an important regulatory role in limiting acetylcholine release. Postjunctional muscarinic receptor
populations in airway smooth muscle are a mixture
of M2 and M3 receptors, the M2 subtype being predominant, particularly in the large airways.
Contraction, however, is primarily mediated by
M3 receptors (even in those smooth muscle preparations where the ratio of M2 : M3 receptors is 90 :
10), the M2 receptor population having at most a
minor supporting role. This is confirmed in airway
preparations from M2 receptor knockout mice,
in which carbachol, a muscarinic agonist, was
hardly less potent than in preparations from wildtype mice.
Cross-talk between Gi-coupled M2 receptors and
Gs-coupled b-adrenoceptors (having opposing effects
on cyclic AMP accumulation or maxi-K channel
opening) has no major effects in modulating muscarinic agonist induced contraction or b-agonist
induced relaxation, at least under physiological circumstances. However, in inflammatory conditions
such as asthma, in which Gi-proteins may be upregulated, the situation may change.
In contrast to M2 receptors, Gq-coupled M3 receptors, generating IP3 and DAG by stimulating
phosphoinositide metabolism, may have a major
influence on b2-adrenoceptor function, even in noninflamed airways. This is due to DAG-induced activation of PKC which may (1) phosphorylate the
b2-adrenoceptor as well as Gs, causing receptor uncoupling and desensitization, and (2) phosphorylate
and activate b-adrenoceptor kinase(s) (bARKs,
which are members of the G-protein receptor kinase
(GRK) family), amplifying homologous, b-agonist
induced desensitization (Figure 1). These processes
may explain the well-known attenuation of b-agonist
efficacy during episodes of severe bronchoconstriction, for example, during exacerbations.
Airway Remodeling
In addition to phosphoinositide metabolism, inhibition of adenylyl cyclase and maxi K-channel activation, stimulation of M3 and/or M2 receptors in
airway smooth muscle cells has been shown to activate different promitogenic signaling pathways,
including the p42/p44 mitogen-activated protein kinase, Rho/Rho kinase, and PI3 kinase pathways. In
vitro studies have revealed that muscarinic agonists
do not induce airway smooth muscle cell proliferation by themselves, but enhance the proliferative
response induced by peptide growth factors. This
effect, which is solely mediated by the M3 receptor,
indicates that acetylcholine may contribute to airway
remodeling as observed in asthma and COPD.
Indeed, in an animal model of chronic asthma it
was recently demonstrated that the long-acting
muscarinic antagonist tiotropium bromide inhibited
increased airway smooth muscle mass, enhanced
airway smooth muscle contractility, and increased
expression of contractile proteins in the lung upon
repeated allergen challenge. This implies that, in addition to their bronchodilating properties, muscarinic
receptor antagonists could be beneficial in the
treatment of asthma by preventing chronic airway
hyperresponsiveness and decline of lung function.
Although not fully established, a more extensive
role of acetylcholine in airway remodeling, including
airway smooth muscle proliferation, contractile protein expression, promitogenic signaling, and regulation of secretory functions as well as cell migration,
has recently been proposed (Figure 2).
Acetylcholine in Respiratory Diseases
As indicated above, the parasympathetic nervous
system represents a major constrictory pathway of
the airways; even basal bronchomotor tone is partly
governed by acetylcholine. Both its release and its
postjunctional effects, including smooth muscle contraction and mucus secretion, are regulated and
mediated, respectively, by muscarinic receptors. So
far, no evidence for upregulation of postjunctional
M3 and M2 receptors has been found in hyperresponsive airways of patients with asthma and COPD.
In contrast, dysfunctional autoreceptors, leading
to exaggerated vagal reflexes in the airways, are well
established in allergic asthma. M1 receptors, facilitating nicotinic neurotransmission in parasympathetic airway ganglia, do not appear to contribute
significantly to bronchomotor tone in humans, either
with or without obstructive airway diseases. Hence,
the principle therapeutic muscarinic receptor target
in asthma and COPD is the M3 receptor. In COPD,
muscarinic receptor antagonists like ipratropium,
a quarternary nonselective antagonist, are very
effective in causing bronchodilatation. A recently introduced antagonist is tiotropium, which acutely
occupies both M2 and M3 receptors; however, while
it dissociates rapidly from M2 receptors during
washout, blockade of M3 receptors persists for
hours. This kinetically based M3 receptor subtype
selectivity could be of therapeutic advantage since
blockade of prejunctional M2 receptors enhances
acetylcholine outflow. Both in COPD and, particularly, in allergic asthma, in which M2 autoreceptors
are already dysfunctional, this is an unwanted side
effect.
ACID–BASE BALANCE 5
ACh
Extracellular matrix
proteins
Migration
Hypercontractility
Chemotaxis
activation
Contraction
Hyperplasia
Figure 2 Proposed mechanisms by which acetylcholine (ACh) could affect airway smooth muscle remodeling. Acetylcholine has been
shown to affect airway smooth muscle contractility, contractile protein expression, promitogenic signaling, and proliferation. In addition,
like several other G-protein-coupled receptor agonists, acetylcholine could also be involved in airway smooth muscle cell migration,
extracellular matrix protein production, and secretion of cytokines and chemokines. Altogether, these effects could contribute to airway
remodeling in asthma and COPD. Reproduced from Gosens R, Zaagsma J, Grootte Bromhaar M, Nelemans SA, and Meurs H (2004)
Acetylcholine: a novel regulator of airway smooth muscle remodelling. European Journal of Pharmacology 500: 193–201, with permission from Elsevier.
See also: Asthma: Overview. Chronic Obstructive
Pulmonary Disease: Overview. Neurophysiology:
Neural Control of Airway Smooth Muscle; Neuroanatomy; Neurons and Neuromuscular Transmission.
Further Reading
Berge RE ten, Santing RE, Hamstra JJ, Roffel AF, and Zaagsma J
(1995) Dysfunction of muscarinic M2 receptors after the early
allergic reaction: possible contribution to bronchial hyperresponsiveness in allergic guinea-pigs. British Journal of Pharmacology 114: 881–887.
Billington CK and Penn RB (2002) M3 muscarinic acetylcholine
receptor regulation in the airway. American Journal of Respiratory Cell and Molecular Biology 26: 269–272.
Coulson FR and Fryer AD (2003) Muscarinic acetylcholine receptors
and airway diseases. Pharmacology and Therapeutics 98: 59–69.
Gosens R, Bos ST, Zaagsma J, and Meurs H (2005) Protective
effect of tiotropium bromide in the progression of airway
smooth muscle remodeling. American Journal of Respiratory
and Critical Care Medicine 171: 1096–1102.
Gosens R, Zaagsma J, Grootte Bromhaar M, Nelemans SA, and
Meurs H (2004) Acetylcholine: a novel regulator of airway
smooth muscle remodelling. European Journal of Pharmacology
500: 193–201.
Racké K and Matthiesen S (2004) The airway cholinergic system:
physiology and pharmacology. Pulmonary Pharmacology and
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airway function. Life Sciences 60: 1061–1068.
ACID–BASE BALANCE
O Siggaard-Andersen, University of Copenhagen,
Copenhagen, Denmark
& 2006 Elsevier Ltd. All rights reserved.
Abstract
The acid–base balance or neutrality regulation maintains a pH
around 7.4 in the extracellular fluid by excreting carbon dioxide
(carbonic acid anhydride) in the lungs and noncarbonic acid or
base in the kidneys. The result is a normal acid–base status in
blood and extracellular fluid, i.e., a normal pH, a normal carbon dioxide tension (pCO2 ), and a normal concentration of
titratable hydrogen ion (ctH þ ). A pH, log pCO2 chart illustrates the acid–base status of the arterial blood. The chart shows
normal values as well as values to be expected in typical acid–
base disturbances, i.e., acute and chronic respiratory acidosis
and alkalosis, and acute and chronic nonrespiratory (metabolic)
acidosis and alkalosis. The chart allows estimation of the concentration of titratable H þ of the extended extracellular fluid
(including erythrocytes), ctH þ Ecf. This quantity is also called
standard base deficit but the term base does not directly indicate
that the quantity refers to the excess or deficit of hydrogen ions.
ctH þ Ecf is the preferred indicator of a nonrespiratory acid–base
disturbance being independent of acute changes in pCO2
in vivo. While pH and pCO2 are directly measured, ctH þ Ecf
6 ACID–BASE BALANCE
is calculated from pH and pCO2 using the Henderson–Hasselbalch equation and the Van Slyke equation.
Description
The acid–base balance or neutrality regulation maintains a pH around 7.4 in the extracellular fluid by
excreting carbon dioxide (carbonic acid anhydride)
in the lungs and noncarbonic acid or base in the
kidneys. The result is a normal acid–base status in
blood and extracellular fluid, i.e., a normal pH, a
normal carbon dioxide tension (pCO2 ), and a normal
concentration of titratable hydrogen ion (ctH þ ).
Figure 1 illustrates the acid–base status of the blood,
especially the relationships among the three key
variables.
pH and the Hydrogen Ion Concentration (cH þ )
pH and cH þ of the plasma are both indicated on
the abscissa of Figure 1. pH is the negative dacadic
logarithm of molal hydrogen ion activity. Concentration of free hydrogen ion (cH þ ) is calculated as
109 pH nmol l 1. pH and pOH are closely related:
pH þ pOH ¼ pKw ¼ 13.622 at 371C, where Kw is the
ionization constant of water. If H þ is considered a
key component of an aqueous solution, then OH is
a derived component. Accounting for H þ and H2O
indirectly accounts for OH as well. It is the author’s conviction that the relevant component is the
hydrogen ion, not hydrogen ion binding groups
(base) nor hydroxyl ions.
The Carbon Dioxide Tension of the Blood (pCO2 )
pCO2 , i.e., the partial pressure of carbon dioxide in
a gas phase in equilibrium with the blood, is shown
on the ordinate on a logarithmic scale in Figure 1.
When pCO2 increases, the concentration of dissolved
carbon dioxide and carbonic acid increases, and
hence the hydrogen ion concentration increases:
CO2 þ H2 O-H2 CO3 -Hþ þHCO
3
The Concentration of Titratable Hydrogen Ion
(ctH þ )
ctH þ is indicated on the scale in the upper left corner
of Figure 1. ctH þ is a measure of added noncarbonic
acid or base. The amount of hydrogen ion added or
removed in relation to a reference pH of 7.40 may
be determined by titration to pH ¼ 7.40 at pCO2 ¼
5:33 kPa ( ¼ 40 mmHg) and T ¼ 371C using strong
acid or base, depending upon the initial pH. Titratable hydrogen ion is also called base deficit, or with
the opposite sign base excess. Unfortunately, the term
‘base’ is ambiguous (it has previously been associated
with cations) and does not directly indicate that the
relevant chemical component is the hydrogen ion.
The term hydrogen ion excess or acronym HX may
also be used. Note: by definition, ctH þ of blood refers to the actual hemoglobin oxygen saturation, not
the fully oxygenated blood.
Acid and base are defined by the equilibrium:
Acidz # Hþ þ Basez1
where Acidz and Basez 1 is a conjugate acid–base
pair. The charge number z may be positive, zero, or
negative. A strong acid, e.g., HCl, dissociates completely: HCl-H þ þ Cl . The anion that follows the
hydrogen ion is called an aprote, nonbuffering, or
strong anion. At physiological pH, even lactic acid is
a strong acid and lactate an aprote anion. A base is a
molecule containing a hydrogen ion-binding group.
A strong base, e.g. OH , associates completely with
hydrogen ion: OH þ H þ -H2O. The cation that
follows the hydroxyl ion is called an aprote, nonbuffering, or strong cation, e.g., Na þ or K þ .
A weak acid (buffer acid) is in equilibrium with its
conjugate weak base (buffer base), e.g.:
H2 CO3 # Hþ þ HCO
3
hemoglobinz # Hþ þ hemoglobinz1
The concentration of titratable hydrogen ion may
be determined for plasma (P), whole blood (B), or a
model of the extended extracellular fluid, i.e., blood
plus interstitial fluid (Ecf). The model consists of
blood diluted threefold with its own plasma to get a
hemoglobin concentration similar to the one obtained if the red cells were evenly distributed in the
whole extracellular volume. An acute increase in
pCO2 in vivo causes a rise in ctH þ B and a fall in
ctH þ P while ctH þ Ecf remains constant. For example, an acute rise in pCO2 from 5.33 to 10.66 kPa
(40–80 mmHg) causes a rise in whole blood ctH þ of
about 5 mmol l 1, a fall in plasma ctH þ of about
3 mmol l 1, while the extracellular ctH þ remains
independent of acute changes in pCO2 . The cause is a
redistribution of hydrogen ions within the extended
extracellular volume. The hydrogen ion concentration increases more in the poorly buffered interstitial
fluid than in the blood plasma, where it increases
more than inside the erythrocytes, where hemoglobin
binds the hydrogen ions. Hydrogen ions diffuse from
the poorly buffered interstitial fluid into the blood
plasma and further into the erythrocytes. Very little
transfer of hydrogen ions occurs between the intracellular space and the extracellular space, so
ctH þ Ecf remains virtually constant during acute
ACID–BASE BALANCE 7
No
rm
–5
0
+5
ex
ce
ss
pCO2 in arterial blood
Concentration of titratable
mmHg
kPa
hydrogen ion in extracellular fluid
20.0
150
mmol l–1
19.0
Siggaard-Andersen 140
0
5
0 5 0
18.0
–1 –1 –2 –2 –3
acid–base chart 130
17.0
H+ deficit
al
120
0
+1
ca
ia
ia
pn
pn
60
11.0
9.0
8.0
7.0
6.0
50
40
s
ces
5.0
Normal
40
Area
30
20
ex
15
ion
12.0
50
Normal
10
en
rog
13.0
10.0
70
it
ic defic
n
ro n
Ch n io
e
g
o
dr
hy
Concentration of
in plasma
+25 bicarbonate
mmol l–1
16.0
15.0
14.0
Hypercapnia
80
er
ca
er
0
+2
90
p
hy
p
hy
ic
on
e
ut
5
+1
100
r
Ch
Ac
H+
110
35
25
e
ut
Ac
3.0
nia
20
onic
2.5
Chr
15
Hypocapnia
ap
oc
p
hy
hyd
roge
n
4.0
3.5
capnia
ion
exc
ess
30
Chronic hypo
yd
te h
Acu
2.0
+30
6.8
pH in arterial plasma
6.9
7.0
140
120
100 90
Concentration of free
hydrogen ion in plasma
nmol l–1
7.1
80
7.2
70
7.3
60
50
Acidemia
7.4
40
35
Normal
7.5
7.6
7.7
30
25
Alkalemia
20
1.5
Figure 1 Acid–base chart for arterial blood with normal and pathophysiological reference areas. The acid–base status is shown as a
point with three coordinates: pH (abscissa), pCO2 (ordinate), and c tH þ (oblique coordinate). The bands radiating from the normal area
(the central ellipse) show reference areas for typical acute and chronic, respiratory and nonrespiratory, acid–base disturbances. Hyper- and
hypocapnia are also called respiratory acidosis and alkalosis, respectively. Hydrogen ion excess and deficit, i.e., increased and decreased
c tH þ , are also called nonrespiratory (or metabolic) acidosis and alkalosis, respectively. Reproduced from Siggaard-Andersen O (1971) An
acid-base chart for arterial blood with normal and pathophysiological reference areas. Scandinavian Journal of Clinical and Laboratory
Investigation 27: 239–245. Copyright & 1970, 1974 by Radiometer Copenhagen A/S, Åkandevej 21, Brønshøj, Denmark.
changes in pCO2 in vivo. ctH þ Ecf is also called
standard base deficit (SBD), or with the opposite sign
standard base excess (SBE), but the term base is deprecated by the author. It is important to use ctH þ Ecf
rather than ctH þ B (whole blood titratable hydrogen
ion) as a measure of a nonrespiratory acid–base disturbance, especially in neonatology where high hemoglobin concentrations and high pCO2 values may be
8 ACID–BASE BALANCE
encountered. The ctH þ B may then be considerably
higher than ctH þ Ecf (as much as 4 mmol l 1) causing an erroneous diagnosis of metabolic acidosis,
when the situation is merely a redistribution of hydrogen ions within the extended extracellular volume.
Projections to the ctH þ scale in the upper left
corner of Figure 1 should be made along the slanting
so-called vivo-CO2 titration curves, which are virtually straight lines (slightly convex upwards). The
slope of the lines depends on the concentration of
nonbicarbonate buffers, i.e., mainly hemoglobin. In
the chart, the slope corresponds to a hemoglobin
concentration of 3 mmol l 1 corresponding to the
hemoglobin concentration of the extended extracellular fluid. Variations in the slope due to variations in
blood hemoglobin concentration are small and generally without clinical significance. Variations in the
concentration of other buffers, e.g., albumin, are
even less significant.
In summary, the hydrogen ion status of the blood
is described by a point in the acid–base chart: the x,y
coordinates indicate cH þ and pCO2 , the oblique
coordinate is ctH þ Ecf.
The Henderson–Hasselbalch Equation
Often a description of acid–base balance is based on
the Henderson–Hasselbalch equation, derived from
the law of mass action:
pH ¼ pK þ log10 ðcHCO
3 =ðaCO2 pCO2 ÞÞ
where pK ¼ 6.10 and aCO2 ¼ 0.23 mmol l 1 kPa 1
¼ 0.0306 mmol l 1 mmHg 1 (solubility coefficient
of carbon dioxide in plasma at 371C). aCO2 pCO2
gives the concentration of H2CO3 plus CO2. pH is
determined by two variables, pCO2 and cHCO3 ,
representing respiratory and metabolic disturbances.
cHCO3 is shown in Figure 1 on a horizontal logarithmic scale along the pCO2 ¼ 5:33 kPa line. Projections to the scale should be made at an angle of
451. However, cHCO3 is not independent of
pCO2 . For this reason, standard bicarbonate was
introduced, i.e., the bicarbonate concentration in
plasma of whole blood equilibrated with a gas mixture with a normal pCO2 (5.33 kPa ¼ 40 mmHg) at
371C. However, even the standard bicarbonate is not
completely independent of acute changes in pCO2 in
vivo, decreasing slightly in acute hypercapnia. To be
independent, the equilibration should be performed
with a model of the extended extracellular fluid.
Projecting from a given point in the chart to the
bicarbonate scale along the slanting vivo-CO2 equilibration lines gives the standard bicarbonate concentration of the extended extracellular fluid.
The Van Slyke Equation
Blood gas analyzers measure pH with a glass electrode and pCO2 with a membrane-covered glass
electrode (Stow-Severinghaus electrode). ctH þ Ecf is
calculated from pH, pCO2 , and cHb (concentration
of hemoglobin) using a model of the titration curve
called the Van Slyke equation (Table 1). The equation calculates the change in buffer base concentration (bicarbonate plus protein anion plus phosphate)
from the value at the reference point:
pHPy ¼ 7:40; PyCO2 ¼ 5:33 kPa; and T y ¼ 37 C
Buffer base (BB) is the difference between the concentrations of buffer anions and buffer cations (the
latter being virtually zero at physiological pH).
Strong ion difference (SID) is the difference between
Table 1 Van Slyke equation for calculation of the concentration of titratable hydrogen ion in the extended extracellular fluid, ctH þ Ecf
þ
c tH þ Ecf ¼ ð1 cHbEcf=cHby Þ ðDcHCO
3 P þ bH Ecf DpHPÞ
c HbEcf ¼ c HbB V B/V Ecf concentration of hemoglobin in the extended extracellular fluid
V B/V Ecf ¼ 1/3 (default value) ratio between the volume of blood and volume of extended extracellular fluid
c Hby ¼ 43 mmol l 1 empirical parameter accounting for an unequal distribution of hydrogen ions between plasma and erythrocytes
Dc HCO3 P ¼ c HCO3 P c HCO3 Py
y
c HCO3 Py ¼ 24.5 mmol l 1 concentration of bicarbonate in plasma at pHPy ¼ 7.40, PCO
¼ 5:33 kPa, T y ¼ 37:0 C
2
DpHP ¼ pHP pHPy
bHþ Ecf ¼ bm Hby cHbEcf þ bP
bm Hby ¼ 2.3 apparent molar buffer capacity of hemoglobin monomer in whole blood
bP ¼ 7.7 mmol l 1 (default value) buffer value of nonbicarbonate buffers in plasma for a normal plasma protein (albumin) concentration
c HbB ¼ rHbB/MmHb (substance) concentration of hemoglobin in blood (unit: mmol l 1) as a function of the mass concentration, rHbB
(unit: g l 1)
MmHb ¼ 16 114 g mol 1 molar mass of hemoglobin monomer
Ecf refers to the extended extracellular fluid, B to whole blood, P to plasma. Replacing cHbEcf by cHbB gives ctH þ B; replacing cHbEcf
by zero gives ctH þ P.
Note: if cHbB ¼ 9.0 mmol l 1 3 rHbB ¼ 14.5 g dl 1, then the Van Slyke equation simplifies to c tH þ Ecf ¼ 0.93 (Dc HCO3 P þ
DpHP 14.6 mmol l 1).
ACID–BASE BALANCE 9
the concentrations of nonbuffer cations and nonbuffer anions (see Figure 2). According to the law of
electroneutrality, the value of BB and SID must be
identical. Buffer base is not a suitable indicator of a
nonrespiratory acid–base disturbance; although independent of pCO2 , it varies with the albumin and
hemoglobin concentrations, which are unrelated to
acid–base disturbances.
Normal Acid–Base Balance
Concentration of ions in arterial plasma (mmol l −1)
Acid–base balance refers to the balance between input (intake and production) and output (elimination)
of hydrogen ion. The body is an open system in
equilibrium with the alveolar air where the partial
pressure of carbon dioxide pCO2 is identical to the
carbon dioxide tension in the blood. pCO2 is directly
proportional to the CO2 production rate (at constant
150
Mg2+
Ca2+
K+
HCO3−
SID+
BB−
Pr
−
100
Na+
Cl −
Cations
Anions
HPO42− +H2PO4−
SO42−
Organic anions
50
Figure 2 Electrolyte balance of arterial plasma showing columns of cations and anions of equal height (law of electroneutrality). The equality of the strong ion difference (SID) and
buffer base (BB) is illustrated. The change in concentration of
buffer base from normal (at pH ¼ 7.40, pCO2 ¼ 5:3 kPa, and
T ¼ 371C) with opposite sign equals the concentration of titratable
hydrogen ion.
alveolar ventilation and CO2 free inspired air) and
inversely proportional to the alveolar ventilation (at
constant CO2 production rate and CO2 free inspired
air). CO2 is constantly produced in the oxidative
metabolism at a rate of about 10 mmol min 1
( ¼ 224 ml min 1) and eliminated in the lungs at the
same rate so that the pCO2 remains at about
5.33 kPa ( ¼ 40 mmHg). Hydrogen ions associated
with any anion other than bicarbonate or exchanging
with a cation are eliminated by the kidneys. In the
oxidative metabolism of sulfur-containing amino acids, hydrogen ions are produced together with sulfate
ions at a rate of about 70 mmol day 1 depending
upon the protein intake. Amino acids are oxidized to
carbon dioxide and water, and the amino nitrogen,
liberated as NH3, combines with carbon dioxide in
the liver via the Krebs urea cycle to form neutral
urea. Therefore, there is no production of base (ammonia) except in the kidneys, where ammonia
formed from glutamine diffuses into the urine where
it binds a hydrogen ion (NH3 þ H þ -NH4þ ) thereby
preventing an excessively low urine pH.
Normal values for the acid–base status of arterial
blood are given in Table 2. The lower pCO2 in
women than men is probably a progesterone effect on
the respiratory center. The values are independent of
age except at birth, where babies tend to have higher
pCO2 , lower pH, and slightly increased ctH þ Ecf,
approaching normal values for adults in the course of
a few hours. In the last trimester of pregnancy, the
pCO2 is lower (about 1 kPa ¼ 7.5 mmHg), compensated by a slightly increased ctH þ Ecf. A protein-rich
diet causes a higher ctH þ Ecf (1–2 mmol l 1) and a
slightly lower pH due to production of sulfuric acid
from sulfur-containing amino acids. A diet rich in
vegetables and fruit causes a lower (negative)
ctH þ Ecf and a slightly higher pH due to organic anions binding H þ in the metabolism to carbon dioxide
and water. High-altitude hypoxia stimulates ventilation; at 5 km above sea level, pCO2 is decreased to
about 3.3 kPa ¼ 25 mmHg. The hypocapnia is compensated by increased ctH þ Ecf, so pH is only slightly
elevated. The values fall in the area of chronic hypocapnia in the acid–base chart (Figure 1).
Table 2 Reference values for arterial blood
Women
þ
cH P
c tH þ Ecf
pCO2
cHCO3 P
Men
1
36.3–41.7 nmol l
(pH: 7.38–7.44)
2.3 to þ 2.7 mmol l 1
4.59–5.76 kPa (33.8–42.4 mmHg)
21.2–27.0 mmol l 1
37.2–42.7 nmol l 1 (pH: 7.37–7.43)
3.2 to þ 1.8 mmol l 1
4.91–6.16 kPa (36.8–46.2 mmHg)
22.2–28.3 mmol l 1
cH þ P: conc. of (free) hydrogen ion in plasma; c tH þ Ecf: conc. of titratable hydrogen ion in extracellular fluid (also called standard base
deficit, SBD); pCO2 : tension of carbon dioxide; cHCO3 P: conc. of bicarbonate in plasma.
10
ACID–BASE BALANCE
Acid–Base Disturbances
Respiratory Acid–Base Disturbances
Acute respiratory acid–base disturbances are characterized by an acute change in pCO2 associated with
an acute change in pH but with unchanged ctH þ Ecf.
The relationship between pCO2 and pH is illustrated
by the oblique in vivo CO2 equilibration lines in the
acid–base chart (Figure 1). Primary increase and decrease in pCO2 are compensated by secondary renal
decrease and increase in ctH þ Ecf, respectively. The
acid–base chart shows the expected values in chronic
hypercapnia and chronic hypocapnia. The effect of
the compensation is a return of pH about two-thirds
towards normal, slightly more in acute hypocapnia.
Nonrespiratory Acid–Base Disturbances
Primary increase and decrease in ctH þ Ecf are compensated by secondary decrease and increase in
pCO2 . A very acute rise in ctH þ Ecf, for example,
due to anaerobic exercise with lactic acid formation,
is only partly compensated because only peripheral
chemoreceptors react promptly to a fall in blood pH.
It takes about an hour before H þ equilibrium between blood and brain extracellular fluid is achieved
and the central chemoreceptors are maximally stimulated. The acid–base values in acute nonrespiratory
acidemia are illustrated in Figure 1 by the area labeled ‘acute hydrogen ion excess’. The outline of the
area is dotted because it is less well-defined than the
other areas of the chart. The compensations in more
slowly developing nonrespiratory acidemia or alkalemia are illustrated by the areas labeled ‘chronic
hydrogen ion excess’ and ‘deficit’, respectively. The
effect of the respiratory compensation is a return of
pH one-third to halfway towards normal.
Once an increase in ctH þ Ecf has been detected,
the question is: what caused the metabolic acidosis?
It may be a production of lactic acid due to anaerobic
metabolism or acetoacetic acid (ketoacidosis) due to
diabetes mellitus or starvation. In both cases the diagnosis may be verified by direct measurement of
blood lactate or acetoacetate. When these analyses
are unavailable, calculation of the concentration of
undetermined anions may be useful, i.e., the sum of
the concentrations of measured cations (Na þ and
K þ ) minus the sum of the concentrations of measured and calculated anions (Cl and HCO3 ). This
equals the sum of the concentrations of unmeasured
anions (mainly Protein , SO24 , HPO24 , fatty carboxylate, lactate, acetoacetate)minus the sum of
the concentrations of unmeasured cations (Ca2 þ
and Mg2 þ ). A metabolic acidosis with a major increase in undetermined anions usually indicates
organic acidosis. A hyperchloremic acidosis may be
a renal acidosis with retention of H þ and Cl or an
intestinal loss of Na þ þ HCO3 with subsequent
intake of saline (Na þ þ Cl ). A hypochloremic
alkalosis may be due to loss of H þ and Cl by
vomiting. Hypokalemic alkalosis is due to inability
of the kidneys to retain hydrogen ions in the presence
of potassium depletion.
See also: Arterial Blood Gases. Carbon Dioxide.
Peripheral Gas Exchange. Ventilation: Overview.
Further Reading
Astup P and Severinghaus JW (1986) The History of Blood Gases
Acids and Bases. Copenhagen: Munksgaard International Publishers.
Davenport HW (1969) The ABC of Acid–Base Chemistry, 5th edn.
Chicago: The University of Chicago Press.
Grogono AW (1986) Acid–base balance. International Anesthesiology Clinics, Problems and Advances in Respiratory Therapy
24(1).
Halperin ML and Goldstein MB (1988) Fluid, Electrolyte, and
Acid–Base Emergencies. Philadelphia: Saunders.
Hills AG (1973) Acid–Base Balance. Chemistry, Physiology, and
Pathophysiology. Baltimore: Williams & Wilkins.
International Federation of Clinical Chemistry and International
Union of Pure and Applied Chemistry (1987) Approved
Recommendation (1984) on Physico-Chemical Quantities and
Units in Clinical Chemistry. Journal of Clinical Chemistry and
Clinical Biochemistry 25: 369–391.
Masoro EJ and Siegel PD (1971) Acid–Base Regulation. Its Physiology and Pathophysiology. Philadelphia: Saunders.
Nahas G and Schaefer KE (eds.) (1974) Carbon Dioxide and Metabolic Regulations. New York: Springer.
Rooth G (1975) Acid–Base and Electrolyte Balance. Lund: Studentlitteratur.
Severinghaus JW and Astrup P (1987) History of Blood Gas Analysis. Boston: Little, Brown and Company.
Shapiro BA, Peruzzi WT, and Templin R (1994) Clinical Application of Blood Gases, 5th edn. St Louis: Mosby – Year Book.
Siggaard-Andersen O (1971) An acid-base chart for arterial blood
with normal and pathophysiological reference areas. Scandinavian Journal of Clinical and Laboratory Investigation 27:
239–245.
Siggaard-Andersen O (1974) The Acid–Base Status of the Blood,
4th edn. Copenhagen: Munksgaard and Baltimore: Williams &
Wilkins Company.
Siggaard-Andersen O (1979) Hydrogen ions and blood gases. In:
Brown SS, Mitchell FL, and Young DS (eds.) Chemical
Diagnosis of Disease, pp. 181–245. London: Elsevier, NorthHolland Biomedical Press.
Siggaard-Andersen O and Fogh-Andersen N (1995) Base excess or
buffer base (strong ion difference) as measure of a non-respiratory acid–base disturbance. Acta Anaesthesiologica Scandinavica 39(supplement 107): 123–128.
Thomson WST, Adams JF, and Cowan RA (1997) Clinical Acid–
Base Balance. New York: Oxford University Press.
West JB (1974) Respiratory Physiology, the Essentials. Oxford:
Blackwell.
West JB (2001) Pulmonary Physiology and Pathophysiology. An
Integrated, Case-Based Approach. Philadelphia: Lippincott
Williams & Wilkins.
ACUTE RESPIRATORY DISTRESS SYNDROME 11
Acute Exacerbations see Asthma: Acute Exacerbations. Chronic Obstructive Pulmonary Disease:
Acute Exacerbations.
Acute Lung Injury see Acute Respiratory Distress Syndrome.
ACUTE RESPIRATORY DISTRESS SYNDROME
G Bellingan, University College London, London, UK
S J Finney, Imperial College London, London, UK
Table 1 Clinical triggers of ARDS
Etiology
Percentage
Sepsis (including pulmonary sepsis)
Aspiration of gastric contents
Pulmonary contusion
Bacteremia
Head injury
Multiple bony fractures requiring ICU admission
Blood transfusion exceeding 10 units in 24 h
Cardiopulmonary bypass
Burns (including smoke inhalation)
Acute pancreatitis
Lung reperfusion injury (e.g., posttransplant)
Near-drowning
43–52
22–36
8–26
4–12
6–11
5–12
5–8
2
2
1
& 2006 Elsevier Ltd. All rights reserved.
Abstract
The acute respiratory distress syndrome (ARDS) is the devastating manifestation of the diffuse pulmonary inflammation that
may occur following a wide range of life-threatening systemic
illnesses. The rapid onset of inflammation and bilateral nonhydrostatic alveolar edema results in severe hypoxemia and reduced pulmonary compliance often mandating mechanical
ventilation. The clinical features, radiology, and pathogenesis
are reviewed in this article. The management of patients comprises primarily of ventilatory support while the lung injury resolves. The techniques of ventilatory support can propagate the
lung injury and adversely affect outcome; the techniques are
discussed in detail here. By contrast, pharmacotherapy has a less
clear role in ARDS. Corticosteroids may be beneficial after the
acute phases, whilst other anti-inflammatory agents have not
proved beneficial. Mortality is determined primarily by the underlying trigger for ARDS, but is approximately 30–40%. Follow-up of survivors has demonstrated that lung function often
improves considerably, whereas nonpulmonary morbidities persist even 12 months after discharge from the intensive care unit.
Introduction
The acute respiratory distress syndrome (ARDS) and
its less extreme manifestation, acute lung injury
(ALI), are devastating conditions that result in sudden bilateral nonhydrostatic alveolar edema and severe hypoxemia. Pulmonary failure is usually so
severe that patients require mechanical ventilation of
their lungs. ARDS and ALI are the consequence of
the diffuse pulmonary inflammation that can be triggered by an insult either to the lung itself or more
commonly at a distant site. As such, they form part
of the spectrum of systemic inflammation that can
occur following many life-threatening insults (see
Table 1).
The constellation of severe respiratory distress, refractory hypoxemia, decreased pulmonary compliance,
The figures in this were based on studies at the university of
Washington and Colorado.
and diffuse radiological changes was first appreciated
by Ashbaugh and co-workers in 1967. They referred to
the clinical scenario as the adult respiratory distress
syndrome. Since an identical condition can also occur
in children, it is now referred to as the acute respiratory distress syndrome. Subsequently, the clinical syndrome has been increasingly recognized and estimates
of the annual incidence of ARDS range from 8 to 70
cases per 100 000 population in developed countries.
Overall, mortality in patients with ARDS is approximately 30–40%, although death is usually attributable to the underlying etiology rather than pulmonary
failure per se.
Etiology
The many possible triggers for ARDS are outlined in
Table 1. Sepsis accounts for the majority of cases in
general intensive care units (ICUs). Patients with multiple risk factors are more likely to develop ARDS.
Intriguingly, ARDS only occurs in a subset of patients
who suffer apparently similar insults. It is not
clear what explains a particular individual going on
12
ACUTE RESPIRATORY DISTRESS SYNDROME
to develop ARDS. Although environmental factors
such as age, sex, smoking history, and intercurrent
pulmonary disease may be important, it is widely
considered that genetic factors are critical. Candidate
genes for which associations have been described include those that encode tumor necrosis factor alpha
(TNF-a), angiotensin-converting enzyme, interleukin-6
(IL-6), surfactant protein B, and Toll-like receptors.
Nevertheless, it has not been possible to draw definitive conclusions since these associations are complicated by tight linkage disequilibrium to other genes,
lack of clear functional effects, and small patient numbers. Moreover, the heterogeneity of ARDS suggests
that many genes with varying phenotypes and penetrance may underpin an individual’s susceptibility.
Functional genomic approaches may help elucidate
these complex genotypes.
After the initial injury, myofibroblasts are observed
in the interstitium and then the airspace, and start to
produce new matrix substance. Indeed, the lung collagen content can double by 2 weeks. With time, type
II alveolar epithelial cells increase in number and
may represent a stem cell population of the lung; they
differentiate into type I alveolar epithelial cells and
repopulate the denuded alveolar basement membrane during healing.
Pulmonary function and computed tomography
(CT) suggest that many patients subsequently return
to have structurally normal lungs although this can
take months. Some patients however develop severe
fibrosis involving both the alveolar space and interstitium. At its most extreme, the ‘honeycomb’ of advanced fibrotic lung disease may form. Many of these
patients succumb to intercurrent infection associated
with a prolonged ICU stay. A few can recover with
incomplete resolution of pulmonary damage.
Pathology
Traditionally, the histological development of ARDS
has been described as having three sequential phases
which affect the lungs in a diffuse manner: exudative,
proliferative, and fibrotic. It is now evident that these
stages overlap considerably and fibrotic changes are
initiated very early.
Within the first 24 h, the lungs appear macroscopically edematous and congested. At this point, light
microscopy reveals edema within the airspaces,
alveolar walls, and septae. Type I alveolar epithelial
cells are swollen, necrotic, and often detached from
the underlying basement membrane. Pulmonary
endothelial cells may also be swollen, with fibrin
thrombi occluding alveolar capillaries. Neutrophils
are also initially observed within alveolar capillaries,
but inflammatory cells then accumulate rapidly in the
edematous alveoli. Over the next few days, the lungs
become more uniformly red as alveolar wall and airspace edema increase and red cells leak into the airspaces. Histologically, hyaline membranes form from
fibrin-rich edema fluid and line the alveoli. The
number of neutrophils increases rapidly as they move
via the interstitium into the airspace and there is increased disruption of vascular structures with further
neutrophil and fibrin plugs occluding capillaries.
Clinical Features
Definition
The diagnosis of ARDS is based on the criteria developed at the 1992 American–European consensus
conference and is illustrated in Table 2. Since the
definition does not consider current management
strategies, the relevance of scenarios in which suboptimal mechanical ventilation and management can
influence whether criteria are met or not is not clear.
The cutoffs for severity of hypoxemia are arbitrary,
the definition of ‘acute’ onset lacks clarity and the
use of the chest X-ray opens the way for individual
interpretation. Further limitations of the definition
include the inclusion of other conditions that probably have different pathological processes (such as
severe pneumocystis carinii infection, diffuse alveolar
hemorrhage), and exclusion of unilateral disease that
may occur following pulmonary lobectomy. Nevertheless, the definition is simple to use and is supported by extensive literature. Post-mortem studies
have demonstrated that the definition is 84% sensitive and 94% specific for diffuse alveolar damage; its
performance in patients who survive is not clear.
Table 2 American–European consensus criteria for diagnosing ALI and ARDS
ALI
Chest radiography
Clinical scenario
Left atrial pressure
Oxygenation
Bilateral airspace shadowing
Acute onset and associated with a condition known to cause ALI/ARDS
No direct or clinical evidence of left atrial hypertension (PAOP o18 mmHg)
PaO2/FiO2 o39.9 kPa (300 mmHg)
PAOP, pulmonary artery occlusion pressure.
ARDS
PaO2/FiO2 o26.6 kPa
(200 mmHg)
ACUTE RESPIRATORY DISTRESS SYNDROME 13
Natural History
The clinical picture of ARDS is dominated initially
by severe hypoxemia due to mismatching of ventilation and perfusion. Indeed, intrapulmonary shunting
may result in oxygen saturations that are relatively
refractory to increases in the inspired oxygen content. Decreased pulmonary compliance increases the
work of breathing and most patients require endotracheal intubation and mechanical ventilation.
From a pulmonary perspective, the high oxygen
requirements persist for some time. Further increases
in the alveolar–arterial oxygen gradient occur with
ongoing pulmonary inflammation, particularly in the
setting of a positive fluid balance, but may also reflect
a superimposed ventilatory pneumonia or pneumothorax. Pneumothoraces tend to occur after the first
week and may tension rapidly. Since the inflamed
lung may tether to the chest wall, pneumothoraces
can be loculated and anterior, and thus easily missed
on plain chest radiography. Thoracic CT may be
required to locate pneumothoraces accurately.
Surprisingly, despite devastating pulmonary failure,
oxygenation often slowly improves allowing withdrawal of mechanical ventilation; for many, this may
take weeks or months and often necessitate temporary tracheostomy. Although disease can remain
compartmentalized with isolated lung failure, ARDS
is usually part of the spectrum of systemic inflammation; hence, patients often demonstrate peripheral
vasodilation, increased cardiac outputs, and systemic
hypotension which may require the administration
of vasopressors such as norepinephrine. Secondary
pulmonary hypertension can occur and result in
acute right ventricular failure. Renal dysfunction is
also common as is the need for acute renal support.
Other organs can also fail as part of this process and
outcome is related to the number of failing organs.
The course of ARDS is not a smooth wave of deterioration and recovery. Rather, it is interspersed by
episodes of deterioration (commonly linked with
intercurrent infection such as ventilator-associated
pneumonia or line-related sepsis) and many patients
need repeated episodes of inotrope and other organ
support prior to final recovery (or demise).
Radiology
The appearance of the plain chest radiograph, although forming part of the consensus definition for
ARDS, can be relatively non-specific. Clues distinguishing ARDS from cardiogenic pulmonary edema
include normal cardiac dimensions, a normal vascular pedicle width, a peripheral distribution of airspace shadowing, and the absence of septal lines.
Chest CT demonstrates that the lungs are affected
in a heterogenous manner. Typically, there is a gradient of opacification from apparently normally
aerated lung, through ground-glass appearances, to
densely consolidated lung. In the supine patient, this
gradient occurs both in ventrodorsal and cephalocaudal directions. These gradients typically reverse
within a few minutes if the patient is moved to the
prone position. Since alveolar edema would not redistribute so quickly, some of these appearance are
not due to increased edema in dependent zones but
due to collapse of these areas due to the weight of the
overlying lung. Thus ground-glass appearances most
likely represent airspace edema, with densely opacified areas representing collapsed edematous lung.
Regions of dense opacification in nondependent areas may signify collapse/consolidation due to infection or retained secretions. At later stages, groundglass appearances may be accompanied by bronchial
dilatation which persists into recovery, suggesting
established fine intralobular fibrosis (Figure 1).
Other Pulmonary Investigations
Bronchoalveolar lavage samples are dominated by the
granulocytic cell population initially. As the disease
evolves, the proportion of granulocytes declines and a
greater proportion of macrophages is seen. Persistent
neutrophilia often portends a poor prognosis.
Since most patients are mechanically ventilated,
there are few data about classical pulmonary function tests in patients with ARDS. Re-breathing techniques have been used during positive pressure
ventilation and have demonstrated marked reductions in the functional residual capacity, carbon
monoxide diffusing capacity (DLCO), and diffusing
coefficient (KCO). Lower values of DLCO and KCO
tend to be associated with nonsurvival.
Disease Severity
There are several scoring systems that evaluate the
severity of ARDS. Although these systems have only
limited clinical utility in individuals, they describe
well the degree of physiological disturbance and act
as useful descriptors of disease severity in clinical
studies. The scoring systems include the acute physiology and chronic health evaluation II and the lung
injury score. The former is used to evaluate all critically ill patients whereas the latter is specifically designed for patients with ARDS.
Pathogenesis
Since neutrophils appear early in histological specimens and dominate in bronchoalveolar fluid samples,
14
ACUTE RESPIRATORY DISTRESS SYNDROME
also key initiators of pulmonary inflammation in
ARDS. Studies of patient groups at risk of ARDS
who do or do not progress to develop refractory
hypoxemia suggest compartmentalized intrapulmonary inflammatory changes (e.g., increased IL-8) may
precede a systemic inflammatory response.
There is widespread activation and/or dysfunction
of many cell types within the lung which results in
the clinical manifestations of ARDS (Figure 2). Thus
endothelial dysfunction and loss of epithelial integrity reduce the barrier function of the alveolar wall
and result in alveolar edema. Alveolar edema is further exacerbated by the loss of epithelial cells which
normally promote fluid transport out of the alveolus
through apical sodium pumps. Surfactant is lost early
during ARDS due to reduced production by damaged
epithelial cells along with neutralization of preexisting surfactant by the protein-rich edema fluid.
Surfactant loss contributes to alveolar collapse, intrapulmonary shunt, and hypoxemia. Endothelial
and smooth muscle cell dysfunction result in impaired hypoxic pulmonary vasoconstriction and,
along with microthrombi, contribute to the development of secondary pulmonary hypertension and also
impacts on outcome.
Animal Models
Figure 1 Typical appearances of ARDS with (a) plain radiology
and (b) computed tomography.
it has been considered that they are important in the
pathogenesis of ARDS. It is possible that activated
and thus rigid 7.5 mm neutrophils may get stuck in
pulmonary capillaries where they release a plethora
of inflammatory mediators that include chemokines,
cytokines, and proteases. Activation may occur at
remote sites and/or by circulating cytokines. However, since ARDS can occur in neutropenic patients,
neutrophils cannot be absolutely required for the
development of ARDS. Circulating inflammatory
mediators (e.g., TNF-a, IL-1b, IL-6, IL-8, leukotrienes) along with the changes that occur within the
coagulation system during systemic inflammation are
Animal models allow the study of the pathophysiology of ARDS and the effects of interventions whilst
tightly controlling both insults and genetic background. However, the multiple models that exist for
ARDS/ALI illustrate that none are ideal. Models in
which the lung is injured directly include hyperoxia,
high tidal-volume mechanical ventilation, and instillation of oleic acid, bleomycin, or thiourea. Alternatively, models mimic systemic sepsis in which the
lung is also injured; these include systemic injections
of lipopolysaccharide and caecal ligation and puncture. Some authors suggest that two-hit models are
more clinically appropriate and combine models of
sepsis with hypovolemia, burns, or high tidal-volume
ventilation.
Management and Current Therapy
The primary management aims for a patient with
ARDS are to maintain adequate oxygenation and
to support any other failing organs whilst the lung
injury resolves. In general, respiratory failure is
sufficiently severe to mandate mechanical ventilation,
the techniques of which significantly influence mortality. By contrast, few, if any, therapies directed at
modifying the pathogenesis and evolution of ARDS,
have been demonstrated to effect outcome. A detailed
ACUTE RESPIRATORY DISTRESS SYNDROME 15
Inflammatory trigger
Mechanical ventilation
E.g., bacterial sepsis, massive transfusion
Stretch-induced
cell signaling
Activated neutrophils
Stretch-induced
cell damage
Soluble mediators
E.g., cytokines, chemokines,
coagulation factors, eicosanoids, ROS
Smooth muscle cells
Endothelial cells
Vascular dysfunction
Reduced HPV
Pulmonary hypertension
Epithelial cells
Barrier dysfunction
Production
Surfactant
Alveolar edema
Neutralization
Alveolar collapse
Figure 2 Pathogenesis of ARDS and ALI. HPV, hypoxic pulmonary vasoconstriction; ROS, reactive oxygen species.
description of optimal mechanical ventilation along
with the pharmacotherapeutic approaches that have
been explored are outlined below.
Conventional Mechanical Ventilation
The manner in which the injured lung is ventilated
influences mortality and may perpetuate lung injury.
The origins of such ventilator-induced lung injury
(VILI) are multifactorial but include the shear
stresses exerted on alveoli during overdistension
and cyclical collapse/re-inflation, and oxygen toxicity. VILI is illustrated by chest CT of those patients
who have survived ARDS: relatively normal lung architecture is often present in previously densely consolidated areas, whereas a reticular pattern of fibrosis
is seen in those regions (often nondependent) that
were exposed to mechanical ventilation. VILI results
in the ongoing release of inflammatory mediators
that may spill over into the circulation and propagate
systemic inflammation, thereby influencing mortality.
Indeed, increased plasma levels of cytokines have
been demonstrated in animal models and in patients
receiving injuriously large tidal-volume ventilation.
This theory has stimulated the development of socalled ‘protective’ strategies for mechanical ventilation which minimize VILI.
Tidal volume Delivery of a normal tidal volume to
extensively consolidated lungs inevitably results in
overdistension of the remaining lung units. Experimental work demonstrated that this may be a significant trigger for VILI, and resulted in the National
Institutes of Health (NIH)-sponsored study of low
tidal-volume ventilation in 861 patients. This landmark study showed that mortality could be reduced
by the use of lower tidal volumes (4–6 ml kg 1, ideal
body mass) or at least the avoidance of more ‘traditional’ tidal volumes (10–12 ml kg 1). The corresponding plateau inspiratory pressures were 25 and
35 cmH2O, respectively. Plasma and bronchoalveolar
lavage cytokine and chemokines levels were greater
in those patients receiving higher tidal volumes. The
study protocol has been adopted by some as the definitive ventilatory strategy. More correctly, it provided excellent evidence for VILI and should form
the basis of lung-protective strategies. Indeed, it has
been suggested that targeting lower-end expiratory
alveolar pressures may be more sensible, since the
specific pulmonary compliance (compliance corrected for accessible lung volume) has been reported
as normal in patients with ALI/ARDS.
Smaller tidal volumes may reduce VILI by virtue of
the reduced cyclical volume per se, or through a reduction in the end-inspiratory volume. It is not clear
16
ACUTE RESPIRATORY DISTRESS SYNDROME
which is the important factor although in vitro experiments on mechanically deformed epithelial layers
have demonstrated that cyclical changes are more
damaging than constant stretch to a high volume.
How these results translate to the in vivo scenario is
not clear.
In the absence of increased respiratory rates,
reductions in tidal volume will reduce alveolar ventilation and result in a hypercapnic acidosis. Permissive hypercapnia is generally well tolerated except in
the setting of a marked metabolic acidosis and increased intracranial pressure. Hypercapnia may also
reduce myocardial contractility, and possibly increase the need for sedation and/or paralysis. The
effects on the immune response are still unclear. The
degree of hypercapnic acidosis allowable is unclear
although many clinicians accept arterial pH values
not less than 7.20. When titrating respiratory rate to
pCO2 , it must be remembered that increases in respiratory rate will have less influence on alveolar ventilation in ARDS due to the increased dead space,
and that reductions in respiratory rate can paradoxically increase CO2 clearance when inspiratory times
are particularly prolonged. Tracheal insufflation of
oxygen and administration of weak bases are sometimes used to combat the acidosis.
Fractional inspired oxygen High fractions of inspired oxygen (FiO2) cause absorption atelectasis
and may be cytotoxic to the lung per se. The standard practice is to titrate the FiO2 to arterial saturations of 88–92%, rather than a specific pO2 which
may be less relevant to oxygen delivery. Lower targets may be appropriate in those patients whose
cardiac output is high or where an improvement
of SaO2 would require an unacceptably injurious
pattern of ventilation (e.g., through a higher FiO2,
positive end expiratory pressure (PEEP), or tidal volume). The benefits of further reductions in FiO2 afforded by the use of inhaled nitric oxide (iNO),
prone positioning, and related maneuvers are unknown and still subject to review (vide infra). Intrapulmonary shunting may result in arterial oxygen
saturations being relatively independent of FiO2 between 0.8 and 1.0.
Positive end expiratory pressure The application of
PEEP during mechanical ventilation increases FRC
and thus prevents the collapse of alveoli at end expiration. This will reduce intrapulmonary shunt and
improve oxygenation. Furthermore, the shear forces
required to repeatedly open collapsed alveoli during
inspiration are considerable and most likely contribute to VILI. Thus, by keeping these alveoli open
throughout the respiratory cycle, PEEP can limit
VILI. Since the lung is heterogeneously affected in
ARDS, excessively high levels of PEEP can lead to
overexpansion of those regions whose compliance is
higher, an effect that may exacerbate VILI. Other
detrimental effects of PEEP include reduced cardiac
preload and cerebral perfusion.
The method to determine the optimal level of
PEEP in ARDS is still not established. A randomized
multicenter trial in 549 patients, titrating PEEP according to the required FiO2, found no outcome differences between a lower and higher PEEP algorithm.
Many clinicians set PEEP by inferring the region of
maximal slope on the pressure–volume curve by assessing maximal tidal volume as PEEP is adjusted
with a constant pressure control. Mathematical
modeling suggests that this is best determined using
a decremental rather than an incremental PEEP trial.
Ventilatory modes The NIH trial of lower tidal-volume ventilation used a mandatory constant flow, volume control mode of mandatory ventilation. There is
considerable interest in the role of descending flow/
pressure control modes since these may promote more
even distribution of gas, as flow is slower at the end of
expiration and thus more likely to be laminar. Pressure-control modes do not influence peak alveolar
pressure, although peak airway pressures may be reduced. The most likely advantage afforded by pressure control ventilation and inverse ratio ventilation is
that these may be associated with a greater proportion of the respiratory cycle spent at plateau inspiratory pressure. Greater plateau times promote more
homogenous ventilation by allowing slow-filling regions to inter-fill from areas with faster time constants. The recruitment of previously collapsed or
partially collapsed slow-filling alveoli reduces shunt
and improves oxygenation. Prolongation of inspiration can also improve CO2 clearance by forcing expiration to be delayed to a point where alveolar pCO2
has risen to a maximum. Faster respiratory rates reduce the efficacy of inverse ratio ventilation by shortening the inspiratory time. Excessive prolongation of
inspiration may reduce expiratory time to a point at
which expiration is not completed. This may result in
dynamic hyperinflation and the generation of autoPEEP. Most clinicians consider auto-PEEP undesirable since levels may be unpredictable thus leading to
cardiovascular compromise.
There are theoretical advantages to modes that allow spontaneous ventilation such as biphasic positive
airway pressure and airway pressure release ventilation. These modes allow continued diaphragmatic
and intercostal function and the ventilation of dependent, better-perfused regions. Although pressure
support modes also allow spontaneous ventilation,
ACUTE RESPIRATORY DISTRESS SYNDROME 17
some argue against them as they preclude any plateau time.
No study to date, excluding those examining
weaning, has demonstrated any outcome differences
according to ventilatory mode.
Drainage of pneumothoraces Multiple pneumothoraces may complicate mechanical ventilation of patients with ARDS. Pneumothoraces can be complex
and localized as the inflamed lung may tether to the
chest wall. Indeed, localization can be difficult by conventional radiography and it may be necessary to insert drains under the guidance of CT. Intercostal drains
are generally left in situ until the patient is well established on a spontaneous ventilatory weaning program.
Supplemental Techniques for Mechanical
Ventilation
Prone positioning Ventilation in the prone position
improves oxygenation in approximately 70% of patients. It is not possible to predict which patients will
respond, and nonresponders may improve on subsequent turns. Experienced teams can easily turn
patients, even with multiple intercostal drains and
intravascular catheters, with few complications. Improvements in oxygenation continue in 50% of patients after they are returned to the supine position
and are probably the consequence of expansion of
previously collapsed lung. More homogenous ventilation may prevent regional overdistension and limit
VILI. Improved secretion drainage and altered diaphragmatic mechanics may also contribute to clinical
improvement. Despite improvements in oxygenation,
prone positioning has not been demonstrated to improve outcome in ARDS.
Recruitment maneuvers Sustained and high airway
pressures of up to 60 cmH2O may be required to
open (or recruit) some regions of collapsed lung.
There has been considerable enthusiasm for the use
of such recruitment maneuvers which, in combination with the application of PEEP, may keep these
newly recruited lung units open. Techniques vary
from sophisticated determination of recruitment and
derecruitment by stepwise alteration of ventilatory
pressures and repeated blood gas sampling through
to simply temporarily increasing PEEP to 30 cmH2O
for 30 s. Improvements in oxygenation have often
been demonstrated, although these may not be sustained. Such high intrathoracic pressures are often
accompanied by brief episodes of reduced cardiac
output and hypotension, but rarely barotrauma. The
role of recruitment maneuvers still has to be clarified
in ARDS.
Partial liquid ventilation Partial liquid ventilation
has been proposed to improve oxygenation and limit
VILI. Perfluorocarbons are dense liquids that have
little biological activity but very low surface tension
and exceptionally high-solubility coefficients for oxygen and carbon dioxide. The lung is filled (partially
or fully) with the perfluorocarbon and then normal
mechanical ventilation applied simultaneously. Since
the perfluorocarbon is volatile, it evaporates over
time through the ventilator circuit. Animal studies
suggest that perfluorocarbons reduce atelectasis,
improve perfusion matching, and improve secretion
clearance. Some studies suggest they may also be
anti-inflammatory, reducing permeability rises and
neutrophil activation. Human studies have revealed
an increase in the incidence of pneumothoraces, mucus plugging, and a disruption of the normal surfactant system. They are ingested by macrophages and
their effect on immune function is unclear. A phase
II/III study of partial-liquid ventilation is underway
in France currently.
Extracorporeal gas exchange Traditionally, extracorporeal gas exchange (ECGE) has only been used
as a last resort in patients in whom oxygenation
cannot be maintained by any other means. Devices
vary from those which oxygenate and support the
whole cardiac output, to those that provide partial
oxygenation, and carbon dioxide removal to supplement conventional ventilation at lower pressures
(extracorporeal lung assist, ECLA). Hemorrhage and
coagulopathies have been the most significant complications, occurring in about 67% of adult patients.
Although no mortality advantage has ever been
shown for ECGE, new technology such as heparinbonded circuits, nonocclusive roller pumps, pumpless circuits, and better anticoagulation control, that
may reduce complications is leading to a re-evaluation of this modality.
Surfactant administration Alveolar surfactant is
lost early in ARDS. The administration of exogenous
surfactant either via a bronchoscope or nebulizer
may reduce surface tension within alveoli and prevent cyclical collapse, improving oxygenation, and
ameliorating VILI. Multiple administrations appear
necessary due to the neutralization of surfactant by
edema fluid. Despite improvements in oxygenation
and phase I/II survival benefits, no large studies of
surfactant have demonstrated any mortality advantage and thus it is not administered to patients with
ARDS. However, there are many outstanding questions regarding the optimal formulation, patient
selection, and dosing regimen.
18
ACUTE RESPIRATORY DISTRESS SYNDROME
Inhaled vasodilators iNO and prostacyclin cause
selective vasodilatation of the regions of the lung
to which they are delivered by ventilation, thus
improving ventilation/perfusion matching and oxygenation. Their short half-lives limit systemic vasodilatation and hypotension. Both agents typically
improve arterial pO2 by about 20% in responders,
and this may be enhanced by the administration of
intravenous almitrine bismesylate, an agent that is
reported to enhance hypoxic pulmonary vasoconstriction. iNO is usually maximally effective at inspired concentrations of less than 10 parts per
million, although the dose–response curve may vary
considerably after 24 h. iNO and prostacyclin may
also favorably influence inflammation, platelet activity, and vascular permeability. Although frequently
used for their beneficial effects on oxygenation, no
survival benefit has been shown for either agent in
controlled studies.
High-frequency ventilation The corollary of the
fact that low tidal-volume ventilation is beneficial is
that high-frequency ventilation may further protect
against VILI. Tidal volumes delivered by jet ventilators and oscillators are less than the anatomical dead
space, and respiratory rates are greater than 1 Hz.
Gas transport occurs via diffusion rather than bulk
flow. To date, no mortality advantage has been demonstrated for these ventilatory techniques in adults.
Noninvasive ventilation Noninvasive ventilation
(NIV) avoids the need for endotracheal intubation
and the associated risks of ventilator-associated
pneumonias, sinusitis, and sedation. The use of
NIV in hypoxemic respiratory failure is controversial and a consensus conference into the role of NIV
in acute respirator failure found little data to support
its use in ARDS. Indeed, the natural history of ARDS
is often longer than the time for which many patients
are able to tolerate tight-fitting masks continuously.
It may thus only delay endotracheal intubation.
Manipulation of the Alveolar Fluid Balance
High pulmonary capillary pressures increase extravascular lung water and result in worsening oxygenation. Indeed, a persistently positive fluid balance is
associated with a worse outcome in ARDS. It is difficult to ascertain whether this association is due to
detrimental effects of administered fluids per se or
simply a reflection of the severity of the underlying
illness requiring fluids for cardiovascular support.
Nevertheless, whilst it is critical to maintain organ
perfusion with an adequate cardiac output, the administration of intravenous fluids in the absence of
evidence of organ hypoperfusion would seem inappropriate. The careful use of vasopressors may allow
the limitation of fluid administration.
Apical sodium and probably chloride channels in
alveolar epithelial cells play an important role in
normal alveoli in maintaining a flux of water into the
circulation. A subgroup of the sodium channels can
be stimulated by b-agonists such as salbutamol and
terbutaline which may be administered either as inhaled preparations or as continuous intravenous infusion. Recent data suggest that salbutamol infusions
can reduce extravascular lung water. How this influences outcome in ARDS is not known and thus these
agents remain experimental.
Manipulation of the Inflammatory Process
Corticosteroids Corticosteroids can reduce inflammation by repressing transcription and destabilizing
pro-collagen mRNA. Initially evaluated in large doses
in patients considered at risk of ARDS or early in
ARDS, they have no beneficial effects on mortality. By
contrast, their administration later when repair and
remodeling have commenced, may be helpful. Possible negative effects include nosocomial infection,
hyperglycemia and an exacerbation of critical illness
poly(myo)neuropathy. In a study of only 24 patients,
corticosteroids caused a significant improvement in
oxygenation and reduced mortality in patients with
unresolving ARDS. Methylprednisolone (2 mg kg 1
day 1) was administered from day 8 for the shorter
of either the duration of mechanical ventilation or 14
days. Although the study was methodologically limited, many clinicians administer a similar regimen to
patients with persistent ARDS after 7 days and in
whom there is little evidence for systemic sepsis. The
results of the North American Late Steroid Rescue
Study (LaSRS) will shortly provide better evidence
regarding the role of corticosteroids in ARDS.
Other agents Other pharmacological agents that
have been assessed in ARDS/ALI are outlined in
Table 3. None have been demonstrated to improve
mortality despite good rationale for their use and
promising preliminary studies.
Outcome
Over the last ten years, mortality has fallen from
around 60% to around 30% in the current major
trials. Although multifactorial in origin, this is in part
due to improvement in the whole critical-care process but a greater appreciation that inappropriate mechanical ventilation can further injure the inflamed
lung. Most patients succumb to multi-organ failure
ADAMs AND ADAMTSs 19
Table 3 Other Pharmacological agents assessed in ARDS/ALI
Agent
Rationale
Effects
Prostaglandin E1
A direct pulmonary vasodilator, inhibitor of platelet aggregation,
and inhibitor of neutrophil adhesion
Mortality unaltered
More rapid improvement in
oxygenation
Dazoxiben
Inhibitor of thomboxane synthase and thus acts as a pulmonary
vasodilator and inhibitor of platelet aggregation
Mortality unaltered
Ketoconazole
Inhibitor of thomboxane synthase and 5-lipoxygenase, thus
reducing production of leukotrienes, neutrophil chemokines
Mortality unaltered
N-acetyl cysteine
Antioxidant that reduces damage by reactive oxygen species
Mortality unaltered
Improved oxygenation and compliance
Lisofylline
Inhibitor of phosphatidic acid which increases cytokine
production and activates neutrophils
Mortality unaltered
or sepsis rather than specific pulmonary failure and
an inability to maintain adequate oxygenation.
Follow-up of patients who survive ARDS has demonstrated progressive improvement in their lung
function that continues to improve even one year
after discharge from the ICU. Lung volumes seem to
improve more rapidly than diffusing capacity and the
distance walked in six min. Radiologically, previously densely consolidated areas frequently appear
normal, with fibrosis of nondependent regions. By
contrast, survivors often have considerable long-term
nonrespiratory morbidities, particularly related to
physical strength and to psychosocial, functional,
and stress indices. Indeed, less than 50% return to
work within 12 months.
See also: Corticosteroids: Therapy. Extracorporeal
Membrane-Gas Exchange. Fluid Balance in the
Lung. Genetics: Gene Association Studies. Hypoxia
and Hypoxemia. Leukocytes: Neutrophils. Lung Imaging. Nitric Oxide and Nitrogen Oxides. Oxygen
Therapy. Oxygen Toxicity. Surfactant: Overview.
Ventilation, Mechanical: Positive Pressure Ventilation.
Further Reading
Brower RG, Lanken PN, MacIntyre N, et al. (2004) Higher versus
lower positive end-expiratory pressures in patients with the
acute respiratory distress syndrome. New England Journal of
Medicine 351: 327–336.
Evans TW, Griffiths MJD, and Keogh BF (eds.) (2002) European
Respiratory Monograph: ARDS, vol. 70(20). Sheffield: ERS
Journals.
Herridge MS, Cheung AM, Tansey CM, et al. (2003) One-year
outcomes in survivors of the acute respiratory distress syndrome. New England Journal of Medicine 348: 683–693.
Meduri GU, Headley AS, Golden E, et al. (1998) Effect of prolonged methylprednisolone therapy in unresolving acute respiratory distress syndrome: a randomized controlled trial. Journal
of the American Medical Association 280: 182–183.
Pinhu L, Whitehead T, Evans T, and Griffiths M (2003) Ventilatorassociated lung injury. Lancet 361: 332–340.
The Acute Respiratory Distress Syndrome Network (2000) Ventilation with lower tidal volumes as compared with traditional
tidal volumes for acute lung injury and the acute respiratory
distress syndrome. New England Journal of Medicine 342:
1301–1308.
Ware LB and Matthay MA (2000) The acute respiratory distress syndrome. New England Journal of Medicine 342:
1334–1349.
ADAMs AND ADAMTSs
C P Blobel, Cornell University, New York, NY, USA
S S Apte, Cleveland Clinic Foundation, Cleveland, OH,
USA
& 2006 Elsevier Ltd. All rights reserved.
Abstract
Proteolysis has emerged as a key posttranslational regulator
of the function of molecules on the cell surface and in the
extracellular milieu. In principle, proteolysis can activate or inactivate a substrate, or can change its functional properties.
ADAM (a disintegrin and metalloprotease) and ADAMTS (a
disintegrin-like and metalloprotease domain with thrombospondin type 1 repeats) proteases are related members of a superfamily of metalloendopeptidases that also includes matrix
metalloproteinases (MMPs) and astacins. ADAMs are integral
membrane proteins that typically cleave other membrane anchored proteins, whereas ADAMTS proteases lack a membrane
anchor, and process both secreted and cell surface molecules.
20
ADAMs AND ADAMTSs
ADAMs are implicated in fertilization, neurogenesis, regulation
of the function of ligands for the epidermal growth factor receptor, and the release of proteins such as the proinflammatory
cytokine tumor necrosis factor alpha (TNF-a) from the plasma
membrane. ADAMTS proteases have key roles in the molecular
maturation of von Willebrand factor and procollagen and are
implicated in the pathogenesis of osteoarthritis. Here, we provide a general overview of the biochemical properties and physiological functions of ADAMs and ADAMTS proteases and
describe their relevance to lung and airway disorders.
ADAMs
Introduction
ADAMs (a disintegrin and metalloprotease) are a
family of membrane anchored glycoproteins that
have been implicated in cleaving and releasing proteins from the cell surface. This process, which is
referred to as protein ectodomain shedding, affects
the function of molecules with key roles in development and disease, including growth factors such as
transforming growth factor alpha (TGF-a), heparinbinding epidermal growth factor (HB-EGF), cytokines such as tumor necrosis factor alpha (TNF-a),
and receptors such as the tumor necrosis factor receptor 1(TNFR1). The first recognized ADAMs were
the two subunits of a sperm protein termed fertilin,
which has a critical role in fertilization. Other
ADAMs were subsequently identified based on their
sequence homology to fertilin, or based on functional
assays, such as their ability to shed the proinflammatory cytokine TNF-a or process and activate the
cell surface receptor Notch.
Structure
The typical domain organization of an ADAM is
shown in Figure 1. Of the over 30 ADAMs that have
been identified to date, only about half contain a
catalytic site consensus sequence and are therefore
predicted to be catalytically active. The remaining
ADAMs that are not catalytically active are thought
to function mainly in cell–cell or cell–matrix interactions.
Regulation of Production and Activity
All catalytically active ADAMs are synthesized
with a pro-domain that helps the metalloprotease
domain fold in the endoplasmic reticulum (ER), and
keeps it inactive until the pro-domain is cleaved,
usually just before reaching the trans-Golgi network.
Once the pro-domain is removed, additional
mechanisms, including phorbol esters, phosphatase
inhibitors, calcium ionophores, and activation of
G-protein-coupled receptors, regulate the catalytic
activity of ADAMs. The disintegrin domain and
cysteine-rich region of ADAMs are thought to have a
role in cell–cell interaction and potentially also in
Cell
ADAMs
Pro
Catalytic
Disintegrin
CRD
EGF-like
TM
TSR
(0−14)
TSR
Cytosolic
ADAMTSs
Pro
Catalytic
Protease domain
Disintegrin-like
CRD
Spacer
Ancillary domain
Figure 1 All ADAMs are membrane-anchored glycoproteins that are characterized by a conserved domain structure: an N-terminal
signal sequence followed by pro (Pro), metalloprotease, and disintegrin domains, a cysteine-rich region (CRD), usually containing an
EGF repeat, and finally a transmembrane (TM) domain and cytoplasmic tail. Only about half of the known ADAMs have a catalytic site
consensus sequence, and are therefore predicted to be catalytically active. The disintegrin domain was first identified in snake venom
toxins that bind to platelet integrins and function as anticoagulants, but is now known to be a characteristic feature of all ADAM and
ADAMTS proteins. ADAMTS proteins are similar to ADAMs in the prometalloprotease domain, but unlike ADAMs, all ADAMTSs are
catalytically active. ADAMTSs have a functionally critical ancillary domain containing specific modules not found in ADAMs, e.g.,
thrombospondin type 1 repeats (TSRs) (see text for details). A critical difference between these protease families is the absence of an
integral membrane segment in ADAMTS proteases.
ADAMs AND ADAMTSs 21
substrate recognition. Finally, the cytoplasmic domain of catalytically active ADAMs usually contains
signaling motifs such as potential phosphorylation
sites and proline-rich Src-homology 3 (SH3) ligand
domains, and several molecules that interact with
the cytoplasmic domains of ADAMs and might
regulate their maturation and function have been
identified.
The catalytic activity and substrate selectivity of
ADAMs has been explored using both biochemical
and cell biological approaches. One important conclusion from biochemical studies was that individual
ADAMs do not have a clear consensus cleavage site
in vitro. Since ADAMs and their substrates are both
membrane anchored, cell-based assays are critical
tools for understanding the substrate selectivity and
regulation of ADAMs in the context of the plasma
membrane. Studies using cells from ADAM knockout mice, or cells treated with small inhibitory RNA
(siRNA) against different ADAMs, have revealed
that these enzymes display substrate selectivity in
cells, although the mechanism underlying this remains to be established. A common feature, however,
is that ADAMs frequently cleave their substrates
close to the plasma membrane, resulting in release or
‘ectodomain shedding’ of the substrate’s soluble ectodomain.
Biological Function
As noted above, ADAM-dependent ectodomain
shedding can profoundly affect the function of the
released substrate protein. Ectodomain shedding can
enable the released molecule to act at a distance from
the cell that it was shed from, which is referred to as
paracrine signaling. For example, processing of the
EGF receptor (EGFR) ligands TGF-a and HB-EGF
by ADAM17 is critical for activation of the EGFR
during development, and therefore mice lacking
ADAM17 resemble mice lacking TGF-a or HB-EGF
or EGFR. Ectodomain shedding is also a key mediator of the role of TNF-a in autoimmune diseases
such as rheumatoid arthritis. Interestingly, receptors
can be either inactivated by ectodomain shedding,
such as the TNFR1, or activated, such as Notch.
Mutations in the cleavage site of the TNFR1 that
decrease its shedding cause accumulation of the receptor, leading to increased susceptibility to TNF in
patients with TNF-receptor associated periodic febrile syndrome (TRAPS).
Function of ADAMs in Lung Development and
in Respiratory Diseases
Currently, ADAM17 is the only ADAM with a clearly
established role in lung development (Figure 2).
Mice lacking ADAM17 are born with respiratory
distress, presumably caused by abnormal alveoli with
septation defects and thickened mesenchyme, as well
as impaired branching morphogenesis and delayed
vasculogenesis, and thus reduced surface for gas exchange. Since similar defects are observed in mice
lacking HB-EGF or the EGFR, the abnormal lung
development in Adam17 / mice is most likely explained by a lack of HB-EGF shedding. With respect
to respiratory diseases, smoking has been implicated
in the activation of ADAMs and the release of EGFR
ligands such as amphiregulin. The resulting activation of the EGFR can presumably contribute to the
pathogenesis of lung cancer by stimulating cell proliferation and DNA replication at the same time that
mutagens are delivered in smoke. Moreover, Grampositive bacteria stimulate the G-protein-coupled
platelet activating receptor (PAR) in patients with
cystic fibrosis, which in turn activates ADAMdependent release of HB-EGF, and thus mucin production. Therefore, inhibitors of ADAMs, such as
hydroxamic acid type metalloprotease inhibitors,
HB-EGF
HB-EGF
ADAM17
Extracellular space
Cell
membrane
Cytoplasm
Figure 2 Proteolytic processing of heparin-binding EGF-like growth factor (HB-EGF) by ADAM17 releases this growth factor from its
membrane tether. The ADAM17-dependent ectodomain shedding of HB-EGF is thought to be critical for the function of this growth factor
during lung and heart development.
22
ADAMs AND ADAMTSs
might be useful in the treatment of cystic fibrosis.
Finally, mutations in the ADAM33 gene have been
linked to asthma susceptibility, although the mechanism underlying the role of ADAM33 in asthma
remains to be determined. In light of the key roles of
ADAMs in regulating signaling via the EGF receptor
and other cell surface signaling pathways, and
the critical roles for ADAMs in lung development
and in asthma and cystic fibrosis, it appears likely
that further studies of this protein family in the context of respiratory disease will uncover novel functions, thus hopefully also providing new targets for
drug design.
ADAMTSs
Introduction
ADAMTS (a disintegrin-like and metalloprotease
domain (reprolysin type) with thrombospondin type
1 repeats) comprises a family of 19 secreted metalloproteases that is distinct from the membrane-anchored ADAMs. The founding member of this
family, ADAMTS1, was described very recently, in
1997. ADAMTS1 was so named because it resembled the ADAMs in the metalloprotease domain and
disintegrin-like module and was thought to be a variant ADAM. Its main point of distinction from
ADAMs, apart from the absence of a transmembrane
segment, was the presence of three modules resembling thrombospondin type 1 repeats (TSRs). Soon
afterwards, it became clear that all 19 ADAMTS
proteases shared these major features.
Structure
A typical ADAMTS consists of prometalloprotease
and ancillary domains. The prometalloprotease domains resemble ADAMs, since the active site
sequence is of the reprolysin (snake venom) type.
Basic amino acid rich sequences providing sites
for removal of the pro-domain by subtilisin-like proprotein convertases (SPCs) are present in the prometalloprotease domain and at its junction with
the catalytic domain. The ancillary domain (from
N-terminus to C-terminus) consists of a disintegrinlike module, a central TSR, a cysteine-rich module, a
cysteine-free spacer, and a variable number of additional TSRs, ranging from 0 (ADAMTS4) to 14
(ADAMTS9 and 20) (Figure 1). An interesting feature of ADAMTS proteases is their clear grouping
into distinct subfamilies. Proteases within a subfamily have an identical modular organization, gene
structure, and active site sequence, suggesting evolution by gene duplication from a common precursor.
Each subfamily has a distinct modular organization,
for example, ADAMTS7 and ADAMTS12, constituting one such subfamily, are the only metalloproteases known to have mucin modules and
glycosaminoglycan attachment sites.
Regulation of Production and Activity
Transcriptional regulation appears to be very important, since many ADAMTS mRNAs are highly
regulated during embryogenesis, for example, ADAMTS2, 3, and 14, or induced in specific circumstances such as inflammation, for example,
ADAMTS1. ADAMTS proteases are synthesized as
zymogens and undergo removal of the prometalloprotease domain by SPCs either within the secretory
pathway or at the cell surface. Subsequent to SPC
processing, proteolysis within the ancillary domain
appears to be an important posttranslational regulator of activity, resulting in further activation or
inactivation of the enzymes. Several ADAMTS proteases bind to the cell surface through unknown
mechanisms that suggest activity as operational cell
surface proteases, and indicate locations at which
posttranslational processing may occur. Unlike
ADAMs, the ADAMTSs typically attack specific
cleavage sites in their substrates. For instance, ADAMTS4 is a glutamyl endopeptidase that processes
Glu–Xaa bonds in many proteoglycans and ADAMTS13 specifically cleaves von Willebrand factor
(vWF) at the Tyr1605–Met1606 peptide bond.
An important concept in the ADAMTS family is
that their catalytic domains alone do not retain
activity towards natural substrates. The ancillary
domain is critical for the recognition and binding of
substrates. In many instances, this requires specific posttranslational modifications in the substrates
(such as glycosylation of proteoglycans, triplehelicity in the case of procollagens, and physical
unfolding of vWF by fluid shear force). In addition,
the minimal substrate binding sites are quite
extended in length and difficult to reproduce in synthetic peptides. This makes the development of
assays for ADAMTS activity and inhibition quite
challenging. The only known ADAMTS inhibitors
are tissue inhibitor of metalloprotease 3 (TIMP-3)
and a2-macroglobulin.
Biological Function
Unexpectedly diverse functions for ADAMTS proteases have been revealed through human genetic
disorders and transgenic animals. All ADAMTS genetic disorders are recessive, as is typical of enzyme
deficiencies. Inherited thrombocytopenic purpura results from ADAMTS13 mutations, with retention of
unusually large polymers of vWF and failure to
ADAMs AND ADAMTSs 23
process these into forms that are optimal for coagulative homeostasis. Acquired thrombocytopenic
purpura may result from circulating anti-ADAMTS13 autoantibodies. Ehlers–Danlos syndrome
(type VIIC or the dermatosparactic type) is a consequence of ADAMTS2 mutations. In this disorder,
tissue (especially skin) fragility results from lack of
complete procollagen processing and a decrease in
structurally competent collagen fibrils. ADAMTS1 is
needed for mouse urinary tract development and fertility, and inhibits angiogenesis in vitro and in vivo
by binding to vascular endothelial growth factor
(VEGF). ADAMTS4 and ADAMTS5 null mice are
developmentally normal, but ADAMTS5 mice are
resistant to both mechanically induced and immune
arthritis, suggesting that ADAMTS5 is a major cartilage-degrading enzyme. ADAMTS10 mutations
cause Weill–Marchesani syndrome (WMS), comprising short stature, brachydactyly, cardiovascular defects, and ectopia lentis. Intriguingly, several aspects
of WMS are the opposite of those seen in Marfan
syndrome, a fairly common inherited connective tissue disorder. ADAMTS20 mediates melanoblast migration from the neural crest and is deficient in a
natural mouse mutant named Belted because of the
belt of white fur across the torso.
Receptors
Although some ADAMTS proteases can bind to
the cell surface, a specific receptor, syndecan-1, has
been identified only for ADAMTS4. Nevertheless,
because of the affinity of many ADAMTS for heparin
and chondroitin sulfate, cell surface proteoglycans
may constitute a broad category of receptors for this
family.
ADAMTS Proteases in Respiratory Diseases
Since thrombocytopenic purpura is a systemic coagulation disorder, patients can develop microthrombi
in their lungs and have sometimes manifested with
acute respiratory distress syndrome. Pneumothorax
has occasionally been reported in Ehlers–Danlos syndrome type VIIC patients and Adamts2 null mice
have widening of their distal air spaces. These mice
show deficiency of procollagen I as well as procollagen III processing. Both collagens are structurally
major components of the lung. Although ADAMTS10 is highly expressed in the lung, WMS cases
are not reported to have lung problems.
See also: Asthma: Overview. Epidermal Growth
Factors. Matrix Metalloproteinases. Tumor Necrosis Factor Alpha (TNF-a ).
Further Reading
Apte SS (2004) A disintegrin-like and metalloprotease (reprolysin
type) with thrombospondin type 1 motifs: the ADAMTS family.
International Journal of Biochemistry and Cell Biology 36:
981–985.
Becherer JD and Blobel CP (2003) Biochemical properties and
functions of membrane-anchored metalloprotease-disintegrin
proteins (ADAMs). Current Topics in Developmental Biology
54: 101–123.
Blobel CP (2005) ADAMs: key players in EGFR-signaling, development and disease. Nature Reviews: Molecular Cell Biology 6:
32–43.
Dagoneau N, Benoist-Lasselin C, Huber C, et al. (2004) ADAMTS10 mutations in autosomal recessive Weill–Marchesani
syndrome. American Journal of Human Genetics 75: 801–806.
Gao G, Plaas AH, Thompson VP, et al. (2004) ADAMTS4
(aggrecanase-1) activation on the cell surface involves C-terminal cleavage by GPI-anchored MT4-MMP and binding of the
activated proteinase to chondroitin sulfate and heparan sulfate on
syndecan-1. Journal of Biological Chemistry 279: 10042–10051.
Kheradmand F and Werb Z (2002) Shedding light on sheddases:
role in growth and development. BioEssays 24: 8–12.
Kuno K, Kanada N, Nakashima E, et al. (1997) Molecular cloning
of a gene encoding a new type of metalloproteinase-disintegrin
family protein with thrombospondin motifs as an inflammation
associated gene. Journal of Biological Chemistry 272: 556–562.
Lapiere CM and Nusgens BV (1993) Ehlers–Danlos type VII-C, or
human dermatosparaxis: the offspring of a union between basic
and clinical research. Archives of Dermatological Research 129:
1316–1319.
Lemjabbar H and Basbaum C (2002) Platelet-activating factor
receptor and ADAM10 mediate responses to Staphylococcus
aureus in epithelial cells. Nature Medicine 8: 41–46.
Lemjabbar H, Li D, Gallup M, et al. (2003) Tobacco smoke-induced lung cell proliferation mediated by tumor necrosis factor
alpha-converting enzyme and amphiregulin. Journal of Biological Chemistry 278: 26202–26207.
Levy GG, Nichols WC, Lian EC, et al. (2001) Mutations in a
member of the ADAMTS gene family cause thrombotic
thrombocytopenic purpura. Nature 413: 488–494.
Porter S, Clark IM, Kevorkian L, and Edwards DR (2005) The
ADAMTS metalloproteinases. Biochemical Journal 386: 15–27.
Shapiro SD and Owen CA (2002) ADAM-33 surfaces as an
asthma gene. New England Journal of Medicine 347: 936–938.
Stanton H, Rogerson FM, East CJ, et al. (2005) ADAMTS5 is the
major aggrecanase in mouse cartilage in vivo and in vitro.
Nature 434: 648–652.
White JM (2003) ADAMs: modulators of cell–cell and cell–matrix
interactions. Current Opinion in Cell Biology 15: 598–606.
Zhao J, Chen H, Peschon JJ, et al. (2001) Pulmonary hypoplasia in
mice lacking tumor necrosis factor-alpha converting enzyme indicates an indispensable role for cell surface protein shedding
during embryonic lung branching morphogenesis. Developmental Biology 232: 204–218.
24
ADENOSINE AND ADENINE NUCLEOTIDES
ADENOSINE AND ADENINE NUCLEOTIDES
R Polosa, University of Catania, Catania, Italy
D Zeng, CV Therapeutics, Palo Alto, CA, USA
& 2006 Elsevier Ltd. All rights reserved.
Abstract
Adenosine is a purine nucleoside. A growing body of evidence
has emerged in support of a proinflammatory and immunomodulatory role for adenosine in the pathogenic mechanisms of
chronic inflammatory disorders of the airways such as asthma
and chronic obstructive pulmonary disease (COPD). The fact
that adenosine enhances mast cell allergen-dependent activation, that elevated levels of adenosine are present in chronically
inflamed airways, and that adenosine given by inhalation causes
dose-dependent bronchoconstriction in subjects with asthma
and COPD emphasizes the importance of adenosine in the initiation, persistence, and progression of these common inflammatory disorders of the airways. These distinctive features of
adenosine have been recently exploited in the clinical and
research setting to identify innovative diagnostic applications for asthma and COPD. In addition, because adenosine
exerts its multiple biological activities by interacting with four
adenosine receptor subtypes, selective activation or blockade of
these receptors may lead to the development of novel therapies
for asthma and COPD. In this article, the evidence for the
roles of adenosine receptors in the pathophysiology of chronic
inflammatory airway diseases such as asthma and COPD are
reviewed.
Introduction
The cardiovascular actions of adenosine were first
described in a classic publication by Drury and SzentGyorgyi in 1929. It was shown that adenosine causes
coronary vasodilation, hypotension, and bradycardia. In 1963, Berne proposed the hypothesis that
adenosine mediates the metabolic regulation of coronary blood flow. Since then, a large body of literature has supported the critical role of adenosine in
modulating numerous cardiovascular functions. Two
well-characterized effects of adenosine have successfully found clinical applications. First, adenosine
causes bradycardia, slows A-V nodal conduction,
and reduces atrial contractility, and is used as a rapid
intravenous bolus for the acute termination of
re-entrant supraventricular arrhythmias. Second,
adenosine causes coronary vasodilation and increased blood flow, and it is used with radionuclide
imaging in the heart to detect underperfused areas of
myocardium.
The action of adenosine in pulmonary diseases
was first discovered in the late 1970s. Holgate and
colleagues observed that adenosine and related synthetic analogs were potent agents in augmenting
IgE-dependent mediator release from isolated rodent
mast cells. A few years later, adenosine (but not its
metabolite inosine or the unrelated nucleoside guanosine) administered by inhalation was shown to be
a powerful bronchoconstrictor of asthmatic but, importantly, not of normal airways.
Asthma and chronic obstructive pulmonary disease
(COPD) are complex syndromes sharing clinical heterogeneity and a number of pathogenic traits, which
include variable degrees of airflow obstruction, bronchial hyperresponsiveness (BHR), and chronic airway inflammation. In addition to its known effect as
a bronchoconstrictor, a growing body of evidence has
emphasized the importance of adenosine in the initiation, progression, and control of chronic inflammation and remodeling of the airways. The key
evidence can be summarized as follows:
1. Adenosine is generated in high concentrations at
sites of tissue injury (e.g., hypoxia and inflammatory cell activation).
2. Elevated levels of adenosine are present in chronically inflamed airways; they have been observed
both in the bronchoalveolar lavage (BAL) fluid
and the exhaled breath condensate (EBC) of patients with asthma, and adenosine concentrations
are also increased after specific allergen challenge
in the plasma of atopic individuals and in the BAL
fluid obtained from sensitized rabbits.
3. The progressive accumulation of adenosine in the
lungs of mice lacking the purine catabolic enzyme
adenosine deaminase (ADA) is strongly associated
with development of lung inflammation, tissue
eosinophilia, airway hyperresponsiveness mucus
metaplasia, airway remodeling, and emphysemalike injury of the lung parenchyma.
4. Adenosine administration by inhalation induces
concentration-dependent bronchoconstriction in
subjects with asthma and COPD whereas the
nucleoside had no discernible effect on airway
caliber in normal individuals.
5. Adenosine augments allergen-induced mediator release from human mast cells in vitro, and potentiates
the immediate response to allergen in asthmatics.
6. Airway hyperresponsiveness to adenosine better
reflects the inflammatory status of the lung in
asthma compared with directly acting bronchospasmogens including methacholine; the exquisite sensitivity of adenosine challenge to detect
inflammatory changes in human airways has been
ADENOSINE AND ADENINE NUCLEOTIDES 25
beneficially exploited to evaluate modifications in
the level of airway inflammation in a prospective
and noninvasive manner.
7. Dipyridamole, a blocker of facilitated adenosine
uptake, may precipitate asthma.
8. Theophylline, an established asthma therapy, is an
adenosine receptor antagonist. Low dose of theophylline blocks AMP-induced bronchoconstriction in asthmatics; this effect is unlikely due to
phosphodiesterase inhibition.
In light of these observations, adenosine has been
proposed to be an important mediator of asthma.
The evidence for the role of adenosine receptor
signaling in the pathogenesis of chronic inflammatory disorders of the airways such as asthma and
COPD is reviewed below.
Adenosine Metabolic Pathways
Adenosine is an endogenous nucleoside consisting of
the purine base, adenine, in glycosidic linkage with
the sugar ribose (Figure 1). Adenosine is present at
low concentrations in the extracellular space and its
levels are greatly increased under metabolically stressful conditions as a result of enzymatic cleavage of the
nucleotide adenosine 50 -monophosphate (AMP) by
the 50 -nucleotidase. This increase in adenosine formation may also occur during inflammation when a
large number of infiltrating inflammatory cells are
competing for a limited oxygen supply. Intracellular
levels of adenosine are kept low principally by its
conversion to AMP by the enzyme adenosine kinase.
Adenosine may also be degraded to inosine by ADA
(Figure 2).
It has been proposed that adenosine may function
as a ‘retaliatory metabolite’. During injury, the imbalance of O2 supply and demand triggers the increased
formation of adenosine. Adenosine may exert its
protective effects by decreasing the energy demand of
the tissue via a direct inhibitory effect on parenchymal
NH2
N
N
HO
N
N
O
OH
Figure 1 Structure of adenosine.
OH
cell function and indirectly by providing a more favorable environment for parenchymal cells, the best
example of which is adenosine-mediated augmentation of nutrient availability via vasodilatation. Moreover, adenosine helps to maintain tissue integrity
by modulating the function of the immune system.
Adenosine Receptors
Adenosine elicits its biological activities by interacting
with four adenosine receptor subtypes designated as
A1, A2A, A2B, and A3 adenosine receptors. The genes
for these receptors have been cloned from humans and
several animal species. Tissue distributions of these
receptors have been determined at the mRNA levels
using Northern blot or in situ hybridization techniques
or at the protein levels using subtype-selective radioligands or antibodies. In general, these receptors are
widely expressed in many tissues. For example, high
levels of A1 receptors are found in brain, adrenal
gland, and adipose tissue whereas high levels of A2A
receptors are found in spleen, thymus, striatum, and
blood vessels. In addition, these receptors are often
found to co-express in the same tissues or even on the
same cells. The relative expression levels of these receptors have been found to be modulated by physiological and/or pathophysiological tissue environments.
Although adenosine is the natural agonist for the
four adenosine receptors, the ability of adenosine to
activate these receptor subtypes varies. In many tissues, A1 and A2A receptors have relatively higher receptor reserves for adenosine, and can be activated by
the physiological levels of adenosine and thus mediate the tonic action of adenosine. On the other hand,
A2B and A3 receptors appear to have relatively lower
affinities and/or receptor reserves for adenosine and
require higher concentrations of adenosine for activation. However, the tissue adenosine levels in many
pathophysiological conditions can reach significantly
high levels to activate the A2B and A3 receptors.
Numerous subtype-selective agonists and antagonists of adenosine receptors have been synthesized
and are used as pharmacological tools. Although
these ligands were classified as selective ligands based
on their differential binding affinities for the four
adenosine receptors, these compounds are often
poorly characterized and not as selective as suggested. There are pharmacological reasons for
the lack of functional selectivity. For example, potencies of an agonist for one given receptor could
vary from one tissue to another depending on the
receptor expression levels and the coupling efficiencies of the cells. As indicated before, adenosine receptors are widely distributed and their expression
levels may be modified during disease processes. This
26
ADENOSINE AND ADENINE NUCLEOTIDES
Ecto-adenosine
deaminase
Ecto-5′-nucleotidase
AMP
Adenosine
Extracellular space
Nucleoside
transporter
Adenosine
deaminase
5′-nucleotidase
AMP
Inosine
Adenosine
Inosine
Adenosine kinase
SAH hydrolase
SAH
Figure 2 Adenosine production and removal occur both intracellularly and extracellularly. Intracellularly, adenosine is formed by the
action of 50 -nucleotidase, which dephosphorylates AMP, or by the action of S-adenosyl-homocysteine (SAH) hydrolase. When adenosine concentrations are high, it is phosphorylated to AMP by adenosine kinase (AK) or degraded to inosine by adenosine deaminase
(ADA). Both 50 -nucleotidase and ADA are found in the extracellular space where they mediate the formation and degradation of
adenosine. The intracellular and extracellular pools of adenosine are kept in equilibrium by the actions of bi-directional nucleoside
transporters (NT). Inhibition of AK, ADA, or NT by drugs or genetic deletions raises the tissue adenosine level.
certainly adds complexity in predicting functional
selectivity of agonists. In the case of antagonists, the
functional blocking effects of competitive antagonists are dependent on the tissue levels of adenosine.
If the levels of adenosine are too low to activate a
given receptor, an antagonist for this receptor will
not have any functional effects regardless of its binding affinity. In addition to these pharmacological
issues, there are pharmacokinetic issues. In many
cases, the half-life and tissue distributions of adenosine receptor ligands are poorly understood, making
it difficult to draw conclusions on the role of receptor
subtypes based on the absence of effects of ‘selective
ligands’ in animal models. In spite of these limitations, selective agonists and antagonists of adenosine
receptors are commonly utilized to establish the
functions mediated by adenosine receptor subtypes.
The four adenosine receptors also differ in their
ability to couple to G-proteins and activate various
intracellular signaling pathways. In most cells, A1 and
A3 receptors couple to Gi/o and inhibit adenylate
cyclase activity whereas A2A and A2B receptors couple
to Gs proteins and increase adenylate cyclase activity
and intracellular cAMP levels. While the adenylate
cyclase–cAMP–protein kinase A axis is the most
well-studied second messenger system involved in
adenosine receptor function, it is clear that adenosine
receptors utilize other signaling pathways as well.
These include members of the mitogen-activated protein-kinase family, such as p38, p42/p44 (ERK 1/2),
and c-jun terminal kinase, as well as various phospholipases, protein phosphatases, and ion channels.
Adenosine Biological Function
Many cell types that play important roles in the
pathogenesis of chronic inflammatory airway diseases are known to express adenosine receptors
and to exhibit relevant effects through adenosine
receptor signaling. These cell types include various
inflammatory cells, such as mast cells, eosinophils,
lymphocytes, neutrophils, and macrophages, and
the structural cells in the lung, such as bronchial
epithelial cells, smooth muscle cells, lung fibroblasts,
and endothelial cells. The role of the adenosine receptors in these inflammatory and structural cells has
been studied extensively using isolated cultured cells
in vitro. In addition, numerous animal models have
been developed and are extremely useful in determining the potential role of adenosine receptor subtypes in pulmonary functions.
A1 Adenosine Receptor
The A1 receptor has been implicated in both pro- and
anti-inflammatory aspects of disease processes. It has
been shown that activation of the A1 receptor can
promote activation of human neutrophils and monocytes and thus, their activation leads to proinflammatory responses. On the other hand, A1 receptors have
been reported to be involved in anti-inflammatory and
protective pathways in experimental models of injury
in the heart, nerves, and kidney.
The early evidence suggesting that the A1 receptor
plays a role in asthma came from experimental
work in rabbits rendered allergic by immunization
ADENOSINE AND ADENINE NUCLEOTIDES 27
protocols with allergen. These animals exhibited a
bronchoconstrictor response to adenosine that was
attenuated by pretreatment with A1 receptor blockers. In the same model, an antisense that targets the
initiation codon of A1 receptor mRNA reduced the
bronchoconstrictor response to adenosine and, more
importantly, to the early response to allergen. However, the relevance of these observations to human
asthma have been questioned due to the fundamental
mechanistic difference between adenosine-induced
bronchoconstriction in the allergic rabbit, which is
due to activation of A1 receptors on the bronchial
smooth muscle, and that in man, which appears to be
dependent on activation of mast cells that do not express the A1 receptor. Furthermore, it has been shown
recently that genetic removal of the A1 receptor gene
from ADA-deficient mice resulted in enhanced pulmonary inflammation along with increased mucus
metaplasia and alveolar destruction, thus indicating
that in this experimental model, the activation of A1
receptors serves an anti-inflammatory and/or protective role in the regulation of pulmonary disorders
triggered by elevated adenosine levels.
A2A Adenosine Receptor
It is now well established that activation of A2A receptors on lymphoid cells leads to inhibition of an
inflammatory response; this is largely due to its ability to induce accumulation of intracellular cyclic
AMP in activated immune cells. Perhaps the strongest evidence for the critical role of A2A receptors in
the regulation of inflammation in vivo originated
from the elegant study of Ohta et al. using mice deficient in A2A receptors. In this model, the absence of
the A2A receptor resulted in enhanced tissue inflammation and damage, thus suggesting a negative regulatory role for the A2A adenosine receptor subtype.
In the airways, A2A receptors are present on most
of the immunoinflammatory cells that have been implicated in asthma. A2A receptors are expressed on
mast cells, and their activation results in increases in
the intracellular cAMP concentrations, which are
known to inhibit the biochemical pathways implicated in the release of histamine and tryptase from
human mast cells. Stimulation of A2A receptors on
neutrophils inhibits neutrophil adherence to the endothelium, prevents upregulation of integrin expression stimulated with formyl-Met-Leu-Phe and
inhibits degranulation of activated neutrophils and
monocytes. Activation of T lymphocytes, which
plays a key role in the recruitment of leukocytes to
the lung in clinical asthma, is also suppressed by A2A
receptor activation. Thus, there are a multitude of
mechanisms by which activation at A2A receptors
could result in suppression of airway inflammation in
asthma and COPD. These findings support the hypothesis that A2A agonists could be potentially useful
in controlling the inflammatory processes.
A2B Adenosine Receptor
The initial evidence for the role of A2B receptors in
asthma and COPD came from selectivity studies of
enprofylline, a methylxanthine structurally closely
related to theophylline. It was shown that enprofylline is a selective antagonist for the A2B receptors
whereas theophylline has similar binding affinities
for A1, A2A, and A2B receptors. The therapeutic concentrations of theophylline and enprofylline match
their affinities for A2B receptors. Thus, it has been
proposed that A2B receptors are possible targets for
the long-term clinical benefit achieved with relatively
low doses of theophylline and enprofylline. On the
other hand, because the anti-inflammatory effects of
endogenous adenosine might be mediated by A1 and
A2A receptors, blockade of A1 and A2A receptors
could be disadvantageous. It has been proposed that
a more selective antagonist of the A2B receptors may
be more effective and safer than theophylline.
Recently, A2B receptors have been shown to have
proinflammatory properties in many pulmonary cells.
For example, functional human adenosine A2B receptors have been identified in mast cells, endothelial
cells, bronchial smooth muscle cells, lung fibroblasts,
and bronchial epithelium. Adenosine, via activation of
A2B receptors, increases the release of various inflammatory cytokines from human mast cells (HMC-1),
from human bronchial smooth muscle cells, human
lung fibroblasts, and human airway epithelial cells.
These cytokines, in turn, induce IgE synthesis from
human B lymphocytes, and promote differentiation of
lung fibroblasts into myofibroblasts. These findings
provide strong support for the hypothesis that adenosine, via activation of A2B receptors, could enhance
airway hyperresponsiveness and the inflammatory responses associated with asthma. Thus, an A2B antagonist could potentially be beneficial in the treatment of
asthma and other pulmonary inflammatory diseases.
A3 Adenosine Receptor
The functional role of the A3 receptor in the pathogenesis of chronic airway inflammatory diseases remains controversial due in large part to differences in
the pharmacology of A3 receptors from different species. For instance, in rodents, mast cell degranulation
and/or enhancement of mast degranulation in response to allergen appears to be dependent on A3 receptor activation. In humans, a relatively high density
of functionally active A3 receptors is expressed in
28
ADENOSINE AND ADENINE NUCLEOTIDES
eosinophils. Transcript levels for the A3 receptor are
elevated in lung biopsies of patients with asthma or
COPD and appear to be involved in the inhibition of
eosinophil chemotaxis when stimulated. Furthermore,
inhibition of important proinflammatory functions of
human eosinophils by the selective A3 receptor agonist, IBMECA, has been reported. Because asthmatic
inflammation is characterized by extensive infiltration
of the airways by activated eosinophils, it is possible
that the elevated adenosine concentrations associated
with asthma could contribute to inhibition of eosinophil activation through stimulation of A3 receptors. In
contrast, in their effort of dissecting out specific
signaling pathways involved in adenosine-mediated
pulmonary inflammation and airway remodeling in
ADA-deficient mice, Young and colleagues have recently demonstrated that mice treated with the selective A3 receptor antagonists MRS 1523 resulted in a
marked attenuation of pulmonary inflammation, reduced eosinophil infiltration into the airways, and
decreased airway mucus production.
Adenosine in Respiratory Diseases
Adenosine may play a critical role in the pathogenesis
of chronic inflammatory disorders of the airways such
as asthma and chronic obstructive pulmonary disease
(COPD). Elevated levels of adenosine are present in
chronically inflamed airways; they have been observed both in the BAL fluid and the EBC of patients
with asthma. Adenosine levels are also raised after
allergen exposure in the plasma and during experimental exacerbation of asthmatic symptoms due to
exercise in atopic individuals. The observed adenosine elevations indicate that adenosine signaling may
regulate aspects of acute and chronic airway disease.
Consistent with the hypothesis of adenosine playing a
critical role in the pathogenesis of chronic inflammatory disorders, mice deficient in ADA develop features
of severe pulmonary inflammation and airway remodeling in association with elevations of adenosine
concentrations in the lung. Features of the pulmonary
phenotype noted include the accumulation of eosinophils and activated macrophages in the airways, mast
cell degranulation, mucus metaplasia in the bronchial
airways, and emphysema-like injury of the lung parenchyma. Although the histology seen in ADA-deficient mice fails to accurately resemble that of human
asthma, since no epithelial shedding, subepithelial
fibrosis, or muscle/submucosal gland hypertrophy
were observed in this model, the ADA-deficient
mouse is a useful tool to study the pathogenetic role
of adenosine in chronic airway inflammation.
The role of adenosine in the pathogenesis of
asthma is not just limited to its biological effects in
Adenosine
A2B receptor
Mast cell
Cytokines
Prostaglandins and
leukotrienes
Histamine
Bronchoconstriction
Figure 3 Mechanism of adenosine-induced bronchoconstriction. Adenosine, possibly via activation of A2B adenosine receptors, enhances the activation of airway mast cells, leading to
increases in the release of potent contractile mediators such as
histamine, leukotrienes, prostaglandins, and cytokines. These
mediators in turn cause bronchoconstriction in human airways.
airway inflammation and remodeling. Adenosine administration by inhalation is known to elicit concentration-related bronchoconstriction in subjects with
asthma and COPD whereas the nucleoside had no
discernible effect on airway caliber in normal individuals. Since these initial observations were made, a
considerable effort has been directed at revealing
the fine mechanisms of adenosine-induced bronchoconstriction, which appear to involve a selective interaction with activated airway mast cells with
subsequent release of preformed and newly formed
mediators (Figure 3).
An important development from adenosine research is the use of an adenosine (or AMP) inhalation
challenge as an innovative diagnostic test for asthma
and COPD. Compared with other noninvasive surrogate markers of airway inflammation, monitoring
of the responsiveness of airways to inhaled adenosine
appears to have the selective ability to probe changes
in airway inflammation and it has been shown to
be very useful when evaluating the effectiveness of
different treatment regimens with inhaled corticosteroids. Because the airway response to inhaled
adenosine is very sensitive to the effect of inhaled
corticosteroids and is a good marker of disease activity, bronchoprovocation with adenosine has been
proposed as a convenient and accurate biomarker to
monitor corticosteroid requirements in asthma and
to establish the appropriate dose needed to control
airway inflammation.
ADHESION, CELL–CELL / Vascular 29
Conclusions and Future Direction
Over the course of 20 years, the initial observation of
the bronchoconstrictive effect of inhaled adenosine
has evolved to provide the basis for a new asthma
therapy as well as a diagnostic test. Recognition of the
potential role of adenosine receptor signaling in the
pathogenesis of chronic airway inflammatory diseases
advocates the principle that modulating adenosine
receptor signaling is likely to constitute a considerable
advance in the management of asthma and COPD.
The clinical evaluation of selective agonists or antagonists for the adenosine receptors should help elucidate the multiple roles of adenosine and its receptors
in the pathophysiology of asthma and COPD.
See also: Asthma: Overview. Bronchoalveolar Lavage.
Chronic Obstructive Pulmonary Disease: Overview.
Further Reading
Blackburn MR, Lee CG, Young HWJ, et al. (2003) Adenosine
mediates IL-13-induced inflammation and remodeling in the
lung and interacts in an IL-13-adenosine amplification pathway.
Journal of Clinical Investigation 112(3): 332–344.
Blackburn MR, Volmer JB, Thrasher JL, et al. (2000) Metabolic
consequences of adenosine deaminase deficiency in mice are associated with defects in alveogenesis, pulmonary inflammation
and airway obstruction. Journal of Experimental Medicine
192(2): 159–170.
Cronstein BN (1994) Adenosine, an endogenous anti-inflammatory agent. Journal of Applied Physiology 76: 5–13.
Cushley MJ, Tattersfield AE, and Holgate ST (1983) Inhaled adenosine and guanosine on airway resistance in normal and asthmatic
subjects. British Journal of Clinical Pharmacology 15(2): 161–165.
Feoktistov I and Biaggioni I (1995) Adenosine A2b receptors evoke
interleukin-8 secretion in human mast cells. An enprofyllinesensitive mechanism with implications for asthma. Journal of
Clinical Investigation 96(4): 1979–1986.
Feoktistov I, Polosa R, Holgate ST, and Biaggioni I (1998) Adenosine A2B receptors: a novel therapeutic target in asthma? Trends
in Pharmacological Sciences 19: 148–153.
Fozard JR (2003) The case for a role for adenosine in asthma: almost
convincing? Current Opinion in Pharmacology 3(3): 264–269.
Fredholm BB, Ijzerman AP, Jacobson KA, Klotz KN, and Linden J
(2001) International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacological
Reviews 53: 527–552.
Ohta A and Sitkovsky M (2001) Role of G protein-coupled
adenosine receptors in down-regulation of inflammation and
protection from tissue damage. Nature 414: 916–920.
Polosa R, Ng WH, Crimi N, et al. (1995) Release of mast cellderived mediators after endobronchial adenosine challenge in
asthma. American Journal of Respiratory and Critical Care
Medicine 151: 624–629.
Rorke S, Jennison S, Jeffs JA, et al. (2002) Role of cysteinyl leukotrienes in adenosine 50 -monophosphate induced bronchoconstriction in asthma. Thorax 57(4): 323–327.
Ryzhov S, Goldstein AE, Matafonov A, et al. (2004) Adenosineactivated mast cells induce IgE synthesis by B lymphocytes: an
A2B-mediated process involving Th2 cytokines IL-4 and IL-13
with implications for asthma. Journal of Immunology 172(12):
7726–7733.
Shryock JC and Belardinelli L (1997) Adenosine and adenosine receptors in the cardiovascular system: biochemistry, physiology and
pharmacology. American Journal of Cardiology 79(12A): 2–10.
Spicuzza L, Bonfiglio C, and Polosa R (2003) Research applications and implications of adenosine in diseased airways. Trends
in Pharmacological Sciences 24(8): 409–413.
Zhong H, Belardinelli L, Maa T, and Zeng D (2005) Synergy between A2B adenosine receptors and hypoxia in activating human
lung fibroblasts. American Journal of Respiratory Cell and
Molecular Biology 32(1): 2–8.
ADHESION, CELL–CELL
Contents
Vascular
Epithelial
Vascular
H M DeLisser, University of Pennsylvania School of
Medicine, Philadelphia, PA, USA
& 2006 Elsevier Ltd. All rights reserved.
Abstract
Four distinct families of adhesion molecules (cadherins, immunoglobulin superfamily members, selectins, and integrins) mediate
vascular cell–cell adhesion. These molecules are required for the
formation of the junctional complexes that enable the assembly of
endothelial cells into functional vascular networks; they mediate
the leukocyte–endothelial adhesive interactions involved in the
trafficking of leukocytes out of the circulation; and they contribute to contacts between pericytes and the endothelium that are
important to the regulation of endothelial cell proliferation. In the
lung these vascular cell–cell interactions are the bases of the host
defense to lung infection or injury, and the regulation of lung
vascular permeability, but are disturbed or dysregulated in diseases such as asthma or the acute respiratory distress syndrome.
30
ADHESION, CELL–CELL / Vascular
Proper cadherin expression and function are essential for normal cell segregation during embryonic
development and are required for the initiation and
maintenance of normal tissue architecture. The extracellular domain has a variable number of homologous repeats of B110 amino acids with putative
Ca2 þ binding sequences (cadherin-repeats), while
the cytoplasmic tail forms complexes with cytoplasmic proteins of the catenin family (a-catenin, b-catenin, g-catenin/plakoglobin, and p120) that mediate
association with the actin cytoskeleton.
Endothelial cells principally express two cadherins: vascular endothelial-cadherin (VE-cadherin), a
molecule expressed almost exclusively by endothelial
cells, as well as the more widely distributed N-cadherin. They appear to be functionally different, with
VE-cadherin mediating endothelial cell–cell adhesion, while N-cadherin may promote endothelial cell
adhesion to pericytes and smooth cells. Other nonclassical cadherins, such as T-cadherin and VE-cadherin-2 (protocadherin 12), have been described on
Description
Families of Adhesion Molecules
A diverse group of protein molecules mediate cell–
cell as well as cell–matrix adhesion. These cell
adhesion molecules are grouped into four major
families: cadherins, immunoglobulin (Ig) superfamily
members, selectins, and integrins (Figure 1).
Cadherins Cadherins were initially identified as a
family of single-pass, transmembrane proteins responsible for mediating calcium-dependent, homophilic,
cell–cell adhesion. These ‘classical cadherins’ are now
recognized to be part of a larger cadherin superfamily
that also includes the structural and functionally
related desmosomal cadherins as well as other subfamilies with cytoplasmic domains that are unrelated
to those of the classical cadherins. (Unless otherwise
specified, the term cadherin will refer to the classical
protein.)
Cadherins
Ig superfamily
Selectins
Integrins
P-selectin
VE-cadherin
E-selectin
PECAM-1
MAdCAM-1
L-selectin
JAM-A
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
Extracellular
repeating elements
lg-like domain
Lectin-like domain
Integrin subunit
Ca2+ binding sites
Mucin-like domain
EGF-like domain
Integrin subunit
Transmembrane
domain
Anchoring domains
Transmembrane and
cytoplasmic domains
Cytoplasmic domain
PDZ binding motif
C
Short consensus repeat
Transmembrane and
cytoplasmic domains
Transmembrane and
cytoplasmic domains
Figure 1 Families of cell adhesion molecules. Ilustrated are structural features that distinguish the members of each of the four major
families of cell adhesion molecules. The extracellular domains of cadherins are composed of a variable number of extracellular repeats
with putative Ca2 þ binding sites, and conserved sequences in the cytoplasmic domain. Immunoglobulin superfamily members share a
common structure in which a variable number of Ig-like homology domains are present in the extracellular domains of the molecule. The
presence of other structural motifs such as mucin-like domains contribute to the structural diversity of these receptors. The selectins are
composed of an NH2-terminal lectin-like binding domain, followed by an EGF-like motif, a variable number of short consensus repeats, a
single-pass transmembrane domain, and a short cytoplasmic tail. Integrins are heterodimeric proteins composed of two noncovalently
linked a and b subunits, each with large extracellular domains and smaller but functionally important cytoplasmic tails.
ADHESION, CELL–CELL / Vascular 31
the endothelium, but their functions are currently not
known.
Immunoglobulin superfamily The Ig superfamily
represents a very diverse group of receptors that
function in a variety of cell types and in a number
of biological processes. Members of this family are
defined by the presence of a variable number of Iglike homology domains in the extracellular domains
of these molecules. The Ig-like motif represents a
sandwich-like structure of two b-sheets each consisting of antiparallel b strands containing 5–10 amino
acids. Appropriately positioned cysteine residues
mediate the formation of intrachain disulfide bonds
and stabilization of each Ig-like domain. The presence of additional structural domains such as mucinlike regions (e.g., mucosal adressin cell adhesion
molecule-1 (MAdCAM-1)) contribute to the structural diversity of this family. Several Ig superfamily
members, including intercellular cell adhesion molecule-1 (ICAM-1), platelet endothelial cell adhesion
molecule-1 (PECAM-1), MAdCAM-1, vascular cell
adhesion molecule-1 (VCAM-1), and the junctional
adhesion molecules (JAMs), are expressed on the
endothelium and play important roles in leukocyte–
endothelial adhesion. Ligand binding for these endothelial Ig superfamily molecules may be homophilic
(PECAM-1 and the JAMs) or involve interactions
with b2 (ICAM-1, JAM-A, and JAM-C) or a4 integrins (MAdCAM-1, JAM-B, and VCAM-1).
Selectins The selectins are a small family of receptors found only on vascular-related cells that mediate
the initial adhesive interactions of circulating leukocytes with the endothelium at sites of inflammation
or in lymphoid tissues. Selectin structure includes an
N2-terminal lectin-like binding domain, followed by
an epidermal growth factor (EGF)-like motif, a variable number of short consensus repeats, a single-pass
transmembrane domain, and a short cytoplasmic tail.
Three selectins (P-selectin, E-selectin, and L-selectin)
have been identified, each differing in their patterns
of expression and regulation. P-selectin is stored in
cytoplasmic granules in platelets and endothelial
cells, but is rapidly mobilized to the cell surface,
where it is transiently expressed (minutes), in response to agonists such as histamine or thrombin.
E-selectin is not present under resting conditions, but
is synthesized and expressed briefly (hours) on vascular endothelial cells following cytokine stimulation.
In contrast, L-selectin is constitutitively expressed on
most leukocytes but is proteolytically cleaved from
the surface following cellular activation.
Selectins recognize sialylated and fucosylated
oligosaccharides (e.g., sialyl Lewis x (sLex) and related
tetrasaccharides), as well as sulfated molecules that
lack sialic acid or fucose, such as sulfatides and heparin glycosaminoglycans. Thus, in vitro, selectins are
able to bind to a variety of glycoproteins on leukocytes
and endothelial cells bearing these structural motifs.
Several of these are mucin-like glycoproteins with
many serine or threonine residues that are potential
sites for attachment of O-linked glycans. In vivo,
however, P-selectin glycoprotein ligand-1 (PSGL-1) is
the dominant ligand for P- and L-selectin in inflammatory settings and may also be physiologically relevant for E-selectin. Recent reports also suggest that
for neutrophils, CD44 may be also be a physiological
E-selectin ligand. Additional L-selectin ligands have
been identified on high endothelial venules (HEV) of
lymphatic tissues and on some activated endothelial
cells. These include CD34, glycosylated cell adhesion
molecule (GlyCAM-1), and podocalyxin.
