Faculté de Médecine
Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM)
Role of P2Y2 nucleotide receptor in the
physiopathology of inflammatory lung
diseases
Caractérisation du rôle des nucléotides extracellulaires et du
récepteur purinergique P2Y2 dans la physiopathologie des
maladies pulmonaires inflammatoires
GILLES VANDERSTOCKEN
Thèse présentée en vue de l'obtention du grade académique de
Docteur en Sciences biomédicales et pharmaceutiques
Promoteur: Dr. Didier Communi
Année académique 2011-2012
Remerciements
3
TABLE OF CONTENT
TABLE OF CONTENT
TABLE OF CONTENT ......................................................................................................................... 5
ABBREVIATIONS ............................................................................................................................... 7
PART I – INTRODUCTION ............................................................................................................. 9
1.
THE RESPIRATORY SYSTEM : CHARACTERISTICS AND FUNCTIONS ........................................................................ 11
2.
INNATE AND ADAPTIVE IMMUNITY : GENERAL OVERVIEW ................................................................................. 12
3.
THE LUNG IMMUNE SYSTEM ........................................................................................................................ 14
3.1. FIRST-LINE OF DEFENCE ................................................................................................................................. 15
3.1.1. MECHANICAL BARRIERS AND REFLEX MECHANISMS ................................................................................... 15
3.1.2. EPITHELIAL CELLS .................................................................................................................................. 15
3.1.3. INTRAEPITHELIAL LYMPHOCYTES ........................................................................................................ 17
3.1.4. ALVEOLAR MACROPHAGES ..................................................................................................................... 17
3.2. SECOND -LINE OF DEFENCE ............................................................................................................................. 18
3.2.1. INNATE IMMUNITY ................................................................................................................................ 18
3.2.1.1. RECOGNITION OF INVADING PATHOGENS : PRRS .............................................................................. 18
Airway epithelial cells ......................................................................................................................... 20
Macrophages....................................................................................................................................... 20
3.2.1.2. RECRUITMENT OF LEUKOCYTES ....................................................................................................... 21
Chemokines ......................................................................................................................................... 21
Adhesion and transmigration through blood vessel walls ................................................................ 22
3.2.1.3. LEUKOCYTES POPULATIONS IN THE LUNG ......................................................................................... 24
Monocytes and macrophages............................................................................................................. 24
Mast cells ............................................................................................................................................ 24
Dendritic cells ...................................................................................................................................... 25
Eosinophils .......................................................................................................................................... 25
Neutrophils ......................................................................................................................................... 25
3.2.1.4. ANTIGEN PRESENTATION BY LUNG DENDRITIC CELLS : LINK BETWEEN INNATE AND ADAPTIVE IMMUNITY.. 26
3.2.2. ADAPTIVE IMMUNITY ............................................................................................................................ 26
3.2.2.1. ANTIGEN PRESENTATION TO T AND B CELLS ...................................................................................... 26
3.2.2.2. EFFECTOR FUNCTION OF T CELLS ...................................................................................................... 27
CD4+ T cells .......................................................................................................................................... 27
CD8+ T cells .......................................................................................................................................... 28
3.2.2.3. EFFECTOR FUNCTION OF B CELLS ...................................................................................................... 28
3.2.2.4. IMMUNOLOGICAL MEMORY ............................................................................................................ 29
4.
EXTRACELLULAR NUCLEOTIDES AND THEIR RECEPTORS IN IMMUNITY AND INFLAMMATION .................................... 30
4.1. EXTRACELLULAR NUCLEOTIDES RELEASE .......................................................................................................... 30
4.2. EXTRACELLULAR METABOLISM ....................................................................................................................... 31
4.3. EXTRACELLULAR NUCLEOTIDE FUNCTIONS ........................................................................................................ 32
4.4. CHARACTERISTICS AND FUNCTIONS OF NUCLEOTIDES RECEPTORS ....................................................................... 32
4.4.1. P1 RECEPTORS ...................................................................................................................................... 34
4.4.2. P2 RECEPTORS ...................................................................................................................................... 34
5
TABLE OF CONTENT
4.4.2.1. P2X RECEPTORS ............................................................................................................................ 34
4.4.2.2. P2Y RECEPTORS ............................................................................................................................ 34
Classification of P2Y receptors ........................................................................................................... 35
Molecular modelling of P2Y receptors ............................................................................................... 36
Function of P2Y2 receptor subtype ..................................................................................................... 36
4.4.2.3. THERAPEUTIC APPLICATION OF P2 AGONISTS AND ANTAGONISTS ....................................................... 38
P2X1, P2Y1 and P2Y12 are target receptor in platelet activation ........................................................ 38
P2Y2 is a target receptor in cystic fibrosis........................................................................................... 39
PART II – AIM OF THE STUDY .................................................................................................. 41
PART III – RESULTS...................................................................................................................... 45
1.
ANIMAL MODELS OF AIRWAY DISEASES IN P2Y2 RECEPTOR KNOCKOUT MICE ....................................................... 47
1.1.
CHARACTERIZATION OF THE ROLE OF THE ATP/P2Y 2R SYSTEM IN ASTHMA .................................................. 47
1.2.
P2Y 2R IS NOT ESSENTIAL IN THE PATHOGENY OF PULMONARY FIBROSIS MODEL INDUCED BY BLEOMYCIN ........ 55
1.3.
PROTECTIVE ROLE OF P2Y2R AGAINST LUNG INFECTION BY PNEUMONIA VIRUS OF MICE ................................ 59
PART IV – DISCUSSION AND CONCLUSIONS ....................................................................... 63
PART V – REFERENCES ............................................................................................................... 73
6
ABBREVIATIONS
ABBREVIATIONS
ADO:
ADP:
AEC:
ALI:
AMP:
AHR:
AM:
AP:
APC:
ASF:
ATP:
ATPS:
BAL(F):
BALT:
BLEO:
CACC:
CAM:
cAMP:
CCR:
CD:
CFTR:
COPD:
CRP:
CXCR:
CX3CR:
DAG:
DC:
dsRNA:
EC:
E-NPP:
E-NTPDase:
Flk-1:
adenosine
adenosine diphosphate
airway epithelial cell
acute lung injury
adenosine monophosphate
airway hyperresponsiveness
alveolar macrophage
alkaline phosphatase
antigen-presenting cell
airway surface liquid
adenosine triphosphate
adenosine 5'-O-(3-thio)triphosphate
broncho-alveolar lavage (fluid)
bronchial-associated lymphoid tissue
bleomycin
Ca2+-activated Cl- channel
cell adhesion molecule
cyclic adenosine monophosphate
CC-chemokine receptor
cluster of differentiation
cystic fibrosis transmembrane conductance regulator
chronic obstructive pulmonary disease
c-reactive protein
CXC-chemokine receptor
CX3C-chemokine receptor
diacylglycerol
dendritic cell
double-stranded RNA
endothelial cell
ecto-nucleotide pyrophosphatase/phosphodiesterase
ecto-nucleoside triphosphate diphosphohydrolase
Foxp3:
fetal liver kinase 1 (also KDR)
forkhead box P3
GPCR:
ICAM:
IEL:
Ig:
IL:
IFN:
IM:
IP:
IP3:
g protein coupled receptor
intercellular adhesion molecule
intraepithelial lymphocyte
immunoglobulin
interleukin
interferon
interstitial macrophage
intraperitoneal
inositol 1,4,5 Trisphosphate
7
ABBREVIATIONS
IP-10:
IPF:
IT:
LFA:
LPS:
LRT:
LTA:
interferon gamma-induced protein 10 (also CXCL-10)
idiopathic pulmonary fibrosis
intratracheal
lymphocyte function-associated antigen
lipopolysaccharide
lower respiratory tract
liptocheic acid
KDR:
kinase insert domain receptor (also Flk-1)
KO:
MCC:
MCP-1:
mDC:
MHC:
MIP:
NLR:
NK:
OVA:
PAMP:
PFU:
PIP2:
PKC:
PLC:
PMN:
PPADS:
PRR:
PVM:
qRT-PCR:
RSV:
RT-PCR:
SPF:
sVCAM-1:
TCR:
TGF:
Th:
TLR:
TM:
Treg:
URT:
UTP:
UTPS:
TNF:
VCAM-1:
VLA-4:
WT:
XCR:
knock-out
mucociliary clearance
monocyte chemotactic protein 1 (also CCL2)
myeloid dendritic cell
major histocompatibility complex
macrophage inflammatory protein
NOD-like receptor
natural killer
ovalbumin
pathogen-associated molecular pattern
plaque-forming unit
phosphatidylinositol 1,4 bisphosphate
protein Kinase C
phospholipase C
polymorphonuclear neutrophil
pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid
pattern recognition receptor
pneumonia virus of mice
quantitative real time polymerase chain reaction
respiratory syncytial virus
Reverse transcription polymerase chain reaction
specific pathogen free
soluble vascular cell adhesion protein 1
T-cell receptor
transforming growth factor
T helper
Toll-like receptor
transmembrane domains
regulatory T-cell
upper respiratory tract
uridine 5'-triphosphate
uridine 5'-O-(3-thio)triphosphate
tumor necrosis factor
vascular cell adhesion protein 1
very late antigen 4
wild-type
C-chemokine receptor
8
PART I – INTRODUCTION
PART I – INTRODUCTION
1. THE RESPIRATORY SYSTEM : CHARACTERISTICS AND FUNCTIONS
The lungs are part of the respiratory system and are located in the chest, inside the rib cage
and above the diaphragm. Lungs are complex organs that consist of spongy, elastic tissue
that is designed for oxygen intake and the expiration of carbon dioxide.
The respiratory system (Fig. 1) include the airways (upper and lower respiratory tracts), the
lung parenchyma, and the respiratory muscles. The upper respiratory tract (URT) includes
the nasal cavity, the pharynx and the larynx, whereas the lower respiratory tract (LRT)
begins with the trachea, which divides into bronchi. The progressive subdivision of the
bronchi gives rise to bronchioles that extend into alveolar ducts, which further branch into
blind-ended alveolar sacs (alveoli). Lung alveoli are made up of thin type I pneumocytes and
more cuboidal type II pneumocytes that produce surfactant and have self-renewal and
differentiation potential. Endothelial cells from lung capillaries are located in close contact
with type I cells, the two cell types being separated only by a common 0,2 mm-thick
basement membrane, allowing the easy diffusion of gases.
Oxygen enters the lungs when we inhale a breath. It is distributed throughout the lungs by a
system called the bronchial tree. The bronchial tree carries oxygen to the alveoli where
oxygen, from air that is inhaled, moves from the lung into the blood stream, and carbon
dioxide, a byproduct of our metabolism, moves from the blood into the lung to be exhaled.
Intake of oxygen and delivery by the blood to tissue is necessary for all of the cells in our
body to function. Removal of carbon dioxide is necessary to maintain the blood's pH at an
appropriate level as part of the body's system of acid-base balance.
Figure 1. The respiratory
system.
Main
anatomical
structures of the respiratory system.
After successive branching of the
bronchi and bronchioles, airways end
into alveoli, in which gas exchanges
occur.
11
PART I – INTRODUCTION
2. INNATE AND ADAPTIVE IMMUNITY : GENERAL OVERVIEW
Immunity is a biological term that describes a state of having sufficient biological defences to
avoid infection, disease, or other unwanted biological invasion.
The human microbial defence system can be simplistically viewed as consisting of 3 levels:
(1) anatomic and physiologic barriers; (2) innate immunity; and (3) adaptive (acquired)
immunity. Failure in any of these systems will increase susceptibility to infection.
Anatomic and physiologic barriers provide the crucial first line of defense against
pathogens. These barriers include intact skin, vigorous mucociliary clearance mechanisms,
low stomach pH, and bacteriolytic lysozyme in tears, saliva, and other secretions.