Integrins The integrins are a family of heterodimeric
membrane glycoproteins composed of noncovalently
associated a and b subunits. To date 18 a and 8 b
subunits have been identified and more than 24 a/b
pairs have been described in vertebrate tissue. The
combination of a and b subunits determines the ligand specificity. Although most integrins bind ligands
that are components of the extracellular matrix, certain integrins (b2 and a4 integrins) bind receptors of
the immunoglobulin superfamily (e.g., ICAM-1,
MAdCAM-1, VCAM-1, and the JAMs). Significant
redundancy, however, exists in that most integrins
are capable of binding several different matrix or
cell surface proteins and many of these ligands bind
to multiple integrins. Through their cytoplasmic domains integrins are able to bind to a complex of
cytoplasmic proteins, including a-actinin, vinculin,
talin, and paxillin, linked to actin filaments. These
integrin–cytoskeletal assemblies in turn form the
foundation for intracellular signaling cascades.
Endothelial Cell–Cell Adhesion
Endothelial cells adhere to one another through two
morphologically distinct junctional structures, adherens junctions (AJs) and tight junctions (TJs), each
composed of characteristic molecules (Figure 2). Although very similar structures are present in epithelial cells, their distribution within intercellular
junction differs in the two cell types. In epithelial
junctions TJs are located toward the apical region
of the intercellular contact with the AJs positioned
below the TJs, while AJs and TJs are intermingled
with each other along the endothelial intercellular
junction. Gap junctions represent a third type of
junctional complex formed by endothelial cells.
32
ADHESION, CELL–CELL / Vascular
Apical/luminal surface
Junctional complexes
Associated
transmembrane proteins
Tight junctions
Occludin, claudins, JAMs
Adherens junctions
Cadherins
Gap junctions
Connexins
Nonjunctional
intercellular proteins
PECAM-1, endoglin
Basal lamina
Figure 2 Adhesion structures found in epithelial and endothelial cells. Shown are intercellular junctional complexes (TJs and AJs) and
their associated transmembrane proteins. Gap junctions mediate intercellular communication and other proteins such as PECAM-1 and
endoglin mediate cell adhesion but are not present in specific junctional complexes.
However, unlike AJs and TJs, these structures mediate cell–cell communication rather than cell–cell
adhesion. Epithelial cells also express another type
of adhesive complex, the desmosone, but these structures are absent from endothelial cells.
Adherens junctions These junctions are formed by
clusters of dimerized cadherin molecules that bind to
cadherin dimers on adjacent cells. Cadherins localize
at AJs only when cells contact one another. The short
cytoplasmic domain of cadherins interacts through a
C-terminal domain with b-catenin and plakoglobin
(g-catenin). b-catenin and plakoglobin associate with
a-catenin which in turn binds to actin microfilaments. This association with the catenins is required
for the adhesive function of cadherins and thus is
essential to the assembly of AJs. An additional binding partner is p120, an src substrate that is homologous to b-catenin and plakoglobin. In contrast to
b-catenin and plakoglobin, p120 binds loosely to a
membrane proximal region of the cadherin cytoplasmic tail, associating with a-catenin or actin cytoskeleton. It appears that p120 is involved in the
regulation of the cadherin turnover and stability.
The formation of AJs provides the cell–cell attachments that are required for the assembly of the endothelium into patent tubular networks. AJs also
contribute to maintenance of endothelial barrier
function, in addition to the regulation provided by
TJs (see below). Anti-VE-cadherin antibodies, both
in vitro and in vivo are found to increase vascular
permeability by disrupting VE-cadherin clustering at
the junctions while leaving other junctional structures
intact. Further, AJs are required for the organization
and/or maintenance of TJs and gap junctions and
thus are critical to the integrity of the entire intercellular junctional complex. In addition, at least two
other nonadhesive functions have been ascribed to
AJs. First, b-catenin, plakoglobin, and p120 are all
able to translocate to the nucleus, where in conjunction with other transcription factors they are able to
modulate gene expression. Consequently, the association of these catenins with VE-cadherin in AJs
may maintain them at the membrane and thus prevent their nuclear translocation. Second, VE-cadherin
engagement promotes endothelial cell quiescence
by modulating vascular endothelial growth factor
(VEGF) receptor-mediated signaling – inhibiting the
receptor-dependent proliferative responses, while activating receptor-mediated survival signals.
Tight junctions These junctions were originally
identified in epithelial cells by electron microscopy
as dense regions in which membrane leaflets between
the adjacent cells are closely opposed such that the
membrane bilayers at the junctions are indistinguishable. In freeze-fracture preparations, TJs have the
appearance of fibrillar strands within the membrane.
They form a continuous seal around the apical region
of the lateral membranes of adjoining cells, subdividing it into apical and basolateral domains. This is
somewhat less so for endothelial cells, where the
position of TJs relative to other intercellular junctional complexes can vary. Further, the level and
extent of TJs depend on the vessel type and location.
Endothelial cell TJs are well developed in arteries
ADHESION, CELL–CELL / Vascular 33
and arterioles, but significantly less so in veins
and postcapillary venules, the principal site for the
extravasation of fluid and inflammatory leukocytes.
TJs are also well developed in the vessels of the brain
where they contribute to the blood–brain barrier, but
are much less organized in the vasculature of other
organs.
Like other junctional structures, TJs are composed
of both transmembrane and intracellular molecules.
Three types of TJ-associated integral membrane proteins have been identified. These are occludin, the
claudins, and several Ig superfamily members, including the JAMs, endothelial cell-selective adhesion
molecule (ESAM), and coxsackie- and adenovirus
receptor (CAR). Occludin and the claudins (of which
more than 20 have been identified) define the presence of this junction and constitute the molecular
basis of the TJ strands, while JAM-A may serve
accessory functions such as the recruitment of
TJ-associated proteins. The intracellular domains of
occludin, the claudins, and the JAMs interact with
zonula occludens-1 (ZO-1), a member of a family of
membrane-associated, PDZ, and guanylate kinase
domain-containing proteins. ZO-1 can bind to actin
filaments and thus anchor TJs to the cell’s cytoskeleton. In addition to ZO-1, other PDZ-containing
and/or actin binding proteins are recruited that contribute to the formation and function of TJs.
Tight junctions restrict both the diffusion of solutes through intercellular spaces (‘barrier function’)
and the movement of molecules between the apical
and basolateral domains of the plasma membrane
(‘fence function’). As such, TJs are the major regulator of cell permeability via the paracellular pathway (i.e., the extracellular space between the lateral
membranes of neighboring cells) and are important
to the maintenance of cell polarity. TJs are also likely
involved in leukocyte transendothelial migration,
possibly through interactions mediated by the JAMs.
Gap junctions Gap junctions constitute another
junctional complex that bridges the intercellular space
between adjacent endothelial cells. However, unlike
the other junctional structures described above, which
mediate cell–cell adhesion, gap junctions promote
intercellular communication. Metabolites, ions, and
second messengers, including Ca2 þ , cAMP, and inositol triphosphate, are able to pass through gap
junction channels from one cell to another, enabling
the coordination of multicellular responses. Gap junction channels are dodecameric structures made up of
the connexin family of proteins. Six individual connexin proteins oligomerize in the plasma membrane
of one cell to form a hemichannel or connexon,
and the docking of two connexons, one from each
opposing cell, results in a complete gap junction
channel.
Other intercellular junction proteins Endothelial
cells express several other adhesive proteins that are
concentrated in intercellular junctions but are not
specifically located in either AJs or TJs. These include (1) PECAM-1, the loss of which compromises
vascular barrier function and angiogenesis; (2)
S-dndo 1, an Ig superfamily member and a mediator of homophilic adhesion; and (3) endoglin, a regulator of transforming growth factor beta (TGF-b)
signaling, the absence of which leads to a phenotype
reminiscent of VE-cadherin-null mice.
Endothelial Cell–Leukocyte Adhesion
The recruitment of leukocytes to sites of infection or
injury begins with a well-defined set of sequential
adhesive interactions between the circulating leukocyte and the activated endothelium (Figure 3). A
similar cascade of events is believed to also mediate
the trafficking of lymphocytes across high endothelial venules in lymphoid tissues.
Endothelial activation Endothelial activation is
characterized by the enhanced expression of endothelial selections (P- and E-selectin) and members
of the Ig superfamiy (ICAM-1, MAdCAM-1, and
VCAM-1). This typically involves de novo synthesis,
with peak expression occurring over several hours:
4–6 h for E-selectin and 12–24 h for VCAM-1 and
ICAM-1. For P-selectin, however, endothelial surface
expression occurs within minutes following stimulation with noncytokine mediators such as thrombin
and histamine as preformed P-selectin is mobilized
from Weibel–Palade bodies. The expression of other
molecules such as PECAM-1 and the JAMs that have
been implicated in leukocyte trafficking do not appear
to be affected by inflammatory mediators.
Leukocyte rolling The initial leukocyte–endothelial
adhesive event is one in which the circulating
leukocyte rolls along the surface of the inflamed
endothelium (and on already adherent leukocytes).
This process involves an initial capture and fast rolling of the leukocyte, followed by a period of slow
rolling and deceleration prior to leukocyte arrest.
This step has long been recognized as principally
mediated by selectins, with each L- and P-selectin
mediating the capture and fast rolling, while E-selectin is preferentially involved in the slow rolling.
Interestingly, L-selectin-mediated capture and rolling
requires a critical threshold of shear stress to occur,
while rolling adhesion mediated P- and E-selectin
34
ADHESION, CELL–CELL / Vascular
Leukocyte rolling
Capture
Fast rolling
Firm adhesion
Diapedesis
Slow rolling
L-selectin
P-selectin
E-selectin
2 integrins/ICAM-1/2
41/IVCAM-1 or 47/MAdCAM-1
PECAM-1
JAMs
CD99
Figure 3 Leukocyte–endothelial interactions. The sequence of adhesive interactions involved in the emigration of leukocytes from the
circulation across the endothelium to extravascular sites is illustrated. The steps of these interactions, in order, are leukocyte rolling, firm
adhesion, and diapedesis (transendothelial migration). Each step is mediated, depending on the cell type and context, by the interactions of specific cell adhesion molecules and their ligands.
demonstrates weak or no dependence on a shear
threshold. The basis for this appears to reside in
the fact that L-selectin engages its ligands through
exceptionally labile adhesive bonds that are only significantly stabilized above a critical shear threshold.
Although originally thought to mediate only firm
adhesion and transendothelial migration (see below),
b2 integrins are now recognized to cooperate with Eselectin in the deceleration of the rolling leukocyte.
Depending on the cell type, there is also evidence
that a4b1, a4b7, and CD44 are able to respectively
promote rolling on VCAM-1, MAdCAM-1, and
hyaluronate-covered surfaces.
Activation–firm adhesion As the leukocyte rolls,
interactions between selectins and their ligands and
between chemokines and chemoattractants associated with the endothelium and G-protein-coupled
receptors on the leukocyte surface, activate b2 (and
probably a4) integrins on the leukocyte surface. This
activation, which involves upregulation of integrin
expression and adhesive activity and the shedding
of L-selectin, enables the arrest and firm adhesion
of the leukocyte to the endothelium. This phase of
leukocyte recruitment is mediated by the binding of
endothelial Ig superfamily members to (activated)
leukocyte integrins (ICAM-1/aMb2, ICAM-1, -2/a1,
b2, VCAM-1/a4b1, and MAdCAM-1/a4b7).
Transendothelial migration (diapedesis) The adherent leukocyte then ‘crawls’ over the luminal surface,
a process that involves the cyclic modulation of
integrin receptor avidity. Once a junction has been
located, the emigrating leukocyte squeezes between
the closely opposed endothelial cells, crossing the
basement membrane to enter the extravascular tissue.
Significantly, this occurs without disrupting the integrity of the endothelium. This process of leukocyte
transendothelial migration or diapedesis is mediated
by at least five cell–cell adhesion molecules: Ig superfamily members PECAM-1 and JAM-A, -B and -C, as
well as CD99, a molecule with a unique structure.
With the exception of JAM-B, all of these molecules
are expressed on both leukocytes and endothelial
cells and thus are capable of homophilic adhesion.
The JAMs also bind leukocyte integrins, heterophilic
interactions that have also been implicated in their
activity in leukocyte trafficking. PECAM-1 appears to
be required for the initial entrance or penetration of
the junction, while CD99 appears to be involved
more distally in the passage through the intercellular
junction. While traversing the junction, homophilic
ADHESION, CELL–CELL / Vascular 35
interactions of leukocyte and endothelial PECAM-1
also upregulate a6b1 on transmigrating leukocytes,
an integrin that is required for the passage of the
leukocyte through the matrix of the basement membrane. Although animal studies have implicated the
JAMs in leukocyte diapedesis, their specific roles
remain to be determined.
Pericyte–Endothelial Cell Adhesion
Pericytes are perivascular cells imbedded within the
basement membrane of the endothelium of capillaries
and postcapillary venules. Contact between the endothelial cell and the pericyte is made by cytoplasmic
processes of the pericyte indenting the endothelial
cell, and vice versa. This results in the so-called ‘peg
and socket’ contact. The normally intervening basal
lamina is absent at these pericyte–endothelial cell
interdigitations and growth factors (e.g., epidermal
growth) factor may concentrate at these contacts. In
some tissues, TJs between pericytes and endothelial
cells have been reported and ‘adhesion plaques’ have
been described in the pericyte membrane.
An inverse correlation exists between endothelial
cell proliferation and the extent of pericyte coverage,
with tissues demonstrating the slowest rate of endothelial cell turnover having the greatest pericyte
coverage. This suggests that pericytes are a major
negative modulator of capillary growth and hence a
promoter of vessel quiescence. This may be mediated
by the pericyte-derived TGF-b, an inhibitor of endothelial cell growth and migration, the activation of
which is dependent on pericyte–endothelial cell contact. Pericytes are also contractile, and thus points
of cell–cell contact may permit contractions of the
pericyte to be transmitted to the endothelial cell
to reduce the caliber of the vessel or alter vascular
permeability.
Vascular Cell–Cell Adhesion in Normal
Lung Function
Lung Defenses
The recruitment of leukocytes is an essential component of the lung’s defense against infection or inflammatory insults. Although data certainly indicate
that the leukocyte–endothelial interactions described
above may operate in the lung to regulate the emigration of circulating leukocytes, there is also evidence that leukocyte recruitment in the pulmonary
circulation may differ, depending on the stimulus,
from that which occurs in postcapillary venules at
extrapulmonary sites. Although the b2 integrins have
been shown to be required for neutrophil emigration
in various models of acute inflammation involving
the systemic vascular bed, including neutrophil recruitment mediated Streptococcus pneumoniae, HCl,
and C5a, these stimuli mediate neutrophil extravasation from the pulmonary circulation that is independent of the b2 integrins. An even more complex
picture is suggested by studies of mice deficient
in both P-selectin and E-selectin or P-selectin and
ICAM-1. In these mutant animals, neutrophil emigration into the peritoneal cavity was completely inhibited during S. pneumoniae-induced peritonitis. In
contrast, neutrophil extravasation into the alveolar
space during S. pneumoniae-induced pneumonia was
intact.
Together these data suggest that leukocyte recruitment from the pulmonary circulation can occur
through pathways that do not require selectins, b2
integrins, or ICAM-1. These observations may be
due in part to the fact that primary site for leukocyte
extravasation is in the pulmonary capillaries and not
in postcapillary venules as occurs in the systemic
vasculature. This is significant because the average
diameter of the leukocyte (particularly the neutrophil) approaches or exceeds that of the capillaries.
As a result, intravascular leukocytes must deform
in order to transit through the pulmonary microvasculature and thus are in close apposition with the
pulmonary endothelium. Consequently, processes
that change the physical properties of the leukocyte
or compromise their ability to deform may be sufficient to slow and arrest the leukocyte in the microcirculation of the lung. Thus, it should not be
surprising that in the lung there may be less stringent requirements for molecular-meditated processes
to capture and attach the intravascular leukocyte to
inflamed pulmonary endothelium.
Lung Permeability
In the lung, as in other vascular beds, endothelial cell
permeability depends largely on the regulation of
paracellular fluid and solute transport through intercellular junctions. The degree of ‘openness’ of this
pathway is governed by the balance of centrally directed contractile forces and opposing forces generated by cell–cell and cell–matrix adhesive complexes
that tether endothelial cells to each other and to the
basement membrane. Of the various junctional complexes, TJs are recognized as the primary determinants of the endothelial barrier function. However,
disruption of VE-cadherin function results in interstitial edema in the lung and there is evidence that in
the setting of hyperosmolarity, E-cadherin expression
may be upregulated on the endothelium of the lung
microvasculature where it acts to promote barrier
function. These data suggest that cadherin-dependent
36
ADHESION, CELL–CELL / Vascular
activity mediated through AJs may also contribute to
the regulation of vascular permeability in the lung.
Barrier integrity varies with vessel type, as the endothelium of the pulmonary microcirculation is
much more restrictive than that of the pulmonary
arteries or veins. This suggests that there may well be
site-specific differences in junctional architecture
and/or cytoskeletal machinery in the pulmonary
vasculature.
Vascular Cell–Cell Adhesion in
Respiratory Diseases
Asthma
Asthma is an inflammatory disease characterized by
increased infiltration of various inflammatory cells in
the bronchial mucosa and airways. In allergic asthma,
antigen exposure results in the activation of mast cells
and TH2 lymphocytes and their subsequent release of
a number of cytokines and lymphocyte or eosinophil
chemoattractants. These inflammatory mediators alter the expression and activity of cell adhesion molecules on these asthma-associated leukocytes and on
the bronchial microvasculature in specific patterns
that target their recruitment out of the circulation
into the bronchial tissues. Analyses of bronchial tissue
from allergic and other asthmatics, as well as studies
of murine models of asthma indicate that interactions
between E-selectin and its ligands, 1CAM-1 and aLb2
and VCAM-1 and a4b1, are involved in the recruitment of eosinophils in the allergic airway response.
It should be noted however, that there are reports in
which acute antibody inhibition of a4b1, b2 integrin,
or ICAM-1 may reduce airway hyperresponsiveness
without reducing eosinophil or lymphocyte accumulation. This suggests that these molecules may mediate processes other than leukocyte–endothelial
interactions, such as leukocyte activation or antigen
presentation, that contribute to the development of
the asthmatic phenotype.
Acute Lung Injury
A large and diverse group of microbial and inflammatory insults may acutely injure the lung culminating in the acute respiratory distress syndrome
(ARDS) and respiratory failure. A key feature of this
syndrome is endothelial cell injury and subsequent
dysfunction manifested by impaired barrier function
and increased vascular permeability. This dysfunction must necessarily involve destabilization of intercellular junctions, but the exact molecular basis is
still being defined. A variety of inflammatory agents
including histamine, lipopolysaccharide, leukotriene
B4, platelet activating factor, nitric oxide thrombin,
as well as cytokines (TNF-a, IL-1, and g-IFN) increase
transendothelial permeability, at least in part, by inducing endothelial cell contraction and the resultant
formation of interendothelial gaps. These alterations
in endothelial morphology require the recruitment
and activation of calcium-dependent cytoskeletal proteins (e.g., actin and myosin) that alter endothelial
cell contour and reorganize intercellular junctional
complexes. The alterations in endothelial permeability induced by inflammatory mediators may involve
changes in the concentration of VE-cadherin and
PECAM-1 in endothelial intercellular junctions as
cytokines have been shown to redistribute these molecules out of endothelial cell–cell contacts with a
concomitant increase in permeability.
An important contributor to the pathogenesis of
endothelial dysfunction in ARDS appears to be
activated neutrophils sequestered in and adherent
to the pulmonary capillaries. These trapped leukocytes release reactive oxygen species and granular constituents that undermine the normal barrier
function of the endothelium. These products may
also contribute the inflammatory cascade by activating monocytes and macrophages leading to release of
additional proinflammatory mediators. Inhibition of
b2 integrins, ICAM-1, and selectins blocks neutrophil recruitment and permeability injury in animal
models of acute lung injury or infection although
there do appear to be alternative pathways as noted
above. This has made these molecules attractive
therapeutic targets for the treatment of ARDS. However, the potential for inhibition of host defenses
and reparative responses continues currently to be
an obstacle to the widespread application of this
approach.
Acknowledgments
This work was supported by the Department of
Defense (PR043482), National Institutes of Health
(HL079090), and the Philadelphia Veterans Medical
Center.
See also: Acute Respiratory Distress Syndrome.
Adhesion, Cell–Cell: Epithelial. Adhesion, Cell–
Matrix: Integrins. Asthma: Overview. Endothelial
Cells and Endothelium. Leukocytes: Neutrophils;
Monocytes; T Cells.
Further Reading
Allt G and Lawrenson JG (2001) Pericytes: cell biology and
pathology. Cells Tissues Organs 169: 1–11.
Angst BD, Marcozzi C, and Magee AI (2001) The cadherin
superfamily. Journal of Cell Science 114: 629–641.
ADHESION, CELL–CELL / Epithelial 37
Bazzoni G and Dejana E (2004) Endothelial cell-to-cell junctions:
molecular organization and role in vascular homeostasis. Physiological Reviews 84: 869–901.
Boitano S, Safdar Z, Welsh DG, Bhattacharya J, and Koval M
(2004) Cell–cell interactions in regulating lung function. American Journal Physiology (Lung Cell Molecular Physiology) 287:
L455–L459.
Dudek SM and Garcia JG (2001) Cytoskeletal regulation of pulmonary vascular permeability. Journal of Applied Physiology
91: 1487–1500.
Hogg JC and Doerschuk CM (1995) Leukocyte traffic in the lung.
Annual Review of Physiology 57: 97–114.
Hynes RO (2002) Integrins: bidirectional, allosteric signaling
machines. Cell 110: 673–687.
Juliano RL (2002) Signal transduction by cell adhesion receptors
and the cytoskeleton: functions of integrins, cadherins, selectins,
and immunoglobulin-superfamily members. Annual Review of
Pharmacology and Toxicology 42: 283–323.
Ley K (2003) The role of selectins in inflammation and disease.
Trends in Molecular Medicine 9: 263–268.
Mandell KJ and Parkos CA (2005) The JAM family of proteins.
Advanced Drug Delivery Reviews 57(6): 857–867.
Muller WA (2003) Leukocyte–endothelial-cell interactions in leukocyte transmigration and the inflammatory response. Trends
in Immunology 24: 327–334.
Sohl G and Willecke K (2004) Gap junctions and the connexin
protein family. Cardiovascular Research 62: 228–232.
Vincent PA, Xiao K, Buckley KM, and Kowalczyk AP (2004) VEcadherin: adhesion at arm’s length. American Journal Physiology Cell Physiology 286: C987–C997.
Vorbrodt AW and Dobrogowska DH (2003) Molecular anatomy
of intercellular junctions in brain endothelial and epithelial barriers: electron microscopist’s view. Brain Research Reviews 42:
221–242.
Epithelial
J K McGuire, Children’s Hospital and Regional
Medical Center, Seattle, WA, USA
& 2006 Elsevier Ltd. All rights reserved.
Abstract
A continuous epithelial sheet lines the respiratory tract to provide a barrier to the outside world and protect against invasion
by microorganisms. Central to these functions are the complex
junctions that regulate adhesion between adjacent epithelial
cells. These junctions create epithelial polarity, regulate paracellular transfer of molecules across the epithelial barrier, and
maintain the integrity of the epithelial layer. Far more than just
‘cellular glue’, the major junctional complexes that regulate
cell–cell adhesion, i.e., tight junctions, adherens junctions, and
desmosomes, are critical in tissue patterning and differentiation
during development, epithelial cell sensing of the pericellular
environment, and modulation of intercellular signaling. Dozens
of different proteins have been associated with cell–cell adhesion functions in mammalian epithelia and only recently have
the structure and function of specific proteins expressed at
cell–cell junctions in the lung and airways been identified.
For example, the claudins are major transmembrane proteins
regulating extracellular interaction at tight junctions, and the
E-cadherin/b-catenin complexes that form adherens junctions
are critical in cell motility and differentiation and their loss may
be associated with epithelial carcinogenesis. However, little is
known about how specific changes in cell–cell adhesion contribute to respiratory disease, and this remains an area of intense
study.
Description
From the upper airway to distal alveoli, an essentially
continuous epithelial sheet comprising of several
specialized cell types lines the respiratory tract to
create a semipermeable barrier to the outside world
and provide protection from invading microorganisms. Essential to these functions are the complex
structures that produce cell–cell adhesion between
individual epithelial cells. Far more than just ‘cellular
glue’ holding epithelial cells together, cell–cell adhesion complexes organize development and differentiation of the lung and respiratory tract, promote and
maintain structural and functional integrity of the
epithelial surface, and regulate cellular responses to
injury and disease. Integral to the consideration of
cell–cell adhesion in respiratory epithelia are the specific structural and functional characteristics of these
cells. Respiratory epithelial cells are polarized, with
distinct apical and basolateral domains, with the
apical domain facing the external environment or
lumen and the basolateral domain forming contacts
with the substratum or neighboring cells. In addition
to regulating cell shape, polarization contributes to
the asymmetric distribution of organelles and molecules within epithelial cells and to the arrangement of
cytoskeletal networks. Cell–cell adhesion complexes
are key determinants of epithelial cell polarity and
are essential in major respiratory epithelial functions
such as maintenance of the barrier to the external
environment and other critical epithelial functions
including secretion and repair.
A high degree of similarity exists across different
species in the structure of cell–cell junctions and as in
most vertebrate epithelia, three major types of junctions mediate intercellular adhesion in respiratory
epithelial cells: tight junctions, adherens junctions,
and desmosomes. Additionally, all epithelia studied
thus far have an adhesive belt called the zonula adherens that encircles the cell just below the apical
surface and that is characterized by an electron-dense
cytoplasmic actin plaque (Figure 1). Along with the
tight junction, which is just apical to the zonula adherens, this adhesive belt delineates the apical and
basolateral epithelial compartments and is an important contributor to regulation of epithelial permeability and barrier function. Although the various
epithelial cell–cell junctions are composed of unique
proteins and serve distinct functions, in general, they
38
ADHESION, CELL–CELL / Epithelial
Tight junction
Adherens junction
Desmosome
Figure 1 Cell–cell junctions in respiratory epithelia. Schematic diagram of the three major types of junctions mediating cell adhesion in
epithelial cells. Tight junction transmembrane components occludin (in red above), claudin, and JAM (blue) interact in the extracellular
space and cytosolic plaque proteins (green) interact with the actin cytoskeleton (pink). Adherens junctions are formed by transmembrane cadherins (orange) that bind the actin cytoskeleton (pink) via the catenins (yellow and blue). Desmosomes are formed by the
desmosomal cadherins (blue and pink) and cytosolic plaque proteins (aqua) that interact with intermediate filaments (green). Electron
micrograph of mouse tracheal epithelium shows ultrastructure of zonula adherens near the apical/luminal epithelial surface.
Table 1 Major cell–cell junction proteins
Junction
Transmembrane proteins
Cytosolic proteins
Cytoskeletal linker proteins
Tight junction
Occludin
Claudins
Junctional adhesion
molecule (JAM)
Cadherins
Desmosomal cadherins
Plaque proteins
PDZ domain proteins (zonula
occludens, PAR)
b-Catenin
Plakoglobin, plakophilins, p0071
a-Catenin
Plakins
Adherens junction
Desmosome
share common structural features; principal among
these are transmembrane receptors, usually glycoproteins that bind proteins on the extracellular surface and determine the specificity of intercellular
interactions. The cytosolic domains of these receptors associate with a variety of cytoplasmic proteins
that link them structurally to the cytoskeleton and
regulate interaction with intracellular signaling pathways (Table 1).
Epithelial Cell–Cell Adhesion in Normal
Lung Function
Tight Junctions
Early electron microscopic assessment of the contacts
between epithelial and endothelial cells identified a
series of apparent fusions between the outer plasma
membranes of adjacent cells where the intercellular
space disappeared. These initial ultrastructural studies suggested and subsequent studies have confirmed
that tight junctions are a central component of
the barrier to the paracellular passage of solutes,
molecules, and inflammatory cells. Thus, an understanding of tight junction structure is important in
considering the regulation of lung barrier function.
Over 40 different proteins have been localized
to the tight junction in epithelial, endothelial, and
myelinated cells that can be further classified as tight
junction integral transmembrane proteins and cytoplasmic plaque proteins. The transmembrane proteins include the tetraspan protein occludin and
members of the claudin family, which contain four
transmembrane domains, two extracellular domains,
ADHESION, CELL–CELL / Epithelial 39
and are oriented with both the N- and C-terminal
ends towards the cytoplasm, and the single transmembrane Ig-domain-containing, junctional adhesion molecule (JAM). All of these interact directly
with cytoplasmic PDZ domain-containing plaque
proteins such as the zonula occludens (ZO) and PAR
proteins, which function as adapters to recruit cytoskeletal or signaling molecules. The tetraspan extracellular domains interact in the paracellular space
with extracellular domains of tight junction proteins
on neighboring cells and occludin, the first integral
membrane protein to be found in tight junctions of
many cell types, also directly binds F-actin via the
last 150 amino acids of its carboxyl tail. The ‘tightness’ of tight junctions appears to be regulated by
changes in the combination and expression ratios of
different claudin family members, which can engage
in homotypic or heterotypic interactions between
several of the 24 distinct claudin gene products thus
far identified in humans. One defined role of claudins
in regulating permeability is the formation of ionselective pores, and different types of claudins appear
to determine the specificity of the pores for individual
ions such as Na þ , Cl , or Ca2 þ . Whereas tight
junctions can form in the absence of occludin, claudins appear to be essential for tight junction formation. It is likely that JAM has an important role
in tight junction assembly and resealing after disassembly in epithelia by engaging in homophilic interactions in the paracellular space.
Tight junction plaque proteins have important
roles in coordinating intracellular signaling by recruiting cytosolic proteins to the tight junction during
assembly, and recent reports of ZO proteins localizing to the nucleus, in addition to their tetraspanbinding at tight junction plaques, suggest that they
transduce signals from the cell membrane to the
nucleus where they may regulate gene expression.
Recruitment of plaque proteins to the tight junction
may also have a role in regulating cell motility and
proliferation. Consequently, although tight junctions
perform key functions in regulating epithelial permeability, it is becoming increasingly clear that tight
junctions are multifunctional complexes involved in
many vital epithelial cell functions. Only recently
have specific patterns of tight junction protein expression in the respiratory tract been characterized,
and much work remains to further define how tight
junctions are regulated in normal and abnormal lung
function.
Adherens Junctions
Adherens junctions are ancient structures present
across eukaryotes and even in related structures in
single-celled organisms such as yeast, and as such,
adherens junctions have critical functions in coordinating cell polarity, cytoskeletal dynamics, cell sorting, and differentiation. In addition to regulating
organization within epithelia, adherens junctions
are also important in transmitting information from
the environment to the interior of cells. At the core
of adherens junctions are the calcium-dependent
transmembrane glycoprotein cadherins, and the major epithelial cadherin is E-cadherin, the best characterized member of the cadherin family and the
primary cadherin expressed in respiratory epithelium. The extracellular domain of E-cadherin, like
other classical cadherins, is comprised of five calcium-binding cadherin repeats that participate in
homotypic interactions with neighboring cells. There
is a single-pass transmembrane domain and the
cadherin cytosolic domain is linked to cytoskeletal
structures via the catenins. Cadherin adhesive activity is regulated in several ways including the level of
cadherin gene expression, which correlates with
the strength of adhesion, and the type of cadherin
expressed, which affects the specificity of cell interaction and plays a role in embryo patterning and
determination of cell fate. Posttranscriptional mechanisms regulating cadherin adhesion include modulation of cadherin clustering at the cell surface
and changes in cadherin interaction with the catenins. Proteolysis of cadherin domains is now recognized as an important means of posttranslational
cadherin modification with extracellular domains
being cleaved by metalloproteinases, the transmembrane domains targeted by g-secretases, and intracellular domains degraded by caspases. Cadherins
are key mediators of morphogenesis, epithelial sheet
integrity, growth control, and maintenance of the
terminally differentiated phenotype. Differential expression of different cadherin classes is likely to be
important in cell sorting and embryo patterning in
development and specific cadherins may be associated with the determination of cell fate. For example,
E-cadherin is the primary cadherin expressed in lung
epithelia, whereas N-cadherin is expressed at cell–
cell junctions in pleural mesothelial cells.
The cytoplasmic E-cadherin domain binds the armadillo family member protein b-catenin, and this
binding induces a tertiary structure that in turn binds
a-catenin, which nucleates the assembly of a multimeric complex that links E-cadherin/b-catenin complexes to the actin cytoskeleton. This process both
stabilizes the intercellular junctions and allows for
coordination of cytoskeletal dynamics with changes
in cell–cell adhesion. These protein interactions
are regulated by phosphorylation events of both the
E-cadherin cytoplasmic tail (increase interaction) and
40
ADHESION, CELL–CELL / Epithelial
b-catenin (decrease interaction) and by the recruitment of various other potential adherens junctionassociated proteins. Therefore, this complexity allows
for many levels of regulation of adherens junctionbased cell adhesion. In addition to its structural role
in adherens junctions, b-catenin also functions as a
noncadherin-dependent signaling factor. In concert
with activation of the Wnt signaling pathway, free
cytoplasmic b-catenin translocates to the nucleus
and binds to transcription factors of the lymphocyte
enhancer-binding factor-1/T-cell factor (LEF-TCF)
pathway to regulate expression of downstream target genes associated with cell migration and proliferation including matrilysin (MMP-7), c-myc, WISP,
and cyclin D1. A comprehensive consideration of
b-catenin in cell signaling is beyond the focus of this
discussion of cell adhesion, but recent studies have
suggested a role for b-catenin in terminal differentiation of alveolar epithelium.
Adherens junctions at the cell surface associate
with several other types of intercellular junctions and
membrane receptors. Ultrastructural studies of epithelial sheets reveal closely apposed membranes
at sites of cell–cell contact where adherens junctions
alternate with desmosomes, and formation of adherens junctions has been shown to be necessary for
desmosome and tight junction formation. Adherens
junction-specific cadherins also associate with other
cell surface receptors that participate in signaling
such as connexins, Notch, receptor tyrosine kinases,
and phosphatases, suggesting that adherens junctions
can be regulated by extracellular cues.
Desmosomes
Desmosomes are specialized junctions that anchor
stress-bearing intermediate filaments at sites of
strong intercellular adhesion. This provides a scaffold that gives integrity to tissues such as skin, heart,
and respiratory epithelium that is subject to mechanical stress. At the core of desmosomes are proteins of
the cadherin (the desmogleins and desmocollins) and
armadillo (plakoglobin, the plakophilins, and p0071)
families, which form membrane-associated complexes that are tethered to intermediate filaments
via the plakins. Analogous to adherens junction cadherins, the single-pass transmembrane desmosomal
cadherin extracellular domains interact in the extracellular space; however, in contrast to adherens junction cadherins, desmosomal cadherins interact in a
heterotypic manner as each desmosome must contain
at least one desmoglein and one desmocollin. Desmocollins and desmogleins are expressed in a tissueand differentiation-specific manner, and thus it
has been proposed that desmosomal cadherins may
function in directing epithelial differentiation. The
cytoplasmic cadherin tails directly bind an armadillo
protein, usually plakoglobin (g-catenin), and this
complex in turn associates with a cytoskeletal linking
protein, e.g., desmoplakin, that mediates attachment
to the intermediate filaments. The result is a series of
connections between adjacent cells that maintains
tissue tensile strength.
Similarities exist between the regulation of adherens junction and desmosomal adhesion; as for
adherens junctions, desmosomal adhesion during
development is regulated by the timing of and type
of desmosomal cadherin or armadillo protein expressed, and as discussed above, desmosome assembly appears to be downstream of and dependent on
adherens junction assembly. Posttranslational mechanisms regulating desmosomal adhesion include proteolytic processing of intracellular cadherin domains
by caspases and cleavage of extracellular domains by
metalloproteinases. Although desmosomes are present throughout the airway and alveolar epithelium,
little is known about the specific regulation of desmosome-based cell adhesion in the respiratory tract.
Cell–Cell Adhesion in Respiratory
Diseases
Although the net permeability of the lung and airways is jointly regulated by the epithelium and endothelium, disruption of paracellular permeability
and leakage of fluid and proteins across the airway or
alveolar epithelium are hallmarks of a variety of respiratory diseases including asthma, allergic rhinitis,
acute lung injury, and pneumonia among others. Furthermore, pathogens may directly target junctional
proteins to facilitate entry into the host. However, the
specific mechanisms by which changes in tight and
adherens junctions and the proteins that regulate
these junctions in diseases of the airways and lung
remain largely undefined. It has been known for a few
years that some types of lung cancer, like other carcinomas, show altered staining patterns for several
adherens junction proteins, and an increasing amount
of evidence suggests that inactivation of E-cadherincatenin complexes plays a significant role in carcinogenesis. Additionally, altered b-catenin signaling has
been implicated in idiopathic pulmonary fibrosis.
Thus, these pathways may be attractive targets for
therapy in these historically difficult to treat diseases.
The available knowledge on the structure, assembly, and function of intercellular junctions in normal
and abnormal processes is rapidly increasing. These
complexes clearly have more than a structural role
and perform critical functions in tissue development,
ADHESION, CELL–MATRIX / Focal Contacts and Signaling 41
homeostasis, and response to injury. However, our
understanding of the regulation of epithelial cell–cell
adhesion in respiratory tract development and maintenance of normal lung function, and in injury and
disease remains limited. Thus, this remains an area of
intense investigation.
See also: Adhesion, Cell–Cell: Vascular. Asthma:
Overview. Basal Cells. Bronchiectasis. Bronchiolitis.
Connexins, Tissue Expression. Contractile Proteins. Defense Systems. Epithelial Cells: Type I
Cells; Type II Cells. Gene Regulation. Lung Development: Overview. Matrix Metalloproteinases. Panbronchiolitis. Signal Transduction. Transcription
Factors: Overview. Tumors, Malignant: Overview;
Metastases from Lung Cancer.
Further Reading
Braga VMM (2002) Cell–cell adhesion and signalling. Current
Opinion in Cell Biology 14: 546–556.
Bremnes RM, Veve R, Hirsch FR, and Franklin WA (2002) The
E-cadherin cell–cell adhesion complex and lung cancer invasion,
metastasis, and prognosis. Lung Cancer 36: 115–124.
Godfrey RWA (1997) Human airway epithelial tight junctions.
Microscopy Research and Technique 38: 488–499.
Gonzalez-Mariscal L, Betanzos A, Nava P, and Jaramillo BE
(2003) Tight junction proteins. Progress in Biophysics and
Molecular Biology 81: 1–44.
Perez-Moreno M, Jamora C, and Fuchs E (2003) Sticky business:
orchestrating cellular signals at adherens junctions. Cell 112:
535–548.
Schneeberger EE and Lynch RD (2004) The tight junction: a multifunctional complex. American Journal of Physiology. Cell
Physiology 286: C1213–C1228.
Wheelock MJ and Johnson KR (2003) Cadherins as modulators of
cellular phenotype. Annual Review of Cell and Developmental
Biology 19: 207–235.
Yin T and Green KJ (2004) Regulation of desmosome assembly
and adhesion. Seminars in Cell and Developmental Biology 15:
665–677.
Zhurinsky J, Shtutman M, and Ben-Ze’ev A (2000) Plakoglobin
and b-catenin: protein interactions, regulation and biological
roles. Journal of Cell Science 113: 3127–3139.
ADHESION, CELL–MATRIX
Contents
Focal Contacts and Signaling
Integrins
Focal Contacts and Signaling
G D Rosen and D S Dube, Stanford University
Medical Center, Stanford, CA, USA
& 2006 Elsevier Ltd. All rights reserved.
Abstract
Focal adhesions (FAs) are contact points for the cell with the
extracellular matrix. These complex structures regulate communication of the cell with the surrounding extracellular environment and signaling through these FAs regulates diverse
cellular processes, including proliferation, migration, apoptosis,
spreading, and differentiation. The principal components of the
FAs are integrins, which are ab heterodimers that regulate cell–
matrix and cell–cell interactions. Focal adhesion kinase (FAK) is
a kinase that localizes to FAs and regulates signaling in FAs.
FAK communicates signals between integrins and intracellular
proteins, which regulate diverse cellular processes such as cell
polarity, migration, and invasion. FAs play a central role in
regulating normal developmental processes such as blood vessel
morphogenesis but increased activity of FA can incite aberrant
events such as tumor cell invasion and pulmonary fibrosis. A
further exploration of the role of FA in health and disease will
provide greater insight into the regulation of normal developmental processes and into the pathogenesis of lung diseases such
as lung cancer, acute respiratory distress syndrome, and pulmonary fibrosis.
Introduction
This article explores the physiology of focal contacts,
and cell signaling in normal and pathological states.
Focal adhesions (FAs) are specialized multimolecular
structures that exist at the points of contact between
the cell membrane and the extracellular matrix. Our
current understanding of the physiological roles of
FAs suggests that in addition to mediating cell adhesion, they also function as a physiological signaling
link between the cytoplasm and the extracellular milieu by promoting the bidirectional transmission of
biochemical signals. Integrins are the core components of FAs whose orderly function underlies the
diverse roles associated with FAs. Integrins are a
family of a/b heterodimeric glycoprotein receptors
that span the cell membrane. Morphologically, these
receptors exhibit an intracellular domain, a transmembrane domain, and an ectodomain that is in association with the cell matrix or other cells. The
42
ADHESION, CELL–MATRIX / Focal Contacts and Signaling
intracytoplasmic domains of integrins can associate
with actin, adaptor proteins, and enzymatic components of signaling cascades and their substrates. This
complex association of the cell matrix, integrins, and
cytosolic proteins forms the molecular conglomerates that are termed FAs and mediates the bidirectional flow of signals between the cell matrix and the
cell. The binding of ligands to integrins in either the
extracellular or intracellular domains induces conformational changes within the integrins that affect
the binding of ligands on the reciprocal end of the
integrin molecule. The activation of FAs by paracrine
and endocrine stimuli culminates in the activation
of specific intracytoplasmic signaling cascades, including tyrosine phosphorylation, focal adhesion
kinase (FAK)-mediated signaling, mitogen-activated
protein-kinase (MAPK), and the generation of phosphatidylinositol-4, 5-bisphosphate. These intracellular signals result in a multitude of cytoplasmic
responses that regulate, for example, cell proliferation, motility, and differentiation. Based on the
multiplicity of functions attributed to FAs, the strict
regulation of their function forms the basis of orderly
organ structure and tissue function.
A better understanding of the mechanisms that are
involved in the regulation of FAs has led to the development of potent antithrombosis and anti-inflammatory therapies. Moreover, the multiple roles of FAs
further promise to aid the elucidation of diverse pathological processes associated with FAs. Some of these
processes include tumor growth, pathological organ
remodeling, cell barrier defects, and embryogenesis
as well as many other pathological entities that are
regulated by cell–matrix and cell–cell contact.
Focal Contacts: Structure and Function
Interference reflection microscopy first identified FAs
as dark areas at the interface between the ventral cell
surface and the extracellular matrix. Diverse types of
focal adhesions have since been described. Subsequently, advances in gene cloning have led to the determination of the primary structures of many of the
constituents of FAs. Early analysis of FA structure
identified integrins as core glycoproteins intimately
associated with the structure of FAs. The elucidation
of the primary structure of integrins revealed three
domains: an extracellular domain, a transmembrane
domain, and an intracellular domain as well as sites
for tyrosine phosphorylation. In parallel with the
elucidation of the primary structure, the solution of
the crystal structures of the components of FAs further advanced testable hypotheses on structure–
function relationships of the individual protein
components of FAs. Studies of the conformational
rearrangements within integrin molecules using diverse tools such as negative stain microscopy, nuclear
magnetic resonance, and spectroscopy have further
aided the understanding of the function of focal
adhesion molecules. These studies have allowed the
evaluation of conformational changes within integrins
using ligand analogs that bind to ligand-induced
binding sites (LIBSs). Site-directed mutagenesis has
also aided the characterization of the roles of submolecular components of protein components of FAs,
while in vivo studies of these mutagenesis studies have
led to the discovery of unique phenotypes, thus further allowing the determination of the functional roles
of individual components of FAs. Most recently, the
use of fluorescent green protein (FGP) tagged proteins
has allowed the determination of stoichiometric relationships of the various components of FAs.
Components of Focal Adhesions:
Integrins and Extracellular Matrix
Integrins are a large family of heterodimeric transmembrane glycoprotein receptors uniquely found
among metazoans whose quaternary structure is characterized by a noncovalent union between a- and
b-subunits. There are 18 a-subunits (MW 120–
180 kDa) and 8 b-subunits (MW 90–110 kDa) that
assemble in various permutations to generate 24 distinct integrin heterodimers. These various subunit
combinations expand the repertoire of intracellular
and extracellular ligands for integrins. In turn, the
expanded receptor capability created by the many
combinations of a- and b-subunits accounts for the
diverse functional roles of integrins. Integrins are
central to the structure of FAs. The significance of
integrins in metazoan cell structure and tissue organization is emphasized by the observation that the
number of integrins parallels the evolutionary complexity of an organism. It has been proposed that the
evolution of metazoans as multicellular organisms
hinged upon the parallel evolution of integrins as anchor apparatus to the cell matrix and intercellular
molecular adhesives. Figure 1 shows the subclassification of the integrin receptor family.
Integrins have a large extracellular domain, a
transmembrane domain, and an intracytoplasmic
domain. The ectodomains of integrins exist in three
major conformational states. The first of these states
is a bent conformation that is similar to the crystal
structure of avb3. This conformation is considered to
be a low-affinity state. The second conformation has
been referred to as a ‘closed head piece’ conformation and is considered to represent an intermediate
affinity conformational state. Finally, there is the
ADHESION, CELL–MATRIX / Focal Contacts and Signaling 43
ΙΙb
Collagen receptors
1
3∗
1∗
9
8
8
2
11
V
6
Leukocyte-specific
receptors
10
5
5
RGD
receptors
2
D
L
M
X
4
3∗
6∗
7∗
7
Laminin receptors
E
4∗
Figure 1 The integrin receptor family. Integrins are ab heterodimers that span the cell membrane. This figure shows the mammalian
subunits and their ab associations. Asterisks denote alternatively spliced cytoplasmic domains. Reproduced from Hynes RD (2002)
Integrins: bi-directional, allosteric signaling machines. Cell 110: 673–687, with permission from Elsevier.
high-affinity ‘open head piece’ conformation, which
is induced by ligand binding. The high-affinity conformation is thought to be acquired by the extension
of the angle between the b- and hybrid domains. A
detailed discussion of the crystal structures of the
various integrins and their interacting partners is
outside the scope of this article. The evidence so far
supports a model whereby the binding of a ligand to
the ectodomain mediates the extension and simultaneous separation of the a- and b-subunits of the integrin heterodimers within the transmembrane and
intracytoplasmic domains. This model also suggests
that the binding of a ligand in the intracellular domain of integrins by adaptor proteins such as talin
also mediates counter conformational alterations
that cause the exposure of LIBS in the ectodomain.
It has further been proposed that these conformational changes are dynamic and dependent on regulatory factors such as growth factors.
Regulation of Integrins
Intracytoplasmic Association between the a- and
b-Chains
A considerable body of experimental data reveals
that the interaction between the a- and b-subunits in
the cytoplasm regulates the activation states of
the integrin receptors. Studies utilizing the platelet
integrin aIIbb3, for example, have revealed that the
aIIb short intracytoplasmic domain negatively regulates the activation of the integrin receptors. The experimental deletion of the intracytoplasmic aIIb
domain or the conserved GFFKR sequence leads to
constitutively activated integrin receptors. Similarly,
deletions of conserved intracytoplasmic b3 sequences
also culminate in constitutively activated integrin
receptors. These observations have led to the conclusion that the interaction between these two cytoplasmic domains negatively controls the activation of
integrins. This interaction seems to occur through a
salt bridge between the R995 of the aIIb and the
D723 in the b3. In support of this idea, the experimental mutation of either amino acid to the charge
neutral alanine leads to a constitutively active integrin. Nuclear magnetic resonance (NMR) studies
have demonstrated that the intracytoplasmic portions of the a- and b-domains interact with each
other. Furthermore, it has been shown that point
mutations in F992A or R995D impair the interaction
of the a- and b-domains.
The Role of Talin in Integrin Activation
Studies support the conclusion that talin, a cytoskeletal actin-binding protein that co-localizes with active
forms of integrins, is a crucial element in the activation of integrins. Talin is a homodimer consisting
of two antiparallel subunits, each approximately
270 kDa. Each of the talin subunits is comprised of
a 220 kDa C-terminal and a 70 kDa globular head,
which is thought to contain the binding sites for
integrins. Talin has been demonstrated to bind the
b1D, b2, b3, b5, and b7 intracytoplasmic integrins at
their N-terminus. Structural analysis of the PTB-like
96 amino acid subdomain of the talin FERM (4.1,
ezrin, radixin, moesin) domain shows a major integrin-binding site. Besides the ubiquitous interaction
44
ADHESION, CELL–MATRIX / Focal Contacts and Signaling
of talin with diverse integrins, the overexpression of a
talin fragment containing the head domain leads to
the activation of integrin molecules. The role of talin
in integrin activation is further supported by the
observation that talin knockdown abrogates aIIb3 activation by endogenous agonists. This observation
suggests that talin is a critical common downstream
target in the activation of at least some integrins.
Molecular mechanisms of the role of talin in the
regulation of integrins are complex and still being
explored. Vonogradova and colleagues have demonstrated that the talin head domain alters the NMR
spectrum for the interaction between the b3 and aIIb
domains. Other investigators have shown that the
talin head domain attenuates the fluorescence resonance energy transfer (FRET) between fluorophoretagged a- and b-integrins within viable cells. These
observations support a model whereby the binding of
the talin phosphotyrosine-binding (PTB) domain to
integrin disrupts the inhibitory interaction between
a- and b-membrane proximal domains.
Regulation of Talin–Integrin Interactions
The preceding section considers that the interaction
of talin with intracytoplasmic domains of integrin
is pivotal to the regulation of integrin function and
hence FAs. A variety of mechanisms have been advanced to account for how these interactions are
regulated. The talin head has a sixfold higher affinity
in comparison to the intact talin molecule. This observation has led to the hypothesis that in the whole
talin molecule, integrin binding sites are obscured.
Therefore, a potential mechanism whereby talin regulates integrin activation is through the modulation
of its conformations by physiological factors, which
then reveal or mask integrin binding sites. Evidence
supporting this possibility accrues from observations
made from the function of the ERM (ezrin, radixin,
moesin) family of proteins. The major integrin-binding site in talin lies within the talin FERM domain,
and binding occurs via a variant of the classical
PTB domain–NPxY interaction. In the ERM family,
the intramolecular interaction of the N-terminal
FERM domain with the C-terminal obscures ligandbinding sites. Intriguingly, ERM phosphorylation,
PIP2 (phosphatidyl inositol 4,5-bis-phosphate), and
rho-dependent pathways abrogate the native inhibition in the unstimulated ERM molecules presumably
by unmasking the interaction of the FERM domain.
Although the role of phosphorylation in the activation of integrins is still debated, it is notable that the
binding of PIP2 to talin has been shown to mediate
a conformational change within the talin molecule
that culminates in the exposure of integrin-binding
sites in the FERM domain. The role of PIP in the
regulation of talin is complicated by the observation
that talin directly stimulates a splice variant of PIP2producing enzyme PIPKIg-90 (phosphatidylinositol
phosphate kinase type Ig-90) thereby increasing PIP2
production. Interestingly, PIPKIg-90 and integrin b
tails have overlapping binding sites on the talin F3
subdomain. This suggests a novel control strategy
whereby PIPKIg-90 and talin may displace each other
from integrin-binding sites following activation.
Another regulatory mechanism for talin–integrin
interaction is thought to involve calpain-mediated
proteolysis of integrin b tails at sites straddling the
NPXY/NXXY motifs that are necessary for the anchorage of integrins to the cytoskeleton and FAs.
Calpains are calcium-dependent thiol proteases that
have a diverse substrate spectrum that includes constituents of the cytoskeletal apparatus and cell-signaling proteins. Structurally, they are composed of an
80 kDa catalytic subunit and a 30 kDa regulatory
subunit. Although calpain was historically considered
to be involved in the detachment of migrating cells,
accumulating evidence suggests that calpain also
functions by regulating actin remodeling during
cell migration. Supportive data have been obtained
through experimental overexpression of calpain in
aortic endothelial cells. These experiments reveal that
overexpression of calpain leads to formation of focal
adhesions. In contrast, when calpain is inhibited in
fibroblasts and endothelial cells, adherence is maintained while cell dispersion is attenuated. Additional
evidence points to the fact that the regulation of actin
filament formation in endothelial cells is dependent
on calpain-mediated proteolysis. The capacity of
calpain to cleave integrins with diverse molecular
structures suggests that calpains recognize higherorder protein structure rather than primary protein
sequence. Calpains also cleave tyrosine kinases (PTK)
such as focal adhesion kinase (FAK) and src and
phosphatases (PTP) PTP-1B, Shp-1, and PTP-MEG.
Other investigators have reported that calpain cleaves
the N-terminal 47 kDa of talin, which is known to be
hyperphosphorylated by protein kinase A.
Focal Adhesion Structure and Signaling
FAs exhibit dynamic spatial–temporal variations in
function. A large group of proteins are core to the
structural behavior of focal adhesions. This is in
keeping with the diversity of roles that are associated
with FAs. The structural integrity of FA molecules
relies on adaptor molecules that perform core scaffolding functions. Multiple other molecules also
interact with the FAs and perform both structural
and enzymatic chores of FAs. The overall function
ADHESION, CELL–MATRIX / Focal Contacts and Signaling 45
and net effect of these scaffolds is to juxtapose
kinases with their substrates and thereby facilitate
FA-mediated signaling. The full repertoire of proteins
that have been observed to interact with FAs is large
and continuously expanding. In general, the relative
expression or activation of different scaffolding proteins depends on the tissue of origin and cellular
stimulus. Therefore, scaffolding proteins not only
confer on FA structural integrity but are also responsible for the functional diversity associated with FAs.
A core component of the FA complex is the
p130Cas protein. The structure of p130Cas includes
an SH3 domain, a proline-rich motif, and a very
complex substrate-binding region that has the
Src-SH2 binding region. The p130Cas SH3 domain
interacts with the FAK possibly through interactions
with the YDYV motif. This same YDYV motif also
binds c-Src. It is thought that c-Src mediates the
phosphorylation of the substrate domain of p130Cas.
It is intriguing to also note that FAK has the capability to phosphorylate an Src binding site within
p130Cas. The phosphorylation of the substrate-binding site within p130Cas achieves two major roles.
First it allows the localization of p130Cas to the FA
and second it allows p130Cas to interact with other
proteins containing the SH2 domain such as protein
tyrosine phosphatase (PTP)-PEST, NcK, and CrK.
Crk/CrkL is an adaptor protein that interacts with
phosphorylated p130Cas via an SH2 domain. The
interaction of Crk with P130Cas leads to the formation of a complex that activates Dock 180, which is
a novel exchange factor. Ultimately, the binding of
Dock 180 to Crk and ELMO culminates in the activation of Rac, which augments the activation of
p130Cas phosphorylation. In general, the association
of p130Cas with Crk activates GTPases that are involved in the maintenance and reorganization of the
actin cytoskeleton, which are important for cell motility and are crucial to establishing cellular structure.
Paxillin is another adaptor molecule that interacts
with a diverse range of proteins such as FAK, PKL,
PTP-PEST, and b1 and a4 integrin intracytoplasmic
tails. Similar to P130Cas, paxillin also complexes
with a wide range of distinct exchange factors in a
phosphorylation-dependent and tissue-specific manner. In summary, scaffold proteins bring into close
proximity protein moieties important for the function of the FAs. The functional diversity conferred by
the combinations afforded through these proteins
results in tissue-specific function.
Kinases
Tyrosine phosphorylation of focal adhesion components is considered to be the core event in the
generation of focal adhesion-mediated cell signaling.
Tyrosine phosphorylation creates sites for binding of
Src-homology domain (SH2-containing proteins).
Moreover, tyrosine phosphorylation directly regulates a variety of kinases and phosphatases. The
principal kinases that regulate focal adhesions are
FAK and Src. Multiple other kinases (ser/thr kinases,
Abl, PYK2,Csk) also probably play a role but their
precise role in regulation of focal adhesions has not
been fully elucidated.
Focal adhesion kinase FAK is a 125 kDa protein
kinase that was initially shown to localize to FAs.
Moreover, it was observed to be phosphorylated
in relation to Src-mediated transformation. FAK is
an FERM domain protein and is known to bind to
many different signaling proteins. In general, it is
considered that phosphorylation of FAK is the sentinel event in FA-mediated cell signaling. In response
to integrin association with a ligand, FAK is activated by autophosphorylation at tyrosine Y397. One
model proposes that subsequent to this autophosphorylation, Y397 acts as a docking site for Src that
in turn mediates the phosphorylation of FAK at
Y576 and Y577 and potentially at other sites. The
phosphorylation of these tyrosines creates molecular contact points for proteins containing SH2
domains. As a result of this, the Ras-regulated
MAPK pathway is activated (Figure 2). In support
of this model, it has been observed that phosphorylation of Y926 in FAK creates an interaction site
(pYNQV) for the SH2 domain of Grb2, an adapter
protein that regulates growth factor signaling
through Ras/SOS through binding of its SH2 and
SH3 domains. This event is considered to be critical
for the activation of the MAPK pathway. Grb2
can be phosphorylated at the Y926 position only
when FAK is dissociated from the focal adhesion
complex. This observation suggests that the phosphorylation of FAK causes it to dissociate from the
FA complex and then phosphorylates its target
proteins and in turn initiates signaling cascades.
The interaction of FAK with FAs occurs through the
FA-targeting domain (FAT) of FAK. Indeed, when the
FAT domain in FAK is mutated, the FAK molecules
are incapable of autophosphorylation and cannot
phosphorylate known endogenous substrates. Although the role of FAK as a regulatory kinase is
widely accepted, it is intriguing that FAK molecules
lacking the kinase domain are still functional in some
cell-signaling pathways. This has led to the proposal
that FAK may predominantly perform a structural
role by acting as a scaffold for diverse constituents of
FAs. However, these same data have been interpreted
46
ADHESION, CELL–MATRIX / Focal Contacts and Signaling
F
K
A
Integrin
Graf
SH3
Pro Y397
371–374 –P
Pro
Pro Y925
712–715 878–881 –P
Paxillin
COOH
SH3-SH2
Src/Fyn
SH2
PI3 kinase
SH2
PI3 kinase
SH2
GRB-2
Paxillin
Grb2
Spreading
Sow/Ras
p130Cas
Erk (MAPK)
Crk/Nek?
Akt?
Apoptosis
Proliferation
Migration
Figure 2 Signaling through focal adhesion kinase (FAK). Association of FAK with integrins activates signaling pathways that regulate
cell proliferation, apoptosis, spreading, and migration. Reproduced from Laboratory of Molecular Biophysics http://biop.ox.ac.uk, with
permission.
to imply that other kinases have the ability to duplicate the kinase functions of FAK.
Src tyrosine kinase The c-Src tyrosine kinase performs a pivotal role in the regulation of the function
of FAs and the cytoskeleton. In transformed cells, vSrc is localized at three distinct locations: perinuclear, the actin cytoskeleton, and focal adhesions. Studies utilizing vSrc mutants in LA 29 rat fibroblasts,
which express a temperature-sensitive (ts) v-src mutant, with D1025 rat fibroblasts, transfected with a ts
mutant of v-fps, have demonstrated that at restrictive
temperatures vSrc is inactive and localizes to the
perinuclear regions where it is intimately associated
with the microtubular cytoskeleton. In contrast, at
more permissive temperatures v-Src localizes to focal
adhesions at the cell periphery where it interacts with
RhoA-induced actin fibers. An interesting observation is that targeting of v-Src is independent of its
own tyrosine kinase function; rather it depends on
its SH3 domain. However, this localization is dependent on the binding of the p85 regulatory subunit
of phosphatidylinositol kinase (P13 K) to the v-Src
SH3 domain as well as on P13 K kinase function.
Although not completely analogous to v-Src, overexpressed c-Src in fibroblasts localizes to a perinuclear location and associates with microtubules and
organelles such as endosomes and the trans-Golgi
network. The localization of c-Src is dependent on
GTPse activity, which directs c-Src to diverse cellular
locations. The extrapolation of the function of c-Src
from the observations of v-Src is limited by the observation that c-Src does not associate with p85 of
p13 K. Thus, the role of c-Src still needs to be fully
defined.
Focal Adhesion Molecules in Respiratory
Disease
A characterization of the role of focal adhesion kinases in normal cellular function and in disease is an
area of active investigation. Focal adhesion molecules such as integrins and FAK play key roles in
the regulation of cell–cell communications, structure,
and remodeling during development of the lung and
other tissues. Perturbations of the tightly controlled
functions of focal adhesions have been shown to
affect tumorigenesis and metastasis but the role of
focal adhesions in diseases of the lung and other organs is less well defined. However, studies have begun to reveal a role for focal adhesions in cellular
function and disease states. For example, focal adhesions have been shown to be important in lymphocyte homing to sites of inflammation as b7 integrins
have been shown to be crucial in the localization of
lymphocytes to sites of inflammation in the gastrointestinal tract. Focal adhesions are also important
ADHESION, CELL–MATRIX / Integrins 47
in hemostasis as GII3b integrins are crucial in platelet
aggregation and their blockade has given rise to a
potent class of antagonists that are used to prevent
platelet aggregation following interventional procedures in cardiology. Also, FAK is required for
blood vessel morphogenesis and migration of vascular smooth muscle cells. FAK has been shown to be
required for microtubule organization, nuclear
movement, and neuronal migration during brain
development and to be a negative regulator of axonal
branching and synapse formation.
In the acute respiratory distress syndrome, perturbation of the cell surface barrier is thought to be
mediated through impaired focal adhesion function
thus leading to increased permeability. Additionally,
TGF-b-induced activation of FAK has been shown to
mediate the TGF-b-mediated suppression of apoptosis in lung fibroblasts, which likely plays a role in
the pathogenesis of idiopathic pulmonary fibrosis.
We among others have shown that FAK is cleaved
during apoptosis and a recent study revealed that
FAK interacts with p53 and inhibits p53-mediated
apoptosis. Further studies will elucidate the precise
role for focal adhesions in normal cellular function
and in disease.
See also: Adhesion, Cell–Cell: Vascular ; Epithelial.
Adhesion, Cell–Matrix: Integrins. Apoptosis. Cell
Cycle and Cell-Cycle Checkpoints. Cytoskeletal
Proteins. Endothelial Cells and Endothelium. Epithelial Cells: Type I Cells; Type II Cells. Extracellular
Matrix: Basement Membranes; Elastin and Microfibrils;
Collagens; Matricellular Proteins; Matrix Proteoglycans; Surface Proteoglycans; Degradation by Proteases.
Leukocytes: Mast Cells and Basophils; Eosinophils;
Neutrophils. Platelets.
Further Reading
Calderwood DA (2004) Integrin activation. Journal of Cell Science
117(5): 657–666.
Calderwood DA (2004) Talin controls integrin activation. Biochemical Society Transactions 32(3): 434–437. (Review: PMID:
15157154: PubMed – indexed for MEDLINE.)
Calvete JJ (2004) Structures of integrin domains and concerted
conformational changes in the bidirectional signaling mechanism of alphaIIbbeta3. Experimental Biology and Medicine
(Maywood) 229(8): 732–744.
Diagne I, Hall SM, Kogaki S, Kielty CM, and Haworth SG (2003)
Paxillin-associated focal adhesion involvement in perinatal pulmonary arterial remodelling. Matrix Biology 22(2): 193–205.
Frame MC, Fincham VJ, Carragher NO, and Wyke JA (2002) vSrc’s hold over actin and cell adhesions. Nature Reviews: Molecular Cell Biology 3(4): 233–245.
Hynes RD (2002) Integrins: bi-directional, allosteric signaling machines. Cell 110: 673–687.
Kanda S, Miyata Y, and Kanetake H (2004) Role of focal adhesion
formation in migration and morphogenesis of endothelial cells.
Cellular Signalling 16(11): 1273–1281.
Mould AP and Humphries MJ (2004) Cell biology: adhesion articulated. Nature 432(7013): 27–28.
Petit V and Thiery JP (2000) Focal adhesions: structure and dynamics. Biology of the Cell 92(7): 477–494.
Schlaepfer DD, Mitra SK, and Ilic D (2004) Control of motile and
invasive cell phenotypes by focal adhesion kinase. Biochimica et
Biophysica Acta 1692(2–3): 77–102.
Schoenwaelder SM and Burridge K (1999) Bidirectional signaling
between the cytoskeleton and integrins. Current Opinion in Cell
Biology 11(2): 274–286.
Takagi J, Petre BM, Walz T, and Springer TA (2002) Global conformational rearrangements in integrin extracellular domains in
outside-in and inside-out signaling. Cell 110(5): 599–611.
Vinogradova O, Velyvis A, Velyviene A, et al. (2002) A structural
mechanism of integrin alpha(IIb)beta(3) ‘‘inside-out’’ activation
as regulated by its cytoplasmic face. Cell 110(5): 587–597.
Wen LP, Fahrni JA, Troie S, et al. (1997) Cleavage of focal adhesion kinase by caspases during apoptosis. Journal of Biological Chemistry 272(41): 26056–26061.
Wozniak MA, Modzelewska K, Kwong L, and Keely PJ (2004)
Focal adhesion regulation of cell behavior. Biochimica et
Biophysica Acta 1692(2–3): 103–119.
Integrins
L M Schnapp, University of Washington, Seattle, WA,
USA
& 2006 Elsevier Ltd. All rights reserved.
Abstract
Integrins are a large family of cell adhesion receptors that mediate cell–cell and cell–extracellular matrix adhesion. Integrin
members are expressed on virtually every cell and provide essential links between the extracellular environment and intracellular signaling pathways. In doing so, integrins play a role in
most essential cell behaviors, including cell survival, apoptosis,
differentiation, and transcriptional regulation. Phenotypes of
integrin knockout mice illustrate important roles of integrins in
lung development, pulmonary fibrosis, and emphysema. Integrins contribute to immune response in the lung by mediating
trafficking of leukocytes to areas of inflammation. In addition,
they function as receptors for a variety of pulmonary pathogens.
Because of their central role in different disease processes, therapeutic agents are being developed to target integrin–ligand
interactions.
Integrins are a major family of cell adhesion receptors. The term integrin was first used in the 1980s to
describe cell surface receptors that ‘integrated’ the
cytoskeleton of one cell with that of another cell or
extracellular matrix protein. Since the first description, it is apparent that integrins are essential for
many important biological processes including, but
not limited to, development, cell proliferation, cell
survival, migration, immune function, and wound
healing. Integrins are found in all metazoa, vertebrates, and invertebrates. No homologs have been
48
ADHESION, CELL–MATRIX / Integrins
Platelet integrin
1
IIb
Leukocyte integrins
E
L
3
3
M
X
2
2
4
5
6
7
D
4
1
V
7
8
5
6
8
9
10
11
Figure 1 Integrin family. a/b associations of mammalian subunits are illustrated. Subunits in red recognize the RGD motif, subunits in
blue are collagen receptors, and subunits in purple are laminin receptors. Some integrins have restricted cell type expression (i.e.,
leukocyte integrins and platelet integrin).
Table 1 Nomenclature of b2 leukocyte integrins
Leukocyte integrin
Names
aLb2
Lymphocyte function-associated
antigen (LFA)-1, CD11a/CD18
Macrophage adhesion molecule
(Mac)-1, CD11b/CD18, CR3
p150,95, CD11c/CD18
CD11d/CD18
aMb2
aXb2
aDb2
identified to date in prokaryotes, plants, or fungi.
The number of integrin members roughly correlates
with the complexity of the organisms. Currently, there
are 18 known a subunits and 8 known b subunits,
which combine to form 24 different heterodimers in
mammals (Figure 1). Different a/b combinations alter
the ligand specificity of the receptor. Additional complexity arises due to alternatively spliced variants of
several integrin subunits. Integrins can be roughly divided into subfamilies according to their shared b
subunit, although a subunits can sometimes bind to
more than one b subunit, thus blurring the original
subfamilies. The receptors are generally named according to the subunit composition (i.e., a8b1). However, older nomenclature still exists (Table 1). For
example, the b1 subfamily of integrins was originally
termed VLA (very late activation) antigens because
they were originally detected on T cells ‘very late
after’ mitogen stimulation (i.e., a4b1 is also referred to
as VLA-4). All mammalian cells express some member of the integrin family. In addition, some integrins
have cell-restricted expression, most notably the
b2-containing integrins, which are found exclusively
on leukocytes, and the integrin aIIbb3 (GPIIb/IIIa),
found exclusively on megokaryocytes and platelets.
Structure
Integrins are heterodimeric glycoproteins that are
composed of two noncovalently associated subunits,
a and b (Figure 2). Each a and b subunit contains a
large extracellular domain, a single pass transmembrane domain, and a short cytoplasmic domain
(20–60 amino acids, except for the b4 subunit cytoplasmic domain, which contains 1000 amino acids).
The cytoplasmic domain interacts with the cytoskeleton and/or cytoplasmic-signaling molecules. A subset of a subunits contains an inserted (I) domain that
is homologous to the A domain of von Willebrand
factor and is important in ligand binding. The extracellular domains of both subunits form the ligandbinding site. Divalent cations such as Ca2 þ or Mg2 þ
are required for functional activity. Divalent cations
can promote or suppress ligand binding, change ligand specificity, or stabilize integrin structure. In general, Mg2 þ promotes cell adhesion, Ca2 þ decreases
it, and Mn2 þ increases ligand affinity.
Insight into the structure of the extracellular domain was obtained from X-ray crystal structure
analysis. The extracellular domain resembles a globular head atop two legs, one from each subunit (Figure 2). The globular heads form the main contact
between the subunits and the ligand. The head of the
a subunit is composed of a seven-bladed b-propeller
ADHESION, CELL–MATRIX / Integrins 49
A domain
-propeller
Hybrid
domain
PSI domain
Thigh
domain
EGF
repeats
Calf
domain
Calf
domain
Transmembrane
domains
domain, followed by an immunoglobulin (Ig)-like
‘thigh’ domain and two ‘calf’ domains. The b subunit
head has a bA domain (similar to the I domain of a
subunits) followed by an Ig-like hybrid domain, PSI
domain (plexin, semaphorin, and integrin), and four
EGF-like domains.
Recently, the crystal structure of the extracellular
domain of avb3 bound to a protypical Arg–Gly–Asp
(RGD) ligand was determined. The RGD peptide is
inserted in a crevice between the heads of the a subunit
and the b subunit. Amino acid residues previously
predicted to be important in ligand binding by mutational analysis were confirmed to participate in ligand
binding. Furthermore, occupation by ligand resulted
in a conformational change of the extracellular domain that is likely to be transmitted to the cytoplasmic
domain and contribute to outside-in signaling.
Regulation of Activity
Cytoplasmic
domains
Figure 2 Schematic representation of integrin based on structure of avb3. Ligand-binding pocket is formed by the b-propeller
and bA domain of a and b subunits, respectively, and is indicated
by the arrow.
Integrins can exist in different conformational states,
and many integrins exist at rest in a low-affinity state
(Figure 3). However, in response to agonists, signaling pathways are activated that cause a conformational change in the integrins to allow high-affinity
binding (‘affinity modulation’). This inside-out signaling refers to the rapid reversible change in integrin
affinity in response to external agonists. The classic
ECM
Extracellular
Intracellular
Actin cytoskeleton
Inactive
low affinity
Conformational
change/
high affinity
Clustering/
increased avidity
Figure 3 Schematic representation of integrin affinity and avidity modulation in response to inside-out signaling. In the resting
(inactive) state, interaction of cytoplasmic tails via salt bridge (black bar) inhibits interaction with cytoskeletal and signaling partners.
After agonist-induced signaling, the salt bridge is broken by cytoplasmic proteins such as talin. This results in a conformational change in
the extracellular domain, allowing high-affinity binding to ligand. Diffusion of integrin within the plasma membrane leads to clustering and
increased avidity.
50
ADHESION, CELL–MATRIX / Integrins
example of inside-out signaling occurs in platelets.
On a resting platelet, the integrin aIIbb3 exists in a
low-affinity state and is unable to bind soluble fibrinogen. After platelet activation (by agonists such
as thrombin, collagen, ADP, or epinephrine), aIIbb3
undergoes a conformational change to a high-affinity
state and binds soluble fibrinogen, causing platelet
aggregation. Regulation of integrin activation is also
critical for leukocyte trafficking. Leukocytes circulate
in a nonadhesive state and require activation (i.e., by
cytokines or chemokines) to allow targeted integrinmediated adhesion to the vascular endothelial cells
and subsequent transmigration. Activation of b2 integrins is also critical for phagocytosis, antigen presentation, and T-cell killer function.
Integrin cytoplasmic domains regulate the activation state of the integrin. Truncation of the a or b
integrin cytoplasmic domain results in a constitutively active receptor. In the resting (inactive) state,
the cytoplasmic tails of a and b subunits are thought
to be in close proximity to each other, connected by a
salt bridge formed between several highly conserved
residues within the cytoplasmic domain. When the
salt bridge is broken, the integrin becomes activated.
Several cytoplasmic proteins can bind to cytoplasmic
tails and activate integrins, including talin, calcium
and integrin binding protein (CIB), and b3 endonexin. Ligand binding can also be strengthened by
increased lateral mobility of the integrin within the
plasma membrane. This results in clustering of the
integrin and increased ligand binding, referred to as
avidity modulation.
as mechanotransducers. Integrins also coordinate
signaling with many receptor tyrosine kinases and
therefore determine cellular responses to soluble
growth factors and cytokines. In addition, integrins
can associate with other transmembrane or membrane-associated proteins (e.g., PDGF-bR, uPAR, and
TM4SF) to influence cell signaling.
Integrin Ligands: More Than Just Glue
Originally described as receptors that mediated adhesion to extracellular matrix proteins such as fibronectin, collagen, and laminin, it is increasingly
evident the integrins play a broader role in homeostasis, and their ligand repertoire is much larger than
originally appreciated. Many integrins recognize the
tripeptide sequence RGD found in many matrix proteins, including fibronectin, vitronectin, and fibrinogen, and other extracellular proteins, including the
latency-associated peptide (LAP)-TGF-b1. Integrins
bind to a variety of counterreceptors (i.e., VCAM,
ICAM, and E-cadherin), illustrating the importance
of integrins in cell–cell adhesion. An increasing
number of different substrates have been identified
as biologically relevant ligands for integrins, including VEGF, matrix metalloproteinase (MMP)-2, sperm
fertilin, and the endogenous angiogenesis inhibitors
angiostatin, endostatin, and tumstatin. Identification
of novel ligands has expanded the spectrum of biological processes that integrins regulate.
Integrins in Respiratory Diseases
Biological Function
Outside-in signaling occurs after ligand binding to
integrins. The cytoplasmic tails do not contain any
intrinsic catalytic activity but act as organizing centers and scaffolding for other signaling components
and cytoskeletal proteins. Most integrins activate focal adhesion kinase, which subsequently activates a
number of classic signaling pathways, such as mitogen-activated protein kinase, PI3-kinase, protein
kinase C, and c-JUN kinase. These pathways regulate diverse cell behaviors, including survival, proliferation, migration, and transcriptional activity.
Integrins are important links to the actin cytoskeleton through binding of the cytoplasmic tails to actinbinding proteins such as talin, filamin, integrin-linked
kinase, and a-actinin. Integrin-mediated adhesion can
activate the Rho family of small GTPases that are
involved in actin cytoskeleton rearrangement. Integrin
activation of RhoA, Rac-1, or CDC42 results in the
formation of stress fibers, lamellopodia, and filopodia, respectively. Thus, integrins are situated to act
The nonredundant, important function of the different integrins is illustrated by the distinct phenotypes
of the knockouts. In addition, several human genetic
diseases illustrate the clinical importance of the integrin family. Leukocyte adhesion deficiency (LAD1) is a rare autosomal dominant disorder in which
patients suffer from recurrent life-threatening bacterial infections, despite normal numbers of circulating
leukocytes. LAD-1 is caused by lack of functional b2
integrins on leukocytes, which results in the inability
of leukocytes to migrate to tissue sites of inflammation and infection. Glanzmann’s thrombasthenia is
an autosomal recessive disorder in which patients
suffer from severe mucocutaneous bleeding due to a
lack of functional aIIbb3 on platelets. Mutations in
a4b6, normally expressed on the basal surface of keratinocytes, cause some cases of the skin blistering
disease epidermolysis bullosa. Mutation of the a7 integrin subunit, expressed primarily on skeletal and
cardiac muscle, is responsible for some cases of congenital myopathy.
ADHESION, CELL–MATRIX / Integrins 51
Because of their diverse roles in many processes,
integrins are attractive targets in many diseases.
Drugs aimed at directly blocking receptor–ligand interactions have been developed and include monoclonal antibodies and small molecule ligand mimetics
(based on RGD). The platelet integrin aIIbb3 antibody, abciximab, was the first anti-integrin drug to
be approved for clinical use. Several agents targeting
integrins are being tested for use in asthma, cancer,
and inflammatory diseases.
Integrins in Lung Development
Early studies using in vitro models of lung branching
showed that administration of RGD peptides decreased branching morphogenesis. These studies
suggested a critical role of cell–matrix interactions,
mediated through integrins in lung development. Further studies on knockout mice have confirmed the
importance of several integrins in lung development.
Mice lacking the a3 integrin subunit exhibit decreased
branching morphogenesis and immature bronchiolar
epithelium, in addition to abnormal kidney development. The underlying epithelial basement membrane
was severely disrupted, demonstrating a critical role
of a3 in basement membrane assembly.
Mice lacking the a9 subunit die soon after birth
due to bilateral chylothorax, suggesting abnormalities in lymphatic development. Recently, VEGF-C
and VEGF-D, key effector proteins in lymphatic development, were identified as novel ligands for a9b1.
a9b1–VEGF interactions mediated endothelial cell
adhesion and migration, suggesting a mechanism for
phenotype found in a9-deficient mice.
The absence of lung phenotype in other integrin
knockouts does not necessarily imply the absence of
a role in lung development. In mice, lung development begins at approximately E12.5 and continues
until approximately 4 weeks after birth. Knockouts
of several potentially important integrins for lung
development were embryonic lethal before lung development was completed (i.e., b1, a5); thus, determining their contribution to lung development is
difficult. In addition, upregulation of other integrins
or related proteins may compensate for the loss of an
integrin subunit during development.
Integrins and Fibrosis
The integrin avb6 is normally expressed at low levels
on airway epithelium. Mice that lack the integrin
subunit b6 develop exaggerated inflammation in the
lung at baseline. Interestingly, following bleomycin
administration, which typically results in lung injury
and fibrosis, the b6-null mice were protected from
development of pulmonary edema and fibrosis. This
was due to a defect in the activation of TGF-b1, an
important mediator of pulmonary injury and fibrosis.
TGF-b1 is secreted in a latent form, complexed with
LAP, and is unable to signal through its receptor. LAP
contains an RGD site that binds to avb6 and results in
activation of TGF-b1. The activation of TGF-b1 by
avb6 requires contact between avb6-expressing cells
(i.e., epithelial cells) and TGF-b receptor-expressing
cells (i.e., macrophages); it also requires an intact
integrin cytoplasmic tail. Like avb6, avb8 also binds
to the RGD sequence in LAP–TGF-b1 and activates
TGF-b1. However, in contrast to avb6, avb8 activation requires metalloproteinase activity and results in
free active TGF-b. Thus, integrin-mediated regulation of TGF-b activity can occur by several distinct
mechanisms.
Another interesting finding in the b6-null mice is
the development of emphysema over time. Backcrossing the b6-null mice onto a MMP-12-null background eliminates the development of emphysema,
suggesting that the development of emphysema is
due to increased expression of MMP-12, a macrophage elastase. The findings suggest a model wherein
active TGF-b1 normally suppresses MMP-12 expression by macrophages. In the absence of avb6-mediated TGF-b activation, MMP-12 expression is no
longer suppressed and ultimately results in a matrixdegrading phenotype in the lung.
Integrins and Asthma
In asthma, integrins, including aLb2 and a4b1,
contribute to trafficking of eosinophils and other inflammatory cells to the lung during an asthma exacerbation. Therefore, integrins are attractive targets
for novel anti-inflammatory therapies in asthma.
Efalizumab, a humanized anti-aL monoclonal antibody, inhibits leukocyte migration to areas of inflammation and is approved for use in moderate to
severe psoriasis. Efalizumab was tested in patients
with mild asthma. Despite a significant decrease in
activated eosinophils in the lung, there was no significant difference in pulmonary function after allergen challenge. There has been much interest in
targeting the a4 integrin subunit in asthma and other
chronic inflammatory diseases, such as Crohn’s
disease, rheumatoid arthritis, and multiple sclerosis.
In multiple sclerosis, treatment with the monoclonal
a4 antibody natalizumab resulted in a significant
reduction in relapses, leading to Food and Drug
Administration approval of natalizumab. However,
since approval, several unexpected cases of progressive multifocal leukoencelphalopathy (PML) have
been reported in patients receiving natalizumab.
PML is a rare, fatal demyelinating disease caused
52
ADHESION, CELL–MATRIX / Integrins
by reactivation of human polyoma virus JC. The
contribution of natalizumab to the development of
PML is not entirely clear, but it likely relates to
preventing migration of immune cells that normally
repress latent virus into the central nervous system.
These findings have raised serious concerns regarding
the safety of a4 antagonists and put into question any
future work targeting the a4 subunit.
Integrins and Acute Lung Injury
The general paradigm for leukocyte trafficking occurs in several steps:
1. selectin-mediated rolling of leukocytes on endothelium,
2. activation of leukocyte b2 integrins by chemoattractants,
3. integrin-mediated firm adhesion of leukocytes to
endothelium, and
4. integrin-mediated transmigration of leukocytes
into the interstitium.
In the lung, leukocyte trafficking differs from the general model in several aspects: the classical selectinmediated rolling does not occur; transmigration of
leukocytes occurs at pulmonary capillaries, not postcapillary venules; and transmigration of leukocytes
into interstitium and alveoli may follow a b2-dependent or b2-independent pathway. In the lung, involvement of b2 integrins for leukocyte migration
depends on the stimulus. For example, leukocyte
trafficking in response to Escherichia coli, Pseudomonas aeruginosa, lipopolysaccharide (LPS), and
IL-1 is dependent on b2 integrins, whereas leukocyte
trafficking in response to Streptococcus pneumoniae,
group B streptococcus, Staphylococcus aureus, and
hydrochloric acid is independent of b2 integrins.
Integrins and Pathogens
Many pathogens utilize host integrins to facilitate cell
attachment and infection (Table 2). Many viruses have
RGD motifs in capsid proteins, which facilitates attachment and internalization of organisms. Hantavirus
Table 2 Virus–integrin interactions
Virus
Integrin receptor
Hantavirus
Echovirus
Rotavirus
Papillomavirus
Coxsackievirus
Foot and mouth disease virus
Adenovirus
HHV-8 (KSHV)
avb3, aIIbb3
a2b1
a2b1, axb2, a4b1
a6b1
avb3, avb6
avb3, avb6
avb3, avb5, avb1
a3b1
causes severe pulmonary disease and thrombocytopenia and infects pulmonary epithelial cells and platelets.
Pathogenic strains of hantaviruses attach to cells via
b3-containing integrins. Another pathogen, foot and
mouth disease virus (FMDV), causes a devastating
disease of cattle. The primary route of infection by
FMDV is through the epithelial cells and associated
lymphoid tissues in the upper respiratory tract, followed by dissemination to other epithelial tissues. The
outer capsid of FMDV contains a highly conserved
RGD tripeptide motif on an exposed loop that interacts with av-containing integrins and facilitates viral
attachment and infection of cells. Adenovirus is important both for the diseases it causes in humans, including respiratory infections, and because replicationdefective variants of adenovirus are being used as vectors for gene therapy. Adenovirus contains a conserved
RGD sequence within a highly variable region in
the penton base protein. In contrast to the viruses just
mentioned, adenoviruses do not use integrins for initial attachment. Instead, adenovirus attaches to cells
through a common coxsackievirus adenovirus receptor
and then the RGD site interacts with av-containing
integrins to facilitate internalization. Likewise, the
causative virus of Kaposi’s sarcoma, human herpes virus 8 (HHV-8), interacts with a3b1 on target cells via
RGD sequence on an outer envelope to facilitate viral
entry and uptake. Thus, viruses and other pathogens
have exploited integrins for their advantage.
Integrins and Malignancy
Most adherent cells are anchorage dependent; that
is, they require integrin-mediated signaling to stimulate cell-cycle progression, promote cell survival,
and respond to soluble growth factors such as
EGF and PDGF. Loss of integrin-mediated signaling
causes a subtype of apoptosis, termed ‘anoikoisis’
from the Greek word meaning ‘homelessness’. Following malignant transformation, the requirement of
integrin ligation is often bypassed by mutations in
oncogenes or tumor suppressor genes, thus allowing
anchorage-independent growth. However, integrin
signaling still contributes to tumorigenesis and tumor
progression. Depending on the integrin, cell type,
and signaling pathway, activated integrins may facilitate tumor growth, metastasis, and attachment at
distant sites or suppress tumor activity. In general,
tumor cells, either through direct signaling mechanisms or through selection process, increase expression of integrins that promote cell proliferation,
survival, and migration and decrease expression of
integrins that promote a quiescent, differentiated
state. Although it is difficult to make generalizations,
a2b1 and a3b1 expression is associated with a tumor
ADRENERGIC RECEPTORS 53
suppressor phenotype, whereas avb3, avb6, and a6b4
expression is associated with tumor progression.
The development of blood supply is essential for
tumor survival and growth, and it is a potential target
of antitumor agents. The endothelial integrin avb3 is
upregulated in tumor neovasculature. In vitro and
animal studies showed that avb3 antagonists (antibodies or peptides) caused tumor regression, inhibited
neovascularization, and induced apoptosis of endothelial cells. Several anti-avb3 agents show promise
as antiangiogenic therapy for cancer treatment.
See also: Adhesion, Cell–Cell: Vascular; Epithelial. Adhesion, Cell–Matrix: Focal Contacts and Signaling.
Asthma: Overview. CD11/18. Epithelial Cells: Type I
Cells; Type II Cells. Extracellular Matrix: Basement
Membranes; Elastin and Microfibrils; Collagens; Matricellular Proteins; Matrix Proteoglycans; Surface Proteoglycans; Degradation by Proteases. Fibroblasts. Matrix
Metalloproteinases.
Further Reading
Calderwood DA (2004) Integrin activation. Journal of Cell Science
117: 657.
Doerschuk CM (2000) Leukocyte trafficking in alveoli and airway
passages. Respiration Research 1: 136.
Guo W and Giancotti FG (2004) Integrin signalling during tumour
progression. Nature Reviews: Molecular Cell Biology 5: 816.
Humphries MJ, McEwan PA, Barton SJ, et al. (2003) Integrin
structure: heady advances in ligand binding, but activation still
makes the knees wobble. Trends in Biochemical Sciences 28: 313.
Hynes R (2002) Integrins. Bidirectional, allosteric signaling
machines. Cell 110: 673.
Plow EF, Haas TA, Zhang L, Loftus J, and Smith JW (2000) Ligand binding to integrins. Journal of Biological Chemistry 275:
21785.
Schwartz MA (2001) Integrin signaling revisited. Trends in Cell
Biology 11: 466.
Sheppard D (2000) In vivo functions of integrins: lessons from null
mutations in mice. Matrix Biology 19: 203.
Sheppard D (2003) Functions of pulmonary epithelial integrins:
from development to disease. Physiological Reviews 83: 673.
Sheppard D (2004) Roles of alphav integrins in vascular biology and
pulmonary pathology. Current Opinion in Cell Biology 16: 552.
ADRENERGIC RECEPTORS
A E Tattersfield, Nottingham University, Nottingham,
UK
& 2006 Elsevier Ltd. All rights reserved.
Abstract
Adrenergic receptors, which includes a, b and dopamine receptors, belong to the large family of G-protein-coupled, seven
transmembrane domain receptors. b-Adrenoceptors are the
best characterized and predominant adrenoceptors in the lung,
with both b1 and b2 receptors being widely distributed. b2Adrenoceptors are an important therapeutic target and their
polymorphisms may influence the response to b2 agonist treatment. Their numbers and functions are regulated by b-agonist
stimulation and by drugs, such as corticosteroids, and cytokines.
a-Adrenoceptors are found on vascular smooth muscle, presynaptic nerve endings, airways, and submucus glands, and they
may help to condition inspired air. There is evidence for D1
dopamine expression on alveolar cells, where they help to clear
lung edema, and for D2 receptors on sensory nerves in the lung,
where they may modulate neurogenic inflammation and reflexmediated symptoms.
Introduction
Adrenergic receptors include a, b, and dopamine receptors; they are part of the large family of G-proteincoupled, seven transmembrane domain receptors.
b-Adrenoceptors are the best characterized and
predominant adrenoceptors in the lung; the role of
a-adrenoceptors and dopamine receptors is less well
established.
b-Adrenoceptors
b1 and b2 receptors were initially identified on the
basis of tissue selectivity to a range of agonists. Both
b-adrenoceptors were subsequently characterized
and cloned, as was a third (b3) receptor and there
are putative claims for a b4 receptor in the heart.
There is 54% homology between b1 and b2 receptors.
Structure
The b2-adrenoceptor contains 413 amino acids with
seven transmembrane-spanning domains, three intraand three extracellular loops, an intracellular C-terminus, and an extracellular N-terminus (Figure 1).
The third intracellular loop is important for linking
to G-proteins.
Site-directed mutagenesis studies show that b-agonists bind to residues on the hydrophobic region
within the cell membrane between the third and sixth
transmembrane domains as shown in Figure 1. The
salmeterol molecule anchors to an exosite on the
fourth transmembrane domain, allowing the molecule to stimulate the receptor repetitively. The intracellular third loop and tail of the b-adrenoceptor
contains phosphorylation sites that are involved in
receptor downregulation.
Polymorphisms
Nine single-base mutations or polymorphisms in
the coding region of the b2-adrenoceptor gene on
54
ADRENERGIC RECEPTORS
Y
Y
2-Agonist
NH2
Cell membrane
I
II
III
IV
V
VI VII
P
s
P
cAMP
PKA
PKC
PTK
P = Phosphorylation
sites
P
P
H
COO
P
+
arrestin
P
ARK
(GRK)
Inhibits
receptor
function
Figure 1 Schematic representation of the human beta 2-adrenoceptor showing the 7 trans membrane domains and the site on the
third, fifth, and sixth domains that are required for beta-agonist binding. P marks the phosphorylation sites concerned with receptor
ancapling. The blue circles indicate the amino acids that are essential for b-agonist binding. Adapted from ‘‘b-adrenergic receptors and
their regulation’’ by PJ Barnes.
chromosome 5Q have been identified, though only
four of these cause amino acid substitutions – at codons 16, 27, 34, and 167. The two most common
polymorphisms are at codon 16, where glycine is
substituted for arginine, and codon 27 where glutamic
acid is substituted for glutamine. Both polymorphisms
are common, with 37% and 23% of subjects being
homozygous for the gly 16 and glu 27 polymorphisms, respectively, in a large general population
study. There is linkage disequilibrium between polymorphisms at codons 16 and 27, that is, they are more
likely to occur together, and this can cause difficulties
when trying to determine the functional effects of a
specific polymorphism.
b-Adrenoceptor Activation
When a b-agonist binds to the b-adrenoceptor/Gs
complex guanosine diphosphate (GDP) is released,
allowing guanosine triphosphate (GTP) to bind and
activate the a-subunit of Gs, which then dissociates
from the b-receptor and is free to stimulate adenylate
cyclase. Adenylate cyclase catalyzes the conversion of
adenosine triphosphate (ATP) to cyclic adenosine
monophosphate (AMP), which in turn activates protein kinase A. Cyclic AMP has a range of actions that
promote smooth muscle relaxation in airways and
other activities such as inhibition of mediator release
in inflammatory cells (see Figure 2 and Table 1).
Most of the effects of b-agonists are mediated via
cyclic AMP and activation of protein kinase A, but
some, such as the opening of maxi K þ channels,
may, in part at least, be due to direct activation by
the Gs a-subunit.
Protein kinase A is also able to phosphorylate and
activate a transcription factor, CREB or cyclic AMP
response element binding protein. By binding to the
cyclic AMP response element (CRE) on the promoter
region of target genes, CREB is able to cause transcription of various genes including the b-receptor
gene. b2-Receptor expression may therefore be increased transiently by b-agonist stimulation. CREB
also interacts with other transcription factors within
the nucleus including some proinflammatory cytokines such as activating protein (AP-1) and nuclear
factor kappa B (NF-kB).
b-Adrenoceptor Regulation and Activity
Until recently, it was thought that b-adrenoceptors
existed in high- or low-affinity states, the high-affinity
state being increased in the presence of b-agonists
and associated with an increased binding affinity for
b-agonists. The affinity for b-adrenoceptor antagonists
did not differ between high- and low-affinity states.
b-Adrenoceptor activation with this model only occurred when an agonist was present. However, recent
work suggests that receptors may have constitutive
activity, that is, activity when no agonist is present.
Some b-adrenoceptor antagonists are now thought to
act by stabilizing this constitutive activity rather than
as a competitive antagonist and hence they can be
described as inverse agonists.
The number of b-adrenoceptors in the airways and
other tissues depends on the rates at which they are
synthesized and degraded; their function also depends on the extent to which they are coupled to the
catalytic unit. Both receptor numbers and functions
ADRENERGIC RECEPTORS 55
K+
-Agonist
Gs
-Adrenoceptor
Gs
ATP
Phosphorylation of
myosin light chain kinase
AC
Inhibition of PI hydrolysis
cAMP
Cell
membrane
Inactive
PKA
Active
PKA
Ca2+ extrusion and
sequestration
Stimulation of
Na+/K+ ATPase
Membrane
hyperpolarization
Smooth muscle
relaxation
Inhibition of
mediator release
Activation of Ca2+ gated
potassium channels
Figure 2 Schematic representation of the signaling pathway following stimulation of the b-receptor; this causes coupling of the
receptor to the Gs protein and stimulation of adenylate cyclase (AC). This in turn causes increased accumulation of cyclic-AMP (cAMP)
and protein kinase A (PKA) and a fall in intracellular calcium. The most important cellular effects for asthma are smooth muscle
relaxation and inhibition of mediator release from inflammatory cells.
Table 1 Main actions of b-adrenergic agonists at different
receptor subtypes
underlie this development:
Tissue
Receptor
Response
Airways
b2 mainly
Heart
Blood vessels
b1/b2
b2
Uterus
Metabolic
b2
b2/b3
Muscle
b2
Bronchodilatation, reduction
in mediator release from
mast cells, increased
mucus production,
increased ciliary
activity
Tachycardia, inotropic action
Dilatation, fall in blood pressure,
compensatory reflex increase
in heart rate
Relaxation
Increase in glucose, insulin,
lactate, pyruvate, nonesterified
fatty acids, glycerol, and
ketone bodies; decrease in
potassium, phosphate,
calcium, and magnesium
Tremor
1. Receptor uncoupling. Exposure of a b-receptor to a
b-agonist causes phosphorylation of sites on the 3rd
intracellular loop and tail of the b-adrenoceptor,
causing the receptor to uncouple from its Gs protein
and catalytic unit. Receptor uncoupling occurs
within minutes and is rapidly reversible. At least
two kinases are involved: the cyclic AMP-dependent
protein kinase A and the G-protein-dependent b-adrenergic receptor kinase (bARK); protein kinase G
may also contribute. Protein kinase A is activated by
fairly low concentrations of b-agonist, as seen with
therapeutic doses, whereas activation of bARK requires higher agonist concentrations and the cofactor
b-arrestin. bARK causes homologous b-receptor desensitization, that is, to b-agonists only, whereas protein kinase A causes heterologous desensitization, so
that the response to other agonists that use the same
catalytic unit, for example, forskolin, is reduced.
2. Sequestration of receptors. Following exposure
to a b-agonist, b-adrenoceptors may be internalized
within the cell, and sequestrated into vesicles. This
can also occur within minutes and is again reversible, the receptors being recycled to the cell surface.
3. Downregulation. With longer term exposure to bagonists, however, b-receptors may be internalized
and degraded and hence are not available for recycling. Polymorphisms at codon 16 and 27 have been
shown to affect b-adrenoceptor downregulation
are modified by b-agonists and by other factors, of
which cytokines and corticosteroids are particularly
relevant to asthma.
Regulation by b-agonists With repeated or continuous exposure to a b-agonist there is a reduction in the
response, a phenomenon known as desensitization or
tolerance. Several mechanisms have been shown to
56
ADRENERGIC RECEPTORS
in vitro. Receptors with the gly 16 polymorphism
have shown increased downregulation whilst those
with the glu 27 polymorphism were protected
against downregulation.
4. Reduced synthesis. b-Adrenoreceptor numbers
may also be lower as a result of a reduction in
receptor mRNA, following continuous exposure.
Regulation by other factors
1. Corticosteroids. Corticosteroids have a number of
effects in vitro that may increase or decrease the
response to a b-agonist. They are able to increase
b-receptor numbers by inhibiting downregulation
of b-receptors, and by increasing b-receptor gene
transcription. However, the activity of CREB can
be inhibited by glucocorticoid transcription factors, thus providing a possible mechanism whereby glucocorticoids might reduce the efficacy of a
b-agonist.
2. Cytokines. The proinflammatory cytokine interleukin 1b has been shown to attenuate b-receptor
responsiveness by uncoupling the receptor from its
catalytic unit; this appears to be due to induction
of the inducible form of cyclooxygenase (COX 2)
and release of inhibitory prostanoids.
Distribution of b-Adrenoceptors
The distribution and type of b-receptor within the
lung has been determined by receptor-binding studies; airway epithelium and alveoli have the highest
receptor density. Some 70% of the b-adrenoceptors
in the lung are of the b2-receptor subtype, the remainder being b1-receptors. Only b2-receptors are
found on airway and vascular smooth muscle
whereas both b1- and b2-receptors are found on submucosal glands and alveoli.
b-Receptors are distributed widely throughout the
body and some of the more important sites are shown
in Table 1.
patients with asthma who are homozygous for arginine at codon 16.
a-Adrenoceptors
Classifying a-adrenoceptors has been less straightforward than classifying b-adrenoceptors. The two
main classes are the a1-adrenoceptors, classic postsynaptic excitatory receptors, and a2-adrenoceptors,
which are usually, though not invariably, presynaptic, where they inhibit neuronal mediator release.
There are three fully defined a1-adrenoceptor subtypes (a1A, a1B, a1D, and a putative a1L) and four
subtypes of the a2-adrenoceptor (a2A, a2B, a2C, and
a2D). Most of the subtypes have now been cloned.
a-Adrenoceptor Activation
The postsynaptic a1-adrenoceptor is coupled to a Gq
protein and produces physiological effects via activation of phospholipase C, which mobilizes intracellular
calcium by increasing inositol 1, 4, 5-trisphosphate
concentrations, and diacylglycerol, which activates
protein kinase C. a1-Adrenoceptors also activate
other pathways such as the mitogen-activated protein kinase pathway. A single base polymorphism has
been described in a1A but no effect on receptor function has been identified as yet. Activation of a2 receptors usually causes inhibition of adenylate cyclase
through coupling with membrane-linked pertussis
toxin-sensitive Gi/o protein.
Distribution
Alpha-adrenoceptors are widely distributed on neuronal and non-neuronal tissues. Within the lung, aadrenoceptors have been located predominantly in
smaller airways, on serous cells in submucus glands,
and on pulmonary blood vessels. a1-Adrenoceptors
are thought to be present on bronchial blood vessels
and a2-adrenoceptors on airway ganglia and presynaptic cholinergic nerve endings.
b-Receptor Numbers and Function in Disease
Positron emission tomography studies indicate that
there are normal numbers of b-receptors in the lungs
of people with mild asthma, and they may even increase in patients dying from asthma. The function
of b-receptors in the lungs of patients with asthma
also appears to be normal, although it may be affected by previous b-agonist exposure and receptor
polymorphisms.
There is no evidence to suggest that the presence of
asthma is associated with b2-receptor polymorphisms or haplotypes. However, there is some evidence that the response to b2-agonists is reduced in
Function
The role of a-adrenoceptors in the lung is not entirely
clear and work is hampered by lack of specificity
of many of the ligands used to investigate a-adrenoceptor subtypes. a1-Adrenoceptors classically
cause smooth muscle contraction and could therefore help control bronchial blood flow and conditioning of inspired air. Other possible roles include
inhibition of histamine release from mast cells and
acetylcholine release from cholinergic nerve endings,
and modulating the content of airway secretions
through serous cell stimulation.
ADRENERGIC RECEPTORS 57
Function in Disease
The possibility that a-adrenergic mechanisms might
contribute to asthma was explored several years ago,
particularly when the density of a-receptors was
found to be higher in patients with asthma. a-Adrenoceptor agonists caused bronchoconstriction in
some but not all studies although whether this is a
specific or non-specific effect is not clear. Similarly
nonselective a-adrenoceptor antagonists such as indoramin caused some degree of bronchodilatation
or protected against induced bronchoconstriction in
some studies, but all the drugs studied had additional
pharmacological effects that could have been responsible. None of the a-adrenoceptor antagonists
available caused worthwhile bronchodilatation in
asthma when given regularly or by inhalation. These
data and the fact that norepinephrine when infused
causes bronchodilatation rather than bronchoconstriction suggest that a-adrenergic mechanisms are
not making an important contribution to the
bronchoconstriction seen in asthma.
Dopamine Receptors
Of the five subtypes of dopamine receptors identified
(D1–D5), D1 and D5 receptors have similar homology
and both stimulate adenylate cyclase. D2, D3, and
D4 receptors are also homologous but they inhibit
adenylate cyclase. Most work relates to the D1 and
D2 subtypes.
Distribution and Function
Dopamine receptors are found in the central and
peripheral nervous system. They have been studied in
the brain predominantly and in the context of the
lung there is interest in the role that central dopaminergic pathways might play in the development
of nicotine addiction.
Within the lung there is evidence for D1 receptors
on alveoli in the rat, which, when stimulated, increase the clearance of lung edema.
D2 receptor mRNA and protein are expressed in
sensory ganglia in the airways and dopamine receptor activation has been shown to inhibit depolarization of the vagus in animals and man, and neuropeptide release from nerve endings. D2 dopamine
receptors may therefore have a role in modulating
neurogenic inflammation and reflex-mediated symptoms such as cough. D2 receptor agonists have reduced cough, mucus production, and tachypnea
in animal models and there is interest in whether
they might reduce symptoms such as cough and
breathlessness in patients with chronic obstructive
pulmonary disease.
Dopamine in low doses stimulates dopamine receptors but as the dose is increased it stimulates b1adrenoceptors followed by a1- and a1-adrenoceptors.
See also: Asthma: Overview. Bronchodilators: Beta
Agonists. G-Protein-Coupled Receptors.
Further Reading
Barnard ML, Ridge KM, Saldias F, et al. (1999) Stimulation of the
dopamine 1 receptor increases lung edema clearance. American
Journal of Respiratory and Critical Care Medicine 160: 982–
986.
Barnes PJ (1986) Neural control of human airways in health and
disease. American Review of Respiratory Diseases 134: 1289–
1314.
Barnes PJ (1995) Beta-adrenergic receptors and their regulation.
American Journal of Respiratory and Critical Care Medicine
152: 838–860.
Birrell MA, Crispino N, Hele DJ, et al. (2002) Effect of dopamine
receptor agonists on sensory nerve activity: possible therapeutic
targets for the treatment of asthma and COPD. British Journal
of Pharmacology 136: 620–628.
Carswell H and Nahorski SR (1983) b-Adrenoceptor heterogeneity in guinea pig airways: comparison of functional and receptor
labelling studies. British Journal of Pharmacology 79: 965–971.
Goidie RG, Papidimitriou SM, Paterson SW, et al. (1986) Autoradiographic localisation of b-adrenoceptors in pig lung using
[125I]-iodocyanopindolol. British Journal of Pharmacology 88:
621–628.
Green SA, Spasoff AP, Coleman RA, Johnson M, and Liggett SB
(1996) Sustained activation of a G protein coupled receptor via
‘anchored’ agonist binding. Molecular localization of the salmeterol exosite within the b-adrenergic receptor. Journal of
Biological Chemistry 39: 24029–24035.
Green SA, Turki J, Innis M, and Liggett SB (1994) Amino-terminal
polymorphisms of the human b-adrenergic receptor impart distinct agonist-promoted regulatory properties. Biochemistry 33:
9414–9419.
Hall IP and Tattersfield AE (1998) b-Adrenoceptor agonists. In:
Barnes PJ, Rodger IW, and Thomson NC (eds.) Asthma. Basic
Mechanisms and Clinical Management, 3rd edn, pp. 651–676.
London: Academic Press.
Koshimizu T, Tanoue A, Hirasawa A, Yamauchi J, and Tsujimoto
G (2003) Recent advances in a1-adrenoceptor pharmacology.
Pharmacology and Therapeutics 98: 235–244.
Lands AM, Arnold A, McAuliff JP, et al. (1967) Differentiation of
receptor systems activated by sympathomimetic amines. Nature
214: 597–598.
Reihsaus E, Innis M, MacIntyre N, and Liggett SB (1993) Mutations in the gene encoding for the b2-adrenergic receptor in
normal and asthmatic subjects. American Journal of Respiratory
Cell and Molecular Biology 8: 334–339.
Schwartz J, Carlsson A, Caron M, et al. (1998) The IUPHAR
compendium of receptor characterisation and classification: dopamine receptors. IUPHAR Media (London) 142–151.
Strader CD, Sigal IS, and Dixon AF (1989) Mapping the functional
domains of the b-adrenergic receptor. American Journal of Respiratory Cell and Molecular Biology 1: 81–86.
58
AEROSOLS
AEROSOLS
S P Newman, Nottingham, UK
& 2006 Elsevier Ltd. All rights reserved.
Abstract
The inhaled route is used to deliver drugs as aerosols for the
maintenance therapy of asthma, chronic obstructive pulmonary
disease, and other conditions. The deposition of aerosol particles in the respiratory tract is an important prerequisite to
obtaining a good clinical effect. Generally, inhaler devices
should deliver particles smaller than approximately 5 mm in
diameter in order to enter the lungs. A variety of inhaler devices
are available for inhalation therapy. Pressurized metered dose
inhalers (pMDIs) have been widely used for 50 years, but
many patients have problems using them correctly. They are
currently being reformulated with ozone-friendly propellants.
Breath-actuated inhalers and spacer attachments may be
useful supplements to pMDIs for some patients. Dry powder
inhalers (DPIs) are easier to use correctly than pMDIs, and
they do not require propellants. Many pharmaceutical companies seem to be prioritizing DPIs above pMDI reformulation, and they are also preferred by many patients. Nebulizers
continue to be used widely, but the limitations of jet and
ultrasonic nebulizers have led to the development of novel
systems, sometimes involving vibrating meshes. Finally, a new
class of inhalers (soft mist inhalers) is emerging, composed
of multidose devices containing liquid formulations, some of
which could challenge pMDIs and DPIs in the portable inhaler
market.
Inhaled Drug Delivery
The pulmonary route may be used to deliver drugs
for the maintenance therapy of some lung diseases,
most notably asthma and chronic obstructive pulmonary disease (COPD). Drugs are also given by inhalation to treat other chest problems, including
respiratory tract infections in cystic fibrosis. In addition, it is hoped that inhaled drugs intended to have
a systemic action in the body (e.g., insulin) will soon
be marketed. The potential benefits of the inhaled
route have long been recognized, but the importance
of good quality inhaler devices that deliver drugs
reliably to the lungs has only been appreciated during
the past 25 years.
Aerosol Properties
An understanding of aerosol properties and aerosol
deposition is an important prerequisite for optimizing inhalation therapy. Drugs are given by inhalation as aerosols of solid particles or liquid droplets,
but for simplicity the term ‘particle’ may be used
to describe both solid and liquid dispersions. The
most important property of an aerosol particle is its
size, and this is best expressed as the aerodynamic
diameter, which also takes into account particle density and shape. For spherical particles, aerodynamic
diameter (Da) and physical diameter (Dp) are related
by the formula Da ¼ DpOr, where r is the specific
gravity of the material from which the particles are
made. In practice, aerosol particles are seldom spherical; for instance, micronized drug particles are often
highly irregular in shape.
Aerosol systems found in medicine are usually heterodisperse, indicating that the particles in a particular spray or cloud have a wide range of sizes.
Monodisperse aerosols, in which all the particles
have approximately the same size, are not normally
found in pharmaceutical products, although they
can be made using specialized equipment. It is preferable to describe the mass or volume distribution of an aerosol rather than the distribution of
particles by number since many small particles
may contain much less drug than a few large particles. In practice, particle size spectra from inhaler
devices often approximate to log-normal distributions. The mass median aerodynamic diameter
(MMAD) may be used to express the average aerosol size. This diameter is such that half the aerosol
mass is contained in larger particles and half in
smaller particles. The spread of particle sizes may
be expressed as a geometric standard deviation
(GSD), a dimensionless quantity. A perfectly monodisperse aerosol has a GSD of 1. A typical pharmaceutical aerosol may contain particles ranging in size
from o0.5 to 410 mm, with an MMAD of 3–4 mm
and a GSD of 2.0–2.5.
As explained later, deposition of aerosols depends
critically on particle size. The fraction of the aerosol
mass contained in particles o5 mm in diameter is
usually termed the respirable fraction or fine particle
fraction (FPF). These are the particles with the greatest likelihood of reaching the lungs in adults, although even smaller particles may be needed for
drug therapies in small children. In adults, particles
o3 mm in diameter are needed in order to deliver
drugs to the alveolated regions – for instance, to deliver inhaled a1 antitrypsin to the alveoli of patients
with emphysema.
Particle size distributions of aerosols intended for
pulmonary delivery may be quantified by several
methods. The approach favored within the pharmaceutical industry is the cascade impactor, through
which the aerosol is drawn by a vacuum pump, and
particles of different sizes are collected on a series
of stages. Each stage can be washed out with a solvent
AEROSOLS 59
so that the amount of drug associated with different
size bands may be quantified by an analytical technique. Supplementary particle size data may be provided by optical methods, the best known of which is
laser diffraction. This involves passing the aerosol
cloud through a laser beam, and the angle of diffraction of the laser light is inversely proportional to
particle size. It is important to remember that these in
vitro measurements are undertaken primarily for purposes of quality control and product release, and they
may not predict accurately drug delivery to the lungs
in vivo.
Deposition of Pharmaceutical Aerosols
Several mechanisms cause aerosol particles to deposit
in the respiratory tract, but the two most important
ones relating to pharmaceutical aerosols are inertial
impaction and gravitational sedimentation.
Inertial impaction takes place mainly in the oropharynx and at the bifurcations between major
airways, when the aerosol particle has too much inertia to follow the air stream as it changes direction.
The probability of inertial impaction occurring is
proportional to D2a Q, where Q is the inhaled flow
rate. Deposition in central lung regions may be
enhanced by the effects of air turbulence, especially
at fast inhaled flow rates. Gravitational sedimentation takes place mainly in smaller conducting airways and in the alveoli, when particles settle onto the
airway surface under gravity either during slow
steady breathing or during breath-holding. The probability of gravitational sedimentation occurring is
proportional to D2a T, where T is the residence time
of the particle in the airways. A third deposition mechanism (Brownian diffusion) is also important for aerosol particles o1 mm in diameter, which
may be pushed in a random direction toward airway walls by collisions with gas molecules. Some
particles (especially those o1 mm in diameter) are
not deposited, and after inhalation they are simply
exhaled.
In addition to particle size, the patient’s inhalation
also plays a major part in determining the site of aerosol deposition. The inhaled flow rate is particularly
important, with slow inhalation usually being recommended in order to reduce impaction losses in the
oropharynx. Deep inhalation and a period of breathholding help to increase gravitational sedimentation in
the peripheral parts of the lungs. For most pharmaceutical aerosols, lung deposition is enhanced by a
combination of aerosol particles o5 mm in diameter
and a slow inhaled flow rate (20–30 l min 1). As
will be explained later, there is an exception to this
rule for dry powder inhalers, where faster inhalation
may preferable. Particles are filtered efficiently from
the inhaled air by the nasal passages, so wherever
practicable it is better to deliver an inhaled aerosol via
a mouthpiece (with mouth breathing) than via a face
mask (with nose breathing).
The airways of the patient who inhales the aerosol
particles also determine the site and extent of deposition in two major ways. First, random variations
in airway geometry between different individuals
will lead to random variations in the deposition pattern. Hence, for aerosols delivered from any inhaler
device, considerable intersubject variability of deposition is to be expected. Second, in patients with
asthma, COPD, and other obstructive conditions, the
airways may be narrowed by bronchospasm, inflammation, and mucus hypersecretion so that aerosol
particles may deposit preferentially in the larger
airways of the lungs, with less deposition in the
peripheral airways.
Both electrostatic charge and humidity affect aerosol deposition in a variety of ways. The most striking
effect of humidity is that dry particles composed of
water-soluble materials are likely to absorb water
when they enter the respiratory tract and, hence, to
increase in size.
The deposition of pharmaceutical aerosols may
be quantified by radionuclide imaging (gamma scintigraphy, single photon emission computed tomography (SPECT), and positron emission tomography
(PET)). SPECT and PET are three-dimensional imaging methods and provide information about the distribution pattern within the lungs. However, PET is
relatively complex and is probably not practical for
use on a regular basis. Certain pharmacokinetic
methods are also useful for assessing delivery of some
drugs to the lungs. For instance, the plasma or urinary concentrations of albuterol in the first 30 min
after inhalation are considered to result solely from
pulmonary absorption.
Pressurized Metered Dose Inhalers
The pressurized metered dose inhaler (pMDI) has
been the backbone of inhalation therapy for asthma
for approximately 50 years, since its introduction by
3 M Riker Laboratories in 1956. Patients and physicians recognized the convenience of the pMDI,
which contains 100–200 doses in a small portable
device that is immediately ready for use (Figure 1).
The pMDI consists of an aluminum can mounted in a
plastic actuator. Individual doses (25–100 ml) are delivered as a spray via a sophisticated metering valve.
The drug is usually a micronized suspension of drug
particles but may be a solution dissolved in propellants, ethanol, or another excipient as a co-solvent.
60
AEROSOLS
Patient presses canister
while breathing in
Canister
Actuator
Drug formulation
in propellants
Spray
plume
Metering
valve
Actuator nozzle
Figure 1 Design and operation of a typical pressurized metered dose inhaler.
The best known pMDI therapies include the b-agonists albuterol, terbutaline, and salmeterol and the
glucocorticosteroids beclomethasone dipropionate,
budesonide, and fluticasone propionate.
Successful pMDI therapy is highly dependent on
the patient’s inhalation technique, and patient education about their use is essential. In most pMDI
products, it is necessary for the patient to press
the pMDI at the same time as inhaling. Failure to
do this is sometimes described as poor coordination
or hand–lung dyscoordination, and it is probably
the most important problem patients have with
pMDIs. A second major problem using pMDIs is
the so-called cold Freon effect, where the patient
stops inhaling when the cold propellant spray is felt
on the back of the throat. Freon is one of the trade
names of chlorofluorocarbon (CFC) propellants. In
order to optimize lung deposition from pMDIs,
patients also need to inhale slowly and deeply and
to hold the breath for several seconds. Even with
perfect inhalation technique, no more than 10–20%
of the dose from a CFC pMDI is deposited in
the lungs, with the majority of the dose being deposited in the oropharynx. However, the lung dose
will vary from product to product according to the
nature of the formulation and the diameter of the
actuator orifice.
Until recently, all pMDIs were formulated in CFC
propellants, giving the pMDI an internal pressure of
approximately 300 kPa (3 atm) and a spray velocity
at the nozzle exceeding 30 m s 1. However, it is
possible to reduce the spray velocity by modifications
to the actuator design, for instance, in the Spacehaler
device (formerly known as Gentlehaler). During the
past few years, the pharmaceutical industry has been
forced to start reformulating pMDIs in non-CFC propellants, consisting of one of two hydrofluoroalkanes (HFA-134a or HFA-227). This challenge arose
following the discovery that the degradation of CFCs
damages stratospheric ozone and has proved to be a
major stimulus to the development of novel inhaler
technologies. The switch to HFA-powered pMDIs
is in progress and will take several more years to
complete. In the meantime, CFCs have been granted
an essential-use exemption in pMDIs under the Montreal Protocol of 1987, reflecting their importance
to the well-being of society. HFAs are greenhouse
gases, and despite the fact that their contribution to
global warming is small, this issue could restrict their
future use.
The development of novel HFA pMDI formulations has not been a simple manner, owing to a range
of technical factors and the need to demonstrate
clinical efficacy and safety for the reformulated products. Individual companies have adopted one of two
strategies. One strategy involves making a product
that is bioequivalent with the CFC pMDI that is to be
replaced so that the HFA pMDI can be used in exactly the same doses as the CFC pMDI. The alternative strategy is to make a product that deposits
drug in the lungs more efficiently than a CFC pMDI.
This usually involves formulating a corticosteroid
product as a solution, enabling a very small particle
size to be achieved as the propellant evaporates. With
such a product, it is also possible to reduce the spray
velocity and to deposit up to half the dose in the
patient’s lung, with greatly reduced oropharyngeal
deposition, so that asthma control may be achieved
using only a fraction of the CFC pMDI dose. A formulation of beclomethasone dipropionate (Qvar)
was the first of these products to reach the market,
and several similar products are either already marketed or in development.
Breath-actuated pMDIs may be helpful in patients
with poor coordination, who cannot actuate the
pMDI at the same time as inhaling. These devices
contain triggering mechanisms that are operated by
the patient’s inhalation via the mouthpiece. However,
it is unlikely that breath-actuated pMDIs confer any
additional benefit on patients who can use a conventional pMDI successfully.
pMDIs with Spacer Devices
Spacer devices are widely used with pMDIs. These
vary greatly in size and shape, with volumes of
commercially available models ranging from 50 to
750 ml. The concept of a spacer is to place some
distance between the point at which the aerosol is
generated and the patient’s mouth, allowing the propellant to evaporate and the rapidly moving aerosol
cloud to slow down before it is inhaled (Figure 2).
The most successful spacers have a one-way valve in
AEROSOLS 61
Formulation:
ordered mixture of
drug and carrier
DPI device
Powder de-aggregated
by patient’s inhalation
Figure 3 Principle of operation of a dry powder inhaler (DPI).
The formulation most frequently consists of an ordered mixture of
micronized drug and carrier lactose, which is de-aggregated by
the patient’s inhalation through the device.
Figure 2 pMDI connected to a large volume spacer device.
the mouthpiece, which allows the pMDI to be actuated into the spacer, with a brief pause before the
patient inhales so that it is not necessary to actuate
and inhale simultaneously. Some spacers function effectively if the patient takes a series of relaxed tidal
breaths from the device immediately after actuating
a dose. Spacers reduce oropharyngeal deposition
of drug and may increase lung deposition, but the
majority of the dose is often deposited on the walls
of the spacer. This may allow the reduction of the
total body burden of inhaled corticosteroids compared
with a standard pMDI. Large volume spacers, such as
the Volumatic and Nebuhaler, have a well-accepted
role in hospital emergency rooms for treating acute
asthmatic attacks. Specially designed spacers with a
volume of 200–300 ml are available for treating young
children.
Most spacer devices are made of plastic, which
may acquire a static charge during handling. This
results in a suspended aerosol cloud being attracted
to the spacer walls, with a marked reduction in the
dose available for inhalation. Specific handling and
washing techniques are usually recommended, and at
least one lightweight metal spacer is available that is
not susceptible to the effects of static charge. With
correct use, including control over electrostatic
charge effects, large volume (4500 ml) spacer devices may deposit more than 30% of the dose from a
CFC pMDI in the patient’s lungs.
Dry Powder Inhalers
Dry powder inhalers (DPIs) have been available
commercially since approximately 1970, although
the earliest prototypes were described several decades earlier. DPIs contain a powder formulation,
which most frequently consists of an ordered mixture
of micronized drug (o5 mm in diameter) and larger
carrier lactose particles that are required to improve
powder flow properties. The patient’s inhalation
through the device is used to disperse the powder
and to ensure that some of the dose is carried into the
lungs (Figure 3). An alternative type of formulation
used in some DPIs consists either of micronized drug
particles alone loosely aggregated into small spherules or of cospheronized drug and lactose.
DPIs are basically of three types: (1) unit-dose devices, in which an individual dose in a gelatin capsule
or blister is loaded by the patient immediately before
use; (2) multiple unit-dose devices, which contain
a series of blisters or capsules; and (3) reservoir devices, in which powder is metered from a storage unit
by the patient before inhalation. Unit-dose devices,
including Spinhaler and Rotahaler, were the only
DPIs available until the mid-1980s. Patients generally
find multiple unit-dose devices, such as the Diskus
(Accuhaler), and reservoir DPIs, such as the Turbuhaler, to be more convenient than unit-dose DPIs
since they provide several weeks’ treatment. DPIs
tend to deposit a greater fraction of the dose in
the lungs compared with CFC pMDIs, but in practice lung deposition varies widely between devices
(Figure 4). Powder formulations are susceptible to
the effects of moisture, and protecting the formulation against these effects is an important part of
DPI design.
By the end of 2004, at least 16 DPIs had been
marketed in different areas of the world for asthma
and COPD therapy, involving a range of unit-dose,
multiple unit-dose, and reservoir systems. A further
20–30 DPIs were also known to be in development.
The anticipated expansion of the generics market for
inhaled asthma and COPD drugs is likely to result in
a number of these novel DPIs reaching the market.
It is interesting to note that the major pharmaceutical
companies with an interest in inhaled asthma and
COPD drugs appear to be prioritizing the DPI over
reformulated HFA pMDIs products. In particular,
62
AEROSOLS
100
90
Percentage of dose
80
70
60
50
40
30
20
10
D
is
kh
U ale
ltr
ah r
al
Pu er
lv
Sp inal
in
ha
Ae ler
ro
liz
e
Ai r
rm
M
AG ax
Tu hal
rb er
uh
Ea aler
sy
ha
C
lic ler
kh
al
e
N
ov r
ol
iz
e
Ta r
ifu
Fl
n
ow
ca
p
Ec s
lip
se
0
Figure 4 Mean percentage of the dose deposited in the lungs from 14 dry powder inhalers (DPIs), obtained in scintigraphic studies.
The high lung deposition from the Flowcaps and Eclipse DPIs probably reflects the properties of the formulation as much as the DPI.
combination products in DPIs containing a longacting b-agonist and a corticosteroid (e.g., Advair
Diskus and Symbicort Turbuhaler) have been very
successful. However, DPIs tend to be more expensive
than pMDIs, and this may limit their use, especially
in developing countries.
DPIs have two major advantages over pMDIs.
First, they do not contain propellants. Second,
all currently marketed models are breath-actuated,
and patients find them easier to use correctly than
pMDIs. However, this second advantage is closely
linked to a disadvantage. In order to disperse the
powder as efficiently as possible, and hence to maximize lung deposition, it may be necessary for patients to inhale as forcefully as possible via the DPI,
and some patients may be either unable or unwilling
to do this. All DPIs exhibit some degree of inhaled
flow rate dependence, with forceful (fast) inhalation
tending to give higher lung deposition than more
gentle (slow) inhalation. For instance, in the Turbuhaler DPI, a reduction in peak inhaled flow rate from
60 to 30 l min 1 was shown to result in a reduction
in lung deposition from 27% to 14% of the dose. In
this respect, DPIs present a paradox since fast inhalation per se is generally associated with enhanced
deposition in the oropharynx, as described previously. Low inspiratory effort through a DPI may result in a reduced emitted dose and poor particle
deaggregation.
The actual magnitude of the peak inhaled flow rate
associated with forceful inhalation will vary between
devices from o30 to 4100 l min 1, according to the
resistance to airflow of each device. Not only the
peak inhaled flow rate achieved through the DPI but
also the time taken to reach the peak flow will determine how efficiently particles are deaggregated. In
practice, it seems that almost all patients with stable
asthma or COPD can inhale sufficiently well via DPIs
to benefit from them.
Several so-called active DPIs have been developed,
in which the powder is dispersed by some mechanism
other than the patient’s inhalation – for instance, by
an internal source of compressed air or by a fan
driven by an electric motor. These active DPIs are
generally more complex than breath-actuated DPIs
and may come to be used primarily for therapies that
require very efficient and reproducible targeting of
drugs to specific lung regions, such as inhaled peptides for systemic therapy.
Sophisticated formulations for use in DPIs are also
in development. These include drug/lactose blends, in
which the surface of the lactose particles has been
smoothed in order to aid dispersion, or particles
made by processes other than micronization. For instance, a spray-dried formulation of the antibiotic
tobramycin is under development for the treatment
and prevention of respiratory tract infections in patients with cystic fibrosis, consisting of low-density
spherical particles that disperse efficiently with minimal inspiratory effort. An advantage of these sophisticated formulations is that often they can be
delivered efficiently to the lungs using very simple
and inexpensive DPI devices.
Nebulizers
Drugs may often be formulated as solutions in water
or ethanol, and they may be delivered by nebulizers
AEROSOLS 63
Signal from piezoelectric
crystal
Drug formulation
in reservoir
Mouthpiece
Baffle
Venturi
Mesh
Drug formulation
in nebulizer cup
Compressed air
To mouthpiece
Figure 5 Design and operation of a typical jet nebulizer.
Figure 6 Principle of operation of a mesh-based nebulizer system. A mesh or grid is vibrated by a piezoelectric crystal, and a
dispersion of micron-sized liquid droplets is formed.
that convert the solution into a spray. A variety
of devices may be used to form the spray, and the
three most common are jet nebulizers, ultrasonic nebulizers, and vibrating mesh nebulizers. An important advantage of nebulizers is that they can be
used with relaxed tidal breathing. This makes them
attractive for delivering inhaled drugs to children,
the elderly, and those undergoing acute asthmatic
attacks, who may not be able to use pMDIs or DPIs
successfully. Currently, nebulizers represent the most
practical way to deliver very large drug doses
(4100 mg) that are occasionally needed for some
inhaled antibiotics.
Jet nebulizers are operated by compressed air
passing through a narrow constriction (a venturi).
A single dose contained in a volume of typically
2–4 ml in a cup within the nebulizer is drawn up a
feed tube and is fragmented into droplets (Figure 5).
Only the smallest droplets are delivered directly to
the patient; larger droplets impact on baffle structures situated close to the nozzle and are returned
to the cup to be nebulized again. Several minutes
are required to nebulize the entire dose, and even
at completion of treatment the majority of the
dose remains within the device as large droplets on
internal walls. There are major differences in performance between different commercially available
nebulizers, with lung deposition ranging from o2%
to 20% of the dose. Jet nebulizers can also be used
to aerosolize micronized suspensions of corticosteroids. Recent developments in technology have
included breath-enhanced nebulizers, in which passage of inhaled air through the device is used to increase aerosol output, and adaptive aerosol delivery
systems, in which aerosol generation is synchronized
to coincide with the first part of the patient’s inhalation. Adaptive aerosol delivery systems seem to be
able to reduce the intersubject variability of aerosol
delivery.
Ultrasonic nebulizers have many properties similar
to jet nebulizers, but the aerosol is formed in a different way. A piezoelectric crystal is located beneath
the cup, and a fountain of droplets is generated. Ultrasonic nebulizers are less popular now than a few
years ago, possibly for several reasons. They may not
handle either suspensions or viscous solutions well,
and there is evidence that they damage some drug
molecules, probably by heat generated during the
nebulization process. Jet and ultrasonic nebulizers
cannot compete with pMDIs and DPIs in the portable inhaler market, partly because they are singledose devices and partly because they generally need
either a compressor or a power source in order to
function.
Several novel nebulizers are available in which the
spray is formed by the passage of drug solution
through a vibrating mesh or grid of micron-sized holes
(Figure 6). The mesh is usually vibrated by a piezoelectric crystal, but unlike ultrasonic nebulizers, there
is no evidence that this process damages drug molecules. Mesh-based systems deliver a higher proportion
of the dose, and achieve higher lung deposition, compared to jet or ultrasonic nebulizers. A smaller percentage of the dose is retained in the device at the end
of treatment, and this can result in less wastage for
expensive drug substances. Nebulization time is short
compared to that of jet and ultrasonic nebulizers,
which should improve patient compliance. Some
vibrating mesh nebulizers are small, compact, and
battery operated, giving them practical advantages
over jet and ultrasonic nebulizers. Careful cleaning of
all nebulizers is essential in order to avoid bacterial
contamination and to ensure that the working parts
(particularly narrow nozzles) function correctly.
64
AEROSOLS
Soft Mist Inhalers
A recent development in inhaler technology has been
the development of low-velocity sprays known as
soft mist inhalers. These devices represent a new
class of multidose inhaler devices and contain liquid
formulations similar to those in nebulizers. A variety
of principles are utilized, including forcing liquid
under pressure through a nozzle array, ultrasonics,
vibrating meshes, and several novel approaches, such
as condensation of vapors to form particle dispersions. Many of these devices are able to achieve extremely high lung deposition (450% of the dose),
and they are capable of delivering drugs to the deepest parts of the lungs. This may allow them to play a
major future role in inhalation therapy, particularly
in situations in which precise aerosol targeting is
needed.
In 2004, one soft mist inhaler (Respimat) was
launched in Europe for asthma and COPD therapy
as a direct replacement for the same drugs given
either in a CFC pMDI or in a DPI. The spray is
formed by passing a metered dose (typically 15 ml)
via a sophisticated nozzle system under pressure. The
velocity of the spray is only a fraction of that found
in a CFC pMDI. This device deposits a greater
percentage of the drug in the lungs compared to a
CFC pMDI (Figure 7), and it is clinically effective
Exhaled
100%
Device
80%
Oropharynx
60%
Lungs
40%
20%
0%
CFC pMDI
Respimat
Figure 7 Fractionation of the dose from a novel Respimat soft
mist inhaler compared to that from a pMDI formulated with
chlorofluorocarbon (CFC) propellants. Data from Newman SP
et al. (1998) Lung deposition of fenoterol and flunisolide delivered
using a novel device for inhaled medicines. Chest 113: 957–963.
using smaller doses. It is probable that other soft
mist inhalers will be marketed in the relatively
near future, and some could mount a significant
challenge to pMDIs and DPIs in the portable inhaler
market.
See also: Asthma: Overview. Bronchodilators: Anticholinergic Agents; Beta Agonists. Chronic Obstructive
Pulmonary Disease: Overview: Emphysema, Alpha-1Antitrypsin Deficiency. Corticosteroids: Therapy. Cystic Fibrosis: Overview. Particle Deposition in the
Lung.
Further Reading
Adjei AL and Gupta PK (1997) Inhalation Delivery of Therapeutic
Peptides and Proteins. New York: Dekker.
Bisgaard H, O’Callaghan C, and Smaldone GC (eds.) (2003) Drug
Delivery to the Lung. New York: Dekker.
Dalby RN, Byron PR, Peart J, and Farr SJ (eds.) (2002) Respiratory Drug Delivery VIII. Raleigh, NC: Davis Horwood.
Dalby RN, Byron PR, Peart J, Suman JD, and Farr SJ (eds.) (2004)
Respiratory Drug Delivery IX. River Grove, IL: Davis Healthcare.
Dolovich M, MacIntyre NR, Dhand R, et al. (2000) Consensus
conference on aerosols and delivery devices. Respiratory Care
45: 588–776.
Hickey AJ (ed.) 2003. Aerosol delivery and asthma therapy (theme
issue). Advanced Drug Delivery Reviews 55, 777–928.
Mitchell JP and Nagel MW (2003) Cascade impactors for size
characterization of aerosols from medical inhalers: their uses
and limitations. Journal of Aerosol Medicine 16: 341–377.
Morén F, Dolovich MB, Newhouse MT, and Newman SP (eds.)
(1993) Aerosols in Medicine: Principles, Diagnosis and Therapy.
Amsterdam: Elsevier.
Newman SP, et al. (1998) Lung deposition of fenoterol and flunisolide delivered using a novel device for inhaled medicines.
Chest 113: 957–963.
Newman SP and Newhouse MT (1996) Effect of add-on devices
for aerosol drug delivery: deposition studies and clinical aspects.
Journal of Aerosol Medicine 9: 55–70.
O’Callaghan C and Barry PW (1997) The science of nebulised drug
delivery. Thorax 52(supplement 2): S31–S44.
Pauwels R, Newman SP, and Borgström L (1997) Airway deposition and airway effects of antiasthma drugs delivered from
metered dose inhalers. European Respiratory Journal 10: 2127–
2138.
Smith IJ and Parry-Billings M (2003) The inhalers of the future? A
review of dry powder devices on the market today. Pulmonary
Pharmacology and Therapeutics 16: 79–95.
Allergic Bronchopulmonary Aspergillosis see Asthma: Allergic Bronchopulmonary Aspergillosis.
ALLERGY / Overview 65
ALLERGY
Contents
Overview
Allergic Reactions
Allergic Rhinitis
Overview
J A Grant and C C Horner, University of Texas
Medical Branch, Galveston, TX, USA
& 2006 Elsevier Ltd. All rights reserved.
Abstract
Allergy is the term used for a collection of diseases mediated by
immunologic mechanisms. Allergic disorders include allergic
rhinitis, conjunctivitis, asthma, urticaria, angioedema, food allergy, drug allergy, and anaphylaxis. The prevalence of allergic
diseases has been increasing in recent times. They are a major
cause of morbidity and decreased quality of life. The development of allergic diseases is influenced by heritable and environmental factors. Diagnosis often involves documenting responses
to allergen such as in skin testing, radioallergosorbant allergen
testing, or bronchial provocation testing. Allergic disorders share
the common pathology of inflammation of affected tissues. Allergy requires sensitization to an allergen and response on reexposure to that same allergen. Pathogenesis involves production
and release of cytokines, chemokines, and lipid mediators which
cause tissue damage and recruit inflammatory cells. Current
therapies include allergen avoidance, antihistamines, leukotriene
modifiers, corticosteroids, phosphodiesterase inhibitors, humanized monoclonal anti-IgE, and immunotherapy.
Introduction
Allergy encompasses a wide variety of disorders that
share immunologic mechanisms. For centuries, patients had allergic symptoms but the causative agent
for sensitivity to allergens was unknown. Allergic
symptoms were first described by Leonardo Bottallo
in sixteenth century Europe. Wyman identified ragweed as the trigger of the ‘autumnal catarrh’ and
Blackely identified hay fever as being initiated by
grass pollen in the 1870s. Twenty years later Behring
described the adverse skin reactions to tubercle bacillus as ‘hypersensitivity’. Portier and Richet attempted
to confer immunity to sea anemone toxin by injecting
dogs with subsequent doses of toxin. They employed
the term ‘anaphylaxis’ (denoting antiprotection) in
1902 to describe a lethal reaction after the dogs received the second dose of the toxin. Von Pirquet used
the term ‘allergy’ in 1906 to describe the skin reaction
to cowpox vaccine at 24 h post-vaccination. His definition of allergy described an organism’s alteration
by contact with an organic agent. Four years later,
Noon observed reactions to pollen extracts on
abraded skin and began immunotherapy with these
extracts. Dale and Laidlaw produced respiratory distress and anaphylaxis in animals by histamine infusion in 1919. Another major discovery was the
identification of cytokines. This class includes chemokines that are important in the recruitment of inflammatory cells.
In 1921, Prausnitz and Kustner demonstrated
that a factor could be transferred to a nonsensitized
person’s skin and confer sensitivity. This factor
was called ‘reagin’ by Coca and Cooke. In 1966,
Ishizaka’s discovery of IgE established a scientific
basis for the specific reactions. Concurrently, S G O
Johannson independently identified a protein in multiple myeloma that was also elevated in allergic patients. Subsequently, the WHO determined that both
proteins were IgE.
In 1968, Gell and Coomb described four classes of
immunologic reactions. These classes are IgE-mediated immediate hypersensitivity (e.g., anaphylactic
shock), IgG- or IgM-mediated cytotoxic reactions
(e.g., immune hemolytic anemia), immune complexmediated reactions (e.g., serum sickness), and delayed
hypersensitivity (e.g., contact dermatitis). Allergic
diseases can be mediated by any of these mechanisms. Allergic disorders include allergic rhinitis,
conjunctivitis, asthma, urticaria, angioedema, food
allergy, drug allergy, and anaphylaxis.
Etiology
Atopy refers to the tendency of patients to be sensitive to allergens. Atopic diseases include eczema,
allergic rhinitis, and asthma. Over the past century,
genetics has been thought to affect allergic disease
occurrence. The incidence of allergic disease in patients with an allergic family history is higher than in
those without one. Atopy is thought to be autosomally transmitted and multigenic. It is probably a
complex trait influenced by both heritable and environmental factors. Environmental exposures likely
have effects on gene expression and the development
of allergic disease. The hygiene hypothesis proposes
66
ALLERGY / Overview
that in Western countries the developing immune
system is deprived of environmental microbial antigens that stimulate Th1 cells. This lack of stimulation
increases the presence of atopic disease. Other factors such as diet and exposure to high pollen counts
at birth may also increase allergic disease.
Linkage studies for allergy and asthma have
produced 420 distinct chromosomal regions with
linkage to asthma or related traits. The lack of distinct
phenotypes, the inexact definitions of allergic diseases,
and the presumed influence of numerous genes have
made positional cloning with linkage studies problematic. The linkages reproduced most frequently are
on 6p, 5q, 12q, and 13q. Linkage on 14q and 7p was
identified in founder populations in Iceland and Finland. Chromosome 2q14–2q32, which includes the
IL-1 gene, has been linked to asthma.
Candidate gene analysis is a promising technique.
Candidate genes encode biochemical markers that
affect allergic diseases. Many of these genes control
IgE and cytokine production. Candidate genes include one loci on 5q near the gene cluster for IL-4,
IL-5, IL-9, and IL-13. Polymorphisms of the IL-9
gene are associated with human asthma. 11q13,
which encodes the b chain of the high-affinity IgE
receptor has been linked to asthma. Polymorphisms
of the IL-4 receptor a chain are associated with
atopic asthma. PHF11, present in the locus for total
IgE on 13q14, is expressed in many immune-related
tissues. It is associated with total IgE and has been
linked with asthma in multiple genome screens. The
ADAM33 gene on 20p13 encodes a disintegrin and
metalloprotease. This gene was mapped in 2002 as
an asthma and airway hyperresponsiveness gene by
Van Eerdewegh and co-workers. A cluster of single
nucleotide polymorphisms (SNPs) was identified in
the ADAM33 gene and demonstrated significant associations with asthma. Howard and co-workers reproduced the association seen by Van Eerdewegh’s
group and lent support to the potential role of
ADAM33 in asthma susceptibility (see Genetics:
Gene Association Studies).
Pathology
Allergic inflammation is present in all allergic diseases.
Most tissues exhibit vasodilation and increased vascular permeability. Eosinophils, neutrophils, CD4 þ
T cells, and basophils eventually infiltrate the site of
allergy. Asthma is a disorder which also involves an
increase in mucous glands, mucus hypersecretion,
smooth muscle hypertrophy, and airway remodeling.
Recruitment of eosinophils is a prominent feature
of allergic diseases (see Leukocytes: Eosinophils).
Eosinophils migrate across blood vessels into tissues
by binding endothelial cell adhesion molecules. Major
basic protein, lipid mediators, and cytokines enable
the eosinophil proinflammatory effects. Eosinophils
may also repair damage to mucosal surfaces with fibrogenic growth factor and matrix metalloprotease.
This repair mechanism may result in remodeling of
airway tissue as seen in asthma.
Mast cells are present in tissues throughout the
body. Features of an allergic reaction vary with
the anatomic site. The site of allergen contact determines the tissue or organ involved. The concentration of mast cells at the site determines the severity.
The wheal and flare is the typical cutaneous allergic
response. When allergen binds to specific IgE on
mast cells, mast cell mediators are released and cause
local blood vessel dilation. These vessels leak fluid
and macromolecules and produce redness and swelling (known as the wheal). Dilation of vessels on the
edge of the swelling causes redness (known as the
flare).
Clinical Features
Patients with allergic rhinitis typically experience
rhinorrhea, congestion, sneezing, and itchy nose.
Patients also may report postnasal drip and associated eye, ear, and throat symptoms. Patients commonly exhibit reactions to dust mite, animal dander,
molds, and pollens. Small molecular weight chemicals can also react with self-proteins and become
allergens. Reactivity to allergens can be determined
by immediate hypersensitivity skin testing. Drops
of allergen are placed into the dermis by various
methods. Most skin testing employs a prick device
composed of metal or plastic to introduce allergen
into the skin. Allergen binds IgE and the mast
cells in the skin release histamine to produce the
wheal and flare response. The skin response can be
compared to controls of saline (negative control)
and histamine (positive control). Radioallergosorbant allergen testing (RAST) quantitates allergenspecific IgE in a patient’s serum. RAST is usually
reserved for patients who have a contraindication to
skin testing or are taking medications that either interfere with testing (antihistamines) or interfere with
treatment should a reaction occur (beta blockers
or ace inhibitors). Nasal cytology can reveal the
presence or absence of inflammatory cells, especially
eosinophils. Adjunct tests such as rhinoscopy and
rhinomanometry can provide further characterization of the nasal airway (see Allergy: Allergic
Rhinitis).
Patients with asthma report symptoms of wheezing, shortness of breath, cough, and chest tightness.
These patients’ airways show hyperresponsiveness
ALLERGY / Overview 67
with reversibility. Patients may experience asthmatic
symptoms in response to allergens, infections, exercise, nonsteroidal anti-inflammatory drugs, gastroesophageal reflux disease, stress, and irritants.
Lung function can be assessed by spirometry before
and after treatment with bronchodilators. Bronchial
hyperresponsiveness can be investigated with bronchial provocation testing such as with methacholine
or exercise challenge testing (see Asthma: Overview).
Urticaria is localized edema in skin or mucous
membranes. Patients have pale to pink wheals that
are extremely pruritic. These lesions are transient
and usually resolve within 24 h. Angioedema involves local edema in deeper areas of skin or mucous
membranes. These lesions are often painful. Urticaria/angioedema can be triggered by temperature,
sun, direct pressure, medication, infections, foods, or
systemic diseases.
Atopic eczema is a skin disorder with pruritis,
erythematous macules or papules, and xerosis. Lesions may become excoriated with crust and exudates. Skin tends to be dry and more permeable to
allergens and bacteria. Chronic irritation may cause
lichenification of the skin and scaling patches or
papules. Young children typically have lesions on the
face, scalp, and extensor surfaces. Older children and
adults have flexor surface involvement.
Food allergy is most frequent in young children.
Symptoms may include urticaria, angioedema, rash,
flushing, rhinitis, wheezing, or anaphylaxis after ingestion of the allergenic food. Some patients experience the oral allergy syndrome which is usually
confined to the oropharynx. Symptoms may consist
of pruritis and angioedema of the tongue, lips, palate, and throat. Patients with birch pollen-induced
rhinitis may develop oral allergy symptoms after eating raw potato, carrot, apple, celery, or hazelnut.
Patients with ragweed pollen-induced rhinitis may
develop symptoms after eating melons or bananas.
Major food allergens in children are milk, egg, peanuts, soybeans, wheat, fish, and tree nuts. Major
food allergens in adults are peanuts, tree nuts, fish,
and shellfish. Conformational and sequential food
epitopes are responsible for food allergy. Patients
with IgE to sequential epitopes react to all forms of a
food and tend to have persistent allergy, whereas
those with IgE to conformational epitopes tolerate
small amounts of food after heating or partial hydrolysis because these conformational epitopes are
destroyed. These patients tend to have clinical tolerance. Prick skin testing for foods can be performed as
described previously. Negative prick skin tests have a
high negative predictive value. Positive skin tests are
not conclusive. RAST may also be performed to aid
in diagnosis.
Drug allergy reactions most commonly consist
of urticaria, morbilliform rash, or anaphylaxis. Skin
testing for drug allergy is not standardized and the
predictive value of this technique is unclear.
Anaphylaxis is an immediate generalized systemic
allergic reaction in response to an allergen. Symptoms
may include flushing, pruritis, urticaria, angioedema,
wheezing, shortness of breath, chest tightness, abdominal pain, nausea, vomiting, diarrhea, laryngeal
edema, arrhythmia, myocardial infarction, and hypotension. Serum tryptase can be measured within 6 h of
a reaction to determine if mast cell degranulation has
occurred during the reaction. Serum histamine and
urinary LTE4 may be quantitated to provide evidence
of anaphylaxis.
Pathogenesis
Immunology plays a large role in allergic diseases. T
lymphocytes are the major effector cells of these immune responses (see Leukocytes: T cells). CD4 þ
lymphocytes are present in two predominant types,
Th1 and Th2 cells. Th1 are helper cells that produce
IL-2 and interferon gamma (IFN-g) and promote cellmediated reactions. Th2 are helper cells that produce
IL-4, IL-5, IL-6, IL-10, and IL-13 and are involved in
humoral immunity and allergic inflammation. Th2
cytokines augment antibody production (especially
IgE), enhance eosinophil production, and are associated with allergic and antibody-driven responses
(Figure 1). Th1 and Th2 cells suppress each other.
Patients with an allergic phenotype generate responses to allergens that favor Th2 responses.
Another type of T cell is the regulatory T cell (T
reg). Thymus-derived CD4 þ CD25 þ regulatory cells
are termed naturally occurring T regs. Adaptive T
regs are T-cell populations induced by in vitro or in
vivo manipulation. Active T-cell suppression by T
regs promotes immunologic tolerance, but the mechanism of this suppression is controversial. There is a
potential role for T regs in the control or prevention
of Th2 responses. CD4 þ CD25 þ T cells and IL-10producing T regs have been shown in humans to
prevent T-cell activation by allergens and are possibly
deficient in atopic patients. T regs may also secrete
the immunosuppressive cytokines IL-10 and TGF-b.
IL-10 inhibits macrophage activation and antagonizes IFN-g while TGF-b inhibits T- and B-cell proliferation.
IgE-mediated allergic responses occur through numerous steps. First, a patient is exposed to an allergen. Then antigen-presenting cells internalize the
allergen which is then processed and presented to
Th2 cells by class II MHC molecules. T-cell receptors
bind the presented allergen thus activating the Th2
68
ALLERGY / Overview
APC
Th2 cell
Allergen
IL-4
Specific IgE
Re-exposure to
allergen
B cell
Arachidonic acid
Prostaglandins,
leukotrienes
Preformed mediators
(histamine, TNF-)
Early response
Mast cell
Vascular leak,
bronchoconstriction,
inflammation, tissue
damage, late
response
Figure 1 Allergen is processed by antigen presenting cells (APCs) and presented to Th2 cells. Stimulated by IL-4, B cells produce
allergen-specific IgE. Upon re-exposure to allergen, allergen cross-links specific IgE on mast cells and activates allergic responses.
cells. The activated cells produce IL-4. Atopic patients have more allergen-specific IL-4 secretory T
cells in circulation than nonatopics and produce more
IL-4 per cell. B cells, in turn, are stimulated by IL-4 to
class-switch to produce IgE, specific to the allergen.
Approximately 20% of an exposed population will
generate specific IgE to an allergen. This IgE binds to
receptors on mast cells. When the patient is subsequently exposed to the allergen, the allergen binds the
IgE already present on the mast cell surfaces and
cross-links the antibodies.
Antibody cross-linking of mast cells activates a variety of responses. The early phase of immediate
hypersensitivity occurs within minutes and involves
the release of preformed mediators from granules.
These mediators include biogenic amines (histamine),
enzymes (tryptase, chymase), carboxypeptidase, cathepsin G, acid hydrolases, and tumor necrosis factor
alpha (TNF-a) (see Leukocytes: Mast Cells and Basophils). Histamine causes endothelial cells to contract
and allow plasma to leak into tissue. Endothelial cells
also produce prostacyclin and nitric oxide which
promote vascular smooth muscle relaxation and lead
to vasodilation. Bronchial smooth muscle constriction is also caused by histamine.
Antibody cross-linking also triggers the initiation
of several pathways which lead to cytokine, prostaglandin, and leukotriene production. Arachadonic
acid is converted to prostaglandin D2 and the cysteinyl leukotrienes (LTC4, LTD4, LTE4) (see Lipid
Mediators: Leukotrienes). These mediators are responsible for vascular leak, bronchoconstriction, inflammation, and tissue damage. These substances
lead to inflammatory changes hours after exposure,
referred to as the late-phase response. The late phase
involves recruitment and infiltration of the mucosa
with neutrophils, eosinophils, basophils, and Th2
cells. TNF-a activates endothelial expression of adhesion molecules E-selectin and ICAM-1 and promotes cell infiltration. IL-3 promotes mast cell
proliferation. IL-5 stimulates eosinophil production
and activation (see Interleukins: IL-5). The chemokines eotaxin and monocyte chemotactic protein-5
from epithelial cells recruit eosinophils. IL-4 and IL13 induce Th2 differentiation. While mast cells are
responsible for the majority of leukotriene production in the early response, basophils and eosinophils
produce most of the leukotrienes in the late-phase
response.
Animal Models
Mouse models have been used in the study of allergy
because mice are readily available, have a well-characterized immune system, and strains are genetically
characterized. Knockout mice can be used to evaluate the role of a cell type or mediator. Many allergists
propose that allergic rhinitis and asthma may represent a continuum of inflammation and should be
considered as a united allergy airway disorder.
Mouse models have been used to investigate this
concept. Mice were systemically sensitized with allergen (most often ovalbumin) and then challenged
with airway allergen. The inflammatory response in
the mouse nose resembles human allergic rhinitis.
Nasal mucosal thickening can be seen on imaging.
Experimental asthma in mice also mimics human
asthma. Bronchial hyperresponsiveness can be documented by plethysmography. Most of the inflammation in mice is seen in the lower airways where the
minority of allergen is deposited. This may indicate
increased sensitivity in the lower airways. Inhaled
ALLERGY / Overview 69
allergen causes an increase in allergen-specific IgE
and eosinophils in the blood and increases bone
marrow eosinopoiesis. Currently, it is unclear why
sensitization causes symptoms in the nose, the lower
airway, or both. There may be a genetic cause so
studying different strains of mice may be useful.
Mouse models have also been used in the study of
atopic eczema. Mice have been sensitized epicutaneously with ovalbumin on shaved skin. Elevated
serum total and specific IgE and IgG1 and increased
dermal IL-4, IL-5, and IFN-g were observed. Deficiencies of these cytokines decreased the eczematous phenotype. For example, IL-4-deficient mice showed Th1biased skin inflammation with decreased eosinophils
and eotaxin mRNA in the dermal infiltrate. IL-5deficient mice similarly had less pronounced epidermal
and dermal thickening and the dermal infiltrate lacked
eosinophils. IFN-g-deficient mice showed only slight
dermal thickening. The necessity of certain lymphocytes in eczema was demonstrated. ab T cells are essential since T-cell receptor a-chain-deficient mice did
not develop a dermal infiltrate or induction of IL-4 or
IgE. Mice that lack gd T cells showed no change in
infiltrate. Likewise, mice that lack B cells still develop
an infiltrate and elevated IL-4.
Numerous animal models have been utilized in food
allergy. Rats and mice have been used to assess foodinduced anaphylaxis. Animals ingested ovalbumin by
gavage or in drinking water and were subsequently
challenged intraperitoneally. The route, dose, and age
of the animal were shown to influence sensitization.
Rats and swine sensitized to allergens and subsequently challenged orally, demonstrated alterations in
small intestinal pathology. Tolerance is dose-dependent
for specific antigens. Mice fed ovalbumin or peanuts
required a 50-fold higher dose of peanuts to develop
tolerance; low doses of peanuts were more likely to
induce sensitization. The importance of an intact mucosal barrier was shown when mice, fed a novel dietary protein while their gastrointestinal tracts were
inflamed, developed sensitization and high serum IgE.
Allergic conjunctivitis has been studied in guinea
pigs, rats, and mice. Mice exposed to ragweed by
topical contact with conjunctival and nasal mucosa
developed signs of allergic conjunctivitis and ragweedspecific IgE. Regulators of vascular permeability are
important in allergic conjunctivitis. Substance
P has been shown to be a mediator of allergic conjunctivitis and acts through NK1 receptors on blood
vessels to produce conjunctival hyperpermeability.
Nitric oxide has been shown to play a major role in
regulating vascular permeability and stimulating prostaglandin E2 production. T-cell adhesion molecules
are integral in allergic conjunctivitis. Guinea pig models with ovalbumin have shown that the integrin very
late activation antigen-4 (VLA-4) plays a critical role
in eosinophil infiltration. Other mice studies showed
that antibodies against the integrin intercellular adhesion molecule-1 (ICAM-1) and its ligand leukocyte
function-associated antigen-1 (LFA-1) inhibited
clinical and histological signs of conjunctivitis. The
significance of IL-1 for inflammatory changes in conjunctivitis was demonstrated using an IL-1 receptor
antagonist in mice exposed to cat dander antigens.
Studies in rats using ovalbumin showed that IFN-g
suppresses the development of allergic conjunctivitis
during the induction phase.
Management and Current Therapy
One of the cornerstones of allergy treatment is avoidance. Avoiding or reducing allergen exposure prevents or minimizes the body’s response to allergen.
Environmental control measures help one decrease
exposure. For example, patients with house dust-mite
allergy can encase their mattresses and pillows in
special covers to minimize exposure to dust mites
while asleep. One study showed that in inner-city
children with atopic asthma, a comprehensive environmental intervention decreased exposure to indoor
allergens and reduced asthma-associated morbidity.
Avoiding allergic foods or drugs can prevent reactions
(Figure 2). Depending on one’s sensitivities, allergen
avoidance may range from simple to extremely difficult. Other therapies available to treat allergic disorders include antihistamines, leukotriene modifiers,
corticosteroids, phosphodiesterase inhibitors, humanized monoclonal anti-IgE, and immunotherapy.
H1-antihistamines have been used for decades for
the relief or prevention of allergic symptoms. Antihistamines have recently received the designation of
inverse agonists because they stabilize the inactive
form of the H1 histamine receptor. First-generation
antihistamines have marked sedation; second-generation antihistamines that are relatively nonsedating
Therapy algorithm
Allergy diagnosis
Asthma diagnosis
Avoidance of allergen
Avoidance of triggers,
allergens
Pharmacotherapy
Pharmacotherapy
Immunotherapy
Immunotherapy
Figure 2 Therapy for both allergic rhinitis and asthma starts
with avoidance of allergens. Pharmacotherapy can be added.
Some patients with allergic rhinitis and asthma benefit considerably from immunotherapy.
70
ALLERGY / Overview
have also been identified. Antiallergic activities include inhibiting the release of mast cell mediators
probably through direct inhibition of Caþ channels.
Anti-inflammatory effects include inhibiting cell adhesion molecule expression and inhibiting inflammatory cell chemotaxis (e.g., eosinophil chemotaxis).
These inhibitions probably involve the downregulation of NF-kB, a transcription factor that regulates
adhesion proteins and proinflammatory cytokines.
Antihistamines have established roles in the treatment
of allergic rhinitis and urticaria. A potential role exists for the treatment of anaphylaxis or asthma.
The cysteinyl leukotrienes and LTB4 are products
of arachadonic acid metabolism by 5-lipoxygenase.
Leukotrienes cause airway inflammation and obstruction by affecting mucus production, smooth
muscle contraction, and vascular permeability. They
may also affect airway remodeling. The effects of
leukotrienes are most likely mediated through the
CysLT1 receptor. The CysLT1 receptor antagonists
montelukast, pranlukast, and zafirlukast block the
actions of LTC4, LTD4, and LTE4. A nonselective
antagonist of the CysLT1 and CysLT2 receptors
is not yet clinically available. Zileuton is a 5-lipoxygenase inhibitor and decreases the production of
leukotrienes. Leukotriene modifiers are useful in allergic rhinitis and asthma. Leukotriene modifiers inhibit an important part of the inflammatory cascade
and decrease eosinophil survival, goblet cell hyperplasia, mucus release, collagen deposition, and airway smooth muscle proliferation.
Corticosteroids block the production of inflammatory cytokines (see Corticosteroids: Therapy). They
may be given topically at the site of inflammation
(nose, lungs, or skin) or delivered systemically.
Glucocorticoids are the most potent therapy for
treating all allergic disorders. They are liposoluble
hormones that enter the cell and bind a cytoplasmic
glucocorticoid receptor (see Corticosteroids: Glucocorticoid Receptors). This receptor translocates to
the nucleus and binds a glucocorticoid-response
element in the promoter region of target genes. The
glucocorticoid receptor can also bind transcription
factors like NF-kB and AP-1 and prevent these
factors from binding their DNA-response elements.
Glucocorticoids control airway inflammation by
inhibiting transcriptional activity of genes encoding
proinflammatory molecules such as cytokines, chemokines, adhesion molecules, and mediator-synthesizing enzymes. They may suppress histone
acetylation and stimulate histone deacetylation. They
may also interfere with signal transduction pathways, such as MAP kinase enzymatic cascades involved in the regulation of transcription factors.
Theophylline is a nonselective phosphodiesterase
inhibitor with a narrow therapeutic ratio and significant drug interactions and has been used exclusively
in asthma (see Bronchodilators: Theophylline). Inhibitors of phosphodiesterase type 4 have recently been
developed. These inhibitors increase the intracellular
concentration of CAMP and exhibit a broad range of
anti-inflammatory effects on effector cells. Blocking
the PDE4B receptor subtype appears responsible for
anti-inflammatory properties of these agents. Cilomast and roflumilast are PDE4 inhibitors that are in
late phase III clinical trials. Roflumilast has demonstrated more selectivity and a superior therapeutic
ratio (Figure 3).
The humanized monoclonal anti-IgE omalizumab
is a new therapy which decreases the amount of IgE
available for reactions and downregulates the number of IgE receptors on mast cells. This therapy is
currently available for the treatment of severe
asthma. Its use in food allergy is being investigated.
Clinical features
• Allergic rhinitis
• Asthma
• Urticaria
• Angioedema
• Atopic eczema
• Food allergy
• Oral allergy syndrome
• Drug allergy
• Anaphylaxis
Rhinorrhea, congestion, sneezing, itchy nose
Wheezing, shortness of breath, cough, chest
tightness
Pruritic wheals
Deep local swelling of skin or mucus membranes
Pruritis, erythematous maculesor papules, xerosis,
lichenification
Urticaria/angioedema, rash, flushing, rhinitis,
wheezing, anaphylaxis
Pruritis/angioedema of tongue, lips, palate, throat
Uticaria, morbilliform rash, anaphylaxis
Flushing, pruritis, urticaria/angioedema, wheezing,
shortness of breath, abdominal pain, nausea,
vomiting, diarrhea, laryngeal edema, arrhythmia,
myocardial infarction, hypotension
Figure 3 Clinical features associated with various allergic disorders.
ALLERGY / Overview 71
Diagnostic workup
Allergic rhinitis
Asthma
History and physical
examination
History and physical
examination
Immediate
hypersensitivity
skin testing or RAST
Spirometry
Medication trial
Nasal cytology
Rhinoscopy or
rhinomanometry
Bronchial
provocation
testing
Figure 4 Diagnostic workups start with the history and physical
exam. Initial testing for allergic rhinitis consists of skin testing or
RAST. Adjunct tests include nasal cytology, rhinoscopy, and
rhinomanometry. Lung function in asthma is assessed by
spirometry and a medication trial is begun. Bronchial provocation
testing can assess bronchial hyperresponsiveness if needed.
Patients with allergic rhinitis and/or asthma who
are (Figure 4) poorly controlled on medications may
find immunotherapy (allergy shots) a feasible alternative. Immunotherapy involves administering injections of allergens to which a patient is sensitive.
Increasing doses of allergen are given weekly until
the patient is at a maintenance dose. This maintenance dose is continued monthly for 3 to 5 years.
Patients receive relief from nasal allergy symptoms
and their asthma may improve. The mechanisms of
immunotherapy are not well defined. Immunotherapy may induce specific T-cell tolerance or a shift
from a Th2 to a Th1 phenotype. Possibly, the Th2
response is inhibited, Th1 response is upregulated, or
both. Immunotherapy also increases the production
of IL-10 and TGF-b by T cells including T regs. IgG
is also produced in response to antigen instead of the
typical IgE. Allergen-specific IgG or blocking antibodies may compete with IgE for allergen and inhibit
IgE activation of mast cells. IgG can also bind
epitopes on allergen that are not recognized by IgE.
This IgG binding may prevent cross-linking of IgE.
Allergen-IgG complexes on antigen-presenting cells
might impair antigen processing or the co-stimulation of T cells and render patients anergic. Immunotherapy is a type of desensitization, where small
amounts of allergen are given until the patient tolerates the allergen. The same theory is used in treatment of drug allergies by giving increasing doses of
drug until a therapeutic dose is tolerated.
Unmethylated CG dinucleotides, or CpG motifs,
are responsible for the immunostimulatory effect of
bacterial DNA and induce a Th1 type response in
humans. Synthetic oligodeoxynucleotides mimic the
bacterial DNA immunostimulatory sequences. These
synthetic nucleotides can be conjugated to allergen to
produce an allergen vaccine that is more immunogenic but less allergenic than allergen alone. These
vaccines are currently under clinical trials.
Recently, there has been an interest in pharmacogenetics. This field recognizes that medications may work
more efficaciously in certain patients because of their
genetic makeup. Single nucleotide polymorphisms
(SNPs) may signal a change in proteins or amino acids in an individual. These changes may alter a drug’s
target, uptake, metabolism, or excretion. A polymorphism in Gly 16 promotes bronchodilator resistance while
the Arg 16 polymorphism potentiates a greater response to bronchodilators. An alternatively spliced
form of glucocorticoid receptor b is present with higher
frequency in corticosteroid-resistant patients. A polymorphism in the 5-lipoxygenase promoter decreases
the response to the 5-lipoxygenase inhibitor zileuton.
Pharmacogenetics is an exciting area that may shape
the future of treatment for allergic diseases.
See also: Allergy: Allergic Reactions; Allergic Rhinitis.
Asthma: Overview. Bronchodilators: Theophylline.
Chemokines. Corticosteroids: Glucocorticoid Receptors; Therapy. Genetics: Gene Association Studies.
Immunoglobulins. Interleukins: IL-5. Leukocytes:
Mast Cells and Basophils; Eosinophils; T cells. Lipid
Mediators: Leukotrienes; Prostanoids. Matrix Metalloproteinases.
Further Reading
Abbas AK and Lichtman AH (2003) Cellular and Molecular
Immunology, 5th edn. Philadelphia: Saunders.
Adelman DC, Casale TB, and Corren J (2002) Manual of Allergy
and Immunology, 4th edn. Philadelphia: Lippincott Williams &
Wilkins.
Broide DH (2001) Molecular and cellular mechanisms of allergic
disease. Journal of Allergy and Clinical Immunology 108: S65–S71.
Cakebread JA, et al. (2004) The role of ADAM33 in the pathogenesis
of asthma. Springer Seminars in Immunopathology 25: 361–375.
Creticos PS, Chen Y, and Schroeder JT (2004) New approaches in
immunotherapy: allergen vaccination with immunostimulatory
DNA. Immunology and Allergy Clinics North America 24:
569–581.
Groneberg DA, et al. (2003) Animal models of allergic and inflammatory conjunctivitis. Allergy 58: 1101–1113.
Gutermuth J, et al. (2004) Mouse models of atopic eczema critically evaluated. International Archives of Allergy and Immunology 135: 262–276.
Hallstrand TS and Henderson WR (2002) Leukotriene modifiers.
Medical Clinics of North America 86: 1009–1033.
Hellings PW and Ceuppens JL (2004) Mouse models of global
airway allergy: what have we learned and what should we do
next? Allergy 59: 914–919.
Helm RM and Burks AW (2002) Animal models of food allergy.
Current Opinion in Allergy and Clinical Immunology 2: 541–546.
72
ALLERGY / Allergic Reactions
Holgate ST (2004) Pharmacogenetics: the new science of personalizing treatment. Current Opinion in Allergy and Clinical Immunology 4: 37–38.
Joad J and Casale TB (1988) Histamine and airway caliber. Annals
of Allergy 61: 1–7.
Kay AB (2001) Allergy and allergic diseases: first of two parts.
New England Journal of Medicine 344(1): 30–37.
Kim DS and Drake-Lee AB (2003) Allergen immunotherapy in
ENT: historical perspective. Journal of Laryngology & Otology
117: 940–945.
Lipworth BJ (2005) Phosphodiesterase-4 inhibitors for asthma and
chronic obstructive pulmonary disease. Lancet 365: 167–175.
Morgan WJ, et al. (2004) Results of a home-based environmental
intervention among urban children with asthma. New England
Journal of Medicine 351: 1068–1080.
Pelaia G, et al. (2002) Molecular mechanisms of corticosteroid
action in chronic inflammatory airway diseases. Life Sciences
72: 1549–1561.
Robinson DS, Larche M, and Durham SR (2004) Tregs and allergic disease. Journal of Clinical Investigation 114: 1389–1397.
Sampson HA (2004) Update on food allergy. Journal of Allergy
and Clinical Immunology 113(5): 805–819.
Shearer WT and Li JT (2003) Primer on allergic and immunologic
diseases. Journal of Allergy and Clinical Immunology 111(2):
S441–S778.
Simons FER (2004) Advances in H1-antihistamines. New England
Journal of Medicine 351: 2203–2217.
Till SJ, et al. (2004) Mechanisms of immunotherapy. Journal of
Allergy and Clinical Immunology 113(6): 1025–1034.
Weiss ST and Raby BA (2004) Asthma genetics 2003. Human
Molecular Genetics 13: R83–R89.
Allergic Reactions
S H Arshad, University Hospital of North Staffordshire,
Stoke-on-Trent, UK
& 2006 Elsevier Ltd. All rights reserved.
Abstract
Allergy is a harmful response to an otherwise innocuous substance. Allergic reactions are immunologically mediated reactions to external substances, usually proteins. These are often,
but not always, mediated by immunoglobulin E (IgE) antibody.
Initial exposure to an allergen results in sensitization with the
production of allergen-specific IgE antibodies. These antibodies
circulate in the blood but are largely bound to high-affinity
receptors on the surface of basophils and mast cells and lowaffinity receptors on eosinophils, macrophages, and platelets.
On further exposure, the allergen reacts with the IgE bound to
mast cells and basophils, causing degranulation and release of
preformed mediators such as histamine and tryptase. These
mediators cause the immediate phase of the type I reaction,
which occurs within a few minutes (immediate hypersensitivity).
Other mediators and cytokines are released and eosinophils are
attracted to the site of activity, precipitating the late phase of the
type I reaction, which starts 4–6 h after exposure. Immediate
hypersensitivity reaction occurs in asthma, rhinitis, and anaphylaxis. In a sensitized asthmatic, early and late phase asthmatic reaction can be observed following bronchial allergen
challenge. Repeated or continued exposure to allergen results in
chronic airway inflammation, which is characteristic of asthma.
Systemic allergic reactions vary in severity from mild (such as
generalized urticaria) to severe and life-threatening reactions
with cardiovascular collapse and death. Anaphylaxis is the clinical syndrome that represents the life-threatening systemic allergic reaction. It results from the immunologically induced
release of mast cell and basophil mediators after exposure to a
specific antigen in previously sensitized individuals. Clinically
indistinguishable reactions, caused by non-IgE-mediated immune mechanisms, are termed anaphylactoid reactions. Common causes of anaphylaxis are foods, drugs, insect stings, latex,
and allergen extracts used for immunotherapy. The symptoms
of anaphylaxis occur within a few minutes of exposure and are
generally related to the skin, gastrointestinal tract, respiratory
tract, and cardiovascular systems. Common manifestations include generalized urticaria/angioedema, nausea, vomiting,
stridor, wheezing, hypotension, and syncope.
Anaphylaxis requires immediate treatment with epinephrine,
given intramuscularly. The dosage for adults is 0.3–0.5 ml, and
for children is 0.01 ml kg 1, of a 1:1000 solution. The dose can
be repeated at 5–15 min intervals. Supportive treatment includes
cardiopulmonary resuscitation (if required), intravenous fluids,
oxygen, antihistamine, and corticosteroids. Following the first
episode, an assessment by an allergist is essential to establish the
cause and for appropriate advice on preventive measures
including avoidance of the offending agent and self-injectable
epinephrine.
Immune Responses
Atopy is defined as the genetic predisposition to form
immunoglobulin (IgE) antibodies on exposure to allergens. The production of IgE is central to the induction of allergic diseases. Allergens are proteins
with the capability to react to the immune system
through their antigenic determinants. Initial exposure to the antigen results in sensitization. Antigens
enter the body through the respiratory and gastrointestinal mucosa and the skin. Allergenic proteins are
engulfed by antigen-presenting cells (APCs) such as
monocytes, macrophages, and dendritic cells inducing primary immune response. The antigen is broken
down to reveal the specific part of the molecule
called antigenic determinant or epitope. Once processed in this way, the antigen is bound to the MHC
class II molecules on the surface of these cells and the
complex is presented to the T lymphocyte cell receptor. Bacterial antigens favor the production of Th1
cells with the secretion of its profile of cytokines,
particularly interferon gamma (IFN-g). In atopic individuals, and in the presence of co-stimulatory signals, naı̈ve T cells are converted to activated CD4 þ
T-helper-2 (Th2) cells.
T lymphocytes play a central role in orchestrating
the allergic reaction. Th2 cells produce cytokines,
such as interleukin-4 (IL-4) and IL-13. These
cytokines cause proliferation and switching of B
cells to IgE producing B and plasma cells, specific to
the antigen (Figure 1). Some of these cells have a long
life and are called memory cells. IL-4 is the most
ALLERGY / Allergic Reactions 73
Antigen
IgE
Antigen-presenting
cell
Naive
T cell
Th2 cell
B cell
Antigen
Release of mediators such as
histamine and tryptase
Mast cell
important pro-allergy cytokine. Apart from switching of B cells to IgE production, it also stimulates T
cells and macrophages, and enhances the expression
of low-affinity IgE receptor (FceR2) on B cells and
adhesion molecules on endothelial cells. This latter
action promotes the movement of cells out of the
blood vessels. It also inhibits other types of immune
reaction, such as antibody-dependent cell-mediated
cytotoxicity. IL-13 has similar but weaker biological
activity though it lasts longer than IL-4. IFN-g inhibits allergic responses and its effects are opposite to
IL-4 and IL-13. Therefore, it is crucial in the regulation of IgE production. Other cytokines, which inhibit allergic responses, are IL10, IL-12, TGF-b, and
IL-8. The direction of immune response on exposure
to allergen depends on the balance of Th1 and Th2
reactivity and ensuing cytokine milieu. In atopic individuals the balance is tilted towards the production
of Th2 type cytokines (IL-4 and IL-13) as opposed to
Th1 type, such as IFN-g. The Th2 differentiation and
production of IgE is also suppressed by regulatory T
cells (CD4 þ CD25 þ ). Thus, an inappropriately weak
T regulatory mechanism would facilitate Th2 dominance and production of IgE and allergic disease.
It is likely that allergen exposure in early childhood results in a lifelong T cell memory pool. In
atopic individuals, the immunological memory is
dominated by Th2 type cells leading to allergic reactivity, whereas in nonatopic subjects, Th1 cells
dominate the memory pool. In addition to genetic
predisposition, environmental factors (infections,
diet, etc.) may also influence the outcome of these
initial responses by altering the cellular and cytokine
milieu within the lymph nodes.
In atopic subjects with their Th2 skew, IgE is
formed specific to the antigenic protein following initial exposure (sensitization). The IgE circulates in the
blood in small quantities but is mostly present in
the tissues bound to high-affinity receptors (FceR1) on
FEV1
Figure 1 A schematic representation of immediate (IgE-mediated) hypersensitivity reaction.
4.5
4
3.5
3
2.5
2
1.5
1
0
1
2
3
4
5
6
7
8
9
10 11 12
Hours (postallergen exposure)
Figure 2 Early and late-phase asthmatic reaction following
allergen inhalation challenge. FEV1, forced expiratory volume.
the surface of mast cells and basophils and FceR2 on
eosinophils, macrophages, and platelets. This IgE can
be detected in the serum by immune assays or in the
skin by allergy skin tests. On further exposure, multivalent antigens bind and cross-link IgE bound to
FceR1 on cell surface leading to the signaling cascade
that causes rapid release of preformed mediators such
as histamine, tryptase, and heparin (Figure 1). These
mediators cause the immediate phase of the type I
hypersensitivity reaction, which occurs within a few
minutes (hence immediate hypersensitivity). There is
also induction of rapid synthesis of arachidonic acid
metabolites such as prostaglandins and leukotrienes
and expression of cytokines (IL-3, IL-4, IL-5, IL-6,
IL-10, IL-13, and tumor necrosis factor alpha
(TNF-a)) and chemokines. Eosinophils are attracted
to the site of activity, precipitating the late phase of
the type I reaction, which starts 4 6 h after exposure
(Figure 2).
Immediate hypersensitivity is central to all IgEmediated allergic reactions and occurs in asthma,
rhinitis, and anaphylaxis. During acute allergic reactions, the process is acute with the release of huge
quantities of histamine causing typical symptoms
and signs, such as acute bronchospasm or anaphylaxis. However, the process may be subacute or
chronic and localized to one site such as lung or nose.
74
ALLERGY / Allergic Reactions
Repeated exposure to allergens leads to the induction
of a more chronic inflammatory process with the influx of inflammatory cells including T lymphocytes
and eosinophils. Cytokines, produced by a variety of
inflammatory cells, including T cells, regulate the
inflammatory process. Proliferation of Th2 subsets
producing predominantly IL-4 and IL-5 results in
differentiation and isotype switching of the naı̈ve B
cells to IgE-producing plasma cells as well as activation and influx of inflammatory effector cells such as
eosinophils. Eosinophils are potentially tissue damaging, particularly after priming with IL-5. Various
cytokines upregulate adhesion molecules on endothelial and epithelial cells, thereby enhancing migration of eosinophils into the mucosa.
Allergic Reaction in the Lung
Allergic reaction in the lung results in airway inflammation. Exposure to allergen is recognized as
important in initiating and maintaining allergic airway inflammation in atopic asthmatics. In the appropriate setting of repeated allergen exposure and
Th2 type immune responses, a cytokine milieu is
created with upregulation of adhesion molecules
and continuous recruitment and activation of inflammatory cells from the bloodstream towards the
bronchial mucosa. The release of cytokines and inflammatory mediators by activated cells causes amplification and persistence of the inflammatory
process. However, structural cells such as epithelial
cells and smooth muscle cells are not merely passive
recipients of immune-related tissue damage but are
active participants of the complex inflammatory cascade, which may well have initiated at the epithelial/
mesenchymal level within the airways. Nonatopics
show similar inflammation in their airways and thus
IgE-mediated allergy in not a prerequisite for airway
inflammation in asthma or rhinitis.
Early Asthmatic Reaction
The effect of allergen exposure can be observed and
studied in a controlled fashion during bronchial allergen challenge. In an atopic asthmatic, inhalation
of allergen to which the patient is sensitized, results
in an immediate hypersensitivity reaction with the
release of mast cell mediators in the bronchial mucosa. These mediators enhance vascular dilatation,
increase permeability of the venule, and increase
mucus secretion, resulting in edema and congestion,
typical of an acute phase reaction. Histamine and
leukotrienes are potent bronchoconstrictors. Histamine stimulates local type c neurones leading to the
release of several neuropeptides, including substance
P, which further increase vascular permeability and
cause stimulation of parasympathetic reflexes augmenting mucous secretion and bronchoconstriction.
These changes manifest clinically in cough, wheeze,
and dyspnea.
Late Asthmatic Response
Clinically, the effect of early asthmatic reaction diminishes after 30 min (Figure 2). This is followed by a
relatively asymptomatic period during which a plethora of cytokines and mediators draw leukocytes to the
tissues. Events initiated during the early response result in vascular dilatation and increased permeability,
edema formation, and the accumulation of cells. IL-5,
secreted from mast cells, lymphocytes, and eosinophils is the most important cytokine for eosinophils.
Besides attracting them to the site of inflammation, it
also causes their proliferation, activation, and increased survival. Other eosinophilic cytokines are IL3, granulocyte-macrophage colony-stimulating factor
(GM-CSF), and chemokines. Upon activation, eosinophils release mediators such as eosinophilic cationic
proteins, major basic proteins, leukotrienes, and prostaglandins. These and other mediators enhance inflammation and cause epithelial damage. This results
in bronchoconstriction clinically 4–12 h later and prolonged bronchial hyperresponsiveness, mucus secretion, and edema formation. Further secretion of a host
of cytokines including IL-3, IL-4 and IL-5, contribute
to an ongoing inflammation.
Chronic Inflammation
With continued or repeated exposure to allergen, a
state of chronic inflammation develops with increased numbers of activated Th2 cells, expressing
mRNA for the secretion of IL-3, IL-4, IL-5, and GMCSF. These cytokines are important in the continuation of inflammation and the attraction of mast cells
and eosinophils. These cells cause further increase
in histamine, prostaglandins, and eosinophilic toxic
products, causing epithelial damage. There is upregulation of intercellular adhesion molecules in the
blood vessels promoting stickiness of the endothelium to leukocytes and facilitating their passage
across, into the tissues. Increased permeability and
cellular infiltration causes mucosal edema.
Even in patients with mild, intermittent asthma, a
state of low-grade inflammation persists, in the absence of symptoms. It is hypothesized that almost
continuous exposure to very small amounts of allergens, such as house dust-mite, or pollen during summer, contributes to this ongoing allergic reaction
without causing symptoms. Under the influence of
IL-4 from mast cells, more B cells are switched to the
ALLERGY / Allergic Reactions 75
production of IgE antibodies, thus maintaining allergic reaction. Bronchoscopy studies reveal increased
numbers of activated inflammatory cells and cytokines in the respiratory mucosa and secretions.
Clinical Effects
The clinical features of asthma are due to the airway
narrowing causing obstruction to airflow. This airway obstruction has three elements:
1. Excessive bronchial smooth muscle contraction. Inflammatory mediators such as histamine,
bradykinin, prostaglandins, and leukotrienes
act directly on their specific receptors to cause
bronchoconstriction. In asthma, the smooth muscles contract easily and excessively following exposure to inflammatory mediators perhaps due to
heightened sensitivity of their receptors. On the
other hand, b2-receptors may have a diminished
response. This feature is called bronchial hyperresponsiveness.
2. Thickening of bronchial wall. Bronchial wall
thickening is due to inflammatory and fibrotic
changes. Increased capillary permeability allows
plasma exudation into the mucosa causing edema
and cellular infiltration. Proliferation of fibroblast
and myofibroblast leads to thickening of the basement membrane with deposition of collagen and
hypertrophy of bronchial smooth muscles (airway
remodeling). This leads to irreversible airway obstruction in chronic asthma.
3. Excessive luminal secretions and cellular debris.
There is excess mucus secretion due to glandular
hyperplasia. The epithelium is fragile and damaged epithelial cells are found in the sputum. Impaired ciliary function encourages retention of
thick mucus in the lumen. During severe exacerbation, the lumen of the airway is blocked by
thick mucus, plasma proteins, and cell debris.
Allergic reactions in the nose follow a similar
process with inflammation of the nasal mucosa, resulting in rhinorrhea, sneezing, and nasal blockage.
Systemic Allergic Reactions and
Anaphylaxis
Allergic reactions vary widely in severity from mild
pruritis and urticaria to circulatory collapse and
death. An acute systemic allergic reaction with one
or more life-threatening features, such as stridor or
hypotension, is termed anaphylaxis. Allergic reactions with troublesome but not life-threatening reactions, such as generalized urticaria/angioedema and
bronchospasm of mild to moderate severity, may be
called severe allergic reactions. Traditionally, the
term anaphylaxis is used for IgE-mediated reactions.
Systemic reactions that clinically resemble anaphylaxis but are caused by non-IgE-mediated mediator
release from mast cells and basophils are referred to
as anaphylactoid reactions. Anaphylaxis occurs in
30/100 000 population/year with a mortality of
1–2%. Offending agents include foods, drugs, insect
stings, and exercise but in 20% of cases no cause can
be found (idiopathic).
Pathogenesis
Systemic allergic reaction occurs as a result of degranulation of mast cells and basophils. Mast cells in
respiratory and gastrointestinal tract, skin, and perivascular tissues are involved in both IgE and non-IgEmediated allergic reaction. IgE-mediated release is
caused by antigen-specific cross-linking of IgE molecules on the surface of tissue mast cells and peripheral
blood basophils. Non-IgE-mediated release may be
due to direct stimulation of mast cells. Mast cells
produce both histamine and tryptase while basophils secrete histamine but not tryptase. Histamine is
a primary mediator of anaphylaxis and signs and
symptoms of anaphylaxis can be reproduced by histamine infusion.
Clinical Features
Anaphylaxis is a rapidly developing generalized
reaction that involves respiratory, cardiovascular,
cutaneous, and gastrointestinal systems. Clinical manifestations vary depending on the cause of anaphylaxis,
route of entry, host factors (such as degree of sensitization, associated factors such as exercise, comorbidity, etc.) and the amount of allergen exposure. The
initial symptoms, such as nasal congestion or pruritis,
can quickly progress to collapse or death. Laryngeal
edema, cardiovascular collapse, and severe bronchospasm are life-threatening features. In one large series
of fatal anaphylactic reactions, 70% of the deaths
were from respiratory causes, and 24% were from
cardiovascular causes. In 10–20% of cases, skin may
not be involved. Anaphylaxis can be confused with
septic or other forms of shock, asthma, airway foreign
body, and panic attacks (Table 1).
Symptoms commonly occur within a few seconds
or minutes of exposure and death may occur within
minutes. Speed of onset of symptoms is indicative of
the severity. Occasionally, the onset of symptoms may
be delayed for 2 h or more. In general, the later the
symptoms begin after exposure to a causative agent,
the less severe the reaction. Food allergens may have
slower onset or slow progression and gastrointestinal
76
ALLERGY / Allergic Reactions
Table 1 Differential diagnosis of anaphylaxis
Acute cardiac event
Vasovagal syncope
Acute angioedema
Acute severe asthma
Pulmonary embolism
Foreign body inhalation
Carcinoid syndromes
Pheochromocytoma
Seizure
Systemic mastocytosis
Panic attack
Vocal cord dysfunction
symptoms are more common. Onset of anaphylaxis to
insect stings or allergen injections is usually rapid:
70% begin in o20 min and 90% in o40 min.
Table 2 Clinical features of anaphylaxis
Symptoms
Physical
examination
Cardiovascular
Faintness, syncope,
palpitations
Throat
Throat tightness,
hoarseness,
inspiratory stridor,
difficulty in
swallowing
Nasal congestion,
rhinorrhea,
sneezing
Pruritis, lacrimation
Generalized warmth,
tingling, pruritis,
rash
Chest tightness,
wheezing, cough,
shortness of
breath
Nausea, vomiting,
abdominal pain,
bloating, cramps,
diarrhea
Dizziness, a sense of
impending
doom,
lightheadedness
Hypotension,
tachycardia,
arrhythmias
Laryngeal edema
Upper
respiratory
tract
Ocular
Skin
Chest
Management
Gastrointestinal
A quick initial assessment should determine the nature and progression of the clinical event (Table 2).
Continuous monitoring is essential as progression
from a mild to a severe episode may occur rapidly.
Epinephrine injected intramuscularly into the thigh
provides the most efficient absorption (Figure 3).
If there is no response to several doses of intramuscular epinephrine, intravenous administration
may be needed, by using a formulation of 1:10 000
(0.1 mg ml 1) at 1 mg min 1, which can be increased
to 2–10 mg min 1. If the response is still inadequate,
transfer the patient to an intensive care unit for close
monitoring and endotracheal intubation, if required.
Specific treatment for coexisting medical conditions
(e.g., coronary artery disease) may be necessary.
There may be complete resolution of the reaction.
However, if there are concerns, continued monitoring for remaining or recurring symptoms is essential.
A short course of corticosteroids may reduce the risk
of recurring or protracted symptoms of a biphasic
reaction but this is not proven. Patients receiving badrenergic blocking agents may not respond adequately to epinephrine. They require continued fluid
replacement and may respond to glucagon. Patients
receiving angiotensin-converting enzyme inhibitors
may also be at increased risk of anaphylaxis and be
more refractory to treatment with epinephrine.
If there is any doubt regarding the diagnosis, blood
should be taken for plasma histamine or serum tryptase levels within the first 4 h after the onset of
symptoms. Elevated serum levels of b-tryptase indicate mast cell activation and degranulation in both
IgE-mediated (anaphylaxis) and non-IgE-mediated
(anaphylactoid) reactions. b-tryptase is useful in differentiating anaphylaxis from other events having
Neurologic
Conjunctival injection
Flushing, urticaria,
swelling of the lips,
tongue or uvula
Wheezing,
tachypnea,
cyanosis,
respiratory arrest
Loss of
consciousness,
seizures
similar clinical manifestations, particularly if hypotension is present. Blood for plasma histamine needs
to be processed immediately to avoid detecting
artificially high levels due to spontaneous basophil
histamine release. If this is not possible, urinary histamine (or metabolites) levels can be checked for up
to 24 h.
After initial treatment of acute anaphylaxis, the
patient should be followed-up closely for the possibility of recurrent episodes. For mild to moderate
episodes and good response to treatment, further
monitoring can be done at home. However, following a severe episode, in-patient monitoring may be
required for late-phase reactions.
Subsequent Assessment
All individuals who have had a known or suspected
anaphylactic episode require a careful allergy evaluation. The aims are to review the diagnosis of anaphylaxis and prevent or minimize the risk of future
anaphylactic episodes by identifying the cause, educate the patient and/or family members regarding
avoidance of the offending agent, education and
training to deal with future inadvertent exposures,
and consideration of desensitization, if appropriate.
The level of confidence in the diagnosis of the original episode should be reviewed with details of the
ALLERGY / Allergic Reactions 77
Evaluate: breathing status, pulse, blood pressure, and level of
consciousness
Recognize life-threatening features such as stridor, shock,
arrhythmia, seizure, and loss of consciousness
• Institute CPR if there is loss of circulation or respiration
• Maintain airway with an airway device or tracheotomy
• Oxygen, if there is circulatory or respiratory compromise
• Epinephrine (1:1000) 0.3−0.5 ml i.m. (children: 0.15 ml),
repeat every 10−15 min, as needed
• Chlorpheniramine 10 mg i.v. (then 6 hourly)
• Hydrocortisone 200 mg i.v.
Improve
Problems persist
Late-phase
symptoms
Monitor
Discharge
home
• Hypotension: intravenous fluids (colloids) and, if needed,
vasopressor agents (e.g., dopamine)
• Bronchospasm: nebulized 2-agonist, consider use of i.v.
aminophylline. Mechanical ventilation may be required
• Urticaria/angioedema: oral/i.v. antihistamines and steroids
Figure 3 Emergency management of anaphylactic and anaphylactoid reactions.
events before and during the episode. Results of any
laboratory tests (e.g., serum tryptase or urine histamine) may be helpful in supporting the diagnosis of
anaphylaxis and differentiating it from other entities.
However, a careful and comprehensive history is the
most useful part of the assessment. Information on
any previous similar episodes, known food or drug
allergy, and medication record should be sought. The
history might suggest a specific cause such as insect
sting or peanut consumption just before the episode.
However, the cause may not be obvious from history
and sometimes no cause can be found despite thorough searching (idiopathic anaphylaxis).
Diagnostic Tests
Skin prick tests (SPTs) or determination of specific
IgE in serum (in vitro test) is helpful in identifying a
specific cause of anaphylaxis in cases of food, insect,
and some cases of drug (penicillin, insulin) allergy.
An SPT is more sensitive than in vitro testing and is
the diagnostic procedure of choice. When possible,
standardized extracts should be used. If skin tests or
in vitro tests do not provide a cause, then challenge
to the suspected agent/agents should be considered.
Challenge procedures are helpful in IgE-mediated allergic reaction where standardized test material is not
available and in non-IgE anaphylactic reactions (such
as to aspirin).
Prevention
Once the offending agent has been identified (e.g.,
food, medication, or insect sting), patients should be
educated regarding the specific exposures and be
counseled on avoidance measures (Table 3). All those
78
ALLERGY / Allergic Reactions
Table 3 Specific measures to reduce allergic reactions to common provoking agents
Provoking agents
Specific preventive methods
Foods
Patients should be taught to read and interpret food labels
They should be encouraged to ask about ingredients in restaurant meals
They should be provided with a list of alternative foods
School personnel should be fully informed of the pupil’s allergy history
Patients should carry a medical identification bracelet or card
Use alternatives, when possible, but avoid crossreactive antibiotics
If penicillin is essential, desensitization could be performed
Remove any hives or nests in the garden
Wear long shoes and full trousers when walking in the fields
Be alert to the presence of insects when outdoors
Discontinue exercise at the earliest symptom
Avoid any exacerbating factors such as aspirin or NSAIDs
Avoid exercise for 4–6 h after eating if there is a history of anaphylaxis after food ingestion
Patients should carry a medical identification bracelet or card
All procedures should be conducted in a latex-free environment
Food with known crossreactivity to latex should be avoided
Consider an alternative procedure that does not require RCM
Use low osmolar RCM
Consider pretreatment with steroids, antihistamine, and ephedrine
Allergen immunotherapy should only be administered under the supervision of a trained allergist
Consider alternative therapy in those who are at higher risk (Table 4)
Care should be taken to avoid dosing errors
Observe patients for at least 20 min after the injection
Consider reducing the dose, if interval between injections had been longer than planned or on opening
of new vials
Adjust dose if large local reaction occurs
Reduce dose in highly sensitive patients or if there is concomitant high allergen exposure
Penicillin
Insect sting
Exercise
Latex
Radiocontrast material
(RCM)
Allergen extracts
with a risk of future anaphylaxis outside the medical settings should carry and be educated in the use
of self-injectable epinephrine and antihistamines.
Self-injectable epinephrine is available in two different strengths (for adults, 0.3 ml of 1:1000 solution
and for children, 0.3 ml of 1:2000 solution) in readyto-use syringes. Antihistamine (such as chlorpheniramine 4–8 mg orally) may be sufficient for a mild
episode but epinephrine should not be held back if
symptoms are severe from the outset or response to
antihistamine is inadequate. Humanized, monoclonal anti-IgE antibody has shown protection against
peanut-induced anaphylaxis.
Allergic Reactions
Foods
Food allergic reactions are common in children and
presents clinically with systemic involvement (as described above), although oral (itching, numbness and
tingling of lips and mouth) and gastrointestinal symptoms may be more prominent. Although any food can
cause a reaction, commonly implicated foods are
milk, egg, peanuts, tree nuts, fish, and shellfish.
Symptoms often occur within minutes of ingestion
and certainly within 2 h. Assessment of specific IgE to
suspected food, either by skin prick test or in vitro
test, in the presence of a suggestive history, is sufficient to make a definitive diagnosis. However, food
challenges (single or double blind) may be required.
Strict avoidance of the offending food is essential.
Patients should also carry, and be trained, in the use
of epinephrine in an emergency following inadvertent
exposure.
Penicillin
Allergic reaction to penicillin is the most common
cause of anaphylaxis. Severe reactions are usually
attributable to parenteral administration. Atopy and
family history of penicillin allergy does not increase
the risk of a reaction. A history of penicillin allergy is
not reliable as nearly 80% of these individuals tolerate penicillin without ill effects. However, these
subjects should have a skin test to major and minor
determinants before penicillin is administered. The
risk of a reaction following a negative test is less than
2%. Skin tests do not resensitize a patient to penicillin. A positive skin test in the presence of a history
of reaction to penicillin indicates a more than 50%
risk of an allergic reaction and penicillin should be
avoided, or if penicillin is mandatory, desensitization
should be considered. Cross-reactivity exists between
ALLERGY / Allergic Reactions 79
cephalosporins and penicillins due to the common
b-lactam ring structure.
Aspirin and Nonsteroidal Anti-Inflammatory Drugs
Aspirin and nonsteroidal anti-inflammatory drugs
(NSAIDs) can induce life-threatening systemic, nonIgE-mediated reaction causing rhinoconjunctivitis,
bronchospasm, urticaria, and laryngeal edema.
In vitro or skin tests are not available and oral challenges are required for confirmation. If the diagnosis
is confirmed, aspirin and NSAIDs should be strictly
avoided. Desensitization may be performed if these
drugs are considered essential.
Insect Sting
Stinging insects belong to the order Hymenoptera.
Common stinging insects include honeybees, wasps,
yellow jackets, hornets, and fire ants. The self-reported
prevalence of insect sting allergy is approximately
1%. Insect venoms contain several well-characterized
allergens that can trigger anaphylactic reactions. Localized reaction may occur at the site of the sting and a
large local reaction may involve, for example, the
whole limb. However, these do not predispose to systemic allergic reaction. Urticaria and angioedema are
key features of systemic allergic reactions caused by
insect sting.
Skin test is the preferred method of confirming allergy and identifying the responsible insect species.
However, careful interpretation is needed, as falsepositive reactions are common. Following a systemic
allergic reaction, only about half of the patients will
react to a future sting. Patients should carry self-injectable epinephrine for early treatment of a sting,
and allergen immunotherapy should be considered,
where risk of future sting is substantial. The immunotherapy is 490% effective but optimal duration is
not known.
Intraoperative Drugs
Common agents responsible for intraoperative anaphylaxis are neuromuscular blocking agents, latex,
antibiotics, anesthesia induction agents, radiocontrast material, and opioids. Neuromuscular blocking
agents and thiopental are responsible for most anaphylactic reactions during general anesthesia. Both
IgE-mediated (muscle relaxants, latex) and direct
stimulation of mast cell (opioids, radiocontrast material) occurs. Clinical manifestations of intraoperative reactions differ from anaphylactic reactions due
to other causes as cardiovascular collapse, airway
obstruction, and flushing is prominent. It may be
difficult to differentiate allergic reaction from the
pharmacologic effects of a variety of medications
administered during general anesthesia. Plasma tryptase is useful in differentiating allergic reaction (due
to mast cell release of mediators) from pharmacologic or other causes. Skin testing is used for diagnosis where IgE-mediated mechanism is suspected.
Otherwise, graded challenge may be required.
Latex
IgE-mediated allergic reactions to natural rubber latex became common during the 1990s due to a sudden increase in the use of rubber gloves. Risk factors
for latex allergy include atopy and previous repeated
exposure to latex (e.g., multiple surgical procedures,
healthcare workers). Skin tests are indicated for
investigation of latex allergy in those who have a
history of possible latex allergic reaction and for
screening those who are at high risk. However, in the
last few years, the use of latex-free gloves and other
products have been effective in reducing the occurrence of latex allergic reactions.
Blood Transfusions
Allergic reactions may complicate 1–3% of blood
transfusions. Most reactions are mild, are associated
with cutaneous manifestations such as pruritis, maculopapular, or urticarial rash and flushing, and require no specific treatment except discontinuation of
transfusion and perhaps antihistamine. However, severe or life-threatening reactions with hypotension
and bronchoconstriction may occur occasionally.
Most of these reactions are due to IgE or IgM antibodies to serum proteins (e.g., albumin, complement
components, IgG, and IgA). Other mechanisms include transfusion of allergen, IgE antibodies, bacterial components or inflammatory substances such as
cytokines, histamine, or bradykinin. Blood components containing large amounts of plasma, such as
fresh frozen plasma, may be associated with more
severe allergic reactions. Serum tryptase, measured
soon after the reaction, may differentiate allergic
from transfusion-related reaction.
Allergen Extract
Allergen extracts are injected during skin test and allergen-specific immunotherapy. The risk of allergic
reaction is extremely low with skin prick test but not
insignificant when these extracts are injected for
treatment. In a survey between 1985 and 1989 in
the US, there were 17 deaths from allergen immunotherapy but none from skin testing. Mild, localized
reactions are relatively common and respond to antihistamine. Factors that increase the risk of an allergic reaction should be kept in mind by those involved
80
ALLERGY / Allergic Rhinitis
Table 4 Risk factors for systemic reactions during allergen
immunotherapy
Severe asthma
Highly sensitive patient
Too rapid increase in the dose of allergenic extract
Starting a new vial of extract
A history of previous systemic reactions to allergen
immunotherapy
Asthmatic symptoms present immediately before receiving an
injection
Administration of pollen extracts during high environmental pollen
exposure
Concomitant treatment with b-adrenergic blocking agents
Fever or an upper respiratory tract infection at the time of
administration of allergenic extracts
Lieberman P (2002) Anaphylactic reactions during surgical and
medical procedures. Journal of Allergy and Clinical Immunology 110(supplement 2): S64–S69.
Moffitt JE (2003) Allergic reactions to insect stings and bites.
Southern Medical Journal 96(11): 1073–1079.
Moneret-Vautrin DA and Kanny G (2002) Anaphylaxis to muscle
relaxants: rational for skin tests. Allergic Immunology (Paris)
34(7): 233–240.
Noone MC and Osguthorpe JD (2003) Anaphylaxis. Otolaryngologic Clinics of North America 36(5): 1009–1020.
Sampson HA (2003) Anaphylaxis and emergency treatment. Pediatrics 111(6): 1601–1608.
Sicherer SH and Leung D (2004) Advances in allergic skin disease,
anaphylaxis, and hypersensitivity reactions to foods, drugs, and
insect stings. Journal of Allergy and Clinical Immunology
114(1): 118–124.
Tang AW (2003) A practical guide to anaphylaxis. American Family Physician 68(7): 1325–1332.
in administration of allergen-specific immunotherapy
(Table 4).
Exercise
Allergic Rhinitis
Exercise-induced anaphylaxis is a physical form of
allergy that may occur in isolation or in combination
with ingestion of food or drug, such as aspirin or
NSAIDs. The episode resembles typical anaphylaxis
and emergency treatment is the same as for anaphylaxis due to other causes. Patients should carry
self-injectable epinephrine. Exercise may also cause
urticaria or bronchospasm without anaphylaxis.
P van Cauwenberge, J-B Watelet, T Van Zele, and
H Van Hoecke, Ghent University Hospital, Ghent,
Belgium
See also: Allergy: Overview. Asthma: Overview. Chemokines, CXC: IL-8. Immunoglobulins. Interferons.
Interleukins: IL-4; IL-10; IL-12; IL-13. Transforming
Growth Factor Beta (TGF-b) Family of Molecules.
Tumor Necrosis Factor Alpha (TNF-a ).
Further Reading
Cockcroft DW (1998) Airway responses to inhaled allergens.
Canadian Respiratory Journal 5(supplement A): 14A–17A.
El Biaze M, Boniface S, Koscher V, et al. (2003) T cell activation,
from atopy to asthma: more a paradox than a paradigm. Allergy
58(9): 844–853.
Gould HJ, Sutton BJ, Beavil AJ, et al. (2003) The biology of IgE
and the basis of allergic disease. Annual Review of Immunology
21: 579–633.
Holgate ST, Davies DE, Puddicombe S, et al. (2003) Mechanisms
of airway epithelial damage: epithelial–mesenchymal interactions in the pathogenesis of asthma. European Respiratory
Journal 44(supplement): 24s–29s.
Joint Task Force on Practice Parameters, American Academy of
Allergy, Asthma and Immunology, American College of Allergy,
Asthma and Immunology, and the Joint Council of Allergy,
Asthma and Immunology (1998) The diagnosis and management of anaphylaxis. Journal of Allergy and Clinical Immunology 101(6 Pt 2): S465–S528.
Kemp SF and Lockey RF (2002) Anaphylaxis: a review of causes
and mechanisms. Journal of Allergy and Clinical Immunology
110(3): 341–348.
& 2006 Elsevier Ltd. All rights reserved.
Abstract
Over the last decades, the prevalence of allergic rhinitis has risen
to epidemic proportions. Nasal symptoms involve sneezing, nasal itch, rhinorrhea, and nasal congestion. These symptoms result from an immunologically mediated (usually IgE-mediated)
inflammation of the nasal mucosa, following allergen exposure
in sensitized patients. Although allergic rhinitis is often trivialized, it has become clear that the disease can cause serious morbidity beyond the nasal manifestations, that it has a significant
impact on quality of life and substantial socioeconomic consequences, and that it is associated with multiple comorbidities,
including asthma, conjunctivitis, sinusitis, and otitis media. Recently, the Allergic Rhinitis and its Impact on Asthma (ARIA)
Working Group proposed a new classification for allergic
rhinitis, based on the duration of symptoms, rather than on
the type of exposure. The severity of the disease is categorized
based on the impact of symptoms on quality of life parameters.
Early and correct diagnosis is the basis for the management of
allergic rhinitis and starts with a thorough clinical history and
physical examination. To confirm the allergic origin of rhinitis
symptoms, allergy tests are performed. The test of first choice is
the skin prick test, which has a good sensitivity and specificity.
Environmental control measures (allergen avoidance), pharmacological treatment, immunotherapy, and education are the
cornerstones of therapeutic management of allergic rhinitis.
Nowadays, many effective pharmacological agents are available
and new potential targets for pharmacotherapy, new routes of
administration, and alterations in treatment dosages and schedules are continuously being investigated. To facilitate and standardize the management of allergic rhinitis and to improve the
patient care, satisfaction, and compliance, several clinical practice guidelines have been developed. Among those, the ARIA
guidelines provide stepwise treatment recommendations, based
on the best available evidence from research.
ALLERGY / Allergic Rhinitis 81
Introduction
Allergic rhinitis (AR) is defined as a nasal disease
with the presence of immunologically mediated
hypersensitivity symptoms of the nose, for example,
itching, sneezing, increased secretion, and blockage.
The great majority of cases are immunoglobulin E
(IgE) antibody-mediated.
In the recent Allergic Rhinitis and its Impact on
Asthma (ARIA) report, AR is considered as a major
chronic respiratory disease because of its high
prevalence in all countries, its significant impact on
quality of life or work performance, its considerable
economic burden, and its association with multiple
comorbidities, asthma in particular.
Several important epidemiological surveys (e.g.,
European Community Respiratory Health Survey,
International Study of Asthma and Allergies in Childhood (ISAAC), and Swiss Study on Air Pollution and
Lung Diseases in Adults) have recently improved our
knowledge about the prevalence of rhinitis. The majority of monocentric studies have reported a prevalence of seasonal AR ranging from 1% to 40% and a
prevalence of perennial rhinitis ranging from 1% to
18%. Furthermore, the ISAAC study phase 1 confirmed this large variation in the prevalence of rhinitis
symptoms throughout the world.
Over the last 40 years the prevalence of AR, like
other allergic disorders, has risen to truly epidemic
proportions. Despite the increasing insight into the
pathophysiology of allergy and the availability of
more effective treatment options, this upward trend
in the incidence of AR continues, being most prominent in countries with a Western lifestyle, especially
among children and adolescents. As a consequence,
great attention is being paid to the identification of
the factors responsible for this disease. It is well established that allergic diseases tend to occur within
families and have a genetic basis. However, numerous generations are needed before changes in the
gene pool occur. Therefore, the recent increase in
prevalence of AR and allergy in general cannot be
explained by genetic factors and is largely attributed
to alterations in the environment.
Diverse environmental and lifestyle factors (prenatal maternal influences, allergen exposure, active and
passive smoking, viral and other respiratory infections, early life microbial exposure, indoor air quality/house dampness, outdoor air pollution, urban vs
farming living environment, socioeconomic status,
dietary factors, etc.) have been identified as possible
risk factors or as protective factors in the pathogenesis of allergy, although the evidence supporting their
involvement varies widely. In order to allow the introduction of individualized primary and secondary
prevention strategies and to meet the challenge of the
growing impact of allergy, further assessment of the
complex interactions within and between genetic and
environmental determinants is required.
Etiology
Allergens are antigens that induce and react through
an IgE-mediated inflammation. The number of identified allergens has expanded enormously. They originate from animals, insects, plants, or fungi. In AR,
airborne allergens are the most common provoking
agents and the increase in time spent indoors in recent years is thought to be responsible for the rise in
incidence of allergic diseases. The most common
indoor allergens are derived from mites, domestic
animals, insects, or plants.
Mites, such as Dermatophagoides pteronyssinus or
D. farinae, usually induce asthma or perennial AR.
They feed on human skin and multiply under hot and
humid conditions. Other types of mite, such as Tyrophagus putrecentiae and Acarus siro, are present in
flour.
The dander and secretions of many animals contain
powerful allergens capable of inducing severe reactions. They can remain airborne for prolonged periods of time. Specific allergens have been identified in
cat, dog, horse, cattle, rabbit, and other rodents.
Outdoor allergens include pollen and molds. Pollen can be categorized into two groups based on their
mode of transport: anemophilous pollen are carried
by the wind and can be transported over long distances, while entemophilous pollen are carried by
insects. The nature and number of pollen vary with
geography, temperature, and climate. Fungi, molds,
and yeasts can liberate large quantities of allergenic
spores into the atmosphere. Their development and
growth are faster in hot and humid conditions. The
main atmospheric molds are Cladosporium and
Alternaria. Finally, it has been noted that inhalation
of insect waste or some bacteria seems to be able to
induce an IgE-mediated reaction. AR can also be
triggered by food and occupational allergens.
Many allergens have enzymatic activity. Simultaneous exposure to both allergenic and proteolytic
activity probably allows greater access to cells of the
immune system and enhances sensitization and allergic inflammatory reactions.
Pathology
Classification of Allergic Rhinitis
Before the publication of the ARIA report, AR was
subdivided into seasonal AR, perennial AR, and by
extension occupational AR, based on the time of
82
ALLERGY / Allergic Rhinitis
exposure to the offending allergen(s). Seasonal AR is
related to a wide variety of outdoor allergens such as
pollens and molds. Perennial AR is most frequently
caused by indoor allergens such as house dust mite,
molds, cockroaches, and animal dander. Occupational AR occurs in response to airborne allegens in
the workplace. Common causes are laboratory animals, wood dust (particularly hard woods), chemicals, and solvents. The distinction between seasonal
and perennial AR, however, is not applicable in all
patients and in all countries because
symptoms of perennial rhinitis may not be present
all year round;
* pollen and molds are perennial allergens in some
parts of the world;
* many patients are sensitized to multiple allergens
and present with symptoms during a number of
periods in the year or even throughout the year; and
* symptoms of seasonal AR do not always occur
strictly within the defined allergen season, due to a
‘priming effect’ and the concept of ‘minimal persistent inflammation’.
*
Therefore, this classification was considered to be
inaccurate and the ARIA Working Group proposed a
major change in the classification of AR. The ARIA
classification for AR uses the terms ‘intermittent’ and
‘persistent’ to describe the duration of symptoms.
Based on the impact of symptoms on quality-of-life
parameters, the severity of the disease is classified as
‘mild’ or ‘moderate–severe’ (Table 1).
Histopathology of Allergic Rhinitis
Pollen-induced rhinitis is the most characteristic
IgE-mediated allergic disease and is triggered by the
interaction of mediators released by cells that are
implicated in both allergic inflammation and nonspecific hyperreactivity. AR is characterized by a
huge inflammatory reaction. The resulting inflammatory cell infiltrate is made up of different cells.
The cellular response begins with chemotaxis, selective recruitment, and transendothelial migration of
cells. These cells can be localized within the different
compartments of the nasal mucosa and represent an
activation state. Their survival is prolonged and they
release a large amount of inflammatory mediators.
They also participate in the regulation of IgE synthesis and communicate with the immune system.
Biopsies from allergic nasal tissue demonstrate a
thicker basement membrane and a greater number of
intraepithelial monocytes, subepithelial eosinophils,
and neutrophils (Figure 1).
Clinical Features
Symptoms of rhinitis include rhinorrhea, nasal obstruction, nasal itch, and sneezing; often, patients
with rhinitis are subdivided into ‘sneezers and runners’ and ‘blockers’, based on the main symptom(s).
Table 1 ARIA classification for allergic rhinitis
‘Intermittent’ rhinitis
Symptoms are present:
p4 days a week
or p4 consecutive
weeks
‘Persistent’ rhinitis
Symptoms are present:
44 days a week
and 44 consecutive
weeks
‘Mild rhinitis’
‘Moderate/severe’
rhinitis
X1 of following items
are present:
None of following items
are present:
sleep disturbance
impairment of daily
activities, leisure,
and/or sport
impairment of work
or school work
troublesome
symptoms
In untreated patients
Adapted from Bousquet J, Van Cauwenberge P, Khaltaev N, Aria
Workshop Group, World Health Organization (2001) Allergic
rhinitis and its impact on asthma. Journal of Allergy and Clinical
Immunology 108(5 supplement): S147–S334.
Figure 1 Hematoxylin-eosin staining (magnification 40) of
normal nasal mucosa demonstrating epithelial cells, seromucous
glands, and subepithelial lymphoid layer.
ALLERGY / Allergic Rhinitis 83
These rhinitis symptoms, however, do not necessarily
have an allergic origin. In the differential diagnosis,
AR must be differentiated from several types of nonallergic rhinitis and other nasal inflammatory conditions (Tables 2 and 3).
The clinical history remains the most essential step
for establishment of the diagnosis. Apart from the
classical rhinitis symptoms, the patient must be questioned about the presence of other symptoms commonly associated with rhinitis, such as loss of smell,
snoring, sleep disturbance, postnasal drip, cough, sedation, conjunctivitis, and lower respiratory symptoms. The history should also contain an evaluation
of the severity and duration of the problem, the impact on daily life, and the response to treatment, and
should document potential allergic and nonallergic
triggers; it must also include a family and occupational history.
Clinical examination starts with a general inspection of the nose, ears, and throat. A complete and
systematic nasal examination is required, especially
in patients with persistent rhinitis. However, anterior
rhinoscopy gives only limited information. Nasal endoscopy is therefore an essential complementary investigation, not to confirm AR, but to exclude other
conditions, such as polyps, foreign bodies, tumors,
and septal deformations.
During allergen exposure, the sinonasal mucosa of
patients with AR can demonstrate a bilateral, but
not always symmetrical, swelling. Often, mucosal
changes in color are seen, from a purplish to a more
common pale coloration. An increase in vascularity
is also commonly noticed. In the absence of allergen
exposure, the nasal mucosa may appear completely
normal, but in patients who have suffered from
rhinitis for several years, irreversible mucosal hyperplasia and/or viscous secretions may also occur
(Figures 2–6).
Table 2 Classification of rhinitis
Allergic rhinitis
Infectious rhinitis: viruses, bacteria, fungi
Occupational rhinitis: allergic and nonallergic
Drug-induced rhinitis, e.g., aspirin
Hormonal rhinitis: puberty, pregnancy, menstruation, endocrine
disorders
Emotional rhinitis
Atrophic rhinitis
Irritant-induced rhinitis
Food-induced rhinitis, e.g., red pepper
NARES: nonallergic rhinitis with eosinophilic syndrome
Rhinitis associated with gastroesophagel reflux
Idiopathic rhinitis
Adapted from International Rhinitis Management Working Group
(1994) International Consensus Report on the Diagnosis and
Management of Rhinitis. Allergy 49 (supplement 9): 5–34.
Diagnostic Evaluation
To confirm the allergic origin of rhinitis symptoms,
the ARIA Working Group states that allergy tests
should be performed. In vivo and in vitro tests for the
diagnosis of allergic diseases are directed towards the
detection of free or cell-bound IgE. Immediate hypersensitivity skin tests are a major diagnostic tool to
demonstrate IgE-mediated allergic reactions. If properly performed, skin tests are the best available
method for detecting the presence of allergen-specific
IgE. The first choice of test is the skin prick test,
which has a good sensitivity and specificity (Figures 7
and 8). It is very important that skin tests are performed carefully and interpreted correctly. Therefore,
Table 3 Differential diagnosis of rhinitis
Mechanical factors
Deviated septum
Adenoidal hypertrophy
Hypertrophic turbinates
Foreign bodies
Choanal atresia
Tumors
Benign
Malignant
Granulomas
Wegener’s granulomatosis
Sarcoid
Infectious (tuberculosis, leprosy)
Malignant – midline destructive granuloma
Ciliary defects
Cerebrospinal rhinorrhea
Adapted from International Rhinitis Management Working Group
(1994) International Consensus Report on the Diagnosis and
Management of Rhinitis. Allergy 49(supplement 9): 5–34.
Figure 2 Profuse watery anterior rhinorrhea in a child with
allergic rhinitis.
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ALLERGY / Allergic Rhinitis
Figure 3 The ‘nasal salute’ refers to habitual rubbing of the
nose due to constant irritation. It usually signifies an underlying
allergic phenomenon of producing a profuse rhinorrhea requiring
frequent wiping. It is most marked in children.
Figure 4 Habitual rubbing of the nose usually produces a horizontal ‘nasal crease’ below the bridge of the nose.
Before allergen challenge
After allergen challenge
Figure 5 Schematic presentation of sinonasal mucosa swelling
before and after allergen challenge.
it is recommended that trained healthcare professionals carry them out. The skin reaction can be affected
by the quality of the allergen extract, the patient’s
age, the use of some pharmacological agents (e.g.,
oral antihistamines and topical skin corticosteroids),
and can also demonstrate seasonal variations. In addition, the possibility of false-positive and false-negative results must be considered (Table 4).
Measurement of total serum IgE lacks specificity
and is of little predictive value in allergy screening in
rhinitis. On the contrary, serum-specific IgE is as
valuable as skin testing. Skin tests, however, are less
expensive, have a greater sensitivity, allow a wide
allergen selection, and give results in less than half an
hour. Serum-specific IgE is indicated in young children, in patients with dermographism or widespread
dermatitis, in patients who are noncompliant for skin
testing, or in those who did not continue with antihistamine treatment (short-acting antihistamines
for 36–48 h, long-acting antihistamines for 4–6
weeks), as this can result in false-negative skin test
results. Serum-specific IgE measurement is also a
safer option in patients who are very allergic and
where an anaphylactic reaction to skin testing is a
possible risk. It is important to remember that positive in vivo or in vitro tests for allergen-specific IgE
must always be interpreted in relation to the entire
clinical presentation. The presence of allergen-specific IgE antibodies is not sufficient for the diagnosis
of allergic disease, as patients can be sensitized in the
absence of (or prior to) the development of any
symptoms.
Nasal challenge tests are used in particular for research purposes and are important in the diagnosis of
occupational rhinitis. The International Committee
on Objective Assessment of Nasal Airways has set up
guidelines concerning the indications, techniques,
and evaluation of nasal challenge tests. In addition to
allergen provocations, nasal challenge tests with aspirin, non-specific agents (histamine, metacholine),
and occupational agents can be performed.
Imaging (sinus plain radiographs, computed tomography, and magnetic resonance imaging) is not indicated for the diagnosis of AR, but may be necessary
to exclude other conditions or complications.
Other diagnostic tests that are employed to assess
the nasal airways include nasal peak flow, rhinomanometry, and acoustic rhinometry, but these are
rarely used in the diagnosis of AR. Objective testing
of a patient’s ability to smell can be performed by
olfactory testing. Mucociliary function can be measured by nasomucociliary clearance, ciliary beat frequency, or electron microscopy, but these tests are of
little relevance in the diagnosis of AR.
ALLERGY / Allergic Rhinitis 85
Figure 6 Nasoendoscopic visualization (right nasal cavity: Hopkins 301) of nasal mucosa swelling in an allergic patient before (a) and
(b) after allergen challenge.
Table 4 Causes of false-positive and false-negative skin tests
Causes of false-positive skin tests:
* Dermographism
* Irritant reactions
* Non-specific enhancement of a nearby strong reaction
* Improper technique/material
Causes of false-negative skin tests:
* Poor initial potency or loss of potency of extracts
* Use of drugs modulating allergic reaction
* Diseases attenuating skin response
* Decreased activity of the skin (infants and elderly patients)
* Improper technique/material
Figure 7 Kit with standardized allergen extracts and positive
and negative control solutions used for allergy skin tests.
Adapted from Bousquet J, Van Cauwenberge P, Khaltaev N, Aria
Workshop Group, World Health Organization (2001) Allergic
rhinitis and its impact on asthma. Journal of Allergy and Clinical
Immunology 108(5 supplement): S147–S334.
Comorbidities
Figure 8 The technique used for skin prick testing involves
introducing a drop of diluted allergen followed by puncturing the
skin with a calibrated lancet (1 mm) at an angle of 451. The drops
should be placed 2 cm apart. All patients undergoing skin prick
testing should also have a positive (histamine) and negative diluent (saline) control test included. Skin tests should be read at the
peak of their reaction by measuring (in mm) the wheal and the
flare around 15 min after pricking. The relevance of skin prick
testing should always be interpreted in the context of the patient’s
history. Positive results can occur in people without symptoms
and, similarly, false-negative results may also occur.
It must be emphasized that AR should be evaluated
as a global systemic disease. Allergic inflammation
does not necessarily limit itself to the nasal airway,
but the treating physician must also be aware of the
possible comorbidities of AR, including asthma, conjunctivitis, sinusitis, and otitis media.
The link between asthma and rhinitis in particular
has gained much interest. Epidemiological studies
have shown that up to 80% of patients with asthma
demonstrate symptoms of rhinitis, while approximately 20–40% of patients with AR have clinical
asthma. There is growing evidence that rhinitis is a
risk factor for the development of asthma, independent of atopy. Additionally, the airway mucosa of nose
and bronchi have many similarities and the clinical
and pathophysiological changes in asthma and AR
are often very comparable. Although there are still
some differences that should be highlighted, the
strong relationship between rhinitis and asthma has
introduced the concept of ‘the united airway disease’.
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ALLERGY / Allergic Rhinitis
Based on these findings the ARIA guidelines recommend that patients with persistent AR should be
evaluated for asthma by history, chest examination,
and, if possible and where necessary, by the assessment of airflow obstruction before and after using a
bronchodilator, whereas patients with asthma should
be evaluated for rhinitis by history and physical
examination.
Another frequent comorbidity of AR is conjunctivitis. It is estimated that 42% of patients with AR
experience symptoms of allergic conjunctivitis and
that 33–56% of the cases of allergic conjunctivitis
occur in association with AR. This coexistence, referred to as ‘rhinoconjunctivitis’, seems to be a typical feature in patients with seasonal pollen allergy.
As eye symptoms substantially contribute to the burden of allergic rhinoconjunctivitis, adequate assessment and treatment of conjunctivitis should be part
of the overall management.
AR is also considered as a contributing factor in
acute and chronic rhinosinusitis. Up to 54% of
adults with chronic rhinosinusitis have symptoms of
AR. Similarly, a high concordance (between 25%
and 75%) of these disorders is found in children.
Conversely, there is a high prevalence of sinus disease
in patients with AR: abnormal sinus radiographs occur in over 50% of adults and children with perennial AR and acute rhinosinusitis occurs often during
the allergy season.
There is still some controversy regarding the etiological role of AR in otitis media with effusion. Most
epidemiological data suggest an association between
these two diseases. However, the available evidence
is compromised by a possible referral bias and by the
lack of prospective, controlled studies. It is still not
clear whether AR predisposes to the development of
otitis or whether nasal dysfunction worsens otitis.
It can be concluded that although the exact pathophysiological links between AR and its several comorbidities still need to be elucidated, AR is not
an isolated disorder but is part of a systemic disease
process. Hence, the ARIA Working Group recommends a coordinated diagnostic and therapeutic
approach instead of a fragmented, organ-based
management.
Pathogenesis
Symptoms of AR develop upon inhalation of allergens in individuals previously exposed to such
allergens and against which they have made IgE antibodies. The pathophysiological process of AR can
be subdivided in two phases: the initial sensitization
phase during which allergen presentation results in
primary allergen-specific IgE antibody formation
by B lymphocyte, and the clinical disease phase during which symptoms in response to subsequent antigen exposure become manifest. The clinical disease
phase, in turn, can be subdivided into two distinct
phases: an early phase largely mediated through mast
cells; and a late phase, which involves cellular infiltration and mediator release.
Sensitization Phase
The development of sensitivity to an allergen requires
IgE antibody production directed at the epitope. After
allergen exposure, antigen-presenting cells present the
allergens to CD4 þ cells. A subset of these CD4 þ
cells, the T-helper type 2 (Th2) lymphocytes, generate
Th2 cytokines, including IL-4 and IL-13, which stimulate IgE synthesis in combination with B-cell–T-cell
ligand–receptor interactions that are pivotal in the
B-cell isotype switching toward IgE synthesis. These
B-cell–T-cell interactions include a major histocompatibility complex class II and T cell receptor/CD3
interaction and a binding of the CD40L on T lymphocytes and CD40 expressed on B cells. As a result,
allergen-specific IgE antibodies are produced and
these sensitize mast cells and other IgE receptor-bearing cells. There is now increasing evidence that IgE is
produced locally in the nasal mucosa since nasal B
cells, in the presence of IL-4 and CD40L positive
mast cells, are able to produce IgE locally.
Clinical Disease Phase
The clinical disease phase consists of two phases: an
early phase and a late phase. The early phase is
largely mediated through mast cells. In sensitized patients, allergen re-exposure mediates cross-linkage, of
adjacent IgE molecules bound to mast cell surfaces. If
a mast cell is activated by IgE cross-linking, it releases
granule products containing histamine, tryptase,
chymase, and cytokines such as interleukin-4 (IL-4),
IL-5, IL-8, IL-13, and tumor necrosis factor alpha
(TNF-a) into the extracellular environment. Second
upon activation mast cells generate arachidonic acid
products including cysteinyl-leukotrienes (LTC4,
LTD4, LTE4) and prostaglandin D2 (PGD2) from
the phospholipid cell membrane. Nasal challenge
with allergens shows the local release within 10–
15 min of histamine, tryptase, PGD2, LTB4, and
LTC4. These mediators cause the characteristic watery rhinorrhea by stimulating gland and globet cell
secretion, vasodilation, and blood vessel leakage,
which is characteristic for the early phase. Histamine
is the most important mediator in AR and induces
symptoms of nasal itching, sneezing, discharge, and
transient nasal blockage whereas leukotrienes appear
to be relatively more important than histamine in
ALLERGY / Allergic Rhinitis 87
inducing nasal blockage. In addition, the mast cell is
thought to contribute to other features in rhinitis,
namely the eosinophilic mucosal inflammation. Eosinophils are present in nasal mucosal biopsies within
the submucosa and epithelium in active rhinitis. They
generate vasoactive mediators and have the capacity
to produce cytotoxic proteins, including major basic
protein, eosinophil peroxidase, eosinophil-derived neurotoxin, and eosinophil cationic protein. Although
the eosinophils feature heavily in the late-phase allergic response, their role in the early-phase allergic
response is not clear.
The late phase starts 4–8 h after allergen exposure.
Clinically, it can be similar to the early phase but, in
general, nasal congestion is more prominent. The late
response involves a process of cellular accumulation.
The expression of adhesion molecules and the presence of cytokines IL-3, IL-4, IL-5, IL-8, granulocytemacrophage colony-stimulating factor, and TNF-a
enhances cell activation, accumulation of neutrophils,
eosinophils, and T lymphocytes, and prolongs the
survival of eosinophils within the nasal mucosal tissue. There is evidence showing that, in persistent
rhinitis, there is an upregulation of the adhesion
mechanisms with increased expression of intercellular
adhesion molecule-1 and vascular cell adhesion molecule-1. During this late phase, activated basophils
are responsible for histamine release. All these events
amplify the allergic inflammatory response, leading
to a real cascade of reactions (Figures 9 and 10).
Although the inflammatory reaction in AR is triggered by allergen exposure, it has been demonstrated
that even in cases of subliminal exposure to the
allergen(s), in the absence of symptoms, a certain
degree of inflammatory infiltration at the mucosal
level persists. This is called the ‘minimal persistent
inflammation’.
The Priming Effect
The ‘priming effect’ refers to the phenomenon where
the amount of allergens necessary to evoke an immediate response decreases with repeated allergen
challenges or exposures. Nasal challenge induces an
immediate clinical response in allergic subjects and a
concomitant appearance of an inflammatory infiltrate. The mucosal inflammation may persist 48–72 h
after allergen exposure. If the subjects are rechallenged within this period the response is more pronounced: the so-called priming effect. This effect is
hypothesized to be a result of the influx and subsiding activity of inflammatory cells during the latephase allergic response. Clinically, this explains the
observation that decreasing allergen quantities are
required to elicit symptoms as the pollen season
progresses. In patients allergic to tree and grass pollen, the tree pollen season has a priming effect on the
subsequent grass pollen season and these patients can
often develop symptoms early in the grass pollen
season when pollen counts are still very low.
Histamine
Concentration
Leukotrienes
Tachykinins
Early phase
Late phase
Time
Blockage
Rhinorrhea
Sneezing
Itching
Figure 9 Nasal allergen provocation results in a significant increase in mediators (histamine, leukotrienes, and tachykinins) and
immediate nasal symptoms (blockage, rhinorrhea, sneezing, and itching) in allergic rhinitis patients. Between 2 and 24 h after allergen
provocation, late-phase nasal symptoms, especially blockage, are observed in allergic rhinitis patients.
88
ALLERGY / Allergic Rhinitis
IL-4
IL-13
Allergen
IgE
Mast cell
IL-5
IL-4
Th2
IL-6
Th0
Eosinophil
IL-4
APC
IL-12
Th1
IFN-
IL-2
IL-18
Figure 10 Allergic inflammation is characterized by a preponderance of T-helper type 2 (Th2) lymphocytes over T-helper type 1 (Th1)
cells. APC, antigen-presenting cell; IL, interleukin; IFN-g, interferon gamma; IgE, immunoglobulin e; Th0, precursor T cell.
Systemic Component in the Allergic Rhinitis
Response
In addition to the local events in the nose, there is a
systemic element to the inflammatory process of AR.
A variety of mechanisms have been proposed to explain the pathophysiological link between the upper
and lower airways, including the loss of nasal protective function, altered breathing pattern, postnasal
drip causing pulmonary aspiration of nasal contents,
the presence of a nasal–bronchial reflex, and the
progression of systemic inflammation. Recent data
suggests that bidirectional systemic inflammation involving the bone marrow is likely to be important.
Local allergen provocation (in the nose or bronchi)
leads to upregulation and release from the bone
marrow of hemopoietic eosinophil/basophil progenitor cells, which migrate to both nose and lungs
where they can undergo differentiation and activation in situ.
Animal Models
Besides clinical and in vitro studies, animal models of
allergic airway disease may provide useful information concerning upper airway inflammation and serve
as a model for relatively invasive experimental procedures. Although AR in animals is rare, chronic
rhinitis associated with Aspergillus infection has been
reported in rodents, poultry, dogs, and horses. In the
absence of a common naturally occurring model of
AR, sensitivity to allergens must be induced in
healthy animals. A number of research groups are
currently applying different experimental protocols to
induce experimental airway allergy. In guinea pigs,
ovalbumin (OVA) is the most common sensitizing
agent. To induce sensitization OVA is repeatedly injected into the peritoneum over the course of several
weeks; this is followed by exposure to aerosolized
OVA, which leads to allergen-specific IgE production.
In addition to systemic sensitization, intranasal applications of OVA may induce allergen-specific IgE
production but this exclusive local sensitization has
been described in only a small number of studies. The
upper airways of mice are less attractive for research
compared to the lower airways, and this is for several
reasons. First, mice are obligatory nose breathers, resulting in a significant baseline inflammation. Second,
eosinophilic inflammation present in the nose of mice
with experimental airway inflammation is rather limited compared to the inflammatory response in the
lower airways.
Several animal models of AR have shed light on
the cellular and cytokine profiles associated with airway inflammation. They demonstrate that the relative importance of mast cells, eosinophils, IgE, and
the cytokines IL-4 and IL-5 in the development of
allergic inflammation varies with the sensitization
protocol used and with the animal strain.
Management and Current Therapy
Environmental control measures (allergen avoidance), pharmacological treatment, immunotherapy,
and education are the cornerstones of therapeutic
management of AR. In select cases, nasal surgery
may be recommended as an adjunctive intervention.
ALLERGY / Allergic Rhinitis 89
Prevention
The treatment of patients with AR starts with the
identification of possible allergens and subsequent
prevention. If possible, allergen avoidance is the first
step in the management of AR. The most common
indoor allergens are dust mites and dog and cat dander. The use of simple measures to avoid the allergens
can relieve the symptoms but there is still a paucity of
data relating to the effectiveness of avoidance. Clinical improvement can be expected within weeks.