Higher vertebrates have developed two interactive protective systems: the innate and
adaptive immune systems (Martin & Frevert, 2005).
The innate immune system is older and consists of soluble proteins which bind microbial
products, and phagocytic leukocytes which float through the bloodstream and migrate into
tissues at sites of inflammation, or reside in tissue waiting for foreign material. The innate
immune system is always active, ready to recognize and inactivate microbial products
entering the body. Speed is a defining characteristic: within minutes of pathogen exposure,
the innate immune system starts generating a protective inflammatory response. Its
specificity is relatively broad, and based on the recognition of common microbial motifs.
Moreover, cytokines and growth factors produced by macrophages and dendritic cells of the
innate immune system drive the specialized antibody responses of the adaptive immune
system: Innate immunity plays a central role in activating the subsequent adaptive immune
response.
Higher animals have evolved an adaptive immune system of T and B lymphocytes that
respond specifically to signals from the innate immune system by producing high-affinity
antibodies to very specific peptide sequences presented on specialized antigen-presenting
cells. In contrast to the limited number of pathogen receptors used by the innate immune
system, the adaptive immune system has an extremely diverse, randomly generated
repertoire of receptors. The benefit of this receptor diversity is that the adaptive immune
system can recognize virtually any antigen. However, this diversity is associated with a risk of
auto-immune diseases and a time delay (up to 5 days) before generating a sufficiently robust
adaptive immune response after the first exposure to an antigen (Turvey & Broide, 2010).
The clonal expansion of T and B cells leads to the development of two main immune
responses: a cellular response characterized by the proliferation, differentiation and
activation of cytotoxic T cells and a humoral response, in which B cells differentiate into
plasma cells and produces antibodies. These antibodies opsonize microbes and viruses and
facilitate their destruction by leukocytes in tissue and lymph nodes. The adaptive immune
system has a memory component lacking in the innate immune system, allowing a more
efficiency response upon a second exposure to the same antigen.
12
PART I – INTRODUCTION
Together, the innate and the adaptive immune systems enable the host to react adequately
to the broad array of microbial and other unwanted harmful products encountered in
everyday life. Table 1 summarizes the main features of innate and adaptive responses.
Cellular elements
Humoral elements
Receptor
characteristics
Ligands recognized
Response time
Immunologic memory
Risk of autoreactivity
Innate immune system
Macrophages, dendritic cells, mast
cells, neutrophils, eosinophils, NK
cells, NK T cells and epithelial cells
Complement proteins, LPS binding
protein, C-reactive protein,
antimicrobial peptides, mannosebinding lectin, …
Invariant, germline encoded
Conserved microbial components
Immediate
None
Low
Adaptive immune system
T and B lymphocytes
Immunoglobulins secreted by
B cells
Generated by random somatic
gene segment rearrangement
Specific details or epitopes of
macromolecules
Delayed by hours to days
Yes
High
Table 1. Overview of defining features of innate and adaptive immunity
(adapted from Martin & Frevert, 2005 ; Janeway & Medzhitov, 2002)
13
PART I – INTRODUCTION
3. THE LUNG IMMUNE SYSTEM
During a normal day, a human breathes nearly 25 000 times: 10 000-15 000 L of air is then
inhaled by the respiratory system. This air contains dust, pollens, bacteria, viruses, smoke,
and volatile chemicals. The lungs have to maintain a system of defence against these
potentially toxic invaders and the observation that respiratory infections are nevertheless
rare is testimony to the presence of an efficient host defence system. Therefore, the lungs
defence system includes immune cells and the secretion of mucus to contain and remove
these unwanted components from the lungs to limit the number, extent and severity of
respiratory tract infections and diseases.
The ciliated epithelium lining the airway prevents colonization by inhaled bacteria in three
general ways: (1) the physical removal by mucociliary clearance, sneezing and cough; (2) the
presence of broad-spectrum antimicrobial mediators in the mucus; and (3) the recruitment
of phagocytic cells and an inflammatory response: It is the first line of defence against
pathogens. Additional protection comes from polypeptide mediators of the innate, nonantibody-mediated host defence and by professional phagocytes. Once the innate host
defence system is activated, also by the cytokine and chemokine pathways,
adaptive/acquired antibody-mediated immune responses and subsequent tissue repair and
remodelling following infection are orchestrated by immunocompetent cells and mediators
(Table 2).
First-line of defence mechanisms
Mechanical barriers and reflex mechanisms
 Sneezing,
 Cough,
 Mucociliary clearance
Epithelial cells
 Physical barrier,
 Transport of IgA,
 Production of antimicrobial mediators
and mucus (defensins, cathelicidins,
lysozyme, lactoferrin, lectins,…)
Intraepithelial lymphocytes
 Lysis of infected cells
Alveolar macrophages
 Phagocytosis,
 Cytokines and chemokines production
Second-line of defence mechanisms
Innate immunity
 Complement,
 Granulocytes,
 Macrophages,
 Dendritic Cells,
Adaptive immunity
 Cellular response (cytotoxic T cells)
 Humoral response (antibody
production)
Table 2. Defence mechanisms of the respiratory system
(adapted from Pilette et al., 2004)
14
PART I – INTRODUCTION
3.1. FIRST-LINE OF DEFENCE
3.1.1. MECHANICAL BARRIERS AND REFLEX MECHANISMS
The density of microbes is greater in the upper respiratory tract (URT) than in the lower
respiratory tract (LRT). In fact, it is usually considered that only a small number of bacteria
are present in the LRT of healthy individuals. This process of exclusion of bacteria is due to
mechanical barriers and reflex mechanisms. The nose can be considered as a first-line
barrier. Its vibrissae, present on the vestibular region of the nasal cavity, are able to filter the
largest particles contained in inhaled air. Nasal mucosa is a type of respiratory mucosa able
to trap other smaller particles by means of its mucus layer. Nasal cilia are able to transport
the mucus toward the oropharynx to be swallowed. LRT airways represent a system with a
physical barrier that is difficult to overcome. Dichotomous branching and angulation of
airways favour the impact of inhaled particles on to the bronchial mucosa surface. At points
of impacts, bronchial-associated lymphoid tissue (BALT) is able to interact with inhaled
airborne microbes and particles, and to start clearance processes via phagocytes and
immune reactions by immunocompetent cells.
A number of reflex mechanisms may help the defence of the respiratory tract. They are
made possible by the presence of irritant and stretching receptors on the mucosa of the
airways of the URT and of the larger LRT. Sneezing is a complex reflex initiated by the irritant
receptors in the nose, usually triggered by inhaled particles, followed by itching, mucus
secretion and ultimately leading to a forceful and sudden expiration through the nose,
preceded by a deep and fast inspiration, able to eliminate the potentially harmful inhaled
particles. In the tracheobronchial tree, the cough reflex plays a similar role in eliminating
foreign inhaled particles.
The constant mechanical clearance of mucus from the airways is considered as a primary
airway defence mechanism. The airway epithelial surface is able to act through ciliary
function and mucus secretion with proper salt/water components in order to maintain the
mucociliary clearance with a mucus "escalator" from the lower airways to the top. With a
mucus layer at the top containing different types of mucins and a largely aqueous layer at
the bottom, the airway secretions are, under normal conditions, able to entrap the vast
majority of inhaled foreign particles and microbes on the mucus layer and to transport the
mucus up to the larger airways to be swallowed or eliminated by coughing. (Balbi et al., 2011
(ERS Handbook)).
3.1.2. EPITHELIAL CELLS
The airway epithelium represents a primary site for the introduction and deposition of
potentially pathogenic microorganisms into the body, mainly through inspired air. The
epithelial layer with its tight junctions constitutes a physical barrier, and epithelial cells are
covered by the electronegatively charged glycocalix. Airways epithelial cells (AECs) play
15
PART I – INTRODUCTION
crucial roles in initiating and augmenting airway host defence mechanisms by regulating
both innate and adaptive immunity through production of functional molecules in the airway
surface liquid (ASF) and via physical interactions with cells of the immune system. Major
antimicrobial products are secreted constitutively and/or inducibly by epithelial cells include
chemokines, cytokines, antimicrobial peptides (lysozyme, lactoferrin, cathelecidins (LL-37),
defensins, collectins, pentraxins), proteinase inhibitors (secretory leukocyte protease
inhibitor (SLPI)), and surfactant proteins (Fig. 2). The pathologic deficiency or inappropriate
regulation of the anti-microbial properties of airway secretions may contribute to epithelial
colonization by microorganisms.
AECs also transport mucosal Immunoglobulin A (IgA) into the airways. Mucosal IgA are
dimeric immunoglobulins which have the particularity to be secreted either by B cells
activated by T cells (monoclonal activation), or by nonconventional B cells (B1 cells) activated
by the direct presentation of the antigen by antigen-presenting cells (APCs), in a T-cellindependent manner. Secreted into the airways, they play a crucial role in the first line
immunity. They neutralize bacteria, by interfering with their motility or their binding to the
airway epithelium, and improve the viscielastic properties of the airway secretions (Diamond
et al, 2000 ; Bals & Hiemstra, 2004 ; Pilette et al, 2004 ; Kato et al, 2007).
Figure 2. Model summarizing the influence of airway epithelial cells on innate and adaptive immune
responses as well as anti-inflammatory processes in the airways. The left part of figure shows the
expression profiles of pattern-recognition receptors (PRRs – see below) in the epithelium. Toll-like receptors
(TLRs) and lactosylceramide recognize pathogens on the cell surface. In contrast, TLR3, NODs and RNA
helicases recognized pathogens intracellularly. After activation of PRRs, epithelial cells produce a wide range of
16
PART I – INTRODUCTION
molecules which enhance innate and adaptive immune responses, cell recruitment and mediate antiinflammatory responses (Kato et al., 2007).
3.1.3. INTRAEPITHELIAL LYMPHOCYTES
Intraepithelial lymphocytes (IELs) are T cells that usually express the  T-cell receptor (TCR)
and CD8α homodimer, in contrast to conventional T cells expressing αβ TCR (complexed to
CD3) and either the CD4 or CD8 αβ coreceptor.  T cells contribute to early stages of
immune responses: they recognize and kill infected epithelial cells that express major
histocompatibility complex class I-like molecules as generic distress signals and thereby also
act in first-line defence. Antiinfectious properties of IELs include direct cytolytic effects (they
are rich in cytotoxic granules), Th1 activity, activation of neutrophils and macrophages and
stimulation of the survival of epithelial cells via the production of epithelial growth factors
(Pilette et al, 2004).
3.1.4. ALVEOLAR MACROPHAGES
Macrophages have a central role in the maintenance of immunological homeostasis and host
defences. In the lungs two main types of macrophages have been described: interstitial and
alveolar macrophages. As a first line of defence, only the role of alveolar macrophages will
be considered in this section.
Pathogen 1 µm in size and smaller (the size of bacteria and viral particles) that are reached
the alveolar space, eluding URT and LRT first-line defences, represents a risk for the host as
its replication and associated alveolar inflammation may damage respiratory structures. So
these particles are carried to the alveolar surface where they interact with soluble
components in alveolar fluids (e.g., IgG, complement, surfactant, and surfactant-associated
proteins) and alveolar macrophages. Alveolar macrophages are phagocytic cells and ingest
all types of inhaled particulates that reach the alveolar spaces. Normally, alveolar
macrophages account for approximately 95% of airspace leukocytes, with 1 to 4%
lymphocytes and only about 1% neutrophils, so that the alveolar macrophage is the resident
respiratory sentinel phagocytes of the lungs (Fig. 3). Other cells in the airways and alveolar
environment can sense microbial products, because pattern recognition receptors (PRRs) in
the Toll-like receptor (TLRs) family are found on alveolar walls and the ciliated epithelium of
the conducting airways (see below).
Remarkably, one of the primary roles of the alveolar macrophage is to keep the airspaces
quiet: they ingest large numbers of inert particulates like amorphous silicates and carbon
graphite particles without triggering inflammatory responses.