However, some allergens require a longer period before they are effectively removed.
Pharmacological Agents
Intranasal steroids Intranasal steroids are a potent
and highly effective treatment for patients with AR.
Their effect is based on localized repression of the
inflammatory response. They reduce the number of
eosinophils, the amount of eosinophilic cationic protein present, and the number of mast cell progenitors.
Their multiple sites of action may account for their
extreme potency. Clinically their efficacy exceeds
that of antihistamines, decongestants, and cromoglycin. Compared to antihistamines they are more effective in reducing nasal blockage. However, they
have a limited effect on associated eye symptoms and
a relatively slow onset of action. There is some evidence that intranasal steroids have a beneficial effect
on asthma symptoms. At the recommended doses,
intranasal steroids cause few side effects. Systemic
side effects have not been observed, and the risk of
systemic absorption and possible effect on growth in
children has been extensively studied but has shown
no effect at the recommended doses. Despite these
findings, nasal steroids should be used at the lowest
possible dose in children. To date, there is no convincing evidence that doses greater than the recommended maximum increase efficacy.
Antihistamines Antihistamines typically reduce itching, sneezing, and rhinorrhea but have no or little effect on nasal congestion associated with the late-phase
reaction. The currently used antihistamines are clearly
less effective than topical nasal steroids. They are
subdivided into first- and second-generation histamines. First- and second-generation antihistamines
differ in their side effects. First-generation antihistamines produce sedation and other central nervous
system symptoms in X20% of patients and may cause
drying of the mouth and urinary hesitancy. Secondgeneration antihistamines also have varying degrees
of anticholinergic, antimuscarinic, and antiadrenergic
effects, but to a lesser extent than first-generation
antihistamines.
Cromoglycate and nedocromil Sodium cromoglycate and nedocromil sodium are both drugs that have
been demonstrated to influence mast cell degranulation in in vitro studies. However, no inhibitory effect
on histamine release has been demonstrated in mast
cells recovered from nasal tissue. These drugs also inhibit the intermediate conductance pathways of mast
cells, eosinophils, epithelial cells, fibroblasts, and sensory neurons. Cromoglycate blocks symptoms of the
immediate and late phase and is effective even when
used shortly before allergen inhalation. Cromoglycate
is more effective than placebo; however, most studies
show it to be less effective than nasal steroids. Both
drugs do not induce any serious side effects.
Ipratropium Ipratropium bromide blocks the activity of atropine and therefore reduces rhinorrhea
when used intranasally. It does not block sneezing, pruritus, or nasal obstruction and is thus of
greatest use in nonallergic rhinitis with predominant
rhinorrhea.
Vasoconstrictors Local vasoconstrictors reduce nasal blockage and are therefore indicated to relieve the
symptoms of AR. However, they create a tachyphylaxis with rebound rhinitis as a symptom that develops after 3–7 days. Excessive consumption of nasal
decongestants can cause rhinitis medicamentosa.
Therefore, nasal decongestants are not recommended
in the treatment of AR and should preferably be used
for a short period when symptomatic vascular decongestion is indicated for the alleviation of nasal
obstruction. Oral pseudoephedrine as a sustained release preparation has been shown to decrease both
the nasal congestion and nasal airway resistance in
patients with hay fever but only at doses where there
is a high risk of systemic side effects.
Leukotriene modifiers While sneezing and nasal
itching correlate best with histamine levels in experimental AR, nasal congestion correlates with LTC4
levels. Leukotriene receptor antagonists provide
some benefits in patients with AR. However, as demonstrated by some recent studies, leukotriene modifiers may be less effective than intranasal steroids. In
children, leukotriene receptor antagonists have been
shown to reduce exercise-induced asthma especially
in combination with inhaled steroids.
Allergen-specific immunotherapy Allergen-specific
immunotherapy is the practise of administering
gradually increasing doses of therapeutic vaccines
of standardized allergen extracts until reaching
an arbitrary dose that is maintained for several
years. Traditionally, allergen-specific immunotherapy
90
ALLERGY / Allergic Rhinitis
is administered subcutaneously. There is now good
evidence that immunotherapy with seasonal and perennial allergens is clinically effective for the treatment
of AR, but it should only be considered in patients
with severe symptoms of AR, when allergen avoidance and pharmacotherapy have failed to reduce
symptoms or when pharmacotherapy has been associated with unacceptable side effects. In addition, specific immunotherapy for AR, when administered early
in the disease process, has been demonstrated to modify the long-term progress of the allergic inflammation
and disease, by preventing the development of new
sensitizations and by preventing the development of
asthma. The exact mechanisms at the basis of the
beneficial effects of allergen-specific immunotherapy
are complex and are still not completely understood.
Recent evidence suggests both a shift away from a
Th2-type response as well as the generation of regulatory T cells. As immunotherapy is not free of risk
and may provoke systemic reactions (severe asthma
attacks and anaphylaxis in particular), it can only be
carried out by or under the supervision of trained
specialists, with direct access to the necessary rescue
medication. Because of these risks, patients must be
closely observed for 20–30 min after injection.
More recently, other administration routes for
allergen-specific immunotherapy have been investigated, including nasal, sublingual-swallow, and oral
immunotherapy. Sublingual administration has shown
to be effective.
Novel treatments Increasing insights into the
pathophysiology of AR, the roles of diverse cells
and their cytokine products, and receptors and mediators involved in allergic inflammation has provided new (potential) targets for pharmacotherapy.
Modulation of allergic response through antimediators and antireceptors has gained much interest (e.g.,
anti-IgE, anti-IL-5, anti-CCR3).
Role of Surgery in the Management of
Allergic Rhinitis
diagnostic tests have been developed, many effective pharmacological agents are currently available,
and new potential targets for pharmacotherapy, new
routes of administration, and alterations in treatment dosages and schedules are continuously being
investigated.
To provide and disseminate this knowledge from
research into practice, to facilitate and standardize the
management of AR, and to improve the patient care
and, consequently, the patient satisfaction and compliance several clinical guidelines have been developed. Before 1998, European and American guidelines
for the management of AR were developed, based on
expert opinion. The European guidelines are in many
respects very similar to the American guidelines, but a
Table 5 Classification schemes of statements of evidence
Category of evidence
Ia: Evidence from meta-analysis of randomized controlled trials
Ib: Evidence from at least one randomized controlled trial
IIa: Evidence from at least one controlled study without
randomization
IIb: Evidence from at least one other type of quasi-experimental
study
III: Evidence from nonexperimental descriptive studies, such as
comparative studies, correlation studies, and case–control
studies
IV: Evidence from expert committee reports or opinions or clinical
experience of respected authorities or both
Strength of evidence of recommendations
A: Directly based on category I evidence
B: Directly based on category II evidence or extrapolated
recommendation from category I evidence
C: Directly based on category III evidence or extrapolated
recommendation from category I or II evidence
D: Directly based on category IV evidence or extrapolated
recommendation from category I, II, or III evidence
Adapted form Shekelle PG, Woolf SH, Eccles M, and Grimshaw J
(1999) Developing guidelines. British Medical Journal 318: 593–
596.
Table 6 Strength of evidence for the treatment of rhinitis
Surgery does not relieve allergic inflammation and
should only be used in case of turbinate hypertrophy
or cartilaginous or bony obstruction, contributing to
or aggravating rhinitis symptoms, especially nasal
obstruction. In these cases, a conchotomy and/or septo(rhino)plasty is recommended. In cases of secondary and independent sinus disease, functional
endoscopic sinus surgery can be performed.
Intervention
Clinical Guidelines for the Management of
Allergic Rhinitis
SIT, allergen-specific immunotherapy.
Adapted from Bousquet J, Van Cauwenberge P, Khaltaev N, Aria
Workshop Group, World Health Organization (2001) Allergic
rhinitis and its impact on asthma. Journal of Allergy and Clinical
Immunology 108(5 supplement): S147–S334.
Our insight into the pathophysiology of AR has increased in recent years. Highly sensitive and specific
Oral anti-H1
Intranasal anti-H1
Cromones
Antileukotrienes
Subcutaneous SIT
Sublingual/nasal SIT
Allergen avoidance
Seasonal AR
Perennial AR
Adults
Children
Adults
Children
A
A
A
A
A
A
D
A
A
A
A
A
A
D
A
A
A
A
A
A
A
A
D
A
A
ALLERGY / Allergic Rhinitis 91
prominent feature of the former is the stepwise approach recommended for the treatment of rhinitis,
which is similar to the Global Initiative for Asthma
guidelines for the treatment of asthma.
In 1999, the ARIA Working Group was founded,
under the initiative of the World Health Organization. Unlike the European and American guidelines,
the ARIA guidelines are formulated on evidencebased medicine, categorized by Shekelle. Based on
these categories of evidence, the strength of evidence
for a certain recommendation is graded from A to D
(Table 5). The evidence for the recommended pharmacotherapy and immunotherapy for AR treatment
is particularly strong (category A evidence strength).
Diagnosis of allergic rhinitis
Allergen avoidance
Intermittent symptoms
Mild
Moderate/
severe
Persistent symptoms
Moderate/severe
Mild
Intranasal corticosteroids
Not in preferred order:
• oral H1-blocker
• intranasal H1-blocker
• and/or decongestant
Review patient after 2− 4 weeks
Not in preferred order:
• oral H1-blocker
• intranasal H1-blocker
• and/or decongestant
• intranasal corticosteroid
• (cromones)
In persistent rhinitis:
review the patient
after 2−4 weeks
If failure: step-up
If improved: continue for 1
month
failure
improved
Step-down
and continue
treatment
for 1 month
Review diagnosis,
compliance, query
infections or other
infections, or other
causes
↑ Intranasal
CS dose
Itch/sneeze:
+H1 blocker
Rhinorrhea:
+ ipratropium
Blockage: + decongestant
or oral CS (short term)
Failure
Surgical referral
If conjunctivitis add :
Oral H1-blocker or intraocular H1-blocker or intraocular cromones (or saline)
Consider specific immunotherapy
Figure 11 Stepwise treatment algorithm for allergic rhinitis in adolescents and adults, as recommended by ARIA. CS, corticosteroids.
Adapted from Bousquet J, Van Cauwenberge P, Khaltaev N, Aria Workshop Group, World Health Organization (2001) Allergic rhinitis
and its impact on asthma. Journal of Allergy and Clinical Immunology 108 (5 supplement): S147–S334.
92
ALVEOLAR HEMORRHAGE
The evidence for allergen avoidance, on the other
hand, is limited (category D evidence strength)
(Table 6).
Similar to the European guidelines, a stepwise approach for the treatment of AR is recommended by
ARIA with the following first-line treatment approaches (Figure 11): oral or intranasal H1-antihistamines, with limited use of decongestants, for mild
intermittent rhinitis; oral or intranasal H1-antihistamines or intranasal corticosteroids, with limited use
of decongestants and cromones, for moderate to severe intermittent and mild persistent rhinitis; and intranasal corticosteroids, with step-down and step-up
options, in conjunction with H1-antihistamines, decongestants, ipratropium, and eventually oral corticosteroids, for moderate to severe persistent rhinitis.
Additionally, specific immunotherapy should be considered in persistent disease and when the symptoms
are moderate to severe and do not respond to conventional treatment. When conjunctivitis is present,
oral or intraocular H1-antihistamines should be used.
Whereas the European guidelines did not take into
account the costs and availability of the treatment
strategies in different countries, the ARIA guidelines
are developed for the whole world and recognize that
the outcome of disease management largely depends
on compliance with the suggested treatment, which
in turn is influenced by the availability and affordability of the specific interventions. Therefore, a flexible stepwise approach is recommended, based on the
four cornerstones of patient education, allergen
avoidance, pharmacotherapy, and immunotherapy,
but modifiable in low-income countries.
See also: Allergy: Overview; Allergic Reactions. Asthma:
Overview. Chemokines. Chymase and Tryptase.
Histamine. Immunoglobulins. Interleukins: IL-4;
IL-5; IL-10; IL-13. Leukocytes: Mast Cells and Basophils; Eosinophils; Neutrophils; T cells. Lipid Mediators:
Leukotrienes; Prostanoids.
Further Reading
Aalberse RC (2000) Molecular mechanisms in allergy and clinical
immunology. Structural biology of allergens. Journal of Allergy
and Clinical Immunology 106: 228–238.
Barnes PJ (2003) Pathophysiology of allergic inflammation. In:
Adkinson F Jr, Yunginger JW, Busse WW, et al. (eds.) Middleton’s Allergy Principles and Practice, pp. 483–499. Pennsylvania: Mosby.
Bousquet J, Van Cauwenberge P, Khaltaev N, Aria Workshop
Group, World Health Organization (2001) Allergic rhinitis and
its impact on asthma. Journal of Allergy and Clinical Immunology 108 (5 supplement): S147–S334.
Howarth PH (2003) Allergic and nonallergic rhinitis. In: Adkinson
F Jr, Yunginger JW, Busse WW, et al. (eds.) Middleton’s Allergy
Principles and Practice, pp. 1391–1410. Pennsylvania: Mosby.
International Rhinitis Management Working Group (1994) International Consensus Report on the Diagnosis and Management
of Rhinitis. Allergy 49(supplement 9): 5–34.
Johansson SG, Bieber T, Dahl R, et al. (2004) A revised nomenclature for allergy for global use: Report of the Nomenclature
Review Committee of the World Allergy Organization. Journal
of Allergy and Clinical Immunology 113: 823–826.
Malm L, Gerth van Wijk R, and Bachert C (1999) Guidelines for
nasal provocations with aspects on nasal patency, airflow and
airflow resistance. Rhinology 37: 133–135.
Salib RJ and Howarth PH (2003) Remodelling of the upper airways in allergic rhinitis: is it a feature of the disease? Clinical
and Experimental Allergy 33: 1629–1633.
Shekelle PG, Woolf SH, Eccles M, and Grimshaw J (1999) Developing guidelines. British Medical Journal 318: 593–596.
Van Cauwenberge P, Bachert C, Passalacqua G, et al. (2000) Consensus statement on the treatment of allergic rhinitis. Allergy 55:
116–134.
Wheatley LM and Platts-Mills TAE (1999) Allergens. In: Naclerio
RM, Durham SR, and Mygind N (eds.) Rhinitis, pp. 45–58.
New York: Marcel Dekker, Inc.
Worldwide variation in prevalence of symptoms of asthma, allergic
rhinoconjunctivitis, and atopic eczema (1998) ISAAC. The International Study of Asthma and Allergies in Childhood
(ISAAC) Steering Committee. Lancet 351: 1225–1232.
Relevant Website
http://www.ginasthma.com – Global Initiative for Asthma. Global
strategy for asthma management and prevention (2003).
ALVEOLAR HEMORRHAGE
O C Ioachimescu, Cleveland Clinic Foundation,
Cleveland, OH, USA
& 2006 Elsevier Ltd. All rights reserved.
Abstract
Bleeding into the alveoli characterizes the syndrome of diffuse
alveolar hemorrhage (DAH) and represents a potential
life-threatening condition. There are many causes of DAH,
including vasculitides, such as Wegener’s granulomatosis, microscopic polyangiitis, Good pasture’s syndrome, connective
tissue disorders, and other conditions. Pathologically, the syndrome is due to pulmonary vasculitis, to a ‘bland’ alveolar
hemorrhage, or it represents the nondominant pathology, as
seen in diffuse alveolar damage from acute respiratory distress
syndrome (ARDS). Most patients present with dyspnea, cough,
hemoptysis (the latter in only 66% of the cases), anemia, and
ALVEOLAR HEMORRHAGE 93
new pulmonary infiltrates. Urgent bronchoscopy and bronchoalveolar lavage is generally required to confirm the diagnosis,
with a superior yield when it is performed in the first 48 h. In
patients with evidence of DAH and renal involvement (pulmonary–renal syndrome), kidney biopsy may be considered to
identify the etiology and direct the therapy. This chapter will
describe the mean features of the DAH syndrome and review in
short the main causes of alveolar bleeding.
Introduction
Hemoptysis is most commonly due to disruption of
the bronchial circulation of various (endo)bronchial
conditions such as bronchitis, bronchiectasis, neoplastic disorders, or (rarely) disorders of the pulmonary circulation. Hemorrhage from a bronchial
source can be rapidly fatal, since a brisk bleeding
can lead to a large amount of blood to occupy anatomic and functional dead space of the respiratory
tract; in this setting the alveoli can also be flooded
quickly, mimicking the true alveolar hemorrhage.
Anatomical injury at the alveolar–capillary basement
membrane level of the pulmonary microcirculation
may cause hemoptysis from arteriolar, venular, or
capillary source. Bleeding into the alveoli characterizes the syndrome of diffuse alveolar hemorrhage
(DAH). Although alveolar hemorrhage may be focal,
generally there are multiple areas affected, hence the
term DAH. Diffuse alveolar hemorrhage should
always be considered a potentially life-threatening
condition; it requires early recognition, care in stabilizing the patient (airway protection, sometimes
selective intubation, mechanical ventilation, etc.) and
specific treatment once the etiology is established.
Synonyms with DAH that can be found in the literature are: (intra)pulmonary hemorrhage, pulmonary
alveolar hemorrhage, pulmonary capillary hemorrhage, alveolar bleeding, and microvascular pulmonary
hemorrhage. The DAH syndrome is relatively rare,
although no studies addressed its specific epidemiology. Currently, our understanding of the etiopathogenesis and the appropriate management relies
mainly on case reports or case series of specific
disorders leading to DAH. Unfortunately, the therapeutic approach is rather non-specific, with a few
notable exceptions (Wegener’s granulomatosis (WG),
Goodpasture’s syndrome, systemic lupus erythematosus (SLE), etc.)
Etiology
Various diseases can lead to DAH syndrome (Table 1).
To date, no prospective studies of DAH have estimated
the relative frequency of various etiologies. A wellknown retrospective review of 34 cases of DAH suggested that the most common cause was WG, which
Table 1 Causes of diffuse alveolar hemorrhage (DAH)
DAH associated with vasculitis
Wegener’s granulomatosis
Microscopic polyangiitis
Goodpasture’s syndrome
Isolated pauci-immune pulmonary capillaritis
Connective tissue disorders
Antiphospholipid antibody syndrome
Mixed cryoglobulinemia
Henoch–Schönlein purpura, IgA nephropathy
Pauci-immune or immune complex-associated
glomerulonephritis
Behc¸et’s syndrome
Acute lung graft rejection
Thrombotic thrombocytopenic purpura and idiopathic
thrombocytopenic purpura
‘Bland’ DAH
Mitral stenosis and mitral regurgitation
Anticoagulants, antiplatelet agents, or thrombolytics;
disseminated intravascular coagulation
Pulmonary venoocclusive disease
Infections: human immunodeficiency virus (HIV) and infective
endocarditis
Toxins: trimellitic anhydride, isocyanates, crack cocaine
Drugs: propylthiouracil, diphenylhydantoin, amiodarone,
mitomycin, penicillamine, sirolimus, methotrexate,
nitrofurantoin, gold, all-trans retinoic acid (ATRA), bleomycin
(especially with high-flow O2), montelukast, zafirlukast,
infliximab
Idiopathic pulmonary hemosiderosis
DAH as nondominant pathology
Diffuse alveolar damage (DAD)
Malignant conditions (e.g., pulmonary angiosarcoma)
Lymphangioleiomyomatosis/tuberous sclerosis
Pulmonary capillary hemangiomatosis
Lymphangiography
accounted for one-third of cases, followed by Goodpasture’s syndrome (13%), idiopathic pulmonary
hemosiderosis (IPH) (13%), collagen vascular disease
(13%), and microscopic polyangiitis (9%). A review
of 29 cases of DAH associated with capillaritis found
that the most common cause was isolated pulmonary
capillaritis. Other conditions associated with DAH are
briefly discussed below. Overall, three different histologic patterns may be seen.
DAH Associated with Vasculitis
This pathologic pattern is characterized by neutrophilic infiltration of the interalveolar and peribronchiolar septal vessels (pulmonary interstitium).
This sequentially leads to anatomic disruption of the
capillaries, and red blood cell extravasation into the
alveoli and interstitium. This leads to neutrophil
fragmentation and apoptosis, with subsequent release
of the intracellular proteolytic enzymes and reactive
oxygen species, which begets more inflammation,
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ALVEOLAR HEMORRHAGE
intra-alveolar neutrophilic nuclear dust, fibrin and
inflammatory exudate, and fibrinoid necrosis of the
interstitium.
DAH Associated with ‘Bland’ Pulmonary
Hemorrhage
This pattern is characterized by intra-alveolar red cell
extravasation without any evidence of inflammation
or destruction of the alveolar capillaries, venules, and
arterioles; the epithelial lesions are usually discrete.
DAH Associated with Other Conditions
(Nondominant Pathology)
Here, the DAH is secondary to diffuse alveolar damage (DAD), lymphangioleiomyomatosis (LAM),
drug-induced lung injury, metastatic neoplasia to
the lungs, mitral stenosis, etc. DAD is the main underlying lesion of the acute respiratory distress syndrome (ARDS), and is characterized by intra-alveolar
hyaline membranes, interstitial edema with minimal
inflammation, and at times by ‘secondary’ DAH.
Clinical Presentation
The syndrome of DAH may present with a constellation of symptoms, signs, and laboratory results that
may suggest the underlying etiology (e.g., WG,
Goodpasture’s syndrome, drug-related vasculitis,
etc.) or only establish the diagnosis of the syndrome
without a specific etiology.
Symptoms
The onset of DAH is most often acute or subacute
(less than 1 week). Dyspnea, cough, and fever are the
common initial symptoms. Some patients, however,
present with ARDS requiring mechanical ventilation.
Hemoptysis may be absent at the time of presentation in up to one-third of patients with DAH syndrome. When present, it ranges from fulminant and
life-threatening to mild and intermittent in nature.
Physical Examination
The lung examination is usually non-specific, unless
there are physical signs of an underlying systemic
vasculitis or collagen vascular disorder (rashes, purpura, eye lesions, hepatosplenomegaly, clubbing, etc.).
The presence of Kerley B lines points toward mitral
valve disease or pulmonary venoocclusive disease.
Computed tomographic imaging studies may show
areas of consolidation alternating with areas of
ground-glass attenuation and preserved, normal
areas. Gallium scan, rarely ordered today for this
purpose, used to be performed in the past to reveal
areas of active vasculitis or inflammatory activity
(rather non-specific and with unclear sensitivity, too).
Other nuclear studies, geared to reveal breakdown of
the microcirculatory integrity and extravasation of
red blood cell out of the vessels, have not been confirmed by the ultimate test of time.
Pulmonary Function Tests
The DAH leads to various degrees of oxygen transfer
impairment and hypoxemia, sometimes severe enough to require ventilatory support. A sensitive
marker for DAH is a sequential increase in diffusing
lung capacity for carbon monoxide (DLCO). In this
setting, due to an increased availability of intraalveolar hemoglobin, capable of binding with highaffinity carbon monoxide, the DLCO is generally
normal or elevated. Unfortunately, the severe condition of these patients and the associated hemoptysis
generally preclude any DLCO measurements. An
obstructive lung disease associated with recurrent
DAH may be due to lymphangioleiomatosis, histiocytosis X, or pulmonary capillaritis in the setting
of microscopic polyangiitis or WG. Recurrent episodes of DAH generally lead to interstitial fibrosis
and ventilatory restrictive defects, as seen in idiopathic pulmonary hemosiderosis.
Laboratory Abnormalities
The blood work generally reveals acute and/or
chronic anemia, ‘stress’ leukocytosis, elevated erythrocyte sedimentation rate, and C-reactive protein
(particularly in the cases of DAH caused by systemic
diseases or associated with a pattern of vasculitis).
Since several causes of DAH may present as pulmonary–renal syndromes (i.e., association of pulmonary
hemorrhage with different types of glomerulonephritis), blood urea nitrogen (BUN) and creatinine concentration elevations, and abnormal urine sediments
(red blood cells/casts, white blood cells/casts, proteinuria of glomerular origin) can be seen.
Imaging Studies
The radiographic findings are generally non-specific
and show new and/or old, patchy or diffuse alveolar
opacities. Recurrent episodes of DAH lead to reticular interstitial opacities due to pulmonary fibrosis, usually with minimal honeycombing (if any).
Diagnosis
DAH represents a medical emergency, since it represents a potentially fatal condition. Despite recent
advances and refinements of the diagnostic and therapeutic tools in DAH, it remains a highly morbid
ALVEOLAR HEMORRHAGE 95
condition, with substantial fatality. One must have a
low threshold to entertain the diagnosis, to confirm it
and to thoroughly look for the underlying etiology.
Methodically, two separate steps are to be followed.
Establish the Diagnosis of DAH Syndrome
The triad of elevated DLCO, hypoxemia, and new
pulmonary infiltrates in the setting of hemoptysis and
dyspnea suggests the diagnosis of DAH. Patients who
present with hemoptysis need to be screened for focal
sources of pulmonary hemorrhage (i.e., bronchitis,
bronchiectasis, infection, neoplastic processes, etc.);
upper airway and gastrointestinal sources must also
be excluded carefully. It is important to remember
that most disorders manifested with severe hemoptysis can cause alveolar infiltrates. In patients
without hemoptysis, the clinical evaluation needs
also to screen for evidence of congestive heart failure,
pneumonia, inhalational or drug-related lung injury,
and other causes of bleeding.
Most patients suspected of having DAH need a
bronchoscopic examination, which serves two purposes: documentation of alveolar hemorrhage by
visual inspection and bronchoalveolar lavage (BAL;
especially if there is no hemoptysis) and exclusion of
an associated infection. This procedure has a higher
yield if it is performed early (within 48 h) rather than
later. An increasing hematocrit or progressively
bloodier aspect of three sequential BAL aliquots
from an affected area is diagnostic of DAH. In subacute or recurrent episodes of DAH, counting the
hemosiderin-laden macrophages (siderophages) as
demonstrated by Prussian Blue staining on a pooled
BAL specimen centrifugate may be useful for diagnosis (generally more than 30% siderophages). BAL
specimens should be sent for routine bacterial, mycobacterial, fungal, viral, and Pneumocystis carinii
microscopic studies and cultures.
The use of transbronchial biopsy in patients with
suspected DAH is of unclear value due to the small
size of the specimens and possible sampling bias.
Unless another clinical condition is suspected, transbronchial biopsy is generally not useful clinically.
Identify the Specific Etiology
Identifying the specific etiology is generally equivalent to the process of differential diagnosis. Serologic
studies need to be performed early during the course
of the disease, although the results are generally not
available in a timely manner for immediate management of the disease. Complement fractions C3 and
C4 and anti-dsDNA, anti-glomerular basement
membrane (GBM), antineutrophil cytoplasmic antibodies (ANCA), and antiphospholipid antibodies
represent the basic panel to be checked in DAH patients (see Autoantibodies).
If the diagnosis of DAH is still not clear or
the underlying etiology is still not known after a
thorough clinical evaluation, imaging studies, serologies, and bronchoscopy, surgical biopsy should
be entertained. Besides, the results of a surgical biopsy may become available faster than the serologic
tests. The ‘ideal’ site to biopsy is dependent on the
pretest probability of the underlying disorder: for
WG a nasal biopsy may suffice, while a kidney biopsy (with immunofluorescent studies) may be less
invasive than others in diagnosing Goodpasture’s
syndrome, microscopic polyangiitis, or systemic
lupus erythematosus. In isolated pulmonary disease
or pauci-immune pulmonary capillaritis a lung biopsy is a mandatory test. Immunofluorescence reveals linear deposition of immunoglobulins and
immune complexes along the basement membrane
in Goodpasture’s syndrome and granular deposits
in SLE, whereas the systemic vasculitides appears
pauci-immune. In cases of pulmonary–renal syndrome, the kidney biopsy shows focal segmental
necrotizing glomerulonephritis. Additionally, skin
biopsy of a lesion can demonstrate leukocytoclastic
vasculitis or Henoch–Schönlein purpura (in the latter
case, immunoglobulin A (IgA) deposits are suggestive
of the diagnostic).
Current Therapy
Corticosteroids are currently the backbone of DAH
syndrome therapy, especially if associated with systemic or pulmonary vasculitis, Goodpasture’s syndrome, and connective tissue disorders. Most authors
recommend intravenous methylpredisolone (up to
500 mg every 6 h, although lower doses seem to have
similar efficacy) for approximately 4–5 days, followed by a gradual taper to maintain doses of oral
steroids. In the setting of pulmonary–renal syndrome, the therapy needs to be initiated as soon as
possible, since the potential of reversibility is lost
exponentially in the first week of disease activity.
Other immunosuppressive drugs can be used in
DAH (e.g., cyclophosphamide, azathioprine, mycophenolate mofetil, etanercept, etc.) depending on the
disease severity, failures to respond to corticosteroids
and the underlying disease (e.g., WG, Goodpasture’s
syndrome). Intravenously administered cyclophosphamide (2 mg kg 1 day 1, adjustable to renal function)
is generally the preferred adjunctive immunosuppressive drug in the initiation phase of the treatment, and
continued several weeks until blood marrow suppression, infections, or other limiting effects require
discontinuation and thereafter switch to consolidative
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ALVEOLAR HEMORRHAGE
and/or maintenance therapy with methotrexate or
other agents.
Plasmapheresis is indicated in the treatment of
DAH associated with Goodpasture’s syndrome or
other vasculitis processes when the titers of pathogenetic immunoglobulins and immune complexes
are very high (e.g., plasmapheresis in the setting of
ANCA-associated vasculitis with overwhelming
endothelial injury and/or hypercoagulable state).
However, its utility in DAH syndromes other than
Goodpasture’s syndrome has not been evaluated in
prospective studies. If intravenous immunoglobulin
(IVIG) therapy adds anything to the treatment of
DAH due to vasculitis or other connective tissue disease is yet unclear.
Besides the treatment of vasculitis and underlying
disorder, stabilization of the patients with moderate
and severe hemoptysis entails supplemental oxygen,
bronchodilators, reversal of any coagulopathy, red
blood cell transfusion, intubation with bronchial
tamponade/protective strategies for the less involved
lung, mechanical ventilation, etc. Several case reports
showed success in treating alveolar hemorrhage due
to allogeneic hemaopoietic stem cell transplantation,
ANCA-associated vasculitis, systemic lupus erythematosus, and antiphospholipid syndrome with recombinant-activated human factor VIIa, which may
become a new tool in the (otherwise poor) armamentarium available for this condition.
Prognosis
Recurrent episodes of DAH may lead to various degrees of interstitial fibrosis, especially in patients with
underlying WG, mitral stenosis, long-standing and
severe mitral regurgitation, and idiopathic pulmonary hemosiderosis. A post-DAH syndrome has also
been described, particularly in microscopic polyangiitis, and idiopathic pulmonary hemosiderosis
and consists of progressive obstructive ventilatory
defects and anatomic emphysema.
Mortality
The disease fatality varies with the underlying cause
of DAH; patients with systemic lupus erythematosus,
anti-GBM antibody disease and several forms of
ANCA-associated vasculitis can have a high mortality (up to 50%) due to the disease activity, infections,
and cardiovascular morbidity (e.g., Churg–Strauss
syndrome).
Experimental Models
Experimental models of different conditions (e.g., microscopic polyangiitis, anti-GBM antibody disease,
etc.) have allowed the indirect study of DAH
syndrome and its close interrelation with iron metabolism. Iron is of crucial importance in the inflammatory and anti-infectious defense of the respiratory
tract and lung parenchymal homeostasis. Local macrophages have a major contribution to iron recycling
from red blood cells, through the phagocytosis of the
intra-alveolar erythrocytes and eventual return of the
electrolyte to the bone marrow erythron, where iron
is incorporated in the structure of heme in maturing
red blood cells. The understanding of alveolar pathology in DAH has been enriched lately by a literature explosion in the field of ferroproteins and
genetics of the regulatory factors involved in the iron
metabolism. It is beyond the scope of this section to
review exhaustively the respiratory iron metabolism,
hence mention will only be made of the main facts.
The free iron’s presence in the extracellular milieu
together with local hypoxia induces reactive oxygen
species and other free radical production, with subsequent peroxidation of the cellular membranes, cell
apoptosis, and preinflammatory cytokine release. The
phagocytosis capacity of the alveolar macrophages
seems to be easily exhaustible, becoming hemosiderin-laden macrophages (siderophages), which in
turn leads to more free iron and heme available in
alveolar spaces and pulmonary interstitium. Recently,
J774 macrophages have been used among other
experimental models to investigate the presence of
different ferroproteins and their role. A recently discovered peptide, hepcidin, seems to have a major ferrostat role in local and general iron overload
syndromes; its role (if any) is yet unclear in the
DAH syndromes. Another interesting protein, ferroportin, seems to be involved in macrophage iron
recycling from engulfed erythrocytes. It is possible
that macrophage’s iron retentive mechanism is abnormal, similar to what is seen in human hereditary
hemochromatosis type 4, or ferroportin disease.
Specific Causes of DAH
Wegener’s Granulomatosis
WG is a systemic disease characterized by necrotizing granulomatous inflammation of the small vessels,
involving predominantly the respiratory tract and
kidneys. The organ involvements can be recalled
using the mnemotechnic JERKS: (1) Joint disease
%
(arthralgias and arthritis); (2) ENT (rhinosinusitis)
% (both upper and
and eye disease; (3) Respiratory
% about 70–95% of cases);
lower respiratory tracts,
(4) Kidney involvement (lesions of focal segmental
%
necrotizing
glomerulonephritis); (5) Skin and other
% of the lung
organs (systemic). The gross appearance
ALVEOLAR HEMORRHAGE 97
ANCA-associated small vessel vasculitis who were
treated with plasma exchange in conjunction with induction immunosuppressive regimens had a remission
of the DAH. Other therapies, such as trimethoprim–
sulfamethoxazole, tumor necrosis factor inhibitors,
and anti-CD20 monoclonal antibodies have been tried
with unproven efficacy or are under scrutiny. Mortality is considerable and most often caused by infections, respiratory failure, and renal failure.
Microscopic Polyangiitis
Figure 1 Diffuse alveolar hemorrhage associated with, necrotizing pulmonary vasculitis (shown) in a patient with Wegener’s granulomatosis.
in WG with DAH is dark red, sometimes associated
with multiple nodules and cavitary lesions (exceptionally solitary pulmonary nodules), and much more
frequently with areas of consolidations and focal or
geographic necrosis (Figure 1). The chest radiograms
show fluffy alveolar and/or interstitial infiltrates reflecting the diffuse microvasculature disorder.
The diagnosis of WG is established based on clinical presentation and serologic confirmation, that is,
positive ANCA antibodies (c- or p-ANCA). In organlimited WG there is ANCA positivity in 60% of
the cases, while in generalized disease in 90–95% of
cases. c-ANCA (directed against proteinase-3) is
present in 85–90% of generalized forms of the disease. If diagnosis is still in doubt, surgical biopsy of
affected organs (kidneys, lungs, nasal mucosa) may be
useful for diagnosis. In WG, the DAH due to pulmonary capillaritis may mark the disease onset or occur
later during the course, in a subclinical and/or recurrent fashion. The progressively bloodier BAL aliquots
with large numbers of red blood cells and hemosiderin-laden macrophages in the absence of an infectious etiology confirm the diagnosis of DAH. The
standard inductive therapy of DAH is high-dose corticosteroids (e.g., 1 g methylprednisolone daily for
3 days) and daily intravenous cyclophosphamide.
Azathioprine generally substitutes cyclophosphamide
in about 6 months or after remission in an inductivemaintenance approach and/or after a cumulative high
dose of cyclophosphamide is reached. IVIG or therapeutic plasma exchange may be used for persistent
disease. In one study, 20 out of 20 patients with
Microscopic polyangiitis (MP), a small-vessel variant
of polyarteritis nodosa, is sometimes difficult to differentiate from WG due to similar clinical presentations, serologic and pathologic findings. The typical
pathologic feature in microscopic pauci-immune
neutrophilic polyangiitis (found in virtually 100%
of cases) is a focal and segmental necrotizing
glomerulonephritis, renal lesion also seen in WG,
Goodpasture’s syndrome, and other connective tissue
disorders. The lack of granulomatous inflammation
differentiates pathologically between WG and MP. A
positive serum perinuclear ANCA (p-ANCA), directed against myeloperoxidase’s epitopes, strongly
supports the diagnosis, but it can be also seen in
20–30% of WG cases. Microscopic polyangiitis
causes relatively frequently a syndrome of severe
DAH. Treatment with corticosteroids and cyclophosphamide followed by azathioprine is similar to
WG therapy. Plasmapheresis and IVIG may be also
useful in difficult-to-treat cases. As in other hemorrhagic conditions, factor VIIa has also been used
with various success. The short-term and long-term
(5-year) mortality rates from DAH in MP are approximately 25% and 40%, respectively.
Goodpasture’s Syndrome
Goodpasture’s syndrome is a form of anti-basement
membrane antibody disease and is characterized by
a combination of DAH and glomerulonephritis.
Anti-GBM antibody, the pathognomonic immunoglobulin, is directed against alpha-3 (IV) collagen
from GBM and is found in the serum of more than
90% of patients and part of the linear immunofluorescent deposits in the glomerular membrane noted
in the disease. The syndrome typically involves DAH
in a smoker (usually a young male, although older
patients, women, and nonsmokers can also have it).
Isolated, renal-sparing DAH, a rare occurrence,
may have anti-GBM both in the serum and in the
glomerular membrane in a typical linear antibody
deposition.
The treatment of Goodpasture’s syndrome includes urgent plasmapheresis and corticosteroids,
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ALVEOLAR HEMORRHAGE
cyclophosphamide, and/or azathioprine. The isolated
DAH seems to respond well to corticosteroids
alone. Alveolar bleeding secondary to Goodpasture’s
syndrome has a 2-year survival rate close to 50%,
while patients presenting with renal insufficiency
have a worse outcome.
Churg–Strauss Syndrome
Churg–Strauss syndrome (allergic and granulomatous angiitis) is a systemic disorder characterized by asthma, peripheral blood eosinophilia, and
systemic vasculitis of small-caliber vessels with extravascular necrotizing granulomas. It involves
mainly the upper respiratory tract, the lungs, and
the peripheral nerves. Tissue eosinophilia may involve the lungs or the gastrointestinal tract.
Pulmonary hemorrhage is rare in this condition,
while radiographic abnormal findings are described
in 50–90% of the cases. p-ANCA positivity occurs in
approximately 35–40%. Histologically, it is distinguished by small- and medium-vessel involvement
and eosinophil-rich infiltrates (sometimes presenting
with an aspect of chronic eosinophilic pneumonia).
Of note, a Churg–Strauss-like syndrome can occur
due to chronic ingestion of carbamazepine, quinine,
or macrolides, while potential risk of aggravation
of the disorder can occur on cysteinil leukotriene
receptor blockers.
Isolated Pauci-Immune Pulmonary Capillaritis
An interesting case of a DAH syndrome is represented
by pauci-immune pulmonary alveolar capillaritis in
the absence of any other systemic involvement or abnormalities. Several cases have been shown to have
elevated serum titers of p-ANCA, but this finding
may reveal in fact a fruste form of MP. Isolated,
pauci-immune pulmonary capillaritis has been also
seen in association with the drug called all-trans retinoic acid (ATRA). Available literature on pauciimmune pulmonary alveolar capillaritis shows that
respiratory failure necessitating mechanical ventilation is frequent in this condition and the response
to corticosteroids and immunosuppressive agents is
favorable.
Connective Tissue Disorders
Diffuse alveolar bleeding has been described in collagen vascular diseases such as SLE, scleroderma,
rheumatoid arthritis, polymyositis/dermatomyositis,
Henoch–Schönlein syndrome, Behc¸et disease, and
mixed connective tissue disorder. In SLE, pulmonary
complications occur in more than 50% of cases,
while DAH can occur in up to 5% of the patients
(although it is the most frequent disorder in this
category presenting with alveolar bleeding); DAH
has generally a poor prognostic connotation (50%
fatality rate) and is due to a process of pulmonary
capillaritis. When DAH occurs in SLE, contrary
to WG, glomerulonephritis is generally absent.
Immunofluorescent studies show granular deposits
of IgG and complement (C3) in the pulmonary
interstitium, alveolar blood vessels, and the GBM. Of
note, the syndrome of DAH is clinically and pathologically distinct from acute lupus pneumonitis,
which presents similarly and may be the inaugural
presentation of SLE. Therapy consists of corticosteroids and cyclophosphamide and/or azathioprine.
Plasmapheresis has no proven benefit. In rheumatoid arthritis, the syndrome of DAH secondary to
pulmonary vasculitis generally occurs late, in ‘burnout’ disease.
Hematologic Conditions
DAD seems to be the dominant lesion in the lungs of
the patients who undergo chemotherapy for leukemia or stem cell transplantation; this lesion can be
accompanied by DAH, sometimes fulminant and fatal, even in the absence of hemoptoic sputa. The offending factors seem to be the chemotherapeutic
agents associated with actinic lesions, thrombocytopenia, and superimposed infections. The therapy includes platelet transfusions, reversal of coagulation
abnormalities, and high-dose corticosteroids.
DAH of the Immunocompromised Patient
Alveolar bleeding can occur in immunocompromised
hosts due to a myriad of factors (infectious, chemotherapeutic, and immunosuppressive drugs, radiation
therapy, thrombocytopenia, pulmonary edema, lung
malignancy, other lung comorbidities, etc.) that have
a common denominator: the endothelial injury. The
exact frequency of the syndrome in the immunocompromised patients is currently unknown, partly because the hemorrhage can be subclinical, and because
empirical therapy is instituted early, since the risk of
invasive evaluation outweighs the benefits. While
subclinical DAH in this setting may have minimal
impact on survival, the alveolar hemorrhage associated with ‘serious’ causes like Kaposi’s sarcoma,
invasive fungal infections (Aspergillus, Pseudoallescheria boydii, etc.), Mycobacterium, Legionella,
or other invasive bacterial species may have catastrophic consequences. In human immunodeficiency
virus (HIV) infection or acquired immunodeficiency
syndrome (AIDS) patients, the cytomegalovirus
(CMV) and Kaposi’s sarcoma represent the main
risk factors for developing DAH. In a study of
ALVEOLAR HEMORRHAGE 99
HIV-infected patients with radiographic infiltrates, in
up to 44% of them more than 20% of the BAL cells
were hemosiderin-laden macrophages.
Drug-Induced DAH
Many drugs have been associated with DAH, including anticoagulants (warfarin, heparin, etc.),
thrombolytic agents, and platelet antiaggregant
agents; as a rule, in order to produce DAH, a ‘second hit’ (infectious, inflammatory, inhalatory, etc.) is
required. Several drugs can produce lung injury and
DAD, with secondary DAH (e.g., sirolimus, methotrexate, nitrofurantoin, etc.). Other drugs causing
DAH trigger a process of pulmonary capillaritis
(e.g., propylthyouracil, phenytoin, mitomycin, and
ATRA). Penicillamine can cause pulmonary capillaritis associated with glomerulonephritis with
granular immunofluorescent deposits (pulmonary–
renal syndrome). Interestingly, these agents are also
responsible for p-ANCA generation. The standard
therapy is discontinuation of the presumed offending
drug and (rarely) plasma exchange.
Other Causes of DAH
Other conditions associated with alveolar bleeding
are: different coagulopathies, toxic exposures (isocyantes, trimellitic anhydride, crack cocaine, etc.)
primary antiphospholipid antibody syndrome, mixed
cryoglobulinemia, Behc¸et’s syndrome, Henoch–
Schönlein purpura, lung transplant acute rejection,
mitral stenosis and regurgitation, pulmonary venoocclusive disease, pulmonary capillary hemangiomatosis, LAM, and tuberous sclerosis Bourneville.
In patients with primary antiphospholipid syndrome
(APS), the thromboembolic complications occur in up
to 15% of cases, pulmonary hypertension in about
2% of the cases, interstitial lung disease in up to 1%
of patients, with DAH in less than 1% of cases.
Idiopathic Pulmonary Hemosiderosis
Idiopathic pulmonary hemosiderosis (IPH) is a diagnosis of exclusion (see Idiopathic Pulmonary Hemosiderosis); it is a rare disease characterized by
recurrent episodes of DAH and ‘bland’ alveolar hemorrhage (Figure 2). IPH occurs most frequently in
children (80% of cases), but adult cases with onset
up to the eighth decade of life have been reported
(20% of cases). The pathogenesis of IPH is largely
unknown. Some cases have been linked to fungi
(Stachybotrys atra), others to environmental insecticides; it seems that several cases have initially been
called IPH, when in fact coagulopathies such as
van Willebrand’s disease have been found upon
Figure 2 Diffuse alveolar hemorrhage without any evidence of
pulmonary vasculitis (‘bland’ alveolar hemorrhage) in a patient
with idiopathic pulmonary hemosiderosis.
further scrutiny. Corticosteroids, hydroxychloquine,
azathyoprine, and other immunosuppressive agents
have been used with favorable effects. Lung transplantation has been reported as rather unsuccessful
in a couple of patients with progressive disease and
significant pulmonary fibrosis, due to recurrent pulmonary bleeding. Survival with IPH varies widely,
although recent data has suggested better outcomes,
most likely due to more aggressive immunosuppressive therapies.
Summary of Therapeutic Options
DAH syndromes represent a serious condition with
possible catastrophic consequences, caused by a myriad of conditions, associated or not with pulmonary
capillaritis. Dyspnea, cough, hemoptysis and new alveolar, fluffy infiltrates in conjunction with bronchoscopic findings of bloody BAL, with numerous
erythrocytes and siderophages make the syndrome
diagnosis evident. Rarely, a surgical biopsy from the
lung or another organ involved by the underlying
condition may be necessary. The advent of ANCA
has revolutionarized the diagnosis of WG, microscopic polyangiitis, Churg–Strauss syndrome, and
other ANCA-associated conditions. The therapy in
DAH targets both the autoimmune destruction of the
alveolar capillary membrane and the underlying condition; corticosteroids and immunosuppressive
agents are still the ‘gold standard’ of therapy in the
majority of cases. Factor VIIa seems to be a promising new therapy for DAH, although further evaluation is needed.
100 ALVEOLAR HEMORRHAGE
See also: Acute Respiratory Distress Syndrome.
Autoantibodies. Bronchoalveolar Lavage. Granulomatosis: Wegener’s Disease. Idiopathic Pulmonary Hemosiderosis. Systemic Disease: Diffuse
Alveolar Hemorrhage and Goodpasture’s Syndrome.
Further Reading
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Medical Journal 90: 237–239.
Afessa B, Tefferi A, Litzow MR, and Peters SG (2002) Outcome of
diffuse alveolar hemorrhage in hematopoietic stem cell transplant recipients. American Journal of Respiratory and Critical
Care Medicine 166: 1364–1368.
Agusti C, Ramirez J, Picado C, et al. (1995) Diffuse alveolar hemorrhage in allogeneic bone marrow transplantation: a postmortem study. American Journal of Respiratory and Critical Care
Medicine 151: 1006–1010.
Bar J, Ehrenfeld M, Rozenman J, et al. (2001) Pulmonary–renal
syndrome in systemic sclerosis. Seminars in Arthritis and Rheumatism 30: 403–410.
Collard HR and Schwarz MI (2004) Diffuse alveolar hemorrhage.
Clinics in Chest Medicine 25: 583–592. vii.
Dhillon SS, Singh D, Doe N, et al. (1999) Diffuse alveolar hemorrhage and pulmonary capillaritis due to propylthiouracil.
Chest 116: 1485–1488.
Dweik RA, Arroliga AC, and Cash JM (1997) Alveolar hemorrhage in patients with rheumatic disease. Rheumatic Diseases
Clinics of North America 23: 395–410.
Dweik RA and Stoller JK (1999) Role of bronchoscopy in massive
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Franks TJ and Koss MN (2000) Pulmonary capillaritis. Current
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Alveolar Proteinosis see Interstitial Lung Disease: Alveolar Proteinosis.
ALVEOLAR SURFACE MECHANICS 101
ALVEOLAR SURFACE MECHANICS
S B Hall and S Rugonyi, Oregon Health & Science
University, Portland, OR, USA
& 2006 Elsevier Ltd. All rights reserved.
Abstract
The major component of the recoil forces that tend to deflate the
lungs is the surface tension of a thin liquid layer that lines the
alveoli. This surface tension is well below the value for a clean
air/water interface, indicating the presence of a surfactant. The
surface tensions in situ specify that the surfactant films must
have certain characteristics. Following large expansions of the
air/water interface during deep inhalations, the surfactant must
adsorb rapidly to form the interfacial film. When compressed by
the shrinking surface area during exhalation, the films must be
sufficiently rigid to resist the tendency to collapse from the interface. Materials washed from the lungs show that pulmonary
surfactant is a mixture containing mostly lipids with some proteins that is synthesized and secreted by the type II pneumocyte.
The disorder in which an abnormality of pulmonary surfactant
most clearly plays a role is the respiratory distress syndrome of
premature babies, although altered surfactant function may also
contribute to the acute respiratory distress syndrome that occurs
in patients of all ages.
Description
The importance of alveolar surface mechanics is
readily evident from the pressure–volume (P–V)
characteristics of the lungs. Pressures required to
maintain any given volume are substantially higher
for lungs inflated with air than with saline. The fundamental difference between the two procedures is
that saline eliminates an air/water interface, the surface tension of which contributes to the inward recoil
forces for lungs inflated with air. The presence of
surface tension implies that the curved surfaces of the
pulmonary airspaces are lined by a layer of liquid.
Electron microscopy has demonstrated that such a
layer coats the alveoli, and that it is thin, with an
average thickness of 0.2 mm, and continuous. The
surface tension resulting from the air/water interface
of this alveolar lining represents the major component of contractile forces in the lungs.
Surface tension results from an imbalance of forces
on molecules close to an interface between two separated phases. For molecules deep within a substance,
attractive forces towards neighboring constituents
are equal in all directions (Figure 1). Within a few
molecular diameters of the interface, however, components experience a reduced attraction towards
constituents in the adjoining phase, resulting in a
net inward pull and a force per unit length, or surface
tension, that tends to contract the interfacial area.
The force along a curved surface, such as in the alveoli, translates directly into a difference in pressures
across the interface. For a spherical interface with
radius R, mechanical equilibrium between surface
tension, s, and the difference in pressures, p p0,
occurs (Figure 2) when
pR2 ðp p0 Þ ¼ 2pRs
which leads directly to the law of Young and Laplace
for a sphere:
Dp ¼ 2s=R
Inflation of lungs with air therefore requires higher
pressure to overcome interfacial forces that are absent
during inflation with saline.
Air
Water
Figure 1 Origin of surface tension: molecules in the bulk water experience an attractive force towards neighboring molecules that is
equal in all directions, resulting in no net force. Within a few molecular diameters of the interface, however, the absence of neighbors
towards the surface results in a net inward pull that tends to shrink the interfacial area. A film of surfactant at the interface tends to
spread, resulting in an opposing force that lowers surface tension.
102 ALVEOLAR SURFACE MECHANICS
p0
p
R
Figure 2 Relationship of surface tension to hydrostatic pressure across a spherical surface. Mechanical equilibrium occurs
when the inward recoil force of surface tension (s) and the opposing force of hydrostatic pressure (p p0) are equal. At the
midsection of a spherical bubble with radius R, surface tension
will produce a force (length s) ¼ 2pRs, and the force from
hydrostatic pressure will be [area (p p0)] ¼ pR2(p p0).
The equality leads directly to the law of Young and Laplace,
Dp ¼ 2s/R.
Several methods have been used to determine surface tension in the lungs. The difference in P–V
curves between air- and saline-filled lungs can provide surface tensions, either with simple assumptions
concerning alveolar geometries and tissue forces, or
with an energetic analysis that makes those assumptions unnecessary. Rinsing the lungs with a series of
detergents or liquids provides an air/liquid interface
with constant known surface tensions, and the intersection of P–V curves from these and normal lungs
indicates points of common surface tension. Fluorocarbon droplets deposited on the interfacial film in
peripheral alveoli spread according to the relative
surface tensions of the fluorocarbon and film, and the
shape of droplets containing different materials has
provided the most direct estimates of surface tensions
in situ. These different approaches have produced
remarkably consistent results. During excursions
through large volumes, surface tension shows a major hysteresis between inflation and deflation that
explains the hysteresis of the P–V mechanics. Deflation from total lung capacity to functional residual
capacity lowers surface tension from B30 mN m 1
to o5 mN m 1. Over tidal volumes, surface tension
varies between 10 mN m 1 and values as low as
1 mN m 1.
These surface tensions are well below the values
for a clean air/water interface, which would be
70 mN m 1, and indicate the presence of a surfactant. Surfactants are the general class of compounds
that have higher concentrations at the surface than in
the aqueous medium. They are amphipathic, with
distinct hydrophilic and hydrophobic regions of the
molecule, and best satisfy the energetics of both portions by orientation across an interface. Once trapped
within the two-dimensional surface, surfactants tend
to spread, resulting in a force that expands the interface and opposes surface tension. Surfactant films with
higher densities produce larger reductions in surface
tension. The very low surface tensions in the lungs
indicate films with particularly high densities.
Surfactants can lower surface tension by adsorbing
to an interface only to a limited extent. Adsorbed
surfactants reach a maximum density, above which
further constituents form a three-dimensional bulk
phase at the interface rather than adding to the twodimensional film. Surface tensions in the lungs reach
values well below this minimum equilibrium value,
and they also vary with volume. These observations
indicate that the low surface tensions and high densities of the films result not from insertion of more
constituents, but from a decrease in surface area
during deflation.
The low surface tensions reached during deflation
are impressively stable in static lungs for prolonged
periods. The low magnitude and particularly the stability of the surface tensions indicate that the compressed films have specific characteristics. When
compressed above the maximum equilibrium density, films that can flow from the surface will collapse
to form the three-dimensional bulk phase, thereby reestablishing equilibrium surface tensions. Only films
that are solid, defined by their inability to flow, can
resist the tendency to collapse and demonstrate the
behavior observed in the lungs.
When compressed sufficiently, even solid films collapse. An interface without surface tension has no
basis for existence, and at sufficiently high densities,
solid films must also rupture. The hysteresis of surface tension, and of hydrostatic pressures, between
deflation and inflation over large volumes reflects at
least partially material lost from the interface during
collapse.
Lavaging the lungs produces the increase in recoil
pressures expected from removal of a surfactant and
a resulting increase in surface tension. Material recovered from the lungs can form films with characteristics indicated by the in situ measurements.
Material obtained from alveolar foam, when suspended in saline, can form small bubbles that persist
for prolonged periods, indicating that the pressuredifference across their surface, which would tend to
dissolve the gas in the surrounding liquid, must be
low, and that according to the law of Young and
Laplace, surface tension must also be small. When
compressed in vitro by changing their surface area,
films formed from lavaged material can reach and
sustain surface tension below the minimum equilibrium value. Material purified from lavaged material
can also restore the P–V mechanics of the original
lungs. These observations provide the basis for the
identification of the surfactant in the lungs.
Material recovered by lavage contains two sets of
phospholipid vesicles that differ in size. The larger
ALVEOLAR SURFACE MECHANICS 103
Air
Liquid layer
Type I cell
Molecular
view
Lamellar
body
Tubular
myelin
Small
vesicles
Surfactant
film
Type II cell
Figure 3 Schematic of pulmonary surfactant in the alveolus. Constituents of pulmonary surfactant are synthesized in type II
pneumocytes and assembled into lamellar bodies, which are secreted into a thin liquid layer that lines the alveolus. The vesicles unravel
and first form tubular myelin, which appears to represent the immediate precursor of a film at the air/water interface. Lavage of the lungs
recovers both the large multilamellar vesicles, which contain mostly phospholipids with small amounts of cholesterol and proteins, and
small unilamellar vesicles, which contain only the lipids.
form has the same morphological appearance as a
subcellular organelle, the lamellar body, of the type II
pneumocytes (Figure 3). Organic extracts of these
larger particles can restore the P–V mechanics of
lavaged lungs, which perhaps best defines these vesicles as ‘pulmonary surfactant’. The smaller particles
may represent material excluded from the interface
during compression to very low surface tensions.
Vesicles of both sizes contain the same set of
phospholipids with small amounts of cholesterol.
The larger particles are much more capable of lowering surface tension in vitro than the smaller vesicles. Four proteins copurify with the larger particles
but are absent from the smaller forms. The organic
extracts of the large particles, which function well in
lavaged lungs, lack surfactant proteins SP-A and
SP-D, and based on a variety of assays, their primary
function appears related to processes other than the
lowering of surface tension. SP-B and SP-C, which
are sufficiently hydrophobic to extract with the lipids
into organic solvents, determine the difference in
surface activity between the large and small particles.
Normal Physiological Processes
To achieve the surface tensions observed in the lungs,
pulmonary surfactant must satisfy conflicting constraints. Vesicles must first adsorb rapidly to form the
interfacial film. Pulmonary mechanics become normal during the first few breaths following the initial
air-inflation of fluid-filled lungs, suggesting that adsorption to the newly created air/water interface
forms a film having the equilibrium density within
seconds. The low surface tensions reached during
deflation indicate that the compressed film avoids
the reverse process of desorption to re-establish the
equilibrium density. Replicating in vitro the full
performance of films in the lungs has been difficult,
and the mechanisms by which pulmonary surfactant
functions in the alveoli remain the subject of active
investigation.
Adsorption
The adsorption of pulmonary surfactant is fundamentally different from the process for other more
common surfactants, which insert into the interface
as individual molecules. Surfactants share the general
characteristic that they exist in solution as individual
monomers only up to a certain concentration, above
which they aggregate into structures such as micelles
and bilayers (Figure 4), the nature of which depends
on the effective shape of the particular molecule. The
‘critical micelle concentration’ at which phospholipids aggregate is less than 10 9 M. Constituents of
pulmonary surfactant therefore exist in the alveolar
lining exclusively as vesicles, which insert as collective units into the interface. Because the vesicles
themselves are stable in aqueous medium, an energy
barrier limits adsorption, which occurs quite slowly
for lipid vesicles without the hydrophobic proteins.
In the alveolar lining, lamellar bodies secreted by
the type II pneumocytes unravel to form a distinct
intermediate structure known as tubular myelin that
apparently represents the direct precursor of the interfacial film (Figure 3). SP-A is required in vitro for
the reconstruction of tubular myelin, which is absent
from extracted surfactants and transgenic animals
that lack SP-A. Extracted surfactants, however, function well in surfactant-deficient lungs, and mice
without SP-A have normal pulmonary mechanics.
The functional significance of tubular myelin for adsorption is therefore unclear. The absence of SP-B,
whether in transgenic animals or in patients with
104 ALVEOLAR SURFACE MECHANICS
Figure 4 Aggregation of surfactants. Above a certain concentration, the hydrophobic portion of surfactants makes them insoluble as
individual molecules, and causes their aggregation into structures that expose their hydrophilic components to the aqueous medium
while sequestering the hydrophobic segments. The particular form of the aggregate depends on the effective shape of the specific
compounds. Single chain surfactants tend to form micelles. Biological phospholipids form bilayers that may stack into the concentric
layers of a multilamellar vesicle.
genetic abnormalities, does produce abnormal pulmonary function, consistent with the crucial role
suggested by in vitro studies of this protein for
adsorption.
Stability of Compressed Film
Under equilibrium conditions, attempts to increase the
density of surfactant monolayers above a maximum
density, either by adding more constituents or by
decreasing interfacial area, instead forms a threedimensional bulk phase that coexists with the twodimensional film. The bulk phase of phospholipids is a
liquid-crystal, in which layers of material stack in the
regularly repeating manner characteristic of a crystal,
but with each layer having the disordered structure
that is characteristic of liquids. Slowly compressed
monolayers of pulmonary surfactant in vitro flow into
these stacked structures (Figure 5), and their ability to
flow indicates that the films are fluid. In the lungs, the
prolonged low surface tensions, well below the minimum equilibrium values, indicate films that resist
flow, and that therefore have the defining characteristic of a solid. The structure of two-dimensional solid
films could be either highly ordered, analogous to a
three-dimensional crystal, or amorphous, like a glass.
At physiological temperatures, a single constituent
of pulmonary surfactant, dipalmitoyl phosphatidylcholine (DPPC), can form highly ordered films
that approach the structure of a two-dimensional
crystal. DPPC has the unusual characteristic relative
to other biological phospholipids that both acyl
chains are fully saturated and that it constitutes an
unusually large amount (30–50%) of pulmonary
surfactant. A widely held view contends that the
functional film in the lung consists of essentially pure
DPPC.
The difference in composition between the secreted vesicles and the hypothetical functional film of
DPPC could result either from selective adsorption of
DPPC or from selective collapse of other constituents. Both processes are difficult to reconcile with
current understandings of how adsorption and collapse occur. One prediction of the model is that P–V
curves should change abruptly over a narrow range
of temperatures. Films of DPPC, like three-dimensional crystals, melt from solid to fluid structures at
specific temperatures. At surface tensions below the
minimum equilibrium value, rates of collapse increase when solid films melt, resulting in increased
surface tensions that would produce higher recoil
pressures. Measurements of the temperature dependence for P–V curves have yielded conflicting results,
and the presence of a highly ordered film remains
unconfirmed. Although films containing only DPPC
would explain the stability of low surface tensions in
the lungs, experimental evidence to support that
possibility is limited.
The solid films that sustain low surface tensions
in situ could also have a structure that resembles a
two-dimensional glass. Three-dimensional liquids, if
cooled fast enough and far enough below their freezing temperatures, retain their disordered structure
but become frozen in place, forming amorphous
solids, or glasses. Two-dimensional fluid films,
defined by their ability to flow into collapsed structures, similarly become jammed into a form that resists collapse if supercompressed to sufficiently high
ALVEOLAR SURFACE MECHANICS 105
Figure 5 Liquid-crystalline collapse. Under equilibrium conditions, surfactant films reach a minimum surface tension at which they
undergo a phase transition to form a three-dimensional bulk phase. Phospholipids form bulk smectic liquid crystals, and compression of
fluid phospholipid films at the minimum equilibrium surface tension can cause the film to flow into stacked structures. Solid films, which
can resist flow and remain at the interface below the minimum equilibrium surface tension, eventually also collapse from the interface at
very low surface tensions, although probably by a process more like fracture.
densities and low surface tensions. These supercompressed films retain their solid behavior and slow rates
of collapse when expanded, even when returned to the
surface tensions at which they originally collapsed. If
they reach low surface tensions in the lungs during a
single exhalation, the films would be transformed, and
their ability to avoid collapse could persist through
multiple cycles of tidal breathing. To achieve low surface tensions, however, the initially fluid films must be
compressed faster than they can collapse. The required rates may occur during normal breathing, but
they are faster than rates in quasi-static experiments
with excised lungs. The supercompressed films, which
would require no compositional change, could explain surface tensions observed in the lungs, but like
the films of pure DPPC, the process by which they
would form remains unclear.
Original views concerning surface tension in the
lungs considered an interfacial film with the thickness of one molecule. Although perhaps difficult to
explain how they might form, monolayers that have
the characteristics of films in the lungs are well described, and more complicated structures were unnecessary to explain the observed behavior. Electron
microscopy, however, has demonstrated that in situ,
at least parts of the interface are occupied by
films that are multilayered. Whether these structures
are formed during adsorption or collapse, and the
extent to which the additional material might affect
the mechanical characteristics of the film, are both
unknown.
Physiological Processes in Respiratory
Diseases
The disorder in which an abnormality of pulmonary
surfactant most clearly represents a major pathogenic factor is the respiratory distress syndrome
(RDS) that occurs in premature babies. Ventilation of
immature lungs that lack adequate amounts of
surfactant injures the alveolocapillary barrier, resulting in pulmonary edema and respiratory failure. Two
mechanisms, both involving shear stresses, could
explain how a deficiency of pulmonary surfactant
would produce the injury. First, elevated surface tension would produce an increased tendency for small
alveoli to collapse, and the shear stresses involved in
reopening the closed airspaces could produce the injury. Second, the meniscus of any fluid column in the
small airways would have an increased surface tension, and the greater pressure-difference across the
interface could rupture the epithelial cells over which
it passes. Elevated surface tensions would also lower
interstitial and pericapillary pressures, resulting in a
greater transmural pressure-difference that would
increase the flow of fluid across the alveolocapillary
membrane. The most direct evidence that deficient
surfactant causes RDS comes from manipulation of
surfactant levels. Subsequent to removing surfactant
by lavage, ventilation of animals produces an injury
that replicates RDS. Conversely, giving exogenous
surfactant to premature babies at risk for RDS prevents or reverses the disorder.
The acute respiratory distress syndrome (ARDS)
was originally called adult RDS to point out the
clinical similarities between adults with injured lungs
and the infants with RDS, and to suggest that the
common presentation resulting from a variety of insults might reflect an abnormality of surfactant acting as the final common pathway. Although the
primary defect in ARDS is an inflammatory process,
abnormal surfactant might perpetuate the initial injury by the same processes that result from an elevated surface tension in RDS.
Surfactant function could be altered in ARDS by
either deficiency or inhibition. Injured lungs have reduced levels of the large active surfactant vesicles,
suggesting a deficiency. Pulmonary surfactant could
also be inhibited by the large number of extraneous
compounds, such as plasma proteins and membrane
lipids, that reach the alveolus in injured lungs and
that can act as surfactants. Direct evidence for elevated surface tensions in ARDS, however, is limited.
The presence of edema complicates the interpretation
106 ALVEOLAR WALL MICROMECHANICS
of P–V mechanics, and the distinction of small lungs,
caused by fluid-filled airspaces that occur with any
pulmonary edema, from stiff lungs, caused by increased surface tension, has been difficult. Evidence
that exogenous surfactants can mitigate ARDS has
also been lacking. The larger doses required to treat
adults with ARDS relative to premature babies with
RDS has limited the therapeutic agents that can be
used. Initial trials with surfactants that lack SP-B
have produced no improvement, just as early attempts to treat babies with RDS using aerosolized
DPPC had no benefit. The role of therapeutic surfactants in ARDS, and of abnormal surfactant in its
pathogenesis, therefore remains unresolved.
See also: Acute Respiratory Distress Syndrome. Alveolar Hemorrhage. Alveolar Wall Micromechanics.
Breathing: Breathing in the Newborn; Fetal Lung Liquid;
First Breath. Bronchoalveolar Lavage. Drug-Induced
Pulmonary Disease. Epithelial Cells: Type I Cells;
Type II Cells. Fluid Balance in the Lung. Infant Respiratory Distress Syndrome. Lung Anatomy (Including the Aging Lung). Lung Imaging. Surfactant:
Overview; Surfactant Protein A (SP-A); Surfactant Proteins
B and C (SP-B and SP-C); Surfactant Protein D (SP-D).
Further Reading
Bastacky J, Lee CY, Goerke J, et al. (1995) Alveolar lining layer
is thin and continuous: low-temperature scanning electron
microscopy of rat lung. Journal of Applied Physiology 79:
1615–1628.
Goerke J and Clements JA (1985) Alveolar surface tension and
lung surfactant. In: Macklem PT and Mead J (eds.) Handbook of
Physiology
The Respiratory System, vol. III, part 1, pp. 247–
261. Washington, DC: American Physiological Society.
Hoppin J, Frederic G, Joseph C, et al. (1986) Lung recoil: elastic
and rheological properties. In: Fishman AE (ed.) Handbook of
Physiology: A Critical, Comprehensive Presentation of Physiological Knowledge and Concepts, pp. 195–215. Bethesda, MD:
American Physiological Society.
Keough KMW (1992) Physical chemistry of pulmonary surfactant
in the terminal air spaces. In: Robertson B, van Golde LMG, and
Batenburg JJ (eds.) Pulmonary Surfactant: from Molecular Biology to Clinical Practice, pp. 109–164. Amsterdam, New York:
Elsevier.
Lewis JF and Jobe AH (1993) Surfactant and the adult respiratory
distress syndrome. American Review of Respiratory Diseases
147: 218–233.
Piknova B, Schram V, and Hall SB (2002) Pulmonary surfactant:
phase behavior and function. Current Opinion in Structural
Biology 12: 487–494.
Robertson B (1984) Pathology and pathophysiology of neonatal
surfactant deficiency (‘‘respiratory distress syndrome,’’ ‘‘hyaline
membrane disease’’). In: Robertson B, Van Golde LMG, and
Batenburg JJ (eds.) Pulmonary Surfactant, pp. 383–418. Amsterdam: Elsevier.
Schürch S, Green FH, and Bachofen H (1998) Formation and
structure of surface films: captive bubble surfactometry. Biochimica et Biophysica Acta 1408: 180–202.
Stamenovic D (1990) Micromechanical foundations of pulmonary
elasticity. Physiological Reviews 70: 1117–1134.
Wilson TA (1981) Mechanics of the pressure-volume curve of the
lung. Annals of Biomedical Engineering 9: 439–449.
ALVEOLAR WALL MICROMECHANICS
F G Hoppin Jr, Brown University, Providence, RI, USA
Introduction
& 2006 Elsevier Ltd. All rights reserved.
The alveolar wall is the site of gas exchange by passive diffusion between inspired gas and the pulmonary capillary blood. Its tiny scale supports passive
diffusion by providing an immense net-diffusing area
and a short path length. Because it is elastically extensible, the gas-exchanging lung units can readily
expand and contract during breathing. How does
this diaphanous structure meet these functional needs
and at the same time avoid structural weakness,
configurational instability, and fluid accumulation?
Abstract
The site of gas exchange in the lung is the alveolar wall. Its
structure supports passive diffusion of the respiratory gases by
spreading the pulmonary capillary bed over an immense surface
area and by having an extremely thin air/blood barrier. It enables the alveolar gas spaces to inflate and deflate by its great
extensibility. Yet it remains stiff enough to withstand the severe
collapsing forces of surface tension acting in tiny confines and to
transmit elastic tensions throughout the network of alveolar
walls, maintaining by those tensions the fine and gross configuration of the lung and a reasonable distribution of ventilation
within the lung. It can modulate local ventilation by activation
of its smooth muscle. The mechanical properties that meet these
exacting requirements can be understood in terms of the behavior of elastic networks and the elastic properties of the wall’s
solid components and of pulmonary surfactant. Dysfunction at
the level of the alveolar wall lies at the core of most disorders of
the lung by impairing diffusion, ventilation, and perfusion.
Normal Mechanics
The lung, like a parachute or a spinnaker, is a tensed
structure (Figure 1). Patency of the airspaces and a
relatively even distribution of expansion and contraction during breathing depend on satisfactory
ALVEOLAR WALL MICROMECHANICS 107
50 µm
E
J
B
Figure 1 Photomicrograph of inflated lung, showing the network of alveolar walls, partitioning the lung into alveolar gas
spaces. The predominant termination of the profile of the alveolar
wall is a triple junction with two other walls (J). Where alveolar
gas spaces open into an alveolar duct (right top), the profiles
either appear bent (B) or simply end (E), and these edges of the
wall are invariably reinforced with connective tissue cables, often
accompanied by smooth muscle. Reproduced from Butler JP,
Oldmixon EH, and Hoppin FG Jr (1996) Dihedral angles of septal
‘bend’ structures in lung parenchyma. Journal of Applied Physiology 81: 1800–1806, used with permission of The American
Physiological Society.
transmission of tensions (lung elastic recoil) throughout. The evidence for the network of alveolar walls
being the major tension-bearing structure in the lung
include the following:
It contains the majority of the lung’s tension-bearing material.
* Its tension-bearing structures are continuous
throughout the lung.
* Distortions of the inflated lung whether by gravity,
local indentation of the surface, or local internal
expansions or contractions behave as expected for
a diffuse, isotropic elastic network.
* Estimates of lung elastic recoil, based on stereological data and elastic properties of the tensionbearing components of the alveolar wall are close
to observed lung-distending pressure differences
over a range of inflation volumes.
*
The other tension-bearing structures, acting mechanically in parallel, include the lung’s outer rind (pleura),
which has been thought to account for only B20% of
the work of inflation, and the fibrous connective tissue
systems of the airway and bronchovascular trees,
which have been estimated to bear B10%.
The network of alveolar walls is a cable/membrane
structure. The membrane portion of the alveolar wall
is tensed at its edges either by other walls or by an
embedded cable of relatively heavy connective tissue.
It is thin, polygonal, and pseudoplanar. It has three
main tension-bearing components: the fine, isotropic
fibrous network; the basement membranes of the epithelium and capillary endothelium; and the air-liquid
interfaces on either side (Figure 2). Along most of its
perimeter (B66%), the wall joins two others (J in
Figure 1). At this junction, there is no cable; the fine
fibrous network, basement membranes, and air-liquid interfaces are directly continuous from one wall
to the adjacent walls. The tensions of the walls at
such junctions are resolved by the balance of the
three walls pulling in three directions. Another mechanism is needed where the alveolar spaces open into
the alveolar duct. At these locations, the wall either
meets one other wall (B24% of its perimeter, B in
Figure 1) or simply ends (B9% of the perimeter, E in
Figure 1) and it requires some ancillary support. This
is provided by relatively heavy connective tissue cables that are curved so as to oppose the wall’s tensions. The fine fibrous network of the walls connects
directly with these cables. The cables of adjoining
walls form a lattice that frames the alveolar duct.
Centrally, their connective tissues merge with the still
heavier connective tissues of the terminal bronchioles. The difference between the E and B configurations, incidentally, probably does not reflect a
functional difference beyond the geometric complexity of space packing of alveoli and of the branching
airway system.
The cables and membranes, being continuous
structures throughout the network, are capable of
transmitting tensions from membrane to membrane
or cable to cable across the lung in any direction.
However, there is also a distinct serial connection
between the cables and membranes at the level of the
alveolar duct and its surrounding alveoli, where the
membranes pull radially against the cables. This is
dramatically apparent as dilation of the ducts of
lungs that have been washed with detergent to increase the wall’s interfacial tension. Normally, however, the elastic properties of these very different
systems are sufficiently matched so that expansion
and contraction of the duct and its surrounding
alveoli are relatively symmetrical during breathing.
Lying alongside most of the cables are bundles of
smooth muscle. Its function relates to its characteristic stiffening when it is activated and passively
length-cycled at typical breathing frequencies. As a
result of the series relationship of the alveolar duct
and its surrounding alveoli (above), stiffening of the
cables reduces the compliance of the respiratory unit.
Indeed, the lung is substantially stiffened in response
to low levels of carbon dioxide (hypocapnia). This
suggests a homeostatic mechanism. Relatively under’ lung units are characteristically
’ Q)
perfused (high V=
hypocapnic. A local response that stiffens the lung
108 ALVEOLAR WALL MICROMECHANICS
Alveolar lining liquid
Epithelial cell
Basement membrane
Interstitium
Basement membrane
Endothelial cell
Epithelial cell nucleus
Fine fiber network
Endothelial cell nucleus
Red blood cell
Plasma
Endothelial cell
Fused basement membrane
Epithelial cell
Alveolar lining liquid
Figure 2 Schematic depiction of the structure of an alveolar wall. It is pseudoplanar and has dimensions on the order of
100 100 10 mm. In the middle is the interstitium, containing the supporting fine fiber network, the marsh-like pulmonary capillary bed,
and small amounts of interstitial fluid. Outside the interstitium on both sides of the wall is a basement membrane supporting the single
layer of thin alveolar epithelial cells and, outside that, a fluid alveolar lining. The capillary itself is a single thin endothelial cell and its
supporting basement membrane, fused on one side of the capillary with the basement membrane of the epithelium.
locally reduces the ventilation of such units. This
hypocapnic pneumoconstriction improves the overall
efficiency of gas exchange, much as hypoxic pulmonary vasoconstriction reduces the perfusion of un’ units.
’ Q)
derventilated (low V=
Tensions in adjoining walls are quite uniform. This
conclusion is drawn from a vector analysis of the
tensions at J and B junctions, the assumption that the
walls are free to rotate relative to each other, and data
for the configuration of fixed inflated specimens. At J
junctions, the distribution of dihedral angles between
walls, extremely narrow around 1201, is consistent
with B2% variation of wall tensions. At B junctions,
the inferred distribution is somewhat wider, but has a
notably sharp cutoff below 1201. Open soap films
supported on complex wire frames show these several
features at their J and B junctions, where they are well
understood to reflect (1) locally uniform film tensions
and (2) a mechanism for adopting the least energy
configuration, that is, minimal surface area. This
finding suggests, intriguingly, that lung structure during growth and development (perhaps even during
remodeling of the adult lung?) responded to these
same physical principles. Local uniformity of wall
tensions, of course, does not imply global uniformity,
which varies systematically over large distances in the
lung, for example, vertically in the gravity field.
The wall is highly extensible, undergoing up to
about twofold change in linear dimensions for the
deepest breaths, that is, about eightfold change of
volume. (Birds followed a different evolutionary
route by having unidirectional air flow through a
relatively stiff gas exchange apparatus.) The risks
inherent in such extensibility include instability, collapse, and gross unevenness of ventilation among the
lung’s different air compartments. These risks are
limited by the wall’s positive compliance and by the
effects of network geometry. A given distortion of the
elastic network stretches the walls on the side away
from the displacement and realigns them closer to the
direction of the displacement. As the walls are positively compliant, the effect is to increase tension on
that side, and the effect of that increase is enhanced
by the realignment of the walls to better oppose the
displacement. The converse occurs on the side towards the displacement. This mechanism acts to stabilize configuration at the fine (alveolus) and gross
(regional) levels.
ALVEOLAR WALL MICROMECHANICS 109
The wall’s positive compliance is conferred independently by both the air–liquid interface and the
solid components (fine fibrous network and basement membranes). Interfacial tension modulated
by pulmonary surfactant varies monotonically from
near 0 to B30 dynes cm 1 over a fourfold change of
surface area. The fine fibrous connective tissues are
collagen fibers (stiff as steel in isolated fibers) and
elastin (the prototypical rubber). They are substantially entangled and cross-linked, and the compressibility of a proteoglycan matrix modifies the extent to
which collagen fibers fold and stretch. The resulting
solid components are capable of more than the about
twofold linear extension, that is, of maintaining tension over the full operating range of lung volume.
The tensions in the interface interact with those of
the solid elements of the wall through curvature.
There is no reason to postulate shear forces between
them. The interface tenses the cables at E and B junctions by curvature that is convex to the airspace, that
is by draping over the cable region like washing on a
clothes line. At the inner corners of the alveolus
(J junctions and the inner side of the B junctions), by
contrast, the curvature of the interface is sharply concave to the airspace, and therefore interfacial tension
reduces the pressure in (i.e., sucks out on) the junctional tissues, transmitting tension to the cable and/or
other septae at that junction. In lungs prepared in
protocols that permit collapse, the freedom to shear
permits the solid elements of the wall to pleat or fold
beneath a smoothly curved interface in the corners of
alveoli. This is probably not the configuration in vivo.
Transiently low distending pressures (o2–3 cmH2O)
permit folding. High distending pressures (416–
20 cmH2O) are required to resolve the folds. With
the appropriate protocols, the wall’s fine fiber network remains quite planar right into the corners of
the alveoli even down to 2–3 cmH2O. This is consistent with the capability of the fine fiber network to
bear tension over the full feasible range of lung inflation, and with its roles of supporting the capillary
network and of maintaining the configuration of the
alveolus, particularly at low inflation volumes.
The pulmonary capillaries are notably affected by
the mechanical state of the wall. The majority of the
capillary bed lies within the flat portions of the wall.
There, the capillary distending pressure (the difference between intracapillary and alveolar gas space
pressures) is in equilibrium with the combined opposing forces of intervening structures, namely the
basement membranes of the capillary and alveolar
epithelium, the fine fiber network, and the air–liquid
interface. The effects are evident in the prominent
bulging of the pulmonary capillaries into the alveolar
space when hydrostatic capillary pressure increases
or interfacial tension is reduced (e.g., in the salinefilled lung). Conversely, the planar forces of the
wall’s solid structures and interfacial tension flatten
the capillaries when the wall is stretched at high lung
volumes. By contrast, the vessels in the corners, for
example, within the J junctions, where the curvatures
of the solid structures and interface are concave to
the alveolar space, are distended at high lung volumes, and may remain open even when the vessels
within the flat portions are compressed. Under those
conditions, blood–flow in such regions is not completely eliminated and there is gas exchange evidence
compatible with very low perfusion units.
Fluid balance is also impacted by mechanical factors, again with different mechanisms in the flat portions of the wall and the corners. Throughout the
body, the volume of fluid in the interstitial spaces is
controlled by the balance between hydrostatic and
osmotic factors across the capillary walls (Starling
equilibrium). The lung, however, is a special case
because of the powerful forces that develop when
interfacial tension acts on very tight curvatures. In
particular, the tissue pressure in the alveolar corners
(J junctions) is lowered. This may be beneficial by
helping to draw interstitial fluid from the flat portions of the alveolar wall into the corners, whence it
may be cleared by the lymphatics. But it is also
counterproductive insofar as it opposes the fluid-absorptive forces across the microvascular walls and
inhibits lymphatic drainage. Of greatest concern, it
can lead to fluid accumulation within the alveolus.
Ordinarily, a simple mechanical negative-feedback
loop limits the accumulation of fluid
any increase
of alveolar fluid initially decreases the curvature of
the interface, reducing the pressure-lowering effect of
interfacial tension and thereby favoring the return of
fluid to the capillaries and lymph. If, however, the
fluid gathers to the point that the corners of the alveolar space are filled, the feedback loop reverses;
any further gathering of fluid increases the curvature,
leading to further alveolar filling or collapse. This
problem is substantially mitigated by the interfacial
tension-lowering effect of pulmonary surfactant,
which has enabled evolution of alveolar walls at a
much smaller scale than would otherwise have been
possible, providing an immense surface area for gas
exchange within a reasonably sized chest.
The Mechanical Role of the Alveolar Wall
in Respiratory Diseases
Mechanical dysfunction of the alveolar wall is at the
core of most major acute and chronic lung diseases,
as readily predictable from the above discussion and
seen in the following examples.
110 ANGIOGENESIS, ANGIOGENIC GROWTH FACTORS AND DEVELOPMENT FACTORS
The essence of emphysema is loss of alveolar walls.
This impairs gas exchange by reducing surface area
of the pulmonary capillary bed and increasing the
path lengths for passive diffusion. It also relaxes the
network of alveolar walls. This has the effect of reducing the dilating effects of the network on the embedded and extrapulmonary airways and blood
vessels. Narrowing of the airways increases their airflow resistance and forces the subject to breathe at
disadvantageously high chest wall volumes in order
to mount sufficient elastic tensions to hold the airways open. Inhomogeneity of damage to the network
of alveolar walls and its effects on airway and blood
vessel calibers cause substantial maldistribution of
ventilation and perfusion, itself a major cause of inefficiency of gas exchange.
A range of mechanical dysfunctions at the level of
the alveolar wall permits blood to shunt through the
lung without exchanging gas. Fluid can gather (pulmonary edema) secondary to the high hydrostatic
pressures of heart failure, or even in normal individuals at very high altitudes. Toxic, infectious, or immunological entities can allow excessive fluid or
protein leakage from damaged capillaries. Airspaces
can collapse (atelectasis) from local underinflation
and gas absorption, from dysfunctional surfactant,
from trauma, or from chest wall dysfunction. High
distending forces (e.g., deep breaths) are required
for reopening. Repeated opening and closing of airspaces in a patient breathing with ventilator support
can have its own direct physical and inflammatory
consequences.
Pulmonary fibrosis due to a wide range of causes
can thicken and stiffen the alveolar wall, impairing
gas exchange and placing a mechanical load on
breathing.
See also: Alveolar Surface Mechanics. Atelectasis.
Chronic Obstructive Pulmonary Disease: Overview;
Emphysema, Alpha-1-Antitrypsin Deficiency; Emphysema, General. Diffusion of Gases. Extracellular Matrix: Basement Membranes; Elastin and Microfibrils;
Collagens; Matrix Proteoglycans. Fluid Balance in the
Lung. Interstitial Lung Disease: Overview; Idiopathic
Pulmonary Fibrosis. Lung Development: Overview.
Myofibroblasts. Occupational Diseases: Overview.
Pulmonary Fibrosis. Smooth Muscle Cells: Airway.
Stress Distribution in the Lung. Surfactant: Overview. Ventilation: Uneven.
Further Reading
Butler JP, Oldmixon EH, and Hoppin FG Jr (1996) Dihedral angles
of septal ‘bend’ structures in lung parenchyma. Journal of Applied Physiology 81: 1800–1806.
Greaves IA, Hildebrandt J, Hoppin FG Jr (1986) Micromechanics
of the lung. In: Fishman AP, Macklem PT, Mead J, and Geiger S
(eds.) Handbook of Physiology. The Respiratory System, vol.
III(1), pp. 217–231. Bethesda: American Physiological Society.
Hoppin FG Jr, Stothert JC Jr, Greaves IA, Lai Y-L, Hildebrandt J
(1986) Lung recoil: elastic and rheological properties. In: Fishman AP, Macklem PT, Mead J, and Geiger S (eds.) Handbook of
Physiology. The Respiratory System, vol. III(1), pp. 195–215.
Bethesda: American Physiological Society.
Mead J (1961) Mechanical properties of lungs. Physiological
Reviews 41: 281–330.
Stamenović D (1990) Micromechanical foundations of pulmonary
elasticity. Physiological Reviews 70: 1117–1134.
West JB (2003) Thoughts on the pulmonary blood–gas barrier.
American Journal of Physiology. Lung Cellular and Molecular
Physiology 285: L501–L513.
Amyloidosis see Interstitial Lung Disease: Amyloidosis.
ANGIOGENESIS, ANGIOGENIC GROWTH FACTORS
AND DEVELOPMENT FACTORS
P A D’Amore, Harvard Medical School, Boston, MA,
USA
M K Sakurai, Boston Children’s Hospital, Boston, MA,
USA
& 2006 Elsevier Ltd. All rights reserved.
Abstract
Angiogenesis is crucial in pulmonary development to establish
the groundwork structure for the lungs’ main function of
gas exchange across a thin blood gas barrier. This network of
blood and air conduits begins to form in early gestation and
continues through early childhood, requiring intricate epithelial–mesenchymal interactions that are regulated by various
angiogenic and growth factors. Disruptions in this complex
system lead to inadequate gas exchange and diseases of respiratory distress. At this time, the precise roles of these growth
factors and interactions between the epithelial and mesenchymal cells are poorly understood. A better understanding of
these processes may lead to improved therapies for various
respiratory diseases.
ANGIOGENESIS, ANGIOGENIC GROWTH FACTORS AND DEVELOPMENT FACTORS 111
Introduction
The basic function of the lung is gas exchange, and
efficient gas exchange requires a thin air–blood interface. Hence, epithelial–mesenchymal interactions
are crucial to the formation of a blood gas. Lung
development, which begins in early gestation and
continues into early childhood, requires complex interactions that are regulated by a number of angiogenic and growth factors. Although it is not
whether mesenchymal development drives epithelial
growth or vice versa, several factors have been identified as important for the development of a functional blood gas barrier. Any disruption to this
intricate and highly orchestrated process can cause
ineffective gas exchange with resulting respiratory
distress. Further knowledge of the factors influencing
pulmonary development may lead to therapeutic interventions for various respiratory diseases.
General Embryology
Lung development is divided into five stages, starting
in early gestation and ending postnatally in early
childhood. Basic organogenesis occurs during the initial embryonic phase. The next three phases – pseudoglandular, canalicular, and saccular – describe the
morphogenesis of primitive lung parenchyma during
those stages. The final alveolar stage extends into
childhood. Concurrent with mesenchymal development, the lung vasculature develops from mesodermal cells by a combination of vasculogenesis and
angiogenesis. Vasculogenesis is the process of de
novo blood vessel development from mesenchymal
precursor cells, whereas angiogenesis involves new
vessels sprouting from existing vasculature. Initially,
the blood lakes, formed by vasculogenesis, create a
capillary plexus and connect to drain into the pulmonary veins. Eventually, the pulmonary arteries
bud off the aortic arches by angiogenesis and grow
into the mesenchyme where they anastamose with
this capillary plexus. This vascular plexus surrounds
the developing airways. The time frame of these
phases overlaps, given the independent development
of each lobe.
During the embryonic stage, the initial lung buds
originate from ventral diverticuli of the primitive
foregut and proliferate into the surrounding mesenchyme. These buds undergo branching morphogenesis to create a complex network of airways that are
lined with columnar epithelium. Simultaneously, vascular lakes of hematopoietic cells that have formed
by vasculogenesis appear in the mesenchyme. Neither the pulmonary artery nor veins have formed at
this time. At first the capillaries only drain into
systemic veins but eventually connect to the pulmonary veins that arise from the primitive cardiac
atrium during the pseudoglandular phase.
Bronchial tree formation and acinar structure
development at approximately 5–17 weeks into gestation mark the pseudoglandular stage. Epithelial–
mesenchymal interactions determine and regulate the
branching patterns. Transplantation experiments
have demonstrated that the mesenchyme directs the
growth and branching of epithelial tubules forming
the conductive airways. In vitro experiments have
shown that removing the mesenchyme from the tip of
a budding epithelial tube prevents further branching
and that transplanted mesenchyme can induce new
budding. During the pseudoglandular stage, differentiation of the cells comprising the airway walls
occurs proximal to distal. These cells become ciliated, nonciliated, goblet, and basal epithelial cells.
At the distal ends, cuboidal epithelial cells line the
forming acini, the mesenchyme begins to thin, and a
network of capillaries develops. During this stage,
the venous system connects to the heart. Later in the
pseudoglandular phase, the pulmonary artery buds
from the aorta and primitive arterial branches begin
to develop alongside the airways. At this point, the
basic airway pattern has been established.
The canalicular stage at gestational weeks 16–26
encompasses acini and pulmonary vasculature development, as well as the initiation of surfactant synthesis. At the crucial gas-exchange region, acini
development involves widening of the peripheral
tubules, further thinning of the mesenchyme and
increased vascularization. The cuboidal epithelium
thins to form the blood gas barrier and differentiates
into type I and type II pneumocytes. The type II
pneumocytes initiate surfactant production.
During the saccular stage, which occurs during
24–38 weeks of gestation, continued expansion of
the air spaces occurs and is accompanied by increased vasculature and decreased interstitial tissue
as the lung prepares for its main function of gas exchange. At this phase, the airways end in thin-walled
terminal sacs, hence the name saccular stage. The
vessels continue to grow and elongate. With the lessening of interstitial tissue, the airspaces begin to approach one another and a capillary bilayer is formed
in the intersaccular septa. The surfactant system
continues to mature in preparation for the function
of gas exchange.
The alveolar stage starts at the end of gestation
and continues for 1–2 years postnatally. Less than
10% of alveoli are present at birth and a majority of
alveolar formation occurs via the process of septation after birth. The walls of the airspaces are called
primary septa and contain a distinct double capillary
112 ANGIOGENESIS, ANGIOGENIC GROWTH FACTORS AND DEVELOPMENT FACTORS
network. Secondary septa form to partition the saccules and form alveoli. This process increases the alveolar surface area and thus the gas-exchange area.
These thick septa transform into a thin epithelial–
endothelial cell lining via a process that is not well
understood. The exact mechanisms that underlie the
five stages of lung development have yet to be elucidated; however, various angiogenic growth factors
and developmental factors have been demonstrated
to play important roles in this process.
Angiogenic Growth Factors,
Developmental Factors, and Their Roles
Epithelial–mesenchymal interactions are crucial in
the formation of a thin blood gas interface during
lung development. Multiple angiogenic and growth
factors interact throughout the five stages of pulmonary development. A variety of in vivo and in vitro
techniques have been used to elucidate the role of
particular growth factors in this process. These studies point to the importance of maintaining a delicate
balance of growth factors during the process of pulmonary development.
Vascular Endothelial Growth Factor
Vascular endothelial growth factor (VEGF) is a wellestablished angiogenic factor. Differential splicing of
a single gene generates three distinct VEGF isoforms
in the mouse and at least five in humans. VEGF acts
to stimulate the proliferation and migration of vascular endothelium as well as to increase vascular permeability. In the lung, VEGF is expressed by type II
pulmonary epithelial cells and localizes to the basement membrane. An analysis of the various VEGF
isoforms in the mouse revealed that the lung produces
primarily VEGF188. This isoform includes two domains, encoded by exons 6 and 7, which are highly
charged and bind with high affinity to the heparan
sulfate, thus accounting for the basement membrane
localization. This specific localization is postulated to
be important for inducing capillary development.
VEGF interacts with tyrosine kinase receptors to exert its cellular functions. These receptors (VEGFR1 or
Flt-1; VEGFR2 or Flt-2 and VEGFR3) are localized
on endothelial cells. Recent studies have revealed that
they are also expressed by a variety of nonendothelial
cell types. VEGF expressed by the pulmonary epithelium induces vascular development via the VEGFR2
on the endothelial precursor cells. Alterations in
VEGF expression lead not only to abnormal vasculature but also to abnormal lung morphology. Additional evidence indicates that VEGF is important for
maintenance of newly formed blood vessels as well.
The tight regulation of VEGF presumably contributes to the formation and maintenance of a normal
blood gas barrier. Experiments involving transgenic
mice have revealed the importance of precise regulation of VEGF expression. Targeted disruption of
VEGF expression results in embryonic mortality,
and inactivation of just a single VEGF allele causes
abnormal blood vessel formation, leading to early
embryonic lethality. Furthermore, overexpression of
VEGF by the respiratory epithelium produces abnormal vascular formation, which also leads to disruption of lung morphology and embryonic lethality.
Hypoxia increases VEGF production by lung epithelial cells, whereas hyperoxia depresses the expression
of VEGF as well as its receptors.
Platelet-Derived Growth Factors
Platelet derived growth factor (PDGF) is composed
of dimers of A and/or B chains and acts via two
tyrosine kinase receptors PDGFR-a and -b. Early in
gestation, PDGF protein localizes to airway epithelial
cells as well as to mesenchymal cells. PDGF is
produced by the epithelium and interacts with PDGF
receptors (PDGFRs) on interstitial mesenchymal
cells. Expression of PDGF appears to change
throughout gestation, increasing during the pseudoglandular phase with minimal production by the
saccular stage. The localization of PDGF as well as
the gestation-dependent expression pattern indicates
an important role during lung development. PDGF is
also involved in the formation of the vessel wall.
PDGF B produced by immature endothelial cells acts
as a mitogen and chemoattractant for undifferentiated mesenchymal, recruiting them to the nascent
vessel. Upon contact with the endothelium, latent
transforming growth factor beta (TGF-b) is activated. The activated TGF-b induces the differentiation of the mesenchymal to become smooth muscle
cells/pericytes. It also acts upon the endothelial cells
to inhibit their proliferation and migration, and it
induces the production of basement membrane.
Taken together, these factors lead to the formation
of a stable, mature vasculature.
PDGF appears to regulate the generation of myofibroblasts. The development and analysis of PDGF A
and PDGF B knockout mice has revealed a role for
PDGF in lung development. Homozygous PDGF A
null mutants do not develop alveolar myofibroblasts
and therefore have reduced elastin deposition, resulting in abnormal alveolarization. Failure of alveolar
septation leads to emphysema in the surviving mutant
mice. PDGF B-deficient mice are embryonic lethal,
due to generalized hemorrhage and edema, most
likely secondary to defective blood wall formation.
ANGIOGENESIS, ANGIOGENIC GROWTH FACTORS AND DEVELOPMENT FACTORS 113
Precise regulation of PDGF levels during each phase
of development is also important. Overexpression of
PDGF A by the respiratory epithelium causes overgrowth of the mesenchyme and results in death from
abnormal lung architecture.
Transforming Growth Factor Beta
The TGF-b family of cytokines regulates cell proliferation, differentiation, recognition, and death
via serine/threonine kinase activity receptors. The
TGF-b1, TGF-b2, and TGF-b3 have been identified
in the developing lung where they have been shown
to stimulate extracellular matrix deposition by lung
fibroblasts and inhibit epithelial cell proliferation.
Spatial and temporal patterns of expression during
lung development suggest a role for TGF-b in branch
morphogenesis. Both mesenchymal and epithelial
cells of the primitive lung express TGF-b1. TGF-b2
expression localizes to the tips of developing bronchioles, while TGF-b3 has been demonstrated in the
proximal respiratory tract epithelium as well as the
terminal growing buds. Signaling via the TGF-b type
I receptor (TGFbRI) directs the formation of branch
points while activation of the TGF-b type II receptor
(TGFbRII) exhibits an inhibitory effect. The inhibitory effect of TGF-b on lung branching is mediated
by the stimulation of the production and deposition
of extracellular matrix proteins and inhibition of
production of collagenase and proteolytic enzymes.
Control over these molecules allows TGF-b to modulate lung branching.
In culture, TGF-b1 and TGF-b2 decrease lung explant size and inhibit branching. TGF-b3 null mice,
which die at birth, have delayed lung development
and exhibit cleft palate as well as pulmonary epithelial, mesenchymal, and vascular dysplasia. In contrast to TGF-b3-deficient mice, TGF-b1 knockout
mice have normal lung development but die by 1
month of age from fulminant pulmonary inflammatory infiltrates. The normal lung development has
been attributed either to functional redundancy provided by the other TGF-b isoforms or to rescue via
transplancental transfer of maternal TGF-b. Overexpression of TGF-b1 results in neonatal lethality and
hypoplastic lungs, with decreased saccular formation
and epithelial differentiation. Conversely, inhibition
of TGFbRIIs stimulates lung development.
Insulin-Like Growth Factors
Insulin-like growth factor-I ( IGF-I) and Insulin-like
growth factor-II (IGF-II) are peptides similar in structure to proinsulin. These growth factors modulate cell
proliferation and differentiation via two receptors,
IGF-IR and IGF-IIR. IGF-IR is a tyrosine kinase receptor, whereas IGF-IIR is a cation-independent
mannose 6-phosphate receptor. In addition, IGF
binding proteins (IGFBPs), which modulate IGF function, have been identified. IGF-I, IGF-II, and IGF-IR
have been localized during early gestation to endothelial cells lining the primary vascular plexus, suggesting a role for these factors in pulmonary vascular
development.
Studies suggest that IGFs may function as survival
factors during pulmonary vascular development.
In vitro experiments involving the treatment of fetal
lung explants with IGF-IR inhibitors exhibit not only
a reduction in endothelial cell number but also an
increase in mesenchymal cell apoptosis. In transgenic
mice, disruption of the IGF-IR gene is lethal postnatally from respiratory failure. Lungs in these mutants
appear hypoplastic although no gross defect in lung
architecture has been observed. Other studies have
suggested that VEGF may act downstream of IGF-1
in vascular development. IGF is important in pulmonary development as a survival factor perhaps via
signaling through other growth factors.
Epidermal Growth Factor and Transforming
Growth Factor Alpha
Epidermal growth factor (EGF) and TGF-a are
members of the EGF family that act through the
EGF receptor (EGFR). EGF and TGF-a are produced
by the mesenchyme and appear to act on the
EGFR located on pulmonary epithelial cells as well
as in neighboring mesenchyme. The source of these
factors and the localization of EGFR indicate an
important role for EGF and TGF-a in epithelial–
mesenchymal interactions. EGF is also expressed
by alveolar epithelial cells and regulates type 2 cell
proliferation in an autocrine manner. Both EGF and
TGF-a affect growth and branching of the pulmonary tree.
In vitro experiments demonstrate that administration of exogenous EGF increases cellular proliferation as well as branching, whereas inhibition of EGF
leads to decreased branching in explants. Targeted
disruption of TGF-a demonstrates no adverse effects,
most likely due to the functional redundancy provided by EGF. However, disruption of EGFR results
in respiratory distress, leading to neonatal mortality.
The lungs of these mutants exhibit septal thickening,
decreased branching, deficient alveolarization, and
reduced epithelial differentiation. Overexpression in
the lung of TGF-a disrupts alveolar development and
causes fibrotic lesions. Clearly the IGFs have an important role in epithelial–mesenchymal interactions
during lung development.
114 ANGIOGENESIS, ANGIOGENIC GROWTH FACTORS AND DEVELOPMENT FACTORS
Fibroblast Growth Factors
The fibroblast growth factors (FGFs) are a family of
proteins that mediate a variety of processes, including cell proliferation, differentiation, cell angiogenesis, and development. These growth factors interact
with specific tyrosine kinase receptors to mediate
their effects. Binding to heparin sulfate proteoglycans
on cell surfaces and within the extracellular matrix
potentiates the effects of some FGFs. Specific FGFs
and FGF receptors (FGFRs) regulated temporally and
spatially during lung development implicate them in
epithelial–mesenchymal interactions.
Abnormal FGF expression and signaling during
pulmonary development lead to anomalous epithelial
branching and differentiation. FGF-10 and keratinocyte growth factor (KGF or FGF-7) induce pulmonary epithelial proliferation and branching. FGF-10
regulates patterning by chemotaxis, whereas KGF influences patterning by promoting epithelial growth.
FGF-10 null mice display perinatal mortality from
aberrant lung development. Disruption of FGFR2
expression results in pulmonary atresia distal to
the main stem bronchi. Interruption of FGFR3 and
FGFR4 signaling leads to excess elastin deposition,
aberrant alveolization, and postnatal lethality. Transgenic overexpression of KGF results in pulmonary
malformations that resemble cystic adenomatoid
malformations. The function of KGF appears to overlap with other factors since KGF knockout mice have
no gross pulmonary abnormalities. FGF signaling is
critical not only for lung development but for postnatal repair following lung injury as well.
Diseases and Therapeutic Considerations
Pulmonary development is a complicated system involving multiple growth factors and extensive epithelial–mesenchymal signaling. Any disruptions in
this system can lead to inadequate gas exchange and
diseases of respiratory distress. Both the vascular and
airway development are affected in diseases with abnormal lung formation such as pulmonary hypoplasia. A better understanding of these developmental
processes may elucidate the mechanisms underlying
various respiratory diseases and thus lead to improved therapies.
Congenital Diaphragmatic Hernia
Congenital diaphragmatic hernia (CDH) is a condition that affects approximately 1 in every 3000–5000
infants and is one of the most common causes of fetal
hypoplastic lungs. In CDH, improper diaphragmatic
development leads to abdominal content herniation
into the thoracic cavity. This results in impaired lung
growth due to the loss of opposition to the lung’s
inherent tendency to collapse. Intrusion of the abdominal viscera into the thoracic cavity is a secondary event that may further hinder lung growth and
development. In addition to being small in size, the
lungs in CDH are also immature. Despite timely repair of the diaphragmatic defect, many infants suffer
postnatally from respiratory compromise due to underdeveloped lungs. Survival and long-term outcome
is dependent on the severity of pulmonary hypoplasia, the extent of bronchopulmonary dysplasia from
ventilatory injury, and other associated anomalies.
Lungs from patients with pulmonary hypoplasia
associated with CDH exhibit significantly decreased
number of airways, decreased vessel density, and
more muscularized arteries. A murine model phenotypically similar to CDH has been developed using
nitrofen exposure. Lungs from nitrofen-treated mice
have decreased levels of VEGF, which may contribute
to their immature state. Nitric oxide, important in
the regulation of smooth muscle cell proliferation,
and nitric oxide synthase are also reduced. The decreased production and diffusion of nitric oxide may
contribute to the muscularized arteries, which in turn
lead to pulmonary hypertension. The possibility of
growth-factor-induced pulmonary growth is currently under investigation in hopes of a therapeutic
intervention for CDH.
Bronchopulmonary Dysplasia
Bronchopulmonary dysplasia (BPD), also known as
neonatal chronic lung disease, is an important cause
of respiratory illness, especially in preterm infants. In
BPD, acute injury of the lung may be caused by
multiple factors, including pulmonary oxygen toxicity, barotrauma from mechanical ventilation and
cellular immaturity. BPD is more common among
premature infants because of higher susceptibility of
the immature lungs to injury. The acute injury causes
an acute inflammatory reaction, which leads to interstitial fibrosis and emphysema with associated
histologic features including airway mucosal metaplasia, smooth muscle hyperplasia, and atelectasis.
Long-term histologic changes include decreased alveolar numbers and surface area.
Airway lavage samples from patients with BPD
contain increased levels of TGF-b, which are associated with an adverse prognosis. As described above,
in vivo experimental models have shown that excessive expression of TGF-b inhibits lung morphogenesis and induces pulmonary fibrosis; EGF and PDGF
are also thought to be involved in the development of
pulmonary fibrosis in BPD. Decreased levels of
VEGF in the presence of alveolar damage are also
ANTICOAGULANTS 115
likely to contribute to the abnormal pulmonary
vasculature associated with lung injury. Manipulation of TGF-b may lead to therapeutic interventions
to prevent the development of chronic pulmonary
fibrosis associated with BPD.
Acknowledgments
The authors were supported by CA45548 (PD’A)
and EY05318 (PD’A).
See also: Bronchopulmonary Dysplasia. Epidermal
Growth Factors. Fibroblast Growth Factors. Insulin-Like Growth Factors. Keratinocyte Growth Factor. Platelet-Derived Growth Factor. Transforming
Growth Factor Beta (TGF-b) Family of Molecules.
Vascular Endothelial Growth Factor.
Further Reading
Alescio T and Cassini A (1962) Induction in vitro of tracheal buds
by pulmonary mesenchyme grafted on tracheal epithelium.
Journal of Experimental Zoology 150: b83–b94.
Burri PH (1984) Fetal and postnatal development of the lung.
Annual Review of Physiology 46: 617–628.
Carmeliet P, Ferreira V, et al. (1996) Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele.
Nature 380: 435–439.
Chinoy MR (2003) Lung growth and development. Frontiers in
Bioscience 8: 392–415.
deMello DE, Sawyer D, et al. (1997) Early fetal development of
lung vasculature. American Journal of Respiratory Cell and
Molecular Biology 16: 568–581.
Gaultier C, Bourbon J, and Post M (eds.) (1999) Lung Development. New York: Oxford University Press.
Han RN, Post M, et al. (2003) Insulin-like growth factor-I receptor-mediated vasculogenesis/angiogenesis in human lung development. American Journal of Respiratory Cell and Molecular
Biology 28: 159–169.
Jankov RP and Keith A (2004) Tanswell. Growth factors, postnatal lung growth and bronchopulmonary dysplasia. Paediatric
Respiratory Reviews 5(supplement A): S265–S275.
Kumar VH and Ryan RM (2004) Growth factors in the fetal and
neonatal lung. Frontiers in Bioscience 9: 464–480.
Shannon JM (1997) Epithelial–mesenchymal interactions in lung
development. In: McDonald JA (ed.) Lung Growth and Development, pp. 81–118. New York: Dekker.
Warburton D and Bellusci S (2004) The molecular genetics of lung
morphogenesis and injury repair. Paediatric Respiratory Reviews 5(supplement A): S283–S287.
Warburton D, Schwarz M, et al. (2000) The molecular basis of
lung morphogenesis. Mechanisms of Development 92: 55–81.
ANTICOAGULANTS
A Günther and C Ruppert, University of Giessen
Lung center (UGLC), Giessen, Germany
& 2006 Elsevier Ltd. All rights reserved.
Abstract
Anticoagulants are widely used for the prevention and treatment of venous and/or arterial thrombosis. Anticoagulants
comprise a chemically heterogeneous group of drugs acting at
different steps within the coagulation cascade. Heparin and heparin-based anticoagulants are indirect anticoagulants that bind
to antithrombin and enhance the inhibitory capacity of this
natural anticoagulant. Coumarin derivatives (e.g., warfarin) interfere with the hepatic synthesis of coagulation factors (vitamin
K antagonists). A third class comprises direct inhibitors of enzymes of the clotting cascade, primarily thrombin. Classic anticoagulants such as unfractionated heparin and coumarins have
some clinical drawbacks. Heparin requires parenteral application, has serious adverse effects, and is difficult to dose and
monitor due to variable and unpredictable pharmacokinetics.
The orally active coumarin derivatives have a narrow therapeutic window and multiple interactions with food and drugs,
necessitating individualized dosing and monitoring. During the
past 10 years, the search for safer and more effective antithrombotic agents resulted in the development or discovery of
new molecules, including fondaparinux and idraparinux (both
selective inhibitors of factor Xa), thrombin inhibitors for parenteral use such as recombinant hirudin and hirulogs, and the
orally active ximelagatran.
The first effective agent for therapy of thromboembolism was unfractionated (UF) heparin. It has become the anticoagulant of choice for the treatment
and prevention of arterial and venous thrombotic
diseases, including pulmonary embolism. Due to the
problems accompanying heparin therapy (unpredictable pharmacokinetics, low bioavailability at low
doses, unpredictable dose response, need for careful
laboratory monitoring, bleeding, and thrombocytopenia), the search for improved anticoagulants resulted in the introduction of low-molecular-weight
(LMW) heparins (Table 1). These heterogeneous
compounds have been shown to produce a more predictable anticoagulant response and fewer adverse
effects than UF heparin, reflecting the improved pharmacokinetic properties (greater bioavailability after
subcutaneous injection (B30%), longer half-life, and
dose-independent clearance). The expectation, however, that these new compounds might eliminate the
risk of bleeding was not confirmed. Another newer
anticoagulant is danaparoid sodium, a heparinoid
that is often used for treatment of heparin-induced
thrombocytopenia.
116 ANTICOAGULANTS
Table 1 Heparins and heparinoids
INN name
Brand name
Manufacturer
Unfractionated heparin
Heparin-sodium
Generic (e.g.,
Liquemin)
Low-molecular-weight heparins
Ardeparin
Normiflo
Wyeth–Ayerst
Certoparin
Mono-Embolex
Novartis
Dalteparin
Fragmin
Pharmacia
Enoxaparin
Clexane
Aventis-Pharma
Nadroparin
Fraxiparine
Reviparin
Clivarine
Sanofi–
Synthelabo
Abbott
Tinzaparin
Innohep
LeoLabs
Heparinoids
Danaparoid
Orgaran
Celltech Pharma
Pentosanpolysulfate
Elmiron
Ortho-McNeal
Hemoclar
Fibrezym
Sanofi Aventis
Bene
Arzneimittel
Oral anticoagulants (4-hydroxycoumarin derivatives), the second major class of traditional anticoagulants, were developed in parallel to heparin,
based on a serendipitous discovery. Today, several
coumarins are used as agents of choice for longterm anticoagulant therapy (Table 2). For historical
and marketing reasons, some countries use more
warfarin (e.g., United States, Canada, and United
Kingdom), others phenprocoumon (e.g., Germany),
and yet others acenocoumarol. Another class of
drugs, called indaniones, are similar to coumarins in
terms of pharmacodynamics and are mostly used in
France.
The traditional anticoagulants UF heparin and coumarin derivatives are highly effective agents and
relatively safe when administered at appropriate
dosages and carefully monitored. LMW heparins
improved and simplified anticoagulant therapy by
obviating the need for routine coagulation monitoring. Newer agents, including fondaparinux and
Method of preparation
Mean molecular
weight
Anti-Fxa:
anti-FIIa
ratio
Extraction from porcine
mucosa
14 000 (range:
5 000–40 000)
1.0
Peroxidative
depolymerization of UF
heparin
Isoamylnitrite
depolymerization of UF
heparin
Nitrous acid depolymerization
of UF heparin
Benzylation and alkaline
depolymerization of UF
heparin
Nitrous acid depolymerization
of UF heparin
Nitrous acid depolymerization
of UF heparin and size
exclusion chromatography
Haparinase digestion of UF
heparin
5 300
2.0
5 200
2.2
6 000
1.9–3.2
4 500
3.3–5.3
4 300
2.5–4.0
3 900
3.6–6.1
6 500
1.5–2.5
6 500
Anti-FXa 4
anti-FIIa
n.a.
Extraction from porine
mucosa
Sulfation of pentosan derived
from beech tree bark
6 000
idraparinux, represent the first agents of a new class
of FXa inhibitors. Both are fully synthetic analogs of
the unique antithrombin-binding pentasaccharide sequence found in UF and LMW heparin. Advances in
molecular biology and biotechnology have led to the
recombinant production of hirudin, the most potent
naturally occurring direct thrombin inhibitor. Hirudin also serves as a standard for the development
of LMW inhibitors of thrombin. Argatroban was
the first synthetic thrombin inhibitor approved for
prophylaxis and treatment of various thrombotic
disorders mainly in patients with heparin-induced
thrombocytopenia. The major drawback of argatroban and hirudins is their requirement for parenteral
administration. Ximelagatran and its active metabolite, melagatran, are members of a class of newly
developed oral direct thrombin inhibitors. These
agents have been approved for anticoagulant treatment very recently and expand the treatment options
for thrombotic disorders. Whether these new agents
ANTICOAGULANTS 117
Table 2 Coumarin and indandione derivatives
INN
Brand name
Manufacturer
Pharmacokinetics
t 12 (h)
Duration of action
(days)
Acenocoumarol (Nicoumalone)
Ethylbiscoumacetate
Dicumarol
Phenprocoumon
Tioclomarol
Warfarin
Sintrom
Tromexane
Generic
Marcumar
Apegmone
Coumadin
Warfilone
Novartis
Ciba-Geigy
Generic
Roche
Merck Lipha
Bristol-Myers Squibb
Merck Frosst
9
2.5
n.a.
150
24
35–45
2–4
1–2
n.a.
7–10
2–3
4–5
Anisindione
Fluindione
Phenindione
Miradon
Previscan
Dindevan
Pindione
Schering
Procter & Gamble
Goldshield
Merck Lipha
9
6
6
3–4
2–3
2–3
will be more effective and safer than traditional
anticoagulants remains to be determined.
Chemical Structure
Heparin and Heparinoids
Heparin is a naturally occurring, highly sulfated
glycosaminoglycan that has been found in mast cells
in a large number of mammalian and nonmammalian
vertebrates. Material for clinical use is derived from
bovine lungs or pig intestinal mucosa and is prepared
either as UF heparin or depolymerized LMW heparin.
Heparin is a heterogeneous mixture of unbranched
polysaccharide chains composed of 15–100 alternating 1-4-linked mucosaccharide units of D-glucosamine and L-iduronic acid or D-glucuronic acid
(Figure 1(a)). Heparin is the most negatively charged
molecule in the human body. The molecular weight of
UF heparin ranges from 5 to 40 kDa with an average
of B14 kDa. LMW heparins are much smaller (mean
MW, B5 kDa) and are produced from UF heparin
by chemical or enzymatic depolymerization. The
chemical composition is similar but not identical.