17
PART I – INTRODUCTION
Figure
3.
The
alveolar
environment in the lungs. Scanning
electron micrograph of a rat lung
showing the images of erythrocytes in
the alveolar wall capillaries and two
alveolar macrophages (MP) in the
alveolar
space
(ALV).
Alveolar
macrophages make up approximately
95% of the leukocytes in the
airspaces of human lungs, and 100%
of the leukocytes in the lungs of
pathogen-free mice.
When bacteria are opsonized by IgG, complement, or surfactant proteins A and D (SP-A and
SP-D) in the epithelial lining fluid, they are ingested by alveolar macrophages and the TLRs in
the phagosomal membrane provide discrimination among the various microbial products
entering the cell. The activation of alveolar macrophages results in a shift in their functional
capacity: they become efficient effector cells, participating in phagocytosis, killing and
coordinating immune response. Alveolar macrophages also have an important role in
producing CC chemokines, such as MCP-1 and RANTES (regulated on activation, normal T-cell
expressed and secreted), which recruit activated monocytes (see below) and lymphocytes
into sites of inflammation in the lungs (Balbi et al., 2011 (ERS Handbook) ; Martin & Frevert,
2005).
The size of the bacterial inoculum, their virulence and resistance and possibly deficits in the
local immune mechanisms of the host may alternatively cause the failure, at least in a first
round, of host defences. This will cause recruitment of additional phagocytes, as neutrophils,
at sites of infection and sustain an immune and inflammatory reaction.
3.2. SECOND-LINE OF DEFENCE
3.2.1. INNATE IMMUNITY
The innate immune response protects the air spaces from the array of microbial
products that enter the lungs on a daily basis.
3.2.1.1. RECOGNITION OF INVADING PATHOGENS : PRRS
The recognition of invading pathogens is a crucial step of innate immunity. The innate
immune response relies on evolutionarily ancient germline-encoded receptors: the patternrecognition receptors (PRRs), which recognize and destroy highly conserved microbial
structures. These PRRs can be expressed on the cell surface, in intracellular compartments,
18
PART I – INTRODUCTION
or secreted into the bloodstream and tissue fluids. This genetically encoded recognition
system enables the host to recognize a broad range of pathogens, without the need for
time-consuming somatic hypermutation of receptors on T cells or immunoglobulin genes.
PRRs recognize microbial components, known as pathogen-associated molecular patterns
(PAMPs), which are essential for the survival of the microorganism and relatively invariant.
The principal functions of pattern recognition receptors include opsonization, activation of
complement and coagulation cascades, phagocytosis, activation of proinflammatory
signaling pathways and induction of apoptosis (Janeway, 1989 ; Janeway & Medzhitov,
2002 ; Turvey & Broide, 2010).
Amongst the PRRs, the Mannan-binding lectin (MBL), C-reactive protein (CRP), and serum
amyloid protein (SAP) are secreted pattern recognition molecules. CRP and SAP are
members of the pentraxin family, and both can function as opsonins or activate the classical
complement pathway. MBL is a member of the collectin family, which also includes
pulmonary surfactant proteins A and D (SP-A and SP-D). MBL binds specifically to terminal
mannose residues, which are abundant on the surface of many microorganisms (Janeway &
Medzhitov, 2002). Transmembrane and intracellular pattern recognition receptors include
the families of Toll-like receptors (TLRs), dectins, and NOD-like receptors (NLRs) (Fig. 4).
Figure 4. Pattern Recognition
Receptors.
Major classes of mammalian
pattern recognition receptors and
their representative microbial
ligands.
MBL,
mannan-binding
lectin;
NLRs, NOD-like receptors; CRD,
carbohydrate
recognition
domain; ITAM, immunoreceptor
tyrosine-based activation motif.
Toll-like receptors are the best characterized receptors of the PRRs. TLRs are a family of
evolutionarily conserved transmembrane receptors, sharing a common cytosolic Toll/IL-1R
domain and an extracellular leucine rich repeat region responsible for recognizing specific
19
PART I – INTRODUCTION
structural motifs of various pathogen, known as pathogen-associated microbial patterns
(PAMPs). Ten human TLRs have been reported and each TLR senses a separate set of ligands
(Table 3).
Table 3. Toll-like receptors and their ligands (Martin & Frevert, 2005).
The interaction of TLRs with their respective ligands help to shape the adaptive immune
response by triggering downstream signalling cascades, which control the induction of proinflammatory cytokines and chemokines, as well as the upregulation of co-stimulatory
molecules to direct the way that dendritic cells instruct T-cells. In the lung, TLRs are higly
expressed by epithelial cells and sentinel cells (alveolar macrophages, myeloid DCs) (Bals et
al., 2004 ; Turvey & Broide, 2010).
Airway epithelial cells
Airway epithelial cells express a variety of TLRs that help them to mount an adequate
response to microbial exposure. Activation of TLR on epithelial cells involved the regulation
of expression of a variety of genes, including those encoding cytokines, chemokines and
antimicrobial peptides. (Bals et al., 2004)
Macrophages
Macrophages express several cell surface receptors such as macrophage mannose receptor
(MMR), macrophage scavenger receptor (MSR) or MARCO, function as pattern recognition
receptors that mediate phagocytosis of microorganisms. MMR interacts with a variety of
gram-positive and gram-negative bacteria and fungal pathogens. The main function of the
MMR is thought to be phagocytosis of microbial pathogens, and their delivery into the
lysosomal compartment where they are destroyed by lysosomal enzymes (Fraser et al.,
1998). MSR belongs to the scavenger receptor type A (SR-A) family and has an broad
specificity to a variety of polyanionic ligands, including double-stranded RNA (dsRNA), LPS,
and liptocheic acid (LTA). Another SR-A family member, MARCO binds to bacterial cell walls
and LPS, and it also mediates phagocytosis of bacterial pathogens (Janeway & Medzhitov,
2002).
20
PART I – INTRODUCTION
3.2.1.2. RECRUITMENT OF LEUKOCYTES
Following the recognition of PAMPs, a set of chemoattractant mediators are released in
order to recruit leukocytes to the inflammatory site, a capital step of the second line of
defence of the lung. Chemotaxis is a fundamental biological process in which a cell migrates
following the direction of a spatial cue. This spatial cue is provided in a form of a gradient of
chemoattractants. Cells are able to sense small differences in chemoattractant
concentrations: usually only a few percents.
Leukocyte chemotaxis is regulated by a number of chemoattractants include bacterial
peptides (fMLP), complement fragment (C5a and C3a), nucleotides, lipid mediators
(leucotrienes, prostaglandins, lipoxins, resolvins) and the superfamily of proinflammatory
cytokines called chemokines. These chemoattractants act as immediate mediators of
inflammatory responses by regulating leukocyte recruitment, infiltration, homing, and
trafficking as well as their development and function.
Chemoattractants bind to their specific cell surface receptors and mainly activate the
heterotrimeric G proteins Gi in leukocytes (Wu, 2005).
Chemokines
Chemokines (chemotactic cytokines) are small, secreted proteins that are mainly involved in
leukocyte chemoattraction. This large family of related molecules is classified on the basis of
structural properties, regarding the number and position of conserved cysteine residues, to
give two major (CXC and CC) and two minor (C and CX3C) chemokine subfamilies and
according to their production in homeostatic conditions and inflammatory conditions. There
is significant redundancy in the chemokine system, as illustrated by the binding of multiple
chemokine (Fig. 5).
Chemokine receptors belong to the G-protein-coupled receptors (GPCRs) superfamily, and
consist of single polypeptide chains with three extracellular and three intracellular loops, an
acidic amino-terminal extracellular domain involved in ligand binding, and a
serine/threonine-rich intracellular carboxy-terminal domain. The external interface
contributes to the specificity of ligand recognition, whereas the conserved transmembrane
sequences, the cytoplasmic loops and the C-terminal domain are involved in receptor
signalling and internalization.
21
PART I – INTRODUCTION
Figure 5. Chemokines and chemokine receptors (Mantovani et al., 2006)
There is significant redundancy in the chemokine system, as illustred by the binding of
multiple chemokines to a particular receptor and the interaction of multiple receptors with a
particular chemokine. At present, 18 chemokine receptors have been defined:
10 CC-chemokine receptors (CCR1 to CCR10), 6 CXC-chemokine receptors (CXCR1 to CXCR6),
1 C-chemokine receptor (XCR1) and 1 CX3C-chemokine receptor (CX3CR1) (Charo &
Ransohoff, 2006 ; Mantovani et al., 2006).
Adhesion and transmigration through blood vessel walls
Leukocyte trafficking (including neutrophil recruitment, lymphocyte recirculation and
monocyte trafficking) requires adhesion to endothelial cells and transmigration through the
vascular endothelium. The adhesion to the endothelium is characterized by three main
steps: rolling, activation and arrest and then the transmigration occurs (Fig. 6).

Rolling: carbohydrate ligands on the circulating leukocytes bind to selectin
molecules, located in inflammatory situations on the luminal membrane of
22
PART I – INTRODUCTION
endothelial cells. These low affinity bonds are reversible, causing the leukocytes to
slow down and roll along the inner surface of the vessel wall.

Activation: activated endothelial cells release chemokines and other
chemoattractant mediators, which bind to glycosaminoglycans and are presented to
leukocytes, resulting in a rapid enhancement of their integrin avidity

Arrest: leukocyte integrins (such as LFA-1, VLA-4, CD11b) strongly interact with
adhesion molecules expressed at the surface of endothelial cells, including the
intercellular- and vascular-cell adhesion molecule-1 (ICAM-1 and VCAM-1
respectively). This strong interaction leads to the arrest of the leukocyte.
Transmigration: once leukocytes are firmly attached to the endothelium, they initiate a step
called “intravascular crawling” characterized by the formation of leukocyte membrane
protrusions into the endothelial cell body and endothelial cell junctions. Then either a
paracellular or a transcellular migration occurs through the vascular endothelium, followed
by migration through the basement membrane.
Figure 6 : The leukocyte adhesion cascade. The three steps of adhesion are shown in bold: rolling, which is
mediated by selectins, activation, which is mediated by chemokines, and arrest, which is mediated by integrins.
Key molecules involved in each step are indicated in boxes (Ley et al., 2007).
The site of recruitment for each leukocyte subset is determined by the specific combination
of selectins, chemokines (and other chemoattractant agents) and their receptors, and
integrins and their ligands expressed respectively by the leukocyte subset and the
endothelial cells at the recruitment site (Ley et al., 2007).
23
PART I – INTRODUCTION
3.2.1.3. LEUKOCYTES POPULATIONS IN THE LUNG
Specific leukocyte populations play distinct roles in host defence in the lung and can be broadly
classified as those that are normal residents of the lung (mast cells, macrophages, dendritic cells)
and itinerant cells that are recruited in response to infection or injury (neutrophils, monocytes, and
lymphocytes)
Monocytes and macrophages
Mononuclear phagocytes are innate immune cells that reside in the bloodstream as
monocytes or in various tissues as macrophages. Monocyte-like precursors colonize
extravascular sites early during embryogenesis to become resident macrophages, acquiring
morphological and functional properties that are characteristic of the tissue in which they
reside (e.g. Kupffer cells in the liver, microglial cells in the brain, alveolar macrophages in the
lungs). In the lungs, recruited monocyte-derived macrophages and resident lung
macrophages play important roles during inflammation. They phagocyte pathogens,
apoptotic cells, release chemokines and cytokines, and act as antigen-presenting cells
(APCs). The proinflammatory cytokines produced by macrophages (notably TNF-, IL-1,
IL-8/CXCL8), and chemokines (such as KC/CXCL1 and MIP-2/CXCL2), initiate a localized
inflammatory response by recruiting neutrophils from the lung capillary networks into the
alveolar space. Macrophages are poor antigen-presenting cells, but carry microbial antigens
into the interstitium and to regional lymph nodes where they are taken up by specialized
dendritic cells and presented to responding lymphocytes to initiate adaptive immune
responses (see chapter 3.2.2 : adaptive immunity) (Balbi et al., 2011 (ERS Handbook) ;
Martin & Frevert, 2005).
Mast cells
Mast cells are key elements in the innate immune system and have been termed the
“antennas” of the immune response for their ability to detect changes in their environment
that they communicate to other cells in the vicinity. Mast cells are located throughout the
body in close proximity to epithelial surfaces, near blood vessels, nerves and glands, placing
them at strategic locations for detecting invading pathogens. In addition, mast cells express
a number of receptors that allow them to recognize diverse stimuli.
In sensitized individuals, Immunoglobulin E (IgE) is bound to Fc receptors (FcRI) expressed
on the mast cell surface and binding of antigen to surface bound IgE induces mast cell
activation. Thus, multiple stimuli of foreign antigens may trigger the same class of receptor.
Human mast cells also express a number of Toll-like receptors including TLR-1, TLR-2, TLR-6,
and TLR-4. Expression of TLRs, in combination with other receptors, allows mast cells to
recognize many potential pathogens and generate specific responses.
Importantly, mast cells are capable of releasing many small molecules that stimulate
inflammation and the adaptive immune response and can polarize T cell subpopulations
towards Th1 or Th2 subtypes. Mast cell products include preformed mediators that are
24
PART I – INTRODUCTION
granule-associated (such as histamine), mediators synthesized de novo (such as leukotriene
C4, platelet-activating factor and prostaglandin D2), an array of cytokines, chemokines, and
growth factors. The strategic location of mast cells in the body and their diversity of
receptors and cytokines indicate an important role for mast cells in regulating innate and
adaptive immunity (Suzuki et al., 2008).
Dendritic cells
See chapter 3.2.2.
Eosinophils
Eosinophils are viewed as effector cells of allergic responses and also contribute to the
elimination of parasites. Eosinophils are bone-marrow derived cells that contain four distinct
granule cationic proteins: major basic protein, eosinophil peroxidase, eosinophil cationic
protein, and eosinophil-derived neurotoxin. Eosinophils play a prominent role in the
pathogenesis of asthma. During allergic inflammation, eosinophils release granule contents
as well as inflammatory mediators including lipid mediators such as leukotriene C4 and
platelet-activating factor (Suzuki et al., 2008).
Neutrophils
The primary function of neutrophils in the innate immune response is to contain and kill
invading microbial pathogens (Nathan, 2006). Neutrophils are the first immune cells to
migrate into infected tissue sites. This recruitment is vital both for direct action against
microorganisms and for attracting lymphocytes able to resolve inflammation over the longer
term. In pulmonary host defence, these phagocytes are preferentially recruited to the lung
in response to infection and inflammation via endothelial expression of adhesion molecules
(selectins and CAMs) and chemokines (CXCL1 (KC) and CXCL2 (MIP-2)). These cells achieve
their antimicrobial function through a series of rapid and coordinated responses that end in
phagocytosis and destruction of the pathogens. Neutrophils have a potent anti-microbial
armamentarium that includes oxidant generating systems, powerful proteinases, and
cationic peptides contained in granules.
CXCR1 and CXCR2 are important for the CXC-chemokine-mediated recruitment of
neutrophils into inflamed lungs. These CXC-chemokines are mainly KC/CXCL1, MIP-2/CXCL2,
ENA-78/LIX/CXCL5, SDF-1/CXCL12, and IL-8/CXCL8. IL-8 is the best characterized chemokines
implicated in the recruitment of neutrophils in human. However, rodents lack a direct
homologue of IL-8, but the chemokines CXCL-1 (KC), CXCL2 (MIP-2), and CXCL5 (LIX) are
regarded as functional homologues of IL-8. Many cells can produce these chemokines
including endothelial cells, epithelial cells, and leukocytes. Additionally, recent studies
showed that endothelial cells possess granules containing CXCL1 (KC), CXCL2 (MIP-2) and
CXCL5 (LIX) (Hol et al., 2010). These granules are mobilized upon demand, enhancing the
role of endothelial cells in neutrophil recruitment. However, resident tissue macrophages
are reported as a major source of CXCL2 (MIP-2) and KC (CXCL1) (De Filippo et al., 2008).
25
PART I – INTRODUCTION
Neutrophils release many cytokines (IL-6, TNF-, …) and chemokines (CXCL1 (KC), CCL3
(MIP1-, …) that modulate innate and adaptive responses (Nathan, 2006).
3.2.1.4. ANTIGEN PRESENTATION BY LUNG DENDRITIC CELLS: LINK
BETWEEN INNATE AND ADAPTIVE IMMUNITY
Adaptive immune reactions start in the lung, with the interaction between antigens and
antigen-presenting cells (APCs). In the lung, at least two types of APCs exist: macrophages
(AMs) and dendritic cells (DCs). DCs are highly efficient antigen-presenting cells and are
pivotally positioned at the interface of innate and adaptive immunity. Immature dendritic
cells are derived from bone marrow precursor cells that travel and reside in the peripheral
tissues, where they actively sample their environment by endocytosis and macropinocytosis.
Upon encountering a pathogen, they undergo a developmental program called dendritic cell
maturation, which includes induction of costimulatory activity, antigen processing, increased
major histocompatibility complex (MHC) class II molecule expression, and migration to the
lymph node, where they can prime naïve antigen-specific T cells, a pivotal process in the
adaptive immune response (Randolph et al., 2008). In this way activation of the adaptive
immune system occurs only upon pathogen recognition by dendritic cells. Pathogen
recognition is mediated by TLRs on the surface of dendritic cells; not surprisingly, these cells
express high levels of most members of the TLR family (Janeway & Medzhitov, 2002).
3.2.2. ADAPTIVE IMMUNITY
The adaptive response is mainly characterized by the proliferation of T or B lymphocytes
specific of one antigen (clonal expansion) and by a memory response. B cell proliferation and
differentiation lead to the development of a humoral response characterized by the
differentiation of plasma cell and the production of antibodies whereas T cells can initiate
either suppressive or cytotoxic responses.
3.2.2.1. ANTIGEN PRESENTATION TO T AND B CELLS
In order to initiate antigen-specific adaptive responses, antigens have to be presented to
lymphocytes, and this antigen presentation differs according to the subset of lymphocytes
concerned. Indeed, for CD4+ T cells, antigens are presented coupled to the major complex of
histocompatibility (MHC) class II, whereas CD8+ T cells, antigens are coupled to MHC class I.
For B cells, the majority of antigens are unable to directly stimulate them without the help of
CD4+ T cells. Therefore, both APCs and B cells themselves capture the antigen and present it,
coupled to MHC class II, to CD4+ T-cells, which in turn interact with B cells (Delves & Roitt,
2000). CD4+ T cells are mainly cytokine-secreting helper cells, whereas CD8+ T cells are
mainly cytotoxic killer cells.
26
PART I – INTRODUCTION
3.2.2.2. EFFECTOR FUNCTION OF T CELLS
CD4+ T cells
Naïve CD4+ T cells, also called Th0 lymphocytes, orchestrate different types of adaptive
immune responses through its differentiation into Th1, Th2, Th17, or regulatory T cells
(Treg), a process called polarization of the immune response. These different responses are
characterized by distinct profiles of cytokine secretion, and exert specific functions (Fig. 7).
Figure
7.
Differentiation
of
helper T cell subsets.
Following activation by
antigen-presenting cells
such as dendritic cells,
naive CD4+ T cells can be
polarized into different
effector T cell subsets —
T helper 1 (TH1), TH2,
TH17 and regulatory T
(TReg) cells — depending
on the local cytokine
environment
(Zou
&
Restifo, 2010).
The type of differentiation of naïve T cells is determined by the pattern of signals they
receive during their initial interaction with antigens. Thereby, distinct sets of cytokines
promoting the differentiation of each lineage have been described:




IL-12/IFN- for Th1;
IL-4/IL-2 (IL-7, TSLP) for Th2;
TGF-/IL-6 (IL-1, IL-21, IL-23) for Th17;
and TGFb/IL-2 for Tregs.
The transcription factors that govern the differentiation of these cells are also well defined:
T-bet/Stat4 for Th1; GATA3/Stat6 for Th2; RORyt/Stat3 for Th17 and Foxp3/Stat5 for Tregs
(Zhu & Paul, 2010; Miossec et al., 2009).
The Th1 and Th2 were the first described responses. Th1 cells produce large amounts of
interferon- (IFN-), induce delayed hypersensitivity reactions, activate macrophages, and
are essential for the defense against intracellular pathogens (such as viruses). Th2 cells
27
PART I – INTRODUCTION
produce mainly IL-4 and are important for the induction of IgE production, the recruitment
of eosinophils to the inflammatory site, and the clearance of parasitic infections. Moreover,
the development of one of these 2 responses cross-inhibits the other: the Th1 cytokine IFN-
inhibits Th2 cells, and secretion of IL-10 by Th2 cells reciprocally inhibits Th1 cells (Delves &
Roitt, 2000; Miossec et al., 2009). Recently, T cells were shown to produce cytokines that
could not be classified according to the Th1-Th2 scheme. Interleukin-17 was among these
cytokines, and the T cells that preferentially produce interleukin-17 but not IFN- or IL-4,
were named Th17 cells. Th17 cells have been implicated in the defence against bacteria and
fungi (Zhu & Paul, 2010; Miossec et al., 2009).
In addition to their roles in the control of pathogens, these three Th effector responses
contribute also to the development of diseases. Specifically, Th2 cells mediate allergic and
asthmatic diseases, whereas Th1 and Th17 cells contribute to acute and chronic
inflammation associated with tissue damage, through the activation of macrophages and
neutrophils.
Finally, a fourth lineage into which naïve T cells can differentiate is regulatory T (Treg) cells.
Treg are a specialized subpopulation of Foxp3+ CD4+ T cells which suppresses activation of
the immune system and thereby maintains tolerance to self-antigens.
CD8+ T cells
CD8+ T cell proliferation and differentiation are a hallmark of the Th1 adaptive response.
CD8+ cytotoxic T cells are able to lyse target cells, such as neoplastic cells or cells infected by
intracellular pathogens. Antigens are presented to MHC class I molecules to CD8+ T cells,
which kill the infected/dysfunctional somatic cells by to different pathways: (a) insertion of
perforins, into the target-cell membrane, producing pores through which granzymes (type of
serine proteases) are “injected”, and (b) induction of apoptosis in target cells following
Fas/Fas ligand interaction (Delves & Roitt, 2000). CD8+ T cells also drive non-cytolytic
effector functions mediated by production of cytokines such as IFN- and TNF- (Ishii &
Koziel, 2008). As for CD4+ T cells, CD8+ Treg cells were described, and participate to the
negative control of immune responses. Similarly to CD4+ Treg cells, Treg cells from the CD8+
lineage may develop intrathymically as well as in peripheral tissues. Their suppressive
activity is mediated by cell-cell contacts as well as soluble factors such as IL-10 and TGF-.
3.2.2.3. EFFECTOR FUNCTION OF B CELLS
The principal functions of B cells are to make antibodies against antigens, perform the role of
APCs and eventually develop into memory B cells after activation by antigen interaction.
When B cells undergo terminal differentiation into plasma cells, they acquire the ability to
produce and secrete high levels of antibodies. These antibodies can be directly protective if
they sterically inhibit the binding of a microorganism or toxin to the corresponding cellular
receptor (neutralizing antibodies). Most antibodies work by binding to an antigen, signalling
to a white blood cell that this antigen has been targeted, after which the antigen is
processed and consequently destroyed. The difference between neutralizing antibodies and
28
PART I – INTRODUCTION
binding antibodies is that neutralizing antibodies neutralize the biological effects of the
antigen, while binding antibodies flag antigens. In most instances, however, antibodies do
not function in isolation but instead their usual role is to focus components of the innate
immune system on the pathogen.