The anticoagulant effect is mediated by a unique
pentasaccharide unit (Figure 1(b)) that binds antithrombin with high affinity and activates the inhibitor
by approximately 1000-fold. This pentasaccharide
sequence is found in B30% of the chains of UF heparin (known as high-affinity heparin) but in only
15–25% of the chains of LMW heparin.
Danaparoid sodium is an LMW heparinoid isolated from porcine mucosa with a mean MW of
6 kDa (range, 4–12 kDa). It consists of a mixture of
84% heparan sulfate (of which 4% has high antithrombin activity), 12% dermatan sulfate, and 4%
chondroitin sulfate. The characteristic disaccharide
repeat units of these glycosaminoglycans are depicted
in Figure 1(c).
Pentosanpolysulfate (PPS) is a highly sulfated,
semisynthetic polysaccharide derived from beech tree
bark with a mean molecular weight of 6 kDa (range,
2–9 kDa). The degree of sulfation is higher than in
heparin, resulting in a higher negative charge density.
Pentosan consists of b-1-4-linked D-xylose units
branched with 4-O-methyl-D-glucuronic acid in a
ratio of 1 uronic to 9 xylose units (Figure 1(d)). In
contrast to heparin and other glycosaminoglycans,
PPS is orally bioavailable.
FXa Inhibitors
Fondaparinux is a fully synthetic analog of the unique pentasaccharide sequence found in heparins. Idraparinux represents the second generation of this
class. The O-methylation and O-sulfation result in a
higher affinity for antithrombin and a higher negative
charge, which in turn accounts for the tight binding
of antithrombin and the prolonged half-life of idraparinux (80 h vs. 17 h for fondaparinux) (Figure 2).
Vitamin K Antagonists
The clinically used vitamin K antagonists are derivatives of either 4-hydroxycoumarin or indan-1,3dione (Figure 3). The substituent at the 3 position
of the coumarin backbone or side-chain variations
influence pharmacokinetic and pharmacodynamic
properties (Table 2). Pharmaceutical preparations
of warfarin, acenocoumarol, and phenprocoumon
contain a racemic mixture of R- and S-enantiomers.
The stereoisomers exhibit different pharmacodynamics and pharmacokinetics since they undergo stereoselective biotransformation in the liver.
118 ANTICOAGULANTS
CH2OSO3k
O
O
COOk
4
1
4
O
OH
1
OH
O
k
NHSO3k
OSO3
(a)
n
Heparin
CH2OSO3k
O
CH2OSO3k
O
COOk
O
O
OH
O
OH
O
k
OSO3
COOk
O
OH
O
OH
OSO3k
NHSO3k
OH
NHCOCH3
CH2OSO3k
O
O
O
NHSO3k
Heparin pentasaccharide
(b)
COOk
O
CH2OSO3k
O
O
O
4
1
4
OH
O
COO
1
4
OH
1
n
O
NHCOCH3
n
Dermatan sulfate
R1O
4
1
1
3
OH
Heparan sulfate
COOk
O
O
O3SO
4
OH
OH
4
CH2OH
O
k
k
OH
CH2OR2
O
Chondroitin sulfate
O
A: R1 = SO3k R2 = H
C: R1 = H
R2 = SO3k
1
3
O
NHCOCH3
OH
(c)
O
O
OSOk
3
OSO3k
O
O
O
n
O
OSOk
3
O
O
OSOk
3
OSO3k
COOk
O
OSOk
3
OSO3k
O
O
OSOk
3
O
OSO3k
O
H3CO OSO3
OSO3k
(d)
Pentosanpolysulfate
Figure 1 Chemical structures of heparin and other members of the glycosaminoglycan family. (a) The major repeat unit of heparin is
depicted, which is found in up to 90% in heparin from beef lung and up to 70% in heparin from pig mucosa. The disaccharide consists of
1-4 linked sulfated L-iduronic acid and sulfated D-glucosamine. (b) The specific pentasaccharide sequence that is responsible for highaffinity binding to antithrombin is depicted. (c) The major repeat units of heparan sulfate, dermatan sulfate, and chondroitin sulfate A are
depicted. (d) A typical sequence of pentosanpolysulfate is shown.
Hirudin and Hirulogs
Hirudin is a naturally occurring inhibitor of thrombin that is a single-chain polypeptide of 65 amino
acid residues with an apparent molecular weight
of 7 kDa. The structure is stabilized by three intramolecular disulfide bridges. In 1986, recombinant
hirudin (r-hirudin) became available. Desirudin
([Val1, Val2]-63-desulfohirudin) and lepirudin
([Leu12]-63-desulfohirudin) are therapeutically used
recombinant hirudins produced in yeast (Table 3).
They are not sulfated and differ only in the first two
amino acids of the N terminus. Both natural and
recombinant hirudins show almost identical pharmacodynamic and pharmacokinetic properties.
ANTICOAGULANTS 119
COOk
O
CH2OSO3k
O
CH2OSO3k
O
CH2OSO3k
O
O
COOk
HO OH
O
OH
O
NHSO3k
O
OSO3k
O
NHSO3k
OH
OH
OH
OCH3
OSO3k
NHSO3k
Fondaparinux
COOk
CH2OSO3k
O
CH2OSO3k
O
O
CH2OSO3k
O
O
COOk
O
H3CO OCH3
OSO3k
OCH3
O
OCH3
OSO3k
OCH3
O
O
OCH3
OSO3
OCH3k
OCH3
OSO3k
Idraparinux
Figure 2 Chemical structure of FXa inhibitors. The structures of fondaparinux and idraparinux are given.
OH
4
12
O
O
1
2
3
3
O
O
4-Hydroxycoumarin
Indan-1, 3-dione
O
O
OH
O
O
O
OH
Warfarin
OH
Phenindione
O
O O
O
OH
O
O
F
Dicumarol
NO2
O
OH
O
OH
Fluindione
Acenocoumarol
O
O O
O
OH
O
Ethylbiscoumacetate
O
O
OCH3
O
Anisindione
Phenprocoumon
Figure 3 Chemical structures of coumarin and indandione derivatives. The lead structures and derivatives are given. Clinically used
coumarin derivatives are substituted at the 3-position, and indandiones are substituted at the 2-position. Asymmetric carbons are
indicated by asterisks.
120 ANTICOAGULANTS
Table 3 Direct thrombin inhibitors
INN name
Brand name
Manufacturer
Route of administration
Half-life
Desirudin
Lepirudin
Bivalirudin
Argatroban
Melagatran
Ximelagatran
Revasc
Refludan
Angiomax
Novastan
Melagatran
Exanta
Aventis/Novartis
Schering, Berlex
The Medicine Company
Texas Biotechnology
AstraZeneca
AstraZeneca
Parenteral (i.v., s.c.)
Parenteral (i.v.)
Parenteral (i.v.)
Parenteral
Parenteral
Oral
2–3 h
1.3 h
25 min
45 min
3–5 h
3–5 h
1
H2N
Val Val Tyr Thr Asp Cys Thr Glu Ser
20
40
Thr Val Cys
Gly
Gly 10
Val Asn Ser Gly Glu Cys Leu Cys Leu Asn Gln
Cys
Gly Gln Gly Asn Lys Cys IIe
Leu 30
Gln Asn Lys Glu Gly Asp Ser Gly
Glu
Gly
65
60
50
Thr Pro Lys Pro Gln Ser His Asn Asp Gly Asp Phe Glu Glu IIe Pro Glu Glu Tyr Leu Gln
COOH
SO3−
44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Gly Thr Pro Lys Pro Gln Ser His Asn Asp Gly Asp Phe Glu Glu IIe Pro Glu Glu Tyr Leu Gln
Hirudin
D−Phe Pro Arg Pro Gly Gly Gly Gly Asn Gly Asp Phe Glu Glu IIe Pro Glu Glu Tyr Leu
Bivalirudin
Figure 4 Structure of hirudin and hirulogs. The amino acid sequences of natural hirudin and the hirulog bivalirudin are given. The
C-terminal sequences blocking the anion-binding exosite (amino acids 54–65 of hirudin) are depicted in blue, and the sequences binding
to the active site of thrombin (amino acids 45–48 of hirudin) are depicted in light blue.
The discovery of the bifunctional inhibition mechanism of hirudin resulted in the development of a
new type of bivalent oligopeptide inhibitors, termed
hirulogs. Bivalirudin, also termed hirulog-1, is a
semisynthetic 20-amino acid peptide consisting of
the N-terminal and the C-terminal active peptide
domains from hirudin joined by a glycine linker
(Figure 4).
mimicking the cleavage site in fibrinogen. The arginine-derived structures are shown in Figure 5. The
pharmaceutical preparation consists of a mixture
of R and S stereoisomers at a ratio of approximately
65:35.
Ximelagatran has a molecular weight of 473.6 Da
and is a pro-drug without intrinsic anticoagulant
activity. Following resorption, it is rapidly converted
into its active metabolite, melagatran (MW, 429.5 Da).
Synthetic Active Site Inhibitors of Thrombin
Argatroban and the orally active ximelagatran are
LMW active site inhibitors of thrombin. They are the
result of structure-based drug design and belong to the
peptidomimetic (arginominetic) group of inhibitors
Mode of Action
The classical and new anticoagulants interact with
different clotting factors at different levels of blood
ANTICOAGULANTS 121
O
H2N
OH
NH
H2N
NH
Arginine
H 3C
NH
O
OH
O
S
O
NH
N
O
O
NH
HN
N
O
CH3
O
HO
O
N
NH2
NH
H2N
CH3
NH
Ximelagatran
Argatroban
Hydrolysis
Reduction
NH
N
O
O
HN
O
OH
HN
NH2
Melagatran
Figure 5 Chemical structures of synthetic thrombin inhibitors. The chemical structures of the arginomimetics argatroban, ximelagatran, and melagatran are shown. Bioconversion of ximelagatran into its active metabolite, melagatran, involves hydrolysis of the
carboxyl ester and reduction of the hydroxyamidino group.
coagulation. A summary of their targets within the
coagulation cascade is shown in Figure 6.
Heparin and Heparinoids
Heparin acts as an accelerator of antithrombin (formerly called antithrombin III). The pentasaccharide
sequence of heparin binds to the lysine site in
antithrombin inducing a conformational change of
the antithrombin molecule, which facilitates binding
to specific clotting factors and accelerates the rate at
which antithrombin inhibits these factors by approximately 1000 times. UF heparin inhibits factors
Xa, IIa (thrombin), IXa, XIa, and XIIa, with FXa
and thrombin representing the most responsive and
most critical factors within the clotting cascade. Heparin, antithrombin, and thrombin form a ternary
complex in which thrombin initially binds to the
122 ANTICOAGULANTS
Extrinsic pathway
Vascular injury
Coumarins, indandiones
Tissue factor (TF)
FVII
Intrinsic pathway
HMW kininogen
Prekallikrein
FXIIa
FXI
FVII
TF
FVIIa
FIX
TFPI, FVIIai
Coumarins, indandiones
Ca2+
FX
FXIa
UF heparin, LMW heparin
Danaparoid, fondaparinux
Surface
FIXa
FVIII
Ca2+, PL
PL, Ca2+
Tenase complex
Antithrombin
FVIIIa
FIXa, thrombin
Hirudin, hirulogs
Argatroban, melagatran
FXa
2+
APC
PL, Ca
Prothrombinase
complex
Prothrombin
FV 2+
Coumarins, indandiones
Thrombin
FXIII
FVa
Ca , PL, FXa
FXIIIa
Heparin cofactor II
APC
Fibrinogen
Danaparoid
Dermatan sulfate
Fibrin
Ca2+
Crosslinked fibrin
Figure 6 Overview of the coagulation cascade and targets of anticoagulants. The targets of anticoagulants within the clotting cascade
are given.
heparin–antithrombin complex in a non-specific
manner to any site of the heparin molecule and then
slides along the surface until it binds to the inhibitor.
The affinity of heparin for the antithrombin–thrombin complex is much lower than that for unreacted
antithrombin. Thus, heparin can dissociate from the
complex and bind to additional unreacted antithrombin molecules, resulting in a continuing anticoagulant effect. The complex, however, is not
effective in inhibiting fibrin-bound thrombin. UF heparin also blocks the thrombin-induced feedback activation of factors V and VIII. The sliding mechanism
requires heparin chain lengths consisting of at least
18 saccharide units. The lower content of glycosaminoglycan chains 418 saccharide units in LMW
heparin accounts for the greater relative activity
against factor Xa. This difference is described in
an activity ratio (anti-FXa:anti-FIIa ratio), which is
1:1 for UF heparin and 1.5:1 to 6:1 for LMW heparin (see Table 1). The anticoagulant activity of
UF heparin is expressed in relation to the fourth international standard: Pharmaceutical preparations
have specific activities of 150–190 U mg 1. The
anticoagulant activity of LMW heparin is expressed
relative to the first international standard for LMW
heparin; the specific activity ranges between 80 and
120 anti-FXa U mg 1 and between 35 and 45 antithrombin U mg 1. In addition to the interaction
with antithrombin, both UF and LMW heparin enhance the release of tissue factor pathway inhibitor
(TFPI), which forms a complex and inactivates
FXa and subsequently FVIIa. UF and LMW heparins also bind to platelet factor 4, which is the predicate for heparin-induced thrombocytopenia (HIT),
a severe complication of heparin therapy that is associated with paradoxic clotting, including deep vein
thrombosis, pulmonary embolism, or occasional arterial thromboses. In HIT, antibodies form against
the heparin–platelet factor 4 complex, leading to
platelet activation and aggregation. LMW heparins
bind less extensively to platelet factor 4 as well as to
plasma proteins, endothelial cells, and macrophages.
However, LMW heparins should not be used in
patients with established HIT since HIT antibodies
have an almost 100% cross-reactivity with LMW
heparin.
ANTICOAGULANTS 123
The heparinoid danaparoid sodium exerts its pharmacological effects in a similar manner via binding
and activation of the serpin inhibitor heparin cofactor
II. Danaparoid has little effect on platelet function and
exhibits low cross-reactivity with heparin-induced
antibodies, making it an option for the treatment
of HIT. Similar to LMW heparins, danaparoid also
exerts a higher inhibitory activity against FXa. The
overall antithrombotic activity is much lower compared to that of UF heparin (anti-FXa activity B10%
and antithrombin activity B1% of that of UF heparin). PPS may act via an antithrombin or heparin
cofactor II-independent pathway, but the exact mechanism of action is not known. It has been used since
the 1960s as an anticoagulant but does not seem to
play a major role in today’s clinical medicine with
regard to anticoagulation. In the United States, PPS is
approved for pain relief in the management of interstitial cystitis.
Fondaparinux and Idraparinux
Fonaparinux and idraparinux are selective indirect
inhibitors of FXa. Their activity also requires the
presence of antithrombin, but inhibition is achieved
solely through binding of the pentasaccharide sequence to antithrombin and induction of a conformational change.
Vitamin K Antagonists
Coumarin and indandione derivatives act as vitamin
K antagonists and exert their anticoagulatory effect
by interfering with the hepatic synthesis of vitamin
K-dependent clotting factors. Synthesis of factors II
(prothrombin), VII, IX, and X involves carboxylation
of glutamate residues to g-carboxyglutamates by a
specific carboxylase, which is required for their biological activity. These g-carboxyglutamate residues
promote binding of the clotting factors to phospholipids, thereby accelerating coagulation. Coumarin
and indandione derivatives interfere with the vitamin
K cycle by inhibiting vitamin K epoxide reductase. By
this mechanism, coumarins decrease the synthesis of
coagulation factors by 30–50%. The anticoagulant
response to coumarins is delayed by 2 or 3 days since
previously formed coagulation factors circulate with
a long half-life (4–6 h for FVII, 20–50 h for factors IX
and X, and 50–70 h for thrombin). A major disadvantage of vitamin K antagonists is the narrow therapeutic index and the need for intensive monitoring.
The anticoagulatory effect is assessed by the international normalized ratio (INR), which is defined as the
ratio of the patient prothrombin time (PT) compared
to the mean PT of normal donors normalized to the
international sensitivity index, a correction factor for
the response of different thromboplastin reagents.
The therapeutic INR range is 2.0–3.0. An INR o2.0
may be ineffective in preventing thrombotic events,
and an INR 43.0 is associated with increased bleeding. The manifold interaction of coumarins with food
and drugs has an important impact on the anticoagulatory response by either increasing or decreasing
anticoagulant activity.
Direct Thrombin Inhibitors (Hirudin, Hirulogs, and
Synthetic Inhibitors)
Hirudin is a highly potent (Ki ¼ 22 fM) inhibitor
specific for the serine protease thrombin. It forms a
stable noncovalent 1:1 stoichiometric complex with
the B-chain of a-thrombin, thereby abolishing its
ability to cleave fibrinogen (Figure 7). Hirudin inhibits both free and fibrin-bound thrombin. During
inhibition, hirudin displaces fibrin from thrombin.
Bivalirudin is a bivalent inhibitor (Ki ¼ 1:9 nM)
blocking both the active site and the substrate recognition site (anion-binding exosite). Argatroban
(Ki ¼ 39 nM) and melagatran (Ki ¼ 2 nM) are peptidomimetics of the sequence of fibrinopeptide A
that interact with the active site of thrombin. They
are reversible, noncovalent, competitive inhibitors
blocking the enzyme’s interaction with its substrate.
The pro-drug ximelagatran has no intrinsic anticoagulant activity but is rapidly converted into melagatran following gastrointestinal resorption.
The direct thrombin inhibitors are recommended
agents for prevention and treatment of HIT. Lepirudin
and argatroban are approved for the treatment of HIT
in the United States. Bivalirudin, which is approved
for anticoagulation during percutaneous coronary intervention, appears to be promising in patients with
HIT. Although displaying pharmacologic advantages,
its use for this indication is only weakly recommended
due to the limited data available.
Novel Anticoagulants
In addition to the previously described anticoagulants,
novel agents that directly target specific steps within
the coagulation cascade have been developed and
tested in clinical phase II or phase III studies or are
currently undergoing preclinical and clinical testing.
They include LMW inhibitors of FXa and recombinant forms of naturally occurring anticoagulants.
Recombinant tissue factor pathway inhibitor (tifacogin) Tifacogin is a recombinant form of the endogenous extrinsic pathway inhibitor TFPI, also referred
to as anticonvertin or lipoprotein-associated coagulation inhibitor. TFPI is a 276-amino acid residue,
high-affinity Kunitz-type serine protease inhibitor.
+
+
+
+
+
+
Fibrin
+
+
+
Hirudin
Heparin recognition site
= anion-binding exosite II
+
+
+
Fibrin
124 ANTICOAGULANTS
Bivalirudin
Thrombin
+
+
+
+
+
+
Substrate recognition site
= anion-binding exosite I
Binding of fibrin(ogen),
PAR-1, heparin cofactor III
thrombomodulin
Binding of heparin,
prothrombin 2 fragment (F2)
platelet gp Ib
+
+
+
Active site
+
+
+
Fibrin
Specificity
pocket
Synthetic inhibitors
(Argatroban, melagatran)
Figure 7 Mechanism of action of thrombin inhibitors. The interaction of hirudin, hirologs, and synthetic thrombin inhibitors with
thrombin is shown.
Inhibition of the tissue factor-mediated extrinsic coagulation pathway occurs in a two-step manner:
TFPI directly binds to FXa at or near its active site
via the second Kunitz domain and produces a FXadependent feedback inhibition of the TF–FVIIa
catalytic complex by formation of a quaternary complex (FXa–TFPI–TF/FVIIa), in which the second
Kunitz domain binds to FXa and the first Kunitz domain binds FVIIa (Figure 8). Despite showing promising results in animal thrombosis models, a phase III
trial failed to demonstrate significant benefit in outcome in sepsis patients.
Recombinant nematode anticoagulant protein
c2 Recombinant nematode anticoagulant protein
c2 (rNAPc2) is a potent 85-amino acid residue anticoagulant protein originally identified in the hookworm Ancylostoma caninum. Like TFPI, rNAPc2
inhibits initiation of the extrinsic coagulation pathway by binding to a noncatalytic exosite on FX or
FXa prior to the formation of the quaternary inhibitory complex with tissue factor–FVIIa. In a phase II
study on patients who underwent knee replacement
surgery, a low dose of rNAPc2 decreased the rate of
deep vein thrombosis and major bleeding.
Active site inhibited factor VII Active site inhibited
factor VII (FVIIai; ASIS) is a recombinant variant of
activated factor VII in which the catalytic function is
irreversibly blocked. The resulting molecule retains
its TF binding capacity but is enzymatically inactive.
FVIIai exerts its effects by competing with plasma
FVII(a) for tissue factor binding on cell surfaces. The
formation of an inactive binary FVIIai–TF complex
attenuates the initiation of coagulation. However,
although recombinant FVIIai was shown to inhibit
TF-mediated injury in animal models, it failed to
elicit a beneficial response in coronary patients in a
phase II trial.
ANTICOAGULANTS 125
3
TFPI
1
2
FXa
Ca2+
TFPI
FXa
Ca2+
TFPI
FVIIa
FVIIa
Ca2+
Ca2+
TF
FXa
TF
Ca2+
Figure 8 Mechanism of action of tissue factor pathway inhibitor (TFPI). TFPI binds to FXa in solution forming a binary complex.
Subsequent binding of FXa–TFPI to the TF–FVIIa complex results in the final quaternary complex inhibiting initiation of coagulation.
Direct FXa inhibitors DX-9065a (Daiichi) is a synthetic, LMW, nonpeptidic, direct reversible and parenteral inhibitor of factor Xa. BAY 59-7939 (Bayer),
razaxaban (Bristol-Myers Squibb), LY 517717 (Eli
Lilly), and YM-60828 (Yamanouchi Pharmaceutical)
are orally active synthetic FXa inhibitors.
Activated protein C (drotrecogin alpha (activated)) The protein C system also regulates coagulation by modulation of the activity of the two
clotting factors, FVa and FVIIIa. Protein C, the key
component of this system, is a vitamin K-dependent
zymogen of an anticoagulant protease, which is
activated on the surface of intact endothelial cells
by thrombin that has bound to the integral membrane protein thrombomodulin. Activated protein C
(APC) can cleave the phospholipid membrane-bound
(activated) clotting factors FVa and FVIIIa, which
blocks the function of the prothrombinase and tenase
complexes and thus inhibits the propagation of coagulation (Figure 9). Activation of protein C is augmented by the endothelial cell protein C receptor, and
the anticoagulant activity of APC is supported by
protein S, a vitamin K-dependent cofactor. The protein C system also has anti-inflammatory and antiapoptotic properties. Drotrecogin alpha (activated) is
a recombinant version of APC. Drotrecogin alpha
was approved for treatment of severe sepsis in adults
based on a phase III trial in which 28-day mortality
was reduced.
Thrombomodulin Thrombomodulin is a complex
multifunctional endothelial cell surface glycoprotein
receptor. High-affinity binding to thrombin involving
EGF domains 4–6 converts thrombin from a procoagulant into an anticoagulant state that can activate protein C. Thrombomodulin not only regulates
hemostasis but also plays an important role in the
126 ANTICOAGULANTS
Protein C
APC
TM
FV
Protein S
FVIIIi
FVIIIa
A
P
C
EPCR
Protein S
Th
FVi
FVa
A
P
C
Figure 9 Protein C pathway: mechanism of action of protein C and thrombomodulin. (Top) Thrombin (Th) generated in the vicinity of
intact endothelial cells binds to thrombomodulin (TM) and activates protein C. This process is enhanced by the endothelial cell protein C
receptor (EPCR). (Bottom) Activated protein C (APC) and protein S form a complex on the membrane of endothelial cells that cleaves
and inactivates FVIIIa (-FVIIIi) and FVa (-FVi) and thus inhibits coagulation. In the case of FVIIIa, this process is further enhanced by
FV, which in this context functions as an anticoagulant cofactor protein.
modulation of inflammation. The EGF 3–6 domains
are involved in activation of thrombin-activatable fibrinolysis inhibitor, a natural anti-inflammatory molecule that inhibits vasoactive peptides such as the
complement anaphalotoxin C5a. Soluble thrombomodulin is a recombinant variant that is undergoing
clinical testing.
Role of Anticoagulants in
Respiratory Medicine
Thromboembolic events, including deep vein thrombosis and pulmonary embolism, are commonly encountered in clinical practice. In addition to the
occlusion of a vessel, ‘periembolic’ processes, such as
activation of circulating platelets and inflammatory
cells at the surface of thrombi and the liberation of
vasoactive mediators (thromboxan, serotonin, and leukotriene B4) and fibrin(ogen)-derived split products,
markedly contribute to cardiopulmonary dysfunction,
with increased pulmonary vascular resistance, elevated
right heart pressure, and gas exchange disturbances.
Anticoagulants are used for prevention and treatment
of a variety of indications, including prevention of
deep vein thrombosis and pulmonary embolism postoperatively after general or orthopedic surgery (e.g.,
hip replacement/fracture), in patients with acute spinal
cord injuries, multiple trauma, ischemic stroke, after
coronary angioplasty, heart valve replacement, and in
many other medical conditions as well as for treatment of established deep vein thrombosis, unstable
angina, and ischemic stroke. In a meta-analysis,
subcutaneous LMW heparin was shown to be as effective and safe as intravenous UF heparin for the
initial treatment of nonmassive pulmonary embolism.
Intravascular clot formation due to predominance
of a procoagulant and suppression of fibrinolytic factors is a key event in a variety of inflammatory and
infectious diseases, such as septic organ failure. Microthrombosis/microembolism is commonly found in
patients with acute respiratory distress syndrome
(ARDS), of which sepsis is the major underlying
cause, and in patients with chronic pulmonary hypertension. The lung microvascular compartment
represents the largest surface of the pulmonary vascular bed and may be of crucial importance in the
dissolution of microthrombi. Under physiological
conditions, patency of capillaries is provided by a
high concentration of antithrombin that is ‘activated’
by glycosaminoglycans (e.g., heparan sulfate) and
thrombomodulin on the surface of endothelial cells.
In addition, clearance of thrombi is achieved through
the production and secretion of tPA and uPA by
microvascular endothelial cells. Under inflammatory
or infectious conditions (e.g., sepsis and ARDS), this
balance between anticoagulation and fibrinolysis regulation is shifted toward coagulation with a decrement in fibrinolysis, mainly due to activation of the
extrinsic tissue factor/FVII-dependent coagulation
pathway and secretion of plasminogen activator inhibitor-1 (PAI-1). In experimental models of acute
lung injury (ALI), APC prevented intravascular coagulation, edema formation, and inflammatory cell
recruitment in the lung. Although administration of
APC has been shown to decrease mortality in patients
ANTICOAGULANTS 127
with severe sepsis, clinical studies demonstrating its
efficacy in ARDS are lacking.
In ALI/ARDS, fibrin deposition occurs in both intravascular and extravascular locations. In patients
with ALI/ARDS, the alveolar hemostatic balance is
shifted toward predominance of procoagulant activity, which is mainly attributable to the extrinsic
pathway enzymes tissue factor and FVII. In contrast,
the fibrinolytic capacity of the alveolar space is
markedly reduced. In lavage fluid from patients with
ARDS, concentrations of urokinase, representing the
predominant plasminogen activator in this compartment, were markedly decreased, whereas elevated
activities of PAI-1 and a2-antiplasmin were consistently encountered. Under these conditions, fibrinogen leaking into the alveolar space due to an
impaired barrier function is rapidly converted into
fibrin. The function of fibrin formation in the alveolar space is largely unsettled. It may well exert
beneficial effects by preventing pulmonary hemorrhage and serve as a primary matrix of wound repair.
On the other hand, alveolar fibrin is a potent inhibitor of surfactant function, thus promoting atelectasis
formation and contributing to the severe impairment
of gas exchange properties. In vitro studies have
demonstrated an incorporation of hydrophobic
surfactant compounds into nascent fibrin strands,
resulting in an almost complete loss of the surface
tension-lowering properties.
Alveolar/interstitial fibrin deposition and aberrant
local fibrin turnover have likewise been shown in patients with chronic interstitial lung disease, including
idiopathic pulmonary fibrosis, sarcoidosis, and hypersensitivity pneumonitis. Alveolar fibrin appears to be a
key factor in triggering the fibroproliferative process in
the lung. Delayed clearance of fibrin from the lung
may promote fibroblast activation, proliferation, and
tissue remodeling (i.e., replacement of the primary fibrin matrix by a secondary collagenous matrix associated with scarring and honeycombing). Thrombin
also acts as a chemoattractant for inflammatory cells
and fibroblasts and stimulates the release of proinflammatory cytokines. Moreover, it induces the release
of growth factors and fibrogenic mediators, thereby
stimulating fibroblast proliferation and connective tissue synthesis. Although the use of anticoagulants is
not part of the standard clinical practice for these
conditions, blockade of the extrinsic coagulation pathway and prevention of extravascular fibrin formation
may be a promising approach for acute inflammatory
and chronic interstitial lung disease.
Experimental studies addressing anticoagulant intervention (e.g., by administration of APC, heparin,
and direct thrombin inhibitor) in animal models of
ALI and pulmonary fibrosis have been shown to
protect the lung and to reduce the fibroproliferative
response. In addition, different anticoagulants are
being tested in ongoing experimental and clinical
studies. A phase II study of recombinant TFPI in
patients with severe sepsis showed improvement in
lung function and a trend toward improved survival
in the subset of patients with ARDS. A follow-up
phase III study, however, failed to demonstrate improved survival. Similarly, the beneficial effects of
recombinant antithrombin observed in animal models could not be confirmed in humans.
Heparin is being evaluated in humans with idiopathic pulmonary fibrosis. In a similar vein, heparin
is considered an alternate agent in the treatment of
allergic asthma. Glycosaminoglycans such as heparan sulfate are expressed as part of a proteoglycan on
cell surfaces and are thought to play a role in the
regulation of the inflammatory response (e.g., by
binding chemokines). Heparin, which is synthesized
by and stored exclusively in mast cells, has been
found to exert anti-inflammatory effects in animal
models and in human disease. Heparin binds and
inhibits a variety of cytotoxic and inflammatory mediators, including eosinophilic cation protein and
peroxidase. Furthermore, heparin has been associated with the inhibition of lymphocyte activation,
neutrophil chemotaxis, smooth muscle growth, and
vascular tone. The first clinical trial with inhaled heparin was performed in 1969, but further human
studies using inhaled heparin were not published until the early 1990s. Three smaller studies of patients
with exercise-induced asthma showed that inhaled
heparin preserved specific airway conductance better
than inhaled cromolyn or placebo following exercise.
Studies investigating the effects of inhaled heparin on
bronchial reactivity following inhaled allergen challenge have yielded mixed results.
Side Effects and Contraindications
The most devastating complication of anticoagulant
therapy is bleeding. The occurrence of bleeding, primarily in the gastrointestinal tract and the central
nervous system, is a major cause of morbidity and
mortality. Consequently, anticoagulants are contraindicated in any case in which the risk of hemorrhage
may be greater than the potential clinical benefit of
anticoagulation. Risk factors for bleeding include
advanced age, serious illness (cerebral, cardiac, kidney, or liver disease), cerebrovascular or peripheral
vascular disease, and an unstable anticoagulant effect. Whereas the action of unfractionated heparin
may be antagonized by protamine sulfate, no specific
antidote is available for LMW heparins, danaparoid,
fondaparinux, or the direct thrombin inhibitors
128 ANTICOAGULANTS
hirudin and ximelagatran. The anticoagulant effect
of coumarin derivatives may be antagonized by low
doses of vitamin K and substitution with fresh frozen
plasma. Other adverse effects of heparins are heparin-induced thrombocytopenia and osteoporosis,
which can occur after long-term treatment and increase the risk for fractures.
An absolute contraindication for coumarins is pregnancy because they cause fetal bone abnormalities
by interfering with g-carboxylation of proteins synthesized in the bone. Heparins do not cross the placental barrier and do not cause these problems.
Long-term use of ximelagatran carries the risk of
severe liver injury. Administration in patients with
hepatic disease is not recommended.
As outlined previously, HIT is an antibody-mediated adverse effect of heparin associated with an increased thrombotic risk. According to the seventh
American College of Clinical Pharmacology consensus conference guidelines, alternative, nonheparin
anticoagulants should be used in patients with
strongly suspected or confirmed HIT. Recommendations include direct thrombin inhibitors as well as
danaparoid. Although withdrawn from the US market, danaparoid remains approved and available for
prevention and treatment of HIT in Canada, Europe,
Australia, and Japan. Because fondaparinux does not
cross-react with HIT antibodies, it may also be useful
for this indication. A general recommendation,
however, cannot be made due to the minimal data
available. Antihirudin antibodies are commonly generated during treatment with lepirudin. Although
they are usually not clinically significant, the European Agency for the Evaluation of Medical Products
recommended the use of nonhirudin anticoagulants
in patients who have previously been exposed to
lepirudin. Coumarins should not be used in patients
with confirmed or strongly suspected HIT because
they can contribute to skin necrosis and venous
limb gangrene. For patients receiving coumarins at
the time of diagnosis of HIT, the use of vitamin K
for reversal of coumarin-induced anticoagulation is
recommended.
See also: Acute Respiratory Distress Syndrome.
Coagulation Cascade: Overview; Antithrombin III;
Factor X; Protein C and Protein S; Thrombin. Interstitial Lung Disease: Overview; Idiopathic Pulmonary Fibrosis. Pulmonary Thromboembolism: Deep Venous
Thrombosis; Pulmonary Emboli and Pulmonary Infarcts.
Thrombolytic Therapy.
Further Reading
Ansell J, Hirsh J, Poller L, et al. (2004) The pharmacology and
management of the vitamin K antagonists: the seventh ACCP
conference on antithrombotic and thrombolytic therapy. Chest
126: 204S–233S.
Bussey H, Francis JL, and the Heparin Consensus Group (2004)
Heparin overview and issues. Pharmacotherapy 24: 103S–107S.
Desai UR (2004) New antithrombin-based anticoagulants. Medical Research Reviews 24: 151–181.
Ginsberg JS, Greer I, and Hirsh J (2001) Use of antithrombotic
agents during pregnancy. Chest 119: 122S–131S.
Happel KI, Nelson S, and Summer W (2004) The lung in sepsis:
Fueling the fire. American Journal of the Medical Sciences 328:
230–237.
Hirsh J and Raschke R (2004) Heparin and low-molecular-weight
heparin: The seventh ACCP conference on antithrombotic and
thrombolytic therapy. Chest 126: 188S–203S.
Hirsh J, Fuster V, Ansell J, and Halperin JL (2003) American Heart
Association/American College of Cardiology Foundation guide
to warfarin therapy. Circulation 107: 1692–1711.
Idell S (2001) Anticoagulants for acute respiratory distress syndrome. Can they work? American Journal of Respiratory and
Critical Care Medicine 164: 517–520.
Idell S (2002) Adult respiratory distress syndrome: Do selective
anticoagulants help? American Journal of Respiratory Medicine
1: 383–391.
Laterre PF, Wittebole X, and Dhainaut JF (2003) Anticoagulant
therapy in acute lung injury. Critical Care Medicine 31(supplement): S329–S336.
Nowak G (2002) Pharmacology of recombinant hirudin. Seminars
in Thrombosis and Hemostasis 28: 415–423.
Srivastava S, Goswami LN, and Dikshit DK (2005) Progress in the
design of low molecular weight thrombin inhibitors. Medical
Research Reviews 25: 66–92.
Warkentin TE and Greinacher A (2004) Heparin-induced thrombocytopenia: Recognition, treatment, and prevention. The seventh ACCP conference on antithrombotic and thrombolytic
therapy. Chest 126: 311–337.
Weitz JI (1997) Low-molecular weight heparins. New England
Journal of Medicine 337: 688–698.
Weitz JI, Hirsh J, and Samama MM (2004) New anticoagulant
drugs. The seventh ACCP conference on antithrombotic and
thrombolytic therapy. Chest 126: 265S–286S.
Wittkowsky AK (2003) Warfarin and other coumarin derivatives:
Pharmacokinetics, pharmacodynamics, and drug interactions.
Seminars in Vascular Medicine 3: 221–230.
Antioxidants see Oxidants and Antioxidants: Antioxidants, Enzymatic; Antioxidants, Nonenzymatic.
ANTIVIRAL AGENTS 129
ANTIVIRAL AGENTS
M A Parniak, E N Vergis, and M E Abram,
University of Pittsburgh, Pittsburgh, PA, USA
& 2006 Elsevier Ltd. All rights reserved.
Abstract
Most respiratory tract infections (RTIs) are viral in origin; numerous different viruses cause RTIs, many of which can be quite
serious. However, only five drugs are approved for their treatment, and these drugs are directed at only two viruses. Four
drugs – amantadine, rimantadine, zanamivir, and oseltamivir –
are used for the prophylaxis and treatment of influenza virus
infection. These drugs target two different stages of influenza
replication. Orally administered amantadine and rimantadine
act only against influenza A and block the ion channel formed
by the viral M2 protein, thereby preventing viral ribonucleoprotein release. Zanamivir and oseltamivir are active against
influenza A and B, and they are competitive inhibitors of the
viral neuraminidase. These drugs block nascent viral release
from the infected cell and thus viral spread by preventing
neuraminidase-catalyzed removal of sialic acid residues from
membrane glycoproteins. Oseltamivir is administered orally,
whereas zanamivir must be delivered topically by inhalation of
the dry powder. The fifth approved drug, the ribonucleoside
analog ribavirin, is administered by nebulization and is approved for the treatment of pediatric respiratory syncytial virus
infection. Its mechanism of action is uncertain, but it may involve alteration of cellular nucleotide pools and inhibition of
viral RNA synthesis.
Introduction
Viruses are the cause of most respiratory tract infections (RTIs); numerous different viruses can infect
the respiratory tract. Many of these infections can be
quite serious, especially in the young, the elderly,
chronically ill, and immunocompromised individuals. However, there are only five drugs approved for
the treatment of viral RTIs, and these drugs are directed at only two viruses. Four of the approved
drugs are used for the treatment or prophylaxis of
influenza, and one is used for the treatment of pediatric respiratory syncytial virus infection.
Drugs for the Treatment of Influenza
Influenza virus mainly infects the upper respiratory
tract, and most individuals recover within approximately 1 week without medical treatment. Nevertheless, RTI due to influenza is a major cause of
morbidity and mortality worldwide. The World
Health Organization estimates that influenza can
account for up to 15% of worldwide upper RTIs
annually, with up to 500 000 deaths. Influenza viruses
that cause human disease are classified into groups
A–C. Influenza A is the most serious pathogen since it
leads to large recurrent epidemics with significant
morbidity and mortality. Four drugs (Figure 1) –
the adamantane derivatives amantadine and rimantadine and the neuraminidase inhibitors zanamivir and
oseltamivir – are used for the treatment or prophylaxis of influenza virus infection. These drugs
target different stages of influenza virus replication
(Figure 2). Amantadine and rimantadine inhibit an
early stage involved in the uncoating of the viral ribonucleoprotein, whereas zanamivir and oseltamivir inhibit a viral enzyme needed to allow nascent virus
release from the infected cell.
Amantadine and Rimantadine
The inhibitory activity of amantadine (adamantan-1ylamine hydrochloride) against influenza A was first
identified in 1964, and this drug was first marketed
in 1966. The close analog, rimantadine (1-adamantan-1-yl-ethylamine hydrochloride), was developed
subsequently due to the adverse neurological side
effects associated with the use of amantadine. These
structurally similar compounds are used primarily
for prophylaxis of influenza A infection, particularly
for individuals with high risk of exposure such as
healthcare workers, and prevent influenza-like illness
in up to 80% of treated individuals. Both drugs can
also reduce the duration of symptoms of established
influenza A infection if therapy is initiated within
48 h of discernible symptoms. The adamantanes are
effective only against influenza A. However, since
influenza A is the source of most serious cases of
influenza-related RTI, the adamantanes are valuable
drugs for the treatment and especially prophylaxis of
influenza infections.
Amantadine and rimantadine are administered
orally, once or twice daily with adult dosages of
100–200 mg per day. For prophylaxis, the drugs are
administered for up to 7 days during high-risk periods
of an epidemic. The two drugs have very different
pharmacokinetic profiles despite their similarity in
structure. Amantadine is rapidly absorbed after oral
delivery, has a half-life of approximately 15 h, is not
metabolized, and is excreted almost entirely in the
urine. In contrast, rimantadine is more slowly absorbed, has a half-life twice that of amantadine, and
undergoes extensive hepatic metabolism. Although
peak plasma levels of rimantadine are less than those
130 ANTIVIRAL AGENTS
H3C
NH2 HCl
NH2 HCl
Rimantadine
Amantadine
O
OH
O
OH
NH
O
H
H
HO
N
H
NH2
NH2
O
HN
HN
OH
OH
O
Zanamivir
O
Oseltamivir
Figure 1 Drugs used for prophylaxis or treatment of influenza virus infection. The drug oseltamivir is shown as the active caboxylic
acid form, although it is administered as the ethyl ester pro-drug.
of amantadine, the former is more highly concentrated in respiratory secretions than amantadine,
thereby providing better access to the viral target.
Mechanism of action Amantadine and rimantadine
target the influenza A virus M2 membrane protein.
The influenza virus enters the cell via endocytosis,
and fusion of the virus membrane envelope with the
endosome membrane provides a mechanism for the
release of the viral ribonucleoprotein (RNP) into
the cytoplasm and thus to the nucleus, where replication of the viral genomic RNA can take place. The
RNP interacts with the viral M1 protein that underlies the viral envelope. Furthermore, M1 masks a
nuclear localization signal on the RNP. Unless the
M1–RNP interactions are disrupted, the RNP can
neither be released into the cytoplasm nor imported
into the nucleus. Disassembly of the complex may
also be important for activation of the viral RNA
polymerase. The influenza M2 membrane protein is
critical in enabling disruption of the M1–RNP complex. The M2 protein is a homotetramer that is considered to form an ion channel in the viral membrane
envelope (Figure 3). During acidification of the viruscontaining endosome, the M2 protein enables
protons to enter the virus particle. Acidification of
the interior of the virus particle results in dissociation
of the RNP from the M1 protein. This allows release
of the RNP into the cytoplasm and subsequent import into the nucleus. Amantadine and rimantadine
bind to the viral M2 membrane protein, blocking
the channel and preventing proton transport into
the viral particle. The M2 protein is not found in
influenza B virus, which accounts for the lack of antiviral activity of amantadine and rimantadine
against influenza B.
Contraindications Central nervous system side effects are noted in significant numbers (up to 10%) of
amantadine-treated individuals. Symptoms include
dizziness, anxiety, difficulty in concentrating, and insomnia, all of which are problematic for healthcare
workers, a major target group for prophylactic treatment during influenza epidemics. Central nervous
system side effects reverse once treatment is stopped.
Severe toxic reactions or death can occur in patients
with renal insufficiency and include serious neurotoxicity (mental status changes, hallucinations, tremor, myoclonus, seizures, and coma) or cardiac
arrhythmias. Dosages of the drugs, especially amantadine, must be lowered and carefully monitored
in such patients. Rimantadine is significantly better
tolerated than amantadine.
ANTIVIRAL AGENTS 131
Zanamivir, oseltamivir
Amantadine,
rimantadine
Figure 2 Influenza virus replication cycle showing stages inhibited by approved therapeutic drugs. Following attachment, endocytosis,
and acidification of the virion-containing endosome, the virion core is also acidified in a process mediated by the viral M2 membrane
proton channel protein. This enables a pH-dependent release of viral ribonucleoprotein (RNP). Adamantane drugs target the virus M2
protein, preventing virion acidification and release of RNP. Nascent virus release from an infected cell requires cleavage of terminal
sialic acid residues from cell surface and virion envelope proteins. Neuraminidase inhibitors target this stage of replication, preventing
spread of virus to uninfected cells.
Neuraminidase Inhibitors
The viral neuraminidase inhibitors zanamivir (5-acetylamino-4-gunadino-6-(1,2,3-trihydroxypropyl)-5,6dihydro-4H-pyran-2-carboxylic acid) and oseltamivir
(4-acetylamino-5-amino-3-(1-ethylpropoxy)-cyclohex1-ene carboxylic acid) were approved for therapeutic
use in 1999. Both drugs are reversible competitive inhibitors of influenza virus neuraminidase. The drugs
are effective against both influenza A and influenza B
and also against strains of influenza A virus resistant to
the adamantane drugs. Zanamivir and oseltamivir
have virtually no activity against human cell neuraminidases at levels that provide potent antiviral
activity. Viral resistance to zanamivir or oseltamivir is
uncommon. Both drugs are approved for treatment of
influenza in adults and children (1 year or older for
oseltamivir and 7 years or older for zanamivir) within
48 h of onset of discernible symptoms. Oseltamivir can
also be used for prophylaxis in adults. Zanamivir has
low oral bioavailability and is administered topically
by dry powder inhalation, whereas oseltamivir can be
132 ANTIVIRAL AGENTS
H+
NH2HCl
V
V
H
A
S H
Viral membrane envelope
H
G
A
S
G
M2 protein subunits
Figure 3 Schematic of the mechanism of adamantane drug inhibition. The drugs interact with specific residues of the influenza A virus
M2 transmembrane protein. This protein forms a channel for the influx of protons into the virion. Only two of the four M2 subunits that
form the channel are shown. Binding of amantadine or rimantadine blocks the channel, preventing intravirion acidification.
administered orally (bioavailability 460%, with a
plasma half-life of approximately 8 h). Oseltamivir is
not metabolized and is excreted almost entirely in the
urine. Oseltamivir is administered as the ethyl ester
pro-drug that lacks antiviral activity. This is hydrolyzed to the active oseltamivir carboxylate by esterases
in the intestinal mucosal epithelia and in the liver.
Mechanism of action Zanamivir and oseltamivir
target the viral enzyme neuraminidase. Neuraminidase, also called sialidase, is one of the two
major influenza viral proteins (the other is hemagglutinin, which is important for binding and fusion
of the virus envelope with the host cell). Neuraminidase is essential for release of newly formed
virus from infected cells and for virus spread
throughout the respiratory tract of the infected host.
Nascent virions bud off from the infected cell encased in an envelope composed of a lipid bilayer derived from the cell plasma membrane with associated
viral envelope proteins. Many cell surface glycoproteins of mammalian cells possess sialic acid as the
terminal sugar, a residue added during glycosylation
events in subcellular Golgi organelles. Influenza virus
envelope proteins are formed in the same subcellular
compartment and are therefore also sialylated. However, the cell surface receptor for influenza virus attachment is sialic acid. Removal of the cell surface
and viral envelope protein sialic acid residues is essential to prevent reattachment of the nascent virions
to the same cell and to prevent aggregation of the
virus particles. Neuraminidase cleaves terminal sialic
acid residues from cellular and viral membrane
glycoproteins, thus destroying the cellular receptors
recognized by the viral hemagglutinin.
Influenza neuraminidase is a tetramer of identical
subunits (Figure 4). The active site in each subunit is
a deep groove on the protein surface that comprises
residues that are apparently identical in all strains of
influenza A and B. The inhibitors zanamivir and oseltamivir result from rational drug design based on
the neuraminidase sialic acid-binding site determined
by crystallography. Both drugs bind with high affinity to the active site of viral neuraminidase (Figure 4),
thus preventing binding of the normal sialic acid
glycoprotein substrate. However, the inhibition
mechanisms of the two drugs differ. Zanamivir is
an analog of sialic acid and acts as a classical competitive inhibitor to prevent substrate binding to the
unliganded enzyme. Oseltamivir is a transition-state
analog of sialic acid cleavage and thus interferes with
neuraminidase conformational changes needed to allow substrate binding. Nonetheless, the result is the
same, and both drugs have similar antiviral potency.
Contraindications Few serious clinical toxicities
have been reported with zanamivir or oseltamivir.
Inhaled zanamivir is generally well tolerated, although exacerbations of asthma may occur. Orally
administered oseltamivir may cause nausea and
ANTIVIRAL AGENTS 133
Figure 4 Structure of influenza B virus neuraminidase bound with the inhibitor zanamivir (in red). The structure was based on
pdb1A4G. Adapted from Taylor NR, Cleasby A, Singh O et al. (1998) Dihydropyrancarboxamides related to zanamivir: a new series of
inhibitors of influenza virus sialidases. 2. Crystallographic and molecular modeling study of complexes of 4-amino-4H-pyran-6-carboxamides and sialidase from influenza virus types A and B. Journal of Medicinal Chemistry 41: 798–807.
gastrointestinal discomfort, although these are transient and less likely if the drug is administered with
food.
NH2
O
N
Drug for the Treatment of Respiratory
Syncytial Virus Infection
N
N
O
Ribavirin
HO
The synthetic nucleoside ribavirin (1-(3,4-dihydroxy5-hydroxymethyl-tetrahydrofuran-2-yl)-1H-[1,2,4]triazole-3-carboxylic acid amide) is a guanosine
analog that is approved for the treatment of pediatric respiratory syncytial virus (RSV) infection
(Figure 5). RSV is the most common cause of severe
lower respiratory tract disease in infants, causing up
to 90% of bronchiolitis and 40% of bronchopneumonia cases. Disease symptoms are much milder in
older children and adults. Although orally administered ribavirin is rapidly absorbed with good bioavailability (approximately 45%), treatment of
pediatric RSV infection requires the drug to be administered by continuous aerosol (nebulizer) for 12–
18 h daily for 3–6 days. This therapy is expensive and
requires specialized equipment and monitoring. Its
use is therefore generally reserved for treatment of
RSV infection in high-risk infants (e.g., premature
infants, immunocompromised children, or infants
with congenital heart disease).
antiviral activity of ribavirin is unclear and may differ for different types of viruses. However, it is generally thought that ribavirin therapy alters cellular
nucleotide pools, thereby affecting viral mRNA
synthesis. It is likely that ribavirin must be phosphorylated by cellular kinases to provide antiviral
activity. Ribavirin-50 -monophosphate inhibits the cellular enzyme inosine-50 -phosphate dehydrogenase,
blocking the conversion of inosine-50 -monophosphate
to xanthosine-50 -monophosphate. This decreases levels of guanosine triphosphate (GTP), which impacts
on both RNA and DNA metabolism. Ribavirin-50 triphosphate may also be a competitive inhibitor of
GTP-dependent 50 -capping of viral mRNA.
Mechanism of action Ribavirin is in fact a broadspectrum antiviral agent that inhibits a wide range
of RNA and DNA viruses. The mechanism for the
Contraindications Aerosolized ribavirin may cause
bronchospasm or conjunctival irritation. It requires close supervision, especially with mechanical
HO
OH
Figure 5 Structure of ribavirin, which is used for the treatment
of high-risk pediatric respiratory syncytial virus infection.
134 APOPTOSIS
ventilation, because precipitation of the drug may
occur. Healthcare workers involved in the administration and monitoring of aerosol ribavirin treatment
may occasionally experience irritation of the eyes
and respiratory tract. Ribavirin is mutagenic, teratogenic, and embryotoxic; thus, aerosolized ribavirin is a risk to pregnant healthcare workers.
See also: Aerosols. Bronchiolitis. Pneumonia: Viral.
Upper Respiratory Tract Infection. Vaccinations:
Viral. Viruses of the Lung.
Further Reading
Abdel-Magid AF, Maryanoff CA, and Mehrman SJ (2001) Synthesis of influenza neuraminidase inhibitors. Current Opinion in
Drug Discovery and Development 4: 776–791.
Broughton S and Greenough A (2004) Drugs for the management
of respiratory virus infection. Current Opinion in Investigative
Drugs 5: 862–865.
De Clercq E (2004) Antiviral drugs in current clinical use. Journal
of Clinical Virology 30: 115–133.
Dreitlein WB, Maratos J, and Brocavich J (2001) Zanamivir and
oseltamivir: two new options for the treatment and prevention
of influenza. Clinical Therapeutics 23: 327–355.
Garman E and Laver G (2004) Controlling influenza by inhibiting
the virus’s neuraminidase. Current Drug Targets 5: 119–136.
Johnston SL (2002) Anti-influenza therapies. Virus Research 82:
147–152.
Kandel R and Hartshorn KL (2001) Prophylaxis and treatment of
influenza virus infection. BioDrugs 15: 303–323.
Lew W, Chen X, and Kim CU (2000) Discovery and development
of GS4104 (oseltamivir): an orally active influenza neuraminidase inhibitor. Current Medical Chemistry 7: 663–672.
Oxford JS, Bossuyt S, Balasingam S, et al. (2003) Treatment of
epidemic and pandemic influenza with neuraminidase and M2
proton channel inhibitors. Clinical Microbiology and Infection
9: 1–14.
Snell NJC (2001) New treatments for viral respiratory tract infections – opportunities and problems. Journal of Antimicrobial
Chemotherapy 47: 251–259.
Tam RC, Lau JY, and Hong Z (2001) Mechanisms of action of
ribavirin in antiviral therapies. Antiviral Chemistry and Chemotherapy 12: 261–272.
Taylor NR, Cleasby A, Singh O, et al. (1998) Dihydropyrancarboxamides related to zanamivir: a new series of inhibitors of influenza virus sialidases. 2. Crystallographic and
molecular modeling study of complexes of 4-amino-4H-pyran6-carboxamides and sialidase from influenza virus types A and
B. Journal of Medicinal Chemistry 41: 798–807.
Torrence PF and Powell LD (2002) The quest for an efficacious
antiviral for respiratory syncytial virus. Antiviral Chemistry and
Chemotherapy 13: 325–344.
APOPTOSIS
A M K Choi and X Wang, University of Pittsburgh,
Pittsburgh, PA, USA
& 2006 Elsevier Ltd. All rights reserved.
Abstract
The phenomenon of programmed cell-death, which is more
commonly known as apoptosis, was first described by C Vogt in
1842. Apoptosis, the term was proposed by Kerr and colleagues
in 1972, is an active process of cellular self-destruction with
distinctive morphological and biochemical features . It is indispensable in the development and maintenance of homeostasis
within all multicellular organisms. The molecular machinery of
apoptosis is also important for the regulation of homeostasis
during adulthood, which is important for the control of neoplasia and autoimmunity. Understanding how apoptosis occurs in
different situations will help to understand the pathogenesis of a
number of human diseases and therefore provide clues to the
treatment.
Apoptotic Pathways
Caspases are involved in various programmed celldeath pathways reported in the literature. They are a
family of cysteine proteases, and many of them are
implicated as important initiators or effectors of the
apoptosis process. To date, at least 14 members of this
family have been identified, but only a subset of them
are partially or fully characterized. Like many enzymes, caspases are synthesized in the cell as inactive
precursors and can be cleaved to active form comprised of two associated heterodimers. They are activated or inactivated through a series of intracellular
steps, or pathways, in response to death or survival
signals, which are subject to multiple regulations.
Clearly, the discovery of diverse apoptosis pathways
involving signals primarily via the death receptors
(extrinsic pathway) or the mitochondria (intrinsic
pathway) using caspases as effector molecules has
dominated the field. Lately, a more sophisticated view
of signaling pathways has arisen which leads to the
observation that cell death can occur even in the absence of caspases.
The death receptor pathway. The extrinsic pathway is initiated at the cell surface through the
APOPTOSIS 135
and caspase-8 are the key components of the Fas
DISC. Once caspase-8 associates with FADD, the
high local concentration of caspase-8 is believed to
lead to its autoproteolytic cleavage to p43/41 and
p20, and activation.
Studies of knockout mice and mutant cell lines established that the DISC is essential for Fas apoptosis
signaling. DISC assembly differs between cell types in
ways that influence the efficiency of Fas signaling. In
‘type I’ cell lines and re-stimulated primary T cells,
the DISC forms efficiently, and apoptosis can be induced with bivalent anti-Fas stimuli without additional cross-linking. The activated caspase-8 directly
cleaves caspase-3 in its downstream. However, in
‘type II’ cell lines and recently activated primary T
cells, the DISC is formed inefficiently, hypercrosslinking of Fas is necessary to induce apoptosis, and
caspase-8 does not activate caspase-3 and cleaves Bid
directly instead. The truncated Bid (tBid) translocates
to mitochondria (Figure 1).
TNF receptor type I (TNFR1) is another important
member in the death receptor family. TNF is a highly
pleiotropic cytokine that elicits diverse cellular
responses ranging from proliferation and differentiation to activation of apoptosis. The different biological activities are mediated by two distinct cell
surface receptors: TNFR1 and TNFR2. TNFR1 appears to be the key mediator of TNF signaling. Upon
binding of its ligand, TNFR1 recruits the adaptor
protein TRADD directly to its cytoplasmic DD. In
turn, TRADD serves as an assembly platform to diverge TNFR1 signaling from the DD: interaction of
Fas/TNF-R1 family protein. Ligation of Fas either by
its ligand, FasL, or by its agonistic antibodies triggers
the homotrimeric association of the receptors. The
clustering of the death domains (DDs) in the intracellular portion of the receptors recruits the adapter
molecule, Fas-associated DD containing protein
(FADD), which then recruits procaspase-8. Activation of procaspase-8 through self-cleavage leads to a
series of downstream events, including activation of
procaspase 3, cleavage of multiple caspase substrates, and induction of mitochondrial damages.
Fas (CD95/APO-1) is the best-characterized member of the tumor necrosis factor (TNF) superfamily
of receptors. Its main and best-known function in
signaling is the induction of apoptosis. Fas receptors
are expressed on the surface of cells as preassociated
homotrimers. Fas is a prototype death receptor characterized by the presence of an 80 amino acid DD in
its cytoplasm tail. This domain is essential for the
recruitment of a number of signaling components
upon activation by either agonistic anti-Fas antibodies or the cognate Fas ligand that initiate apoptosis.
The complex of proteins that form upon triggering of
Fas is called the death-inducing signaling complex
(DISC). The DISC consists of an adaptor protein and
initiator caspases and is essential for induction of
apoptosis. A number of proteins have been reported
to regulate formation or activity of the DISC. In
the DISC, the adaptor molecule FADD is bound to
Fas. FADD has been shown to interact with several
proteins through its death effector domain (DED),
including caspase-8. These two molecules, FADD
FasL
Fas
Membrane
}
FADD
Caspase-8
Activated caspase-8
DISC
Bid
Bax
tBid
Activated-Bax
Mitochondria
Cytochrome c
Caspase-9
Caspase-3
Cell death
Figure 1 A schematic diagram outlining the cell death pathways.
136 APOPTOSIS
TRADD with RIP-1 and TRAF-2 leads to nuclear
factor kappa B (NF-kB) activation. Alternatively,
TRADD can recruit FADD and procaspase-8, which
is subsequently activated to initiate apoptosis.
Under native conditions, a controversy has been
reported about the formation of a TNFR1-associated
DISC at the plasma membrane. It was reported that
TNFR1-induced apoptosis involves two sequential
signaling complexes. The initial plasma membrane
bound complex (complex I) consists of TNFR1, the
adaptor TRADD, the kinase RIP1, and TRAF2 and
rapidly signals activation of NF-kB. In a second step,
TRADD and RIP1 associate with FADD and caspase-8, forming a cytoplasmic complex (complex II)
in human fibrosarcoma cells, HT1080. Recently, it
has been reported that the DISC forms at the plasma
membrane in several cell lines. We agree with this
report in that the difference between the results of
Micheau and co-workers and Harper and co-workers
may be based on the different compositions of the
lysis buffers used and the fact that different labeling
and precipitation protocols are employed; we also
discovered that the DISC formed at the plasma
membrane in primary cultured hepatocytes (our unpublished results), which is consistent with the results of Schneider-Brachert and co-workers.
The mitochondrial pathway. Many death stimuli
do not seem to depend on the death receptor pathway. Instead, the death signals are transmitted to mitochondria through unique intracellular signaling
pathways, where release of cytochrome c is induced.
Cytochrome c activates Apaf-1, in the presence of
dATP, which in turn activates procaspase-9. Activated
caspase-9 can then cleave downstream effector caspases. Mitochondria apoptosis pathway is involved
in many types of cell death induced by stress signals,
such as irradiation, DNA-damaging drugs, hormone,
or growth factor withdrawal. It has to be pointed out
that cytochrome c release and caspase activation may
not be the only effects caused by the various insults
on mitochondria. Others include mitochondrial
depolarization and free-radical generation. Death
stimuli transmitted through the Fas/TNFR1 death receptor family are mainly mediated directly by caspase
cascades in cytosol. However, in certain types of cells,
such as hepatocytes, the effector caspases may not be
efficiently activated by caspase-8 and thus the mitochondria pathway mediated by Bid becomes critical. As mentioned above, Bid is cleaved by caspase-8
and translocated to mitochondria to induce cytochrome c release.
The mitochondria pathway is subject to regulation
by Bcl-2 family proteins. This family of proteins consists of both death antagonists (Bcl-2, Bcl-XL, Bcl-W,
Bfl-1, and Mcf-1) and death agonists (Bax, Bak, Bid,
Bim, Bnip3, Bnip3L (Nix), Bad, Bik, and Bok). They
share structural homology in Bcl-2 homology (BH)1,
2, 3, and 4 domains, although not all members have
all domains. The BH1 and BH2 domains of the
antagonists are required to heterodimerize with the
death agonists and repress cell death. Conversely, the
BH3 domain of the death agonists is required for
them to heterodimerize with the death antagonists
and to promote cell death. Bcl-2 family proteins can
regulate caspase activation through the regulation of
cytochrome c release from mitochondria, which is
inhibited by the death antagonists (Bcl-2 or Bcl-XL),
and promoted by the death agonists (Bax or Bid).
However, the exact mechanisms by which Bcl-2 proteins modulate apoptosis are still subject to much
debate and controversy. One hypothesis is that both
proapoptotic and antiapoptotic Bcl-2 proteins bind
directly to components of the mitochondrial pore,
leading to either its opening or closure, respectively. A
second hypothesis is that upon activation, proapoptotic members such as Bax and Bak insert into the
outer mitochondrial membrane where they oligomerize to form a protein-permeant pore of their own.
Regulation of Bcl-2 proteins can occur at multiple
levels. For example, Bid is cleaved by caspase-8 to
form tBid, which then translocates to the mitochondrion and induces permeability transition, suggesting a
linkage between the extrinsic and intrinsic pathways.
In the context of signal transduction, phosphorylation also plays a critical role in regulating Bcl-2 proteins. In general, serine phosphorylation of Bad by
multiple kinases causes its sequestration and hence
inactivation by 14-3-3 proteins, and phosphorylation
of Bim can target it for proteosomal degradation (13).
The other apoptosis is the endoplasmic reticulum
(ER)-involved pathway. As mentioned above, two
major apoptotic cascades are triggered by specific
initiator caspases: the death receptor pathway and the
mitochondrial pathway. These are activated by
caspase-8 and caspase-9, respectively. The key caspase in the ER pathway is caspase-12. Proper folding
of polypeptide into a three-dimensional structure is
essential for cellular function, and protein malfolding
can threaten cell survival. Various conditions can
perturb the protein folding in the ER, which is collectively called ER stress. In order to adapt ER stress
conditions, the cells respond in three distinct ways
such as transcriptional induction of ER chaperones,
translational attenuation, and ER-associated degradation. After ER functions are severely impaired, the
cell is eliminated by apoptosis via transcriptional induction of CHOP/GADD153, the activation of c-Jun
NH2-terminal kinase, and/or the activation of caspase-12. This type of cellular stress is receiving increased attention because it is considered a cause of
APOPTOSIS 137
pathologically relevant apoptosis and is especially
implicated in neurodegenerative disorders. ER stress
activates caspase-12 on the surface of the ER, and
caspase-12-deficient cells are resistant to ER stress
inducers, indicating that caspase-12 is significant in
ER stress-induced apoptosis. However, the mechanism responsible for caspase-12 activation is largely
unknown, unlike in the case of both caspase-8 and
caspase-9 whose activation mechanisms have been
revealed at the molecular level.
Organelle Dysfunction
Mitochondria. As the main energy source for all
cellular processes, the proper function of the mitochondrion is important to the survival of the cell.
The dysfunction of this organelle can usually be attributed to depolarization of the membrane, resulting
in the loss of its property of selective mitochondrial
membrane permeability (MMP), usually resulting in
cell death. Many proapoptotic signals and antiapoptotic defenses converge in the mitochondrion. The
congregation of proteins like Bax on the outer membrane cause alteration in the membrane permeability
and compromise the integrity of the mitochondrial
membrane, where MMP ensue. On the other hand,
the mitochondrion can mount a defense through the
antideath members of the Bcl-2 family, namely Bcl-2
and Bcl-XL. For example, these proteins usually reside at the mitochondrial membrane and can inhibit
the function of Bax and consequently prevent the
onset of MMP.
It has been reported that caspase-8 resides in
mitochondria. In the human breast carcinoma cell
line MCF7, caspase-8 predominantly colocalizes
with and binds to mitochondria. After induction of
apoptosis through Fas or TNFRI, active caspase-8
translocate to plectin, a major cross-linking protein
of the three main cytoplasmic filament systems,
whereas the caspase-8 prodomain remain bound to
mitochondria.
Golgi apparatus. To date, there is no direct evidence suggesting that the Golgi apparatus is directly
involved in the initiation of any apoptotic pathway.
However, it has been reported that many apoptosissignaling proteins are enriched at the Golgi membrane. Among them are caspase-2, TNFR, Fas, and
TNF-related apoptosis-inducing ligand receptor 1.
Recently, we have found that the DISC (Fas/FADD/
caspase-8) forms first in Golgi complex, and then
translocates to plasma membrane. The blockage of
the DISC transport dramatically decreases the DISC
level in plasma membrane.
Plasma membrane. The plasma membrane is the
first line of defense for the cell against extracellular
stimuli. With the right type of stimulus, the corresponding receptor will instigate a signal cascade that
results in cell death. To date, three different types of
death receptors have been identified: the TNFR, the
Fas, and the APO-3. There was a report that Fas in
type I lymphocytes associates with glycosphingolipid-enriched detergent-resistant membrane microdomains termed lipid rafts. Although there is a
controversy if the DISC formation is involved in lipid
raft, we have found that the DISC formation localizes in the lipid raft of lung endothelial cells exposed
to hypoxia/reoxygenation. The lipid-based machinery may be involved in the formation of carriers
trafficking from the Golgi complex to the cell
surface.
Bcl-XL
It is well known that Bcl-XL protects cells from
apoptosis mediated by a variety of stimuli, but the
mechanisms are not fully understood. Overexpression of Bcl-XL reportedly confers protection upon
mitochondria, making it more difficult for numerous
stimuli to induce permeability transition pore opening; or Bcl-XL tends to form small channels that
assume a mostly closed conformation, preferring
cations; Bcl-XL sequesters BH3 domain-only molecules in stable mitochondria complexes, preventing
the activation of Bax. Bcl-XL was reported to block
Bax translocation. Studies by others reported that
Bcl-XL could not block activation of Fas by FasLinduced apoptosis, especially caspase-8 activation. A
role for caspase-8 mediating Bid cleavage accompanied by cytochrome c release was observed in focal
cerebral ischemia endothelial cells and focal cerebral
ischemia in rats. It has been established that Bcl-XL
blocks Bid cleavage and functions downstream of
caspase-8 to inhibit Fas-induced apoptosis of MCF7
breast carcinoma cells. Recently, we have found that
Bcl-XL inactivates caspase-8 by disrupting DISC formation in the plasma membrane, Golgi complex, and
nucleus. Bcl-XL retains the DISC in mitochondria
where caspase-8 is inactivated. Bcl-XL breaks the
physical association of Fas and caspase-8 with
GRASP65, a Golgi-apparatus-related protein. This
indicates that Bcl-XL downregulates the transfer of
DISC to the plasma membrane by the Golgi component, at the same time diverting the DISC formation
to the mitochondria. FLIP (FLICE (Fas-associated
death-domain-like IL-1beta-converting enzyme)-inhibitory protein)) was identified as an inhibitor of
Fas signals. At least four splice variants have been
identified (31–34). The largest variant FLIP long
(FLIPL), a protein of 440 amino acids, is highly homologous to caspase-8. In fact, FLIPL contains a
138 APOPTOSIS
caspase-like domain at the carboxy-terminus but, as
indicated by the name, was originally characterized
as a molecule with inhibitory activity on caspase-8,
in which FLIPL inhibits the final cleavage between
the prodomain and the p20 subunit of the p43/41
intermediate.
In addition to inhibition of receptor-mediated
apoptosis, more recently, it has been reported that
FLIP also promotes activation of NF-kB and Erk
signaling pathway. Therefore, FLIP is not simply an
inhibitor of death-receptor-induced apoptosis but
also mediates the activation of NF-kB. The death
effector domain (DED) of FLIP binds to Fas/FADD
complexes and inhibits the recruitment and activation of procaspase-8 and therefore acts as antiapoptotic molecule. We have reported that, in addition to
inhibiting the recruitment of caspase-8 into the DISC
by decreasing DISC formation in the plasma membrane, FLIP blocks the transfer of the DISC formed
in the Golgi to the plasma membrane. FLIP expression also inhibits Bax activation and Bax-induced
apoptotic cell death by promoting the association of
the inactive form of PKC to Bax, which inactivates
Bax.
The Effect of Tyrosine Kinases on
Apoptosis
Tyrosine kinases are important mediators of the
signaling cascade, determining key roles in diverse
biological processes such as growth, differentiation,
metabolism, and apoptosis in response to external
and internal stimuli. Tyrosine kinases are a family
of enzymes, which catalyze phosphorylation of select
tyrosine residues in target proteins, using ATP. Tyrosine kinases are primarily classified as receptor tyrosine kinases, such as epidermal growth factor
receptor (EGFR), platelet-derived growth factor receptor (PDGFR) and c-Met, and nonreceptor tyrosine
kinases, such as SRC, ABL, FAK, and Janus kinase.
The receptor tyrosine kinases are not only cell surface
transmembrane receptors, but also enzymes having
kinase activity. The structural organization of the receptor tyrosine kinase exhibits a multidomain extracellular ligand for conveying ligand specificity, a
single pass transmembrane hydrophobic helix, and a
cytoplasmic portion containing a tyrosine kinase domain. The kinase domain has regulatory sequence
both on the N- and C-terminal end. Nonreceptor
tyrosine kinases are cytoplasmic proteins, exhibiting
considerable structural variability. The nonreceptor
tyrosine kinase has a kinase domain and often possesses several additional signaling or protein–protein
interacting domains such as Src homology (SH)2,
SH3, and the phosphotyrosine homology (PH)
domain. Generally, receptor tyrosine kinases activate
many signaling pathways to inhibit apoptosis by regulating Bcl-2 family member protein expression and
their phosphorylation. For example, receptor tyrosine
kinases activate the serine/threonine kinase Akt,
which inhibits apoptosis through the Bad phosphorylation. Here, we focus on recent novel findings that
tyrosine kinases are involved in apoptotic regulation.
The initiation of Fas-mediated apoptotic pathway
is to stimulate Fas activities through Fas phosphorylation. The phosphorylation at the Fas tyrosine is
thought to be prerequisite for Fas membrane trafficking and DISC formation in hepatocytes exposed
to hyperosmolarity and Fas ligand in endothelial cells
of the lung exposed to hypoxia/reoxygenation (our
unpublished results). EGFR is reported to associate
with Fas and induce Fas phosphorylation at the
tyrosine site through EGFR tyrosine kinase activity.
Hepatocyte growth factor (HGF) receptor, c-Met,
can inhibit Fas-mediated apoptosis by physically
binding to Fas, which may block the Fas conformation change required for trafficking and DISC formation in hepatocytes. HGF can upregulate the
c-Met/Fas association through c-Met-signaling pathway in lung epithelial and endothelial cells (our
unpublished results). HGF also inhibits Bax and Bid
activation by activating p38 MAPK that induces
Bax and Bid phosphorylation, which decreases their
conformation change and activation.
The Src kinase family comprises three ubiquitously
expressed members (Src, Fyn, Yes), which share functional domains such as an amino-terminal myristoylation sequence for membrane targeting, SH2 and SH3
domains, a family member-specific unique region, and
a kinase domain and a carboxy-terminal noncatalytic
domain. Recent studies on vascular smooth muscle
cells and endothelial cells indicated an involvement of
c-Src, but not of Fyn, in the c-Jun NH2-terminal kinase (JNK) activation in response to oxidative stress.
Also, oxidative stress-induced activation of Erk-5 in
fibroblasts was shown to be Src-dependent, but not
Fyn-dependent, whereas Fyn, but not Src, was required for activation of p90 ribosomal S6 kinase
by reactive oxygen species. It has been reported that
hydrophobic bile acids rapidly activate Yes but not
Fyn, Src, or Lck. Activated Yes associates with the
EGFR in a protein kinase A-sensitive way and acts as
an EGFR-activating kinase, thereby triggering a decisive event in bile acid-induced apoptosis.
Phosphorylation of the p53 tumor suppressor
protein is a critical event in the upregulation and
activation of p53 during cellular stress. It was
demonstrated that the signaling pathway linking
oxidative stress to p53 through PDGFb receptor.
Hydrogen peroxide-induced phosphorylation of the
APOPTOSIS 139
PDGFb receptor and p53 activation was inhibited by
kinase-inactive forms of the PDGFb receptor.
Concluding Remarks
Apoptosis plays an important role in cell growth,
differentiation, and homeostasis. It is obvious that
our current knowledge in terms of the mechanism
of apoptosis and its regulation is far from complete.
For example, we do not have conclusive answers to
questions such as: how does the DISC formation
transport from Golgi complex to plasma membrane?
What is the importance/significance of the DISC
formed first in Golgi complex? How does FLIP affect
the Bax/mitochondria apoptotic pathway? What is
the mechanism of the dual function (antiapoptosis
and proapoptosis) of tyrosine kinases? Much more
effort is required to dissect and document these
pathways. Studies of the apoptotic regulation will
help understand the mechanisms of cell death or
cancer development. We must better characterize the
novel pathways of cell death and further our understanding of the pathologies underlying a variety of
human health problems.
See also: CD14. Cysteine Proteases, Cathepsins.
DNA: Repair. Extracellular Matrix: Collagens. Hepatocyte Growth (Scatter) Factor. Ion Transport:
Overview. Myofibroblasts. NADPH and NADPH Oxidase. Oncogenes and Proto-Oncogenes: jun Oncogenes. Transcription Factors: AP-1; NF-kB and Ikb.
Tumor Necrosis Factor Alpha (TNF-a).
Further Reading
Anto RJ, Mukhopadhyay A, Denning K, and Aggarwal BB (2002)
Curcumin (diferuloylmethane) induces apoptosis through activation of caspase-8, BID cleavage and cytochrome c release: its
suppression by ectopic expression of Bcl-2 and Bcl-xl. Carcinogenesis 23: 143–150.
Bin L, Li X, Xu LG, and Shu HB (2002) The short splice form of
casperc-FLIP is a major cellular inhibitor of TRAIL-induced
apoptosis. FEBS Letters 510: 37–40.
Cao XX, Mohuiddin I, Chada S, et al. (2002) Adenoviral transfer
of mda-7 leads to BAX up-regulation and apoptosis in mesothelioma cells, and is abrogated by over-expression of BCL-XL.
Molecular Medicine 8: 869–876.
Chen K, Albano A, Ho A, and Keaney JF Jr (2003) Activation of
p53 by oxidative stress involves platelet-derived growth factorbeta receptor-mediated ataxia telangiectasia mutated (ATM)
kinase activation. Journal of Biological Chemistry 278: 39527–
39533.
Cheng EH, Wei MC, Weiler S, et al. (2001) BCL-2, BCL-X(L)
sequester BH3 domain-only molecules preventing BAXand BAK-mediated mitochondrial apoptosis. Molecular Cell 8:
705–711.
Creagh EM, Conroy H, and Martin SJ (2003) Caspase-activation
pathways in apoptosis and immunity. Immunological Reviews
193: 10–21.
Eramo A, Sargiacomo M, Ricci-Vitiani L, et al. (2004) CD95
death-inducing signaling complex formation and internalization
occur in lipid rafts of type I and type II cells. European Journal
of Immunology 34: 1930–1940.
Gniadecki R (2004) Depletion of membrane cholesterol causes
ligand-independent activation of Fas and apoptosis. Biochemical
and Biophysical Research Communications 320: 165–169.
Harper N, Hughes M, MacFarlane M, and Cohen GM (2003) Fasassociated death domain protein and caspase-8 are not recruited
to the tumor necrosis factor receptor 1 signaling complex during
tumor necrosis factor-induced apoptosis. Journal of Biological
Chemistry 278: 25534–25541.
Hsu H, Shu HB, Pan MG, and Goeddel DV (1996) TRADDTRAF2 and TRADD-FADD interactions define two distinct
TNF receptor 1 signal transduction pathways. Cell 84: 299–308.
Huang DC, Hahne M, Schroeter M, et al. (1999) Activation of Fas
by FasL induces apoptosis by a mechanism that cannot be
blocked by Bcl-2 or Bcl-x (L). Proceedings of the National
Academy of Sciences, USA 96: 14871–14876.
Kataoka T, Budd RC, Holler N, et al. (2000) The caspase-8 inhibitor FLIP promotes activation of NF-kappaB and Erk signaling pathways. Current Biology 10: 640–648.
Kerr JF, Wyllie AH, and Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue
kinetics. British Journal of Cancer 26: 239–257.
Kroemer G and Reed JC (2000) Mitochondrial control of cell
death. Nature Medicine 6: 513–519.
Krueger A, Schmitz I, Baumann S, Krammer PH, and Kirchhoff S
(2001) Cellular FLICE-inhibitory protein splice variants inhibit
different steps of caspase-8 activation at the CD95 death-inducing signaling complex. Journal of Biological Chemistry 276:
20633–20640.
Liou AK, Clark RS, Henshall DC, Yin XM, and Chen J (2003) To
die or not to die for neurons in ischemia, traumatic brain injury
and epilepsy: a review on the stress-activated signaling pathways
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Micheau O and Tschopp J (2003) Induction of TNF receptor Imediated apoptosis via two sequential signaling complexes. Cell
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Momoi T (2004) Caspases involved in ER stress-mediated cell
death. Journal of Chemical Neuroanatomy 28: 101–105.
Muppidi JR and Siegel RM (2004) Ligand-independent redistribution of Fas (CD95) into lipid rafts mediates clonotypic T cell
death. Nature Immunology 5: 182–189.
Paul MK and Mukhopadhyay AK (2004) Tyrosine kinase – role
and significance in Cancer. International Journal of Medical
Science 1: 101–115.
Peter ME and Krammer PH (2003) The CD95(APO-1/Fas) DISC
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Plesnila N, Zinkel S, Amin-Hanjani S, Qiu J, Korsmeyer SJ, and
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AQUAPORINS
A S Verkman and Y Song, University of California,
San Francisco, CA, USA
& 2006 Elsevier Ltd. All rights reserved.
Abstract
Aquaporins (AQP) are a family of water transporting proteins
expressed in many epithelial, endothelial, and other tissues.
AQP1 is expressed in microvascular endothelia throughout the
lung/airways, AQP3 in basal cells in large airways, AQP4 at the
basolateral membrane of epithelia throughout the airways, and
AQP5 at the apical membrane of type I alveolar epithelial cells
and submucosal gland acinar cells. The expression of some of
these aquaporins increases near the time of birth and appears to
be regulated by growth factors, inflammation, and osmotic
stress, suggesting a role of aquaporins in lung physiology. However, studies in transgenic mouse models of AQP deficiency have
provided evidence against an important physiological role for
aquaporins in many lung functions. Although AQP1 and AQP5
provide the principal route for osmotically driven alveolar water
transport, alveolar fluid clearance in the neonatal and adult lung
is not affected by AQP deletion, nor is lung CO2 transport or
fluid accumulation in experimental models of lung injury. In the
airways, AQP3 and AQP4 facilitate water transport; however,
airway hydration, airway surface liquid layer volume and composition, and airway fluid absorption are not impaired by
AQP3/AQP4 deletion. In airway submucosal glands, AQP5 deletion significantly reduced the rate and increased the protein
content of fluid secretions. Thus, although AQPs have many
important extrapulmonary physiological functions, in the lung/
airways AQPs appear to be important mainly in airway
submucosal gland function. The substantially slower rates of
fluid transport in airways, pleura, and lung compared to renal
and some secretory epithelia may account for the apparent lack
of functional significance of AQPs at these sites. However, the
possibility remains that AQPs may play a role in lung physiology under conditions of stress/injury not yet tested or in
functions unrelated to transepithelial fluid transport.
Description
Fluid movement between the distal airspace and interstitial and vascular compartments of the lung/airways is important in the maintenance of airspace
hydration, the absorption of airspace fluid near the
time of birth and in pulmonary edema, and the secretion of fluid onto the airway surface by submucosal glands. Aquaporins (AQPs), a family of small
(B30 kDa monomer) integral membrane water
channel proteins, are expressed in many cell types
involved in fluid transport. There are more than 10
aquaporins in mammals, at least four of which are
expressed in the respiratory system (Figure 1). AQP1
is expressed in microvascular endothelia near airways and alveoli, as well as in microvessels and mesothelial cells of visceral and parietal pleura. AQP3 is
expressed in the basolateral membrane of basal epithelial cells lining the trachea and large airways, and
at the basal membrane in human (but not rodent) alveoli. AQP4 is expressed in the basolateral membrane
AQUAPORINS 141
AQP3
AQP4
AQP5
Nasopharnyx
(upper airways)
Submucosal
gland
Airway
surface
liquid
Microvessels
AQP1
Trachea
AQP4
AQP3
Pleura
Lung
Distal
airway
AQP1
Alveolus
Type I
Microvessels
Type II
AQP5
AQP1
Figure 1 Aquaporin expression in lung and airways. AQP1 is expressed in microvascular endothelial and pleural membranes; AQP3
in large airway epithelia; AQP4 at the basolateral membrane in epithelia throughout the airways; and AQP5 at the apical membrane of
alveolar type I cells and of serous acinar cells in submucosal glands.
High osmolality
Low osmolality
Water
Figure 2 Tetrameric structure of AQPs. Each AQP tetramer consists of four B30 kDa monomers, each of which contains a narrow
water-transporting pore. Water moves across AQP pores in response to osmotic gradients.
of ciliated columnar cells in bronchial, tracheal, and
nasopharyngeal epithelia. AQP5 is expressed in the
apical membrane of type I alveolar epithelial cells and
of acinar cells in submucosal glands. AQP5 has also
been reported in human lung to be expressed in
bronchial and nasopharyngeal acinar and ciliated
duct columnar cells. This expression pattern in fluid
transporting cells provides indirect evidence for a role
of aquaporins in lung/airway physiology. AQPs 1, 4,
and 5 are water selective, and consist of tetramers of
four monomeric B30 kDa subunits, each of which
contains an independent narrow pore pathway for
water transport (Figure 2), whereas AQP3 (an ‘aquaglyceroporin’) transports both water and glycerol.
142 AQUAPORINS
Normal Physiological Processes
Developmental Regulation of Aquaporin
Expression
Rodent studies have shown developmental regulation
of lung aquaporin expression with distinct patterns
for each aquaporin. AQP1 is detectable just before
birth in rodents, increasing several-fold perinatally
and into adulthood. Functional measurements in
rabbit showed significantly increased lung water permeability in the perinatal period in parallel to increasing AQP1 expression. AQP1 expression is also
upregulated by treatment with corticosteroids. In
contrast, little AQP5 is expressed at birth and gradually increases until adulthood, whereas AQP4 expression strongly increases just after birth and is
upregulated by -agonists and glucocorticoids.
Regulation of Aquaporin Expression in Adult Lung
Regulated aquaporin expression is also seen in the
adult lung. As in prenatal lung, AQP1 expression can
be upregulated by corticosteroids. AQP1 and AQP5
expression are reduced in rodent lung following
adenoviral infection, and AQP5 expression is increased after bleomycin exposure. Reduced AQP5 expression was found after exposure of a mouse lung
epithelial cell line (MLE-12) to tumor necrosis factor
alpha (TNF-a), suggesting a possible mechanism for
its downregulation in viral infection in vivo. AQP5
expression was increased in MLE-15 and alveolar
epithelial cells exposed to hypertonicity. Although
potentially interesting, the physiological relevance of
many of these observations is unclear, as the airway/
lung is probably not exposed to significant hypertonicity, and regulated AQP expression is a general
phenomenon not specific to the lung/airways.
Role of Aquaporins from Functional Studies in
Knockout Mice
Transgenic mice lacking each of the lung aquaporins
(AQP1, AQP3, AQP4, and AQP5) were generated in
our laboratory both individually and in combinations.
Comparative studies were done in wild-type and AQPdeficient mice to investigate the physiological role of
aquaporins in the lung/airways. These mice have been
quite informative in elucidating important roles of
various aquaporins outside the lung. For example,
mice lacking AQPs 1–4 are defective in their ability to
concentrate urine, mice lacking AQP1 have impaired
angiogenesis and cell migration, mice lacking AQP3
have reduced epidermal hydration, mice lacking AQP4
have altered brain water balance and impaired neural
signal transduction, mice lacking AQP5 have impaired
saliva secretion, and mice lacking AQP7 manifest progressive fat accumulation and adipocyte hypertrophy.
Fluid Transport in Distal Lung
The proposed aquaporin functions in distal lung include alveolar fluid absorption at the time of birth
and in the adult lung, gas (CO2, O2) exchange, and
formation/resolution of lung edema in response
to acute and subacute lung injury. AQP5 is expressed
in type I alveolar epithelial cells and AQP1 in microvascular endothelial cells. Osmotic water permeability between alveolus and capillary was B10-fold
reduced in lungs of AQP1 and AQP5 null mice
compared to wild-type mice, and 30-fold reduced
by AQP1/AQP5 codeletion (in double knockout
mice). Interestingly, AQP1 deletion mildly reduced
hydrostatic lung edema in an isolated perfused
lung preparation, and computerized tomographic
analysis of two humans lacking AQP1 showed a
blunted increase in airway wall thickness following
saline infusion compared to control subjects. Reduced
hydrostatic lung edema in AQP1 deficiency may be
related to an abnormality in microvasculature deficiency, since on theoretical grounds hydrostatic driving forces should be unable to produce significant net
fluid movement across a water-only pathway.
Alveolar fluid clearance is an important function
of the alveolar epithelium. Fluid absorption is the
result of sodium absorption through the epithelial
sodium channel (ENaC) in response to the electrochemical driving force created by the basolateral
membrane Na-K-ATPase of type II alveolar epithelial
cells. The consequent osmotic imbalance drives
water absorption primarily through the type I cells.
Alveolar fluid clearance is measured in fluid-filled
lung models from the kinetics of increasing concentration of an airspace volume marker such as radiolabeled albumin. Remarkably, even with maximal
stimulation of alveolar fluid absorption with betaagonists and pretreatment with keratinocyte growth
factor (to increase the number of type II cells), AQP1
or AQP5 deletion did not reduce alveolar fluid clearance. Further, the rapid absorption of fluid from the
airspace just after birth was not impaired by aquaporin deletion, nor was lung edema following acidinduced epithelial cell injury, thiourea-induced
endothelial cell injury, or hyperoxic subacute lung
injury. The much slower rate of maximal alveolar
fluid absorption (0.016 ml min 1cm 2) compared to
fluid absorption in kidney proximal tubule or saliva
secretion in salivary gland (410 ml min 1 cm 2)
may account for the lack of effect of AQP1 and
AQP5 deletion on alveolar fluid clearance. Because
rates of fluid transport are relatively low in lung, the
low intrinsic (aquaporin independent) water permeability of the alveolar epithelial and capillary
membranes appears to be adequate to allow fluid
AQUAPORINS 143
transport to occur without impairment under normal
physiological conditions and in response to clinically
relevant stresses.
Fluid Transport in the Pleura
Fluid is continuously secreted into and reabsorbed
from the pleural space. Little fluid is present in the
pleural space (0.2 ml kg 1) despite its large surface
area (4000 cm2 in man, 10 cm2 in mouse). Fluid entry
into the pleural space involves filtration across microvascular endothelia near the pleural surface, and
movement across a mesothelial barrier lining the
pleural space, whereas fluid clearance is thought to
occur primarily by lymphatic drainage. Pleural fluid
can accumulate in pathological conditions such as
congestive heart failure, lung infection, lung tumor,
and the acute respiratory distress syndrome. AQP1 is
expressed in microvascular endothelia near the visceral and parietal pleura and in mesothelial cells in
visceral pleura. Osmotic water permeability across the
pleural barrier, measured from the kinetics of pleural
fluid osmolality changes after instillation of hypertonic or hypotonic fluid into the pleural space, was
rapid in wild-type mice (50% osmotic equilibration in
2 min), and slowed by fourfold in AQP1 knockout
mice. However, the clearance of saline instilled in the
pleural space was not affected by AQP1 deletion, nor
was the accumulation of pleural fluid in a fluid overload model produced by intraperitoneal saline administration or in a thiourea model of acute endothelial
injury. Thus, although rapid osmotic equilibration
across the pleural surface is facilitated by AQP1, as
found in distal lung, AQP1 does not appear to play a
major role in physiologically important mechanisms
of pleural fluid accumulation or clearance.
Fluid Transport in the Airways
Potential functions of aquaporins in the airways include humidification of inspired air, regulation of airway surface liquid (ASL) volume and composition,
and absorption of fluid from the airways. Evaporative
water loss in the airways is thought to drive water
influx from capillaries and interstitium into the ASL by
the generation of an osmotic gradient. The depth and
ionic composition of the ASL should depend theoretically on the ion transporting properties of the airway
epithelium and the rate of evaporative water loss, as
well as the water permeability of the airway-capillary
barrier. Osmotic water permeability in upper airways,
measured by dilution of an airway volume marker in
response to an osmotic gradient, was reduced in mice
lacking AQP3 and/or AQP4. However, there was little
effect of AQP3/AQP4 deletion on humidification of
lower airways, as measured from the moisture content
of expired air during mechanical ventilation with dry
air through a tracheotomy, or of upper airways, as
measured from the moisture content of dry air passed
through the upper airways in mice breathing through a
tracheotomy. Also, the depth and salt concentration
of the ASL in the trachea, as measured in vivo using
fluorescent probes and confocal microscopy, was not
altered by AQP3/AQP4 deficiency. Finally, active isosmolar fluid absorption, measured in nasopharyngeal
airways (using a volume marker as done for alveolar
fluid clearance) was not impaired by aquaporin deletion. Thus, although AQP3/AQP4 facilitate osmotic
water transport in the airways, they play at most a
minor role in airway humidification, ASL hydration,
and isosmolar fluid absorption. Interestingly, one
study showed increased airway reactivity in response
to bronchoconstricting agents in AQP5 null mice. The
mechanism of this phenotype was not established, but
may be related to indirect effects of AQP5 deletion on
agonist-induced fluid secretion from submucosal
glands as described below.
Fluid Secretion by Airway Submucosal Glands
Submucosal glands in mammalian airways secrete a
mixture of water, ions, and macromolecules onto
the airway surface. Glandular secretions are important
in establishing ASL fluid composition and volume,
and in antimicrobial defense mechanisms. Abnormally
viscous gland secretions in cystic fibrosis have been
proposed to promote bacterial adhesion and inhibit
bacterial clearance. Submucosal glands contain serous
tubules, where active salt secretion into the gland lumen creates a small osmotic gradient driving water
transport across a water-permeable epithelium, as well
as mucous cells and tubules, where viscous glycoproteins are secreted. AQP5 is expressed at the luminal
membrane of the serous epithelial cells. Pilocarpinestimulated fluid secretion was found to be reduced by
twofold in AQP5 null mice, as determined by nasopharyngeal fluid collections and video imaging of fluid
droplets (covered with mineral oil) secreted by individual submucosal glands. Analysis of secreted fluid
showed a twofold increase of total protein concentration in AQP5 null mice, suggesting intact protein and
salt secretion across a relatively water-impermeable
epithelial barrier. There was no significant difference
of submucosal gland morphology or density in wildtype versus AQP5 knockout mice. AQP5 thus facilitates fluid secretion in submucosal glands, indicating
that the luminal membrane of serous epithelial cells is
the rate-limiting barrier to water movement.
CO2 Transport by Aquaporins in Lung
AQP1-dependent CO2 transport has been proposed
based on measurements in AQP1-overexpressing
144 ARTERIAL BLOOD GASES
Xenopus oocytes; however, subsequent studies
showed unimpaired CO2 permeability in AQP1-deficient erythrocytes, where rapid CO2 transport
occurs by a passive membrane solubility-diffusion
mechanism. Measurements of CO2 movement in the
perfused and in vivo mouse lung showed no effect of
AQP1 deletion, providing direct evidence against the
role of AQP1 in lung CO2 transport.
Aquaporins and Respiratory Disease
A small number of AQP1-deficient humans have been
identified. Although initially reported to have no phenotype, subsequent studies showed that they manifest a urine concentrating defect that is qualitatively
similar to that found in AQP1-deficient mice. As
mentioned above, AQP1-deficient subjects have also
been found to have a small reduction in the increase
in bronchiolar wall thickness following intravenous
volume overload compared to normal controls,
though other lung phenotypes have not been reported. The significance of this observation is unclear.
Together with a considerable body of data in
transgenic mice, the functional studies suggest that
aquaporins play at most a minor role in normal lung
physiology and clinically relevant states of lung injury, with the possible exception of AQP5 in submucosal fluid secretion. It remains unresolved why
aquaporins are expressed at multiple sites of fluid
movement in the lung/airways, and why the expression of lung aquaporins appears to be altered in states
of stress. When available, nontoxic aquaporin-selective inhibitors will be useful to examine effects of
acute aquaporin inhibition, which may reveal lung/
airway phenotypes that might not be manifest in mice
or humans with chronic aquaporin deficiency. If significant aquaporin-dependent phenotypes are found,
then pharmacological modulation of aquaporin function may have clinical applications, such as AQP5
inhibition in reducing glandular fluid secretions in
allergic and infectious rhinitis, or increasing AQP5
expression/function to reduce secretion/ASL viscosity
in cystic fibrosis.
See also: Basal Cells. Fluid Balance in the Lung.
Further Reading
Agre P, King LS, Yasui M, et al. (2002) Aquaporin water
channels from atomic structure to clinical medicine. Journal
of Physiology 542: 3–16.
Bai C, Fukuda N, Song Y, et al. (1999) Lung fluid transport in
aquaporin-1 and aquaporin-4 knockout mice. Journal of Clinical Investigation 103: 555–561.
Borok Z and Verkman AS (2002) Lung edema clearance: 20 years
of progress. Invited review: role of aquaporin water channels in
fluid transport in lung and airways. Journal of Applied Physiology 93: 2199–2206.
Dobbs L, Gonzalez R, Matthay MA, et al. (1998) Highly waterpermeable type I alveolar epithelial cells confer high water
permeability between the airspace and vasculature in rat lung.
Proceedings of the National Academy of Sciences, USA 95:
2991–2996.
Folkesson H, Matthay MA, Frigeri A, and Verkman AS (1996)
High transepithelial water permeability in microperfused distal
airways: evidence for channel-mediated water transport. Journal
of Clinical Investigation 97: 664–671.
King LS, Nielsen S, and Agre P (1996) Aquaporin-1 water channel
protein in lung-ontogeny, steroid-induced expression, and distribution in rat. Journal of Clinical Investigation 97: 2183–
2191.
King LS, Nielsen S, Agre P, and Brown RH (2002) Decreased
pulmonary vascular permeability in aquaporin-1-null humans.
Proceedings of the National Academy of Sciences USA 99:
1059–1063.
Krane CM, Fortner CN, Hand AR, et al. (2001) Aquaporin-5 deficient mouse lungs are hyperresponsive to cholinergic stimulation. Proceedings of the National Academy of Sciences, USA 98:
14114–14119.
Ma T, Fukuda N, Song Y, Matthay MA, and Verkman AS (2000)
Lung fluid transport in aquaporin-5 knockout mice. Journal of
Clinical Investigation 105: 93–100.
Saadoun S, Papadopoulos M, Hara-Chikuma M, and Verkman AS
(2005) Targeted AQP1 gene deletion impairs angiogenesis and
cell migration. Nature 434: 786–792.
Song Y and Verkman AS (2001) Aquaporin-5 dependent fluid secretion in airway submucosal glands. Journal of Biological
Chemistry 276: 41288–41292.
Verkman AS (2002) Physiological importance of aquaporin water
channels. Annals of Medicine 34: 192–200.
ARTERIAL BLOOD GASES
J W Severinghaus, UCSF, San Francisco, CA, USA
& 2006 Elsevier Ltd. All rights reserved.
Abstract
Blood gas analyzers consist of three electrodes measuring pH,
PCO2 , and PO2 at 371C. They were introduced in about 1960
following inventions by R Stow (CO2) and L Clark (PO2) both
dating from 1954. From these outputs, internal computers calculate O2 saturation, base excess, bicarbonate, and other derived
variables such as the compensation by the body for acid–base
abnormalities. Arterial PO2 and PCO2 can be approximated using
heated skin surface ‘transcutaneous’ electrodes, which are
commonly used in premature infants and nurseries. Hemoglobin oxygen saturation, SO2%, is also directly measured by
ARTERIAL BLOOD GASES 145
multiwavelength blood oximeters. Arterial SO2 is approximated
by pulse oximeters, which detect the arterial pulsatile variations
in red and infrared light penetrating a finger, ear, or other tissue,
a method invented by T Aoyagi in Tokyo in 1973 that became
commercially available in 1983. Interpretation of blood gases
and acid–base balance is briefly discussed. Figures include
schema of the three electrodes, a pulse oximeter probe, an
acid–base compensation diagram, and photographs of the first
three-function blood gas analyzer, a combined PO2 PCO2 transcutaneous electrode in use on a child, and a pulse oximeter probe
on a finger.
Henderson–Hasselbalch (HH) equation:
pH ¼ pK0 þ log½HCO
3 =SPCO2 In plasma, pK0 ¼ 6.1, the effective dissociation
constant of H2CO3 (carbonic acid), calculated as S
PCO2 . S is CO2 solubility, 0.031 mM l 1 Torr 1.
HCO3 is plasma bicarbonate content calculated as
plasma [CO2 content – SPCO2 ].
Introduction
Electrodes
Arterial blood gas analyzers directly measure pH,
PCO2 and PO2 , (mmHg or Torr) and calculate standard base excess (SBE), bicarbonate (HCO3 ), oxygen
saturation (SO2), and other variables useful in diagnosis and clinical management of patients in emergencies, anesthesia, surgery, recovery, and intensive
care. These tests were rarely done until the 1950s
when electrodes were invented and developed. Understanding of acid–base and blood gas theory depended on discoveries in physical chemistry.
pH Electrode
Ionic Theory
Electrochemistry and physical chemistry of solutions
of acids, alkalis, metals, and salts were transformed from empiricism to theory by the 1884 thesis
of Svante Arrhenius in Uppsala, Sweden. Wilhelm
Ostwald then used a platinum electrode to measure
hydrogen ion strength electrically. In 1893,
Ostwald’s student Walther Nernst applied the longestablished laws of gases to ions in solution to calculate the electrical potential of batteries or cells.
Buffers
Shortly after 1900, Lawrence J Henderson at Harvard adapted the mass action law to relate [H þ ] to
PCO2 and HCO3 :
In 1905, Max Cremer noted that hydrogen ions permeated some kinds of very thin glass, developing
electrical potential gradients across the glass. Fritz
Haber and Z Klemensiewicz made the first glass pH
electrode in 1909. Its potential was a linear function
of pH, not H þ ion concentration. In 1925, the first
glass cup-shaped blood pH electrode was produced
by Phyllis T Courage. By 1933, capillary blood pH
electrodes were being made commercially. Blood pH
was corrected to 371C with the Rosenthal factor
( 0.0147 1C 1) until thermostatted blood pH electrodes became available in the 1950s. A pH and reference electrode is schematically shown in Figure 1.
Details Special glass compositions permit hydrogen
ions to diffuse through imperfectly annealed submicroscopic cracks, probably exchanging loci with
loosely bound alkali cations, especially lithium. Some
pH electrodes are sensitive to very high Na þ concentrations. At 371C, the electromotive force (EMF)
across the glass measured with reference electrodes
(usually silver–silver chloride) is 61.5 mV per pH unit
change (a 10-fold change in H þ ion concentration).
Sample
K ¼ ½Hþ ½HCO
3 =H2 CO3
Glass body
He demonstrated how respiration could buffer metabolic acids by reducing PCO2 . The kidneys can also
change blood and extracellular fluid buffer base
to partially normalize pH in respiratory acidosis or
alkalosis.
AgCl internal reference
H+ permeable
glass
Liquid junction
Saturated KCl
Origin of pH
In order to define H þ ion concentrations such as
0.000 000 04 moles liter 1 (e.g., in blood) more elegantly, in 1907, S P L Sørensen suggested defining
pH as the negative log of hydrogen ion concentration
(or activity). In 1915, K A Hasselbalch converted
Henderson’s equation to log form, later dubbed the
AgCl reference
Sample
Figure 1 Schema of a blood pH electrode with a liquid junction
to a reference electrode in a thermostatted cuvette.
146 ARTERIAL BLOOD GASES
For use in blood, the reference electrode usually contacts blood via a liquid junction containing saturated
KCl. Rapid diffusion of K þ into red cells causes an
often-ignored error of 0.01 pH at the liquid junction when calibration is done with aqueous buffers.
PCO2 Measurement
Until the worldwide polio epidemics of 1950–52,
PCO2 was measured by the HH equation. This required measuring plasma CO2 content by acidification and extraction in a manometric Van Slyke
apparatus and measuring pH and correcting it to
371C. In Copenhagen’s communicable disease hospital in 1950–51, sometimes up to 100 patients at a
time were manually ventilated by volunteers using a
bag and mask with O2. The laboratory director Poul
Astrup, needing a faster analytic method than the
HH equation, devised a new simple method. He
measured pH before and after equilibrating the sample with known PCO2 . He then computed patient
PCO2 by extrapolation. His method became the
standard for the rest of the decade.
PCO2 Electrode
At Ohio State University in 1954, while also trying to
resolve the polio problem, Richard Stow invented a
PCO2 electrode. He covered a glass bulb-shaped pH
electrode with a rubber glove (through which CO2
can diffuse but H þ cannot), over a film of distilled
water. However, Stow’s electrode drifted because
various cations in pH glass altered the distilled water
pH. This electrode was stabilized by John Severinghaus at the National Institute of Health (NIH) by
adding bicarbonate and salt, and was manufactured
by many firms from 1959 onwards. A blood PCO2
electrode is illustrated in Figure 2.
Details An internal, nearly flat, glass pH electrode
is separated from the sample by a membrane permeable to CO2 but not ions (e.g., Teflon or silastic).
Under the membrane, a spacer (e.g., lens-cleaning
tissue paper) holds a film of electrolyte containing
about 10 mM NaHCO3 and usually 0.1 M salt or
KCl. Thermostatted to 37oC, the output signal is a
log function of PCO2 , about 30 mV per decade in
distilled water (Stow’s design), doubling to 61 mV
per decade with bicarbonate electrolyte.
Oxygen Electrode
In 1950, Leland Clark at Antioch College, Ohio used
perfused isolated liver to study steroid metabolism.
He needed oxygenated blood so he built a bubble
oxygenator and discovered how to defoam the blood
using silicone oil on glass wool. The journal Science
rejected his paper on the basis that he hadn’t measured the PO2 in the oxygenated blood; to this end,
Clark invented a polarographic oxygen electrode. He
covered a platinum disc cathode sealed in glass with
cellophane to keep blood protein from poisoning the
cathode. It served his purpose but was not accurate,
requiring very high flow past the sensor due to the
depletion of oxygen at the membrane surface due
to its consumption by the cathode. In October 1954,
a sudden inspiration led Clark to substitute a less
O2-permeable and electrically insulating polyethylene membrane for cellophane, by mounting a reference electrode and cathode in electrolyte in a sealed
probe (Figure 3). His invention was presented at a
meeting of the FASEB in April 1956.
Details In a polarographic oxygen electrode, a negatively biased platinum cathode donates electrons to
Stow–Severinghaus PCO2 electrode
Sample
AgCl external
reference electrode
AgCl internal reference
H+ permeable
glass
0.1M KCl + 0.01M KHCO3
Glass body
Teflon
or
silastic
membrane
Electrolyte
in paper spacer
Sample
Figure 2 Schema of a blood PCO2 electrode with Teflon CO2 permeable membrane and spacer to contain electrolyte for pH measurement.
ARTERIAL BLOOD GASES 147
Clark-type oxygen electrode
25 µm polypropylene membrane
Sample
0.1 m KCl electrolyte
10 µm Platinum wire
Solid glass
AgCl reference
Sample
Figure 3 Schema of Clark’s polarographic PO2 electrode, after
cathode size was greatly reduced to avoid ‘stirring’ effect.
dissolved oxygen:
O2 þ 4e þ 2H2 O ) 4OH
The only major functional change since Clark’s original invention has been to reduce the cathode diameter from 2 mm to about 10 mm, requiring far more
sensitive current analysis, which was not available 50
years ago. This almost eliminated the need for the
sample to be rapidly stirred. The electrolyte usually
contains KCl, and may have added agents for viscosity. No separator is needed between cathode and
membrane. The cathode is biased to about 0.65 V
at which all oxygen molecules reaching the cathode
are reduced. Cathode current is a linear function of
the membrane surface PO2 .
Figure 4 The first blood gas analyzer containing three electrodes in a water bath at 371C with tonometer for preparing blood
for calibration of PO2 electrode. Reproduced from Severinghaus
JW (2002) The invention and development of blood gas apparatus. Anaesthesiology 97: 253–256, with permission from
Lippincott Williams & Wilkins.
Blood Gas Analyzers
In 1958, Severinghaus and Bradley created the first
three-function blood gas analyzer by mounting a
Clark PO2 electrode with a tiny stirring paddle, a
Stow–Severinghaus PCO2 electrode, and a commercial pH electrode in a water bath at 371C (Figure 4).
A small tonometer was included in which blood
could be equilibrated with air or a known gas to
calibrate the Clark electrode. Modern blood gas
analyzers compute many variables from the three
measured values.
Transcutaneous PO2
PaO2 can be estimated transcutaneously using a flat
Clark type PO2 electrode, typically internally heated
to about 431C. Heating causes sufficient dermal vasodilation to raise skin capillary PO2 to nearly equal
arterial PO2 . Heating also raises blood PO2 by about
7% per degree, while skin oxygen consumption reduces surface PO2 , these two factors approximately
canceling each other out. No correction factors for
Figure 5 Transcutaneous combined PO2 and PCO2 electrode
monitoring a patient recovering from anesthesia.
temperature or skin metabolism are thus needed. Introduced in the mid-1970s, these devices are widely
used on premature and term infants to help control
oxygen therapy and prevent blindness following retinal vascular growth interference (Figure 5).
Transcutaneous PCO2
Arterial PCO2 can also be estimated transcutaneously
using flat CO2 electrodes heated (e.g., to 431C) to
increase skin capillary blood flow. The signal must be
corrected 4.7% per degree to 371C, and reduced
about 4 Torr to compensate for skin metabolism and
electrode surface cooling.
148 ARTERIAL BLOOD GASES
Hemoglobin Oxygen Saturation
Prior to about 1970, this was measured by vacuum
extraction of oxygen from blood, before and after
equilibrating a sample with air.
Multiwavelength Oximetry
Compared with oxygenated blood, desaturated
blood strongly absorbs red light (at about 660 nM
wavelength). At the isobestic point, 805 nM (near
infrared), absorption is unaffected by oxygenation.
Multiwavelength oximeters use the ratio of optical
density of a thin film of hemolyzed blood at red and
infrared wavelengths to calculate saturation and
hemoglobin concentration, with small corrections
for other pigments such as bilirubin, detected at
other wavelengths. A typical laboratory ‘bench’ oximeter using at least five wavelengths can be precise to
at least 0.1% saturation. Some oximeters use filters
to select wavelengths while others use more stable
and precisely defined diffusion-grating monochromators, avoiding the need for user calibration. Confirmatory testing with dyes is recommended. Some
blood gas analyzers include a multiwavelength oximeter (often termed CO-oximeter because it also
measures the fraction of hemoglobin bound to carbon monoxide).
Pulse Oximetry
Light passing through a finger or ear is partly absorbed by the blood in its path. Arteries expand with
each pulse, absorbing a bit more light. Pulse oximeters measure the amplitude of the pulsatile variation
of light as a fraction of total transmitted red
(e.g., 660 nM) and infrared (e.g., 900–950 nM) light
(Figures 6 and 7). The ratio of these two ratios was
shown by T Aoyagi in 1973 (Nihon Kohden Co,
Tokyo) to be a unique function of arterial oxygen
saturation, theoretically independent of venous or
capillary saturation, or skin color, tissue thickness, or
other pigments.
In the late 1940s, Earl Wood (Mayo Clinic) modified G Millikan’s 1942 original ear oximeter by adding a pneumatic pressure capsule. Wood showed that
when blood was readmitted to a pressure-blanched
ear, the ratios of the decreases in red and infrared
light passing through the ear were unique functions
of oxygen saturation.
Indications
Blood gas analysis has become so commonplace that
its use is nearly universal in diagnosis during admission, in emergencies, trauma, intensive care, anesthesia, and surgery. It is considered by physicians to
Figure 6 Pulse oximeter probe taped on fingertip with red and
infrared LEDs on nail side and a photo diode on the dorsal side.
Photodiode
Finger
Red and infrared LEDs
Figure 7 Schema of pulse oximeter probe on a finger.
be the most useful and important diagnostic procedure available. Its indications are global and will not
be listed here.
Common Patterns of Results and
Interpretation
Diagnostic Terminology
A pH of less than 7.35 is called acidemia, while that
over 7.45 is termed alkalemia. A PCO2 over 45 Torr
indicates respiratory acidosis or hypercapnia, while
values under 35 (males) or 30 (females) indicate
respiratory alkalosis or hypocapnia. A standard base
excess (SBE) more negative than 5 mM is metabolic
acidosis and over þ 5 mM is metabolic alkalosis.
Presence of compensatory responses to chronic acid–
base respiratory or metabolic imbalances can be predicted and used in diagnosis (Figure 8).
Normal PO2 at sea level in young adults is 90–
100 mmHg. It falls with age to 60–70 mmHg at age
80. There is no consensus on what PO2 level is defined as ‘hypoxia’. Arterial oxyhemoglobin ‘functional’ saturation (100 x HbO2/[HbO2 þ HHb]) is
normally 97–98% (i.e., 2–3% deoxyhemoglobin,
ARTERIAL BLOOD GASES 149
30
7.7
7.6
7.5
7.4
Metabolic SBE (mM)
M
20
CR
10
0
AR
AR
−10
CR
−20
M
7.3
Metabolic
alkalosis 7.2
Metabolic
acidosis 7.1
7.0
pH
−30
10
20
Respiratory
alkalosis
Respiratory
acidosis
30
40
50
60
70
Respiratory PaCO2 (Torr)
80
90
Figure 8 Acid–base compensation diagram predicting in vivo
compensation for respiratory and metabolic acid–base imbalance. AR and CR, acute and chronic respiratory, respectively; M,
metabolic. Reproduced from Schlichtig R, Grogono AW, and
Severinghaus JW (1998) Human PaCO2 and standard base excess compensation of blood gas apparatus. Critical Care Medicine 26: 1173–1179, with permission from Lippincott Williams &
Wilkins.
HHb). Carboxyhemoglobin or methemoglobin or
other abnormal forms, if present, reduces ‘fractional’
saturation (or % oxyhemoglobin) computed as 100 x
HbO2/total Hb but is not counted in ‘functional’
saturation. The terms ‘hypoxia’ or ‘hypoxemia’ generally imply that SaO2 is at least 5% lower than expected (at that age and altitude).
Temperature Correction
Clinicians often ask whether they should instruct the
laboratory to correct blood gas values to patient temperature. In general, this is not necessary. The appropriate pH and PCO2 for optimal physiologic function
is the same function of temperature as are the in vitro
temperature correction factors. At 301C in a hypothermic patient, the appropriate pH is 7.4 measured at
371C, or 7.55 corrected to 301C. Animals with a normal body temperature of 301C have a pH of 7.55.
Fish in Antarctic waters at 01C have a pH of 8.0, as
does normal human blood cooled in vitro to 01C.
Blood at 90% SaO2 with PO2 ¼ 60 Torr will have
PO2 ¼ 30 Torr at 251C, but delivers oxygen at least as
effectively to tissues in hypothermia (as shown by decreased tissue lactate). However, physiologists who
wish to study pulmonary gas transport and compare
alveolar and arterial blood gas tension gradients must
correct blood values from the laboratory (at 371C) to
the body temperature under study.
SID or SBE?
The interpretation of acid–base balance divides
clinicians into two ‘schools of thought’. Strong ion
difference (SID) can identify the causes of many metabolic abnormalities in addition to obtaining an approximation of the degree of plasma metabolic acid–
base abnormality, provided that all anions and cations are measured. This unfortunately is not a measure of the whole body extracellular fluid acid–base
condition. For that one needs the SBE, which is
computed from directly measured arterial pH, PCO2 ,
and PO2 without separate ion measurements. SBE is
thus a quantitative analysis of imbalance while SID is
an approximation of imbalance with additional causative suggestions.
Hydrogen Ion Concentration or pH?
pH is used widely both for convenience and because
chemical activities and potentials are log functions of
concentrations. All animals no matter what their
normal body temperature maintain their pH 0.6 unit
above neutrality, i.e., a ratio of [OH ]/[H þ ] of
about 16 whereas [H þ ] varies by a factor of more
than 4 between 01C (fish) and 401C (hummingbirds).
Buffering is a linear function of pH, an exponential
function of [H þ ] making straight pH lines on acid–
base compensation plots.
Some clinicians prefer to interpret acid–base abnormalities using an approximation of Hendersen’s
equation:
Hþ ¼ 24PCO2 =HCO
3
using nM l 1 for H þ , mmHg for PCO2 and mM l 1
for HCO3 . This requires a constant 24 combining
the dissociation constant K, CO2 solubility, equating
carbonic acid with dissolved CO2, and a factor for
the three different units.
Oxygen Dissociation Curve Computations
and Corrections
Blood gas analytic apparatus commonly provides a
computed value of SO2 (oxygen saturation). The observed PO2 is first corrected from observed pH to
pH ¼ 7.4 by
obs
obs
ÞÞ
logP7:4
O2 ¼ logPO2 ð0:48ð7:4 pH
At pH ¼ 7.40, 371C, the relationship of SO2 to PO2
is most simply and accurately expressed by
SO2 % ¼ 100ð23; 400ðP3O2 þ 150PO2 Þ1 þ 1Þ1
No temperature correction (e.g., to patient temperature) is needed, all measurements being at 371C.
150 ARTERIES AND VEINS
SO2 in a blood sample does not vary with sample
temperature.
State of the Art
Electrodes versus Optical Sensors
Optodes can measure pH, PCO2 and PO2 using dyes,
fluorescence, quenching, and other optically responsive material, permitting the use of extremely small
sensors on optical fiber tips in blood at catheter tips
or inside tissue cells. They rival electrodes in accuracy and cost, in particular providing portable and
disposable sensors (e.g., for cardiac bypass oxygenator control, bedside and field blood gas analysis).
See also: Acid–Base Balance. Diffusion of Gases.
Diving. Erythrocytes. High Altitude, Physiology and
Diseases. Oxygen–Hemoglobin Dissociation Curve.
Permeability of the Blood–Gas Barrier. Ventilation:
Control.
Further Reading
Geha DG (1990) Blood gas monitoring. In: Blitt CDK (ed.) Monitoring in Anesthesia and Critical Care Medicine. New York:
Churchill-Livingston.
Nunn JF (1993) Nunn’s Applied Respiratory Physiology, 4th edn.
Oxford: Butterworth-Heinemann.
Schlichtig R, Grogono AW, and Severinghaus JW (1998) Human
PaCO2 and standard base excess compensation for acid-base
imbalance. Critical Care Medicine 26: 1173–1179.
Severinghaus JW (1979) Simple, accurate equations for human
blood O2 dissociation computations. Journal of Applied Physiology 46: 599–602.
Severinghaus JW (2002) The invention and development of blood
gas apparatus. Anaesthesiology 97: 253–256.
Severinghaus JW and Astrup PB (1987) History of Blood Gas
Analysis. International Anesthesiology Clinics, vol. 25(4).
Boston: Little Brown.
Severinghaus JW, Astrup P, and Murray J (1998) Blood gas analysis and critical care medicine. American Journal of Respiratory
Critical Care Medicine 157: S114–S122.
Severinghaus JW and Bradley AF Jr (1958) Electrodes for blood
PO2 and PCO2 determination. Journal of Applied Physiology
13: 515–520.
ARTERIES AND VEINS
D E deMello, Saint Louis University Health Sciences
Center, St Louis, MO, USA
& 2006 Elsevier Ltd. All rights reserved.
Abstract
The lung’s vasculature is different from that of most other
organs because it has a double arterial supply and a double
venous drainage system. The pulmonary artery which carries
deoxygenated blood to the lungs branches alongside the airways
into conventional and supernumerary arteries before it empties
into the vast capillary network in the alveolar walls where
gas exchange occurs across air–blood barriers. Conventional
and supernumerary pulmonary veins drain oxygenated blood
to the left atrium of the heart. The smaller (intra-acinar) vessels
develop at the same time as do the acini of the lung and a
major component of acinar lung development occurs postnatally. Alveoli increase from about 20 million at birth to
about 300 million by the time lung growth is complete in
adolescence. Consequently, a large component of intra-acinar
vessels develop postnatally and during this critical growth phase
are susceptible to local influences such as increased blood flow
from intracardiac left to right shunts which can curtail vessel
growth.
Arterial wall structure is composed of endothelial cells that
line the lumen, smooth muscle cells that make up the media, and
fibroblasts that contribute the adventitial fibrous sheath. The
wall structure changes from proximal to distal vessels in the
lung, and alterations in the normal structure may occur during
intrauterine life or postnatally in response to a variety of stimuli.
Altered wall structure results in functional changes reflected in
pulmonary artery pressure and resistance.
Blood vessel assembly begins in primitive mesenchymal cells
that undergo a complex series of steps before the mature vessel
structure and function is attained. These stages in vessel development are under the control of a large number of transcriptional and growth factors. The timing and dose of some of these
‘angiogenic’ factors is critical to normal embryonic and fetal
development; absence or even reduced expression of some factors is lethal for the developing embryo.
Anatomy, Histology, and Structure
Unlike most other organs, the lung, because of its gas
exchange function, has a double arterial supply and
double venous drainage (Figure 1). One of the arterial systems, the pulmonary arterial tree, serves as a
conduit for deoxygenated blood from the body to the
alveoli where it drains into a vast capillary network
within which gas exchange and oxygenation occur.
From the alveoli, oxygenated blood is transported to
the left atrium of the heart via the pulmonary veins.
The other arterial system is the bronchial arterial tree
which serves a nutrient function to the airways and
perihilar structures. The arterial supply to the pleura,
except at the hilar region, is from the pulmonary
artery. For the intrapulmonary structures there are
no bronchial veins, so all intrapulmonary structures
drain to the pulmonary vein, resulting in a small
amount of venous admixture in the left atrium. The
hilar structures, however, drain to true bronchial
ARTERIES AND VEINS 151
Pulmonary artery
Alveoli
Respiratory
bronchiolus
Pulmonary
vein
Bronchioli
Bronchial artery
Bronchi
Precapillary shunts
To left
atrium
Capillary bed
To right
atrium
Azygos vein
Figure 1 The lung’s vasculature consists of a double arterial and a double venous system. The pulmonary artery supplies the alveoli
whereas the bronchial artery supplies the airways, the pulmonary artery, and the gas exchange region. Both systems drain via the
pulmonary veins to the left atrium, except for the ‘true’ bronchial veins at the hilum which drain via the azygos veins to the right atrium.
Reproduced from Reid L, Fried R, Geggel R, and Langleben D (1986) Anatomy of pulmonary hypertensive states. In: Bergofsky EH (ed.)
Abnormal Pulmonary Circulation, vol. 4, pp. 221–263. New York: Churchill Livingstone, with permission from Elsevier.
veins and then to the azygos system and the right
atrium.
Segmental hilum
Lateral pathway
Conventional and Supernumerary Arteries
The main pulmonary artery that forms the outflow
path of the right ventricle divides into the right and
left pulmonary arteries which enter the lung at the
hilum. Within the lung the pulmonary arteries travel
alongside the airway branches within a connective
tissue sheath. The arterial branching pattern is similar
to that of the airway it accompanies, but dissection
has revealed many more branches from the pulmonary artery than from its accompanying airway. Two
types of arterial branches exist. Those that branch
dichotomously next to the airways are conventional
branches, the longest of which are the axial pathways
running the longest possible course from hilum to
distal pleura. The lateral branches of the conventional
pulmonary arteries supply the alveoli near the axial
pathways (Figure 2). The other type of branch is the
supernumerary artery which is short, arises at right
angles to the axis of the pulmonary artery, and supplies the alveoli in the immediate vicinity of the artery
(Figure 3). Supernumerary arteries are more numerous toward the periphery of any arterial pathway.
Over the preacinar length of an axial artery, the ratio
of the number of supernumerary to conventional
Axial pathways
Pleura
Figure 2 The path of axial and lateral airways in the human
lung. The pulmonary arteries are alongside the airways within the
bronchoarterial sheath and follow the same path. Reproduced
from Davies P, deMello DE, and Reid LM (1990) Structural
methods in the study of development of the lung. In Gil J (ed.)
Models of Lung Disease: Microscopy and Structural Methods,
vol. 47 of Lung Biology in Health and Disease, pp. 409–472. New
York: Dekker, with permission.
branches is of the order of 3 : 1 and the lumen crosssection area of the supernumerary side branches is
about one-third of the total cross-sectional area of all
side branches (Figure 4). Supernumerary arteries have
152 ARTERIES AND VEINS
19-week fetus
Conventional artery
Supernumerary
Airway
Hilum
1
3
1
2
2
3
4
5
4
6
5
6
7
8
7
8
9
10
10
Airways
Conventional arteries
Supernumerary arteries
Figure 3 The anatomy of the pulmonary arterial tree. Conventional arteries divide alongside the airways at acute angles to the
main axis and supply the respiratory region at the end of the axial
pathway. Supernumerary arteries are short, arise at right angles
to the main axis, and supply the airspaces adjacent to the axial
vessel. Modified from deMello DE and Reid L (2002) Vascular
development of lung. In: Tomanek R (ed.) Assembly of the
Vasculature and its Regulation, vol. 2, pp. 211–237. Boston:
Birkhauser, with permission.
a special sphincter at their point of origin which
probably serves a functional purpose in recruitment.
This valve is responsive to vasoconstrictive agents
suggesting that it may be responsible for regulation of
blood flow into the supernumerary artery. Because
the diameter of the supernumerary vessel is smaller
than that of the conventional artery from which it
arises, it has a higher critical opening pressure, providing a built-in mechanism for recruitment as pulmonary artery pressure rises. It is noteworthy that the
two types of pulmonary artery have different pharmacological profiles; for example, supernumerary arteries are 30 times more sensitive in their response
to a vasoconstrictor such as 5-hydroxytryptamine
(5-HT) compared to conventional arteries, and endogenous nitric oxide selectively attenuates the vasoconstrictor response to 5-HT in the supernumerary
but not in the conventional artery. In pathological
conditions, these short vessels provide an alternate
route for blood in the conventional artery obstructed
by thromboemboli. The capillary bed between an artery proximal to the block can open to conduct blood
to an arterial bed distal to the block. Blood flow can
produce remodeling of a vessel whether artery or
vein, and injection techniques have demonstrated that
such compensatory or collateral flow produces arcades between axial pulmonary arteries in a number
of pathological states (Figure 5).
11
11
12
12
13
14
14
18
19
21.23 21
23
25
9
15
15
16
17
17
20
20
22
24
Terminal
bronchiolus
Figure 4 Posterior basal lung segment in a 19-week fetus.
Airway and accompanying conventional artery branches are
numbered. The more numerous supernumerary arteries (58) are
also shown. Reproduced from Hislop A and Reid L (1972) Intrapulmonary arterial development in fetal life: branching pattern
and structure. Journal of Anatomy 113: 35–48, with permission
from Blackwell Publishing Ltd.
Conventional and Supernumerary Veins
The venous drainage resembles the arterial pattern in
that there are more venous tributaries than airway
branches. The ‘conventional’ veins arise from the points
of division of an airway and pass to the periphery of the
unit, combining as tributaries to form progressively
larger venous conduits. The supernumerary veins are
short and drain small regions around the conventional
veins. Additional tributaries to the pulmonary veins
arise from pleura and connective tissue septa within the
lung. Lung development is unique in that there is a set
timetable for the growth and maturation of its major
tissue components, airways, alveoli, and blood vessels,
and that a large portion of its development continues
postnatally until about 12 years of age.
Laws of Lung Development
The template for the growth pattern of the lung is
reflected in the three ‘laws of lung development’.
ARTERIES AND VEINS 153
Airway
Artery
Hilum
Vein
m
Preacinar
Resistance
artery
TB
Intraacinar
RB
AD
A
pm
Precapillary
unit
Postcapillary
unit
nm
Capillary
Figure 5 Lung arteriogram from a premature infant who died at 9
months of age with pulmonary hypertension and vascular hypo
plasia. Note dilated, tortuous vessels and interpulmonary artery
arcades (arrows). Reproduced from Rendas A et al. (1980) American Review of Respiratory Disease 121: 873–880, Official Journal
of the American Thoracic Society, & American Thoracic Society,
with permission.
Figure 6 Human lung arteriograms. Top, newborn; bottom left,
18 months; right, adult. Increase in radiodensity with age reflects
progressive postnatal increase in number of intra-acinar arteries.
Reproduced from Reid L (1978) The pulmonary circulation: remodeling in growth and disease. The 1978 J Burns Amberson
Lecture. American Review of Respiratory Disease 119: 531–546,
Official Journal of the American Thoracic Society, & American
Thoracic Society, with permission.
These generalizations are also predictive of the effect
of perturbation at different times during development and they offer a framework against which to
assess vascular growth patterns and to interpret
anomalous development.
Law I – Airways. The airways (i.e., bronchi and
bronchioli) are present by the 16th week of intrauterine life.
Figure 7 The arteriovenous loop between the right and left side
of the heart. The acinus is a unit of lung and includes that portion of
lung that is subtended by the terminal bronchiole, i.e., respiratory
bronchioles, alveolar ducts, and alveoli. The pulmonary
artery that accompanies the airways is shown, including the resistance segment that is immediately precapillary in the newborn
child when the acinus is 1 mm in diameter. In the adult the acinus
is 1 cm in diameter and the resistance vessels are further downstream at the level of the alveolar ducts and alveoli. m, muscular;
pm, partially muscular; nm, nonmuscular; TB, terminal bronchiole;
RB, respiratory bronchiole; AD, alveolar duct; A, alveolus. Reproduced from deMello DE and Reid LM (1991) Arteries and veins. In:
Crystal RG, West JB, Barnes PJ, Cherniack NS, and Weibel ER
(eds.) The Lung: Scientific Foundations, pp. 767–777. New York:
Raven Press, with permission from Lippincott Williams & Wilkins.
Law II – Alveoli. At birth the ‘alveolar’ spaces are
more primitive than in the adult. They have been
described as ‘primitive saccules’. Of these saccules
about 20 106 are present at birth. After birth the
alveoli multiply, so that by the age of 8 the number
is about 300 106 and within the adult range.
Law III – Vascular. The ‘vascular law’ reflects the
first two. The preacinar branches of the pulmonary artery (i.e., those that accompany bronchi or
bronchioli), as well as the preacinar venous tributaries, appear at virtually the same time as do the
accompanying airways. The intra-acinar vessels
appear as alveoli grow (Figure 6).
Preacinar Arteries: Structure and Size
The cellular structure of any vessel is composed of
three cell types that make up the three coats of a
vessel: the intima is constituted by endothelial cells,
the media is composed of smooth muscle cells or
their precursors, intermediate cells or pericytes, and
the adventitia is made up of fibroblasts. Intercellular
products also contribute to the composition of each
coat. Regional and organ specificity dictate functional differences. A number of mediators, cytokines,
growth factors, or hormones determine and modulate these differences.
154 ARTERIES AND VEINS
Conventional branches
Diameter of axial artery and
side branches (µm)
Supernumerary branches
1000
External diameter of
axial pathway
500
Acinus
Lobule
Figure 8 The relative sizes and numbers of conventional and supernumerary pulmonary arteries. Reproduced from deMello DE and
Reid LM (1991) Arteries and veins. In: Crystal RG, West JB, Barnes PJ, Cherniack NS, and Weibel ER (eds.) The Lung: Scientific
Foundations, pp. 767–777. New York: Raven Press, with permission from Lippincott Williams & Wilkins.
Partially
muscular
Muscular
Non
muscular
Capillary
(a)
M
I
P
E
Artery
(b)
Lumen
M
(c)
Figure 9 (a) Diagram depicting the light microscopic appearance of the structure of a pulmonary artery. In its path from the hilum to the
periphery, the muscle coat gradually disappears so that the most peripheral segment still precapillary, is nonmuscular. (b) Diagrammatic
illustration of the ultrastructural features of the pulmonary artery. From the hilum toward the periphery, muscle cells (M) are replaced by
intermediate cells (I), and in the immediate precapillary segment, pericytes (P) are present. (c) Same as (a), in cross-section. Reproduced from deMello DE and Reid LM (1991) Arteries and veins. In: Crystal RG, West JB, Barnes PJ, Cherniack NS, and Weibel ER
(eds.) The Lung: Scientific Foundations, pp. 767–777. New York: Raven Press, with permission from Lippincott Williams & Wilkins.
ARTERIES AND VEINS 155
Fetus
100
60
Percentage arterial population
20
Child
100
60
NM
M
PM
20
Adult
100
60
20
100
200
300
400
Arterial size (ED, µm)
Figure 10 The structure of vessels of different sizes at different ages in the human. The distribution of muscular (M), partially muscular
(PM), and nonmuscular (NM) arteries is similar in the fetus and adult; however, in the child, larger arteries are nonmuscular and partially
muscular. ED, external diameter. Reproduced with permission from the BMJ Publishing Group Davies G and Reid L (1970) Thorax 25:
669–681.
In the lung, the vessels proximal to the capillary
bed are arteries and those distal to the capillary bed
are veins. The immediate pre- and postcapillary vascular segments are functionally specialized and serve
to protect the capillary bed (Figure 7). They are the
key reactive site for remodeling in disease. Preacinar
artery structure is established early in fetal life as
elastic, transitional, or muscular and the program for
structure is a regional or topographic one. In the adult
along an axial pathway, the first seven generations
which are vessels down to a diameter of 3000 mm
(distended) are elastic. Vessels from 3000 to 2000 mm
in diameter representing generations seven to nine
have a transitional structure and smaller vessels down
to a diameter of 30 mm have a muscular structure.
The largest partially muscular and nonmuscular arteries are found in diameters up to 150 and 130 mm
respectively (Figure 8). Along any arterial pathway, a
complete muscular coat gives way to an incomplete
coat which at first consists of a spiral before disappearing from vessels, still larger than capillaries, to
produce a nonmuscular artery (Figure 9). On the arterial side of the capillary bed, nonmuscular, partially
muscular, and muscular small arteries are identified.
On the venous side a similar arrangement occurs. By
electron microscopy intermediate cells and pericytes,
each a precursor of the smooth muscle cell, are identified: the intermediate cell in the nonmuscular part of
the partially muscular artery and the pericyte in the
nonmuscular wall. Whereas the pericyte lies within
the basement membrane of the endothelial cell, the
intermediate cell, like a smooth muscle cell, has its
own basement membrane, but, unlike the latter does
not have dense bodies.
Characteristics of an artery include size, structure,
and the thickness of the media as well as its position within the branching pattern. Because vessel
remodeling occurs with growth and in disease, it is
best to characterize a pulmonary artery by its position in the branching pattern defined by the structure
of the accompanying airway, a landmark that remains relatively constant from fetal life. In arteries,
the relative circumferences of the internal and external elastic laminae appear to influence the size and
morphology of the endothelial cell and its microenvironment. For example, in vessels larger than
200 mm, the external lamina is shorter than the internal suggesting that it is the former that restricts
distension and that the internal lamina may never be
smoothed out. In the smaller arteries the situation is
156 ARTERIES AND VEINS
reversed suggesting that the internal lamina is the
limiting structure. The balance between these is
changed in disease. The functional significance of
this, in pulsatile flow and propagation of the pulse
wave, has not been established.
Postnatally, the length and diameter of the entire
pulmonary vascular tree changes, but structural
changes occur mainly at the sites of alveolarization
in the acinar region, that is, in vessels distal to the
level of the terminal bronchiole. In the newborn
lung the resistance arteries, that is, vessels with a
smooth muscle wall, are upstream from the alveolar
wall. Normally before birth pulmonary arterial muscularization stops at the beginning of the acinus, so
that the peripheral vessels are muscularized later
(Figures 10 and 11). In conditions such as idiopathic
pulmonary hypertension of the newborn, congenital
heart lesions associated with increased pulmonary
artery flow or pressure before birth, and pulmonary
hypoplasia, a well-developed muscle coat is found in
more peripheral and smaller arteries than is normal.
The functional correlate of this structural change
is an elevation in pulmonary artery pressure and
resistance.
AD
Species differences in structure and rate of lung
growth occur and need to be considered when interpreting experimental results. In general, animals like
the sheep that can walk or run within hours of birth
have a prenatal burst in lung development, preparing
the animal as it were for considerable, immediate
postnatal activity. In mammals like the rat, mouse,
and pig a similar burst in lung growth occurs postnatally, and in the human, postnatal lung growth
continues for several years until adolescence.
Postacinar Veins: Structure and Size
The veins are present at the periphery of an acinus
within their own connective tissue sheath. As additional tributaries are added in their course toward
the hilum, they increase progressively in size. Conventional axial veins enter the larger veins at an acute
angle and are the main pathways from periphery to
hilum. Their numbers are equivalent to the airway
generations and to conventional arteries. The supernumerary tributaries are shorter, drain the lung immediately around the axial vein, and connect with
RB
TB
Pleura
Alv
Species Variation
Normal
Fetus
3 days
10 months
3 years
10 years
19 years
Cases
PPHN
Mec Asp (fatal)
HLHS
IDTAPVR
Figure 11 Top: diagram of an acinus of the lung which includes the terminal bronchiole (TB), respiratory bronchioles (RB), alveolar
ducts (AD), and alveoli (alv). Below: the open bars indicate the level beyond which at different ages the arteries are nonmuscular. The
blue bars indicate the extent of muscularization (‘precocious muscularization’) in different disease processes: PPHN, persistent pulmonary hypertension of the newborn; Mec Asp (fatal), fatal cases of PPHN with meconium aspiration; HLHS, hypoplastic left heart
syndrome; IDTAPVR, infradiaphragmatic total anomalous venous return. Reproduced from Reid L, Fried R, Geggel R, and Langleben D
(1986) Anatomy of pulmonary hypertensive stages. In: Bergofsky EH (ed.) Abnormal Pulmonary Circulation, vol. 4, pp. 221–263. New
York: Churchill Livingstone, with permission from Elsevier.
ARTERIES AND VEINS 157
Pathway 1
1200
∗
800
External diameter of
axial pathway
Diameter of axial vein and tributaries (µm)
400
Hilum
Pathway 2
1200
800
+
+
+
400
Hilum
Pathway 3
Conventional veins
400
Supernumerary veins
Figure 12 Diagrammatic reconstruction of conventional and supernumerary venous tributaries in a 20 week fetus. Pathway 2 is
9.31 mm in length. *Point where pathway 2 joins pathway 1. þ Point where pathway 1 joins pathway 2. z Point where pathway 3 joins
pathway 2. There is a higher density of venous tributaries at the periphery. Reproduced from Hislop A (1971) The fetal and childhood
development of the pulmonary circulation and its disturbance in certain types of congenital heart disease. PhD Thesis, London
University, with permission from A A Hislop.
the conventional veins at right angles (Figure 12).
The preacinar tributaries are developed by 20 weeks
gestation, and subsequent tributaries arise within the
acinus. Supernumerary tributaries exceed conventional, their ratio being 3.5 : 1, and more vessels
drain from the capillary bed than enter.
Postcapillary veins have an endothelial lining, but
no muscle coat. Larger veins contain an internal
elastic lamina with only occasional medial smooth
muscle fibers. The medial thickness increases in yet
larger veins, but even the largest veins do not have
a well-defined external elastic lamina. Elastin and
collagen are present between the smooth muscle
cells. The largest nonmuscular veins are approximately 150 mm in diameter and as with the arteries,
overlap in size groups occurring between muscular,
partially muscular, and nonmuscular veins.
At 20 weeks gestation, the vein structure consists
of endothelial cells resting on collagen mixed with
an occasional elastic fiber. Even the largest veins have
no muscle in their wall. Scattered muscle fibers appear by 28 weeks, but a continuous layer is only
developed at term, and even then, no elastic lamina
is present. Postnatally, the thickness of the muscle
158 ARTERIES AND VEINS
increases but not as much as in arteries. At birth the
smallest muscular vein measures about 105 mm in
diameter, at 3 years the smallest is 130 mm, and at 10
years 70 mm. No significant structural change occurs
after this time.
Intra-Acinar Arteries and Veins: Structure and Size
By 5 years of age, axial pathways have finished development and future growth occurs in the more peripheral and smaller arteries that appear as alveoli
are formed, but in these vessels, muscularization is
slower, so that during childhood the transition
from muscular to partially to nonmuscular occurs
in vessels of a larger size than in the fetus or adult
(Figure 10). Therefore, in the child, the structure of
intra-acinar arteries cannot be predicted by their size.
Bronchial Arteries
Early in gestation, primitive bronchial arteries arise
from the dorsal aorta in the neck region near the
celiac axis and are distributed with the early
branches of the airways (Table 1). When lobar or
segmental airway branches are present at about the
6th week of embryonic life, the central bronchial artery branches disappear. Between the 9th and 12th
weeks, definitive bronchial arteries arise from the
aorta, pass along the superior surface of the airways,
and communicate with the pre-existing capillary bed
in the distal airways. Sometimes the primitive bronchial arteries persist and migrate with their point of
origin, to a site below the diaphragm, still near the
Table 1 The development of the bronchial arteries
Week of
gestation
Airway
Blood vessels
4th
Main
5th
Lobar
6th
9th to 12th
Segmental
Primitive ventral aorta
Pulmonary vein links to heart
6th arch supplies lung
Paired systemic arteries from
dorsal aorta
Only blood from right ventricle to
pulmonary artery
Systemic arteries disappear
Bronchial arteries enter
peribronchial plexus
The primitive paired bronchial arteries arise from the dorsal aorta
near the celiac axis in the neck. These have usually disappeared
before the 5th week; if they persist, they then migrate with the
celiac axis to below the diaphragm as seen in certain abnormal
conditions (e.g., sequestered segment).
Reproduced from deMello DE and Reid LM (1997) Arteries and
veins. In: Crystal RG, West JB, Barnes PJ, Cherniack NS, and
Weibel ER (eds.) The Lung: Scientific Foundations, 2nd edn.,
pp. 1117–1127. Philadelphia: Lippincott-Raven Press, with
permission from Lippincott Williams & Wilkins.
celiac axis. A portion of the lung bud may remain
attached and manifests later as a lung sequestration.
Arteries and Veins in Normal Lung
Function
Blood Vessel Assembly
Two processes are involved in the development of
blood vessels: (1) angiogenesis, the branching of new
vessels from pre-existing ones, and (2) vasculogenesis,
the formation of blood lakes that undergo transformation into vessels. In the lung, blood vessel development must be coordinated temporally and spatially
with airway and alveolar development. Angioblasts
are the precursor cells from which blood vessels are
derived and they are of mesodermal origin. In the
development of the pulmonary vasculature, angioblasts must interact with epithelial and mesenchymal
components of the lung. Central events determine for
a given vessel, the direction of blood flow, its distribution or drainage and its central connections, and
local or peripheral events serve as modifiers. These
determine the fine structure of the vessels as they supply the ultimate functioning unit of the lung. Vessel
branching occurs at the end of a pathway and lengthening or widening results from cell multiplication.
Chimeric experiments have shown that angioblasts
migrate widely and to considerable distances from
their sites of origin. Biologic differences in vessel cells,
for example, endothelial cells from different sites in
the vascular tree, exist, reflecting the influence of the
local milieu. The transcription factor TAL1/SCL is a
marker of angioblasts. In the presence of extracellular
matrix, endothelial cell precursors form protrusions
that result in the formation of vascular cords and then
vessels. This process requires integrin-mediated adhesions. Vascular endothelial growth factor (VEGF)
plays a role as a mitogen and as a vascular morphogen
in vasculogenesis. Whereas vascular smooth muscle
cells arise through progenitors within the mesoderm,
they are also derived from endothelial cells.
In the fetal mouse, transmission electron microscopy shows that between 9 and 10 days, primitive
angioblast precursors within the mesenchyme surrounding the lung bud, form vascular lakes that have
hematopoietic cells in their lumen (Figures 13(a)–
13(c)). Mercox pulmonary vascular injections combined with scanning electron microscopy of the
vascular casts indicate that conventional and supernumerary branches from the main pulmonary artery
are derived by angiogenesis and that the more distal
vessels, those of the future alveolar region, arise by
vasculogenesis, that is, from a lumen that appears
ARTERIES AND VEINS 159
Figure 13 Transmission electron micrographs of fetal mouse thoraces. (a) 9 day fetus: a space between densely packed mesenchymal cells contains membrane-bound vesicles. Magnification 8750. (b) 10 day fetus: mesenchymal cells around intercellular
spaces appear thin and endothelial-like. Magnification 5250. (c) 10 day fetus: some intercellular spaces contain hematopoietic precursor cells. Magnification 5250. Reproduced from deMello DE, Sawyer D, Galvin N, and Reid LM (1997) Early fetal development of
lung vasculature. American Journal of Respiratory Cell and Molecular Biology 16: 568–581, Official Journal of the American Thoracic
Society, & American Thoracic Society, with permission.
locally within the mesenchyme: at 12 days, there are
four generations of central arterial branches but no
luminal connection is seen between these vessels and
the lakes in the peripheral lung mesenchyme, where
already a dense collection of lakes containing hematopoietic cells is present. By 14 days, five to seven
generations of central artery branches, supernumerary and conventional, are present. There is now a
connection between the central vessels and the peripheral system so that casts of the peripheral vessels
are obtained by central injection (Figures 14(a)–
14(c)). Between 15 days and term, there is increasing
complexity of the peripheral vascular casts reflecting
an increase in the connections between the central
and peripheral systems (Figure 14(d)). Whereas the
processes of angiogenesis and vasculogenesis occur
separately but concurrently, a third process, fusion,
between these two systems is necessary for the circulation to be established.
In the human, examination of serial sections of
embryos and fetuses of different ages in the Carnegie
Collection of Human Embryos housed in the Carnegie Institute of Washington, DC (now located in
the Museum of Human Development in the Armed
Forces Institute of Pathology in Washington, DC)
revealed that the blood lakes are the first to form and
are present in the primitive mesenchyme around the
lung bud in the neck between stages 14 and 18 (32–
44 days of gestation). As gestation progresses, abundant lakes appear in the subpleural mesenchyme. At
stage 23 (56.5 days), pulmonary artery branches accompany the airways but lag behind the airway by
two to three generations. The thick-walled arteries
end blindly in a solid cord of cells. Between 12 and
16 weeks, an extensive capillary network is present
in the subpleural mesenchyme surrounding the most
distal airway buds but separated from them by mesenchyme. By 22 to 23 weeks, the capillary network
160 ARTERIES AND VEINS
S
S
R
L
R
PA
PA
L
PA
PA
Aorta
Aorta
13 days
12 days
(a)
(b)
PA
S
L
R
PA
L
PV
R
PV
CL
15 days
(c)
16 days
(d)
Figure 14 Photomicrographs of pulmonary arterial mercox casts of mouse fetuses. (a) 12 day fetus: up to four generations of arterial
branches are present. (b) 13 day fetus: isolated patches of peripheral vessels are filled with mercox. (c) 15 day fetus: mercox filling of
more peripheral vessels results in visualization of several generations of arterial branches with an increase in diameter. (d) 16 day fetus:
mercox filling of the expanded peripheral vascular network results in a markedly dense cast that obscures the proximal vessels. S,
systemic; R, right; L, left; PA, pulmonary artery; PV, pulmonary vein; CL, cardiac lobe. Reproduced from deMello DE, Sawyer D, Galvin
N, and Reid LM (1997) Early fetal development of lung vasculature. American Journal of Respiratory Cell and Molecular Biology 16:
568–581, Official Journal of the American Thoracic Society, & American Thoracic Society, with permission.
approaches the airway epithelium and bulges into the
air space indicating that blood barriers for future gas
exchange have formed. At this time, the pulmonary
artery accompanies even the most distal airway
branch just beneath the pleura. So from this study,
it appears that in the human also, the three processes
of vascular development, that is, angiogenesis, vasculogenesis, and fusion, contribute to the establishment of the pulmonary circulation. Other studies
using markers for endothelial cell precursors in
mouse embryos and three-dimensional reconstruction of serial sections of human embryos suggest that
the intrapulmonary vascular tree is predominantly
developed by the process of vasculogenesis.
Controlling Mechanisms: Genes and Factors
Involved in Vessel Assembly
The path from angioblast precursor within primitive
mesenchyme to mature blood vessel is complex and
ARTERIES AND VEINS 161
Primitive
mesenchyme
Hemangioblasts
Endothelial cell
commitment
('blood island')
Migration and
tube formation
Smooth muscle
recruitment and
differentiation
Mature
blood
vessel
dHAND
MEF2C
SMAD5
SmLIM
LKLF
Proteases
SCL/tal-1
Ets-1
Fra1
Vezf1
ARNT
ELF-1
EPAS
Fli-1
GATA2
GATA3
HIF-1
HOXD3
NERF-2
Ets-1
AML-1
COUP-TFII
HESR1
HOXB3
PPAR-
Transcription factors
Growth factors and receptors
bFGF
Flk-1
Flt-1
integrin V3
P/GH
TGF-
TIE2
VEGF
Angiopoietin-1
TIE2
Figure 15 The multiple steps involved in vessel assembly from primitive mesenchyme requires the sequential action of numerous
factors. Reproduced from Pediatric and Developmental Pathology vol. 7, 2004, pp. 422–424, A matter of life and breath: context article,
deMello DE, figure 1, with kind permission of Springer Science and Business Media.
involves a number of steps including commitment to
differentiate into an endothelial cell, the action of
proteases, migration and tube formation, recruitment
and differentiation of smooth muscle cells, and acquisition of mature vessel structure and function.
This complex process is under the coordinated control and influence of a large number of genes and
growth factors (Figure 15), which have been identified by experiments involving overexpression or
knockout of growth factors or genes. A fine-tuned
and delicate balance in the temporal and spatial expression of a variety of genes and factors is essential
for both normal intrauterine vessel development and
postnatal vessel maintenance. For example, knockout of the VEGF gene or even its reduced expression
in heterozygosity results in lethal defects in vessel
formation in the mouse embryo, whereas overexpression of VEGF in the lung results in the formation
of oversized vessels and disordered airway morphogenesis. Even the relative ratios of VEGF isoforms is
critical for normal vessel and airway development.
This was demonstrated in the VEGF 120 mouse in
which the other VEGF isoforms, VEGF 164 and
VEGF 188, are not expressed. Homozygous VEGF
120 mice have a severe reduction in the number
of intra-acinar arteries and air–blood barriers and a
delay in lung development resulting in hypoplastic
lungs (Figures 16 and 17).
Knockout of the Flt-1 (VEGF receptor) gene produces a lethal defect in angiogenesis and knockout of
the Flk-1 (VEGF receptor) gene results in a lethal
failure of vasculogenesis. Transforming growth factor beta (TGF-b) is important for the eventual wall
structure of the developing vasculature and influences growth, migration, and differentiation of endothelial, smooth muscle, and mesenchymal cells and
pericytes. The receptor tyrosine kinase gene (tie-1)
and receptor (Tie -2) are involved in the regulation of
vasculogenesis and angiogenesis.
Transitional Circulation and Perinatal Adaptation in
Humans
At birth, as the lung expands with air, structural
changes occur within the resistance segment of the
pulmonary arteries producing an increase in compliance, and drop in resistance and pulmonary artery
pressure as a result of an increase in external diameter and decrease in wall thickness. By 4 months the
resistance arteries lose their fetal wall thickness, but
in vessels less than 200 mm in diameter, wall thickness
gradually increases.
162 ARTERIES AND VEINS
1 mm
E13.5
1 mm
E15.5
1 mm
1 mm
1 mm
E18.5
E18.5
E18.5
Figure 16 Photographs of fetal mouse lungs from three gestational ages: top, E13.5; middle, E15.5; bottom, E18.5. Lungs of wild-type
(VEGF þ / þ , left) and heterozygous (VEGF 120/ þ , middle) fetuses do not differ in size. At all gestational ages, the lungs of homozygous fetuses (VEGF 120/120, right) are smaller than the lungs of wild-type and heterozygous littermates. Reproduced from
Galambos C, Ng Y-S, Ali A, et al. (2002) Defective pulmonary development in the absence of heparin-binding vascular endothelial
growth factor isoforms. American Journal of Respiratory Cell and Molecular Biology 27: 194–203, with permission.
Structure
The distribution of preacinar elastic and transitional
arteries is unchanged during childhood, although the
size range varies with age. Intra-acinar arteries which
develop as new alveoli are formed postnatally, are nonmuscular initially, and acquire muscle coats gradually
with time. By 10 years of age, even alveolar wall level
arteries have muscle coats.
Arteries and Veins in Respiratory
Diseases
Abnormalities of pulmonary arteries or veins can result from disorders of vessel assembly or from postnatal events that interfere with postnatal vascular
growth or cause structural remodeling of the existing
vasculature.
Veins
Disorders of Vessel Assembly
During childhood, the veins increase in size and peripheral veins increase in number. Within an acinus,
the number of veins exceeds that of the arteries
presumably to facilitate blood flow through the capillary bed.
These disorders are the consequence of aberrations
in the processes involved in vessel assembly, that
is, angiogenesis or vasculogenesis and result in failure
of growth, overgrowth, or structural/functional
abnormalities.
ARTERIES AND VEINS 163
1 mm
(a)
1 mm
(b)
1 mm
(a)
1 mm
(c)
1 mm
(b)
1 mm
(c)
Figure 17 Lung mercox vascular casts of VEGF 120/120 fetal mouse littermates. Genotypes: (a), wild-type; (b), heterozygous; (c),
homozygous. Gestational ages: top, E17; bottom, E18. The homozygous fetal casts are smaller, and fewer peripheral vessels make the
casts less dense than those of the wild type or heterozygous littermates. Reproduced from Galambos C, Ng Y-S, Ali A, et al. (2002)
Defective pulmonary development in the absence of heparin-binding vascular endothelial growth factor isoforms. American Journal of
Respiratory Cell and Molecular Biology 27: 194–203, with permission.
Aberrant angiogenesis
Absence of the main pulmonary artery or its
branches The main pulmonary artery or one of its
branches fails to grow and the lung is supplied instead by collateral vessels from the systemic circulation. Usually the pattern of intrapulmonary artery
branching is unaffected so it can be presumed that
central sprouting or angiogenesis fails, but that peripheral vasculogenesis is normal and the systemic
collaterals assume the role of the intrapulmonary arterial tree.
Misalignment of blood vessels This disorder often
accompanies another serious condition, alveolar capillary dysplasia (Figure 18). The central pulmonary
arteries and veins are ‘misaligned’ so that the veins
are displaced from their normal location at the periphery of a lung lobule and instead share the
bronchoarterial sheath. When present in all lobes of
the lung and in association with alveolar capillary
dysplasia, the condition is fatal.
Hypoplastic vascular tree: dwarfism and congenital
diaphragmatic hernia The small thoracic cage in
most forms of dwarfism or skeletal dysplasia is associated with small lungs. The main structural components of the lung, airways, alveoli, and vessels are
hypoplastic but additional abnormalities suggest a
direct metabolic effect as well.
In congenital diaphragmatic hernia, the restricted
space available for fetal lung growth produces hypoplastic lungs with a reduced number of airways and
alveoli. Because the pulmonary arteries travel and
branch with the airways, their number is also reduced and the size is small but appropriate for the
smaller lung volume. Arterial structure is also altered
so that the walls are thicker and muscle extends into
smaller, more peripheral vessels.
164 ARTERIES AND VEINS
(a)
(b)
(c)
Figure 18 (a) Post-mortem lung barium angiograms. (left) Preterm neonatal lung reveals filling of preacinar and intra-acinar arteries.
(right) Lung of a term infant with alveolar capillary dysplasia; the angiogram has the look of a ‘pruned tree’ because of a reduced number
of intra-acinar arteries. (b) In the normal lung (left), the pulmonary artery (long arrow) is present within the bronchovascular sheath and
the vein (short arrow) lies within the pulmonary septum at the periphery of the lung lobule. In misalignment of the pulmonary vessels
(right), the pulmonary arteries (long arrows) and veins (short arrows) lie within the same bronchovascular sheath. The pulmonary
arteries are filled with barium (post-mortem injection) and the veins are empty. Movat pentachrome stain. Magnification 100. (c) The
normal term lung (left) has numerous air–blood barriers (arrows) within alveolar walls. In alveolar capillary dysplasia (right) air–blood
barriers are absent and vessels larger than normal capillaries are present in the middle of thickened alveolar septa (arrows). Movat
pentachrome stain. Magnification 200. Reproduced from deMello DE (2004) Pulmonary pathology. Seminars in Neonatology 9: 311–
329, with permission.
Disordered vasculogenesis
Alveolar capillary dysplasia In this rare and fatal
disorder, air–blood barriers which are critical for gas
exchange fail to form within alveolar walls. When
the entire lung is affected, the condition is incompatible with independent air-breathing existence
(Figure 18). Sometimes, however, a single lobe is
affected suggesting failure of a local inductive mechanism for triggering capillary growth.
Fusion Intrapulmonary arteriovenous malformations point to aberrant connections between the central and peripheral pulmonary vasculature during
fetal development. This is usually a circumscribed
ARTERIES AND VEINS 165
lesion suggesting that while overall angiogenesis and
vasculogenesis has occurred normally, the mechanism that regulates fusion (? via chemotaxis) is faulty
and results in communications between arteries and
veins. Rarely, the process may involve an entire lobe.
Postnatal Disorders
Idiopathic (persistent) pulmonary hypertension of
the newborn (PPHN), with or without meconium
aspiration (also known as meconium aspiration
syndrome) In both instances, the overall pattern
of arterial growth is normal, but significant functional abnormalities at birth result from structural
alterations in preacinar and intra-acinar arteries. The
vessels are often small and have thick muscle walls
and adventitial coats. There is precocious muscularization of intra-acinar arteries so muscle coats are
present within smaller, normally nonmuscularized
arteries.
Congenital heart disease with left-to-right shunts In
many types of congenital heart disease, the pulmonary circulation develops normally in utero,
but adaptational changes occur after birth. An increase in pulmonary blood flow from left to right
shunts interferes with postnatal intra-acinar artery
growth and if left uncorrected will result in a
reduction in intra-acinar artery number, increased
pulmonary vascular resistance, and pulmonary hypertension. Systemic arteriovenous anastomoses in
which high pulmonary blood flow occurs before
birth will result in an abnormal vascular structure
at birth.
See also: Acute Respiratory Distress Syndrome.
Alveolar Hemorrhage. Angiogenesis, Angiogenic
Growth Factors and Development Factors. Arterial
Blood Gases. Bronchial Circulation. Bronchopulmonary Dysplasia. Chronic Obstructive Pulmonary
Disease: Acute Exacerbations. Coagulation Cascade:
Factor VII. Diffusion of Gases. Epithelial Cells:
Type I Cells; Type II Cells. Hypoxia and Hypoxemia.
Infant Respiratory Distress Syndrome. Lung Anatomy (Including the Aging Lung). Lung Development:
Overview; Congenital Parenchymal Disorders; Congenital
Vascular Disorders. Lymphatic System. Neonatal
Circulation. Nitric Oxide and Nitrogen Oxides. Oxygen Therapy. Oxygen Toxicity. Pediatric Pulmonary Diseases. Permeability of the Blood–Gas
Barrier. Pulmonary Circulation. Pulmonary Edema.
Pulmonary Vascular Remodeling. Smooth Muscle
Cells: Vascular. Surgery: Transplantation. Systemic
Disease: Diffuse Alveolar Hemorrhage and Goodpasture’s Syndrome. Vascular Disease. Vascular Endothelial Growth Factor. Vasculitis: Overview;
Microscopic Polyangiitis.
Further Reading
Davies G and Reid L (1970) Thorax 25: 669–681.
Davies P, deMello DE, and Reid LM (1990) Methods in experimental pathology of pulmonary vasculature. In: Gil J (ed.)
Models of Lung Disease: Microscopy and Structural Methods,
vol. 47 of Lung Biology in Health and Disease, pp. 843–904.
New York: Dekker.
deMello DE (1999) Structural elements of human fetal and neonatal lung vascular development. In: Control Mechanisms in the
Fetal and Neonatal Pulmonary Circulation, Proceedings of the
Ninth Pulmonary Circulation Conference, Sedalia, CO, Oct
1999, pp. 37–64.
deMello DE (2004) A matter of life and breath: context article.
Pediatric and Developmental Pathology 7: 422–424.
deMello DE (2004) Pulmonary pathology. Seminars in Neonatology 9: 311–329.
deMello DE and Reid L (2000) Embryonic and early fetal
development of human lung vasculature and its functional
implications. Pediatric and Developmental Pathology 3:
439–449.
deMello DE and Reid L (2004) Pre- and post natal development of
the pulmonary circulation. In: Haddad GG, Abman SH, and
Chernick V (eds.) Basic Mechanisms of Pediatric Respiratory
Disease, pp. 77–101. Ontario: BC Decker.
deMello DE and Reid LM (1991) Arteries and veins. In: Crystal RG,
West JB, Barnes PJ, Cherniack NS, and Weibel ER (eds.) The
Lung: Scientific Foundations, pp. 767–777. New York: Raven
Press.
deMello DE and Reid LM (1991) Pre and post-natal development of the pulmonary circulation. In: Chernick V and
Mellins RB (eds.) Basic Mechanisms of Pediatric Respiratory
Disease: Cellular and Integrative, pp. 36–54. Philadelphia: BC
Decker.
deMello DE and Reid LM (1997) Arteries and veins. In: Crystal
RG, West JB, Barnes PJ, Cherniack NS, and Weibel ER (eds.)
The Lung: Scientific Foundations, 2nd edn., pp. 1117–1127.
Philadelphia: Lippincott-Raven.
deMello DE and Reid LM (2002) Vascular development of lung.
In: Tomanek R (ed.) Assembly of the Vasculature, vol. 2, pp.
211–237. Boston: Birkhauser.
deMello DE, Sawyer D, Galvin N, and Reid LM (1997) Early fetal
development of lung vasculature. American Journal of Respiratory Cell and Molecular Biology 16: 568–581.
Galambos G, Ng YS, Ali A, et al. (2002) Defective pulmonary
development in the absence of heparin-binding vascular endothelial growth factor isoforms. American Journal of Respiratory Cell and Molecular Biology 27: 194–203.
Hall SM, Hislop AA, Pierce CM, and Haworth SG (2000) Prenatal
origins of human intrapulmonary arteries. American Journal of
Respiratory Cell and Molecular Biology 23: 194–203.
Hislop A (1971) The fetal and childhood development of the pulmonary circulation and its disturbance in certain types of congenital heart disease. PhD Thesis, London University.
Hislop A and Reid L (1972) Intra-pulmonary arterial development
during fetal life: branching pattern and structure. Journal of
Anatomy 113: 35–48.
Hislop A and Reid L (1973) Fetal and childhood development of
the intrapulmonary veins in man: branching pattern and structure. Thorax 28: 313–319.
Hislop A and Reid L (1973) Pulmonary arterial development during childhood: branching pattern and structure. Thorax 28:
129–135.
Oettgen P (2001) Transcriptional regulation of vascular development. Circulation Research 89: 380–388.
166 ASTHMA / Overview
Reid L (1978) The pulmonary circulation: remodeling in growth
and disease. The 1978 J Burns Amberson Lecture. American
Review of Respiratory Disease 119: 531–546.
Reid L, Fried R, Geggle R, and Langleben D (1986) Anatomy of
pulmonary hypertensive states. In: Bergofsky EH (ed.) Abnormal Pulmonary Circulation, Vol. 4, pp. 221–263. New York:
Churchill Livingstone.
Rendas A, et al. (1980) American Review of Respiratory Disease
121: 873–880.
Schachtner SK, Wang YQ, and Baldwin SH (2000) Quantitative analysis of embryonic pulmonary vessel formation.
American Journal of Respiratory Cell and Molecular Biology
22: 157–165.
Asbestos see Occupational Diseases: Asbestos-Related Lung Disease.
ASTHMA
Contents
Overview
Allergic Bronchopulmonary Aspergillosis
Aspirin-Intolerant
Occupational Asthma (Including Byssinosis)
Acute Exacerbations
Exercise-Induced
Extrinsic/Intrinsic
Overview
P Chanez, Hôpital Arnaud de Villeneuve, Montpellier,
France
& 2006 Elsevier Ltd. All rights reserved.
Asthma is one of the most frequent chronic diseases.
It is responsible for absenteeism from school and
work, thereby handicapping daily life. Its management is based on drug therapy, control of the environment, therapeutic education, and management of
triggering factors.
Abstract
Asthma is one of the most prevalent chronic diseases in most of
the countries in the world. Its continuous increase is clearly
described in most places, confirming the potential importance of
the environment. These environmental factors interact in a susceptible individual with some complex polygenic background,
to reveal the asthma phenotype. Several triggers including inhaled allergens, viruses and some indoor and outdoor pollutants
have been pointed as potential culprits to induce, perpetuate, or
exacerbate asthma. Inflammation and structural changes are
hallmarks of asthmatic airways occurring from the nose to the
distal part of the lung. The relationships between those structural changes and clinical and functional abnormalities clearly
deserve further investigations. Those findings led to the reinforcement of the use of inhaled corticosteroids as the pivotal
treatment for asthma. The adjunction of long-acting b2 agonists
has been shown to be the next logical step of pharmacology, in
case of poor control when using inhaled steroids alone. The
long-term management should include some tailor-made environment control and educational measures leading to a better
partnership with the patients. In some occasions, the addition of
leukotriene receptor antagonists, specific immunotherapy, and
more recently, subcutaneous anti-IgE may offer better control
for a subset of patients. A better understanding of the mechanisms, especially in the most severe forms of the disease, is
paramount to develop better preventive strategies and innovative therapies.
Physiopathology
Asthma is an inflammatory disorder accompanied
by remodeling of the airways. This inflammation is
secondary to polymorphic inflammatory infiltration,
rich in mast cells, and eosinophils. In genetically
predisposed subjects, this inflammation may cause
symptoms which, in general, are related to diffuse
variable bronchial obstruction that is spontaneously
reversible or subsides under the influence of treatment. This inflammation is also associated with
bronchial hyperresponsiveness to a wide variety of
stimuli. This definition of asthma is supported by the
physiopathological and clinical findings on the disease. The variability and reversibility of the airflow
impairment, under the influence of bronchodilators
and glucocorticoids, distinguish asthma from other
bronchial disorders. Chronic rhinosinusitis is very
often associated with asthma (approximately 80%
of cases) and must be investigated, not only by
questioning, but also by careful nasal examination.
ASTHMA / Overview 167
Several arguments support a link between persistent
rhinosinusitis and asthma: the common characteristics of inflammation and tissue reorganization, similar epidemiology and chronicity, higher risk of
asthma in cases of rhinitis, higher risk of bronchial
hyperreactivity in patients suffering from rhinitis,
occurrence of bronchial inflammation after nasal
challenge with an allergen, and the same aggravating
and triggering factors. The efficacy of local corticoid
therapy constitutes another link. On the other hand,
there is no solid evidence in favor of the bronchial
efficacy of nasal treatment. Systemic treatments also
require additional evaluation of their joint efficacy in
rhinosinusitis and asthma.
Diagnosis
Positive Diagnosis
This must be based on the definition of asthma. The
two characteristic elements are clinical (chronicity,
variability, and reversibility of symptoms), and functional. Tests of respiratory function clearly confirm
the diagnosis provided that:
1. spirometry shows a reversible obstructive ventilatory disorder from 12% to 15% in comparison
with theoretical values, or at least 180 ml in
absolute value, with short-acting b2-mimetics;
20% or 250 ml during a 10-day test with glucocorticoids;
2. peak expiratory flow (PEF) has a variability
X20%; and
3. bronchial hyperreactivity is another feature of the
diagnosis.
There have been few studies on the sensitivity and
specificity of the functional signs of asthma and their
predictive values seem insufficient. Those maintained
in the international recommendations are cough,
dyspnea, sibilant rhonchi (wheezing), chest tightness,
and expectoration.
There may be one or several symptoms. Symptoms
may be absent at the time of the examination. Definition of reversibility or its significance is only obtained from expert opinions or by consensus.
The same is true for the usefulness of the corticosteroid test to identify asthmatic patients; a short
course of oral corticosteroid is used generally. The
dose and duration of steroid treatment given in the
literature are highly variable. However, these criteria are generally considered to be sufficient to
propose the diagnosis of asthma and to potentially
qualify nonresponding asthmatic patients as steroidresistant.
Table 1 Differential diagnosis of asthma in the child and adult
In the child
In adults
Obliterating bronchiolitis
Cystic fibrosis
Foreign body
Tracheobronchomalacia
Bronchial inhalations
Vocal cord dysfunction
Upper airway abnormalities
Immunodeficiency (IgA, IgG2, IgG4)
Ciliary dyskinesia
Abnormal aortic arch
Bronchiectasis
Cystic fibrosis
Foreign body
Tracheobronchomalacia
Bronchial inhalations
Vocal cord dysfunction
COPD
Heart failure
Bronchial amyloidosis
Abnormal aortic arch
Tracheobronchial cancer
Bronchiolitis
Alternative Diagnosis
The main differential diagnoses are summarized in
Table 1.
Clinical Definitions
From the point of view of the terminology, three essential terms must be used and seem to be operational in clinical medicine and for the follow-up of
asthmatics – the seriousness of the asthma which refers to the current state of the patient (serious acute
asthma), the control of the asthma which refers to
recent events (symptoms of brief duration and exacerbations), and the severity of the asthma which is
mainly evaluated over the past year.
Exacerbations
The definition of an exacerbation is variable and it is
based on the unanticipated use of drugs, the persistent symptoms (repetition of short-lasting symptoms
generally on two consecutive days), the increased
bronchial obstruction, and the requirement for substantial change in treatment with oral corticoids being the most frequent.
Exacerbations form part of poor control of the
asthma, but differ from it. The permanence of shortlasting symptoms with return to the baseline state between these symptoms defines simple poor control,
whereas an exacerbation is characterized by a persistent worsening without a return to the baseline state.
Control
Several asthma evaluation control questionnaires
have been developed and concern the events occurring during the 1–4 weeks before the visit. Some even
cover a period of 3 months, or even the time since the
previous visit. There are no data in the literature
pointing to one or the other of these periods.
168 ASTHMA / Overview
Juniper showed that her questionnaire (ACQ) was
more discriminating than a daily diary. The authors
clearly specified that this comparison concerned clinical trials and no conclusion could be deduced
for daily clinical practice. These indexes are often
correlated with generic and specific quality of life
questionnaires: AQLQ, SF 36, Saint Georges questionnaire. The definition of optimal (or excellent)
and suboptimal (or acceptable) control is based on
experts’ agreement and their clinical experience.
Table 2 Factors aggravating controlled asthma
Severity
Table 3 Examples of candidate genes implicated in the development of asthma
The notion of severity was initially based on the same
parameters as control, though these were assessed retrospectively over a longer period, often including the
number of successful anti-inflammatory treatments
and the best level of respiratory function. At present,
the assessment of asthma severity must be made more
objectively, according to the control and the quantity
of drugs required to obtain and maintain it.
Difficult Asthma
Difficult asthma may only be defined after answering
questions about its diagnostic reality, patient adherence to treatment, and the existence of major comorbidity which may interfere with management,
making it impossible to obtain an acceptable control.
Positive answers to these questions aid in diagnosing
severe asthma. Severe asthma is characterized by
frequent and serious exacerbations (stays in intensive
care units), the persistence of airway obstruction,
resorting to high doses of inhaled corticosteroids or
even oral steroid dependence, and in some cases, by
lack of steroid sensitivity. It should be pointed out
that patients, nursing staff (nurses, educators in
asthma schools etc.) , general practitioners, and even
respiratory physicians are not always familiar with
these notions of seriousness, control, exacerbations,
and severity. They, therefore, often differ in their assessment of the same clinical situation. Efforts to
provide information, education, and coherence are
therefore required more than ever.
Precipitating Factors
The risk factors of loss of control of asthma are
summarized in Table 2.
Serious Acute Asthma
From the patient’s point of view, the seriousness of an
attack may be defined by the following characters:
*
*
it is unusual and a doctor must be called;
it leads to the discontinuation of current activity
(work, school, or play); and
Interruption of anti-inflammatory treatment
Viral infections
Administration of aspirin or a betablocker
Hormonal factors with premenstrual recurrence in woman
Contact with allergen
Atmospheric pollution
Domestic pollution
Meteorological factors
Stress
Chromosome
Candidate gene
5q
6q
Th2 related cytokines
TNF-a
11q
Clara cell protein
CC10
FceRIb: IgE highaffinity receptor
Interferon g
12q
14q
16q
T-lymphocytes
receptor
IL-4 receptor a
20q
ADAM 33
*
Potential relation with
asthma
Airway inflammation
Airway inflammation,
treatment
Airway inflammation,
treatment
Airway inflammation
Airway inflammation,
treatment
Airway remodeling
Certain authors note that an attack leading to a
change in primary therapy must be considered to
be potentially serious.
For the doctor, it is always a potentially fatal medical emergency. Table 3 presents the clinical signs of
serious acute asthma (SAA).
Various items have been recognized as essential to
management of SAA:
1. Value of evaluation protocols.
2. Requirement to measure respiratory function
(PEF) and blood gases.
3. Efficacy of treatment with inhaled short-acting b2mimetics, systemic glucocorticoids, inhaled anticholinergics (48 h mainly in the child), and oxygen
therapy.
4. Value of second-line therapy with intravenous b2mimetics, nebulization using helium oxygen as
vector (heliox), intravenous magnesium.
5. Requirement for regular clinical re-evaluation.
6. The criteria for hospitalization are not validated,
though they are widely used and consist of PEF
o60% of the predicted value or o100 l min 1, no
clinical improvement, severe underlying asthma,
ASTHMA / Overview 169
Table 4 Clinical signs of serious acute asthma
Table 6 Why new therapies are needed in asthma?
Threatening syndrome
Worsening in a few days
Increase in the frequency of attacks
Increase of severity of attacks
Resistance to treatment
Increase in drug consumption
Disease-free intervals less and less frequent
Progressive decrease in PEF
Therapy
Intermittent
mild
persistent
Moderate
persistent
Severe
persistent
Side effects
None
Few potential
Efficiency
Compliance
High
Low
Variable
Variable
Highest
frequency
Low
Usually better
Signs of immediate seriousness
Unusual and/or progressive dyspnea
Difficulty in speaking or coughing
Agitation
Sweats and/or cyanosis
SCM muscle permanently tensed
RR 430 min 1
HR 4120 min 1
Paradoxical pulse 420 mmHg
PEF o150 l min 1
Gain in PEF under treatment o60 l min
PaCO2 440 mmHg
Table 7 Goals for asthma treatment
Minimize symptoms
Achieve best lung function
Prevent asthma exacerbations
Obtain treatment with the best therapeutic balance
Educate patients for best partnership
Avoid decline in lung function
1
Signs of distress
Consciousness disorders
Collapsus
Pause in breathing
Respiratory silence
PEF, peak expiratory flow; HR, heart rate; RR, respiratory flow;
SCM, sternocleidomastoid.
Table 8 ICS side effects usually not clinically significant if low
doses are used
Frequent and mild
Oral candidosis
Hoarseness
Potential and mild
Cough
Skin bruising
Table 5 Risk factors of mortality in serious acute asthma
Severe asthma (bronchial obstruction during intercritical period or
frequent exacerbations)
Patients presenting major rapid variations in bronchial obstruction
(variation in PEF 430%)
Major reversibility under bronchodilators
Psychosocial instability
Use of three drugs (or more) for asthma
Frequent resort to ER for SAA, with hospitalization and/or
intensive care
Poor perception of symptoms
Elderly patients
Patients with hypereosinophilia
Poor compliance and denial of disease
and psychosocial problems. The same is true for
criteria concerning the return home (PEF 4 60%
or 300 l min 1).
After an episode of SAA, oral glucocorticoid therapy must be prescribed for from 7 to 14 days, combined with inhaled glucocorticoid therapy and
follow-up with therapeutic education. The risk factors for SAA mortality are given in Tables 4 and 5.
Suissa showed that the death rate due to asthma decreased by 21% per additional vial of inhaled corticoids received during the previous year. The death
rate due to SAA therefore decreased if low doses of
Rare and potentially severe
Surrenal insufficiency
Biologically proven but long-term clinical significance unknown
Osteoporosis
Impaired growth velocity
Cataract
inhaled corticoids were given as primary therapy.
These data validate a fundamental aspect of the longterm therapeutic management of asthma (Tables 6, 7,
and 8).
Asthma Associations
Bronchopulmonary allergic aspergillosis This entity is defined by the following major symptoms:
1. recurrent lung infiltrates with eosinophils, sensitive to corticoids,
2. highly characteristic proximal bronchiectasis,
3. sometimes high blood eosinophilia (41000),
4. increased total IgE and specific IgE (measured
by RAST) and IgG (precipitins) immune reaction
against Aspergillus fumigatus, and
5. Aspergillus can sometimes be present in sputum.
170 ASTHMA / Overview
These point to severe asthma, usually justifying
long-term oral corticoid therapy and, in certain circumstances, antifungal treatment.
Churg–Strauss syndrome This is a granulomatous
and necrosing vasculitis. It is a rare form of asthma
characterized by the severity of the respiratory symptoms, the levels of blood eosinophilia (generally
above 1500 mm3), and the existence of extrarespiratory signs (neurological and cutaneous). It is often
oral steroid-dependent and sometimes requires
immunosuppressive agents.
Triggering and Aggravating Factors
Asthma is a multifactorial syndrome. It is more appropriate to speak of triggering factors rather than
etiological factors. The diagnostic procedure should
take place in two stages – first, by investigating for
the presence of such a factor in a patient and second,
by evaluating its role in the global evaluation of the
asthma (seriousness, control, and severity).
Genetic Component
The genetic component is indisputable. It is well
known that asthma runs in families. Conventionally,
this hereditary constituent seems to be associated
with atopy. However, recent work has demonstrated
that even asthmatics with no demonstrable personal
or family allergic factor (intrinsic asthma) had asthmatics in their family. Asthma is almost certainly
polygenic in origin. Association with numerous polymorphisms have been reported at different loci in
relation with or not with the known physiopathological data for the disease. The finding that different
patients have different responses to antiasthmatic
treatments has made it possible to develop potentially promising strategies for pharmacogenetic studies. For instance, data concerning the polymorphism
of b2-mimetic receptor and the potential severity of
the disease represent an interesting approach. Some
examples of candidate genes implicated in the development of asthma are reported in Table 3.
Environmental Component
Inhaled allergens These are allergens present in the
ambient air. When inhaled in small quantities, they
are capable of sensitizing subjects and triggering
symptoms when they reach the level of the respiratory mucosa.
Dermatophagoides pteronissimus. These form one
of the major allergens of household dust.
Animal proteins. Proteins produced by pets, laboratory experiments, or leisure activities.
Arthropods. They are insects such as locusts or
cockroaches, which can cause asthma.
Atmospheric molds and yeasts. They are an important source of allergens.
Allergens present in insalubrious habitats are associated with more severe asthma.
Food allergens Food and drinks may cause respiratory reactions after allergic sensitization, but nonallergic reactions or toxic reactions may also occur
due to non-specific histamine release.
Drugs Very often, drugs behave as haptens. Atopic
subjects and asthmatics do not have any predisposition to hypersensitivity to drug products but when
these occur, they are more violent.
Aspirin intolerance This sometimes involves severe
asthma, often with a late-onset with pan-rhino sinusitis with polyposis and systemic reactions, often
with anaphylactic shock after ingestion of aspirin or
nonsteroidal anti-inflammatory drugs.
Occupational sensitizers They may be allergenic,
but they may also act by toxic, irritative, or pharmacological mechanisms. The most important directly allergic allergen is wheat flour, responsible for
bakers’ asthma. Another classical example of occupational asthma is due to work in contact with isocyanates. Demonstration that the asthma is caused
by an occupational agent requires the use of a coherent, clinical, and functional diagnostic procedure,
sometimes including bronchial challenge tests in the
laboratory.
Other factors
Importance of viruses This is variable as a function
of age in the triggering of asthma exacerbations. In
the infant, wheezing bronchitis is usually caused by
viruses though the possibility of asthma must not be
over- or underestimated. Certain studies show that
more than 60% of asthma exacerbations are related
to a viral respiratory infection.
Viral infections can be involved at various levels:
occurrence of exacerbations,
prevention of the occurrence of asthma,
* induction of asthma, and
* persistence and development of chronic asthma
due to chronic infections.
*
*
Bacterial infection This plays a secondary role in
the physiopathology of asthma and as a triggering
factor in exacerbations. The presence of endotoxins
in the environment may be involved in the triggering
ASTHMA / Overview 171
of exacerbations in asthmatic patients, though it may
also potentially protect an individual from the development of asthma symptoms (hygiene theory).
Pollution SO2, NO2, and ozone act in synergy to
trigger an attack. These different pollutants are responsible for exacerbations, sometimes serious or
even very serious, especially in a child. The main
sources of pollution are boiler plants (using fuel-oil
and coal), household and industrial refuse incinerators, and motor vehicles (diesel engine). Particulate
pollution has been shown to be associated in industrialized areas with an increase in asthma exacerbations. Diesel exhaust particles worsen allergic
symptoms by amplifying the release of inflammatory
mediators.
Indoor pollution should not be forgotten and in
particular that produced by home boilers. There are
several evidences indicating that indoor pollution indicating a potential role for endotoxins, cockroaches,
and parents’ smoking are major contributors for
asthma exacerbations and lack of control in children
with asthma. There is no definitive argument proving
the involvement of atmospheric pollution in the acquisition of the asthma phenotype of a given subject.
Gastroesophageal reflux Gastroesophageal reflux
(GER) is more frequent in asthmatics than in a normal population. The acidity of the lower esophagus
induces reflex bronchoconstriction. Contamination
of the bronchi may also occur following incompetence of the cardia. Controlled studies have not
shown the value of systematic treatment of GER to
improve the asthma, even in the most severely affected patients.
Psychological and/or psychiatric factors These are
certainly involved, both in the expression of symptoms and beliefs on treatment, and therefore therapeutic compliance. They are, however, always
difficult to evaluate.
Influence of endocrine factors This is probable and
the role of sex hormones is always mentioned first
(effect of puberty, episodes of genital life in woman).
Premenstrual exacerbations are rare events which
can contribute to the severity of the disease. It has
been shown recently that severe asthma is more frequent in women than in men. Menopause is an important period for asthma revival, poor control, or
late-onset, but the exact sustaining mechanisms are
yet to be understood. Sexual female hormonal replacement has been shown in one epidemiological
study to be associated with an increase in emergency
room attendance for asthma.
Exercise Induced Asthma
This is characterized by the occurrence of bronchial
obstruction at the end of exercise. In certain cases,
the asthma occurs during exercise though it is then
possible to ‘run through the asthma’ if the exercise is
continued. This is practically always present in children and adolescents and it may constitute a marker
of bronchial hyperreactivity and inflammation. It is
essential to control this form of asthma to allow
normal physical activity and a harmonious physical
and psychological development. Intercritical bronchial obstruction may be responsible for exertional
dyspnea though this is not exertional asthma in the
strict sense of the term.
Smoking
Tobacco smoke (active and passive) by itself induces
inflammation of the airways with hypersecretion,
paralysis, and ciliary destruction, and infiltration of
the distal aerial spaces by neutrophils. Active smoking is associated with a more severe asthma which
responds less well to corticosteroids. It is not always
easy to distinguish between asthma in the smoker
and chronic obstructive pulmonary disease (COPD)
with a certain degree of variability of the respiratory
function.
Maternal smoking may induce more asthma or
atopy-associated diseases in children. Passive smoking by children is clearly responsible for uncontrollable asthma and this factor must be always looked
for in the child. Many asthmatic adolescents are
smokers and they have a high risk of loss of control
and SAA. Adapted educational strategies must always be proposed.
Treatment
Asthma treatment is based on three main axes: drug
therapy, control of aggravating and triggering factors,
and education of patients and health professionals.
The objectives must be discussed with the patient,
though the main aim is to obtain an acceptable control of the disease.
Pharmacology
Corticocorticosteroids The efficacy of treatment
with inhaled corticocorticosteroids is well established. It decreases functional signs, exacerbations,
deaths, and costs, and improves the level of respiratory function and bronchial reactivity. All these effects have been demonstrated in comparison with
placebo. Inhaled corticoids considerably improve the
risk–benefit ratio of long-term treatment (Figure 1).
They are indicated in persistent, mild, moderate, or
172 ASTHMA / Overview
severe asthma. The local side effects of inhaled corticoids (candidiasis, hoarse voice) are effectively
prevented by rinsing the mouth after inhalation or
by the use of a spacer. At recommended dosages, in
persistent mild to moderate asthma (and up to
1500 mg day 1 of beclomethasone equivalents), the
risks of systematic adverse effects (adrenal insufficiency, osteoporosis) are low. The main adverse
effects are susceptibility to bruises and skin atrophy.
In the child, the mean dosage of 400 mg day 1 of
budesonide does not modify growth. However, questions still remain about inhaled corticosteroids their
efficacy in modifying the severity of asthma, their
interaction with the natural history of the disease, or
the long-term systemic side effects are still a topic of
debate.
100
75
Side effects
Efficacy
50
25
0
Mild
Moderate
Severe
Figure 1 Risk–benefit ratio potential for drugs in the treatment
of persistent asthma.
Bronchodilators b2-mimetics are very potent
bronchodilators. They stimulate bronchial smooth
muscle b2-receptors. Short-acting b2-agonists (from
4 to 6 h) may be distinguished from long-acting
b2-agonists (12 h and more). This class of drug has
no or very few anti-inflammatory effects, and causes
little tachyphylaxis.
Side effects are minor with mainly tremors, which
disappear during treatment, and usually nonserious
tachycardia-type palpitations.
In persistent asthma, on-demand treatment with
short-acting b2-agonists must be combined with
continuous anti-inflammatory treatment. In case of
suboptimal or unacceptable control in patients requiring daily doses of these short-acting b2-agonists
and/or if symptoms occur at night, a long-acting
b2-agonist may be prescribed. The use of shortacting b2-agonists is then reserved for the treatment
of episodes of dyspnea occurring in spite of wellconducted treatment. A high consumption is then
associated with loss of control. It is better to give
them on demand, rather than systematically, and
they are not associated with a risk of an increased
death rate. The value of long-acting b2-agonists is
demonstrated by a high level of evidence. These
agents improve the control of asthma when inhaled
corticoid therapy is insufficient alone. Their combination with inhaled corticoid therapy is better than
a double dose of the latter drug. This strategy improves the control of the asthma, decreases the incidence and seriousness of exacerbations, and
improves patient quality of life. At present, LABAs
should never be considered as the sole treatment for
asthma (Figures 2 and 3).
BHR
FEV1
Hours
Days
Weeks
Symptoms
∆% DEP
Months
Exacerbations
Figure 2 Improvement of control components after first treatment initiation in asthma.
…Years
ASTHMA / Overview 173
+ High-dose ICS
± OCS
± Theophylline
± AntiIGE
± Steroid sparing
± Ipratropium
± Leukotriene
antagonists
+ LA 2 agonists
+ Moderate-dose ICS
± Leukotriene antagonists
+ Low-dose ICS
SA 2 agonists as required
Mild controlled
Severe uncontrolled
Education
Environment control ± specific immunotherapy
Comorbidities treatment
Figure 3 Treatment of asthma according to control.
Figure 4 Pathology of asthma.
Inhaled corticoid–long-acting b2-agonist combination Combination in a same inhaler of a corticoid
and a long-acting b2-agonist follows recommendations for the treatment of persistent, moderate to
severe asthma, with facilitated administration, and
the objective of improving compliance, notably of
corticoid therapy. Two combinations have been developed: fluticasone–salmeterol and formoterol–
budesonide. The benefit of these fixed combinations
in comparison with separate combination of longacting b2-agonists and inhaled corticocorticosteroids
is not completely established and nor is the value of a
fixed combination administered systematically or
modulated on demand (Figure 4).
Cysteinyl–leukotriene receptor antagonists The
cysteinyl–leukotriene receptor antagonists used at
present are montelukast and zafirlukast. They are
used orally and potentially can treat asthma and
rhinosinusitis. These agents have been shown to be
useful in exercise induced-asthma, as well as a singleagent therapy of mild or moderate asthma. They lead
174 ASTHMA / Overview
to bronchodilation and have been able to improve
asthma control in long-term placebo-controlled studies. Their equivalence with low-dose inhaled corticoid
therapy (400 mg day 1 of beclomethasone) is suspected but not fully demonstrated. The equivalence
of a combination of an antileukotriene in comparison
with a long-acting b2-agonist in noncontrolled patients treated with inhaled corticoid therapy alone is
not proven. Their role in severe asthma is uncertain;
it should be interesting, though, to follow their
benefit in aspirin-induced asthma, which was recently
demonstrated in some clinical studies.
Theophylline This is a less potent bronchodilator
than the b2-mimetics. It also has numerous other
actions and in particular an anti-inflammatory activity. Its effects are strictly dependent on its serum
concentration, which must be maintained in a relatively narrow range between 10 and 20 mg l 1. Side
effects are also dose-dependent. At present it is not
used much for primary treatment of asthma, except
in the most severely affected patients.
Anticholinergics Ipratropium and oxitropium are
antagonists of muscarinic receptors. They are
bronchodilators but are less potent and slower to
act than b2-agonists. They are indicated in the treatment of SAA and exacerbations of chronic asthma as
a complement to short-acting b2-agonists. Their efficacy beyond the first 48 h of the treatment is unknown. Proof of their efficacy exists especially for
treatment in children. There are few adverse effects.
Their efficacy in the management of chronic asthma
has not been evaluated.
Tioptropium (Spiriva) is a long acting anticholinergic drug, which has been developed for the
treatment of COPD. Its potential interest in asthma
and specifically severe asthma deserves further investigations.
Control of the Environment
Although environmental control seems useful, there
is no complete evidence of its efficacy. It comprises
the reduction of exposure to allergens and non-specific irritants and the prevention of active and passive
smoking.
Allergy is one of the major factors associated with
asthma and the most important for children of
school age. Prevention of exposure to allergens is
logically the first measure to be taken. This obviously
concerns pets though this is not necessarily well accepted by patients or close relatives and friends. It is
possible to try to eradicate house-dust mites though
this is rarely complete. Diagnosis of occupational
asthma with a maximum of evidence may force a
patient to change jobs. It is more difficult to adopt
outdoor pollution-control measures which mainly
concern the authorities.
Immunotherapy
Specific immunotherapy is of some help in allergic
asthmatic patients if simple rules are applied. Patients
with persistent severe rhinitis and persistent mild
asthma benefit most from this treatment. It is contraindicated in uncontrolled and severe asthma. On the
other hand, the development of anti-IgE strategies may
give hope to better control in severely allergic patients.
Education
Education of patients is indicated in all asthmatics,
though it should be adapted to the severity, importance of triggering factors, and specific personality of
each patient. It must be based on a precise educational diagnosis. Objectives must be defined in partnership with the patient. The educational methods
used depend on these objectives. This therapeutic
education may be individual or may be provided in
institutions such as asthma schools.
Conclusion
Asthma is a bronchial inflammatory disease that alters the structure of the airways including the upper
airways, which may sometimes have systemic repercussions. It is chronic, variable, and reversible and
also a multifactorial, polygenic, and environmental
disease. Management must be based on a long-term
strategy. The major objective is to obtain satisfactory
control to allow an optimal quality of life. Treatment
involves use of a potent and well-tolerated pharmacopoeia, but may only be implemented in partnership
with the patient.
See also: Asthma: Allergic Bronchopulmonary Aspergillosis; Aspirin-Intolerant; Occupational Asthma (Including
Byssinosis); Acute Exacerbations; Exercise-Induced; Extrinsic/Intrinsic. Bronchodilators: Anticholinergic Agents.
Capsaicin. Carbon Monoxide. CD14. Chemokines.
Chymase and Tryptase. Cilia and Mucociliary Clearance. Cyclic Nucleotide Phosphodiesterases. DNA:
Repair. Dust Mite. Environmental Pollutants: Oxidant
Gases. Eotaxins. Epidermal Growth Factors. Extracellular Matrix: Surface Proteoglycans. Fibroblasts.
Gastroesophageal Reflux. G-Protein-Coupled Receptors. Histamine. Immunoglobulins. Interferons. Kinins and Neuropeptides: Vasoactive Intestinal
Peptide; Other Important Neuropeptides. Leukocytes:
Eosinophils. Lipid Mediators: Overview; Leukotrienes; Prostanoids; Platelet-Activating Factors. Oxidants
and Antioxidants: Oxidants. Pediatric Pulmonary
ASTHMA / Overview 175
Diseases. Proteinase-Activated Receptors. Signs of
Respiratory Disease: Lung Sounds. Symptoms of
Respiratory Disease: Dyspnea. Tumor Necrosis Factor Alpha (TNF-a ).
Further Reading
Abramson MJ, Puy RM, and Weiner JM (2003) Allergen immunotherapy for asthma. Cochrane Database of Systematic Reviews
4: CD001186.
Agertoft L and Pedersen S (2000) Effect of long-term treatment
with inhaled budesonide on adult height in children with
asthma. New England Journal of Medicine 343: 1064–1069.
American Thoracic Society (1995) Standardization of spirometry,
1994 update. American Journal of Respiratory and Critical Care
Medicine 152: 1107–1136.
ANAES (2004) Recommandations pour le suivi médical des patients asthmatiques adultes et adolescents. Paris 169pp (with an
english translation on site www.anaes.fr).
Anderson SD and Holzer K (2000) Exercise-induced asthma: is it
the right diagnosis in elite athletes? Journal of Allergy and Clinical Immunology 106: 419–428.
Barnes PJ (1995) Inhaled glucocorticoids for asthma. New England Journal of Medicine 332: 868–875.
Barnes PJ (2004) Corticoresistance in airway disease. Proceedings
of the American Thoracic Society 1: 264–269.
Bateman ED, Boushey HA, Bousquet J, et al. (2004) GOAL
Investigators Group. Can guideline-defined asthma control be
achieved? The gaining optimal asthma control study. American
Journal of Respiratory and Critical Care Medicine 170: 836–844.
British Thoracic Society (2003) British guideline on the management of asthma. Thorax 58(supplement 1): S1–S83.
Chung KF, Godard P, Adelroth E, et al. (1999) Difficult/therapyresistant asthma: the need for an integrated approach to define
clinical phenotypes, evaluate risk factors, understand pathophysiology and find novel therapies. European Respiratory
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Allergic Bronchopulmonary
Aspergillosis
P Wark, Southampton University, Southampton, UK
& 2006 Elsevier Ltd. All rights reserved.
Introduction
Allergic bronchopulmonary aspergillosis (ABPA) is a
complex condition that results from hypersensitivity
to the fungus Aspergillus fumigatus (Af). ABPA was
first described in the UK in 1952, and has been estimated to occur in 1–2% of chronic asthmatics and
up to 15% of patients with cystic fibrosis. It is unclear whether the prevalence of ABPA has declined
with the widespread use of inhaled corticosteroids to
treat asthma. There is an excessively high prevalence
of ABPA in cystic fibrosis where evidence suggests
that ABPA may cause a faster decline in lung function. In chronic asthma, ABPA follows a more variable course with recurrent exacerbations and, at
least in some patients, it leads to proximal bronchiectasis and irreversible fibrotic lung disease.
Exposure to Af can cause a wide range of pulmonary diseases, which are summarized in Figure 1,
including: severe life-threatening pneumonia in the
immunosuppressed; subacute infections such as the
formation of aspergilloma in those with pre-existing
pulmonary cavities; a form of extrinsic allergic alveolitis; and hypersensitivity diseases. Subjects with
asthma or cystic fibrosis (CF) may become sensitized
to Af following inhalation of spores; once sensitized
this results in a type I, IgE-mediated reaction and a
spectrum of clinical responses from acute exacerbations of asthma to a more sustained and intense
inflammatory response that shows features of both
type I and type III hypersensitivity and leads to
ABPA. ABPA is unique in these disorders as there is
evidence of persistence of the organism within the
airways resulting in an intense local and systemic
immune response that is associated with mucus
impaction and may lead to bronchiectasis and pulmonary fibrosis.
Etiology
Fungi belonging to the genus Aspergillus are ubiquitous spore-forming filamentous fungi and while any
member of the group can cause allergic sensitization
most disease is attributable to Af. The organism is
present in outdoor environments where it readily
grows in decomposing organic matter, such as decaying vegetation in soil, mulches, wood chips, freshly
mown grass, and sewage treatment debris. Indoors,
Af can be found in damp areas especially on walls or
ceilings where water damage has occurred; in addition, spores can be found in very large quantities in
bird excreta. Acute inhalation of spores is known to
trigger acute asthma in sensitized asthmatics, while in
subjects with ABPA inhalation of Af antigens leads to
a dramatic worsening of the acute and late-phase response with falls in FEV1 of 50–79%. Exacerbations
of clinical disease have been reported to occur coincident with high outdoor spore counts but a direct
temporal relationship to inhalation and an acute exacerbation of ABPA remains unclear.
A genetic predisposition to ABPA is also possible,
with reports of familial occurrence. Two small studies have shown a higher carriage rate of at least one
mutation of the cystic fibrosis transmembrane regulator (CFTR) gene in subjects with ABPA, compared
to subjects who are skin-test positive to Af and those
with asthma alone. The alleles HLA-DR2 and -DR5
have also been linked to severity of disease in ABPA.
Most recently, a polymorphism of surfactant proteins
has been shown to be associated with increased serum IgE and blood eosinophilia in patients with
ABPA. These findings suggest there may be a link
between ABPA and an inability to effectively clear
the organism in a group whose immune response is
likely to be one associated with atopy and a T-helper
(TH)-2 response.
ASTHMA / Allergic Bronchopulmonary Aspergillosis 177
No previous
lung disease
Asthma
Cystic
fibrosis
Cavitatary
lung disease
Immunocompromised
Inhale Af spores
Acute asthma
(those sensitized up
to 30%)
No immune response
Colonization with Af
Mucoid impaction
extrinsic allergic
alveolitis
(all extremely rare)
ABPA
(1− 2%)
ABPA
(10 −15%)
Aspergilloma
Invasive
pulmonary
aspergillosis
Figure 1 A summary of the wide range of pulmonary diseases caused by exposure to Aspergillus fumigatus. Aspergillus fumigatus is a
ubiquitous environmental fungus and spores are regularly inhaled. For most individuals with no previous lung disease, this appears to
cause no immune response and no symptoms. High-dose exposure or exposure of susceptible individuals may trigger extrinsic allergic
alveolitis or, more rarely, colonization leading to an intense immune response as in ABPA with areas of mucoid impaction and
inflammation. Up to 30% of subjects with asthma are sensitized to Af. In those individuals exposure to Af may initiate an acute asthma
response. A proportion of asthmatics seem to be particularly prone to the effects of Af and are unable to clear the organism. It persists in
the lower airways and initiates the intense immune response resulting in ABPA, which occurs in 1–2% of asthmatics. In CF, particularly
in those with atopy or a history of asthma-like symptoms, sensitization to Af occurs. In at least 10–15% of CF individuals the organism is
not cleared and colonization ensues. This initiates an immune response like that seen in asthma but is associated also with the
endobronchial inflammation and chronic infection already present in CF. An aspergilloma (fungal ball) consists of masses of fungal
mycelia, inflammatory cells, fibrin, mucus, and tissue debris, these usually developing in a preformed lung cavity. In a study of 549
subjects with pulmonary cavities due to old tuberculosis, aspergillomas were present in 11%. Subjects with immunosuppression
subsequent to chemotherapy or the use of immunosuppressives or with disorders such as chronic granulomatous disease are unable to
clear Af. In these cases the immune response is so ineffective that direct fungal invasion occurs, leading to a severe acute pneumonia,
sepsis, and often death.
Pathology
In the early stages of ABPA, pulmonary lesions consist
of bronchial mucoid impaction, and areas of eosinophilic pneumonia. In mucoid impaction, bronchi are
dilated and filled with mucus that contains necrotic
eosinophils, Curschman’s spirals, Charcot-Leyden
crystals, and occasionally fungal hyphae. Eosinophilic
pneumonia is synonymous with radiographic infiltrates, composed of focal areas of alveoli filled with
eosinophils and macrophages. As the disease progresses, endobronchial inflammation increases, which
begins as a mixed eosinophilic/neutrophilic obliterative bronchiolitis. This then progresses to the complete
replacement of bronchial structures by inflammatory
cells (histiocytes, lymphocytes, and plasma cells),
known as bronchocentric granulomatosis. At the center of these areas are necrotic inflammatory cells and
Af hyphae. Destruction of the bronchial wall matrix
and subsequent scarring and repair are thought to account for the development of bronchiectasis and fibrosis. The intensity of the inflammatory infiltrate
present in the airways of subjects with ABPA certainly
correlates with the severity of bronchiectasis present.
Pathological features of ABPA are demonstrated in
Figure 2.
Clinical Features
Diagnosis and Disease Staging
The criteria for diagnosis were standardized in 1977
by Patterson and co-workers and are summarized in
178 ASTHMA / Allergic Bronchopulmonary Aspergillosis
Table 1 Diagnostic criteria for ABPA
Essential criteria
Asthma
Immediate skin-prick test (SPT) positive to Af
Total serum IgE 4 1000 ng ml 1 (400 IU ml 1)
Serum specific IgG and IgE antibodies to Af (or positive
precipitins)
Presence of bronchiectasis
Yes, define as ABPA-CB
No, define as ABPA-S
(a)
(b)
(c)
Figure 2 (a) Hyphae of Aspergillus fumigatus grown in vitro; (b)
mucoid impaction. The dilated bronchus is filled with mucus that
contains eosinophils, cell debris, and the products of eosinophil
degranulation (Charcot-Leyden crystals). The bronchial epithelium is heavily infiltrated by inflammatory cells. (c) Bronchocentric
granulomatosis: the epithelium of the bronchus becomes completely replaced by a granulomatous infiltrate of histiocytes,
lymphocytes, plasma cells, and eosinophils. In addition, there are
necrotic inflammatory cells and mucus within the dilated airway
and marked generalized bronchial wall thickening. Slides Courtesy of Dr B Addis, Southampton General Hospital.
Table 1. They require the patient to have a pre-existing diagnosis of asthma (or cystic fibrosis), immediate-type skin reactivity to Af, and, at least during
exacerbations or in the absence of treatment,
Nonessential criteria
Transient pulmonary infiltrates on chest radiograph
Blood eosinophilia
Precipitating antibodies to Af
Expectoration of mucus plugs
peripheral blood eosinophilia, precipitating antibodies to Af antigen, elevated serum IgE, and IgG
antibodies against Af. Radiological evidence of proximal bronchiectasis is frequently found with ABPA,
but is no longer felt to be a prerequisite for diagnosis.
However, the presence of bronchiectasis along with
skin-test reactivity and eosinophilia is quite specific
for ABPA.
ABPA has been subdivided into five stages. Stage I
is the initial acute presentation, with eosinophilia,
immediate-type skin reactivity to Af, total serum IgE
greater than 2500 ng ml 1, and pulmonary infiltrates
on a chest radiograph. Stage II is the disease in remission, where there is persistent immediate-type
skin reactivity and precipitating antibodies to Af antigens. In stage III there is an exacerbation of symptoms with all the characteristics of stage I but with a
twofold rise in serum IgE and new pulmonary infiltrates. Stage IV patients have asthma where control
of symptoms is dependent on chronic use of highdose corticosteroids. In stage V, chronic disease has
progressed to predominately fixed airflow obstruction with extensive bronchiectasis and fibrosis.
Skin-prick testing is a useful screening test to identify potential patients with ABPA. It is highly
sensitive but not specific. Consequently, a negative
skin-prick test for Af can rule out ABPA whereas a
positive test warrants further investigation, particularly in the case of an asthmatic with frequent exacerbations or parenteral corticosteroid dependence.
Patients with a positive skin test should be evaluated
with serology including measurement of total serum
IgE, specific IgG, and IgE antibodies to Af and precipitins. Patients with an IgE 4 1000 ng ml 1 (400
IU ml 1) and positive IgE Af and IgG Af are likely to
have ABPA and warrant further investigation with a
high-resolution computed tomography (CT) scan of
the chest to determine the presence of bronchiectasis.
Patients with central bronchiectasis are classed as
ASTHMA / Allergic Bronchopulmonary Aspergillosis 179
ABPA-CB (ABPA with central bronchiectasis) and
those without are classed as ABPA-S (ABPA serology). In milder disease, a high index of suspicion is
required, with the criteria fulfilled only during an
exacerbation or when off parenteral corticosteroids.
Radiology
During acute disease flares chest radiographs may
demonstrate transient pulmonary infiltrates that occur
as a result of eosinophilic pneumonitis and areas of
mucoid impaction that appear as areas of atelectasis
with toothpaste shadows and the finger-in-glove appearance of bronchi filled with inspissated mucus.
Permanent radiographic changes are a feature of more
advanced disease with the development of central
bronchiectasis and in some areas of parenchymal fibrosis. While bronchial wall thickening and minimal
bronchiectasis (limited to 1–2 segments) has been described in asthma, the presence of widespread central
bronchiectasis, which is best seen on high-resolution
CT scans, is characteristic of ABPA. In addition, areas
of bronchial wall thickening, mucoid impaction, circular opacities, and areas of atelectasis are common.
ABPA and Cystic Fibrosis
Diagnosis of ABPA in CF is more difficult though the
criteria have recently been reviewed and standardized
by a consensus conference; these criteria are summarized in Table 2. The diagnosis in CF is complicated
by the pre-existing presence of bronchiectasis and
chronic endobronchial infection. A multiple regression analysis of over 14 000 individuals with CF determined that wheezing, a diagnosis of bronchial
asthma, and colonization with Pseudomonas aeruginosa were independent risk factors for ABPA. The
development of ABPA in someone with cystic fibrosis
heralds a more complicated clinical course and is associated with more bacterial colonization, lower lung
function, and an increase in pulmonary complications.
Pathogenesis
Af is an effective pathogen in humans as intrinsic
qualities of the organism enhance its ability to infect
Table 2 Diagnostic criteria for ABPA in cystic fibrosis
An acute or subacute deterioration in clinical symptoms (cough,
wheeze, exercise capacity, change in pulmonary function, or
increase in sputum) not attributable to another cause
Immediate skin test reactivity to Af
A total serum IgE 4 400 IU ml 1
Along with one of the following:
precipitins to Af or specific IgG to Af
new or recent infiltrates, mucus plugging, or proximal
bronchiectasis on either chest radiograph or CT scan
the lungs and cause disease. The spores of Af are 2–
5 mm in size with a hydrophobic coat that allows
them to be inspired into the lungs. Once inspired, the
conidia of Af are able to bind to surfactant molecules
in the distal airway lumen, as well as complement
(C3) and fibrinogen. The conidia germinate within
the airways and form focal areas of mycelia. The
mature organisms are then capable of releasing
allergens, virulence factors, and proteases. These factors contribute to: (1) impaired mucociliary clearance; (2) impaired action of fungicidal proteins and
complement in the airway lining fluid; and (3) inhibition of phagocytosis and the killing capacity of
phagocytic cells (macrophages, neutrophils).
As the organism is ubiquitous in the environment,
sensitized individuals are likely to regularly inhale
spores and this will always represent a fresh source
of antigenic stimulus. However, Af is unique as an
aeroallergen and inhalation alone is insufficient to
lead to ABPA. Persistence of viable Af within the
airways appears to be an important factor in determining the development of ABPA. Viable Af has been
found growing on and between bronchial epithelial
cells, despite an intense inflammatory cell infiltrate
while Af proteases lead to the release of proinflammatory mediators from epithelial cells. These proteases also have the ability to detach epithelial cells
from their basement membrane and are particularly
potent. This loss of epithelial integrity may lead to
exposure of the underlying matrix, to which Af can
also adhere allowing direct damage of the airways
and the development of bronchiectasis; in addition,
the disruption of mucosal integrity enhances antigen
uptake and activation of T cells.
In addition to its direct effect on the airway epithelium, Af appears to be able to elicit a powerful
TH2 immune response. The presence of Af infection
in mice induces a TH2 lymphocyte response, while
the extracts from Af when co-cultured with B cells
elicit the release of IgE and subjects with ABPA develop specific TH2 CD4 þ cells in response to exposure. Epithelial cell activation and a TH2 immune
response favors recruitment and activation of eosinophils. Eosinophil activation and release of toxic
granular proteins are likely to contribute to damage
to both the epithelium and airway matrix. In asthma
TH2 cells also release IL-4 and IL-13, which induces
the release of TGF-b from epithelial cells, leading to
a repair response with myofibroblast activation, fibrosis, and airway wall remodeling.
This intense recruitment of inflammatory cells to
the airway lumen and the development of progressive
airway wall damage is in keeping with airway inflammation found in patients with ABPA and the
development of radiographic bronchiectasis.
180 ASTHMA / Allergic Bronchopulmonary Aspergillosis
1
Mucoid
impaction
Af conidia
Tenacious
mucus
Alveolar
macrophage
3
2
Proteases
Epithelial
detachment
Mycelia
Fibroblast
8
Allergens
Inflammatory
cytokines/chemokines
4
6
5
IgE
IL-5
B cell
TH2
lymphocyte
IL-4 & IL-13
Eosinophil
7
Figure 3 A model for the pathogenesis of ABPA. 1: The conidia of Af are just 2–5 mm in diameter and are easily respired into the distal
airways. 2: The conidia are trapped within the tenacious mucus bilayer, binding to surfactants, complement, and fibrinogen. The conidia
attach to bronchial epithelial cells. Here they germinate and mature, forming mycelia or small fungal balls. 3: Mature Af produces
proteases and allergens. Aspergillus proteases are extremely potent. They activate epithelial cells, releasing proinflammatory cytokines
and chemokines, leading to the hypersecretion of mucus, thus damaging epithelial cells, which results in widespread epithelial detachment and the loss of mucosal integrity. This allows the proteases to damage the airway matrix also. In addition, proteases can block
the ability of alveolar macrophages to ingest Af, the net effect being impaired mucociliary clearance and facilitated colonization. 4:
Activated epithelial cells release proinflammatory mediators, such as interleukin-8 (IL-8), IL-6, RANTES, and MCP-1, activating and
attracting lymphocytes, eosinophils, and neutrophils to the airways. 5: Aspergillus allergens are potent activators of TH2 type immune
response from TH cells. These cells release IL-5, which recruits and activates eosinophils. 6: Eosinophils migrate to the airways and
infiltrate. Once activated, they release toxic granular proteins that further damage the epithelium and airway matrix. 7: Activated TH2
lymphocytes produce IL-4 and IL-13, which induce isotype switching of bells to produce IgE antibodies. In addition, IL-4 and IL-13 have
been shown to induce epithelial cells to secrete the anti-inflammatory but pro-fibrotic mediator TGF-b. 8: Epithelial and airway matrix
damage elicits a wound-repair response in the airways. Release of TGF-b has been shown to induce fibroblasts to release growth
factors and collagen. This is thought, at least in part, to induce airway wall remodeling in asthma.
A model for the pathogenesis of ABPA is proposed
in Figure 3. ABPA appears to develop in susceptible
individuals who are sensitized to Af; they are unable to
clear the organism, which then adheres to the bronchial epithelium releasing allergens and proteases. This
activates an intense TH2 immune response and leads
to progressive airway destruction and remodeling.
Animal Models
A number of investigators have employed animal
models to determine the role of Af antigens in triggering the immune response and in determining the
constitutive components responsible for this response.
Most investigators have used crude Af extract to sensitize animals, often followed by ongoing exposure to
viable conidia or Af spores. Uniquely, sensitization
with Af extract does not require an adjunct; it is
thought that Af proteases effectively disrupt the
mucosal barrier enhancing antigen presentation.
Recently, recombinant antigen components of Af
extract have been used; identifying Asp f 1, 3, and 4
as independent inducers of airway inflammation and
bronchial hyperresponsiveness (BHR).
The most widely employed animal model has been
murine. As in humans, sensitization to Af leads to a
ASTHMA / Aspirin-Intolerant
Table 3 Components of the immune response to Aspergillus
elucidated by murine models of ABPA
Immune mediator
Response observed in the model
Block IL-13
Reduced pulmonary eosinophilia and
goblet cell hyperplasia
No change to subepithelial fibrosis
Reduced IgE and pulmonary eosinophila
More pronounced TH1 response and
consequent pulmonary inflammation
Inhibit all aspects of the TH2 inflammatory
response to Af
Reduced pulmonary eosinophils
Airway inflammation occurs with monocyte
infiltrate and BHR still increased
(a) Treated at time of Af sensitization,
increased airway inflammation, and BHR
(b) Treated at 14–30 postsensitization,
reduced airway inflammation, and BHR
Increased TH2-mediated inflammation
Increased IgE production
Reduced TH1 response with lower levels of
interferon-g and reduced pulmonary
neutrophils
Reduced clearance of Af
Block IL-4
Addition of IL-10
Block IL-5
Inhibit MCP-1 or
inactivate
receptor CCR2
CCR2 knockout
mice
potent response with production of IgE, IgG1, pulmonary eosinophilia, and TH2 activation and their
recruitment to the airways. In addition, such models
have identified Af as potent inducers of BHR, airway
remodeling, goblet cell hyperplasia, and mucus hypersecretion. The ability to use knockout animals and
block specific cytokines, chemokines, and their receptors in murine models has allowed a greater understanding of the specific components of the immune
response to Af; the most important of these are summarized in Table 3. The profound TH2 immune response to Af has been well demonstrated in these
models along with the suggestion that such a response
is relatively ineffective in clearing the organism.
See also: Chemokines, CXC: IL-8. Cystic Fibrosis: Overview; Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Interleukins: IL-4; IL-13.
Transforming Growth Factor Beta (TGF-b) Family of
Molecules.
Further Reading
Bateman ED (1994) A new look at the natural history of aspergillus hypersensitivity in asthmatics. Respiratory Medicine 88:
325.
Bosken CH, Myers JL, et al. (1988) Pathological features of allergic bronchopulmonary aspergillosis. American Journal of
Surgical Pathology 12(3): 216–222.
Chetty A (2003) Pathology of allergic bronchopulmonary aspergillosis. Frontiers in Bioscience 8: e110–e114.
Greenberger P, Miller T, et al. (1993) Allergic bronchopulmonary
aspergillosis in patients with and without evidence of bronchiectasis. Annals of Allergy 70: 333–338.
181
Grunig G and Kurup VP (2003) Animal models of allergic
bronchopulmonary aspergillosis. Frontiers in Bioscience 8:
e157–e171.
Kauffman HF (2003) Immunopathogenesis of allergic bronchopulmonary aspergillosis and airway remodelling. Frontiers in
Bioscience 8: e190–e196.
Kauffman HF and Tomee JF (1999) Inflammatory cells and airway
defense against Aspergillus fumigatus. Immunology and Allergy
Clinics of North America 18: 619–640.
Kurup VP and Banerjee B (1996) Allergic aspergillosis: antigens and
immunodiagnosis. Advances in Medical Mycology 2: 133–154.
Patterson R, Greenberger P, et al. (1982) Allergic bronchopulmonary aspergillosis: staging as an aid to management. Annals of
Internal Medicine 96: 286–291.
Soubani AO and Chandrasekar PH (2002) The clinical spectrum
of pulmonary aspergillosis. Chest 121: 1988–1999.
Stevens D, Moss RB, et al. (2003) Allergic bronchopulmonary
aspergillosis in cystic fibrosis
state of the art: Cystic Fibrosis
Foundation Consensus Conference. Clinical Infectious Diseases
37(supplement 3): S225–S264.
Varkey B (1998) Allergic bronchopulmonary aspergillosis: clinical
perspectives. Immunology and Allergy Clinics of North America
18(3): 479–501.
Wark PAB and Gibson PG (2001) Allergic bronchopulmonary
aspergillosis: new concepts of pathogenesis and treatment. Respirology 6: 1–7.
Wark PAB, Gibson PG, et al. (2003) Azoles for allergic bronchopulmonary aspergillosis associated with asthma. Cochrane Database System Review 4(CD001108).
Aspirin-Intolerant
A P Sampson, University of Southampton School of
Medicine, Southampton, UK
& 2006 Elsevier Ltd. All rights reserved.
Abstract
Aspirin-intolerant asthma (AIA) is a phenotype experienced by
10–20% of persistent asthmatics, in whom acute bronchoconstriction is induced by ingestion of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs). These drugs share
the ability to inhibit synthesis of prostanoids by blockade of
cyclooxygenase (COX). Acute reactions to NSAIDs can be life
threatening and may be associated with rhinoconjunctival and
dermal symptoms. Drugs that selectively inhibit COX-2 appear
to be better tolerated than nonselective inhibitors of COX-1 and
COX-2. Patients with AIA usually have persistent underlying
asthma, often associated with nasal polyposis. Pathologically,
the bronchial and nasal airways of AIA subjects show chronic
eosinophilia, with evidence of activation of eosinophils and
mast cells during acute reactions. The etiology of AIA is unclear,
but the proposed mechanism focuses on the inhibition by
NSAIDs of the synthesis of a prostanoid, putatively prostaglandin E2, that would normally suppress local inflammatory reactions. The consequent synthesis of cysteinyl-leukotrienes and
other leukocyte-derived mediators contributes to bronchoconstriction and other acute features. Treatment of AIA involves
avoidance of NSAIDs combined with conventional management
of underlying asthma, with 75% of AIA patients requiring corticosteroids. Controlled desensitization with regular doses of an
NSAID can provide protection against acute reactions.
182 ASTHMA / Aspirin-Intolerant
Introduction
The classical aspirin-intolerant asthma (AIA) syndrome was described by Samter and Beers in 1968 as
a triad of rhinosinusitis (often with nasal polyps),
asthma, and aspirin sensitivity. Patients with fullblown AIA are about twice as likely to be female as
male, but no more likely to be atopic than the general
population. Patients typically present in early middle
age with nasal congestion, anosmia, and rhinorrhea,
and many develop nasal polyps, often with secondary
infection of the paranasal sinuses. Bronchoconstriction and airway inflammation usually emerge later,
and this becomes severe, perennial asthma, with
about 75% of patients needing oral or inhaled corticosteroids to maintain control of their symptoms.
Acute respiratory reactions to nonsteroidal anti-inflammatory drugs (NSAIDs) may be accompanied by
rhinoconjunctival symptoms and by dermal symptoms such as facial flushing and exacerbation of preexisting urticaria.
Aspirin challenges reveal that the tendency to
bronchoconstrict in response to therapeutic doses of
NSAIDs is more prevalent than the full-blown ‘aspirin triad’ recognized clinically. Based on patient history alone, the prevalence of NSAID intolerance in
adult asthmatics is 3–5%, but it rises to 19% when
consecutive asthmatic patients are challenged with
oral aspirin. Prevalence in asthmatic children is less
than 2% based on history alone, but 13–16% of
postpubertal asthmatic children respond adversely
when aspirin challenged. NSAID intolerance is overrepresented in the severe asthmatic population.
Among patients who have experienced a near-fatal
asthma exacerbation requiring treatment in the intensive care unit, 24% are aspirin-sensitive based on
history alone. Most life-threatening acute exacerbations in AIA patients are directly due to inadvertent
use of NSAIDs, and in a few cases the precipitating
NSAID was prescribed by the patient’s own physician. However, over 40% of life-threatening asthma
exacerbations in AIA patients cannot be attributed to
NSAID ingestion, illustrating the severity of the underlying chronic asthma in these patients.
Etiology
Aspirin (acetylsalicylic acid) and other members of
the family of NSAIDs inhibit formation of the prostanoid family of lipid mediators by blocking isozymes of cyclooxygenase (COX). They are used
therapeutically for their mild analgesic, antipyretic,
and anti-inflammatory actions. NSAIDs are variably
associated with adverse reactions including prolonged bleeding, nephritis, and gastric ulceration.
Table 1 Chemical classes of some nonsteroidal anti-inflammatory drugs (NSAIDs)
Acetic acids
Indomethacin
Sulindac
Diclofenac
Fenamates
Mefenamic acid
Oxicams
Piroxicam
Propionic acids
Ibuprofen
Pyrazoles
Phenylbutazone
Benoxaprofen
Oxyphenbutazone
Salicylates
Aspirin (acetylsalicylic
acid)
Sodium salicylate
In 1919, Cooke recognized that in some patients
with asthma, aspirin may also precipitate life-threatening acute exacerbations. Such reactions have now
been recognized in response to a wide variety of
NSAIDs of diverse chemical classes (Table 1).
A family history of AIA is reported by only 6%
of AIA patients, suggesting that any genetic influences are subtle and expressed phenotypically only
in the presence of relevant environmental factors.
Leukotriene C4 synthase is the terminal enzyme
for the synthesis of the bronchoconstrictor cysteinylleukotrienes postulated to contribute to airway
narrowing in AIA. A biallelic polymorphism in the
promoter region of the LTC4 synthase gene has
been reported, involving an A to C transversion at
a position 444 bp upstream of the transcription
start site. The variant
444C allele may lead to
enhanced transcription of the LTC4 synthase gene
in relevant cells. The prevalence of the variant allele
has been reported to be significantly elevated in AIA
patients from a Polish population, but this has
not been replicated in US Caucasian or Japanese
populations.
Pathology
A classification system for allergic and pseudoallergic
reactions to NSAIDs was proposed by Stevenson,
Sanchez-Borges, and Szczeklik in 2001. AIA is classified as a type 1 pseudoallergic reaction in which
asthmatic patients with a high frequency of sinusitis
and nasal polyps experience lower respiratory tract
reactions and/or rhinoconjunctival symptoms after
exposure to therapeutic doses of aspirin and other
NSAIDs. Reactions are dose-dependent and may be
life threatening. The type 2 category describes urticarial reactions or angioedema induced by NSAIDs in
patients with pre-existing chronic urticaria, and
types 3 and 4 describe urticarial or sporadic reactions in patients who are otherwise normal. In contrast to types 1–4 above, in which patients show
extensive cross-reactivity to many NSAIDs, patients
classified in types 5–8 react adversely only to a single
ASTHMA / Aspirin-Intolerant
drug. These reactions include urticaria/angioedema,
anaphylaxis, aseptic meningitis, and hypersensitivity
pneumonitis.
A consistent, although not diagnostic, pathological
finding in AIA patients is chronic eosinophilia in the
blood, nasal polyps, and bronchoalveolar lavage
(BAL) fluid. Immunohistochemical studies in bronchial biopsies have confirmed a marked bronchial
mucosal eosinophilia in AIA patients, with eosinophil counts three- to fourfold higher than in aspirintolerant asthmatics and 10- to 15-fold higher than in
normal subjects. After NSAID challenge, further increases in eosinophils, their activation marker ECP
(eosinophil cationic protein), and histamine have
been described in the nasal airways, BAL fluid, and
plasma of AIA patients, suggesting activation of
eosinophils and mast cells. In the nasal airway, aspirin challenge of AIA patients is associated with increments in nasal tryptase and histamine, strongly
suggesting mast cell activation. Tryptase and histamine levels also rise in the serum of patients experiencing systemic reactions to oral aspirin, but not in
those with localized respiratory reactions. Release of
tryptase is a recognized marker of mast cell activation in the pulmonary airway, and occurs within
5 min of allergen challenge in allergic asthmatics.
However, tryptase did not rise in the BAL fluid of
AIA patients challenged with inhaled or endobronchial lysine-aspirin. In contrast, a rise in urinary levels of the PGD2 metabolite 9a, 11b-PGF2 following
endobronchial lysine-aspirin challenge has been interpreted as evidence for mast cell activation in AIA.
Together, the evidence suggests that inflammatory
mediators released both from mast cells and from
eosinophils contribute to the pulmonary and rhinoconjunctival symptoms following NSAID exposure
in AIA patients.
Clinical Features
As there are no acceptable in vitro tests for AIA,
confirmation of aspirin intolerance can only be obtained by NSAID challenge under controlled conditions. Lung function is monitored while patients
ingest incremental oral doses of aspirin or inhaled
doses of a lysine-aspirin conjugate or sulpyrine. Concomitant use of b2-adrenergic agonists, cromones,
and inhaled corticosteroids may mask responses to
NSAID challenge, leading to a high rate of false-negative results. In a 3-day oral challenge protocol, incremental doses of aspirin are given at 3-hourly
intervals up to a maximum of 650 mg. The challenge
is terminated when forced expiratory volume in 1 s
(FEV1) falls by at least 20%. Reactions begin around
183
50 min after oral aspirin ingestion, ranging from 20
to 120 min. A shorter protocol involves the inhalation
of incremental aerosolized doses of lysine-aspirin, a
soluble and nonirritant form (this is not available in
the US). Respiratory reactions to inhaled lysineaspirin often occur within 1 min, so shorter dosing
intervals of 30–60 min allow the entire challenge to
be completed within 1 day. Inhaled lysine-aspirin
challenges are safer than oral challenges as reactions
are localized to the airways and are easily reversible
with inhaled b2-agonists. The sensitivity of inhaled
lysine-aspirin challenge is similar to oral aspirin
challenge.
Pathogenesis
The Cyclooxygenase Theory
The most successful model to explain acute respiratory reactions to NSAIDs is the cyclooxygenase
theory promulgated by Szczeklik in 1975. This postulates that reactions are related directly to the pharmacological activity of NSAIDs in inhibiting
isozymes of COX, the key enzymes in the synthesis
of the prostanoid family of lipid-inflammatory mediators. Intolerance to an individual NSAID in vivo
was shown to be predictable by its potency in inhibiting COX in vitro, with strong inhibitors (including
aspirin, indomethacin, mefenamic acid, ibuprofen,
and piroxicam) being common precipitants of adverse reactions, while weak inhibitors (such as sodium salicylate) precipitated reactions rarely or only
at high doses. The concept that inhibition of prostanoid synthesis is the trigger for NSAID-induced
reactions is recognized as a key advance.
The Role of Cysteinyl-Leukotrienes: The Shunting
Hypothesis and the PGE2 Brake Hypothesis
The second key advance was the elucidation in 1979
of the structure of slow-reacting substance of anaphylaxis (SRS-A) as a mixture of potent bronchoconstrictor products of a related family of lipid
mediators, the cysteinyl-leukotrienes (cysteinyl-LTs).
The cysteinyl-LTs – LTC4, LTD4, and LTE4 – are now
recognized to have important bronchoconstrictor and
proinflammatory roles in many phenotypes of asthma. Their particular relevance to AIA emerged from
the recognition that their biosynthetic pathway, the 5lipoxygenase (5-LO) pathway, shares arachidonic
acid as a common precursor with the prostanoid
pathway (Figure 1). The leukotriene pathway is not
inhibited by NSAIDs. Specific blockade of the prostanoid pathway by NSAIDs was therefore proposed
to shunt arachidonate away from conversion into
184 ASTHMA / Aspirin-Intolerant
NSAIDs
Membrane phospholipids
Phospholipase A2
−
Cyclooxygenase-1
Arachidonic acid
−
Zileuton
5-lipoxygenase
FLAP
LTA4
PGG2
PGH2
Cyclooxygenase-2
Prostanoid synthases
& isomerases
LTC4 synthase
LTC4
LTD4
LTE4
(Cysteinyl-leukotrienes)
−
Montelukast
Pranlukast
Zafirlukast
CysLT1 receptors
PGE2
TXA2
PGD2
PGF2
Prostanoid receptors
(EP1−4, TP, DP, CRTH2, FP)
Bronchoconstriction
Vascular permeability
Edema
Mucus secretion
Eosinophil migration
Figure 1 The 5-lipoxygenase and cyclooxygenase pathways of eicosanoid metabolism.
prostanoids, which have relatively little bronchoconstrictor activity, towards the formation of cysteinylLTs, which are highly potent bronchoconstrictors.
This ‘shunting’ hypothesis is superficially attractive
but measurements of lipid mediators in isolated
leukocytes treated with NSAIDs argues against such
a simple mechanism. It has therefore been superseded
by an alternative notion, the ‘PGE2 brake’ hypothesis.
This proposes that NSAIDs block the formation of
an anti-inflammatory prostanoid, PGE2, which is
otherwise known to suppress leukotriene synthesis
by leukocytes. Exposure to NSAIDs may thus liberate
the 5-LO pathway from suppression by endogenous
PGE2 in vivo, at least in susceptible individuals
(Figure 2).
There are three main lines of experimental evidence supporting this model:
1. The triggering effect of NSAIDs on LT synthesis
can be mimicked in vitro in a number of inflammatory leukocyte subtypes. Endogenous PGE2
suppresses, and NSAIDs consequently enhance,
leukotriene synthesis in eosinophils, neutrophils,
basophils, and macrophages, but apparently not
in human lung mast cells. Eosinophils themselves
generate sufficient PGE2 to suppress their own
LTC4 synthesis by about 90%. Treatment of
eosinophils with indomethacin inhibits endogenous PGE2 synthesis and stimulates LTC4 release. Replacement experiments showed that
exogenous PGE2 restores the braking effect, returning LTC4 synthesis to the levels seen before
indomethacin treatment. PGE2 may act in an
autocrine or paracrine manner at EP2 receptors on
the cell surface, followed by an increase in intracellular cAMP and activation of protein kinase A,
but the subsequent steps by which 5-LO activity is
inhibited are unknown.
2. Eicosanoids can be measured in biological fluids,
including bronchoalveolar lavage (BAL) fluid, and
urine. Endoscopic challenge with lysine-aspirin
reduces PGE2 and thromboxane A2 levels in the
BAL fluid of AIA patients and aspirin-tolerant
asthmatics, but a dramatic rise in BAL fluid cysteinyl-LTs is seen only in the AIA group. Following
challenge with oral aspirin or inhaled lysine-aspirin, urinary LTE4 levels, used as a marker of
whole-body cysteinyl-LT production, rise three to
sevenfold in AIA patients, but not in aspirin-tolerant asthmatics; this response is not seen after
methacholine-induced bronchoconstriction or placebo challenge. At the same time, there is a fall in
urinary markers of prostanoid synthesis, such as
11-dehydro-thromboxane A2. In AIA patients,
preinhalation of PGE2 before challenge with
inhaled lysine-aspirin completely ablates the rise
in urinary LTE4 and prevents the consequent
bronchoconstriction, providing strong evidence
that cysteinyl-LT synthesis has a functional role
in the NSAID-induced airway narrowing. The
ASTHMA / Aspirin-Intolerant
185
Mechanism of acute NSAID reactions
Mast cell / eosinophil
NSAID
COX-1
FLAP
5-LO
LTC4
synthase
LTC4
cAMP
ECP
Histamine
Tryptase IL-5
EP2
PGE2
Bronchoconstriction
Eosinophillia
Vasodilation / edema
Remodeling / BHR
Figure 2 In this model, PGE2 derived from constitutive cyclooxygenase-1 (COX-1) normally acts via cell surface EP2 receptors to
suppress the activity of mast cells and eosinophils, including the activity of the 5-lipoxygenase pathway. However, when an NSAID
ingested by a susceptible individual inhibits COX-1, the reduced synthesis of PGE2 is insufficient to suppress the leukocyte effectively.