3.2.2.4. IMMUNOLOGICAL MEMORY
When B cells and T cells are activated some will become memory cells. Throughout the
lifetime of an animal these memory cells form a database of effective B and T lymphocytes.
Upon interaction with a previously encountered antigen, the appropriate memory cells are
selected and activated. In this manner, the second and subsequent exposures to an antigen
produce a stronger and faster immune response. Indeed, the memory response is more
rapid, produces a higher number of lymphocytes, and in the case of B cells, induces higher
levels of antibodies with greater affinity to the antigen.
29
PART I – INTRODUCTION
4. EXTRACELLULAR NUCLEOTIDES AND THEIR RECEPTORS IN IMMUNITY
AND INFLAMMATION
Human health is under constant threat of a wide variety of dangers, both self and nonself.
The immune system protects the host against such dangers in order to preserve human
health. For that purpose, the immune system is equipped with a diverse array of both
cellular and non-cellular effectors that are in continuous communication with each other.
Nucleotides constitute an intrinsic part of this extensive immunological network through
purinergic signalling, which are widely expressed throughout the body. ATP and other
nucleotides thus constitute “danger signals”. The role of these nucleotides during the course
of inflammatory and immune responses in vivo is complex: the nature of their effects may
shift from immunostimulatory to immunoregulatory or vice versa depending on extracellular
concentrations as well as on expression patterns of purinergic receptors and ecto-enzymes.
(Bours et al., 2006)
The first description of the extracellular signalling by nucleotides was by Drury & SzentGyörgyi in 1929. Purinergic receptors were defined later in 1976 by Burnstock.
4.1. EXTRACELLULAR NUCLEOTIDES RELEASE
Extracellular nucleotides, induces significant functional changes in a wide variety of cells and
tissues. They can be released from the cytosol of damaged cells, from exocytotic vesicles
and/or granules contained in many types of secretory cells and from transmembrane
transporters (Fig. 8) (Dubyak & el-Moatassim, 1993).
The dynamic regulation of extracellular nucleotides manifested by the balance between
release, catabolism, and interconversion in the extracellular space has imposed a layer of
complexity to the understanding and quantification of the pharmacologically active
nucleotides.
Figure 8. Nucleotidereleasing pathways.
Along with massive
nucleotide
leakage
upon
cell
damage,
nucleotides can appear
in the external milieu
via various non-lytic
pathways,
including
electrodiffusional
movement
through
ATP release channels,
facilitated diffusion by
nucleotide-specific
transporters
and
vesicular exocytosis.
(Yegutkin, 2008)
30
PART I – INTRODUCTION
Concentrations of nucleotides in the extracellular space ranging from 0,1 to 10 µM are
required for activity. Furthermore, stress or mechanical stimulation of many cell type results
in a massive elevation in the concentration of extracellular nucleotides. Indeed, ATP and
probably other nucleotides are stored at high concentrations in dense granules in platelets,
synaptic vesicles in neurons and granules. The concentration of nucleotides in these vesicles
could reach up to 150 to 1000 mM. There exist efficient extracellular mechanisms for the
rapid metabolism of released nucleotides by ecto-ATPases and ecto-5'-nucleotidases (see
below) (Dubyak & el-Moatassim, 1993). Physiological ATP concentrations in plasma are
normally submicromolar (400–700 nM) (Coade & Pearson, 1989; Ryan et al., 1996).
4.2. EXTRACELLULAR METABOLISM
Extracellular concentrations of ATP can rise under several conditions, including
inflammation, hypoxia and asthma (Bodin & Burnstock, 1998 ; 2001; Lazarowski et al., 2003,
Idzko et al., 2007). Concentrations of ATP in the extracellular compartment are controlled by
enzymes catalyzing their conversion (Fig. 9) (Zimmermann, 2000 ; Moriwaki et al., 1999;
Goding, 2000). These so-called ecto-enzymes are located on cell surfaces or may be found in
soluble form in the interstitial medium or in body fluids. The currently known ecto-enzymes,
include four families that partially share tissue distribution and substrate specificity: (1) the
ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family (such as NTPDase1
or CD39), (2) the ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) family, (3)
alkaline phosphatase (AP), and (4) ecto-5′-nucleotidase (CD73).
Figure
9. Overview of
conversion pathways of
ATP
and
adenosine.
Purinergic
receptors
bind
extracellular ATP and the
reaction products that result
from its enzymatic hydrolysis
by
ectonucleotidases.
P2
receptors bind ATP and ADP,
whereas P1 receptors bind
adenosine. The metabolism of
extracellular ATP is regulated
by several ectonucleotidases,
including
members
of
the
E-NTPDase
(ectonucleoside triphosphate
diphosphohydrolase)
family
and
the
E-NPP
(ectonucleotide
pyrophosphatase/phosphodie
sterase)
family.
Ecto-5′nucleotidase (Ecto-5′-NT) and
alkaline phosphatase (AP)
catalyse
the
nucleotide
degradation to adenosine.
(Field & Burnstock 2006)
31
PART I – INTRODUCTION
The first family catalyzes the sequential degradation of extracellular nucleotide tri- and
diphosphates. It includes seven members (NTPDase1 to -6 and NTPDase8) of which
NTPDase1, -2, -3 and -8 are involved in the breakdown of ATP and adenosine 5′-diphosphate
(ADP) to adenosine 5′-monophosphate (AMP). The second family consists of 3 members
(NPP1, -2 and -3), which catalyze the hydrolysis of cyclic AMP (cAMP) to AMP, ATP to AMP
and ADP to AMP. A splice variant of NPP2: E-NPP2 (autotaxin), is involved in the conversion
of AMP to adenosine. The third family comprises a protein family of non-specific
ecto-phosphomonoesterases catalyzing the degradation of nucleotide tri-, di- and
monophosphates. Finally, the fourth family is represented by CD73 which catalyzes the
hydrolysis of AMP to adenosine. According to cellular location and kinetic properties, this
enzyme can be grouped into four forms, of which a membrane-bound form and a soluble
form are involved in extracellular metabolism of AMP. Besides the enzymes that degrade
extracellular nucleotides, enzymes catalyzing the generation and interconversion of
extracellular adenine and uridine nucleotides have also been described (Harden et al., 1997;
Lazarowski et al., 1997 ; 2000 ; Yegutkin, 2008).
Taken together, a wide variety of enzymes are involved in the control of extracellular
nucleotide and nucleoside levels. These enzymes are essential to the regulation of purinergic
signalling by nucleotides (Bours et al., 2006).
4.3. EXTRACELLULAR NUCLEOTIDE FUNCTIONS
The nucleotides are present in every living cell of the human body. Extracellular ATP, UTP,
ADP, and UDP play key roles in a diversity of tissue functions such as inflammation,
developmental processing, pulmonary function, nociception, auditory and ocular function,
the apoptotic cascade, astroglial cell function, metastasis formation, bone and cartilage
disease, platelet aggregation/hemostasis and several role in the nervous system including
neuroprotection, central control of autonomic functions, neural–glial interactions, control of
vessel tone and angiogenesis, pain and mechanosensory transduction, …. (see reviews :
Williams & Jarvis, 2000 ; Burnstock, 2006).
4.4. CHARACTERISTICS AND FUNCTIONS OF NUCLEOTIDES RECEPTORS
Receptors for purines are subdivided into P1 (adenosine (Ado)) and P2 (ATP) receptors
(Burnstock, 1978), and later subdivision of P2 receptors into P2X and P2Y subtypes was
made on the basis of pharmacology (Burnstock & Kennedy, 1985). It was recognized that
some P2Y receptors responded to pyrimidines as well as purines (Seifert & Schultz, 1989).
After cloning of P2 receptors and studies of transduction mechanisms in the early 1990s, the
basis for subdivision into P2X and P2Y receptor families was confirmed and extended
(Abbracchio & Burnstock, 1994) and seven subtypes of P2X receptors and eight subtypes of
P2Y receptors are currently recognized (Ralevic & Burnstock, 1998).
32
PART I – INTRODUCTION
Figure 10. Receptors for extracellular nucleotides and adenosine. The P1 family of receptors for
extracellular adenosine are G-protein-coupled receptors that signal by inhibiting or activating adenylate cyclase
(a). The P2 family of receptors bind extracellular ATP or ADP, and are comprised of two types of receptor (P2X
and P2Y). The P2X family of receptors are ligand-gated ion channels (b) and the P2Y family are G-proteincoupled receptors (c). S–S, disulphide bond; e1–e4, extracellular domain loops 1–4; i1–i4, intracellular domain
loops 1–4. (Fields and Burnstock, 2006).
33
PART I – INTRODUCTION
4.4.1. P1 RECEPTORS
The P1 receptors or adenosine receptors are a class of purinergic receptors belonging to the
superfamily of seven transmembrane-spanning receptors (Fig. 10, a). There are four
different adenosine receptors, denoted A1, A2A, A2B, and A3 (Ralevic & Burnstock, 1998;
Fredholm et al., 2001). These receptor subtypes bind extracellular Adenosine (Ado) with
different affinities. The A2A and A2B receptors preferably interact with members of the Gs
family of G proteins (activation of adenylate cyclase) and the A1 and A3 receptors with Gi/o
proteins (inhibition of adenylate cyclase).
4.4.2. P2 RECEPTORS
Purinergic P2 receptors family are subdivided into two categories, the ionotropic channel:
P2X receptor and the metabotropic G protein-coupled: P2Y receptors.
4.4.2.1. P2X RECEPTORS
P2X receptors are a family of cation-permeable ligand gated ion channels that open in
response to the binding of extracellular ATP. To date, seven genes coding for P2X subunits
have been characterized and named to as P2X1 through P2X7 (Khakh et al., 2001; North,
2002). All subunits of P2X receptors share a common topology, possessing two plasma
membrane spanning domains, a large extracellular loop and intracellular carboxyl and amino
termini (Fig. 10, b).
4.4.2.2. P2Y RECEPTORS
The first P2Y receptors were cloned in 1993 (Lustig et al., 1993; Webb et al., 1993). They
corresponded to receptors previously characterized by pharmacological criteria: P2Y1
(formerly P2Y) and P2Y2 (formerly P2U). Nowadays, there are eight accepted human P2Y
receptors: P2Y1,2,4,6,11,12,13, and P2Y14 (Abbracchio et al., 2003). The missing numbers
represent either nonmammalian orthologs or receptors having some sequence homology to
P2Y receptors but for which there is no functional evidence of responsiveness to
nucleotides.
In the 90s my host laboratory in the IRIBHM has cloned and characterized several human
P2Y receptors.
Human P2Y4 receptors have been cloned and characterized in 1995 and UTP is the most
potent activator (Communi et al., 1995 ; Nicholas et al., 1996). Human P2Y6 receptors are
UDP receptors cloned and characterized in 1996 (Communi et al., 1996). Among P2Y
receptors, the human P2Y11 has some unique properties (Communi et al., 1997): it is coupled
to Gs (cAMP pathway) whereas P2Y1, P2Y2, P2Y4 and P2Y6 receptors are coupled to Gq/11
(inositol phosphate/Ca2+ pathway), its affinity for its natural ligand ATP is low , it is the only
34
PART I – INTRODUCTION
P2Y receptor gene that contains an intron in the coding sequence (P2X family receptors
contain introns as well) and there is no functional rodent ortholog of P2Y11. The human P2Y12
receptors have been identified and characterized in 2001 and ADP is the natural agonist of
this receptor (Hollopeter et al., 2001). The human P2Y13 receptors have been identified and
characterized in 2001. ADP is the naturally agonists of the receptor (Communi et al., 2001).
Finally The P2Y14 receptor is activated by UDP-glucose (Chambers et al., 2000).