The result is a surge in the synthesis and release of LTC4 and other mediators, leading to bronchoconstriction and inflammations, AIA
patients may be susceptible to this response because they have exaggerated LTC4 synthase expression, and/or a defect in the PGE2
brake, based perhaps on insufficient COX-1 activity or more likely on impaired signaling from EP2 receptors.
protective effect of inhaled PGE2 does not correlate with its relatively weak bronchodilator activity, confirming that PGE2 preinhalation protects
by restoring the suppression of cysteinyl-LT synthesis, not by dilating airways directly.
3. The effector role of cysteinyl-LTs in adverse respiratory and rhinitic reactions to NSAIDs in most
AIA patients has been confirmed with placebocontrolled clinical trials of specific leukotriene
modifier drugs. The 5-LO inhibitors zileuton and
ZD-2138 markedly blocked the rise in urinary
LTE4 and the fall in FEV1 following oral aspirin
challenge of AIA. Rhinoconjunctival and dermal
reactions to oral aspirin are also blocked by
zileuton. Antagonists of CysLT1 receptors block
oral NSAID-induced respiratory reactions in AIA
patients.
The PGE2 Brake is Derived from COX-1
The cyclooxygenase theory and the PGE2 brake hypothesis are thus crucial in understanding how
NSAIDs trigger LT synthesis and bronchoconstriction, but they do not explain why only some asthmatics are susceptible to these responses. AIA patients
tolerate selective COX-2 inhibitors including nimesulide, meloxicam, and rofecoxib, and respond
adversely most often to NSAIDs with a greater selectivity for COX-1, such as aspirin and indomethacin.
This suggests that cytoprotective/anti-inflammatory
PGE2 in the lung is produced by constitutive COX-1.
COX-1 is expressed in a large number of cell types,
including mast cells, eosinophils, macrophages,
vascular endothelial cells, bronchial epithelium, and
bronchial smooth muscle. The exact cellular sources
of the putative PGE2 brake remain unclear. AIA patients may overproduce cysteinyl-LTs chronically and
acutely after NSAID exposure because of a defect in
endogenous PGE2 synthesis. However, inhaled lysineaspirin equieffectively inhibits airway PGE2 synthesis
both in AIA patients and in aspirin-tolerant asthmatics. Baseline levels of PGE2 and other prostanoids are
not consistently different in the BAL fluid and urine of
AIA patients and control groups. Immunohistochemical studies of AIA bronchial biopsies have found little
evidence for a meaningful anomaly in the cellular expression of COX isozymes. One possibility is that a
defective PGE2 brake may derive not from a failure of
PGE2 synthesis, but of leukocytes to be suppressed by
PGE2. An anomaly in EP2 receptor structure, expression, or signaling might be one explanation, and this
would also be consistent with the lack of clinical
benefit shown in AIA patients treated with the stable
PGE1 analog misoprostol.
Suggestive evidence of an anomaly within the cysteinyl-LT biosynthetic pathway itself is that baseline
cysteinyl-LT synthesis appears to be two- to sevenfold higher in AIA patients than in control groups,
even in the absence of exposure to NSAIDs, as demonstrated by measurements of cysteinyl-LTs in BAL
fluid, induced sputum, and urine (Figure 3). The increases seen after NSAID exposure are superimposed
upon this chronically elevated baseline. Immunohistochemical analysis of bronchial biopsies from
Cys-LT production
186 ASTHMA / Aspirin-Intolerant
NSAID
AIA
ATA
N
0
1
2
3
Time (h)
Figure 3 Schematic diagram showing that cysteinyl-leukotriene
production is chronically elevated in patients with aspirin-intolerant asthma (AIA) compared to those with aspirin-tolerant asthma
(ATA) and normal subjects (N). A further rise in cysteinyl-LT
production after exposure to a nonsteroidal anti-inflammatory
drug (NSAID) occurs only in the AIA group.
AIA and aspirin-tolerant asthmatic subjects shows
that counts of cells immunostaining for LTC4 synthase, the terminal enzyme for cysteinyl-LT synthesis,
were fivefold higher in AIA biopsies than in aspirintolerant asthma biopsies and 18-fold higher than
in normal biopsies. The numbers of LTC4 synthasepositive cells in the bronchial mucosa correlated with
elevated levels of cysteinyl-LTs in the BAL fluid and
with bronchial responsiveness to inhaled lysine-aspirin. Persistent overproduction of cysteinyl-LTs in
steady-state AIA may therefore be related to overexpression of LTC4 synthase in the bronchial wall,
much of it within eosinophils and mast cells. This
may also contribute to the further surge in cysteinylLT synthesis when PGE2 suppression is removed by
NSAIDs.
Animal Models
Although the anti-inflammatory actions of NSAIDs
and their effects of prolonged bleeding, nephritis,
and gastric ulceration can be replicated in animal
models, there has been relatively little interest in developing animal models of AIA. This may reflect, at
least in part, the difficulty of replicating the diverse
immunopathological features of asthma, especially
chronic remodeling of structural airway tissues, and
also of replicating the typically high degree of disease
severity and glucocorticoid dependency observed in
AIA patients.
Management and Current Therapy
National and international management guidelines for
asthma (e.g., GINA guidelines) are based on disease
severity and make no therapeutic distinctions between
AIA and other asthma phenotypes. Asthmatics should
avoid using NSAIDs either prescribed inadvertently or
purchased over the counter. NSAID-induced acute reactions in susceptible asthmatics can be treated with
nebulized b-2 adrenergic agonists, repeated frequently
over several hours where necessary. Decongestants
and antihistamines (topical or oral) can be used for
associated rhinoconjunctival symptoms. In the most
severe cases, intubation and mechanical ventilation in
the intensive treatment unit may be required.
Treatment of chronic AIA focuses on anti-inflammatory therapy of the upper and lower airways using
topical and inhaled/insufflated corticosteroids. Antibiotics may be required when purulent nasal secretions indicate infection, and many AIA patients
require repeated polypectomies. Some AIA patients
may require NSAIDs for concomitant inflammatory
disease such as arthritis, and in such cases, aspirin
desensitization may be an option. Acute respiratory
reactions to NSAIDs in AIA patients are always followed by a refractory period lasting 2–5 days during
which further reactions to NSAIDs cannot be induced. Sensitivity to NSAIDs re-emerges within a
week, but regular low-dose NSAIDs can maintain the
refractory state indefinitely. Daily or alternate-day
dosing of NSAIDs is therefore used clinically to
desensitize AIA patients to inadvertent ingestion of
NSAIDs and to allow NSAID therapy of concomitant diseases such as arthritis. Cross-desensitization
occurs such that repeated dosing with one NSAID
provides protection against adverse reactions to
other NSAIDs. Chronic desensitization reduced the
numbers of acute exacerbations and hospital admissions in AIA patients compared to a control group of
AIA patients who avoided NSAIDs, associated with
reductions in corticosteroid use. The mechanism of
desensitization is unknown, but is tentatively linked
to reductions both in the quantity of cysteinyl-LTs
synthesized following NSAID exposure, and with reduced bronchial responsiveness to cysteinyl-LTs. In
the nasal airway, desensitization has been linked to a
reduction in expression of the CysLT1 receptor on
infiltrating leukocytes.
In small studies in AIA patients, the antiallergic
cromone sodium cromoglycate, the antiviral agent
acyclovir, the antibiotic roxithromycin, and the longacting b2-agonist salmeterol have all been reported to
block not only the bronchial responses to inhaled
NSAIDs, but also the associated rise in urinary LTE4
excretion. The mechanism of these drugs is difficult
to understand in this context, but may involve prevention of mast cell degranulation. On the basis of
the pathophysiological evidence of a central role of
cysteinyl-LTs in AIA, clinical trials of 5-LO inhibitors
and CysLT1 receptor antagonists have been reported.
Zileuton improved lung function and rescue beta-2
ASTHMA / Occupational Asthma (Including Byssinosis) 187
agonist use and restored the sense of smell in a 6week study of 40 AIA patients. In an 8-week crossover trial of montelukast in 80 AIA patients, there
were significant improvements in lung function, use
of rescue therapy, symptom scores, night-time awakenings, and asthma quality-of-life (QOL) scores
compared with placebo. In the clinic, treatment response to leukotriene modifiers is variable, but a
treatment trial is advised in patients with AIA uncontrolled by topical corticosteroids.
See also: Allergy: Overview. Asthma: Overview. Bronchoalveolar Lavage. Bronchoscopy, General and
Interventional. Chymase and Tryptase. Genetics:
Overview. Histamine. Immunoglobulins. Leukocytes:
Mast Cells and Basophils; Eosinophils; Neutrophils. Pulmonary Function Testing in Infants.
Further Reading
Drazen JM, Israel E, and O’Byrne PM (1999) Treatment of asthma
with drugs modifying the leukotriene pathway. New England
Journal of Medicine 340: 197–206.
Pavord ID and Tattersfield AE (1994) Bronchoprotective role for
endogenous prostaglandin E2. Lancet 345: 436–438.
Samter M and Beers RF (1968) Intolerance to aspirin: clinical
studies and consideration of its pathogenesis. Annals of Internal
Medicine 68: 975–983.
Sanak M and Szczeklik A (2000) Genetics of aspirin-induced
asthma. Thorax 55(supplement 2): S45–S47.
Stevenson D, Sanchez-Borges M, and Szczeklik A (2001) A classification of allergic and pseudoallergic reactions to drugs that
inhibit cyclooxygenase enzymes. Annals of Allergy, Asthma and
Immunology 87: 1–4.
Stevenson DD, Simon RA, and Zuraw BL (2003) Sensitivity to
aspirin and nonsteroidal antiinflammatory drugs. In: Adkinson
NF, Bochner BS, Yunginger JW, et al. (eds.) Middleton’s Allergy:
Principle and Practice, 6th edn., pp. 1695–1710. St Louis, MO:
Mosby.
Szczeklik A, Gryglewski RJ, and Czerniawska-Mysik G (1975)
Relationship of inhibition of prostaglandin biosynthesis of analogues to asthma attacks in aspirin sensitive patients. British
Medical Journal 1: 66–69.
Szczeklik A and Stevenson DD (2003) Aspirin-induced asthma:
advances in pathogenesis, diagnosis, and management. Journal
of Allergy and Clinical Immunology 111: 913–921.
Occupational Asthma
(Including Byssinosis)
D J Hendrick, Royal Victoria Infirmary, Newcastle
upon Tyne, UK
& 2006 Elsevier Ltd. All rights reserved.
Abstract
Respirable agents in the workplace are responsible for about
10% of asthma arising in adult life, and for an annual incidence
of occupational asthma (OA) of 25–100 per million employed
in industrially developed countries. For 5–10% of cases, toxic
mechanisms are responsible, and asthma is the result of accidents that release agents such as chlorine, sulfur dioxide, and
acetic acid into occupational environments. For the remaining
90–95%, asthma appears to arise through hypersensitivity
mechanisms. Many of the several hundred causal ‘asthmagens’
are reactive chemicals of low molecular weight, though some
are naturally occurring allergens of high molecular weight.
Some agents (e.g., di-isocyanates, epoxy resins, flour) have such
sensitizing potency that at current exposure levels in some occupations (e.g., spray painting and baking) the risk of OA exceeds that of asthma arising spontaneously. OA is thus
important in an epidemiologic sense, and it serves as a useful
model of asthma in general.
Asthma of occupational origin is like asthma resulting from
any other cause. However, when due to hypersensitivity mechanisms, it has one unusual characteristic. If the diagnosis is recognized within 6–24 months and exposure ceases, there is a
meaningful possibility that active disease will resolve. Therefore, the onus is on physicians to consider the possibility of an
occupational cause in every adult presenting with asthma.
Introduction
It was not until the twentieth century that investigatory techniques acquired the sophistication to distinguish airway disorders from those of the lung
parenchyma/interstitium. The definition of asthma,
the elucidation of its clinical characteristics and pathogenic mechanisms, and the evolution of strategies for
its recognition, management, and prevention have
consequently been comparatively recent events. Nevertheless, it is likely that asthma was the explanation
for many of the respiratory disorders of antiquity and
the middle ages that were recognized to afflict (and
sometimes devastate) workers in certain trades and
industries. The realization that asthma may arise as a
direct consequence of inhaled occupational agents has
been the focus of particular attention over the last
three to four decades, and our understanding of occupational asthma (OA) has largely arisen during this
period.
OA has no recognized differences from asthma in
general with respect to its pathology and genetic etiology, and so this article will focus on its features of
special interest.
Definitions and Classification
Asthma is a disease of the intrathoracic airways that is
characterized, and often defined, by its means of clinical expression (diffuse airway obstruction that varies
in degree over time) and its underlying pathogenic
basis (a state of enhanced airway responsiveness).
OA is caused by exposure to agents, almost exclusively airborne, that are encountered primarily in
the workplace. Such exposure in susceptible individuals causes the level of airway responsiveness to
188 ASTHMA / Occupational Asthma (Including Byssinosis)
increase into the asthmatic range. Two distinct mechanisms are recognized. First, hypersensitivity mechanisms appear responsible since there is a latency
period of presumed sensitization between exposure
onset and symptom onset; this accounts for 90–95%
of cases. Second, acute toxicity is the initiating event;
this is generally a consequence of inhalation of toxic
agents during an industrial accident. Asthma following the latter is sometimes identified as the reactive
airways dysfunction syndrome (RADS) or, more simply, irritant asthma. Neither term is fully satisfactory.
The first may be interpreted, incorrectly, to indicate a
disorder other than asthma, while the second may be
mistaken for pre-existing asthma that is exacerbated
non-specifically at work (e.g., because of exertion in
cold air); this is ‘work-aggravated’ not ‘occupational’
asthma, and is not associated with an increase in
airway responsiveness.
It is currently unclear whether repeated exposures
to toxic agents at dose levels insufficient to provoke
clinical reactions might nevertheless cause minor increases in airway responsiveness. If so, the cumulative effect might elevate airway responsiveness to a
level at which asthma eventually becomes inevitable
(‘low-level RADS’). This would simulate the latency
period association with presumed hypersensitivity
mechanisms, and ongoing exposure might then provoke acute symptoms in a non-specific fashion. This
would appear to simulate hypersensitivity mechanisms also, but such exposure should provoke reactions in any asthmatic individual with a sufficient
level of airway responsiveness.
Key Clinical Features
When hypersensitivity mechanisms are responsible,
further exposures to the particular inducing asthmagen may provoke specific asthmatic reactions. Symptoms are dependent on the degree of hypersensitivity,
the magnitude of the provoking stimulus, the level
of airway responsiveness, and the ease with which
the affected individual perceives changing respiratory
sensations.
Airway responsiveness varies in level from subject
to subject over a considerable range, and can be
quantified throughout the population at large. Its
distribution (like that of most biologic parameters) is
unimodal, and it follows the common biologic pattern of a ‘bell-shaped’ curve. The tail in which responsiveness is most marked gives rise to asthma, but
the adjacent segment implies vulnerability, and the
opposite tail implies comparative impunity. It is misleading to think of asthmatic and nonasthmatic subjects being fundamentally different because of the
presence or absence of airway hyperresponsiveness;
the issue, rather, is whether an individual’s level of
airway responsiveness is sufficiently high to make
meaningful bronchoconstriction likely when there
are appropriate provoking stimuli.
Whether the resulting degree of bronchoconstriction is perceived to be distressing (or is perceived at
all), is very dependent on psychological factors. This
adds an important further level of complexity and
variability, since at all degrees of asthmatic severity
as defined physiologically, there will be a wide spectrum of perceived disability. This is particularly so in
OA because of resentment, even anger, over the possible liability of a third party (the employer), and the
possibility of compensation.
Longstanding OA, like asthma generally, is commonly associated with airway obstruction that has a
reduced capability for reversal. It may come to simulate chronic obstructive pulmonary disease (COPD).
Epidemiology
Over recent decades, OA has proved consistently to
be the commonest type of newly diagnosed occupational lung disease in industrially developed countries, though the various disorders attributable to
asbestos have an equal cumulative incidence. Between them, asthma and asbestos account for
65–70% of all incident respiratory disorders of occupational origin. In most outbreaks of OA no more
than a few per cent of exposed workers become affected, but there are examples with both pathogenic
pathways of prevalences approaching 50%.
The reported incidence of OA varies widely at a
global level, depending on local employment patterns
and diagnostic criteria. Estimates from statutory notification schemes, compensation registers, voluntary
surveillance schemes, and general population surveys, suggest that 5–15% (median 9–10%) of asthma
beginning in adult life is occupational. When asthma
arises for the first time in a working adult, the background odds favoring an occupational cause over a
nonoccupational cause are consequently of the order
1 in 10 only. This is consistent with the estimate from
SWORD (Surveillance of Work-Related and Occupational Respiratory Disease) data that about 40
cases of OA per million employed workers arise in
the UK each year.
SWORD has usefully considered the incidence of
new cases within specific working groups for which
there are particular occupational exposures. For example, among spray painters, who may use asthmagenic di-isocyanate, epoxy resin, and acrylic paints,
the average annual incidence of OA from 1992 to
1997 was 1464 per million – more than threefold the
UK national average for all cases of incident asthma.
ASTHMA / Occupational Asthma (Including Byssinosis) 189
Thus, in the case of spray painters developing asthma
in adult life, the background odds favor an occupational cause over a coincidental cause. Other settings
associated similarly with greater than even odds include baking, metal treatment, chemical processing,
and plastics manufacture. Most national estimates
range from 25 to 100 million per year.
Table 1 Commonly reported causes of occupational asthma
Animal sources
Arthopods/
insects
Birds
Mammals
Marine species
Bees, locusts, mites, silkworms, weevils
Feather bloom, excreta
Rodent urinary protein, pancreatic enzyme
supplements
Corals, crabs, fishmeal, oysters, prawns,
sponges
Etiology
Many occupational agents (some 400) have been reported to be definite or probable sensitizers capable
of inducing asthma. Some of the most prominent are
classified in Table 1, and the most common reports to
SWORD over a 9-year period are listed in Table 2.
Notable agents reported to cause RADS over recent
years have been acetic acid, chlorine/chlorine dioxide/hydrochloric acid, di-isocyanates, dinitrogen tetroxide, endotoxin, fire smoke, freons, hydrobromic
acid, Iraq/Iran war gases, pentamidine, phosphoric
acid, silo and swine confinement gases, sulfur dioxide/sulfuric acid, tear gas, and welding fume.
Clinical Features
Once a diagnosis of asthma is suspected from the
clinical history or physical examination and confirmed objectively (by the demonstration of reversible airway obstruction or the measurement of airway
responsiveness), the diagnostic issue turns to whether
it has arisen occupationally.
If the toxicity pathway has provided the mechanism, the diagnosis is clear and needs no further investigation. Asthma becomes evident during the
recovery phase from what is usually a combination
of conjunctivitis, rhinitis, pharyngolaryngitis, tracheobronchitis, and (possibly) pneumonitis induced
by a major exposure to a toxic chemical or organic
dust. In survivors, full recovery is the rule. If asthma
arises, it is generally the only persisting respiratory
problem, though occasionally bronchiectasis or an
obliterative bronchiolitis is also detectable.
If hypersensitivity has provided the mechanism, the
diagnosis is more challenging and may be extremely
difficult. This is partly because the latency period
during which sensitization occurs (usually 3–24
months) may be very short (days or weeks) or very
prolonged (several years), and partly because in most
working environments associated with OA, most
cases of incident asthma are coincidental and quite
unrelated to occupation. What follows addresses this
diagnostically more challenging variety of OA.
Clinical History
The history provides an obvious starting point, but
may be importantly distorted. Affected workers
Vegetable sources
Beans
Castor, coffee, soy
Colophony
Pine resin solder
Enzymes
Bromelain (pineapple), papain (papaw)
Grain/flour
Gums
Acacia, arabic, karaya
Mushrooms
Oil mists
Tea
Tobacco
Wood
Iroko, latex, redwood, western red cedar
Microbial sources
Endotoxin
Gram-negative organisms
Enzymes
Bacillus subtilis (alcalase, maxatase,
subtilisin)
Spores
Chemical sources
Inorganic
Salts/oxides of aluminum, chromium, cobalt,
nickel, platinum, vanadium
Stainless-steel welding fume
Organic
Acid anhydrides/phthalates, acrylates,
amines, azodicarbonamide, benzalkonium,
chloramine, di-isocyanates, fluxes,
formaldehyde, furfuryl alcohol,
glutaraldehyde, isothiazolinones,
organophosphate pesticides, plexiglass,
polyvinyl pyrolysis fume, reactive dyes,
styrene, sulphone chloramides, tannic acid
Pharmaceutical sources
Intermediates
6-amino penicillanic acid
Drugs
Cimetidine, cephalosporins, ispaghula
(psyllium), penicillins, piperazine,
sulphonamides, tetracycline
Table 2 Causes of occupational asthma reported to SWORD
over 9 years
Agent
1990–98 (%)
1998 (%)
Di-isocyanates
Laboratory animals/insects
Flour/grain
Fluxes/glues/resins/solders
Aldehydes
Wood dust
Welding fume
Enzymes
Latex
Others
Total
16
9
8
7
5
5
2
13
12
7
9
5
47
100
The figures are rounded to the nearest whole number.
14
6
34
100
190 ASTHMA / Occupational Asthma (Including Byssinosis)
anxious to remain employed may deny or minimize
relevant symptoms; they may also exaggerate or falsify critical aspects. An overwhelming belief that occupational exposure (and/or employer negligence) is
responsible may, curiously, make a diagnosis of OA
the ‘independent variable’ on which the symptoms
depend: ‘‘if improvement during holidays/vacations
is a cardinal diagnostic feature of OA then, yes, this
must be so in my case since I know I have OA.’’
For a classical case, there is exposure to a known
asthmagen and other exposed workers are affected.
For the affected individual, there is recognition that
symptoms arise or worsen after a minimum of 1–2
hours and a maximum of 8 h from the onset of each
sufficiently strong exposure, and persist for hours or
days. Such ‘late’ asthmatic reactions are characteristically associated with increases in airway responsiveness and so are much more definitive of OA
than ‘immediate’ reactions, which may simply result
non-specifically through ‘irritant’ mechanisms.
Mild late reactions may resolve within hours so
that there is full recovery by the following day, but
more commonly the rise in airway responsiveness is
sufficient to worsen asthmatic severity for several
days. A weekend away from work may therefore be
insufficient to allow full recovery, and the association
between occupational exposure and symptoms may
not be recognized until there is a 2-week period of
vacation (or sick leave). Even then gradual recovery,
particularly cessation of disturbed sleep, may not become obvious until the second week, only to be reversed within a matter of days of returning to work.
Serologic Investigations
Laboratory investigation for diagnostic IgE antibodies to relevant asthmagenic agents has proved disappointing for two reasons. First, many occupational
asthmagens are reactive chemicals of low molecular
weight. They are not thought to act as sensitizers
until coupled with appropriate haptogenic body proteins, and it has proved difficult to produce suitable
complexes for antibody detection. Second, exposed
individuals may develop IgE responses without any
apparent ill effect. Antibodies appear to correlate
more closely with exposure than disease. Nevertheless, when radioallergosorbant allergen testing to
relevant asthmagens is available, positive tests increase the probability of OA, even if sensitivity and
specificity are limited.
Peak Expiratory Flow
More popular and more readily available is peak
expiratory flow (PEF) monitoring. Test subjects
take measurements on several occasions each day
for periods of several weeks, so that any differences
in pattern between work days and rest days can be
detected. Statistical software can aid interpretation.
The unsupervised recordings may lack reliability and
precision, and work-related changes may reflect nonspecific ‘irritant’ reactions rather than specific hypersensitivity responses. There is consequently some
difference of opinion over the value of PEF monitoring. In practice, however, it provides the most widely
used diagnostic tool for OA. Figure 1 illustrates a
typical positive outcome.
Inhalation Challenge Tests
The greatest diagnostic confidence comes from laboratory inhalation challenge tests that are monitored
by both supervised serial measurements of spirometry and repeated measurements of airway responsiveness. When there is deteriorating ventilatory
function and increasing airway responsiveness, and
the changes can be evaluated statistically, a diagnosis
of OA can be considered ‘confirmed’ with considerable confidence, especially if the outcome is shown to
be repeatable and the tests are carried out in a double-blind fashion. Thus, neither test subject nor the
immediately supervising physician knows whether
the challenge exposure involved the suspected asthmagen or a dummy ‘placebo’. These principles, with
two methods of statistical evaluation, are illustrated
in Figures 2 and 3. In practice, such tests require
sophisticated equipment, are very time-consuming,
pose potential risks, and are inevitably restricted to a
few centers. They involve a fraction of 1% of all
cases, but are particularly valuable (arguably indispensable) when hitherto unrecognized occupational
asthmagens are first investigated. Figure 3 illustrates
one such case involving a novel asthmagen.
Return-to-Work Studies
A useful, and practical, compromise is the ‘return-towork’ challenge test. For this, the test subject is kept
from work (or at least from exposure to the suspected
asthmagen) for a period of 2–3 weeks, during which
time any asthmatic medication is reduced to a minimum (ideally discontinued). In true OA, some improvement is likely (or there is no deterioration with
treatment reduction), and can be demonstrated by serial measurements of spirometry and airway responsiveness. Hourly spirometric monitoring over the 3
days prior to the return-to-work generates confidence
limits, and so allows the detection of any statistically
significant deterioration subsequently (usually within
a few days only if the asthma is occupational). If airway responsiveness too increases significantly, OA in
the individual is reasonably ‘confirmed’. The method
Diurnal
variation (%)
ASTHMA / Occupational Asthma (Including Byssinosis) 191
50
20
460
440
420
PEF (l min–1)
400
380
360
340
320
300
280
M T W T F S S M T W T F S S M T W T F S S M T W T F S S M T W T F S S M T W T F S S
18 19 20 21 22 23 24 25 26 27 28 29 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
11
12
Readings 9 9 9 8 9 8 7 9 8 7 9 8 8 7 5 9 9 8 8 8 8 9 8 3 6 8 6 7 8 9 9 7 7 9 8 6 9 7 8 8 7 6
W W
W
W W
Comments
Figure 1 Serial PEF measurements in a laboratory technician sensitized to a floor cleaning material. Serial 2-hourly PEF measurements, waking to sleeping, for 6 weeks. Top panel: diurnal variation expressed as % predicted. Middle panel: daily maximum, mean,
and minimum PEF. Bottom panel: date and number of PEF readings per day. W, days without waking measurement. Dotted line:
PEF ¼ 359 represents the predicted PEF. The shaded columns represent work days. Reproduced from Hendrick DJ, Burge PS, Beckett
WS, and Churg A (eds.) (2002) Occupational Disorders of the Lung: Recognition, Management, and Prevention, p. 54. London:
Saunders, with permission from Elsevier.
does not confirm the identity of the asthmagenic
agent, and is only suitable if the test subject is
still employed and the employer cooperates fully. In
many cases the possibility of an occupational cause
does not arise until after the affected individual has
ceased employment or the work environment has
changed. Figure 4 illustrates this methodology in a
newly appointed factory safety officer who found
himself working with di-isocyanates.
FEV1 (l)
4
3
2
0
−40 0
Animal Models
80 2
Min
4
6
8 10 12
24
H
Figure 2 Inhalation challenge test with nebulized ceftazidime
(3.2 mg) in a production worker. Upper plot: mean FEV1 at each
hourly measurement point from 3 control days. Middle plot: lower
95% confidence band for the hourly control FEV1 derived from
the pooled variance. Lower plot: FEV1 following nebulized ceftazidime administered in a double-blind fashion. The lower confidence band is breached for well over an hour, indicating a
significant decrement consistent with a late asthmatic reaction.
Reproduced from Hendrick DJ, Burge PS, Beckett WS, and
Churg A (eds.) (2002) Occupational Disorders of the Lung: Recognition, Management, and Prevention, p. 64. London: Saunders,
with permission from Elsevier.
Several animal species have provided invaluable insight as to how asthma arises following exposure to
occupational agents. ‘Sensitization’ has been achieved
by inhaled, dermal, and/or peritoneal routes for many
occupational asthmagens (notably acid anhydrides,
colophony, di-isocyanates, latex, plicatic acid), and
both immediate and late asthmatic reactions have
been provoked by subsequent inhalation challenge.
The airways are then characterized by inflammation,
eosinophil infiltration, mucus hypersecretion, and
hyperresponsiveness. Hypersensitivity mechanisms
have been confirmed by transferring lymphocytes or
serum from affected to unaffected animals, which
then respond to inhaled challenge in a similar fashion
192 ASTHMA / Occupational Asthma (Including Byssinosis)
5.0
4.0
FEV1 (l)
3.0
2.0
1.0
0
−40
(a)
0
60
Min
2
4
6
8
10 12
24
H
FEV1 area decrement 2−12 h
(l × h)
9
8
Confidence
limits
7
6
5
99%
4
3
95%
2
1
0
1
(b)
2
Control days
3
SINOS (32 µg)
Figure 3 Inhalation challenge test with nebulized SINOS (32 mg) in a research and development technician. (a) The FEV1-time plot
following challenge with a newly developed low-temperature bleach-activating agent, SINOS. The shaded zone defines the 2–12 h area
decrement (2–12 hAD). It provides a summary measure to quantify a late asthmatic reaction. The line demarcating the upper boundary
represents the mean of the measurements during the 40 min before challenge. (b) Comparison of the 2–12 hAD following the SINOS
challenge with those following three double-blind control challenges with nebulized solvent alone. It exceeds their 95% and 99%
confidence limits, confirming that a significant late asthmatic reaction has occurred. The outcome was reproduced when the procedure
was repeated. Reproduced from Hendrick DJ, Burge PS, Beckett WS, and Churg A (eds.) (2002) Occupational Disorders of the Lung:
Recognition, Management, and Prevention, p. 64. London: Saunders, with permission from Elsevier.
to the donor animals. Specific IgE antibodies to the
inducing agents (or hapten conjugates) have been evident commonly, but not invariably. Occasionally specific IgG antibodies are reported. There is involvement
of both CD4 þ and CD8 þ T cells, with a dominant
T-helper-2 cells (Th2) response. The mechanisms include deposition of excess extracellular matrix (and
increased activity of matrix metalloprotease, MMP)
and activation of the vascular endothelium associated
with the release of vascular endothelial growth factor
(VEGF). Experiments with inhibitors of MMP and
VEGF have shown substantial reductions of the markers of asthmatic activity, possibly pointing a way to
novel strategies for future management.
Animal models have additionally confirmed that
airway inflammation induced by exposure to toxic
levels of certain reactive chemicals may also induce
airway hyperresponsiveness. This simulates the RADS
pathway. Ozone and chlorine exert their effects
through oxidative stress, which is associated with increased expression of inducible nitric oxide synthase
and an increase in airway responsiveness. Ozone has
also been shown to suppress Th1 responses (possibly
thereby enhancing Th2 responses), and acute exposure to ozone or nitrogen dioxide amplifies asthmatic
responses to allergenic agents in already-sensitized
animals.
Animal models have also been used to investigate
the exposure threshold levels at which airway inflammation and hyperresponsiveness first develop.
The importance of this for occupationally induced
asthma in humans is readily evident, though extrapolation from animals is necessarily fraught with
uncertainty. The threshold levels of exposure that
trigger meaningful responses once sensitization has
occurred may differ critically from those that are responsible for initial sensitization, and are likely to
differ appreciably from individual to individual.
ASTHMA / Occupational Asthma (Including Byssinosis) 193
3.00
FEV1 (l s–1)
2.00
Test day 3
1.00
Mean of three control days
Lower boundary for control days
Work
0.00
07:30 08:30 09:30 10:30 11:30 12:30 13:30 14:30 15:30 16:30 17:30 18:30 19:30 20:30 21:30 22:30 23:30
Time (h)
Figure 4 Return-to-work study of a safety officer employed by a factory using di-isocyanates. He developed asthma within months of
employment onset. He was kept from work for several weeks during which airway responsiveness improved steadily (PD20.methacholine; 3.2-25.0 mg). An increase in PD20 of twofold or more indicates a significant change. The figure illustrates the third
successive day of the return-to-work study. The lowered initial FEV1 measurement reflects an equivocal late reaction from the preceding
day. On Day 4, following the 3 days at work, the PD20 was significantly decreased to 9.0 mg (i.e., to less than half the pretest value of
25.0 mg).
Management and Current Therapy
Drug and ancillary therapies for OA are those of
asthma of any cause, but OA arising by the hypersensitivity route offers an additional and critical
means of management. If the relevance of an occupational sensitizer is recognized within 6–24 months
of symptom onset, and if exposure then ceases, there
is a meaningful probability of the asthmatic state resolving entirely (i.e., the level of airway responsiveness falls into the nonasthmatic range). This is almost
unknown for adults with nonoccupational asthma,
unless it is drug induced (beta-blockers, nonsteroidal
anti-inflammatory drugs (NSAID). There is inevitably much variability from individual to individual,
but if exposure ceases within as short a period as 6
months, the probability of complete resolution may
exceed 50%. If the period exceeds 24 months, it is
more likely that active asthma will continue, even in
the absence of ongoing exposure.
Most individuals developing OA have unskilled or
semiskilled jobs. Their susceptibility to develop
asthma with exposure to sensitizing agents will inevitably put them at a disadvantage in the labor market,
and this will be enhanced if active asthma persists. It
is unfortunate that most lose their current jobs and
never become gainfully employed again. This poses
several management challenges. A correct diagnosis is
the cornerstone, since mild asthma rarely leads to job
loss of itself. A false-positive diagnosis may have
devastating but unnecessary economic consequences
for both the affected individual and the employer. A
dilemma arises when asthma is occupational, but is
mild, has been active for several years, and does not
improve much after an experimental period away
from the workplace. If the affected individual cannot
find (or does not wish for) alternative employment,
the risk of continuing exposure needs to be assessed.
It will certainly diminish the long-term probability of
regaining a nonasthmatic level of airway responsiveness, but this may be minimal anyway. More importantly it may cause a further and permanent increase
in airway responsiveness, and hence worsening
asthma with the possibility of remodeling and fixed
airway obstruction. A period of close surveillance,
with objective serial measurements of spirometry and
airway responsiveness, may help the affected individual and his medical advisors to judge whether the
194 ASTHMA / Occupational Asthma (Including Byssinosis)
obvious benefits justify the uncertain risks. There is
anecdotal evidence that tolerance develops in some
affected individuals and that they have little to lose by
continued exposure.
Complete cessation of exposure offers the best
outcome, especially if this can be achieved by using
an alternative, nonsensitizing agent for the particular
industrial process. If this is not possible or practical,
it may be that exposure levels can be reduced by
modifications to job plans, task sharing/exchanging,
improved industrial hygiene (ventilation/extraction),
or the use of respiratory protection equipment.
Prevention
Prevention offers the best means of control. When a
risk of OA is identified, surveillance programs may
identify emergent cases at a point when exposure
cessation is likely to produce cure. This assumes such
programs are efficient. Some employers rely primarily on environment measurements, and reassure
themselves (inappropriately) that if they have complied with regulatory limits, any emergent cases of
asthma must be nonoccupational. When a clear risk
of OA is recognized, the onus should lie with disproving the diagnosis when new cases of asthma
arise, rather than the converse. It is the very nature of
‘allergy’ that some affected individuals become sensitized at exposure levels that are harmless to the
majority, and that lie within regulatory limits.
Compensation
Compensation is inevitably an issue for affected
workers, particularly those who become disabled
and unemployed. Equally inevitably, compensation
systems differ from country to country. Regretfully,
few include provision for retraining and re-placement. The physician managing OA needs to be familiar with local procedures in order to offer proper
advice. At a time of increasing litigation, the physician too may find him- or herself the focus of a legal
(negligence) suit if he or she has failed to provide
correct advice.
Byssinosis
The respiratory effects of dust from cotton and other
biologic fibers became prominent in the nineteenth
and early/mid-twentieth centuries. They appeared
initially to be specific to the cloth manufacturing industry and the appellation, byssinosis, arose to identify the disorder characterized by them. This has had
the misleading consequence of segregating cotton
dust asthma from the many other types of OA that
were identified later, and of disguising the other respiratory disorders that may result from cotton dust.
A very characteristic feature of reported byssinosis
has been a prominence of symptoms on the first work
day following periods without exposure. These tend
to diminish or even cease on subsequent days, at least
in some individuals, despite similar levels of ongoing
exposure. While chest tightness and breathlessness
may be a consequence of asthma, some affected individuals describe an influenza-like reaction with
fever and malaise (‘Monday fever’). The latter may
occur on the very first employment day, that is, before any possibility of sensitization. Such symptoms
have also been generated in experimental conditions
when naı̈ve volunteers are exposed. The same febrile
reaction may occur in subjects heavily exposed to
metal fume or the fume arising from certain polymers. It is closely related to neutrophil inflammation
and the release into plasma of pyrogenic interleukin6 (IL-6).
These non-specific systemic symptoms are features
of ‘inhalation fevers’ generally. They occur widely
with exposure to a variety of organic dusts, and in
these contexts are now known more commonly by
the term ‘organic dust toxic syndrome (ODTS)’.
Products from microbial overgrowth appear to be
responsible, and high airborne concentrations of respirable spores and endotoxins are characteristically
seen. This is consistent with the recognition that
byssinosis is most prevalent among those who harvest, transport, store, sort, process, and clean cotton
before its ‘decontaminated’ fibers are woven into
cloth. The matter is complex since heavy exposure of
naı̈ve subjects to organic dust may also provoke
cough, chest tightness, and bronchoconstriction.
These symptoms are rarely severe, and rarely last
for more than a day or two. Nevertheless, this response simulates RADS, and no clear sensitizer has
been incriminated in the pathogenesis of byssinosis.
Some individuals affected by other forms of OA
may also have more prominent symptoms on Mondays that peter out as the working week progresses. A
further ‘characteristic’ of byssinosis has also proved
to be non-specific, namely the development of COPD.
Early studies of byssinosis were necessarily cross-sectional, and it was assumed that subjects with COPD
had initially experienced the typical acute and reversible features (i.e., asthma and/or inhalation fever),
which had progressed to produce ‘chronic byssinosis’.
It seems more likely now that COPD and asthma (and
inhalation fever/ODTS) are independent disorders
arising from cotton dust exposure, which result from
different levels of susceptibility within exposed workers and different patterns of cellular response. Even
so, it is to be expected that COPD might develop
ASTHMA / Acute Exacerbations 195
because of asthma as well as independently of it, and
there is convincing evidence to support this from recent longitudinal studies.
Acute Exacerbations
S L Johnston, Imperial College London, London, UK
& 2006 Elsevier Ltd. All rights reserved.
See also: Chronic Obstructive Pulmonary Disease:
Overview. Interleukins: IL-6. Occupational Diseases:
Asbestos-Related Lung Disease.
Further Reading
Beach JR, Young CL, Avery AJ, et al. (1993) Measurement of
airway responsiveness to methacholine: relative importance of
the precision of drug delivery and the method of assessing response. Thorax 48: 239–243.
Brooks SM, Weiss MA, and Bernstein IL (1985) Reactive airways
dysfunction syndrome (RADS). Persistent asthma syndrome after high level irritant exposures. Chest 88: 376–384.
Burge PS, Pantin CFA, Newton DT, et al. (1999) Development of
an expert system for the interpretation of serial peak expiratory
flow measurements in the diagnosis of occupational asthma.
Occupational and Environmental Medicine 56: 758–764.
Cote J, Kennedy S, and Chan-Yeung M (1990) Outcome of patients with cedar asthma with continuous exposure. American
Review of Respiratory Diseases 141: 373–376.
Glindmeyer HW, Lefante JJ, Jones RN, et al. (1994) Cotton dust
and across-shift change in FEV1 as predictors of annual change
in FEV1. American Journal of Respiratory and Critical Care
Medicine 149: 584–590.
Hendrick DJ and Burge PS (2002) Occupational asthma. In:
Hendrick DJ, Burge PS, Beckett WS, and Churg A (eds.) Occupational Disorders of the Lung – Recognition, Management,
and Prevention, pp. 33–76. London: Saunders.
Herrick CA, Xu L, Wisnewski AV, et al. (2002) A novel mouse
model of diisocyanate-induced asthma showing allergic-type inflammation in the lung after inhaled antigen challenge. Journal
of Allergy and Clinical Immunology 109: 873–878.
Lee KS, Jin SM, Kim SS, and Lee YC (2004) Doxycycline reduces
airway inflammation and hyperresponsiveness in a murine
model of toluene diisocyanate-induced asthma. Journal of Allergy and Clinical Immunology 113: 902–909.
Lee YC, Kwak YG, and Song CH (2002) Contribution of vascular
endothelial growth factor to airway hyperresponsiveness and
inflammation in a murine model of toluene diisocyanate-induced
asthma. Journal of Immunology 168: 595–600.
Meyer JD, Holt DL, Cherry NM, and McDonald JC (1999)
SWORD ’98: surveillance of work-related and occupational respiratory disease in the UK. Occupational Medicine 47: 485–489.
Newman Taylor A (2000) Asthma. In: McDonald JC (ed.) Epidemiology of Work Related Diseases, 2nd edn., ch. 8, pp. 149–
174. London: BMJ Books.
Schilling RSF, Hughes JPW, Dingwall-Fordyce I, et al. (1995) An
epidemiological study of byssinosis among Lancashire cotton
workers. British Journal of Industrial Medicine 12: 217–227.
Stenton SC, Avery AJ, Walters EH, and Hendrick DJ (1994) Technical note: statistical approaches to the identification of late
asthmatic reactions. European Respiratory Journal 7: 806–812.
Stenton SC, Dennis JH, Walters EH, and Hendrick DJ (1990) The
asthmagenic properties of a newly developed detergent ingredient – sodium iso-nonanoyl oxybenzene sulphonate. British Journal of Industrial Medicine 47: 405–410.
Venables KM, Topping MD, Howe W, et al. (1985) Interaction of
smoking and atopy in producing specific IgE antibody against
a hapten protein conjugate. British Medical Journal 290:
201–204.
Abstract
Acute exacerbations of asthma are acute worsenings of asthma
symptoms accompanied by reductions in lung function, normally provoked by some external event or combination of
events. Exacerbations may be relatively mild or severe. The
most severe may lead to asthma death. Symptoms include increased breathlessness, wheeze, cough, or chest tightness. Severity is graded on a combination of symptoms, clinical signs,
and lung function. The majority of asthma exacerbations, particularly in children, are precipitated by acute respiratory tract
viral infections. These may interact with a number of other cofactors such as allergen exposure, air pollution, and exercise.
Exacerbations are more likely to occur on a background of
poorly controlled rather than well-controlled underlying disease. Pathology involves increased airway inflammation with
most inflammatory cell types implicated in pathogenesis. Most
prominent, however, are neutrophils, lymphocytes, and eosinophils. A wide variety of acute inflammatory mediators are increased during exacerbations. Severity of virus infection is the
major determinant of severity of exacerbation. Asthmatics have
increased susceptibility to viral and bacterial infection. Treatment includes optimal control of underlying disease, inhaled/
oral steroids, and bronchodilators. The role of anti-infective
therapy is under investigation.
Introduction
Asthma is itself a heterogeneous disease. Asthma exacerbations also, therefore, are by definition heterogeneous as exacerbations are defined as a worsening
of a pre-existing state. Given that the etiology (see
below) of exacerbations is also heterogeneous, heterogeneity of both the underlying cause and of the
etiology of the exacerbation makes the exacerbation
itself very varied in its presentation. One of the difficulties in clinical practice and clinical research is
defining exacerbations accurately and differentiating
them from poor control of the underlying disease.
There is no agreed definition of an exacerbation, but
most clinicians and clinical researchers would agree
on something like ‘episodes of relatively sudden onset and rapidly progressive increase in symptoms of
shortness of breath, cough, wheeze, or chest tightness
accompanied by reduction in lung function and normally provoked by some external event or combination of events’.
Exacerbations of asthma are very important as they
are the major cause of morbidity and mortality in
asthma and are also responsible for the greater part of
healthcare costs associated with asthma treatment.
Currently available treatments for asthma exacerbations include supportive care, oxygen if required,
bronchodilators, and oral/inhaled corticosteroids.
196 ASTHMA / Acute Exacerbations
This treatment is at best only partially effective. Understanding the causes and mechanisms of asthma
exacerbations is therefore of great importance as
there is an urgent need for more effective preventive
and interventional therapy.
Etiology
In infants and young children the vast majority (approaching 100%) of exacerbations are precipitated
by acute respiratory tract virus infections. Respiratory syncytial virus (RSV) is the major cause of acute
wheezing illness severe enough to require hospitalization in infants and 1–2-year-old children. There is,
however, increasing evidence that rhinoviruses also
play an important role and most recent data suggest
RSV is implicated in 70–80% of hospitalized wheezing lower respiratory tract infections and rhinoviruses in around 40%. The incidence of dual infection
with these two viruses and with a variety of other
respiratory viruses is likely to be as high as 20–30%.
There is much less data available on community
lower respiratory wheezing illness but birth cohort
studies have now reported that rhinoviruses are the
dominant cause of acute attacks of wheezing in this
age group, perhaps causing around three times as
many episodes of wheezing illness as RSV and other
respiratory viruses.
In school-age children and teenagers, viruses precipitate at least 80% of acute asthma exacerbations.
Rhinoviruses again are by far the most common virus
implicated accounting for around two-thirds of viruses detected. In adults, viruses likely cause around
two-thirds to three-quarters of asthma exacerbations. In adults in particular where virus loads tend to
be much lower than in children, the evidence is less
strong than it is in children and further work is required to clarify the role of viruses.
In all these age groups, viruses can interact with
a number of other factors in provoking asthma exacerbations. There are good data in both adults and
children that virus infection and exposure to an allergen to which the patient is sensitized interact in a
synergistic manner in increasing the risk of exacerbation. There is also evidence that air pollution
interacts with virus infection in increasing the risk of
lower respiratory illness when infected and it is
possible that a number of other cofactors also play
a role.
Poor control of the underlying disease is a major
risk factor for asthma exacerbation and treatment
with prophylactic therapy, principally with inhaled
corticosteroids, but also with leukotriene receptor
antagonists and long-acting beta-agonists is a major
protective factor against exacerbation.
Asthma exacerbations show marked seasonality in
all age groups, peaking 1–2 weeks after school return, most especially in the autumn and most especially in school-age children. This seasonal pattern is
important as it emphasizes the times at which prophylactic therapy is most needed.
Asthmatics have recently been shown to have increased susceptibility to virus infection through impaired innate immunity and there is a biological
rationale that they are also likely to have impaired
acquired antiviral immunity. Clinical studies confirm
that asthmatics are more susceptible to virus infection and bacterial infection, having a greater risk of
invasive pneumococcal disease. To date, very little is
known about susceptibility to infection in asthma
but this is an important area for future research.
Genetic susceptibility is also an underresearched area
and very little is known in this regard.
Finally, there is increasing evidence that chronic
infection with the atypical bacteria Chlamydophila
pneumoniae and Mycoplasma pneumoniae may play
a role in exacerbations of asthma and a recent study
confirms therapeutic benefit of treating asthma exacerbations with an antibiotic active against these
organisms. Further studies are required to confirm
these findings and to shed further light on the role of
chlamydia and mycoplasma.
Pathology
Relatively little is known about the pathology of
asthma exacerbations for an obvious reason: sampling the lower respiratory tract during an exacerbation is extremely difficult. In one study asthmatic
patients were bronchoscoped during naturally occurring colds, and eosinophilic, neutrophilic, and CD8 þ
T-cell inflammatory responses were found. Studies of
post-mortem samples from patients dying of asthma
exacerbation also reveal CD8 þ T cells, activated
CD8 þ T cells, increased perforin expression, and
impaired ratios of interferon gamma (IFN-g) to interleukin-4 (IL-4), possibly implicating impaired
antiviral immunity in asthma death. Noninvasive
methods of sampling the lower airway include measurement of exhaled breath condensate or exhaled
nitric oxide, and sputum sampling. Studies with sputum again show both eosinophilic and neutrophilic
inflammation with increased levels of IL-8 and fibrinogen. Markers of both eosinophil and neutrophil
activation are also increased and sputum lactate dehydrogenase (LDH) as a marker of virus-induced
cytotoxicity is also markedly elevated. Levels of sputum LDH and eosinophil cationic protein were associated with longer hospital stay, indicating that
virus-induced cell damage was the major predictor of
ASTHMA / Acute Exacerbations 197
the severity of asthma exacerbation and that eosinophilia as a consequence of either viral and/or allergen
exposure was also an important contributor.
Clinical Features
Asthma exacerbations present with a sudden worsening of wheeze, cough, shortness of breath, and
chest tightness with a reduction in lung function.
Exacerbations can range from mild to severe, the
most severe leading to asthma death. Diagnosis is
normally made on the clinical history with the assistance of peak expiratory flow measurement and/or
spirometry. Clinical examination will normally reveal a distressed, anxious patient with tachycardia,
tachypnea, and audible wheeze on auscultation
(though in the most severe exacerbations the chest
can be almost silent). Patients will have prolonged
expiration, and signs of severe exacerbation include
inability to talk in complete sentences, tachycardia
above 110 beats min 1, a respiratory rate above 25
breaths min 1, and a peak expiratory flow below
50% of predicted or best. Presence of pulsus paradoxus also indicates a severe attack and life-threatening attacks are suggested by a silent chest, presence
of cyanosis, bradycardia, or hypotension, and a peak
expiratory flow below 30% of predicted or best.
Measurement of blood gases should be carried out.
Hypoxia is usual. In milder exacerbations, hypocapnia is common due to hyperventilation while in
more severe and life-threatening exacerbations, PCO2
starts to rise. A raised PCO2 or rising PCO2 is an indication for intensive care. Chest X-rays should be
performed to exclude pneumonia and pneumothorax. The response to therapy and the progress of the
exacerbation are monitored with serial peak expiratory flow or spirometry testing. Routine hematology
and biochemistry are indicated but other blood testing is not normally required.
Pathogenesis
Asthma exacerbations are always mixed in their
pathogenesis. They involve a mixture of acute and
chronic inflammation provoked by virus infection and
other stimuli including allergen exposure, air pollution, tobacco smoke, etc. Much of the information we
have gained regarding the pathogenesis of asthma
exacerbations has come from experimental studies of
rhinovirus infection in asthmatic and normal subjects.
These studies have implicated neutrophils, CD4 þ and
CD8 þ lymphocytes, and eosinophils, but in addition
mast cells, macrophages, and mediators of acute inflammation are also shown to play important roles,
including leukotrienes. Most cytokines/chemokines
that have been investigated have been found to be
elevated in exacerbations. Perhaps the most prominent are interleukin-8, interleukin-1b, tumor necrosis
factor alpha (TNF-a), the regulated upon activation
normally T-cell expressed and secreted factor (RANTES), and IFN-g. The mechanisms of induction of
lower airway inflammation in the context of respiratory virus infection are of great interest as these represent potential targets for interventional therapy.
Nuclear factor kappa B (NF-kB) is very strongly implicated in virus-induced inflammation, as is activating protein 1 (AP-1). However, a number of other
transcription factors are also implicated. Mechanisms
of induction of mucus secretion are also of great interest – complex pathways are involved, but NF-kB,
Sp1 transcription factors, and the epidermal growth
factor receptor signaling pathway are all implicated.
Airway obstruction is a result of acute smooth
muscle contraction, chronic smooth muscle hypertrophy, mucus secretion, acute tissue edema, and
chronic tissue inflammation with airway fibrosis/remodeling. An important aspect of pathogenesis is
host resistance to infection as virus infection is the
major precipitant to exacerbation. Recent studies
have confirmed that asthmatic bronchial epithelial
cells mount defective apoptotic and IFN-b innate
immune responses to rhinovirus infection. In consequence, rhinovirus infection is robust while in normal epithelial cells infection is largely abortive. The
clinical studies indicating increased susceptibility to
virus infection in asthma are consistent with this biological evidence, and more recent evidence that
asthmatics are also susceptible to invasive pneumococcal disease possibly indicates a more generalized
immune deficit. There is an urgent need to increase
our understanding of host immunity to both viral
and bacterial infection in asthma.
Experimental Models
There are no small-animal models of rhinovirus infection as rhinoviruses only infect humans and nonhuman primates. There are animal models of other
virus infections including influenza, RSV, and parainfluenza, and these models have been combined in
some instances with allergen exposure to try to
increase our understanding of the pathogenesis of
virus-induced asthma exacerbations. These studies
indicate that impaired T helper (Th)-1 immune responses increase susceptibility to virus infection and
that airway inflammation, airway obstruction, and
bronchial hyperreactivity are increased when virus
infection occurs in the presence of ongoing allergic
inflammation. Other studies have confirmed an increased risk of developing allergen sensitization if
198 ASTHMA / Acute Exacerbations
allergen exposure occurs in the context of an acute
respiratory virus infection. Studies with RSV indicate
that the major protective immune responses are production of neutralizing antibody, natural killer (NK)
cell, and CD8 þ T-cell IFN-g responses. A great deal
of further work is needed to increase our understanding of the interaction between virus infection
and allergen exposure in the context of asthma exacerbations.
In the absence of an animal model, investigators
have carried out human rhinovirus experimental infections in asthmatic and normal subjects, and have
demonstrated bronchial hyperactivity and airway
obstruction in asthmatic volunteers. These studies
have also implicated Th1 responses in increased susceptibility to virus infection and further work in
these models is ongoing to try to increase our understanding of the pathogenesis of virus-induced
asthma.
In vitro models of host immunity to virus infection
and of virus-induced lower airway inflammation
include infection of airway epithelial cells and macrophages with a variety of respiratory viruses including rhinoviruses, RSV, and influenza. These
studies demonstrate that virus infection induces
many proinflammatory cytokines and chemokines,
dependent upon transcription factor activation such
as NF-kB, AP-1, and NF-IL-6. In turn, much of the
activation of transcription factors is dependent upon
activation of oxidant pathways. Production of nitric
oxide appears to exert some degree of protection
against virus infection.
Management and Current Therapy
Perhaps the most important aspect to management of
asthma exacerbations is prevention of asthma exacerbations. Optimal control of underlying disease
with optimal therapy including inhaled corticosteroids, leukotriene modifiers, and long-acting betaagonists has been shown to reduce exacerbation frequency by approximately 50%.
Once exacerbations occur, the initial response is to
give short-acting bronchodilators, if mild via inhalation through a spacer, and if more severe by nebulization. Short-acting beta-agonists are initial
therapy. The addition of anticholinergic bronchodilators has been shown to further improve lung function and to reduce the need for hospitalization. In
mild exacerbations, increasing the dose of inhaled
corticosteroids can reduce the severity of the exacerbation and speed recovery. However, evidence indicates that doubling the dose is usually not adequate
and that quadrupling the dose or giving high-dose
therapy may give better responses. In moderate to
severe exacerbations, oral/systemic corticosteroid
therapy is indicated. Intravenous magnesium has
been shown to be of some benefit in more severe
exacerbations failing to respond to initial therapy.
Oxygen and supportive care should be given to all
exacerbations where hypoxia is a feature. Leukotriene modifiers have been shown to be more beneficial in exacerbations in infants and young children.
Their intravenous use in the acute setting has also
been shown to produce some benefit, though there is
not much evidence available as yet. Use of standard
antibiotics in two placebo-controlled studies has produced no evidence of benefit. However, a recent
study with an antibiotic active against atypical bacteria and with anti-inflammatory properties has
shown clinically significant benefit over placebo.
The most severe exacerbations require high dependency unit or intensive care monitoring and some require invasive ventilation.
Hospitalization with acute exacerbation is a major
risk factor for asthma mortality, and preventive therapy including inhaled corticosterioids and self-management plans are strongly indicated in such patients.
See also: Chemokines. Leukocytes: Eosinophils;
Neutrophils. Viruses of the Lung.
Further Reading
British Thoracic Society, Scottish Intercollegiate Guidelines Network (2003) British guidelines on the management of asthma.
Thorax 58(Supplement 1): 11–94.
Corne JM, Marshall C, Smith S, et al. (2002) Frequency, severity,
and duration of rhinovirus infections in asthmatic and nonasthmatic individuals: a longitudinal cohort study. Lancet
359(9309): 831–834.
Gern JE (2002) Rhinovirus respiratory infections and asthma.
American Journal of Medicine 112(Supplement 6A): 19S–27S.
Gern JE and Busse WW (2002) Relationship of viral infections to
wheezing illnesses and asthma. Nature Reviews: Immunology
2(2): 132–138.
Gern JE and Lemanske RF Jr. (2003) Infectious triggers of
pediatric asthma. Pediatric Clinics of North America 50(3):
555–575.
Green RM, Custovic A, Sanderson G, et al. (2002) Synergism between allergens and viruses and risk of hospital admission with
asthma: case-control study. British Medical Journal 324(7340):
763. (Erratum British Medical Journal 324 (7346): 1131.)
Johnston SL and Martin RJ (2005) Chlamydophila pneumoniae
and Mycoplasma pneumoniae: a role in asthma pathogenesis?
American Journal of Respiratory and Critical Care Medicine,
doi: 10.1164/rccm.200412–1743pp.
Message SD and Johnston SL (2002) Viruses in asthma. British
Medical Bulletin 61: 29–43.
Message SD and Johnston SL (2004) Host defense function of the
airway epithelium in health and disease: clinical background.
Journal of Leukocyte Biology 75(1): 5–17.
Talbot TR, Hartert TV, Mitchel E, et al. (2005) Asthma as a risk
factor for invasive pneumococcal disease. New England Journal
of Medicine 352(20): 2082–2090.
ASTHMA / Exercise-Induced 199
Wark PA, Johnston SL, Bucchieri F, et al. (2005) Asthmatic bronchial epithelial cells have a deficient innate immune response to
infection with rhinovirus. Journal of Experimental Medicine
201(6): 937–947.
Wark PA, Johnston SL, Moric I, et al. (2002) Neutrophil degranulation and cell lysis is associated with clinical severity in virusinduced asthma. European Respiratory Journal 19(1): 68–75.
Exercise-Induced
N C Thomson and M Shepherd, University of
Glasgow, Glasgow, UK
& 2006 Elsevier Ltd. All rights reserved.
Abstract
Exercise-induced asthma (EIA) is described in asthmatics, elite
athletes, and the general population alike. Difficulties reconciling the variety of presentations complicate our understanding of
the disease process and prevalence. Exercise-induced bronchospasm (EIB) can be measured during and after exercise in susceptible individuals, but whether this signifies EIA is unclear.
Exercise can cause an inflammatory response in the airway that
appears to be dose dependent and exaggerated by breathing cold
dry air. Classical understanding of the pathogenesis of EIA suggests that drying of the airway mucosa leading to an osmotic
gradient across the epithelium and basement membrane combined with cooling of the bronchial wall triggers bronchospasm
in EIA. Modern investigation suggests that airway surface stress
can be communicated to the bronchial wall by chemical signals
arising from the epithelium. Managing a patient with EIA requires careful investigation to ensure the correct diagnosis and
consideration of alternative disease processes should be made.
Pharmacological intervention can both treat and limit the extent
of EIB, but behavioral modifications can also be introduced.
Finally, clinicians managing EIA should be aware of the conflicts
that arise when regular asthma medications are used by patients
engaging in competitive sport.
Introduction
Increased airway resistance triggered by vigorous exercise is variously called exercise-induced asthma
(EIA) or exercise-induced bronchospasm (EIB). Although a consensus exists on the criteria for making
a diagnosis of EIB, the range of circumstances in
which it has been recognized mean a precise clinical
definition of EIA is difficult to achieve. EIB has been
described in asthmatics and those with no history of
asthma and subjects ranging from school children to
elite athletes. It is not clear whether the disease is the
same in all cases. While it may be more accurate to
reserve the term EIA for exercise-induced, symptomatic bronchial narrowing occurring in previously
diagnosed asthma we will use the term broadly to
describe asthmatic symptoms or physiological
changes consistent with asthma developing during
or immediately after exercise.
Epidemiology
EIA is common in known asthmatic subjects and exercise has been recognized as a trigger for ‘asthma’
since the seventeenth century. At least 90% of asthma
sufferers will experience a fall in forced expiratory
volume (FEV1) or peak flow during or shortly after an
appropriate exercise challenge. Some authorities believe that all asthmatics will have demonstrable
bronchospasm following sufficient exercise challenge.
In atopic patients who suffer only from allergic rhinitis
prevalence drops to 40%. Studies of other groups are
complicated by the definition of EIA used. For example, comparing the prevalence of EIB, hypersensitivity
to methacholine, and self-reported symptoms of EIA
consistently identify different groups within a given
population. Most of the prevalence data provided are
therefore relatively generalized (Table 1).
EIB is a useful model for clinical asthma and it has
been used as a screening tool. These cohorts are frequently drawn from populations engaged in regular
exercise so may not represent a truly unselected population. Based on these studies the prevalence of EIA
in the general adult population is estimated at between 6% and 13% and this varies geographically
with higher levels in the UK and Australia and lower
levels in developing countries. In cohorts of normal
school children challenged with exercise then tested
by spirometry up to 12% are shown to have EIA.
The identification of exercise-induced bronchial narrowing is often a surprise to both child and adult
subjects, who may not have been aware of any such
problem. A poor perception of airflow restriction is
generally recognized in asthma but also casts some
doubt on the definition of EIA based on spirometry
alone. It is not known if otherwise normal subjects
who experience a fall in FEV1 with exercise will
benefit from asthma therapy or will go on to develop
clinical asthma later in life.
Failure to recognize potentially reversible EIA may
not be a trivial matter. Children who suffer distressing
Table 1 Estimated prevalence (percent study population) of
exercise induced asthma based on a number of studies using
different methodology
Study population
Children
(o18 years) (%)
Adults (%)
General population
Asthma
Atopic nonasthmatic
Athletes
Elilte
Recreational
9
45–80
15
4–20
70–90
40
19
4.2–20
11–50
EIA prevalence has been reported using physiological based parameters and self-reporting questionnaires.
200 ASTHMA / Exercise-Induced
symptoms will tend to avoid exercise leading to possible psychosocial and neurological development
problems. Similarly, the importance of correct identification of EIA is highlighted by studies demonstrating lower self-esteem scores in children who
perceive themselves to be socially disadvantaged by
asthma.
A surprisingly high prevalence of EIA is reported
in both recreational and elite athletes (Table 2). Anecdotally, this association has been explained by the
focus of some asthmatic children on ventilatory control and on athletic pursuit to achieve this. Whatever
the cause, around 23% of recreational athletes report
asthma induced by exercise while within the elite
ranks prevalence rates of between 16% in summer
outdoor events and 50% in winter events such as
cross-country skiing or ice skating are reported.
While asthmatics frequently state that swimming
tends to cause less bronchospasm than other sports, a
particularly high prevalence of EIA has been observed in elite swimmers in whom up to 79% have
been reported to have demonstrable bronchial hyperresponsiveness to methacholine and 33% have
asthma.
The prevalence of EIA reported in elite athletes
has raised concern among sporting governing bodies
regarding the potential for inappropriate use of
performance enhancing medications. As a result a
considerable body of research has focused on EIA
developing in elite athletes. However, elite athletes
are subject to a variety of additional possible triggers
of EIB including the stress of competition. Thus,
whether this research is equally applicable to recreational athletes or nonathletic sufferers from EIA is
not known.
Therefore, it appears that EIA, defined by a fall in
FEV1, occurs in a relatively high proportion of the
general population. This prevalence is increased
by underlying atopy, asthma, and athletic pursuit,
Table 2 The prevalence of EIA based on sporting pursuit
Athletes
Season
Olympians
Summer
Winter
Elite/events
Summer
Winter
All seasons
a
Activity
Asthmaa
(%)
EIB
(%)
23
22
Track/field
Indoor
Nordic skiing
Skating
Nonendurance
Swimming
19
12
60
6.4
1
44–50
12
50
35
Definition of asthma varies among reports and includes selfreporting of ever diagnosed asthma and current or ever used
asthma medications.
particularly outdoor winter endurance events. Accurate assessments of prevalence are hampered by difficulty in definition stemming from the range of
possible criteria for diagnosis and the variety of
presenting complaints, which will be outlined later.
Further difficulties stem from poor perception and
variable self-reporting. This difficulty in precise definition has also limited understanding of the pathology underlying EIA.
Pathogenesis
Pathophysiology
The normal respiratory responses to exercise result
in an increase in ventilation of up to 200 l min1 .
In order to facilitate the increased airflow, mild
bronchodilation occurs early during an exercise
event and this can be measured using spirometry.
Classically, EIA was described as taking place after
the exercise challenge was completed, but it is clear
that airway narrowing can occur during exercise
lasting more than 12 min and in the postexercise period. The ‘stop–start’ nature of some sporting events
means that symptoms developing during exercise are
entirely consistent with a diagnosis of EIA. A variable
period of protection from further exercise-induced
bronchial narrowing known as the refractory period
is well described. A refractory period may last from
30 min to 2 h and may be due in part to the bronchodilator properties of prostaglandin E2 (PGE2).
The cause of EIA and the refractory period are not
fully understood; however, considerable evidence
points to a cellular response to increased ventilation
triggering an inflammatory reaction in the airway. EIA may therefore be considered as a subset
of chronic asthma in which exercise is the trigger to
inflammation. While this hypothesis is attractive, a
variety of studies have produced conflicting data regarding the inflammatory character of EIA.
The three-stage model proposed by R Gotshall
serves as a useful framework to consider the pathogenesis of EIA. In this model an exercise challenge
serves as a trigger sensed in the airway that ultimately signals to the cells of the airway controlling
caliber. Hyperventilation alone can cause bronchoconstriction in human and canine subjects and has
been identified as the key element of exercise that
triggers EIA. How this trigger leads to bronchoconstriction is a matter of controversy.
Airway Cooling and Hyperosmolarity
In 1864, H H Slater noted that cold air triggered
asthma and offered pulmonary vascular congestion
as a possible explanation. This theory has been
ASTHMA / Exercise-Induced 201
developed by McFadden and others. Airway cooling
in response to inspired cold air may trigger vasoconstriction of the bronchial vasculature. Subsequent
reflex vasodilation and hyperemia with extravasated
fluid leading to mucosal edema could lead to airway
narrowing. Although attractive, this theory fails to
acknowledge the capacity of the upper airways to
warm inspired air such that it is unlikely that cold air
is delivered to medium-sized airways, the main site of
narrowing in asthma. However, cooling of the airways could take place if evaporation of mucosal water exceeded the replacement capacity of the airway.
Exercise is associated with mouth breathing, which
bypasses the humidifying effect of the nasal mucosa,
and dry air has been shown to increase the capacity
of an exercise challenge to trigger EIB. Evaporation
of water from the upper airways might take place as
a result of a large increase in ventilation of relatively
dry air. This would cause an increase in the osmolarity of the airway mucosa. This concept of
airway hyperosmolarity has been developed by Anderson and colleagues and has become popular in
current literature. Inhaling hyperosmolar mannitol
or saline solutions can trigger bronchospasm in the
absence of an exercise stimulus. It has been difficult
to separate the specific elements of these two processes and it may be that both are partly responsible
for the effect of hyperventilation on the airway in
EIA. Since cold air holds less moisture than warm air,
this may add to the drying of the airway.
Alternative mechanisms for transducing the proasthmatic stimulus to a bronchoconstrictive response
can be postulated. These include stretching of the
airway wall cells and altered pressure dynamics of
the airway lumen. Ultimately, a signal to alter the
diameter of the airway lumen is generated that results in a fall in FEV1. The nature of this signal is also
controversial but biochemical mediators associated
with inflammation offer a possible explanation.
Inflammation in EIA
It has become clear that an inflammatory process in
the airway leads to bronchial hyperresponsiveness
and chronic asthma. Exacerbations of asthma can be
caused by a variety of inflammatory triggers such as
allergen exposure or viral infection. While less intuitive it seems possible that EIA may also have an
inflammatory basis. Table 3 details the variety of inflammation markers that have been studied with reference to EIA.
Biological markers of inflammation A variety of
studies have demonstrated elevated inflammatory
cells in the airway lumen and bronchial wall from
athletes engaged in a wide spectrum of athletic pursuits. These cells include eosinophils, T lymphocytes,
macrophages, and mast cells all of which are recognized as key cellular elements of the asthmatic
inflammatory response. Changes in airway inflammatory cell number correlate with changes in bronchial reactivity and some groups have demonstrated
that the degree of bronchospasm in response to exercise challenge more closely reflects the level of airway eosinophilia than the prechallenge methacholine
sensitivity. Bronchial biopsies taken from resting
cross-country skiers demonstrated increased inflammatory cells in the bronchial wall compared to nonathlete controls. Regular exercise may therefore lead
to a persistent airway inflammation beyond the acute
effects of exercise challenge. Cell-based studies appear to support the role of exercise as a trigger for
airway inflammation.
The study of winter athletes described above demonstrated that airway wall inflammation occurred
even in the absence of symptomatic EIB. This suggests that airway inflammation is not sufficient to
cause EIA and that exercise per se can induce inflammation in normal subjects. Unfortunately, none
of these studies offer an explanation for why some
athletes are susceptible to the inflammatory effects of
exercise while others are not.
Inflammatory cells in the airway are presumed to
cause bronchial hyperresponsiveness by the production of chemical mediators of inflammation. A variety of such mediators have been studied in asthma
and EIA (Table 3). Early studies investigating the role
of the mast cell stabilizers such as nedcromil and
sodium cromoglycate identified histamine as a likely
Table 3 Inflammatory mediators in EIA
Mediator
Role in asthma
Induced by exercise
Efficacy of inhibitor
Histamine
Leukotrienes
Prostaglandin D2
Bronchoconstrictor
Bronchoconstriction, smooth muscle proliferation, chemoattractant
þ
þ
?
þþ
Variable
Various inflammatory mediators have been investigated for their capacity to induce or maintain airway inflammation in EIA. Evidence for
induction during exercise comes from studies looking at plasma, urine, or sputum levels of the mediator or product. The role of histamine
has recently been challenged due to the overriding effect of leukotriene antagonists with no additional effect of antihistamine and a
consistent difficulty in demonstrating exercise induction.
202 ASTHMA / Exercise-Induced
trigger for EIA although this has been challenged
recently. The role of other chemical mediators of inflammation including cysteinyl leukotrienes (LTD4
and LTC4) and prostaglandins (PGD2 and PGF2)
have been supported by the discovery of increased
levels in postexercise plasma and urine and by the
effects of specific antagonists.
The role of inflammatory cells and their products
in the development of EIA is supported by studies
demonstrating the capacity of exercise to increase
their availability to airway cells. However, not all
investigators have found increased levels of proasthmatic mediators in response to exercise, and the
presence of a particular agent does not imply a causal
link. Further evidence is available from interventions
aimed at reducing airway inflammation.
Do Anti-Inflammatory Therapies Prevent EIA?
Inhaled corticosteroids (ICs) are the most widely
used airway-delivered anti-inflammatory agents.
Among well-controlled asthmatic subjects who use
regular ICs to abolish normal symptoms 50% will
still experience EIB on testing. This suggests that EIA
is relatively resistant to standard doses of IC therapy.
This hypothesis is supported by early studies demonstrating only a 50% reduction in exercise-induced
fall of FEV1 when ICs were introduced. When a
short-acting b-adrenergic receptor agonist was added
to the IC therapy, this further reduced EIB by 30%,
hinting that increased airway resistance in EIA is a
complex phenomenon.
The introduction of modern anti-inflammatories
has reinforced the theory that EIA is an inflammation-mediated phenomenon. Leukotrienes and in
particular LTD4 are regarded as among the most
potent proasthmatic inflammatory mediators. Leukotriene receptor antagonists (LTRAs) inhibit the
effect of these mediators at their specific receptors.
The introduction of LTRAs to common practice
suggested that they might have efficacy in treating
EIA, a suggestion supported by subsequent examination. LTRA therapy provides a dose-dependent
protection from EIB and shortens the time to recovery of FEV1. In contrast antihistamine therapy does
not appear to add any further protection over LTRA
medication.
Several lines of evidence support the role of inflammatory processes in EIA. Exercise can trigger
and maintain airway inflammation that is associated
with bronchial hyperresponsiveness. Intervention
studies show that combating inflammation can improve EIA. What is unclear, however, is what predisposes an individual to develop exercise-induced
airway inflammation and why otherwise potent antiinflammatory agents are only partially effective in
preventing EIA despite improved control in underlying asthma.
Finally, various chemical triggers of asthma have
been investigated for their ability to trigger or enhance bronchoconstriction on exercise challenge. Of
these adenosine, the product of ATP breakdown,
seems worthy of further investigation. Exercise increases circulating adenosine levels suggesting that
any proasthmatic activity of this chemical could
then be enhanced by exercise. A correlation between
the fall in FEV1 following exercise challenge in asthmatic subjects and plasma adenosine levels has been
reported. Adenosine is believed to trigger inflammatory cell degranulation, and in the context of asthma
this may enhance a mild asthmatic response making
it considerably more severe. Such studies may
ultimately explain why exercise can be the only trigger for symptomatic EIA in otherwise nonasthmatic
subjects.
Integrating the Two Pathogenic Theories
It can be concluded that exercise-induced hyperventilation can trigger physical changes in the airway
that are subsequently transduced to an inflammatory
signal in the bronchial wall, which can be assumed to
lead to bronchoconstriction and possibly chronic inflammation in susceptible people. Can physical
changes at the surface of the airway communicate
to the bronchial wall to cause inflammation and
bronchial hypersensitivity? Evidence for pathways
communicating signals between different airway
compartments has been accumulating over the last
decade, offering possible mechanisms to complete
the model. As yet these pathways are untested in the
context of EIA but do offer intriguing possibilities.
It is becoming clear that airway epithelial stress
can cause the release of inflammatory mediators that
promote airway inflammation. Recent research supports the development of an integrated epithelium–
mesenchyme complex (the epithelial mesenchymal
trophic unit) reminiscent of the embryonic lung. Epithelial monolayers in vitro can trigger inflammatory
signals in fibroblasts and smooth muscle cells of the
airway supporting such a communication. Inflammatory cells found superficially in the airway, such as
eosinophils, can be activated by osmolar stress and
release mediators capable of eliciting an asthmatic
response offering a more direct line of communication. Thus, drying and cooling of surface epithelium
could in theory at least trigger generalized airway
inflammation as seen in chronic asthma. The hyperemia following airway rewarming would support an
inflammatory process by supplying exudates and inflammatory cells (Figure 1).
ASTHMA / Exercise-Induced 203
Hyperventilation/exercise
Cooling of the airway & bronchial
vasoconstriction
Reactive vasodilitation &
mucosal hyperemia
Hyperosmolarity of airway
mucous layer
Mucosal congestion &
exudate formation
Accumulation of inflammatory
cells in the airway lumen
Osmotic stress
communicated to superficial
cells of the airway
Degranulation
Degranulation of
inflammatory cells – release
of inflammatory mediators
LTD4 and PGE2
Release of proinflammatory
cytokines from epithelial cells
Bronchial wall inflammation, airway smooth muscle
contraction, mucosaledema, possible epithelial cell
shedding
Figure 1 Summary diagram of the proposed pathogenesis of EIA. A series of pathological events following increased ventilation in
exercise are proposed that trigger an asthmatic response in susceptible individuals.
Clinical Presentation
Symptoms associated with exercise can arise from a
variety of sources including the lungs, heart, and gastrointestinal tract. A careful history can help to identify the most likely source, but EIA may present with
classical symptoms such as dyspnea, wheeze, and
cough or with more obscure symptoms making the
diagnosis difficult. Complaints such as headache, abdominal pain, chest pain, cramps, and severe fatigue
may all respond to treatment for EIA. Symptoms may
develop early during an exercise event or following its
completion. The association with exercise should
alert the clinician to the possibility of EIA although
the complaint may seem unrelated to the lungs. Frequently patients believe they are simply ‘out of condition’ and even well-controlled asthmatics may not
associate their exercise-induced symptoms with asthma. EIA can be the only manifestation of hyperresponsive airways so the absence of a formal diagnosis
of asthma does not exclude the possibility of EIA.
Differential Diagnosis
Since the diagnosis of EIA is not always straightforward to make a high index of clinical suspicion, it
204 ASTHMA / Exercise-Induced
Table 4 Differential diagnosis of the patient presenting with
exercise-induced breathlessness
System
Disease
Respiratory
EIA
COPD
Pulmonary fibrosis
Pulmonary vascular hypertension
Angina
Ventricular dysfunction
Dysrhythmia
Poor fitness
Cardiovascular
General
COPD, chronic obstructive pulmonary disease; EIA, exerciseinduced asthma.
should be combined with an open mind regarding the
variety of other potential sources of symptoms. In
particular, true vocal cord dysfunction can be difficult to distinguish from EIA when symptoms predominantly present with exercise. A list of alternative
diagnoses for exercise-induced dyspnea is presented
(Table 4). It is not yet clear if patients who have
demonstrable exercise-induced bronchoconstriction
but no symptoms will benefit from pharmacological
treatment.
Examination of the lungs of a subject with exerciseinduced dyspnea will usually be normal in the context
of asthma or lone EIB. The presence of wheeze or
hyperinflation might point to chronic airflow obstruction that has gone unrecognized. A full examination might point to an alternative diagnosis as
listed. Again quiescent asthma or lone EIB will usually be associated with normal pulmonary function
tests while abnormalities may point to alternative
pulmonary diagnoses or chronic airflow obstruction.
Investigation
The need for further investigation will be directed by
the clinical findings but may fall into two groups. In
the first a pragmatic trial of pharmacological intervention prior to a predictable exercise trigger of
symptoms can be the most useful and easiest test. In
some cases, however, detailed pulmonary function in
response to exercise is desirable either to monitor
treatment, to make a difficult diagnosis, or if the
presence of EIA would limit the performance of essential life-saving work (American Thoracic Society
(ATS) Guideline). Other uses for detailed exercise
testing include the diagnosis of asthma in elite athletes for drug monitoring or performance testing.
In a pragmatic trial a patient should be prescribed
either a preventative or a reliever inhaled therapy and
advised to use this prior to or during exercise that
would normally trigger the reported symptoms. Antiinflammatory drugs such as inhaled corticosteroids
or cromoglycate have been shown to have some preventative efficacy in this setting. b2-adrenergic agonists have been shown to relieve exercise-induced
bronchoconstriction but their efficacy is reduced in
EIB. Patients should be able to record the effect of
treatment on symptoms or on peak flow with a small
degree of training.
Where it is desirable to direct investigations towards a specific diagnosis of EIA, a variety of alternative procedures, ranging from exercise provocation
to bronchial challenge, are available. Various procedures have been described that are capable of triggering airway narrowing in asthma consistent with a
diagnosis of EIA. The principal element of all these
tests is to raise minute ventilation as occurs with exercise. It is possible to trigger bronchial narrowing by
hyperventilation and this has been recommended as a
surrogate for more formal exercise challenge. More
exercise specific tests have been developed for both
field and laboratory. While testing in the field has the
advantage of replicating the conditions that normally
trigger asthma in the subject, the equipment required
to make useful measurements has to be portable.
This has led to the development of protocols designed to measure pulmonary responses to exercise
under laboratory conditions that replicate the essential features of outdoor activity. Guidelines recommending the best practice for performing these tests
are available, the essential features of which are
summarized below.
Patient Instructions
Subjects should avoid bronchoprotective or bronchoreliever medication for 48 h and have eaten only a
light meal. Antihistamines and caffeine should also
be avoided. Exercise should be kept to a minimum
prior to the test, as around 50% of EIA sufferers will
experience a refractory period of up to 4 h after vigorous exercise. Any medical or orthopedic contraindications to exercise should be considered and the
ability of the patient to fulfill the physiological requirements should be ensured.
Exercise
Exercise can be performed on an electronic treadmill
or stationary bicycle as both methods have been
validated. The desired level of exercise is based on
80–90% of maximal heart rate (based on an HR 220
– age) or 50–60% of maximal voluntary ventilation.
The degree of exercise required to achieve this level
of exercise response will vary considerably among
subjects and it is advised that a period of rapid progressive increase in workload is used to achieve these
targets and then maintained for at least 4 min. The
ASTHMA / Exercise-Induced 205
total exercise time for adults should be around 6–
8 min. During the exercise, heart rate and where
possible minute ventilation should be measured to
ensure that an adequate stimulus to the airway is
being achieved.
Climatic Considerations
Outdoor exercises are generally better than indoor
exercises at triggering bronchospasm. This is believed to be due to lower humidity and temperature
and cool dry air is known to trigger asthma in exercise sensitive subjects better than indoor room air.
Maintaining the ambient temperature at 20–251C
and relative humidity at 50% is satisfactory. Exercise
should be performed with a nose clip to prevent nasal
humidification of inspired air.
Severe responses to exercise have been described in
susceptible people and it is advisable to have medical
supervision available to monitor patients’ responses
and administer bronchodilator therapies as required.
Measurements
FEV1 (% fall)
FEV1 is the most useful and convenient measurement
to make in the laboratory. Where an alternative diagnosis is suspected full flow-volume loops can offer
additional information, while peak expiratory flow
might be used if field testing is performed. FEV1
should be measured before exercise and following
the exercise challenge. Intervals of 5, 10, 15, 20, and
30 min are recommended by the ATS; however, additional measurements can be taken and should be in
the event of severe symptoms of bronchial obstruction. Changes in FEV1 can then be observed by plotting percent resting FEV1 against time (Figure 2).
20
18
16
14
12
10
8
6
4
2
0
What constitutes a ‘positive’ exercise test is controversial due to the variety of techniques described for
measurement. Most authorities consider a fall of more
than 10% of resting FEV1 as abnormal especially as
the ‘normal’ response to exercise is bronchodilation.
Some groups require a greater than 15% fall in FEV1
to diagnose EIA and this appears to be the most frequently quoted value. Plotting the FEV1 against time
allows an area under the graph calculation to be made
that improves the reliability of the test.
Alternative Testing Procedures
Other techniques to mimic the airway response to
exercise have been described. These are generally
thought to be more convenient than full exercise
challenge, but are necessarily less specific. Osmotic
drying of the airway has been achieved by inhalation
of mannitol and this method has received some attention from testers from the field of elite sport.
Eucapnic hyperventilation achieves a ventilation rate
that mimics that achieved by exercise and has also
been used as an outpatient measure of the airway
responses.
Management
The efficacy of asthma medications for exerciseinduced symptoms will be dealt with elsewhere.
Patients should be encouraged to manage their symptoms rather than stop the exercise activity that
triggers it. Occasionally, altering the exercise environment is helpful, for example, changing to indoor
from outdoor activity. Some elite athletes have
learned to manage their asthma by introducing a
targeted warm up to trigger a mild episode of EIA
and the resultant refractory period elite may allow
the completion of the competitive element of the activity. Sporting authorities restrict the use of most
inhaled therapeutic agents for asthma. It is sensible
for athletes and their coaches to be aware of these
restrictions, which can be accessed easily. A range of
websites for agencies involved in regulating drugs in
sport is given at the end of this article.
Conclusion
0
10
20
30
40
50
60
70
Time from start of exercise (min)
Figure 2 A plot of change in FEV1 as percent baseline with
time exercised. Exercise begins at time 0 and ceases at the point
marked by the arrow. FEV1 is measured and the percent change
is calculated.
, change in FEV1;
, the effect of an agent
that changes maximum fall in FEV1;
, the effect of an agent
that reduces the duration of EIB. As can be seen it is frequently
more accurate to represent changes to EIB by describing the
area under the curve than any single element of the plot.
EIA may be a specific disease entity in some circumstances or may be the only manifestation of latent
asthma. It is common in asthma sufferers and in
athletes of all capabilities. The basis for the development and persistence of exercise triggered airway
pathology is not fully understood but probably reflects the ability of hyperventilation to trigger airway
inflammation. Diagnosis can be complicated by the
variety of presentations and detailed investigations
206 ASTHMA / Extrinsic/Intrinsic
are sometimes required. Asthma management is not
always successful at controlling symptoms and behavioral changes may have to be made to achieve
control of the disease.
See also: Exercise Physiology. Lipid Mediators:
Leukotrienes.
Further Reading
Anderson SD and Brannan JD (2002) Exercise-induced asthma: is
there still a case for histamine? Journal of Allergy and Clinical
Immunology 109(5): 771–773.
Anderson SD and Daviskas E (2000) The mechanism of exerciseinduced asthma is. Journal of Allergy and Clinical Immunology
106(3): 453–459.
Gotshall RW (2002) Exercise-induced bronchoconstriction. Drugs
62(12): 1725–1739.
Milgrom H (2004) Exercise-induced asthma: ways to wise exercise. Current Opinion in Allergy and Clinical Immunology 4:
147–153.
Seale JP (2003) Science and physicianly practice: are they compatible? Clinical and Experimental Pharmacology and Physiology 30(11): 833–835.
Storms WW (2003) Review of exercise-induced asthma. Medicine
and Science in Sports and Exercise 35(9): 1464–1470.
Tan RA and Spector SL (2002) Exercise-induced asthma: diagnosis
and management. Annals of Allergy, Asthma, and Immunology
89(3): 226–235.
which many cells and cellular elements play a role, in particular,
eosinophils, mast cells, T lymphocytes, neutrophils, and epithelial cells. Some patients develop structural changes of the airway, a process known as remodeling, possibly due to ongoing
inflammation and abnormal repair processes. Susceptible individuals experience recurrent episodes of wheezing, breathlessness, chest tightness, and cough, particularly at night and in the
early morning. These episodes are usually associated with widespread but variable airflow obstruction, which is often reversible, and bronchial hyperresponsiveness to a variety of stimuli.
Acute asthma is a common medical emergency and requires
prompt assessment and treatment. Advances in the understanding of the genetic and environmental factors that account for
asthma and its pathogenesis should lead to improved management strategies.
Introduction
Historical Perspective
The symptoms of asthma were described by Aretaeus
over 2000 years ago. However, despite significant
progress in our understanding of its pathogenesis and
considerable improvements in pharmacological treatment, we have been unable to halt the relentless increase in prevalence that has taken place over the last
30 years.
Definition
Relevant Websites
http://www.olympic.org – Home page of the International Olympic Movement, offering insight into the role of antidoping authorities in elite sport. It includes useful links to national
Olympic committees detailing specific national guidelines for
athletes.
http://www.wada-ama.org
Homepage of the world antidoping
authority. Including details of the use of prescribed proscribed
therapeutics in sport.
http://www.asthma.org.uk
Asthma UK link to exercise induced
asthma with tips for patients and some general information
regarding school and preschool sporting activities for asthma
sufferers.
Extrinsic/Intrinsic
N C Thomson and G Vallance, University of
Glasgow, Glasgow, UK
& 2006 Elsevier Ltd. All rights reserved.
Several different definitions have been devised that
describe the asthma phenotype. In 1997 the National
Asthma Education and Prevention Program Expert
Panel Report defined asthma as: ‘‘A chronic inflammatory disorder of the airways in which many cells
and cellular elements play a role, in particular, mast
cells, eosinophils, T lymphocytes, neutrophils, and
epithelial cells. In susceptible individuals, this inflammation causes recurrent episodes of wheezing, breathlessness, chest tightness, and cough, particularly at
night and in the early morning. These episodes are
usually associated with widespread but variable airflow obstruction that is often reversible either spontaneously or with treatment. The inflammation also
causes an associated increase in the existing bronchial
hyperresponsiveness to a variety of stimuli.’’
Epidemiology
Prevalence of Asthma and Atopy
Abstract
Asthma is one of the most common chronic diseases, affecting
300 million people worldwide. There has been a significant increase in prevalence over the last 30 years, particularly in the
West. Complex relationships between genetic and environmental factors, such as viral infections, allergens, and occupational
agents, influence the origin and progression of the disease.
Asthma is a chronic inflammatory disorder of the airway in
Asthma is one of the most common chronic conditions affecting 300 million people worldwide. The
Global Initiative for Asthma (GINA) estimates that
one in 20 people in the world now have asthma, with
a significant increase in the prevalence of disease over
the last 30 years. This is in parallel with an increase in
other atopic diseases, such as allergic rhinitis and
Country
ASTHMA / Extrinsic/Intrinsic
207
Scotland
Jersey
Guernsey
Wales
Isle of Man
England
New Zealand
Australia
Republic of Ireland
Canada
Peru
Trinidad & Tobago
Costa Rica
Brazil
United States of America
Fiji
Paraguay
Uruguay
Israel
Barbados
Panama
Kuwait
Ukraine
Ecuador
South Africa
Finland
Malta
Czech Republic
Ivory Coast
Colombia
Turkey
Lebanon
Kenya
Germany
France
Japan
Norway
Thailand
Sweden
Hong Kong
United Arab Emirates
Philippines
Belgium
Austria
Saudi Arabia
Argentina
Iran
Estonia
Nigeria
Spain
Chile
Singapore
Malaysia
Portugal
Uzbekistan
FYR Macedonia
Italy
Oman
Pakistan
Tunisia
Latvia
Cape Verde
Poland
Algeria
South Korea
Bangladesh
Morocco
Occupied Territory of Palestine
Mexico
Ethiopia
Denmark
India
Taiwan
Cyprus
Switzerland
Russia
China
Greece
Georgia
Romania
Nepal
Albania
Indonesia
Macau
0
5
10
15
20
25
30
35
40
Prevalence of asthma symptoms (%)
Figure 1 Ranking of the prevalence of current asthma symptoms in childhood by country: written questionnaire. Reproduced with
permission from GINA (2004) Self-reported wheezing in the previous 12 month period in 13- to 14-year-old children. Global Burden of
Asthma, p. 6.
atopic dermatitis. These increases have been most
noticeable in affluent countries with a mild climate
such as the UK, New Zealand, Australia, and North
America (Figure 1) and correlate with urbanization
and the adoption of a westernized life style. It is more
common in children than in adults. The clearest risk
factor for the development of asthma is atopy. Atopy
is the genetic predisposition for the development
of an IgE-mediated response to common aeroallergens. Complex relationships between atopy and
environmental factors such as viral infections, allergens, and occupational agents influence the origin
and progression of the disease.
Morbidity
The morbidity from asthma is considerable. Surveys
of patients with asthma indicate that many have
poorly controlled symptoms, impaired indices of
quality of life, and are often receiving inadequate
208 ASTHMA / Extrinsic/Intrinsic
treatment. Hospital admission rates for asthma, particularly in children, increased from the 1970s until
the mid 1980s and have since then remained stable.
In the US during 2002 there were 13.9 million outpatient visits, 1.9 million emergency room visits, and
484 000 hospitalizations. Many children and adults
with asthma lose time from school and work, respectively. The financial impact of asthma is considerable; in the US the estimated total cost exceeds $6
billion per annum, with hospitalization and emergency room visits making up 50% of that figure.
Classification
Asthma can be classified on the basis of severity and
etiology.
Severity
The severity of asthma can be graded by assessing the
frequency and severity of symptoms and measurements of lung function before treatment is started or
by the level of treatment required to achieve asthma
control (Table 1).
Etiology
Mortality
International mortality figures for asthma are often
unreliable due to misclassification of the cause of
death. In Western countries, where studies were restricted to the 5–34 years age group, the mortality
from asthma increased steadily from the mid-1970s
to the late 1980s. More recent studies suggest a plateau or decline in deaths from asthma. Risk factors
for increased morbidity and potential mortality
include socioeconomic deprivation, ethnicity, urban
dwelling, and comorbid issues such as drug abuse.
The vast majority of deaths occur among those with
chronic severe asthma; few deaths occur among those
with previously mild disease. Deaths are associated
with inadequate treatment with inhaled or oral steroid and with poor follow-up and monitoring.
Natural History
The findings of the Tucson Children’s Respiratory
Study suggested three clinical phenotypes of childhood asthma. Transient infant wheezing occurs during infancy, but not after the age of three years. These
children have no family history of atopy and have a
good prognosis. The second phenotype is the nonatopic wheeze of the toddler and early school years,
after an early lower respiratory tract infection. The
third phenotype is persistent atopic wheeze, which
describes children who continue to wheeze at age 10
and have associated atopy and airway hyperresponsiveness. Many children have a favorable outcome
with spontaneous remission in their adolescence.
Risk factors for progression into adulthood include early onset with severe symptoms, poor lung
function, and airway hyperresponsiveness. It occurs
more commonly in girls with associated atopy. Most
adults with mild-to-moderate asthma appear to continue to have symptoms of a similar severity. Remission of adult asthma is rare. Irreversible airflow
obstruction can develop in nonsmokers with asthma
particularly in those individuals with severe symptoms and mucus hypersecretion. Smokers with
asthma have an accelerated decline in lung function.
In 1947, Rackeman was the first to subdivide asthma
into intrinsic and extrinsic asthma. He noted that
extrinsic asthma, now described as allergic asthma,
started before the age of 30 years and was associated
with atopy. Intrinsic or nonallergic asthma was noted
to begin in middle age and was not associated with
allergy, but with nasal polyps. It is more common in
women.
Etiology
Genetic
Twin and family studies have demonstrated that
atopic diseases cluster in families and have a genetic
basis. Genome-wide scans have shown that many
genes determine the risk of asthma. The region of
chromosome 5q31-33 controls the production of interleukin (IL)-4 and IL-13 and has been linked to
atopy. Linkage studies have implicated other candidate genes on chromosomes 2, 3, 4, 6, 7, 11, 12, 13,
17, and 19. Classical positional cloning approaches
have led to the identification of new genes of potential significance such as ADAM33, IL4RA, and
CD14. This may contribute to our understanding
of the mechanism of disease, such as the role of
ADAM33 in airway hyperresponsiveness. They may
also cast light on different individual responses to
therapy, as there are polymorphisms of a number of
common drug targets. The delineation of the precise
relationships between these genetic factors and environmental agents is now required.
Environment
Hygiene hypothesis In 1989, Strachan noted an inverse relationship between family size and hay fever,
with children with more siblings having a lower risk
of atopy. It has been suggested that childhood exposure to infection may protect against risk of atopy.
Low levels of infection in infancy, associated with
improvements in public health, may deprive the
immune system of the Th1 stimulus that normally
balances the Th2 predominance of the neonate. This
ASTHMA / Extrinsic/Intrinsic
209
Table 1 Classification of asthma severity by daily medication regimen and response to treatment
Patient symptoms and lung function on current
therapy
Current treatment step
Step 1: intermittent
Step 2: mild persistent
Step 3: moderate
persistent
Level of severity
Step 1: intermittent
Symptoms less than once a week
Brief exacerbations
Nocturnal symptoms not more than twice a
month
Normal lung function between episodes
Intermittent
Mild persistent
Moderate persistent
Step 2: mild persistent
Symptoms more than once a week but less
than once a day
Nocturnal symptoms more than twice a month
but less than once a week
Normal lung function between episodes
Mild persistent
Moderate persistent
Severe persistent
Step 3: moderate persistent
Symptoms daily
Exacerbations may affect activity and sleep
Nocturnal symptoms at least once a week
60%oFEV1 o80% predicted OR
60%oPEFo80% of personal best
Moderate persistent
Severe persistent
Severe persistent
Step 4: severe persistent
Symptoms daily
Frequent exacerbations
Frequent nocturnal asthma symptoms
FEV1 p60% predicted OR PEFp60% of
personal best
Severe persistent
Severe persistent
Severe persistent
Reproduced with permission from GINA (2004) Global Strategy for Asthma Management and Prevention, Chapter 7, Part 4A, Figures
5–7, p. 7.
unrestrained Th2 response is postulated to predispose to allergic disease. European studies showing
lower levels of atopic disease amongst children raised
in rural communities have suggested that exposure to
bacterial endotoxin may play a role in the development of tolerance to common allergens. However,
the high levels of asthma associated with cockroach
allergy in inner-city areas of the US, and the simultaneous increase in Th1 diseases, such as type 1 diabetes, illustrate that our understanding of the
relationship between environmental exposure and
disease is not yet complete.
Allergen exposure Sensitization to the house dust
mite Dermataphagoides pteronnysinus is the most
common risk factor for the development of asthma in
adults and children. Early exposure to the dust mite
antigen Der p1 has been shown to increase the risk of
asthma fivefold. Peat elegantly demonstrated the doseresponse relationship between dust mite level and severity of asthma symptoms across six different regions
of Australia with increasing humidity levels. Dust
mites thrive in a humid environment, and improved
westernized building construction techniques, favoring colonization by house dust mites, may have contributed to the increase in asthma. Studies at altitudes
where the levels of house dust mite are extremely low
have suggested that a low allergen environment does
improve symptoms of asthma. However, simple measures such as mattress covers and carpet cleaning have
shown little effect on reducing the domestic burden of
dust mite and asthma symptoms.
Exposure to other indoor allergens from animal
dander and cockroach are also important risk factors
for asthma. Removal of the pet from the family is
advised to reduce exposure to the allergen. However,
early exposure to cat allergen has recently been demonstrated to protect against development of asthma
and the full intricacies of the relationship remain to
be elucidated.
Cockroach infestation has become rampant in inner-city areas of the US and sensitization to the
210 ASTHMA / Extrinsic/Intrinsic
German cockroach Blatella germanica has become
an important risk factor for asthma.
Outdoor air environment Sensitization to the fungus Alternaria is a risk factor for asthma. It has been
suggested that outbreaks of asthma after thunderstorms are related to release of fungal spores of
Cladosporium.
The relationship between air pollution and asthma
is complex as there has been considerable reduction
in air pollution over the time period when asthma
prevalence has increased. Extremely high levels of
asthma have been noted in the rural Highlands of
Scotland. It has been suggested that particulate pollution may protect against incidence but exacerbate
symptoms in those sensitized.
Diet Changes in the modern processed diet have
been linked with an increased risk of asthma. High
levels of omega-3 oils associated with eating fresh
fish have been shown to reduce the risk of asthma.
Breast-feeding to 3 months has also been associated
with lower levels of asthma.
Cigarette smoking There is much evidence to suggest that exposure to tobacco smoke in utero and in
early childhood increases the risk of allergy and
wheeze. Nevertheless, as the total numbers of smokers
in the UK have fallen during the last 30 years, it is not
the whole answer to the rise in asthma prevalence.
Pathology
Fatal Asthma
In cases of fatal asthma the lungs are hyperinflated
due to air trapping caused by plugging of the medium
to small airways with mucus and inflammatory cells,
particularly eosinophils. Histologically, the airways
show characteristic changes: an intense infiltration
by inflammatory cells, particularly eosinophils and T
lymphocytes, sloughing of the surface epithelium,
thickening of the reticular basement, increase in the
airway smooth muscle mass, increased numbers of
epithelial goblet cells, vasodilatation, and edema
(Figure 2). Neutrophils are found also in those who
die suddenly from acute asthma.
Chronic Asthma
Information on the pathology of asthma has been
obtained from bronchial biopsies obtained from
patients with mainly mild asthma. The histological
changes are similar although less pronounced than
those obtained from cases of fatal asthma. The
similarity of the histological changes in allergic or
extrinsic and nonallergic or intrinsic asthma suggests
a final common pathogenic mechanism in both types
of asthma. Neutrophils are found more commonly in
patients with severe asthma.
Clinical Features
Acute Asthma
Acute asthma is a common medical emergency. It is
characterized by a progressive increase in dyspnea,
cough, or wheeze. The decrease in expiratory airflow
can be quantified by a fall in peak expiratory flow
(PEF) or forced expiratory volume in 1 s (FEV1). Deterioration usually progresses over hours to days, although in some cases it may be more sudden and
require rapid treatment within minutes. The severity
of exacerbation is highly variable. Respiratory tract
Mucus
Epithelial cells and
goblet-cell hyperplasia
Thickening of sub-basement
membrane
Cellular infiltrate
Hypertrophy of smooth
muscle
Vascular congestion
Figure 2 Autopsy specimen of airway from a subject who died from acute asthma showing characteristic histological changes.
Hematoxylin and Eosin, 40. Photograph courtesy of Dr F Roberts, Department of Pathology, Western Infirmary Glasgow.
ASTHMA / Extrinsic/Intrinsic
infections are thought frequently to precipitate attacks of asthma, particularly in children. Infections
are mainly viral, especially human rhinoviruses but
also respiratory syncytial virus, adenoviruses, parainfluenza, and influenza viruses. The role of infection
in provoking asthma attacks in adults is less certain.
Clinical assessment
Clinical features A short history of the features of
an exacerbation should be elicited. The aims are to
ascertain duration and severity of symptoms, with
the perspective of current medication and prior admissions. Assessment must be rapid and accurate to
permit prompt treatment. The history is usually one
of increasing breathlessness and wheeze. Patients often have difficulty speaking and sleep is disturbed by
the severity of these symptoms. There is increasing
need for bronchodilator treatment, which becomes
less effective. The patient may show signs of exhaustion and reduced conscious level. There is invariably
an associated tachycardia, increase in respiratory
rate, and auscultation of the chest may reveal severe
wheeze or absent breath sounds that indicates very
severe airflow obstruction. The chest becomes hyperinflated and patients may use accessory respiratory
muscles.
Investigations Measurement of pulse oximetry is
necessary in acute asthma to evaluate oxygen saturation. The aim is to maintain SpO2492%. Arterial
blood gas analysis is necessary if SpO2o92%. If oxygenation remains inadequate despite supplemental
oxygen, additional complications should be considered, particularly pneumonia. The earliest abnormality is respiratory alkalosis and hypocarbia, but
normal oxygen tension. As airflow obstruction increases there is uneven distribution of inspired air
and changes in the normal ventilation-to-perfusion
ratio. As severity increases, hypoxemia develops. The
presence of normal levels of arterial carbon dioxide
tension is ominous as it indicates the patient is becoming exhausted. Monitoring response to treatment
should be on the basis of PEF and clinical examination. A chest radiograph may show evidence of
hyperinflation, mucus plugging, and atelectasis in an
acute exacerbation, but these findings may add little
to management. A chest radiograph should be performed if pneumothorax is suspected.
Levels of severity Asthma guidelines have been developed to ensure prompt, systematic history and
examination, and ensure accurate assessment of severity. The British Guideline on the Management of
Asthma defines a moderate exacerbation as one
presenting with increasing symptoms of wheeze,
211
dyspnea, or breathlessness and a fall in PEF to
50–75% of the best or predicted. However, if the PEF
falls to 33–55% best or predicted and is accompanied
by an inability to speak in complete sentences, tachypnea of 425 breaths per min, or tachycardia of
110 beats per min, the exacerbation is classified as
severe. Life-threatening features are a PEF less than
one-third of best or predicted or hypoxia demonstrated by arterial oxygen saturations of less than
92% on air or arterial partial pressure of less than
8 kPA. Normal levels of CO2, a silent chest on examination, or feeble respiratory effort, cyanosis,
bradycardia, dysrhythmia, hypotension, exhaustion,
confusion, or coma are all signs of a near fatal
episode.
Chronic Asthma
The diagnosis based on a history of episodic respiratory symptoms especially after exercise or during
the night is usually not difficult. Demonstration of
reversible airflow obstruction gives a simple, reliable,
and objective diagnosis of asthma. Evidence of reversibility can be found through the history of symptoms of episodic cough, wheeze, chest tightness, or
dyspnea, measurement of PEF, or spirometry, and
trials of therapy. Conditions to be considered in the
differential diagnosis are listed in Table 2.
Clinical assessment
Clinical features Asthma may present with wheeze,
shortness of breath, cough, or chest tightness. The
hallmark of asthma is that these symptoms tend to be
variable and intermittent. They are often worse at
night and early morning and provoked by triggers
such as allergens or exercise (Table 3). Less common
factors are rhinitis, bacterial sinusitis, menstruation,
gastroesophageal reflux, and pregnancy. When cough
is the predominant symptom without wheeze, this is
Table 2 Differential diagnosis of asthma
Disease
Children
Adults
Cystic fibrosis
Gastroesophageal reflux
Bronchiectasis
Ciliary dyskinesia
Developmental disorder of the airway
Inhaled foreign body
Chronic obstructive pulmonary disease
Left ventricular function
Pulmonary thromboembolism
Vocal cord dysfunction
Upper airway obstruction
Pulmonary eosinophilia
Bronchial carcinoid
O
O
O
O
O
O
O
O
O
O, Diagnosis should be considered.
O
O
O
O
O
O
O
O
O
O
O
O
212 ASTHMA / Extrinsic/Intrinsic
Table 3 Triggers of asthma
Infections, particularly viral
Allergens, e.g., house dust mite, pollens, animals
Occupational agents, e.g., isocyanate-containing paints, flour
Environmental pollutants, e.g., cigarette smoke, sulfur dioxide
Drugs, e.g., beta-blockers
Exercise
Cold air
Hyperventilation
Foods
Psychological factors
referred to as cough-variant asthma. The physical
sign of wheezing (usually expiratory, bilateral, polyphonic, and diffuse) is associated with asthma but
has low sensitivity and specificity, and in many patients examination will be normal.
Investigation A simple measure of pulmonary function by PEF is helpful, not only for initial assessment,
but also to monitor symptoms, alert to deterioration
in airflow obstruction, and evaluate response to
treatment. Twice-daily recording of PEF for two
weeks is a simple and cheap method of demonstrating variation in airflow obstruction, with a diurnal
variation of greater than 15% confirming the diagnosis. However, in patients with mild asthma, the
PEF may show normal variability. Spirometry demonstrates an obstructive pattern with reduction of the
ratio of FEV1 to forced vital capacity (FVC). The
key feature is that this airway obstruction may be
reversible. Administration of a bronchodilator typically causes an increase in FEV1 of 12–15%. However, failure to demonstrate reversibility does not
exclude asthma, or prove irreversible disease.
Airway hyperresponsiveness is a characteristic feature of asthma and can be demonstrated by bronchial
provocation techniques. The most common methods
are provocation by inhalation of methacholine or
histamine and exercise challenge. Fall in FEV1 is
measured by serial spirometry after inhalation of increasing concentrations of methacholine. Results are
expressed as the concentration of the agent that elicits a fall of 20% in FEV1. This concentration defines
the degree of bronchial responsiveness and severity
of disease. Skin prick testing, measurement of total
and specific IgE levels, and blood eosinophilia are
difficult to interpret in asthma because they have
variable sensitivity and specificity. Routine chest radiographs in asthma may yield no new information
and may be normal in chronic asthma.
Assessment of control Asthma control can be determined by assessing symptoms, inhaled b2 adrenoceptor agonists use, lung function as well as rate
of exacerbations, number of emergency consultations
for asthma, and hospital admissions. Good control is
described as the presence of minimal symptoms during day and night, minimal need for reliever medication, no exacerbations, no limitation of physical
activity, and normal lung function. It may not be
possible to achieve good asthma control in patients
with moderate or severe persistent asthma (Table 1).
Specific clinical problems
Asthma during pregnancy The course of asthma
during pregnancy varies, with a similar proportion of
women improving, remaining stable, or worsening.
The risk of an exacerbation of asthma is high immediately postpartum, but the severity of asthma
usually returns to preconception level after delivery.
Changes in b2-adrenoceptor responsiveness and
changes in airway inflammation induced by high
levels of circulating progesterone have been proposed
as possible explanations for the effects of pregnancy
on asthma.
Gastroesophageal reflux Gastroesophageal reflux
can trigger attacks of asthma although the incidence
is unclear. The mechanism is unknown; possibilities
include aspiration or an esophagio-bronchial reflux
triggered by acid irritation of the esophageal mucosa.
Pathogenesis
The pathogenesis of asthma involves acute and chronic
inflammation as well as remodeling (Figure 3). The
underlying process is one of inflammation involving
eosinophils and T lymphocytes, with the release of
various mediators and cytokines, although recent
evidence indicates a role for other cells including
mast cells, neutrophils, macrophages, and epithelial
cells. Some patients develop structural changes of the
airway, a process known as remodeling. The mechanisms involved in remodeling remain to be clarified
but probably consist of ongoing inflammation and
abnormal repair processes. Remodeling occurs in
many asthmatic patients, although the extent varies.
It is thought that remodeling may play an important
role in causing symptoms and loss of lung function in
severe asthma, although this hypothesis remains to
be established.
Airway Inflammation
There are two distinct responses to inhalation of an
allergen. The immediate hypersensitivity reaction, in
which wheeze occurs within minutes, is comparable
to the wheal-and-flare response of skin. Further
wheeze is caused by the late phase response, mounted
between 6 and 9 h after allergen provocation.
ASTHMA / Extrinsic/Intrinsic
Inducers of asthma
213
Inflammatory mediators and asthma
Allergen
Antigen
presenting
cell
Dendritic cell
Lymph node
Acute
airway
inflammation
Chronic
airway
inflammation
Th2
lymphocyte
Airway
remodeling
Cytokines
(e.g., IL-4, IL-5, IL-13)
Symptoms
Exacerbations
Disability
Figure 3 In susceptible individuals allergens, occupational
agents, and other known and unknown inducers of asthma
cause airway inflammation. The inflammation may be acute and
resolved, but in most individuals the inflammation is chronic and
may be associated with airway remodeling. Airway inflammation
and remodeling cause asthma symptoms, exacerbations, and
disability.
The immediate reaction involves the activation of
mast cells by allergen cross-linking two IgE antibodies (Figure 4). IgE antibodies are produced by B cells
in response to processed antigen, which is presented
by airway dendritic cells in the draining lymph node.
The IgE antibodies bind mast cells by their high-affinity receptors (FceRI). Cross-linking by allergen of
IgE antibodies initiates signal transduction by the
FceRI effecting degranulation of the mast cell. This
promotes release of mediators such as histamine,
tryptase, eicosanoids, and reactive oxygen species.
These spasmogens cause secretion of mucus, smooth
muscle constriction, and vasodilation. Leakage of
plasma protein causes edema of the airway wall, impedes clearance of mucus, and causes formation of
plugs; all result in reduced airway conductance.
The late phase reaction includes the accumulation
of activated eosinophils, lymphocytes, macrophages,
neutrophils, and basophils. The ability of cytokines
to induce the expression of adhesion molecules provides a mechanism for cell migration from the circulation to the airway.
Eosinophils are thought to play a central role in
the pathogenesis of chronic asthma. IL-5 controls the
production of eosinophils by the bone marrow and
their subsequent release into the circulation. They
migrate from circulation to the airway under the
influence of chemokines and release toxic granule
proteins, including major basic proteins, eosinophil
peroxidase and eosinophil cationic protein, Th2
Mast cell
(e.g., leukotrienes,
histamine,
tryptase)
Epithelium
Eosinophil
(e.g., prostaglandins,
IL-6, IL-8)
(e.g., leukotrienes,
major basic proteins)
Inflammatory mediators
BronchoMucosal
constriction edema
Mucus
hypersecretion
Bronchial
reactivity
Airway
remodeling
Symptoms of asthma
Figure 4 Possible pathways in the development of airway inflammation in asthma following exposure to allergen.
cytokines, and leukotrienes. Major basic proteins
causes direct airway damage with epithelial shedding. Leukotrienes increase vascular permeability
and constrict smooth muscles. Challenge of the airway with allergen increases the local levels of IL-5,
which correlates directly with the degree of airway
eosinophilia. Recently, doubts have arisen about
the role of eosinophils in causing airway hyperresponsiveness in asthma. Treatment of patients with
allergic asthma using anti-interleukin-5 monoclonal
antibody does not prevent allergen-induced bronchoconstriction or airway hyperresponsiveness, despite
markedly suppressing eosinophil numbers within the
airways.
T cells are found in abundance in the inflamed
airways of asthma patients and it is widely held that
the Th2 subset is a driving force in allergic inflammation. The T helper subsets were characterized by
Mosman on the basis of their signature cytokines.
Th2 cell cytokines include IL-4, IL-5, and IL-13. IL-5
is involved in eosinophil maturation and activation,
whereas IL-4 and IL-13 control synthesis of IgE.
However, the Th2 paradigm for allergic asthma is
now thought to be too simplistic and an additional
role for Th1 cells has been postulated.
214 ASTHMA / Extrinsic/Intrinsic
The mast cell degranulation is crucial to the acute
response to allergen, with release of histamine and
synthesis of mediators effecting early recruitment,
adhesion, and proliferation of cells. It may also contribute to remodeling as it contains proteoglycans
with various functions, including support structures
for remodeling.
The role of other inflammatory cells in the pathogenesis of asthma including neutrophils and macrophages is less clearly characterized. There are also
complex interactions between neural control of airways and inflammation.
Airway Remodeling
There has been considerable evidence that inflammation alone may not explain the full pathophysiology of asthma. Two large longitudinal studies of
inhaled corticosteroids have shown little effect on the
natural history of asthma. Acute airway inflammation should be resolved to permit resumption of normal function. In chronic asthma, a state of increased
injury and repair is thought to lead to remodeling.
It is coordinated by inflammatory cells, such as
eosinophils, mast cells and T cells, and structural
cells such as fibroblasts. Growth factors are secreted
in response to epithelial damage and cause thickening of the smooth muscle and basement membrane,
resulting in airway narrowing. It is thought that a
primary abnormality of the epithelium predisposes to
such damage by toxins. The interaction between the
thickened basement membrane and altered submucosa, known as the epithelial-mesenchymal trophic
unit, is postulated to be important early in the origins
of disease. This hypothesis is supported by the recent
identification of ADAM33 as an asthma susceptibility gene expressed abundantly by the smooth muscle
and fibroblasts, but not by inflammatory cells.
Animal Models
Animal models have been used to study both the
pathogenesis and treatment of asthma. Asthma does
not occur spontaneously in animals although both
horses and the Berenji greyhound develop a respiratory condition that has some features of asthma.
Most animal models involve sensitization to an allergen such as ovalbumin or house dust mite allergen
challenge. The species commonly used include mice,
guinea pigs, sheep, rats, monkeys, and dogs. The
mouse is often used in these models, mainly because
this species allows for the application in vivo of gene
deletion technology as well as the low cost and
availability of inbred species with known characteristics. Animal models of allergen-induced airway
inflammation and hyperresponsiveness have provided important information on the acute inflammatory response to allergen exposure but have been less
relevant to the study of mechanisms of chronic
asthma and airway remodeling. The interpretation of
data from experiments in animal models is influenced
by the protocol used to sensitize and challenge the
animal and the strain of animal used.
See also: ADAMs and ADAMTSs. Allergy: Overview.
Angiogenesis, Angiogenic Growth Factors and Development Factors. Arterial Blood Gases. Asthma:
Overview; Allergic Bronchopulmonary Aspergillosis;
Aspirin-Intolerant; Occupational Asthma (Including Byssinosis);
Acute
Exacerbations;
Exercise-Induced.