The P2Y receptors display the general motif of a single-polypeptide chain forming seven
helical transmembrane domains (TM), which are connected by three extracellular and three
intracellular loops. The ends of the chain form an extracellular amino-terminal region and a
cytoplasmic carboxyl-terminal region, as shown for the P2Y1 receptor (Fig. 10, c).
Classification of P2Y receptors
Alignment of the amino acid sequences of the cloned P2Y receptors has shown that the
human members of this family are 21 to 48% identical (Table 4). The highest degree of
sequence identity is found among the second subgroups of P2Y12,13,14. Furthermore, the
eight P2Y receptors identified so far have a H-X-X-R/K motif in TM6. The P2Y1,2,4,6,11
receptors share a Y-Q/K-X-X-R motif in TM7, whereas in P2Y12,13,14 receptors, this motif is
substituted with K-E-X-X-L (Abbracchio et al., 2003). These positively charged residues in TM
6 and 7 are crucial for receptor activation by nucleotides (Erb et al., 1995 ; Jiang et al., 1997).
They probably interact with the negative charges of the phosphate groups of nucleotides.
Therefore, from a phylogenetic and structural point of view, two distinct P2Y subgroups
characterized by a relatively high level of sequence divergence can be identified. The first
subgroup includes P2Y1,2,4,6,11 subtypes and the second subgroup encompasses the P2Y12,13,14
subtypes. Finally, these two P2Y receptor subgroups also differ in their primary coupling to
transductional G proteins. In particular, receptors in the first subgroup (P2Y 1,2,4,6,11) all
principally use Gq/G11 to activate the PLCβ/IP3 pathway and release intracellular calcium,
whereas receptors in the second subgroup (P2Y12,13,14) almost exclusively couple to members
of the Gi/o family of G proteins.
Receptor
P2Y1
P2Y2
P2Y4
P2Y6
P2Y11
P2Y12
P2Y13
P2Y14
Agonist
(human)
ADP
ATP/UTP
UTP
UDP
ATP
ADP
ADP
UDP-glucose
P2Y1
/
P2Y2
38
/
P2Y4
44
41
/
Percentage of Identity
P2Y6
P2Y11
P2Y12
46
32
24
41
29
25
43
32
25
/
34
24
/
22
/
P2Y13
24
26
26
24
21
48
/
P2Y14
27
26
28
23
23
47
47
/
Table 4 : classification of P2Y receptors into two subsets
35
Amino Acid Motifs
TM6
TM7
HXXK
QXXR
HXXR
KXXR
HXXR
KXXR
HXXK
KXXR
HXXR
QXXR
HXXR
KEXXL
HXXR
KEXXL
HXXR
KEXXL
PART I – INTRODUCTION
Molecular modelling of P2Y receptors
The two distinct subgroups of P2Y receptors were successfully modelled by homology
modelling, with the high-resolution structure of bovine rhodopsin serving as a template
(Moro & Jacobson, 2002). More recently computational models of all of the P2Y receptors
were derived from a multiple-sequence alignment based on a combined manual and
automatic approach, which takes into account not only the primary structure of the proteins
but also the three-dimensional information deducible from the secondary and tertiary
structures of the template (Fig. 11). Ligand docking modelling was performed on the P2Y1
and P2Y12 receptor models. The results suggested that ADP binds to the cavity delimited by
TM1, TM2, TM3, TM6 and TM7 and capped with extracellular loop 2 (Costanzi et al., 2004).
Figure
11.
Theoretical
structures
of
the
putative
nucleotide
binding sites of P2Y1
(A) and P2Y12 (B)
receptors based on
mutagenesis
and
molecular
modeling
experiments (Costanzi
et al., 2004).The large
figures
show
the
binding sites as viewed
from the plane of the
plasma membrane with
docked
nucleotide
ligands. Key residues
found to interact with
the
ligand
are
indicated.
color coded:
cyan:
orange:
green:
magenta:
blue:
red:
gray:
TM1;
TM2;
TM3;
TM4;
TM5;
TM6;
TM7
Function of P2Y2 receptor subtype
Amongst the P2Y receptors family, P2Y2 receptors, previously known as P2U, have been
cloned and pharmacologically characterized in 1993 (Lustig et al, 1993). P2Y2 is a ubiquitous
36
PART I – INTRODUCTION
receptor: mRNA has been detected in human skeletal muscle, heart, brain, spleen,
lymphocytes, macrophages, bone marrow, liver, stomach, pancreas and lung (Moore et al,
2001). P2Y2 receptors are fully activated by equivalent concentrations of ATP and UTP,
whereas ADP and UDP are less effective agonists (Lustig et al, 1993; Parr et al, 1994;
Lazarowski et al, 1995a). UTPS has been shown to be a potent hydrolysis resistant agonist
of P2Y2 receptors (Lazarowski et al, 1995b). Suramin acts as a competitive antagonist of
human and rat P2Y2 receptors (Charlton et al, 1996).
P2Y2 receptors can directly couple to PLC1 via Gq/11 protein to mediate the production of
Inositol 1,4,5 Triphosphate (IP3) and diacylglycerol (DAG), second messengers for calcium
release from intracellular stores and PKC activation, respectively (Fig. 12).
Figure 12. The Dual Signalling Pathway: IP3,
DAG & Calcium (http://www.utm.utoronto.ca)





37
PLC = Phospholipase C
PIP2 = Phosphatidylinositol 1,4
bisphosphate
IP3 = Inositol 1,4,5 Trisphosphate
DAG = Diacylglycerol
PKC = Protein Kinase C (C = Kinase)
PART I – INTRODUCTION
Site-directed mutagenesis of the P2Y2 receptor has been used to demonstrate that
replacement of positively charged amino acids in TM helices 6 and 7 with neutral amino
acids decreases the potencies of ATP and UTP, suggesting that these domains play a role in
binding the negatively charged moieties of nucleotide agonists (Erb et al., 1995).
inflammation and immunity
The P2Y2 receptor contains the consensus integrin-binding motif, Arg-Gly-Asp (RGD) in its
first extracellular loop that facilitates P2Y2 receptor colocalization with V3/5 integrins (Erb
et al., 2001). The V3/5 integrins are known to regulate angiogenesis and inflammatory
responses including cell proliferation, migration, adhesion, and infiltration (Hutchings et al.,
2003), responses also mediated by P2Y2 receptor activation (Wilden et al., 1998 ; Seye et al.,
2002 ; Greig et al., 2003a ; 2003b ; Schafer et al., 2003 ; Bagchi et al., 2005 ; Kaczmarek et al.,
2005), suggesting that nucleotides may transactivate integrin signalling pathways by virtue
of P2Y2 receptor binding to integrins.
In addition, P2Y2 receptor up-regulation in endothelial cells increases the binding of
monocytes to endothelial cells due to P2Y2 receptor-mediated increases in the endothelial
expression of vascular cell adhesion molecule (VCAM)-1 (Seye et al., 2003). Up-regulation of
VCAM-1 was shown to be dependent on P2Y2 receptor-mediated transactivation of vascular
endothelial growth factor receptor-2 (KDR/Flk-1), a response that was inhibited by deletion
of the proline-rich, SH3 binding sites (PXXP) from the P2Y2 receptor, demonstrating a
mechanism whereby P2Y2 receptors can cause inflammatory responses (Seye et al., 2004).
The effects of P2Y2 receptor signalling is extended to the mobilization of cells engaged in the
destruction of inhaled pathogens, including monocytes/macrophages and leukocytes. In
most instances, these actions are essential to resolve airway infection. On the other hand,
chronic or exaggerated activation of these functions lead to irreversible damage to the
respiratory system. Whether P2Y2 receptors are beneficial or detrimental in these two
scenarios was examined using models of acute and chronic lung complications with
knockout mice (see PART III - Results).
4.4.2.3. THERAPEUTIC APPLICATION OF P2 AGONISTS AND
ANTAGONISTS
P2X1, P2Y1 and P2Y12 are target receptor in platelet activation
Platelets express three nucleotide receptors: the P2X1 cation channels activated by ATP, and
two GPCRs, P2Y1 and P2Y12, both activated by ADP. Each of these receptors has a selective
role during platelet activation (Hechler et al., 2005), which has implications for their role in
thrombosis (Gachet & Hechler, 2005). The deficiency of P2X1 does not modify the platelet
responses to ADP whereas P2Y1 and P2Y12 coactivation is necessary for normal ADP-induced
platelet aggregation since separate inhibition of each of them by selective antagonists
results in dramatic inhibition of aggregation (Jin & Kunapuli, 1998 ; Gachet, 2001 ; Hechler et
38
PART I – INTRODUCTION
al., 2005). Then it’s not hard to understand that one of the most interesting therapeutic
applications of P2Y receptors is currently represented by the use of antagonists of these
receptors as antithrombotic agents in the prevention of recurrent stroke and heart attacks.
The agents currently on the market (Ticlopidine and Clopidogrel) target the P2Y12 receptor
(Thebault et al., 1975 ; Savi et al., 1998) . The thienopyridines (Ticlopidine and Clopidogrel)
inactivate the P2Y12 receptor irreversibly via covalent binding of an active metabolite
generated in the liver (Thebault et al., 1975 ; Savi et al., 1998).
P2Y2 is a target receptor in cystic fibrosis
In healthy lungs, the balance between Cl- secretion and Na+ absorption represents a major
mechanism of signalling to regulate mucociliary clearance activities. P2Y2 receptor activation
increases Cl- secretion and inhibits Na+ absorption in epithelial cells.
In cystic fibrosis, a disease that is caused by genetic defects in the gene for the CFTR (Cystic
fibrosis transmembrane conductance regulator), a major epithelial anion channel, P2Y2 has
promising perspectives as a therapeutic target to promote CaCC (Ca 2+-activated Cl- channel)
activity improving poor Cl- production in the airway associated with defective CFTR (Clarke &
Boucher, 1992 ; Parr et al., 1994) (Fig. 13).
Unfortunately, today, cystic fibrosis treatment like Denufosol Tetrasodium from Inspire
Pharmaceuticals, failed to beat placebo in phase 3 clinical trials despite a good start
(Accurso, et al 2011).
Figure 13. Purinergic regulation of ion transport. ATP promotes P2Y2 receptor mediated CaCC activity, and
inhibition of the epithelial Na+ channel ENaC. Potentially, the P2Y2 receptor promotes PKC-mediated CFTR
activation. ATP hydrolysis results in adenosine accumulation (Ado), which in turns activates the A2b receptor,
leading to cyclic AMP (cAMP) and protein kinase A (PKA)-mediated CFTR activation. CFTR : Cystic fibrosis
transmembrane conductance regulator. (Lazarowski et al., 2009).
39
PART I – INTRODUCTION
P2Y2R knockout mice are as viable as wild-type mice. The fact that the airways are not
obstructed in these mice is consistent with the distinct roles of P2Y2R-CaCC and A2BR-CFTR
signalling pathways in mucociliary clearance (MCC). Under steady-state conditions, the
surface Ado concentrations partially activate A2BRs, which maintains sufficient CFTRmediated fluid secretion for MCC. In contrast, the surface ATP concentrations periodically
reach the activation threshold of P2Y2Rs during normal tidal breathing, as a result of
mechanical stress-induced nucleotide release. This weak and transient activation stimulates
fluid and mucin secretion to maintain a thin shield above the ciliated epithelium. Together,
these Ado- and ATP-mediated signaling events continuously produce and evacuate this
protective film along the ciliary escalator. On the other hand, an alarm situation, sensed by
the interaction of an irritant or pathogen with the epithelium, initiates a robust and
transient MCC response through ATP release and P2Y2R activation. The rapid metabolism of
ATP into Ado by ectonucleotidases triggers a second wave of fluid secretion through
enhanced A2BR-CFTR activity and ciliary beating, both gradually returning to baseline with
Ado concentration. Hence, P2Y2R-mediated MCC only predominates during alarm situations.
40
PART II – AIM OF THE STUDY
PART II – AIM OF THE STUDY
The P2Y2 receptor is a ubiquitous receptor that is fully activated by ATP and UTP. His
implication is then really considerable in several biological mechanisms and process.
Therefore, we decided to study and characterize the role of this receptor in physiological and
pathological situations using wild-type and knock-out mice for the P2Y2 receptor. An
interesting paper on the role of ATP in asthma by Idzko and colleagues, in 2007 (see results)
put us to study the involvement of P2Y2 receptor in this disease. The choice of lung diseases
was furthermore supported by the fact that P2Y2 is implicated in diseases like cystic fibrosis,
infection of the lung by Pseudomonas aeruginosa and acute lung injury and constitutes an
attractive candidate for the development of new therapeutic drugs.
The aim of our work is to identify potential roles of the P2Y2 receptor in inflammatory lung
diseases, using a set of models for various lung diseases in mouse. To assess these questions,
we used the P2Y2 knock-out mice and their wild-type controls in several models such as
asthma, acute lung injury, viral pneumonia, … Some of these models were performed in the
laboratory, whereas others were made in collaboration with University of Liège (ULg) for
models of viral pneumonia (Laboratory of Pathology, Prof. D. Desmecht).
43
PART III – RESULTS
PART III – RESULTS
1. ANIMAL MODELS OF AIRWAY DISEASES IN P2Y2 RECEPTOR
KNOCKOUT MICE
The mouse is often used as a model to improve our knowledge on human physiological and
physiopathological conditions. Among the reasons for the choice of this specific mammalian
species, there are the brreding efficiency, the possibility to work with genetically identical
cohorts (inbred strains), and the existence of many genetically modified strains. As a
consequence, mouse models have been widely used in many aspects of biomedical research,
which has led in particular to a growing understanding of the mouse immune system and its
involvement in pathological conditions (Bates et al., 2009). However, as mice are not
humans, no mouse model completely recapitulates all features of the corresponding human
disease and the applicability to human depends on the understanding of the similarities and
differences between two species (Mitzgerd & Skerrett, 2008).
P2Y2 receptor knockout mice are fertile, undistinguishable from their wild-type littermates,
and show no abnormality in their organs, including the heart, lung, pancreas, intestines,
kidneys and trachea.
1.1.
CHARACTERIZATION OF THE ROLE OF THE ATP/P2Y2R
SYSTEM IN ASTHMA
Asthma is one of the commonest chronic diseases of affluent societies. Indeed, according to
the Global Initiative for asthma (GINA), approximately 300 million people, regardless of age
and ethnicity, suffer from asthma.
A major issue with asthma is its heterogeneity and the multiplicity of different and
overlapping phenotypes. Cardinal features of asthma include airway inflammation,
reversible airflow obstruction, airway hyperresponsiveness (AHR) and tissue remodelling
with CD4+ helper cells, mast cells, and eosinophils. Asthma immunopathology is dominated
by a T helper (Th) cell response skewed towards a Th2 cytokine/chemokine pattern that also
involves airway smooth muscle (ASM) cells and tissue remodelling. Pathogenesis is
orchestrated by a complex interplay between several immune and inflammatory cells and
their secreted products.
Asthmatic patients maintain normal airway ATP levels, but these concentrations rise tenfold
in response to an allergen. The consequences of this excess ATP were examined in the OVA
sensitization/challenge model of allergic asthma (Idzko et al, 2007). The mice are sensitized
by intra-peritoneal injection, and then challenged by nebulization 10 days later. After 24 h,
their BAL fluid had accumulated eightfold higher ATP concentrations than in the
saline-challenged control animals, as reported in the allergen-challenged asthmatic patients.
They also developed all major features of allergic asthma, including eosinophil-dominant
47
PART III – RESULTS
lung infiltrates, goblet cell metaplasia, hyperresponsiveness to methacholine and Th2
inflammation in the lymph nodes (IL-4, IL-5 and IL-13). These complications were
significantly reduced when the OVA challenges were conducted in the presence of an
ATP-metabolizing enzyme (apyrase) or P2Y receptor antagonist (PPADS or suramin). Through
a series of in vivo and in vitro protocols, Idzko et al. demonstrated that the excess ATP
generated after an allergen challenge triggers airway inflammation by the activation of
resident dendritic cells, and their mobilization to the lymph nodes to trigger a Th2 type
inflammation. Consequently, this study supports the therapeutic potential of P2Y receptor
antagonists for the treatment of airway inflammation in asthmatic patients.
Figure 14. The Physiopathology of asthma. Inhaled allergens activate sensitized mast cells by crosslinking
surface-bound IgE molecules to release several bronchoconstrictor mediators. Allergens are processed by
myeloid dendritic cells, which are conditioned by thymic stromal lymphopoietin (TSLP) secreted by epithelial
cells and mast cells to release the chemokines CC-chemokine ligand 17 (CCL17) and CCL22, which act on
CC-chemokine receptor 4 (CCR4) to attract T helper 2 (Th2) cells. Th2 cells have a central role in orchestrating
the inflammatory response in allergy through the release of interleukin-4 (IL-4) and IL-13 (which stimulate B
cells to synthesize IgE), IL-5 (which is necessary for eosinophilic inflammation) and IL-9 (which stimulates
mast-cell proliferation). Epithelial cells release CCL11, which recruits eosinophils via CCR3. (Barnes, 2008).
Following the very interesting study of Idzko and colleagues, and the well establish role of
the P2Y2 receptor in the lung disease, we start our own model of asthma on P2Y 2-deficient
mice. These data were published in the “Journal of Immunology” in 2010.
48
PART III – RESULTS
49
PART III – RESULTS
50
PART III – RESULTS
51
PART III – RESULTS
52
PART III – RESULTS
53
PART III – RESULTS
54
PART III – RESULTS
1.2.
P2Y2R IS NOT ESSENTIAL IN THE PATHOGENY OF
PULMONARY FIBROSIS MODEL INDUCED BY BLEOMYCIN
Like in asthma, Idiopathic pulmonary fibrosis (IPF) is an inflammatory lung disease associated
with elevated airway ATP concentrations (Riteau et al., 2010). IPF is a chronic progressive
and ultimately fatal lung disease of the lower respiratory tract, characterized by
inflammation, abnormal and excessive deposition of collagen (fibrosis) in the interstitium
accompanied by an excessive production of extracellular matrix proteins. IPF is the
consequence of recurrent epithelial damage with alveolar epithelial cells injury and
hyperplasia and epithelial–mesenchymal transition, leading to destruction of the alveolar
architecture. By the time patients seek medical treatment for symptoms, the disease process
is generally advanced, and the evolution of the process is uncertain. The situation with IPF is
even more complicated, since the etiology of the disease is unclear and no single trigger is
known that is able to induce IPF.
Different models of pulmonary fibrosis have been developed over the years. Most of them
mimic some, but never all features of human IPF, especially the progressive and irreversible
nature of the condition. Common methods include radiation damage, instillation of
bleomycin, silica, and transgenic mice. The bleomycin model of pulmonary fibrosis is the
best characterized murine model in use today (Adamson & Bowden, 1974).
Bleomycin is a chemotherapeutic antibiotic, produced by the bacterium Streptomyces
verticillus (Umezawa et al., 1966). This antibiotic was found to be effective against squamous
cell carcinomas and skin tumors (Umezawa, 1974). Its use in animal models of pulmonary
fibrosis is based on the fact that fibrosis is one of the major adverse drug effects of
bleomycin in human cancer therapy. Bleomycin acts by causing single and double-strand
DNA breaks in tumor cells and thereby interrupting the cell cycle (Claussen & Long, 1999 ;
Chaudhary et al., 2006 ; Grande et al., 1998).
In mice, bleomycin causes inflammatory and fibrotic reactions within a short period of time,
even more so after intratracheal instillation. The initial elevation of pro-inflammatory
cytokines (interleukin-1, tumor necrosis factor-α, interleukin-6, interferon-) is followed by
increased expression of pro-fibrotic markers (transforming growth factor-β1, fibronectin,
procollagen-1), with a peak around day 14. The “switch” between inflammation and fibrosis
appears to occur around day 9 after bleomycin (Chaudhary et al., 2006). Moreover,
histological hallmarks, such as intra-alveolar buds, mural incorporation of collagen and
obliteration of the alveolar space, are present in bleomycin-treated animals similar to IPF
patients (Usuki & Fukuda, 1995).
In our fibrosis model induced by bleomycin we did not succeed to observe any differences
between P2Y2+/+ and P2Y2-/- mice. To assess the role of P2Y2R, 8-week-old wild-type (WT) and
P2Y2-/- mice were inoculated intratracheally with 0,05U (0,05mg) of bleomycin and were
55
PART III – RESULTS
monitored daily for survival and weight loss (Fig. 15A). This dose of bleomycin allows fibrosis
to settle without mortality. WT and P2Y2-/- mice began to lose weight right after the
inoculation of bleomycin and then they all recover around day 10 post-inoculation. We also
tried a higher dose of bleomycin (0,15U = 0,15mg) to monitor bodyweight and to observe
survival in extreme conditions. Once again, no differences were observed between the 2
groups of mice regarding the bodyweight and survival (Fig. 15B and C). Despite the lack of
differences in the bodyweight, we analysed leukocytes recruitment into the BALF of mice at
days 6 and 21 after bleomycin inoculation. As mentioned above, days 6 (Fig. 16) after
bleomycin inoculation is before the “switch” between the inflammation process and the
fibrosis whereas days 21 (Fig. 17) post inoculation is during the fibrosis process. We also
stained lungs of WT and P2Y2-/- mice with Masson’s trichrome (Fig. 18). Masson's trichrome
is a three-colour staining protocol which produces red muscle fibres, blue collagen, and black
cell nuclei. Unfortunately, all these investigations did not succeed and we did not observe
any phenotypic change between WT and P2Y2-/- mice.
Figure 15. Bodyweight and survival curves of P2Y2+/+ and P2Y2-/- mice. Following intranasal inoculation
of bleomycin (0,05 and 0,15U), P2Y2-/- and wild-type (WT) C57BL/6 mice were monitored daily for weight loss
and survival. A, Bodyweight loss with a low dose of bleomycin (0,05U). B, Bodyweight loss and survival curves
(C) with a high dose of bleomycin (0,15U).
56
PART III – RESULTS
Figure 16. Comparable airway inflammation in bleomycin-inoculated P2Y2+/+ and P2Y2-/- at day 6. WT
and P2Y2-/- mice were inoculated with 0,05U of bleomycin and sacrificed at day 6. A, BALF was collected and the
total number of cells was evaluated. Cytospin preparations of BALF (B) from WT and P2Y2-/- were counted (D).
C, Flow-cytometry quantification of macrophages, lymphocytes and eosinophils in the BALF of P2Y2+/+ and
P2Y2-/-.
Figure 17. Comparable airway inflammation in bleomycin-inoculated P2Y2+/+ and P2Y2-/- at day 21.
WT and P2Y2-/- mice were inoculated with 0,05U of bleomycin and sacrificed at day 21. A, BALF was collected
and the total number of cells was evaluated. B, Flow-cytometry quantification of macrophages, lymphocytes
and eosinophils in the BALF of P2Y2+/+ and P2Y2-/-.
57
PART III – RESULTS
Figure 18. Pathology seen with bleomycin. A, Trichrome staining of a lung section on day 21. The blue
staining represents collagen deposition. B, Quantification of the collagen deposition with the plugin Threshold
Colour from ImageJ software.
We assessed the role of P2Y2 receptor in the fibrotic lung response using the
bleomycin-induced model. Clearly, our results showed no differences between P2Y2+/+ and
P2Y2-/- mice.
In the absence of clear phenotype for P2Y2-/- mice, these models were not investigated further.