Bronchoalveolar Lavage. Bronchodilators: Anticholinergic Agents; Beta Agonists. Carbon Dioxide.
Chymase and Tryptase. Corticosteroids: Therapy.
Dendritic Cells. Dust Mite. Endothelial Cells and
Endothelium. Environmental Pollutants: Overview.
Epidermal Growth Factors. Genetics: Overview; Gene
Association Studies. Histamine. Immunoglobulins. Leukocytes: Eosinophils; Neutrophils; Monocytes; T cells;
Pulmonary Macrophages. Lipid Mediators: Overview.
Matrix Metalloproteinases. Neurophysiology: Neural
Control of Airway Smooth Muscle. Oxygen Therapy.
Pneumothorax. Respiratory Muscles, Chest Wall,
Diaphragm, and Other. Signs of Respiratory Disease: Breathing Patterns; General Examination; Lung
Sounds. Smooth Muscle Cells: Airway. Symptoms of
Respiratory Disease: Cough and Other Symptoms.
Tumor Necrosis Factor Alpha (TNF-a ). Upper Airway
Obstruction. Upper Respiratory Tract Infection.
Further Reading
Barnes P, Drazen J, Rennard S, and Thomson NC (eds.) (2002)
Asthma and COPD – Basic Mechanisms and Clinical Management. London: Academic Press.
Bel E (2004) Clinical phenotypes of asthma. Current Opinion in
Pulmonary Medicine 10(1): 44–50.
Bousquet JP, Jeffery Busse W, Johnson M, and Vignola A (2000)
Asthma – from bronchoconstriction to airways inflammation
and remodeling. American Journal of Respiratory and Critical
Care Medicine 161: 1720–1745.
British Thoracic Society (2003) British guideline on the management of asthma. Thorax 58: i1–i94.
Busse W (2001) Asthma. New England Journal of Medicine 5:
350–362.
GINA (2004) Self-reported wheezing in the previous 12 month
period in 13- to 14-year-old children. Global Burden of Asthma,
p. 6.
GINA (2004) Global Strategy for Asthma Management and Prevention, Chapter 7, Part 4A, Figure 5–7, p.7.
Kay AB (2001) Allergy and allergic diseases. New England Journal
of Medicine 344: 30–37.
Kips JC, Anderson GP, Fredberg JJ, et al. (2003) Murine models of
asthma. European Respiratory Journal 22(2): 374–382.
Larche M, Robinson R, and Kay AB (2003) The role of T lymphocytes in asthma. Journal of Allergy and Clinical Immunology
111: 450–459.
ATELECTASIS 215
McFadden E (2003) Acute severe asthma. American Journal of
Respiratory and Critical Care Medicine 168: 740–759.
National Asthma Education and Prevention Program Expert Panel
Report: guidelines for the diagnosis and management of asthma,
Update on Selected Topics – 2002. Journal of Allergy and clinical Immunology 110(5 pt 2): S141–S219.
NHLBI/WHO (2004) Global initiative for asthma: global strategy
for asthma management and prevention. NHLBI/WHO Workshop Report NIH 02-3659. Bethesda: NIH.
O’Byrne PM and Thomson NC (eds.) (2001) Manual of Asthma
Management, 2nd edn. London: W B Saunders.
Rodrigo G, Rodrigo M, and Jesse B (2004) Acute asthma in adults:
a review. Chest 125(3): 1081–1102.
Tattersfield A, Knox A, Britton J, and Hall I (2002) Asthma. Lancet 360: 1313–1322.
Thomson NC, Chaudhuri R, and Livingston E (2004) Asthma
and cigarette smoking. European Respiratory Journal 24:
822–833.
ATELECTASIS
P A Kritek, Brigham and Women’s Hospital at Harvard
Medical School, Boston, MA, USA
& 2006 Elsevier Ltd. All rights reserved.
Abstract
Atelectasis is the loss of volume resulting from decreased gas in
a given portion of lung. The mechanisms that cause atelectasis
can be divided into three categories: passive, adhesive, and resorptive. Passive atelectasis results from space-occupying lesions
in either the pleural space or the parenchyma compressing adjacent normal lung tissue. Adhesive atelectasis is caused by a
decrease in the level or activity of surfactant leading to an increase in surface tension in the alveolus and subsequent collapse. Resorptive atelectasis ensues when there is partial or
complete occlusion of flow of gas between alveoli and the trachea. Oxygen, carbon dioxide, and nitrogen will diffuse from
the alveolus into the capillary until all gas is removed from the
alveolar space. Small areas of atelectasis can be found in normal
lungs due to the effects of gravity. Pneumothoraces or large
bullae can result in passive atelectasis. Adhesive atelectasis is a
feature of respiratory distress syndrome of the neonate as well
as acute respiratory distress syndrome in adults. Resorptive atelectasis is found distal to an obstructive lesion, such as a tumor
or mucus plug. Atelectasis associated with anesthesia is complex
and caused by a combination of these mechanisms.
thereby creating a smaller space in which to maintain
the inflated lung (Figure 1). As a result, adjacent lung
tissue will lose gas and subsequently collapse. This
form of atelectasis is referred to as passive because the
collapsed lung is not inherently abnormal but is being
affected by an adjacent pathologic process. If the inciting cause of the atelectasis is resolved (e.g., a
pneumothorax is evacuated), the underlying atelectatic lung should reexpand and return to normal function. It should be noted, however, that after a segment
of lung has collapsed, there are local changes in permeability, inflammatory markers, alveolar macrophage function, and surfactant associated with all
types of atelectasis that may predispose to abnormal
function upon reinflation.
Adhesive Atelectasis
Atelectasis is the loss of volume resulting from decreased gas in a given portion of lung. These changes
can be small, affecting only subsegmental regions, or
more dramatic, leading to collapse of an entire lung.
The mechanisms that result in atelectasis can be divided into three categories: passive, adhesive, and resorptive. Each of these is discussed individually. Note
that although discussions of radiographic descriptions
of atelectasis are included, the discussion is grounded
in these pathophysiologic aspects of atelectasis.
Adhesive atelectasis results from the absence, loss, or
decreased activity of surfactant within the alveoli
(Figure 2). Surfactant, produced by type II alveolar
cells, decreases surface tension as alveolar surface
area decreases and balances the retraction forces of
the lung in order to avoid end-expiratory alveolar
collapse. When surfactant is decreased or inactivated, the balance is upset and atelectasis ensues. In
this situation, there is loss of gas volume based on
mechanical forces within the alveolus as opposed to
external compression.
This form of atelectasis can occur in the neonatal
infant in the setting of immature type II alveolar cells
and decreased surfactant production. Alternatively,
certain adult disease states, including ventilator-associated pneumonia and acute respiratory distress
syndrome (ARDS), are associated with decreases in
absolute surfactant levels or its activity.
Passive Atelectasis
Resorptive Atelectasis
Passive atelectasis results from a space-occupying lesion within the pleural space or the parenchyma
Resorptive atelectasis results when there is partial or
complete occlusion of flow of gas between alveoli
Description
216 ATELECTASIS
Pneumothorax
Pleural space
Normal alveolus
Passive atelectasis
Figure 1 Normal alveolus and passive atelectasis.
Edema and
decreased
surfactant
Normal alveolus
Adhesive atelectasis
Figure 2 Normal alveolus and adhesive atelectasis.
and the trachea (Figure 3). As the section of obstructed lung continues to be perfused, the partial
pressure of oxygen in the alveolus equilibrates with
that in the alveolar capillary. The loss of oxygen in
the alveolus leads to increased concentrations of nitrogen and carbon dioxide in the alveolus, and subsequent gradients result in movement of both gases
from the alveolus into the capillary. This process will
continue until all gas has been removed from the
airspace, a phenomenon that takes 18–24 h in normal volunteers with a completely occluded lobe.
Resorptive atelectasis can result from any cause of
obstruction to airflow. Common etiologies include
tumor, mucus plug, and foreign body. The process of
resorptive atelectasis is thought to occur more quickly
in the setting of oxygen-rich gas because the first step
of oxygen absorption is more rapid and there is a
larger portion of gas that is absorbed initially.
Atelectasis in Normal Lung Function
By definition, atelectasis is caused by loss of gas from
a normally gas-filled section of lung. However, there
is a form of passive atelectasis that is commonly seen
in ‘normal’ lungs that is referred to as dependent
atelectasis. This is the loss of volume in alveoli and
ATELECTASIS 217
Tumor
Normal alveolus
Resorptive atelectasis
Figure 3 Normal alveolus and resorptive atelectasis.
small airways in the lower lung zones based on gravity-related decreases in transpulmonary pressures.
Although these areas of atelectasis may have physiologic consequences in patients with underlying lung
disease (e.g., ARDS), the small loss of volume most
likely has no physiologic consequence in normal subjects. The increased use of computed tomography
(CT) has led to a greater awareness of these changes.
Dependent atelectasis will resolve with position
change, as demonstrated by repeated tomography
with the patient in the prone position.
Atelectasis in Respiratory Diseases
reserved for collapse of lung associated with pleural
thickening, most commonly found in association
with asbestos exposure. The etiology of rounded
atelectasis is not well understood but is thought to
result from pleural fibrosis contracting and causing
adjacent lung to curl upon itself and collapse. The
radiographic findings are best characterized on chest
tomography. The lesions are classically peripheral,
subpleural, uniform in density, and mass-like in appearance. They often have a curvilinear opacity of
bronchi and vessels (termed a comet tail) extending
toward the hilum. Care needs to be taken in diagnosing a lung nodule as rounded atelectasis purely on
CT findings because there are many reports of malignancy masquerading as rounded atelectasis.
Passive Atelectasis
Passive atelectasis can occur in a generalized or localized manner. In the setting of a large pneumothorax, the majority of the parenchyma of the ipsilateral
lung will undergo passive atelectasis. In contrast,
lung tissue adjacent to a bleb or a parenchymal cyst
can collapse in a more localized form of passive atelectasis. Both examples illustrate a space-occupying
phenomenon that causes subsequent loss of gas and
collapse. With relief of a large space-occupying lesion, such as a pneumothorax, the underlying lung
may develop reexpansion pulmonary edema. This
process is thought to result from changes in pulmonary capillary permeability but is not well understood. The pulmonary edema is usually transient and
resolves with supportive care.
There is a unique form of passive atelectasis
termed rounded atelectasis. This description is
Adhesive Atelectasis
The classic example of absorptive atelectasis is that
of respiratory distress syndrome (RDS) of the neonate. As discussed previously, type II alveolar cells
are often immature and not fully functional in the
preterm infant, predisposing the infant to RDS.
There have been significant decreases in morbidity
and mortality in RDS with the initiation of surfactant
(natural or synthetic) replacement in the immediate
neonatal period. This improvement is in part due to
the marked decrease in atelectasis and resulting improved gas exchange with surfactant therapy.
Many studies demonstrate decreased surfactant
levels in adults with ARDS. There is also experimental evidence for decreased surfactant activity in
the setting of leakage of plasma proteins into the alveolar space. At the same time, studies using CT have
218 ATELECTASIS
demonstrated areas of atelectasis in ARDS. The etiology of this atelectasis is likely multifactorial. There
are increased changes in dependent lung zones suggesting a component of passive atelectasis accompanying the adhesive atelectasis, the latter presumably
from decreased quantity or activity of surfactant.
Some believe that the recurrent opening and closing of small, atelectatic lung units contributes to
ventilator-induced lung injury (termed ‘atelectrauma’). In response to this, there has been increasing
research on how to avoid or overcome the atelectasis
associated with ARDS. Efforts to replace surfactant
through a variety of modalities have not demonstrated a mortality benefit, although some studies
have shown improved gas exchange. Lung ventilation strategies aimed at maintaining an ‘open lung’,
such as recruitment maneuvers, high-frequency jet
ventilation, and conventional ventilation with high
positive end expiratory pressure (PEEP), have shown
transient rises in oxygenation but no improvement
in mortality. There is no conclusive evidence supporting any specific treatment of atelectasis associated with ARDS.
Resorptive Atelectasis
Endobronchial tumors are a common cause of resorptive atelectasis, often resulting in segmental or
lobar collapse. One retrospective review reported an
incidence of atelectasis in one of five patients with
small cell lung cancer. Because these are gradual
processes occurring over weeks to months, the atelectatic lung often does not completely reexpand
or function normally if the obstruction is relieved.
However, because there is a risk for postobstructive
pneumonitis and there is evidence for increased bacterial growth in atelectatic lung, clinicians will often
attempt to minimize the obstruction.
In contrast to the more gradual development of
atelectasis with tumor growth, resorptive atelectasis
can occur rapidly with acute occlusions of large airways. Mucus plugs, in both patients with asthma and
those with altered secretion clearance, have been described as causes of resorptive atelectasis. Resorptive
atelectasis has also been reported with malpositioned
endotracheal tubes that selectively intubate the right
lung. In a short period of time, the entire left lung can
undergo atelectasis that will resolve with repositioning of the endotracheal tube.
by CT) and is often clinically significant. Lobar
atelectasis can result in parenchymal shunt and be
manifest as marked hypoxemia. There is evidence of
a positive correlation between the amount of atelectatic lung and the degree of hypoxemia.
The atelectasis associated with anesthesia is caused
by multiple mechanisms. The first contributor is
dependent atelectasis (a form of passive atelectasis)
from prolonged recumbent positioning with exaggerated effects of gravity. There is also evidence of
changes in diaphragm position and shape with supine
positioning for anesthesia contributing to dependent
atelectasis. These effects are more pronounced when
neuromuscular blockade is used in conjunction
with anesthesia. All these changes are especially pronounced in morbidly obese patients. Some anesthesiologists advocate for intermittent use of large
tidal volume breaths to overcome atelectasis intraoperatively, whereas others advocate for elevated
levels of PEEP with ventilation.
Administration of supplemental oxygen as part of
mechanical ventilation contributes to resorptive atelectasis. Results are mixed with regard to whether
higher inspired fractions (80–100%) during induction and maintenance of anesthesia increase the
likelihood of atelectasis compared to lower oxygen/
nitrogen mixtures. There is also evidence of early
airway closure during anesthesia contributing to the
resorptive mechanism of atelectasis. It has been suggested that repeated episodes of atelectasis, from the
previously mentioned means, can also lead to impaired surfactant function resulting in a component
of adhesive atelectasis as well.
The issues of atelectasis often persist into the postoperative period, resulting in persistent hypoxemia.
The resorptive atelectasis associated with higher inspired fractions of oxygen persists after extubation
despite attempts at recruitment at the end of the operation. Dependent atelectasis, which in the normal
subject will resolve with changes in position and deep
breathing, often persists due to decreased mobility,
pain, and sedation in the postoperative period. These
conditions also commonly lead to impaired clearance
of secretions predisposing to obstruction of airways
and resorptive atelectasis. Deep breathing, incentive
spirometry, vibration beds, chest physiotherapy, noninvasive ventilation, and therapeutic bronchoscopy
all seem to have similar results in terms of resolution
of postoperative atelectasis.
Atelectasis Associated with General Anesthesia
Atelectasis associated with general anesthesia deserves special mention because it occurs at rates as
high as 90% of anesthetized patients (as detected
See also: Alveolar Surface Mechanics. Breathing:
Breathing in the Newborn. Diffusion of Gases. Lung
Imaging. Mucus. Oxygen Therapy. Peripheral Gas
Exchange. Physiotherapy. Signs of Respiratory
AUTOANTIBODIES 219
Disease: Lung Sounds. Surfactant: Overview. Ventilation, Mechanical: Positive Pressure Ventilation.
Further Reading
Brower RG, et al. (2003) Effects of recruitment maneuvers in patients with acute lung injury and acute respiratory distress syndrome ventilated with high positive end-expiratory pressure.
Critical Care Medicine 31(11): 2592–2597.
Fraser RS and Parâe PD (1999) Fraser and Parâe’s Diagnosis of
Diseases of the Chest, 4th edn. Philadelphia: Saunders.
Gunther A, et al. (2001) Surfactant alteration and replacement in
acute respiratory distress syndrome. Respiration Research 2(6):
353–364.
Hallman M, Glumoff V, and Ramet M (2001) Surfactant in respiratory distress syndrome and lung injury. Comparative Biochemistry and Physiology Part A: Molecular & Integrative
Physiology 129(1): 287–294.
Hedenstierna G and Rothen HU (2000) Atelectasis formation
during anesthesia: causes and measures to prevent it. Journal of
Clinical Monitoring and Computing 16(5–6): 329–335.
Kreider ME and Lipson DA (2003) Bronchoscopy for atelectasis in
the ICU: a case report and review of the literature. Chest 124(1):
344–350.
Magnusson L and Spahn DR (2003) New concepts of atelectasis
during general anaesthesia. British Journal of Anaesthesia 91(1):
61–72.
Peroni DG and Boner AL (2000) Atelectasis: mechanisms, diagnosis and management. Paediatric Respiratory Reviews 1(3):
274–278.
Rouby JJ, et al. (2003) Acute respiratory distress syndrome: lessons from computed tomography of the whole lung. Critical
Care Medicine 31(4 supplement): S285–S295.
Spragg RG, et al. (2004) Effect of recombinant surfactant
protein C-based surfactant on the acute respiratory distress
syndrome. New England Journal of Medicine 351(9): 884–
892.
Vaaler AK, et al. (1997) Obstructive atelectasis in patients with
small cell lung cancer. Incidence and response to treatment.
Chest 111(1): 115–120.
Woodring JH and Reed JC (1996) Types and mechanisms of
pulmonary atelectasis. Journal of Thoracic Imaging 11(2):
92–108.
AUTOANTIBODIES
O C Ioachimescu, Cleveland Clinic Foundation,
Cleveland, OH, USA
& 2006 Elsevier Ltd. All rights reserved.
Abstract
Autoimmunity represents an immune response directed against
self-antigens, which may be (artificially) separated into T-cell
and B-cell responses. The importance of the B cells in autoimmunity is correlated to the production of several autoantibodies,
which have a role in diagnosis, pathogenesis, guiding therapy,
and predicting outcome of the patients with these conditions.
Among these autoantibodies, antineutrophil cytoplasm antibodies (ANCAs) have been distinguished as very important in
several conditions, such as Wegener’s granulomatosis, microscopic polyangiitis, Goodpasture’s syndrome, drug-related vasculitides, etc. We review in this article the importance of
different classes of autoantibodies and their relationship to underlying disorders.
Introduction: Autoimmunity and Lung
Disorders
The phenomenon of autoimmunity has been the object of exploration for over a century. Recent technical and methodological advances in the study of
cellular and biochemical processes of autoimmunity
have led to a publication ‘big bang’. Nevertheless,
the etiopathogenesis of most autoimmune disorders
remains, to date, largely unknown. In this article, we
discuss the basic mechanisms leading to autoimmunity, as we understand them today, and then the
relevance of several classes of autoantibodies in various lung conditions.
An autoimmune disorder is a pathologic condition
caused by an autoimmune response, which is critically
dependent upon antigen quality, concentration, and
persistence, and the magnitude of the interaction between self and foreign components. The autoimmune
response leads to end-organ organ-damage through
cellular or humoral mechanisms. The humoral response is represented by antibody-mediated injury,
which could be differentiated in direct, cytotoxic antibody damage or immune complex-related damage.
Despite their diverse etiology, there are several pathogenetic mechanisms which are common to all autoimmune conditions. With few exceptions, they require
the presence of self-reactive CD4 positive lymphocytes.
The immune system is naturally endowed with
myriad mechanisms responsible for recognition of
and defense against foreign assaults. Direct and rapid
responses can be mediated by a set of germline-encoded receptors, called Toll-like receptors (TLRs),
which recognize specifically the molecular determinants of various pathogens. Activation of the innate
immune defense mechanisms leads to a response
from different cell types (local dentritic cells, natural
killer lymphocytes, neutrophils, monocytes, macrophages, basophils, eosinophils, and mastocytes),
ranging from production of chemokines, cytokines,
adhesion molecules, antimicrobial, pro- and antiapoptoic factors. This response is reproducible (acts
220 AUTOANTIBODIES
by the same intensity at each antigenic exposure), is
not antigen specific, and creates a pool of memory
cells for long-term immunity. The adaptive immunity
requires contributions from cells whose receptors
are generated by VDJ recombination, the T and B
lymphocytes. Furthermore, inducible expression of
TLRs in B cells may provide a link between the innate and adaptive branches of the immune system.
Genetic susceptibility to autoimmune diseases may
occur at several levels: the major histocompatibility
complex (MHC) haplotype and polymorphisms
of genes involved in establishing self-tolerance and
immune regulation, for example, the autoimmune
regulator (AIRE), the T-cell immunoglobulin and
mucin-domain-containing (TIM) family, and cytolytic T-lymphocyte-associated antigen 4 (CTLA-4).
There are several major pathways that can initiate
or modulate autoimmunity:
1. molecular mimicry of self-proteins by viral agents
can activate specific T cells that attack both the
specific virus and the host;
2. bacterial infections can cause intense inflammation, with secondary polyclonal activation of ‘bystander’ T cells, which will act in concert in
intensifying the local injury;
3. epitope exposure and maintenance in the local
milieu as a result of self local damage may perpetuate the immune response; and
4. drug metabolism or infection can cause the formation of proteins which are seen as ‘foreign’ and
thus result in a neo-antigen-specific response to
the modified self.
There is also a range of possible posttranslational
modifications (PTMs) of proteins that can allow immune recognition of neo-self epitopes. The most direct way the posttranslational changes can influence
T-cell reactivity is to generate a new peptide–MHC
(pMHC) complex that can stimulate stronger binding to T lymphocytes through T-cell receptors.
In general, the immune recognition can be modified by posttranslational changes of the antigens in a
number of ways:
1. Different additions (glycosylation, methylation,
phosphorilation, etc.), as in collagen-induced
arthritis.
2. Enzymatic or spontaneous conversions (deamidation or citrullination) as in celiac disease or rheumatoid arthritis (RA). In the latter, multiple
autoantibodies have been described: antiperinuclear factor, antifilaggrin, and antikeratin antibodies. The target antigens are the result of PTM,
namely deimination of the natural aminoacid
arginine to the amino acid citrulline by the activity of peptidylarginine deiminase (PAD). This
discovery led to the development of a new serologic test for RA, that is, antibody against cyclic
citrullinated peptide (CCP), which has a high
specificity (497%) for the disease. Anti-CCP
binding also seems predictive of progression to
RA in recent-onset arthritis, or retrospectively in
blood samples positive for rheumatoid factor
(RF), donated years before onset of symptoms of
the disease. There is also evidence that citrullination may play a role in T-cell autoreactivity in RA
by enhancing the strength of the bonds to MHC II
molecules. Therefore, expression of PAD4 and
citrullinated proteins, presence of anticitrulline
antibody-secreting plasma cells in the inflamed
synovium, the strong MHC–peptide bonds, and
the predictive strength of anti-CCP testing, all
prove that this autoimmune cascade is involved in
the progression of RA.
3. Extracellular modifications by proteolysis or antibody binding.
The current understanding is that differential
PTMs might therefore provide the means of provoking a local autoaggressive immune response as a
consequence of an infection. This would be an alternative explanation of the autoimmunity by self-mimicking microbial antigens, for which definitive proof
remains elusive in humans as yet.
ANCAs and Associated Lung Disorders
In 1982, antineutrophil cytoplasm antibodies
(ANCAs) were first described in patients with
pauci-immune glomerulonephritis. By 1985, ANCA
had already been linked to Wegener’s granulomatosis
(WG); within a few more years, a link with microscopic polyangiitis (MP), and ‘renal-limited’ vasculitis has been made. As of today, ANCAs have become
very important factors in the diagnosis, pathogenesis, and classification of vasculitides. ANCAs can
be determined in two different ways: indirect
immunofluorescence assay (IIA) and enzyme-linked
immunosorbent assay (ELISA). While IIA is more
sensitive than ELISA, the latter is clearly more specific; hence, in clinical testing for ANCA it is recommended to screen with IIA, and to confirm all
positive results with ELISA directed against the specific antigens, if possible in a reference laboratory.
Furthermore, and unfortunately, there is a significant
subjective component to the interpretation of the
immunofluorescence tests, so confirmation by other
tests is needed before releasing the definitive ‘positive
test’. The main target antigens for ANCAs are
AUTOANTIBODIES 221
proteinase 3 or PR3 (PR3–ANCA) and myeloperoxidase or MPO (MPO–ANCA). PR3 and MPO are
located in the azurophilic granules of neutrophils,
and the peroxidase-positive lysosomes of monocytes,
respectively. Other azurophilic granule proteins can
cause p-ANCA autoantibodies: lactoferrin, elastase,
cathepsin G, bactericidal permeability inhibitor, catalase, lysozyme, azurocidin, beta-glucuronidase, etc.
specimens, because formalin-fixed neutrophils do
not cluster charged cellular components around the
nucleus.
* Mixed or atypical pattern, seen mostly in patients
with other immune-mediated conditions than systemic vasculitis (e.g., connective tissue disorders,
inflammatory bowel disease, and autoimmune
hepatitis). Such patterns may be confused with
p-ANCA patterns.
Immunofluorescence Assay
When the sera of patients with ANCA-associated
vasculitis (AAV) or other conditions are incubated
with ethanol-fixed human neutrophils, three major
IIA patterns are observed (Figure 1):
c-ANCA pattern (cytoplasmic), with diffuse staining of the cytoplasm. In most cases, the responsible antibodies are PR3–ANCA in the setting of
WG; rarely, MPO–ANCA can present in the
cytolasmic, uniform immunofluorescence.
* p-ANCA pattern (perinuclear), which results from
staining around the nucleus, which in fact is fixation artifact when ethanol is used. With ethanol
fixation, positively charged granule constituents
cluster around the negatively charged nuclear membrane, leading to perinuclear fluorescence. The
usual antibody responsible for this pattern is MPO–
ANCA (rarely PR3–ANCA), generally in the setting
of MP. A positive p-ANCA immunofluorescence
staining pattern may also be detected in a wide
variety of inflammatory illnesses; it has a low
specificity for vasculitis. The p-ANCA pattern on
IIA can be similar to that caused by antinuclear
antibodies (ANAs), the reason why individuals
with ANA frequently have ‘false-positive’ results on
ANCA testing by immunofluorescence. Rigorous
IIA testing procedures for ANCA (especially when
a diagnosis of vasculitis is entertained) entails the
use of both formalin- and ethanol-fixed neutrophil
*
Enzyme-Linked Immunosorbent Assay
Specific ELISA kits for antibodies directed against
PR3, MPO, and other azurophilic granule components are commercially available. PR3–ANCA and
MPO–ANCA should be part of any standardized
approach to the testing for ANCA; they are associated with substantially higher specificities and positive predictive values than their corresponding IIA
patterns (c- and p-ANCA, respectively).
Antineutrophil cytoplasm antibodies are mainly
associated with Wegener’s granulomatosis, microscopic polyangiitis, Churg–Strauss syndrome (CSS),
‘renal-limited’ vasculitis (pauci-immune glomerulonephritis without evidence of extrarenal disease), and
drug-induced vasculitides. In these conditions, there
is either PR3–ANCA or MPO–ANCA, but almost
never both. ANCA with different antigen specificities
may be detected in various other rheumatologic and
gastrointestinal disorders.
Wegener’s Granulomatosis
The histopathologic hallmark of WG is necrotizing
granulomatous inflammation that involves the respiratory tract, kidneys, skin, and/or the joints. Nearly
90% of patients with generalized, active WG are
ANCA-positive, while in limited disease (restricted to
upper or lower respiratory tract disease and no renal
involvement) only 40–60% of patients may be
ANCA-positive. Thus, the absence of ANCA does
Figure 1 Indirect immunofluorescence assay patterns on ethanol-fixed human neutrophils. (a) Diffuse, cytoplasmic cANCA;
(b) perinuclear pANCA; (c) antinuclear (ANA) illustrated as control.
222 AUTOANTIBODIES
not exclude the diagnosis; among WG patients with
ANCA positivity, 80–95% have PR3–ANCA by
ELISA, the rest nearly always have MPO–ANCA.
The diagnostic performance of PR3–ANCA for WG
(positive and negative predictive values) is related
mainly to disease prevalence in the population
searched, and the disease activity. Persistently, high
or rising titers of ANCAs are often associated with
disease relapses. However, this association may not
occur in 10–30% or more of those with such ANCA
profiles during one or more years of follow-up.
Microscopic Polyangiitis
Approximately 70% of patients with MP are ANCA
positive. Most ANCA-positive MP patients have
MPO–ANCA, with a minority having PR3–ANCA.
ANCA serologies are useful in distinguishing MP
from classic polyarteritis nodosa (PAN), a vasculitis
of medium-sized muscular arteries. In general, PAN
is associated with neither PR3–ANCA, nor MPO–
ANCA. Since PR3–ANCA or MPO–ANCA may occur in both WG and MP, it is important to know that
these diseases cannot be distinguished solely on
ANCA tests. However, distinction between MP and
WG is not clinically that important, since their treatment and prognosis are similar.
Churg–Strauss Syndrome
Both PR3–ANCA and MPO–ANCA have been detected in patients with CSS; overall, 50% of CSS
patients are ANCA positive, whereas in those with
active, untreated disease the percentage is even
higher. So far, no identification has been made of
any consistent clinical differences with therapeutic or
prognostic implications between ANCA-positive CSS
and ANCA-negative CSS.
Renal-Limited Vasculitis
Pauci-immune vasculitis limited to the kidney is
characterized by focal and segmental glomerular inflammation and necrosis with little or no deposition
of immunoreactants (IgG, IgA, IgM, and complement fractions). Almost all patients are ANCA positive, and up to 80% of them have MPO–ANCA.
Some consider this disorder as part of the WG/MP
spectrum because the renal histologic findings are
indistinguishable, and because some patients with
renal-limited vasculitis eventually develop extrarenal
manifestations of either WG or MP.
positive. One study, for example, found that 38 of
100 sera with anti-GBM antibodies also had ANCA;
of these, 25 had MPO–ANCA, 12 had PR3–ANCA
and 1 both types of ANCAs. Almost all such patients
have both ANCAs and anti-GBM antibodies at
the first serum examination. The clinical significance
of combined ANCA and anti-GBM serologies is unclear. In some, the titers of ANCAs are low and there
are no clinical manifestations of vasculitis. In others,
however, there are disease features of anti-GBM
antibody disease, but quite typical of systemic
vasculitis, including purpura, arthralgias, and granulomatous inflammation, suggesting the concurrence
of two disease processes. The incriminated self-antigen for the anti-GBM antibodies is the same as in
patients with anti-GBM antibody disease alone, suggesting that the inciting epitopes are the same. Alternatively, the production of ANCA could precede
that of anti-GBM antibodies, with pulmonary or renal damage caused by ANCA leading to secondary
anti-GBM antibody formation.
Other Rheumatologic Disorders
ANCAs have been reported in virtually all rheumatic
diseases, including RA, systemic lupus erythematosus
(SLE), Sjogren’s syndrome (SS), scleroderma, inflammatory myopathies, dermatomyositis, relapsing polychondritis, and the antiphospholipid syndrome. In
most cases, the IIA pattern is p-ANCA. Many reports
of ANCAs in these diseases preceded the era of reliable ELISA assays for PR3– and MPO–ANCA, and
used only IIA. Various target antigens have been described in these disorders, such as lactoferrin, elastase, lysozyme, cathepsin G, and others. In other
cases, the specific target antigens have not been identified yet.
Cystic Fibrosis
Non-MPO p-ANCAs are common in patients with
cystic fibrosis (CF), particularly among those with
bacterial airway infections. The ANCA is generally
directed against BPI (bactericidal/permeability-increasing) protein. In one series of 66 patients with
CF, BPI-IgG and BPI-IgA ANCAs were found in 91%
and 83%, respectively. Anti-BPI titers were directly
related to the severity of airway destruction. It is
unclear whether this relationship represents an epiphenomenon or a response to overwhelming infection, with major release of endotoxins.
Anti-GBM Antibody Disease (Goodpasture’s
Syndrome)
Others
Up to 40% of patients with antiglomerular basement
membrane (anti-GBM), antibody disease are ANCA
ANCA has also been observed in isolated patients
with autoimmune hepatitis, Bürger’s disease,
AUTOANTIBODIES 223
(pre)eclampsia, subacute bacterial endocarditis, leprosy, malaria, and chronic graft-versus-host disease.
ANCA positivity is seen in 60–80% of patients with
ulcerative colitis and in primary sclerosing cholangitis. It can be observed in only 10–27% of patients
with Crohn’s disease, in whom only low titers are
present. The p-ANCA is the predominant appearance, and is directed against a myeloid cell-specific
50 kDa nuclear envelope protein. Other reported
antigens include BPI, lactoferrin, cathepsin G, elastase, lysozyme, and PR3. The pathogenetic significance of these antibodies is unclear. The titers of
ANCAs do not vary with the activity or severity of
the disease and, in ulcerative colitis, do not fall even
after colectomy.
Drug-Induced ANCA-Associated Vasculitis
Several medications can induce various forms of
AAV. Most patients diagnosed with drug-induced
AAV have high titers of MPO–ANCA. In addition,
most have also antibodies to elastase or lactoferrin.
Relatively few have PR3–ANCA positivity. Many
cases of drug-induced AAV are associated with constitutional symptoms, arthralgias/arthritis, and cutaneous vasculitis. However, the full range of clinical
features associated with ANCAs, including crescentic
glomerulonephritis and alveolar hemorrhage, can
also occur. Discontinuation of the offending agent
may be the only intervention necessary for mild cases
of AAV induced by medications; such cases have in
general only constitutional symptoms, arthralgias/arthritis, and/or cutaneous vasculitis. Some patients,
however, require high doses of corticosteroids and
even cyclophosphamide because of more severe manifestations. The most renowned medications capable
of causing AAV are propylthiouracil (PTU), carbimazole, thiamazole, hydralazine, procainamide,
minocycline, penicillamine, allopurinol, phenytoin,
and clozapine. Drug-induced AAV is quite rare;
consequently, the above medications should not be
incriminated until other etiologies have been thoroughly excluded.
Propylthiouracil
PTU may be the most common offending agent causing drug-induced AAV. Generally, the medication is
taken for long periods of time before the complication occurs (months to years). Vasculitis is a rare
complication, whereas a much higher percentage develops serological evidence of ANCA. In a cross-sectional study, 27% of patients receiving long-term
treatment with PTU developed MPO–ANCA. The
postulated mechanism by which PTU leads to AAV
stems from the observation that PTU accumulates in
neutrophils and then it binds to MPO altering its
structure, which could lead to ANCA production in
susceptible individuals. Discontinuation of the offending drug may be the only intervention necessary
in mild cases. Some patients, however, require high
doses of corticosteroids and even cyclophosphamide,
while others require maintenance therapy. ANCA
titers usually persist at low levels, even after active
vasculitis goes into resolution, which points out that
previous ingestion of PTU is an important piece of
information in the history of patients with other
pathologies and prior PTU-induced, ‘by-stander’
ANCA.
Hydralazine
Hydralazine may cause drug-induced lupus and
drug-induced AAV. Unlike hydralazine-induced lupus
syndrome, hydralazine-induced AAV is frequently
associated with a pauci-immune glomerulonephritis, antibodies to double stranded DNA (dsDNA),
high titers of MPO–ANCA, and is a more ‘serious’
condition.
Minocycline
Minocycline had produced fever, livedo reticularis,
arthritis, and ANCA in a seven-patient case series.
The p-ANCA IIA pattern associated with this disorder is usually directed against ‘minor’ antigens
such as cathepsin G, elastase, and bactericidal/permeability increasing (BPI) protein, rather than
against ‘major’ antigens, such as MPO. Symptoms
typically resolve after minocycline discontinuation,
and recur with drug rechallenge. Some patients require treatment with corticosteroids for short periods
of time. More serious manifestations of minocyclineinduced AAV are crescentic glomerulonephritis,
lupus-like syndrome, and cutaneous ‘classic’ PAN.
Pathogenesis of ANCA-Associated Disorders
Substantial evidence in animal models and human
observations supports a significant pathogenetic
role of ANCA in producing widespread tissue damage. The hypothesis is that antibodies produce a necrotizing vasculitis by inciting a respiratory burst
with diffuse endothelial damage, chemotaxis, and
degranulation of neutrophils and monocytes. If autoantibody (ANCA) generation is secondary to a cryptic epitope exposure or a primary event, it is still
unclear. After a cryptic antigen exposure, the epitope
spread may occur, leading to a more generalized, systemic reaction (Figure 2). The number of activated B
lymphocytes seems to correlate with the activity score
of the disease (e.g., Birminham vasculitis score),
which supports the hypothesis that B lymphocytes
224 AUTOANTIBODIES
AAT
ANCA
Insult
PR3 or MPO
Macrophage
MMP-12
Neutrophil
TNF-
IL-8
Monocyte
Endothelial cell
Figure 2 A simplified model of ANCA pathogenesis and neutrophil/monocyte priming. Of note, alpha-1 antitrypsin (AAT) is a natural
tissue inhibitor of proteinase 3 (PR3), myeloperoxidase (MPO), or other lysosomal enzymes. In addition, binding ANCA to the cell
surface is mediated by specific FcgR receptors for immunoglobulins. IL-8, interleukin-8; MMP-12, matrix metalloproteinase 12; TNF-a,
tumor necrosis factor alpha. Copyright (2005) from Journal of COPD by Ioachimescu O and Stoller J. Reproduced by permission of
Taylor & Francis Group, LLC., http://www.taylorandfrancis.com.
are involved in the disease pathogenesis. Furthermore, while in normal conditions MPO and PR3
mRNA transcripts are found almost exclusively in
early promyelocytes, it was noted that in AAV
disorders (and not in SLE or other conditions), both
MPO and PR3 mRNA are found in high concentrations in the peripheral neutrophils, and this correlates
well with neutrophil total number and disease
activity, but not with ANCA titer, ‘left shift’, or cytokine levels, including tumor necrosis factor alpha
(TNF-a). Cell membrane PR3 and MPO expression
has been found in the affected glomeruli of the
ANCA-associated diseases (e.g., WG, MP), but not in
normal glomeruli, suggesting that local factors also
play an important role in gauging the organ involvement. In CSS, it is still unclear what the eosinophil’s
contribution to the disease pathogenesis is.
Autoantibodies in Connective
Tissue Disorders
Laboratory screening is commonly used for evaluation of connective tissue disorders (CTDs), although
there are rare tests which are sensitive or specific
enough to establish the diagnosis. For example,
Westergren sedimentation rate and C-reactive protein are commonly elevated in infections, malignant,
or inflammatory conditions; therefore, a high value
does not add too much towards the diagnosis, while
a low titer may make an active CTD seem unlikely.
Table 1 illustrates a synopsis of autoantibodies found
in different immune conditions.
Table 1 The main pathogenic autoantibodies in different lung
conditions
Antibody
Lung disease
ANCA
ANA
Anti-Sm,
Anti-dsDNA
Anti-U1-RNP
RF
CCP
Anticentromere
Anti-Scl
Anti-Jo1
Cryoglobulins
Anti-GM-CSF
WG, MP, CSS, drug-related AAV
SLE, SS, scleroderma, RA, MCTD, UCTD
SLE
MCTD
RA, SLE, MCTD, UCTD
RA
CREST syndrome
Scleroderma
DM/PM – ILD
Essential and secondary cryoglobulinemia
PAP
ANAs are found positive in most of CTD patients,
with different frequencies (from 30% in RA, to 95%
in SLE and scleroderma). On the other hand, other
lung conditions such as idiopathic pulmonary fibrosis
and coal worker’s pneumoconiosis may have positive
ANAs in titers above 1:40 in up to 33% of the cases,
as opposed to 2–3% of the general population. An
extractable nuclear antigen (ENA) panel is available
in most of the reference laboratories, which will
guide the testing against common antigens if ANA is
positive. Anti-dsDNA and anti-Smith (Sm) antibodies are relatively specific for SLE: 95–97%, and
50–99%, respectively. While anti-dsDNA antibodies
seem to correlate with the disease activity, anti-Sm
antibodies tend to persist after normalization of
anti-dsDNA titers. An interstitial lung disease (ILD)
AUTOANTIBODIES 225
identical to usual interstitial pneumonia (UIP) or
non-specific interstitial pneumonia (NSIP) can be
seen in SLE, RA, scleroderma, or dermatomyositis,
while bronchiolitis obliterans with organizing pneumonia (BOOP) can be frequently seen in RA. In SLE,
the most frequent pulmonary complications are pleurisy and pleuritis, lupus pneumonitis, shrinking lung
syndrome, bacterial pneumonia, diffuse alveolar
hemorrhage (DAH), thromboembolic disease, and
pulmonary arterial hypertension.
Traditionally, RF is found with higher frequency in
patients with RA and long-standing disease, with
multiple extra-articular manifestations. In other
lung conditions, such as hypersensitivity pneumonitis
(bird fancier’s lung) or idiopathic pulmonary fibrosis
(IPF), RF can be positive in up to half of the cases.
Since part of the workup for interstitial lung disorders is exclusion of associated rheumatic conditions,
reliance on more specific testing is generally required
to diagnose a systemic disease like RA. This can be
achieved by anticyclic citrullinated peptide (CCP)
antibodies, which have a much higher specificity, of
91–98%. In RA, the lung involvement can take the
form of ILD (UIP, NSIP, BOOP), DAH, pleuritis
and pleural effusion, rheumatoid nodules (follicular,
constrictive, or obliterative) bronchiolitis, cricoarythenoid arthritis with upper airway obstruction
or drug-induced pneumonitis (methotrexate, gold,
cyclophosphamide, rituximab etc.).
In the workup of disorders associated with myopathy or myositis, the determination of muscle
creatinphosphokinase (or aldolase) is important. Polymyositis (PM) and amyotrophic dermatomyositis
(ADM) can both present with lung disease in more
than 65% of cases, as one study found at the time of
diagnosis; it is important to know that, contrary to
rheumatoid lung disease, in PM or ADM, the pulmonary involvement may be inaugural. In DM/PM,
ANA is positive in up to 30% of the patients; one in
three patients will also have antibodies against an
ENA called histidyl tRNA synthetase, or Jo-1 antibodies. Other antiaminoacyl tRNA synthetases have
been identified to date: anti-PL7 or anti-treonyl
tRNA synthetase, anti-PL12 or anti-alanyl tRNA
synthetase, anti-OJ or anti-isoleucyl tRNA synthetase, anti-EJ or anti-glycyl tRNA synthetase, anti-KS
or anti-asparaginyl tRNA synthetase, and anti-Wa
directed against a 48 kDa protein yet uncharacterized, but known to be bound to acetylated tRNA.
It has been noted that patients with anti-synthetase
syndrome (arthritis, Raynaud’s syndrome, mechanic’s hands, and anti-Jo-1 antibodies) have a much
higher incidence of pulmonary disease. Pathologic
pictures of NSIP, UIP, or BOOP can be seen in these
settings.
The interstitial lung disease in scleroderma can
occur with both limited and diffuse disease. ANA
is generally positive in both forms of the disease,
anticentromere or anti-TO/TH antibodies found in
limited disease, while anti-Scl antibodies are more
often found in the diffuse variants of scleroderma.
The cellular and fibrotic form of NSIP can together
account for 70% of patients with scleroderma, suggesting a high rate of pulmonary involvement.
Sjogren’s syndrome is an autoimmune exocrinopathy and disorder of the epithelia characterized by
lymphocytic infiltration of the glandular and nonglandular subepithelial tissue. It presents with xerostomia
(dry mouth), xerophtalmia (dry eyes) and keratoconjunctivitis, xerotrachea (dry trachea, presenting as dry
cough and frequent infections), and arthritis. The pulmonary involvement in SS manifests as lymphocytic
bronchitis, lymphocytic follicular bronchiolitis, bronchial-associated lymphoid tissue lymphoma, lymphocytic interstitial pneumonia (LIP) or cystic lung
disease. The serologic markers of this condition
(either primary or secondary SS) are represented by
SS-A (anti-Ro) and SS-B (anti-La) antibodies.
Mixed connective tissue disorder (MCTD) is an
overlap syndrome with features of RA, SLE, scleroderma, and DM/PM, which do not meet the criteria
for the individual disorders, in the presence of antiribonucleoprotein (anti-RNP) antibodies on ENA
testing, directed against U1 RNP, which is rich in
uridine. Pulmonary involvement is common in patients with MCTD, and has feature of UIP or NSIP,
with significant septal thickening, less ground-glass
attenuation and honeycombing on CT scans.
‘Incomplete’ rheumatic conditions can occur and
are usually called undifferentiated connective tissue
disorder (UCTD) and may involve the respiratory
tract in a fashion similar to MCTD, although without any evidence of anti-RNP antibodies.
In cryoglobulinemia, a condition characterized by
cryoglobulins in the serum, there is a systemic inflammatory response with involvement of small and
medium-size vessels, and generated by cryoglobulincontaining immune responses. By definition, cryoprecipitation is a phenomenon of protein precipitation at temperatures lower than 371C, and it can be
present when serum proteins or plasma proteins
precipitate (cryofibrinogens and cryoglobulins respectively). Cryoglobulins are a mixture of immunoglobulins and complement components, which
generally precipitate upon refrigeration of the serum. Three types of cryoglobulinemic conditions
have been described (Brouet’s classification): type I,
characterized by isolated monoclonal immunoglobulins (Ig) and seen mostly in hematologic conditions;
type II with polyclonal Ig seen in hepatitis C, HIV
226 AUTOANTIBODIES
and other viral conditions; and type III, with mixed
cryoglobulins, encountered in CTDs. Pulmonary
involvement may be commonly seen in type III
cryoglobulinemia and, rarely, in type I, and is mostly
subclinical. In up to 50% of cases, there may be
cough, pleuritic chest pain, and/or dyspnea. Pulmonary function tests usually show evidence of reactive
small airway disease, and occasionally a decreased
diffusing lung capacity for CO. Pulmonary vasculitis,
DAH, or BOOP can also be found (though rarely).
Another rare condition is hypocomplementemic
urticarial vasculitis syndrome, which presents with
urticarial vasculitis, arthritis, glomerulonephritis,
and obstructive lung disease; it is thought to be
caused by serum IgG against C1q fraction of the
complement, which will decrease significantly (similar to SLE, but more commonly, with angioedema
and eye inflammation).
Autoantibodies in Other Conditions
Lung Cancer
The antibodies responsible for the paraneoplastic
syndromes are discussed in articles Tumors, Malignant: Overview; Bronchogenic Carcinoma.
Sarcoidosis
A possible relationship between sarcoidosis and autoimmunity was described more than a century ago,
although is still not accepted to be an autoimmune
condition. Good preliminary results of the CD20
targeting in several autoimmune conditions (sarcoidosis, SLE, RA, type II cryoglobulinemia, neuropathies, WG, Goodpasture’s syndrome, etc.) have
opened important avenues for research, conceivably
capable of improving our understanding of the
pathogenesis of these conditions and the effectiveness of pathogenesis-directed therapy.
Pulmonary Alveolar Proteinosis
Animal and human studies have confirmed a pivotal
role played by granulocyte-macrophage colony-stimulating factor (GM-CSF) in pulmonary alveolar proteinosis (PAP) pathogenesis. A decreased GM-CSF
pathway activity seems to be the common pathogenic
pathway. It was shown that a neutralizing (or blocking) anti-GM-CSF IgG antibody can be found in
bronchoalveolar lavage fluid and sera of patients
with idiopathic PAP. The sensitivity of the serum
anti-GM-CSF assay is close to 100% and the
specificity too is close to 100% when using a cutoff titer of 1:400. Furthermore, anti-GM-CSF antibodies are increasingly used as a diagnostic tool in
PAP. To what degree a high end-titer of anti-GM-CSF
represents an indication to treat more aggressively, or
with a larger dose of GM-CSF, remains to be proven.
Conclusions
During the past century, much has been accomplished in defining, understanding, and treatment of
autoimmune conditions, yet more is to be learned.
Autoantibodies play an important role in these immune processes, and of particular importance are
ANCAs, involved in the pathogenesis of the so-called
ANCA-associated vasculitides. The importance of
the autoantibodies stems not only from their contribution to the diagnosis of different conditions, but
also from their role in pathogenesis and the importance in monitoring the disease progression.
See also: Cryoglobulinemia. Cystic Fibrosis: Overview. Granulomatosis: Wegener’s Disease. Interstitial
Lung Disease: Overview. Systemic Disease: Diffuse
Alveolar Hemorrhage and Goodpasture’s Syndrome.
Toll-Like Receptors. Tumors, Malignant: Overview.
Vasculitis: Overview.
Further Reading
Bonfield TL, Russell D, Burgess S, et al. (2002) Autoantibodies
against granulocyte macrophage colony-stimulating factor are
diagnostic for pulmonary alveolar proteinosis. American Journal of Respiratory Cell and Molecular Biology 27: 481–486.
Csernok E (2003) Anti-neutrophil cytoplasmic antibodies and
pathogenesis of small vessel vasculitides. Autoimmunity Reviews 2: 158–164.
Imbert-Masseau A, Hamidou M, Agard C, Grolleau JY, and
Cherin P (2003) Antisynthetase syndrome. Joint, Bone, Spine
70: 161–168.
Ioachimescu O and Stoller J (2005) A review of alpha-1 antitrypsin
deficiency. Journal of COPD 2(2): 263–275.
Mahadeva R, Dunn AC, Westerbeek RC, et al. (1999) Anti-neutrophil cytoplasmic antibodies (ANCA) against bactericidal/
permeability-increasing protein (BPI) and cystic fibrosis lung
disease. Clinical and Experimental Immunology 117: 561–567.
Miescher PA, Zavota L, Ossandon A, and Lagana B (2003) Autoimmune disorders: a concept of treatment based on mechanisms
of disease. Springer Seminars in Immunopathology 25(supplement 1): S5–S60.
Paran D, Fireman E, and Elkayam O (2004) Pulmonary disease in
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B
BASAL CELLS
M J Evans, University of California, Davis, CA,
USA
& 2006 Elsevier Ltd. All rights reserved.
Despite differences in basal cell distribution, the unifying feature in all animal species is that the number
of basal cells present is related to the height of the
columnar epithelium. This relationship is associated
Abstract
Basal cells are an integral part of pulmonary airway epithelium.
They exist as a separate layer of cells covering most of the
basement membrane zone. In this central position, they can interact with columnar epithelium, neurons, the basement membrane zone, and underlying mesenchymal cells. In addition, they
interact with inflammatory cells, lymphocytes, and dendritic
cells. The interactions with trafficking leukocytes and neurons
take place in the lateral intercellular space between basal cells. In
this central position basal cells become a very important part
of the epithelial–mesenchymal trophic unit of larger airways.
Structurally, basal cells function in attachment of columnar
epithelium with the basement membrane zone. Failure of attachment between columnar and basal cells is thought to be
responsible for sloughing of the columnar epithelium in asthmatics. They also function in regulation of fibroblast growth
factor-2 signaling from the basement membrane zone, neurogenic inflammation, the inflammatory response, transepithelial water movement, and oxidant defense of the tissue and
formation of the lateral intercellular space for airway epithelium.
A subpopulation of basal cells (parabasal cells) has the potential
to function as progenitor cells. Clinically, basal cells are probably
also involved with the formation of squamous cell carcinoma
and, possibly, the progression to organized carcinoma.
LIS
LIS
*
LIS
* BC
BC
PCB
*
BC
BMZ
(a)
Introduction
Basal cells are derived from undifferentiated columnar epithelium in the developing airway. They are
characterized by their basal position in the columnar
epithelium, the presence of hemidesmosomes (characterized by alpha 6 beta 4 integrins), cytokeratins 5
and 14, and the nuclear protein p63 (Figure 1(a)).
The distribution of basal cells varies by airway level
and animal species. Airways that are larger in diameter have more basal cells than airways with smaller
diameters. For example, the largest numbers of basal
cells are found in the trachea. As the airway decreases in diameter, the number of basal cells also
decreases, and none are present in the terminal bronchioles. As basal cells increase in number, they displace columnar cells on the basement membrane
zone (BMZ). In human airways, 90–95% of the
BMZ is covered by basal cells from the trachea
to bronchioles 1–3 mm in diameter (Figure 1(b)).
(b)
Figure 1 (a) Electron micrograph of tracheal epithelium from a
young rhesus monkey demonstrating basal cells (BC), parabasal
cells (PCB), the lateral intercellular space (LIS), and the basement membrane zone (BMZ). The lateral intercellular space is the
open area between cells (asterisks). The lateral intercellular
space is reduced or absent between ciliated cells and ciliated
cells next to secretory cells (arrowheads). Magnification 1500.
Copyright 2001 from Cellular and molecular characteristics of
basal cells in airway epithelium. Experimental Lung Research 27:
401–415 by Evans MJ, Van Winkle LS, Fanucchi MV, et al. Reproduced by permission of Taylor & Francis, Inc. (b) Scanning
electron micrograph of a sheep tracheal whole mount treated with
EDTA. The columnar epithelium has been released leaving a
layer of basal cells attached to the BMZ. The basal cells cover
between 90% and 95% of the BMZ. Magnification 1100.
230 BASAL CELLS
with the role of basal cells in attachment of columnar
epithelium to the BMZ.
Functionally, large airway epithelium is stratified,
with a layer of basal cells attached to the BMZ and a
layer of columnar epithelium attached to the basal
cells. Thus, basal cells act as a separate layer of cells
in a central position, that can interact with columnar
epithelium, neurons, BMZ, and the underlying mesenchymal cells. In addition, they can interact with
inflammatory cells, lymphocytes, and dendritic cells
in the epithelium. These interactions take place in the
lateral intercellular space. The lateral intercellular
space is a distinct space between basal cells, basal
and adjacent secretory cells, and secretory cells
(Figure 1(a)). The lateral intercellular space is hydrated with the aid of the proteoglycan hyaluronan,
which is bound to CD44 adhesion molecules on the
surface of basal cells. In this central position, basal
cells become a very important and integral part of the
epithelial–mesenchymal trophic unit of large airways. The large number of receptors found on basal
cells that bind growth-regulating proteins and trafficking leukocytes (Table 1) supports this concept.
Basal Cell Functions in the Normal Lung
Junctional Adhesion
The structural role of basal cells in the airways is for
attachment of columnar epithelium to the BMZ. Epithelial cells are attached to the BMZ by hemidesmosomes and cell adhesion molecules. Cytokeratins 5
and 14 link anchoring junctions of basal cells with the
cytoskeletons of adjacent cells through desmosomes
and to the BMZ with hemidesmosomes. This arrangement of junctional adhesion provides mechanical
stability to a group of cells or tissue. In airway epithelium, basal cells are the only cells that form hemidesmosome junctions with the BMZ. Columnar cells
are attached to the BMZ via desmosome attachment
with basal cells. The significance of basal cells in
junctional adhesion can be demonstrated by treating the tissue with ethylenediaminetetraacetic acid
(EDTA). Desmosome junctions are dependent on
calcium. When the tissue is treated with EDTA, the
calcium is removed from the desmosome, and the
columnar epithelium is released leaving the basal cells
attached to the BMZ (Figure 1(b)).
The number of basal cells present at a particular
airway level and their morphology is related to their
role in junctional adhesion. When the columnar epithelium increases in height, there is an increase in the
size and shape of basal cells along with a corresponding increase in desmosome attachment with
the columnar epithelium and hemidesmosome attachment with the BMZ. These changes maintain a
Table 1 Cellular and molecular characteristics of basal cells
Cell surface characteristics
Alkaline phosphatase
Aquaporin 3 transmembrane water channels
b-adrenergic receptors
CD44 transmembrane glycoproteins
Epidermal growth factor receptor
Fibroblast growth factor receptor-1
Fas receptors and ligand
ICAM-1
IgE receptor
Integrins (a6b4)
Lectins
LEEP-CAM
MRP transmembrane transporters
MUC 1,4,8
Neurokinin-1 receptor
Neutral endopeptidase
Syndecan-4
4-1 BB receptor
Intracellular characteristics
Adrenomedullin receptor (mRNA)
Autotaxin (mRNA)
Annexin II
Bcl-2 protein
Extracellular SOD (mRNA)
Leukemic inhibitory factor
P63 nuclear protein
Reproduced from Evans MJ, Van Winkle LS, Fanucchi MV, et al.
(2001) Cellular and molecular characteristics of basal cells in
airway epithelium. Experimental Lung Research 27: 401–415.
constant amount of junctional adhesion between the
columnar epithelium and the BMZ. Thus, the relationship between basal cell junctional adhesions and
height of the epithelium is constant and not related to
airway level or animal species.
Fibroblast Growth Factor-2 Signaling
Fibroblast growth factor-2 is stored in the BMZ of
the airways where it binds with perlecan, a heparan
sulfate proteoglycan that is an intrinsic constituent
of the BMZ. Fibroblast growth factor-2 is released
from perlecan in response to various conditions and
becomes an important cytokine within the local microenvironment of the epithelial–mesenchymal trophic unit. In airway epithelium, basal cells are the only
cell type involved with fibroblast growth factor-2
signaling. Fibroblast growth factor-2 signals by forming a ternary complex with fibroblast growth factor
receptor-1 (FGFR-1) and syndecan-4. When the fibroblast growth factor-2 ternary complex is formed, it
initiates tyrosine kinase signaling associated with cell
proliferation, migration, and differentiation. Basal
cells express the cell surface receptors FGFR-1 and
syndecan-4 whereas columnar cells do not (Figure 2).
Presumably, fibroblast growth factor-2 is stored in
airway BMZ as an intact growth factor to aid in rapid
cellular responses to changes in local environmental
BASAL CELLS 231
Syndecan-4
FGFR-1
Basal cell
FGF-BP
FGF-2
Perlecan
Collagen I, III, & V
Perlecan
Basement membrane zone
Figure 2 Ternary signaling complex in airway epithelium. In
this illustration, BMZ-bound FGF-2 is released, and formation of
the FGF-2 ternary complex with basal cells occurs via diffusion or
binding with FGF-binding protein (FGF-BP). This is an example
of how basal cells may function in growth factor signaling, neurogenic inflammation, and the inflammatory response through
interactions with substances in the lateral intercellular space.
Reproduced from Evans MJ, Fanucchi MV, Baker GL, et al.
(2003) Atypical development of the tracheal basement membrane zone of infant rhesus monkeys exposed to ozone and
allergen. American Journal of Physiology: Lung, Cellular and
Molecular Physiology 285: L931–L939, with permission from The
American Physiological Society.
conditions such as sloughing of damaged columnar
epithelium or damage to the BMZ by leukocyte
trafficking.
Neurogenic Inflammation
Basal cells are involved with regulation of neurogenic
inflammation. In response to various inhaled foreign
materials, axons in the airway epithelium release
neuropeptides into the lateral intercellular space, initiating the process of neurogenic inflammation (increased vascular permeability, neutrophil adhesion,
vasodilatation, gland secretion, ion transport, smooth
muscle contraction, increased cholinergic transmission, and cough). Basal cells contain the protein leukemic inhibitory factor. Leukemic inhibitory factor is
thought to function in neurogenic inflammation by
stimulating the release of neuropeptides (tachykinins)
from axons and the formation of neurokinin receptors. Neutral endopeptidase is a cell surface enzyme
also associated with the process of neurogenic inflammation. The enzyme neutral endopeptidase is
expressed mainly on the surface of basal cells. The
enzyme neutral endopeptidase cleaves neuropeptides
in the lateral intercellular space. Cleavage of neuropeptides by neutral endopeptidase modulates the
neurogenic inflammatory responses in the airways.
Inflammation
Basal cells participate in the inflammatory response
by upregulating expression of receptors for migratory
inflammatory cells and lymphocytes. Human basal
cells upregulate intercellular adhesion molecule-1.
Human basal cells can also upregulate expression of
IgE receptors indicating that they may be involved
with allergic responses of the airway. An unusual cell
adhesion molecule, lymphocyte endothelial–epithelial
cell adhesion molecule, is expressed in the basal cell
layer of human bronchial epithelium. Lymphocyte
adhesion to epithelia and endothelia is mediated by
lymphocyte endothelial–epithelial cell adhesion molecule. Human basal cells also express the receptor
4-1BB. The receptor 4-1BB is a member of the tumor
necrosis factor receptor super-family and is associated
with T cell activation. Human basal cells express the
Fas receptor and its ligand FasL. Ligation of the Fas
receptor by migratory inflammatory cells can lead
to their apoptosis. Expression of these molecules by
basal cells may play an important role in regulation of
the inflammatory response. They interact with inflammatory cells when the latter are moving through
the lateral intercellular space of airway epithelium.
Transepithelial Water Movement
Basal cells have the aquaporin water channel AQP3,
which is not found in the columnar epithelial cells.
Instead, columnar epithelial cells have the aquaporin
water channel AQP4. Water transfer between cells
and the matrix occurs through these water channels.
AQP3 is found in a basolateral position in the airways and in other tissues, implying movement of
water between the extracellular matrix and epithelium. The presence of AQP3 water channels in the
membranes of basal cells of normal airway epithelium demonstrates a unique role for the basal cell in
fluid modulation of airway surface liquids and also in
the lateral intercellular space. The cellular distribution of AQPs 3 and 4 in airway epithelium implies
the presence of cell-specific pathways for transcellular water movement between the extracellular matrix
and epithelium. The cystic fibrosis transmembrane
conductance regulator protein is a regulator of AQP3
water channels in basal cells suggesting a role in this
disease. Downregulation of AQP 3 is thought to play
a role in the pathogenesis of bronchiectasis.
Oxidant Defense of the Tissue
Basal cells in normal human airway epithelium
express extracellular superoxide dismutase mRNA.
Extracellular superoxide dismutase is a secreted protein found in the extracellular matrix responsible for
metabolizing superoxide free radicals. The physiologic
functions are not fully defined but it is thought to be
critical for the protection of extracellular matrix elements against oxidative damage. The multidrug resistance-associated protein transmembrane transporter is
232 BASAL CELLS
found in bronchial epithelium and plays a major role
in cell detoxification and defense against oxidant stress
via efflux of glutathione conjugates into the lateral
intercellular space. The pattern of multidrug resistance-associated protein transmembrane transporter
expression differs markedly according to cell type. In
basal cells it is distributed over the entire circumference of the cell whereas in ciliated cells it is restricted
to the basolateral surface. Expression of extracellular
superoxide dismutase and multidrug resistance-associated protein transmembrane transporter by basal cells
in normal subjects indicates that basal cells participate
in defense of the tissue against oxidative stress.
Progenitor Cells
The basal cell has the capacity to be the progenitor of
columnar airway epithelium. This has been demonstrated in studies where denuded airways were repopulated with enriched populations of basal cells, in
biphasic organotypic cultures, and in vivo following
loss of the columnar epithelium. Under these conditions, basal cells dedifferentiate into a highly proliferative cell phenotype from which a mucociliary
epithelium redifferentiates.
In vivo there are two populations of proliferating
basal cells (basal and parabasal cells). Basal cells have
their nuclei next to the basement membrane. Parabasal
cells are taller, and their nuclei are above the layer of
basal cell nuclei. Basal cells make up 31% of the cell
population in large airways and the taller parabasal
cells make up 7%. However, parabasal cells have a
proliferative fraction 4 to 5 times greater than basal
cells. The higher proliferative fraction in parabasal
cells suggests they may be more active in reparative
proliferation following injury when compared with
basal cells. Parabasal cells probably are the intermediate cells seen with electron microscopy. Intermediate
cells are known to be the primary proliferating cells
following injury to the columnar epithelium.
During growth of the airway, the basal cell has a
high rate of proliferation. The purpose of such proliferation in the growing airway is for the formation
of new basal cells. The increase in basal cells per
millimeter is related to their role in attaching columnar epithelium to the BMZ. Increased proliferation of
parabasal cells during development is probably associated with formation of the columnar epithelium.
In the normal adult airway epithelium, the rate of
basal cell proliferation is low. Such proliferation may
be for replacement of dying basal cells. When basal
cells are lost due to injury or apoptosis, proliferation
of surviving basal cells occurs, and they are replaced.
Proliferation of parabasal cells is most likely associated
with normal turnover of the columnar epithelium.
Basal Cells in Respiratory Disease
Asthma
Defining the mechanism of columnar cell attachment
to the BMZ was critical to the understanding of
asthma and other disease conditions associated with
sloughing of epithelium. In conditions where columnar cells are sloughed from the epithelium of larger
airways, basal cells remain attached to the basal lamina. Desmosomal attachment between the columnar
epithelium and basal cells represents a plane of cleavage between the two cell populations. Failure of desmosomal attachment between columnar and basal
cells is thought to be responsible for sloughing of the
columnar epithelium in asthmatics. The mechanism of
desmosomal failure between airway cells is not
known. However, it may not be failure per se but
rather a specific protective function of basal cells.
Shortly after the columnar epithelium has been
sloughed, basal cells flatten out and form a protective
barrier. This is considered to be an important protective function of the basal cell along with the ability to
quickly release desmosomal attachments to damaged
columnar epithelial cells. It has also been speculated
that stimulation of the epithelial neural system causes
edema of the lateral intercellular space with a subsequent local sloughing of the epithelium. In a similar
manner, an influx of inflammatory cells could overwhelm the lateral intercellular space and cause local
sloughing of the epithelium. However, these scenarios
are speculations and the reasons for or mechanisms of
epithelial sloughing are not known at this time.
Bronchiogenic Cancer
The p63 nuclear protein is specific for basal cells. Its
functions are not clear, however, it has been used as a
marker for tumors of basal cell origin in several tissues. In the lung, p63 was shown to be expressed in
basal cells of normal tissue, in areas of squamous
metaplasia, and in squamous cell carcinomas of the
bronchi. In poorly organized carcinomas, p63 is expressed in most of the cells. However, it is only found
in the basalar external cells of organized carcinomas.
In adenocarcinomas, p63 is present occasionally but
is not present in small cell carcinomas. These findings
indicate that basal cells are probably involved with
the formation of squamous cell carcinoma and, possibly, the progression to organized carcinoma. Basal
cells have also been implicated in abnormal epithelium found in idiopathic pulmonary fibrosis when
p63 was used as a marker.
See also: Adhesion, Cell–Cell: Vascular; Epithelial. Adhesion, Cell–Matrix: Focal Contacts and Signaling; Integrins. Aquaporins. Cell Cycle and Cell-Cycle
BREATHING / Breathing in the Newborn 233
Checkpoints. Epidermal Growth Factors. Extracellular Matrix: Basement Membranes; Elastin and Microfibrils;
Collagens; Matricellular Proteins; Matrix Proteoglycans; Surface Proteoglycans; Degradation by Proteases. Fibroblast
Growth Factors. Stem Cells.
Further Reading
Boers JE, Ambergen AW, and Thunnissen FB (1998) Number and
proliferation of basal and parabasal cells in normal human airway epithelium. American Journal of Respiratory Critical Care
and Medicine 157(6 Pt 1): 2000–2006.
Evans MJ, Fanucchi MV, Baker GL, et al. (2003) Atypical development of the tracheal basement membrane zone of infant rhesus monkeys exposed to ozone and allergen. American Journal
of Physiology: Lung, Cellular and Molecular Physiology 285:
L931–L939.
Evans MJ and Moller PC (1991) Biology of airway basal cells.
Experimental Lung Research 17: 513–531.
Evans MJ and Shami SG (1989) Lung cell kinetics. In: Lenfant C
and Massaro M (eds.) Lung Cell Biology, vol. I. of the series
Lung Biology in Health and Disease, pp. 1–36. New York:
Marcel Dekker, Inc.
Evans MJ, Van Winkle LS, Fanucchi MV, and Plopper CG (1999)
The attenuated fibroblast sheath of the respiratory tract epithelial-mesenchymal trophic unit. American Journal of Respiratory
Cell and Molecular Biology 21: 655–657.
Evans MJ, Van Winkle LS, Fanucchi MV, et al. (2001) Cellular and
molecular characteristics of basal cells in airway epithelium.
Experimental Lung Research 27: 401–415.
Ford JR and Terzaghi-Howe M (1992) Basal cells are the progenitors of primary tracheal cultures. Experimental Cell Research
198: 69–77.
Inayama Y, Hook GE, Brody AR, et al. (1988) The differentiation potential of basal cells. Laboratory Investigation 58:
706–717.
Johnson NF and Hubbs AF (1990) Epithelial progenitor cells in rat
trachea. American Journal of Respiratory Cell and Molecular
Biology 3: 579–585.
Mercer RR, Russell ML, Roggli VL, and Crapo JD (1994) Cell
number and distribution in human and rat airways. American
Journal of Respiratory Cell and Molecular Biology 10:
613–624.
Persson CGA and Erjefalt JS (1997) Airway epithelial restitution
after shedding and denudation. In: Crystal RG, West JB, Weibel
ER, and Barnes PJ (eds.) The Lung, pp. 2611–2627. Philadelphia: Lippincott-Raven.
Basement Membranes see Extracellular Matrix: Basement Membranes.
Berylliosis see Occupational Diseases: Hard Metal Diseases – Berylliosis and Others.
Bradykinin see Kinins and Neuropeptides: Bradykinin.
BREATHING
Contents
Breathing in the Newborn
Fetal Breathing
Fetal Lung Liquid
First Breath
Breathing in the Newborn
J A Adams, Mount Sinai Medical Center, Miami Beach,
FL, USA
& 2006 Elsevier Ltd. All rights reserved.
Abstract
The complex physiology of breathing in the newborn has important developmental and maturational aspects. Early in fetal
life the respiratory system and controllers respond to intrauterine
stimuli (PaCO2 , PaO2 , and lung inflation). The complex
network of control of breathing can be summarized into three
basic components: (1) controllers, (2) effectors, and (3) sensors.
At birth a multitude of inputs are responsible for initiation
of rhythmic ventilation, and adaptation of the pulmonary circulation to extrauterine life. The normal physiology of breathing in
the newborn is affected by posture, sleep state, and gestational
age. Pathophysiological aspects of breathing in the newborn
can be divided into: (1) fetal pathologies that occur during
fetal life and ultimately result in abnormal lung development,
234 BREATHING / Breathing in the Newborn
(2) immediate postnatal events that occur primarily as a result
of poor or abnormal transition to extrauterine life, and (3) neonatal events occurring in the neonatal period that lead to
intrinsic lung disease or deranged control of breathing such as
apnea. This article emphasizes the general importance of understanding the influence of age, posture, sleep state, and maturation
on breathing in the newborn, and provides a general overview of
these. Paramount to the interpretation of normative and study
data is the understanding of the influence of these factors.
Description
The undertaking of a complex vital physiological
function such as breathing in the newborn is a marvelous feat. Initiation of breathing in the newborn
requires a multitude of appropriately synchronized
events and signaling pathways. In preparation for
extrauterine life and in order to assume the responsibility for extrauterine gas exchange, an anatomical,
maturational, and functional process must occur. The
alveoli are developed by the 25th week of gestation
and by the 35th week of gestation, adequate quantities
of surfactant (surface active material that keeps alveoli
open and maintains alveoli stability) are present. Pulmonary circulation parallels alveolar development,
and thus by the 25th week of gestation alveolar gas
exchange can take place, but requires assisted ventilation. During fetal life, respiration is present in
human fetuses from 10 weeks onward. These fetal
breathing movements are not as well synchronized as
those occurring in the newborn period. In fetal life,
breathing is discontinuous and becomes continuous
after birth. The fetus spends nearly 30% of its time
engaged in discoordinate form of breathing associated
with rapid irregular electrocortical activity, as seen in
active sleep state. In addition, since the entire airways
are filled with amniotic fluid, at term almost 600 ml of
amniotic fluid is ‘inhaled’ per day. Maintenance of
normal amniotic fluid volume, and respiratory activity, are crucial in the development of the airway, and
maturation of lung structure and function. The fetus is
also capable of modulating breathing movements in
response to: (1) PaCO2 (hypercarbia increases fetal
breathing), (2) PaO2 (hypoxia abolishes fetal breathing
in sleep, and (3) pulmonary reflexes (inflation reflex of
Herring–Bauer lung distension with saline infusion
decreases frequency of breathing).
The overall framework for the control of breathing
can be schematically viewed as: (1) controller (central
nervous system and brainstem); (2) effectors (respiratory muscles and airway); and (3) feedback
(chemoreceptor, mechanoreceptors, i.e., stretch receptors) (Figure 1). Functional maturity of the controller, effectors, and feedback mechanism allows for
extremely premature newborns to survive, albeit requiring ventilatory assistance. The maturational process continues during gestation until term at 38–40
weeks of gestation, but even after birth the continual
adaptation to air-breathing is an ongoing process.
The First Breaths
During vaginal birth but less so during Cesarean
section delivery, the thoracic cage is compressed to as
much as 160 cmH2O pressure; this produces an ejection of tracheal fluid via the airways. The recoil of
the chest wall causes a passive inspiration and establishes an air–liquid interface. The first active
breath is made slightly easier by the fact that some
fetal lung fluid is retained in the alveoli and smaller
airway, thus requiring less distending pressure than
a totally collapsed lung. In addition, in near-term
and term newborns, surfactant produced by type II
pneumocytes decreases alveolar surface tension,
which prevents the lungs from total collapse at the
lower transpulmonary pressures that occur in the
subsequent breaths (Figure 2).
The stimuli for the first active inspiration is debatable but is likely to be a multifactorial set of events
with which the newborn is confronted including
change in temperature, light, noise, gravity, hypercapnea, sudden change in PaO2 , etc. A complete discussion on the control of breathing during the first
breaths is beyond the scope of this article, but suffice
it to say that environmental nonrespiratory stimuli
will enhance or facilitate the general tone of the respiratory neurons.
In addition to the mechanical effects of the first
breath, the pulmonary circulation that parallels lung
development and is maintained at high pulmonary
vascular resistance during fetal life must also transition from a fetal to a newborn circulation. The latter
is characterized by a gradual lowering of the pulmonary vascular resistance and eventual closure and redirection of blood flow via the foramen ovale and
ductus arteriosus, and a change from a parallel circulation to one in series (Figure 3). The resultant
effect is ultimately to match ventilation to perfusion
for the most efficient oxygen extraction and delivery
and carbon dioxide removal.
Sleep state modulates control of breathing. The
sleep and waking cycles begin to develop during fetal
life. In the premature newborn at 24 weeks’ gestation,
rapid eye movement (REM) periods account for
nearly 80% of the total sleep time. At term, REM
sleep is 60–65% of sleep time, while in adulthood it
accounts for only 20–25%. There are major differences in the control of breathing between awake and
sleep, which are independent of age. During sleep the
brain is controlled more by stimulation related to gas
exchange than any other stimuli. When comparing
the breathing patterns and pulmonary mechanics
BREATHING / Breathing in the Newborn 235
Controllers
Effectors
Cerebrum
Cerebellum
Midbrain
Sensors
Brainstem
Chemoreceptors
Lung receptors
Upper airway
Spinal cord
Proprioreceptors
Vagal resp. motor efferents
Upper airway
receptors
Resp. muscles
Lung
Figure 1 Schematic representation of the control of breathing. Adapted from Givan DC (2003) Physiology of breathing and related
pathological processes in infants. Seminars in Pediatric Neurology 10: 271–280, with permission from Elsevier.
among newborns, measurements should always be
made in the same sleep state and in the same posture.
Noninvasive methods to quantify these breathing
patterns over various sleep states can serve as a basis
for comparison.
Breathing is affected by gestational age. Maturation of ventilatory response to chemical and mechanical stimuli is also influenced by gestational age.
The ventilatory response to hypoxia in newborns is
biphasic. It is characterized by an initial phase of
hyperventilation followed by hypoventilation below
baseline levels. The etiology of such biphasic response is unclear, and may be related to metabolic
demands or neurotransmitters (Figure 4). This response is clearly different to that which occurs in
older infancy and adults. The ventilatory response to
carbon dioxide in premature infants is also different
from that in term newborns. In premature infants the
ventilatory response to hypercapnea is quantitatively
and qualitatively different from that in older term
neonates and adults. Term infants and adults increase
ventilation through an increase in tidal volume
and frequency; premature infants do not increase
frequency in response to hypercarbia, and have a
prolonged expiration. This response appears to be
centrally mediated at the level of the brainstem and
probably involves the inhibitory neurotransmitter
gamma-aminobutyric acid (GABA). Furthermore,
the ventilatory response to hypercapnea is influenced
by sleep state in term newborns. There is a greater
response in minute ventilation during quiet state
compared to REM (Figure 5).
Breathing is affected by posture in newborns. The
characteristics of a very compliant chest wall in newborns have a significant mechanical effect on breathing as efficiency of ventilation is reduced due to
increased chest wall compliance. In full term infants, a
change from supine to prone posture increases minute
ventilation and respiratory drive, with a concomitant
decrease in thoracoabdominal asynchrony. In premature infants (o35 weeks’ gestation) supine posture
is associated with higher respiratory rate, lower
arterial oxygen saturation, lower ventilatory response
to hypercapnea, and increased thoracoabdominal
236 BREATHING / Breathing in the Newborn
Saline
200
Air
Deflation
Volume (ml)
150
100
50
Inflation
0
0
4
8
(a)
12
16
20
Pressure (cmH2O)
200
After surfactant
Volume (ml)
150
100
50
Before
surfactant
0
0
4
8
12
16
20
-50
(b)
Pressure (cmH2O)
Figure 2 (a) Pressure–volume curves after saline and air expansion of the lung. Lower pressures are required to expand a saline-filled
lung compared to air. The deflation curve in air is not superimposable on the inflation curve (hysteresis) due to mobilization and
orientation of surfactant during deflation decreasing alveolar surface tension as the alveolar surface contracts. Adapted from Nelson NM
(1999) The onset of respiration. In: Avery GB, Fletcher MA, and McDonald MG (eds.) Neonatology Pathophysiology & Management of
the Newborn, pp. 257–278. Philadelphia: Lippincott Williams & Wilkins, with permission from Lippincott Williams & Wilkins. (b) Pressure–
volume loops of a normal lung (red) and surfactant-deficient lung. Note that the slope of the loop is decreased in the surfactant-deficient
lung. A greater amount of pressure is required for a lower change in lung volume. (Compliance ¼ D volume/D pressure.)
synchrony. Thus, posture appears to play a role in
pulmonary mechanics, primarily in the efficiency of
ventilation. Whether these changes occur as a result of
conferring greater chest wall stability or diaphragmatic mechanical advantage in the prone posture in both
full term and preterm infants remains to be clearly
elucidated. In spite of this, conferring a mechanical
advantage in the prone posture for full term infants
does not justify its use in the care of the full term
newborns upon hospital discharge. Sudden infant
death rates have clearly decreased in the US as a result
of the ‘Back to Sleep Campaign’.
Pathophysiology
In order to understand the effects of various pathological states on breathing in the newborn it is useful
to describe three distinct time periods for occurrence
of pulmonary pathology: (1) fetal, (2) immediate
postnatal, and (3) neonatal.
Fetal
To a large extent, lung growth is dependent on amniotic fluid production and volume, and the mechanical
BREATHING / Breathing in the Newborn 237
Pulmonary vascular resistance
Pulmonary venous ret
Left atrial pressure
PaO2
(>30)
Ventilation
Closed
ductus
arteriosus
Closed
ductus
venosus
Closed
foramen
ovale
L–R shunt
ductus
Right atrial
pressure
Umbilical
venous ret
Peripheral vascular resistance
Figure 3 Schematic of the transition to neonatal circulation once ventilation is established. L–R, left to right; ret, return. Multiple factors
play a role in the conversion of this circulation, including prostaglandins, endothelin, and endothelial-derived nitric oxide. Adapted from
Nelson NM (1999) The onset of respiration. In: Avery GB, Fletcher MA, and McDonald MG (eds.) Neonatology Pathophysiology &
Management of the Newborn, pp. 257–278. Philadelphia: Lippincott Williams & Wilkins, with permission from Lippincott Williams &
Wilkins.
% Change in baseline
30
20
10
0
−10
−20
Control
60
180
300
−30
Time (s)
Figure 4 The biphasic ventilatory response to hypoxia. Note the increase in ventilation, followed by a marked decrease in ventilation
below baseline values. Adapted from Martin RJ, Di Fiore JM, Jana L, et al. (1998) Persistence of the biphase ventilatory response to
hypoxia in preterm infants. Journal of Pediatrics 132: 960–964, with permission from Elsevier.
shear it exerts on the primitive airways and alveolar
ducts. Thus, a spectrum of pulmonary hypoplasia due
to multiple etiologies is the major pathological problem in the fetal period.
Immediate Postnatal
Failure to transition from a fluid-filled to a gas-filled
pulmonary tree and to reabsorb the fetal lung
fluid results in a transient condition called transient
238 BREATHING / Breathing in the Newborn
Min vent (ml kg–1 min–1)
1000
pulmonary hypertension with increased pulmonary
vascular resistance and right to left shunting via the
patent ductus arteriosus and foramen ovale. This
condition has several underlying etiologies including
intrauterine hypoxia, pneumonia, and other pulmonary vascular pathologies.
Quiet
750
REM
500
Neonatal
250
45
50
55
60
65
% Increase in min
vent from BL
PET CO2
100.0%
Quiet
REM
80.0%
60.0%
40.0%
20.0%
0.0%
Figure 5 Ventilatory response to hypercapnea. Note the difference in slope of the line between active state (REM) and quiet
state in full term infants. End tidal CO2 ¼ PET CO2. Reproduced
from Cohen G, Xu C, and Henderson-Smart D (1991) Ventilatory
response of the sleeping newborn to CO2 during normoxic rebreathing. Journal of Applied Physiology 71(1): 168–174, with
permission from The American Physiological Society.
tachypnea of the newborn (TTN or respiratory distress syndrome (RDS) type II). This condition is
characterized by the early appearance of tachypnea
and a variable degree of respiratory distress in an
otherwise ‘healthy appearing’ newborn. The greater
occurrence of TTN in newborns delivered by Cesarean section make this condition rather common.
Onset of respiratory distress after the initial newborn
transitional period (about 4–6 h) usually signifies intraparenchymal lung pathology due to pneumonia
(viral or bacterial).
Medications administered during labor and delivery can also have an effect on breathing; these include narcotics or magnesium sulfate, both of which
can depress respiratory drive.
In the premature infant, failure to transition from
fetal to extrauterine life can be due to pulmonary
insufficiency secondary to RDS type I (surfactant deficiency). Surfactant administration has markedly
improved mortality and morbidity of this disease
process and has radically changed the outcomes for
premature newborns. In both premature and term
newborns, failure of the pulmonary circulation to
transition from intrauterine fetal circulation to that
of the newborn results in the well known persistent
pulmonary hypertension of the newborn (PPHN) or
persistent fetal circulation (PFC) characterized by
In premature infants, particularly those born at less
than 34 weeks’ gestation who have immature lungs
or an intrinsic pulmonary pathology, a frequent respiratory problem is apnea. Apnea is typically defined
as cessation of breathing for more than 15 s; however, the literature is replete with various definitions
for the duration of cessation of breathing. Apnea is
most commonly seen in premature infants, and the
younger the gestational age the more common its
occurrence (Figure 6). Apnea in the premature infant
can be caused by intracranial pathology, metabolic
derangements, and infection, among others. The diagnosis of apnea of prematurity is one of exclusion
and thus an exhaustive undertaking of diagnostic
tests should be done prior to categorizing apnea as
such (Figure 7). In addition to cessation of breathing
or ineffective ventilation, clinically relevant apneas
decrease arterial oxygen saturation and cause bradycardia. With the advent of computerized monitoring and noninvasive technologies to measure
breathing and arterial oxygen saturation, long-term
studies in newborns have become possible in the
home environment. It has been found that apneas
can occur at any gestational age and that their severity can only be detected if various physiological
parameters are simultaneously monitored.
Apnea has been typically classified into three types:
(1) central (no respiratory efforts and thus no airflow), (2) obstructive (respiratory efforts against a
partially or totally occluded airway), and (3) mixed
(combination of central and obstructive within the
same event). With the advent of more refined monitoring techniques, it is evident that purely obstructive
apneas in the newborn are rare, and that most apneas
in premature infants have a mixed/obstructive
characteristic. Cessation of airflow is present followed by obstructive breaths or obstructive breaths
are followed by cessation of respiratory effort. Perhaps the most clinically relevant and important aspect of apnea relates to the changes in arterial oxygen
saturation that it can cause and bradycardia (Figures
8(a)–8(c)). Using respiratory inductive plethysmography that measures both ribcage and abdominal
excursions and can be calibrated in the newborn to
provide a relative estimate of tidal volume, we have
been able to study apneas in premature newborns. In
BREATHING / Breathing in the Newborn 239
Relative incidence of apnea
Apnea of prematurity
Prematurity
Term 1 mo 2 mo Term 1 mo 2 mo 6 mo
Age
Figure 6 Relative incidence of apnea as a function of maturation. Note the dramatic decrease in apnea after term gestation.
Reproduced with permission from NeoReviews, Vol. 3, pages e66–e70, Copyright 2002.
Immaturity
Hypercapnic response
Inhibitory reflexes
Hypoxic depression
Apnea
Seizures
Infection
IVH
Hypoxemia or anemia
Drugs
Reflux
Head & body position
Figure 7 A schematic representation of the etiology of apnea in
premature newborns. Examples of cause of apneas are highlighted in yellow. IVH, intraventicular hemorrhage. Adapted with
permission from NeoReviews, Vol. 3, page(s) e66–e70 and NeoReviews, Vol. 3, page(s) e59–e65, Copyright 2002.
addition to apneas, newborns also experience hypopnea (decreased tidal volumes below 25% of the
unimpeded baseline value). Hypopnea is a commonly
recognized problem in sleep disorder breathing in
adults but not commonly appreciated in newborns
due to the lack of ability to measure changes in tidal
volume over prolonged periods. The end result of
both apneas and hypopneas is to cause decreased
functional residual capacity (FRC). Since newborns
typically have relatively low FRC and oxygen stores,
the concomitant decrease in arterial oxygen saturation during apneas or hypopneas is not surprising.
Additionally, we have found using noninvasive methods that during periods of apnea in the premature
newborn the cardiac output can decrease by as much
as 50%. Cerebral blood flow fluctuations have also
been shown to occur to a greater degree during periodic breathing, apnea, and REM sleep. Whether
cardiac output and episodic arterial oxygen desaturation and changes in cerebral blood flow will have
untoward long-term neurological effects needs to be
determined.
Conclusion
Disordered breathing in the premature and term infant can be ascribed to a multitude of causes, which
240 BREATHING / Breathing in the Newborn
30.5 mm s–1
NIMS respi. events
9.6 mm s–1
NIMS respi. events
400 %Vt
Vt
400 %Vt
RC
400 %Vt
AB
(a)
300 %Vt
Vt
300 %Vt
RC
300 %Vt
AB
180 deg
EP Ang
0 deg
5.00 mV
ECG
200 B/M
HR
0 B/M
0
5
10
15
20
s
(b)
Figure 8 Examples of respiratory inductive plethysmographic recordings: (a) normal tidal breathing in the newborn; (b) central apnea
of 14 s duration; and (c) mixed/obstructive apnea of 21 s duration (note a decrease in HR to 90 bpm, followed by a decrease in arterial
oxygen saturation to a nadir of 80%). Vt, the tidal volume from calibrated respiratory inductive plethysmography derived as a percent of
the calibration value. RC, the volume contribution of the ribcage to tidal volume. AB, the abdominal volume contribution to tidal volume.
EPANG, the phase angle between the RC and AB, obtained from a plot of RC vs. AB; values of 180o denote complete paradoxical
motion of the RC and AB in opposite directions. ECG, electrocardiogram. HR, heart rate derived from the electrocardiogram. OxiP, the
arterial pulse obtained from the pulse oximeter. SAT, percent arterial oxygen saturation.
BREATHING / Breathing in the Newborn 241
5.6 mm s–1
300 %Vt
NIMS respi. events
Vt
300 %Vt
RC
300 %Vt
AB
Apnea
2.00 mV
ECG
200 B/M
HR
50 B/M
255 cu
OxiP
0 cu
100 %
SaO2
50 %
0
5
10
15
20
25
30
35
s
(c)
Figure 8 (Continued )
have their basis in pulmonary developmental problems, intrinsic pulmonary disease, or deranged control of breathing. The influence of sleep state,
posture, and gestational age are paramount in the
interpretation of both normative and study data.
See also: Breathing: Fetal Breathing; Fetal Lung Liquid;
First Breath. Infant Respiratory Distress Syndrome.
Sudden Infant Death Syndrome.
Further Reading
Adams JA, Zabaleta IA, and Sackner MA (1994) Comparison of
supine and prone noninvasive measurements of breathing patterns in fullterm newborns. Pediatric Pulmonology 18: 8–12.
Adams JA, Zabaleta IA, and Sackner MA (1997) Hypoxemic
events in spontaneously breathing premature infants: etiologic
basis. Pediatric Research 42: 463–471.
Adams JA, Zabaleta IA, Stroh D, Johnson P, and Sackner MA
(1993) Tidal volume measurements in newborns using respiratory inductive plethysmography. American Review of Respiratory Diseases 148: 585–588.
Avery GB, Fletcher MA, and McDonald MG (1999) Neonatology
Pathophysiology & Management of the Newborn. Philadelphia:
Lippincott Williams & Wilkins.
Baird TM, Martin RJ, and Abu-Shaweesh JM (2002) Clinical
associations, treatment, and outcome of apnea of prematurity.
NeoReviews 3(4): e66–e70.
Campbell AJ, Bolton DP, Taylor BJ, and Sayers RM (1998) Responses to an increasing asphyxia in infants: effects of age and
sleep state. Respiration Physiology 112: 51–58.
Chernick V (1981) The fetus and the newborn. In: Hornbein T
(ed.) Regulation of Breathing, Part II, pp. 1141–1179. New
York: Dekker.
Cohen G, Xu C, and Henderson-Smart D (1991) Ventilatory response of the sleeping newborn to CO2 during normoxic
rebreathing. Journal of Applied Physiology 71(1): 168–174.
Curzi-Dascalova L (1992) Physiological correlates of sleep development in premature and full-term neonates. Neurophysiology
Clinincs 22: 151–166.
Fanaroff AA and Martin RJ (2002) Neonatal–Perinatal Medicine
Disease of the Fetus and Infant. St Louis: Mosby.
Givan DC (2003) Physiology of breathing and related pathological
processes in infants. Seminars in Pediatric Neurology 10: 271–280.
Guilleminault C and Robinson A (1996) Developmental aspects of
sleep and breathing. Current Opinion in Pulmonary Medicine 2:
492–499.
Martin RJ, Abu-Shaweesh JM, and Baird T (2002) Pathophysiologic mechanisms underlying apnea of prematurity. NeoReviews
3: e59–e65.
Martin RJ, Di Fiore JM, Jana L, et al. (1998) Persistence of the
biphasic ventilatory response to hypoxia in preterm infants.
Journal of Pediatrics 132: 960–964.
Mortola JP and Saiki C (1996) Ventilatory response to hypoxia in
rats: gender differences. Respiration Physiology 106: 21–34.
Nelson NM (1999) The onset of respiration. In: Avery GB, Fletcher
MA, and McDonald MG (eds.) Neonatology Pathophysiology
& Management of the Newborn, pp. 257–278. Philadelphia:
Lippincott Williams & Wilkins.
Polin RA and Fox WW (1992) Fetal and Neonatal Physiology.
Philadelphia: Saunders.
Rigatto H (1992) Maturation of breathing. Clinical Perinatology
19: 739–756.
Stocks J, Sly PD, Teppers RS, and Morgan WJ (1996) Infant Respiratory Function Testing. New York: Wiley-Liss.
242 BREATHING / Fetal Breathing
Fetal Breathing
R Harding and S B Hooper, Monash University,
Melbourne, VIC, Australia
C A Albuquerque, Santa Clara Valley Medical Center,
San Jose, CA, USA
& 2006 Elsevier Ltd. All rights reserved.
Abstract
Fetal breathing movements (FBMs) are breathing-like movements that occur episodically in healthy mammalian fetuses. As
with postnatal breathing, FBMs are centrally organized rhythmic contractions of the diaphragm, but may also involve other
skeletal muscles such as those of the chest wall and upper respiratory tract. Owing to the airways being filled with liquid,
FBMs cause only minor changes in lung volume but typically
lower intrathoracic pressure by up to 5 mmHg and alter the
shape of the fetal chest. By altering intrathoracic pressure they
affect blood flow within the fetus and fluid movement within the
fluid-filled fetal airways. FBMs are characteristically highly variable in frequency and amplitude, but become more organized
with increasing gestational age and relate to fetal behavioral
states. They are inhibited by fetal hypoxia and hence can be
used in the diagnosis of fetal compromise. FBMs are critical for
normal in utero lung growth and development as they maintain
the fetal lung in an expanded state by opposing lung recoil: in
the absence of FBMs the fetal lungs tend to ‘deflate’ leading to
lung hypoplasia. At birth, breathing becomes continuous, possibly by removal of inhibitory substances produced by the placenta or fetal brain and by increased carbon dioxide production.
Introduction
Fetal breathing movements (FBMs) are breathinglike movements that occur episodically in healthy
fetuses during much of gestation. FBMs have been
observed in many mammalian species, including
man, as well as in birds and reptiles. As with postnatal breathing, FBMs are centrally organized rhythmic contractions of the diaphragm, but may also
involve other skeletal muscles such as those of the
chest wall and upper respiratory tract. They play no
role in fetal gas exchange, as the fetal ‘airways’ are
filled with liquid; in sheep, they typically lower intrathoracic pressure by up to 5 mmHg.
Most of the available information on FBMs has
been obtained from two species, humans and sheep.
In humans, FBMs can be detected by ultrasonography from about 10 weeks of gestation. They are
observed as rhythmic descending movements of the
diaphragm and are usually coincident with inward
movement of the chest wall and outward movement
of the abdominal wall. In chronically catheterized
fetal sheep, FBMs can be detected as electrical activity of the diaphragm muscle, rhythmic reductions in
intratracheal or intrathoracic pressure, or fluid movement within the trachea.
Two other types of inspiratory efforts have been
recognized in the fetus; isolated deep inspiratory
efforts and asphyxial gasping. Isolated deep inspiratory efforts, often termed hiccups, are commonly
observed in healthy fetal humans, pigs, and sheep;
typically, these occur in low-frequency bouts. Asphyxial gasps are strong inspiratory efforts initiated
by fetal asphyxia and involve intense activation of
many respiratory muscles.
Central and Peripheral Control of FBMs
FBMs are an expression of rhythmic activation of
neurons in the fetal brainstem. These brainstem neurons generate rhythmic bursts of activity in phrenic
motoneurons and in vagal preganglionic fibers destined for dilator muscles of the upper respiratory
tract. With development, episodes of FBMs become
temporally associated with fetal behavioral states
involving body movements, rapid eye movement
(REM) sleep, or arousal. During the last third of
gestation, FBMs are usually infrequent or absent in
association with the fetal state resembling quiet, or
non-REM, sleep (low activity state). The periods of
FBM/REM sleep/activity and the intervening periods
of apnea/non-REM sleep/inactivity are of similar duration, both occupying B50% of the time.
Compared to postnatal life, respiratory drive in the
fetus is relatively low and is regulated mainly by fetal
CO2 levels, as the incidence and amplitude of FBMs
are increased by elevated fetal PaCO2 (partial pressure
of carbon dioxide in arterial blood) levels and decreased by low fetal PaCO2 levels. It is assumed that
this CO2 drive is mediated by acidification of the fluid
surrounding the central chemoreceptors, as altering the
pH of fetal blood or cerebrospinal fluid (CSF) can have
effects similar to those of alterations in PaCO2 levels.
FBMs are inhibited by moderate to severe hypoxia
(Figure 1), by what is thought to be a central inhibitory mechanism that also inhibits other forms of fetal
skeletal muscle activity and alters fetal behavioral
state favoring reduced activity. This inhibitory mechanism may override the stimulatory effects of mild
hypoxia. Although peripheral chemoreceptors are active in the fetus and do respond to hypoxia, they do
not apparently play a role in the hypoxic inhibition of
FBMs. Recent studies suggest that central adenosine
receptors play a major role in the central inhibition of
FBMs and alteration in fetal behavior by hypoxia.
Effects of FBMs on Airway Fluid
Movement and Lung Volume
As FBMs alter fetal intrathoracic pressure, it is to be
expected that they affect fluid movement within the
BREATHING / Fetal Breathing 243
50
Incidence of FBMs (min h–1)
40
30
20
∗ ∗
10
∗
∗
∗
∗ ∗ ∗
∗
∗
24 h normoxia
∗ ∗
24 h hypoxia
0
−4
0
4
8
12
16
20
24
28
Period of hypoxia (h)
Figure 1 Effects of 24 h of fetal hypoxia on the incidence of FBMs in a late gestational fetal sheep. Hypoxia causes a profound but
transient inhibition of FBMs, with normal values returning after 14–18 h of hypoxia. Asterisks show values that differ significantly from
control values. Reproduced from Hooper SB and Harding R (1990) Changes in lung liquid dynamics induced by prolonged fetal
hypoxemia. Journal of Applied Physiology 69: 127–135, used with permission from the American Physiological Society.
respiratory tree. Oscillatory fluid flows synchronous
with diaphragm movements have been detected in
the fetal trachea and at the nose in humans. Although
there is some controversy as to the volume of fluid
moved with each fetal ‘breath’, it is clear that ‘tidal
volume’ in the fetus is much smaller than after birth,
which can be attributed to the much greater viscosity
of liquid relative to air. In fetal sheep, for example,
‘tidal volumes’ measured by flow meters in the trachea are much less than 1 ml, whereas after birth, in
the same species, tidal volumes are typically 30–
50 ml. Thus, FBMs are essentially isovolumic, causing only very small changes in thoracic volume with
each breath. However, in human fetuses, ‘tidal volumes’ of about 2 ml have been estimated by ultrasound, in the trachea and at the nose. The reason for
the larger ‘tidal volume’ in humans is unknown but
may be due to the slower breathing rates of human
fetuses compared to fetal sheep.
Although FBMs individually may have little effect
on volume flow within the airways, it is clear that
episodes of FBM can result in the movement of much
greater volumes, relative to total lung fluid volume.
This effect on fluid movement can be largely attributed to FBM-related changes in transpulmonary
pressure and upper airway resistance. Studies in
sheep have shown that net fluid movement to and
from the fetal lungs is affected by FBM episodes such
that most of the efflux occurs during these episodes;
during episodes of FBM the laryngeal dilator muscles
are rhythmically active (in phase with the diaphragm)
and laryngeal adductor muscles are largely quiescent.
Hence, the resistance to tracheal fluid movement offered by the upper airway is lowered during FBMs,
allowing fluid that has accumulated within the airways to flow into the pharynx, from where it is either
swallowed or flows into the amniotic sac. During
periods of fetal apnea, the laryngeal dilator muscles
become quiescent and adductor tone is usually
present, resulting in raised resistance of the upper
airway. Together with a lack of inspiratory muscle
activity, this results in low rates of fluid efflux from
the trachea, although increased efflux may occur
with fetal postural adjustments or other activities,
including those resembling straining movements or
Valsalva maneuvers that are common in the fetus.
The fetal upper airway plays a critical role in regulating the flow of fluid to and from the lungs, thereby
preserving lung volume and the composition of liquid
within the airways. Removal of the influence of the
upper airway by creating a tracheo-amniotic bypass
results in a major loss of lung liquid during periods of
apnea and a large ingress of amniotic fluid during
FBM episodes. This loss of upper airway function
not only allows near total lung deflation during
periods of fetal apnea, it also allows large influxes
of amniotic fluid during FBM episodes. This brings
the alveolar epithelium into contact with undiluted
amniotic fluid with potentially harmful effects, especially if the amniotic fluid contains meconium.
Although the net flow of liquid within the fetal
trachea is away from the lungs, primarily due to
continuous liquid secretion, periods of influx may
occur, especially in association with periods of vigorous FBMs. This may explain the entry of substances into the lungs following their deposition in
the amniotic fluid.
Effects of FBMs on Fetal Blood Flows
As FBMs alter intrathoracic pressure, it is to be expected that they will affect blood flows within the
fetus. FBM-related changes in blood flows have been
observed in the umbilical vein, fetal vena cava,
foramen ovale and, more recently, in the pulmonary
artery. In the pulmonary artery, episodes of vigorous FBMs are associated with increased blood flow,
likely to be a consequence of reduced resistance in
the pulmonary capillaries as a result of an altered
transmural pressure.
Functional Importance of FBMs
A major function of FBMs is to maintain lung liquid
volume, and hence lung expansion, which is known
to be essential for normal growth and structural
maturation of the fetal lung. This role of FBMs has
been demonstrated in experimental models that have
eliminated FBMs while preserving the integrity of the
diaphragm. The long-term abolition or suppression
of normal FBM results in lung hypoplasia and structural immaturity of the lungs, which is probably due
to a chronic reduction in lung expansion rather than
abolition of the small phasic movements of the chest
wall. A chronic reduction in fetal lung expansion
leads to a reduction in lung tissue growth and alterations in the structure of the alveolar wall and epithelium; similar changes in lung development are
caused by the abolition of FBMs. Abolishing the diaphragmatic movements that cause FBM is thought to
reduce the force that normally opposes the inherent
elastic recoil of the lungs, thereby allowing the fetal
lungs to ‘deflate’; a further reduction occurs if the
resistance offered by the upper airway is removed
(Figure 2).
Use of FBMs in Clinical Assessment
In the healthy human fetus, FBMs occur at an average frequency of 60 per min and are accompanied
by increased body movements and heart rate variability. FBMs are first detectable at about 10–12
weeks of gestation when they are usually irregular
and sporadic. From about 28 weeks of gestation onwards, FBMs become more regular and organized
Lung volume (% of intact fetus)
244 BREATHING / Fetal Breathing
100
80
60
40
20
0
Intact
fetus
No
No FBMs Collapsed FRC
FBMs
&
lung
newborn
no UA
Figure 2 Lung luminal volumes in fetal and neonatal sheep,
expressed in relation to values in the intact, late gestation ovine
fetus in utero. Lung liquid volume, and hence lung expansion, is
reduced by the prolonged absence of FBMs (no FBMs) induced
by phrenic nerve blockade or a high section of cervical spinal
cord. A further reduction occurs if the fetal upper airway is bypassed, allowing direct continuity between the fetal lungs and the
amniotic sac (no FBMs & no UA). Lung luminal volume is reduced
further when the lungs are removed from the fetus (collapsed
lung) due to unopposed recoil of the fluid-filled lung. Functional
residual capacity (FRC) in the air-breathing newborn is shown for
comparison. Data from Harding R and Hooper SB (1996)
Regulation of lung expansion and lung growth before birth.
Journal of Applied Physiology 81: 209–224.
into discrete episodes. Using color Doppler and spectral ultrasonography analyses, the breath-to-breath
interval and the inspiratory phase of the respiratory
cycle have been shown to increase from 22 to 35
weeks gestation, but then decrease towards term.
FBMs are used as one component of the ‘fetal biophysical profile’ which is widely used to assess fetal
health; the occurrence of FBMs is observed, together
with assessments of fetal heart rate, amniotic fluid
volume, fetal body movements, and fetal body dimensions. If FBMs are not detected, the fetus may be
hypoxic, may have a neural disorder affecting the
brainstem and phrenic motor nerves, or it may have
an abnormality of skeletal muscle function. However, it must be recognized that the inhibition of
FBMs by chronic fetal hypoxia may be only transient, as has been shown in sheep (Figure 1). FBMs
have also proven to be useful for the diagnosis of
intrauterine infection in patients with preterm
rupture of membranes as they have a high negative
predictive value for intra-amniotic infection; a
depression in FBM incidence is non-specific but is
suggestive of infection. Studies of patients with premature rupture of membranes have shown a decrease
in FBM incidence in those patients positive for amniotic fluid infection, clinical chorioamnionitis, or
neonatal sepsis. This may be related to inflammatory
cytokines affecting fetal behavioral states.
BREATHING / Fetal Breathing 245
Fetal Breathing, Amniotic Fluid Volume,
and Lung Growth
There is conflicting evidence on the role of FBMs in
the development of pulmonary hypoplasia in human
fetuses with preterm rupture of membranes and reduced amniotic fluid volume (oligohydramnios). Studies examining the role of FBM in the lung hypoplasia
associated with premature rupture of membranes
have shown either no change or a reduction in the
incidence of FBM. However it is likely that both the
oligohydramnios following membrane rupture and a
reduced incidence of FBMs may contribute to the associated fetal lung hypoplasia. In humans, it has been
shown that a prolonged reduction in FBMs at critical
periods of development (i.e., o24 weeks of gestation)
can lead to pulmonary hypoplasia; similarly, pulmonary hypoplasia can develop in human fetuses after
prolonged (more than 6 days) membrane rupture. It is
likely that oligohydramnios leads to lung hypoplasia
due to increased flexion of the fetal trunk, which
compresses the fetal lungs causing their deflation and
reduced growth rates.
Effects of Labor on FBMs
At term, the incidence of FBMs decreases. In sheep,
the incidence of FBMs decreases 2–3 days before the
onset of labor and remains reduced until delivery.
This may be related to increased circulating concentrations of prostaglandin E2, which is known to
inhibit FBMs. Similarly, in humans, fetal apnea of
20–60 min duration is considered to be a reliable indicator of premature labor with delivery within
48–72 h. During spontaneous or induced labor, the
incidence of FBMs is decreased to less than 10% of
the time during the latent phase and is further decreased during the active phase of labor.
In human pregnancy, the rupture of fetal membranes at term does not appear to affect FBMs.
However, a study before term showed a significant
reduction in FBMs for the first 2 weeks of membrane
rupture, compared to controls, with a return to near
normal by the third week. With increased uterine
contractility there is a decrease in the incidence of
FBMs.
Maternal Smoking and Fetal Breathing
As with most drugs taken by the mother, tobacco
smoking has been shown to affect fetal behavior.
Nicotine readily crosses the placenta, and is thought
to reduce utero-placental and/or umbilico-placental
blood flow, contributing to fetal hypoxemia; furthermore, the formation of carboxyhemoglobin as a result
of maternal smoking reduces the oxygen carrying
capacity of fetal blood. FBMs are inhibited by maternal tobacco smoking, and the inhibition may continue
for up to an hour following a single cigarette. It is
thought that this effect is due mostly to fetal hypoxemia, or more specifically to reduced cerebral oxygen delivery. Maternal smoking has recently been
shown to increase the resistance in the fetal cerebral
artery, and this may also contribute to reduced cerebral oxygen delivery. The inhibition of FBMs by maternal smoking may contribute to the known adverse
effects of smoking on fetal lung development. The
same may apply to other drugs taken by the mother,
such as narcotics, sedatives, or analgesics.
Respiratory Transition at Birth
The physiological mechanisms underlying the transition from discontinuous fetal breathing to continuous postnatal breathing are complex and not fully
understood. During parturition and when the umbilical cord is cut at birth, the neonate may become
profoundly hypoxemic, hypercapnic, and acidemic.
It is also exposed to lower environmental temperatures, leading to increased heat loss, and to a greatly
increased degree of external sensory stimuli, thereby
changing the behavioral state to one of arousal. It is
also possible that the removal of circulating inhibitory or suppressive substances that originate in the
placenta (e.g., prostaglandin E2, adenosine, neuroactive progesterone metabolites) may contribute to
the onset of continuous breathing. For example, their
removal may lead to an increase in metabolic activity
(e.g., by stimulating thermogenesis), and hence an
increase in the rate of CO2 production, or to an increase in the sensitivity of central chemoreceptors to
CO2. It has also been suggested that endocrine
changes at birth, such as a large increase in circulating catecholamines, lead to respiratory stimulation,
possibly via an increase in fetal metabolism and
hence CO2 production. Studies of fetal sheep maintained ex utero by extracorporeal oxygenation also
support the notion that CO2 plays a crucial role in
the maintenance of continuous breathing after birth.
The integrity of the vagus nerves has been shown
to be essential for the onset of adequate breathing at
birth. Although the critical pathways have not yet
been identified, it is likely that volume receptive
feedback from the lungs is involved. Studies in neonatal lambs have shown that a reduction in end-expiratory lung volume (functional residual capacity,
FRC), which reduces vagal sensory traffic from pulmonary stretch receptors, results in profound hypoventilation, periodic breathing, and active glottic
adduction during periods of apnea. This indicates
246 BREATHING / Fetal Lung Liquid
that receptive vagal feedback of lung volume at endexpiration, which is normally maintained by an adequate FRC, is essential for continuous breathing in
the newborn, and explains, at least in part, the benefits of positive end-expiratory pressure (PEEP) in the
treatment of infantile apnea.
See also: Breathing: Fetal Lung Liquid. Lung Development: Overview.
stretch stimulus that is essential for normal fetal lung growth. If
the distending influence of fetal lung liquid is absent, the lungs fail
to grow, which is the primary mechanism for fetal lung hypoplasia
in humans. At birth, the airways are cleared of liquid to allow the
entry of air and the onset of air breathing, due to a reversal of
the transepithelial ionic gradient that promotes reabsorption into
the lung parenchyma; this is stimulated by the release of stressrelated hormones during labor. Increases in transpulmonary pressure, due to changes in fetal posture, are likely to also contribute
to the clearance of fetal lung liquid at birth.
Further Reading
Description
Bissonnette JM (2000) Mechanisms regulating hypoxic respiratory
depression during fetal and postnatal life. American Journal of
Physiology. Regulatory, Integrative and Comparative Physiology 278: R1391–R1400.
Bocking AD (2003) Assessment of fetal heart rate and fetal movements in detecting oxygen deprivation in-utero. European Journal of Obstetrics and Gynecology 110: S108–S112.
Champagnat J and Fortin G (1997) Primordial respiratory-like
rhythm generation in the vertebrate embryo. Trends in Neuroscience 20: 119–124.
Cosmi EV, Anceschi MM, Cosmi E, et al. (2003) Ultrasonographic
patterns of fetal breathing movements in normal pregnancy. International Journal of Gynaecology and Obstetrics 80: 285–290.
Harding R (1997) Fetal breathing movements. In: Crystal RG,
West JB, Weibel ER, et al. (eds.) The Lung, Scientific Foundations, pp. 2093–2103. Philadelphia: Lippincott-Raven.
Harding R and Bocking AD (2001) Fetal Growth and Development. Cambridge: Cambridge University Press.
Harding R and Hooper SB (1996) Regulation of lung expansion
and lung growth before birth. Journal of Applied Physiology 81:
209–224.
Hooper SB and Harding R (1990) Changes in lung liquid dynamics
induced by prolonged fetal hypoxemia. Journal of Applied
Physiology 69: 127–135.
Laudy JA and Wladimiroff JW (2000) The fetal lung. 1: Developmental aspects. Ultrasound in Obstetrics and Gynecology 16:
284–290.
Rigatto H (1996) Regulation of fetal breathing. Reproduction,
Fertility and Development 8: 23–33.
Throughout embryonic and fetal development, the
future airways of the lung are filled with liquid and
the lungs take no part in gas exchange. This liquid
(fetal lung liquid) is a unique secretory product of
the lung and is quite unlike fetal plasma or amniotic
fluid in composition; it has a low pH, a high Cl concentration, and low protein content. Fetal lung
liquid is secreted across the pulmonary epithelium
and leaves the lungs by flowing out of the trachea.
Fetal Lung Liquid
S B Hooper and R Harding, Monash University,
Melbourne, VIC, Australia
& 2006 Elsevier Ltd. All rights reserved.
Abstract
Fetal lung liquid is secreted across the pulmonary epithelium and
enters the future airspaces due to an osmotic gradient created by
the transepithelial flux of ions into the lung lumen. This liquid
leaves the lungs by flowing out of the trachea, whereby it is either
swallowed or contributed to amniotic fluid volume. The high resistance to liquid movement through the fetal upper airway promotes the retention of lung liquid within the future airways,
which provides a small (1 or 2 mmHg) internal distending pressure on the lungs. This acts as an internal hydrostatic splint
that maintains the fetal lungs in a distended state and provides a
Control of Fetal Lung Liquid Secretion
Fetal lung liquid is formed by the net movement of
Cl and Na þ across the epithelium into the lumen,
which provides an osmotic gradient for the movement of water in the same direction. This mechanism
is thought to be driven by Na þ /K þ ATPase, which
creates an electrochemical gradient for Na þ to enter
epithelial cells via the Na þ /K þ /2Cl cotransporter.
The resultant Na þ -linked Cl entry across the basolateral membrane increases intracellular Cl concentrations that passively exit the cell, down its
electrochemical gradient, via selective channels located in the apical surface. The net movement of Cl into the lung lumen generates a small transepithelial
potential difference (lumen negative) that also promotes the movement of Na þ . As a result, water
moves down an osmotic gradient generated by the
net movement of Na þ and Cl (Figure 1). However,
water movement across the epithelium must also
depend on the intraluminal hydrostatic pressure
existing, which will oppose its movement into the
lung lumen. At rest, a small hydrostatic distending
pressure (1 or 2 mmHg above ambient pressure) is
present within the lung lumen and, therefore, the secretion of lung liquid must normally occur against a
small hydrostatic pressure. When intraluminal pressures are increased above 5 or 6 mmHg, lung liquid
secretion ceases, indicating that the osmotic pressure
driving lung liquid can be counterbalanced by a
hydrostatic pressure of 5 or 6 mmHg. On the other
hand, when the fetal lungs are partially deflated and
the intraluminal hydrostatic pressure is reduced, fetal
lung liquid secretion rates increase.
BREATHING / Fetal Lung Liquid 247
Interstitial
space
Epithelial
cell
Lung
lumen
H2O
Na+
K+
2Cl−
Cl−
2K+
3Na+
Na+
Figure 1 The proposed mechanism for fetal lung secretion
across the pulmonary epithelium. Na þ /K þ ATPase, located on
the basolateral surface of pulmonary epithelial cells, provides the
free energy for Na þ to enter the cell via the Na þ /K þ /2Cl cotransporter, which promotes the entry of Cl against its electrochemical gradient. Cl exits the cell across the apical membrane
down its electrochemical gradient, which causes a transepithelial
potential difference (lumen negative) that promotes the movement of Na þ into the lung lumen. The combined net flux of Na þ
and Cl into the lung lumen provides an osmotic gradient for
water to flow in the same direction.
Although the mechanisms for fetal lung liquid