58
PART III – RESULTS
1.3.
PROTECTIVE ROLE OF P2Y2R AGAINST LUNG INFECTION BY
PNEUMONIA VIRUS OF MICE
In this study, we used a viral pneumonia model induced by the pneumonia virus of mice (PVM). PVM,
along with the human respiratory syncytial virus (hRSV), are viruses of the paramyxoviridae family
(subfamily Pneumovirinae), which are enveloped, negative sense, single-stranded RNA viruses
(Easton et al., 2004). Pneumovirus virions are generally spherical but PVM virions are predominantly
filamentous, 100 to 120 nm in diameter, and up to 3 µm in length. Pneumoviruses have genomes of
approximately 15 kB. The encoded proteins are well conserved among pneumoviruses and play
important functions for attachment and entry into host cells, viral replication, ….
The pneumovirus virion is surrounded by a lipid envelope derived from the plasma membrane of the
host cell, into which the three viral glycoproteins, the attachment (G), fusion (F), and small
hydrophobic (SH) proteins, are inserted (Fig. 19). The helical nucleocapsid is located within the Mprotein layer, and includes the single-stranded RNA genome in association with the virus
nucleoprotein (N) and large (L) proteins and the phosphoprotein (P).
Figure 19. Schematic representation of the pneumovirus particle. The RNA genome associates with viral
proteins to form the helical nucleocapsid structure (represented on the right and in the center of the virion on
the left). The proteins consist of the nucleocapsid protein (N), the phosphoprotein (P), and the large
polymerase (L) protein. The nucleocapsid structure is surrounded by the matrix (M) protein, which forms a link
between the nucleocapsid and the lipid membrane of the virus particle. Embedded in the lipid membrane are
the attachment (G) glycoprotein, the fusion (F) protein, and the small hydrophobic (SH) protein (not shown).
Besides their phylogenic proximity and genomic similarities, another shared characteristic of the
PVM and the RSV pneumoviruses is their capacity to enhance airway hyperreactivity and Th2
response induced after an allergic sensitization and challenge by ovalbumin in mouse (Siegle et al.,
2010). Indeed, PVM infection acts similarly to early-life hRSV infection in human, which is known to
increase the risk of subsequent development of childhood asthma. According to these similarities,
59
PART III – RESULTS
PVM is considered as the mouse counterpart of human RSV, and presents the advantage of being a
natural rodent pathogen. Indeed, as a human pathogen, there is little if any replication of hRSV in
mice, so that murine models using this virus require a very large inoculum (>104 PFU). By comparison,
intranasal inoculation of fewer than 30 PFU of PVM results in clear-cut replication in mouse lung
tissue, yielding titers as high as 108 PFU/g (Easton et al., 2004).
PVM infection induces a disease that begins on day 6 post-infection and inoculation of more than
300 PFUs is generally lethal by day 9 post-infection (Domachowske et al., 2004). The primary targets
of PVM in vivo are respiratory epithelial cells (Bonville et al., 2006). Infection of alveolar
macrophages has been reported (Panuska et al., 1992) but efficient viral replication is observed only
in the cell line monocyte-macrophage RAW 264.7, and not in alveolar macrophages (Dyer et al.,
2007). In infected mice, virus replication is accompanied by a profound inflammatory response with
recruitment of neutrophils and eosinophils, marked edema, mucus production, and airway
obstruction, leading to significant morbidity and mortality. This is associated with marked respiratory
dysfunction and by local inflammatory mediators production including MIP-1 (CCL3), MIP-2 (CXCL2),
MCP-1 (CCL2) and IFN- (Bonville et al., 2006). Additionally, microarray analysis of transcripts from
lung tissue indicates that PVM infection promotes up-regulation of pro-inflammatory genes, like IFN, MCP-3 (CCL7), RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted – CCL5),
and eotaxin (CCL11) (Domachowske et al., 2002). Among granulocytes, as in enhanced RSV infection
in human, eosinophils are recruited to the lung of PVM-infected mice and represent about 3% of
total BALF cells at day 6 post-infection (Garvey et al., 2005). Eosinophils contribute to the
inflammatory state by synthesis of pro-inflammatory cytokines and chemokines (IL-6, IP-10, MIP-1,
MIP-2) (Dyer et al., 2009). However, their role seems to be limited, as infected mice deficient in
eosinophils present similar viral titers and weight loss (Percopo et al., 2009).
Subsequently, a predominant Th1 adaptive response occurs from day 8 post-infection, with a
pronounced influx of CD8+ cytotoxic T cells (Frey et al., 2008). This cytotoxic response is enhanced by
type I interferon production (IFN- and IFN-) and plays a crucial role in anti-PVM immunity, as it
contributes to control PVM replication and is correlated to the severity of the diseases in a viral dosedependent fashion. Thereby, it has been shown that T cell-deficient mice do not develop signs of
disease despite failure to control the virus (Frey et al., 2008).
Finally, besides these cellular responses, it has been demonstrated that PVM infection can also
induce an efficient humoral response, characterized by the production of anti-PVM antibodies. This
humoral response is IFN- independent, being identical for wild-type and IFN--deficient mice (Ellis et
al., 2007).
In our study, we demonstrated a protective role of P2Y2R in this model. Even though the underlying
mechanisms are still under investigation, our data suggest that P2Y2R might be considered as a
potential therapeutic axis for future anti-viral and anti-inflammatory therapies. These results were
included in a manuscript submitted for publication to the “Journal of Immunology”. The revision
process is ongoing.
60
PART III – RESULTS
ARTICLE PVM
61
PART IV – DISCUSSION AND CONCLUSIONS
PART IV – DISCUSSION AND CONCLUSIONS
According to the World Health Organization (2008 report), respiratory diseases (excluding
lung cancer) are the third cause of mortality worldwide.
World
Deaths in millions % of deaths
Ischaemic heart disease
7.25
12.8%
Stroke and other cerebrovascular disease
6.15
10.8%
Lower respiratory infections
3.46
6.1%
Chronic obstructive pulmonary disease (COPD)
3.28
5.8%
Diarrhoeal diseases
2.46
4.3%
HIV/AIDS
1.78
3.1%
Trachea, bronchus, lung cancers
1.39
2.4%
Tuberculosis
1.34
2.4%
Diabetes mellitus
1.26
2.2%
Road traffic accidents
1.21
2.1%
Table 1. The 10 leading causes of death (World Health Organization - 2008)
For decades, these kinds of reports have suggested that cardiac and infectious illnesses are
the main causes of death in the developed and developing world. If respiratory illnesses,
including pneumonia, bronchitis, chronic obstructive pulmonary disease, cancer and
tuberculosis in the respiratory system, among others, were grouped separately, the sum
would reach 17% of all deaths and would be the top cause of death in the world.
The European Lung Foundation (ELF) predicts a further increase in the number of deaths
from lung diseases between now and 2020, in particular from chronic obstructive pulmonary
disease (COPD), lung cancer and tuberculosis. In 2020, out of 68 million deaths worldwide,
11.9 million will be caused by lung diseases (4.7 by COPD (+43.3%), 2.5 by pneumonia, 2.4 by
tuberculosis (+79.1%) and 2.3 million by lung cancer (+65.5%)).
It is important that these common health problems are recognized not just as health-care
issues but also as financial problems affecting the productivity of those suffering from them.
As reported by the European Lung Foundation the total financial burden of lung disease in
Europe amounts to nearly €102 billion. COPD contributes to almost one-half of this figure,
followed by asthma, pneumonia, lung cancer and tuberculosis.
Amongst respiratory diseases, inflammatory lung diseases constitute a major part of public
health problem. As a consequence, investigating the immune mechanisms that contribute to
the pathogenesis of these diseases is essential to identify candidate targets for the
development of new therapeutic drugs. Furthermore, over the past 20 years, the growing
awareness that purinergic signalling events shape the immune and inflammatory responses
to infection and allergic reactions warranted the development of animal models to assess
their importance in vivo in acute lung injury and chronic airway diseases. The pioneer work
conducted with the adenosine deaminase-deficient mouse provided irrefutable evidence
that excess adenosine (ADO) accumulating in the lungs of asthmatic patients, constitutes a
powerful mediator of disease severity (Blackburn et al., 1998). Therapeutic targets are
currently evaluated using knockout mice and agonists/antagonists for each ADO and ATP
65
PART IV – DISCUSSION AND CONCLUSIONS
receptors. In general, these mice present no apparent phenotype or lung complication,
unless they are subjected to pathological conditions.
The field of purinergic inflammation formulated the unifying concept that ATP is released as
a «danger signal» to induce inflammatory responses upon binding purinergic receptors. The
most convincing evidence that the P2Y2 receptor is engaged during alarm situations comes
from the studies conducted on the knockout mice about cystic fibrosis (see Intro 4.4.2.3).
Chronic respiratory diseases are commonly associated with elevated airway ATP
concentrations, as reported in cystic fibrosis, but also in idiopathic pulmonary fibrosis (IPF)
and chronic obstructive pulmonary disease (COPD) patients (Riteau et al., 2010 ;
Lommatzsch et al., 2010), and they are raised by allergens in asthmatic patients (Idzko et al.,
2007). Asthma is one of the commonest chronic diseases of western societies. The striking
increase of asthma over recent decades and the rarity of this disease in less affluent
populations confirm the importance of environmental factors in the cause of asthma –
although which environmental factors are responsible is still not clear. Family studies show
that genetic factors are also important in determining individual susceptibility. There is no
mouse model that completely reproduces all the features of the human disease, but a
number of murine asthma models mimic the features of asthma and allow the study of
underlying cellular and molecular mechanisms. The Th2-driven airway changes in these
models are mainly evoked by exposure of the airways to archetypic antigen ovalbumin (OVA)
for several days after systemic sensitization. This widely used model reproduce the airway
eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma. Idzko
and colleagues demonstrated that the excess ATP generated after an allergen challenge
triggers airway inflammation by the activation of resident dendritic cells, and their
mobilization to the lymph nodes to trigger a Th2 type inflammation (see PART III – Results,
chapter 1.1) (Idzko et al., 2007).
In this work, the P2Y2 receptor was investigated in several mouse models to assess his role in
the physiopathology of inflammatory lung diseases.
Role of the P2Y2 receptor in the case of Th2-related disease:
Ovalbumin-induced asthma model
Because of the well-established role of ATP which is a major mediator of lung inflammation
in asthma and a potent agonist at the P2Y2 receptor, and because of the availability of the
P2Y2 knock-out mice in the lab, we decided to evaluate the possible contribution of this
receptor in an ovalbumin(OVA)-induced asthma model.
In our model of asthma, we demonstrate that allergic lung inflammation is decreased in
animals lacking the P2Y2 receptor. Indeed, we have observed a significant reduction in
vascular cell adhesion molecule(VCAM)-1 expression on lung endothelial cells and the level
of soluble VCAM-1 (sVCAM-1) was strongly reduced in the bronchoalveolar lavage fluid
(BALF) of P2Y2-/- as well. Concomitantly a defective accumulation of eosinophils was
66
PART IV – DISCUSSION AND CONCLUSIONS
observed in P2Y2-deficient lungs. Finally, we have observed that ATP increased leukocyte
adhesion on lung endothelial cells through P2Y2 receptor activation and VCAM-1 increase
(Vanderstocken et al., 2010). These findings support a role for the P2Y2R-mediated
up-regulation of VCAM-1 in the purinergic stimulation of lung eosinophilia. This molecule is a
major chemoattractant and adhesion molecule for leukocytes, such as eosinophils which are
among the major effector cells in asthma. Additionally, sVCAM-1 was described as an
inducer of eosinophil chemotaxis (Ueki et al., 2009) and allergen-induced accumulation of
eosinophils was previously associated with increased levels of this form of VCAM-1 (Fernvik
et al., 1999). VCAM-1 is expressed by endothelial cells, smooth muscle cells, fibroblasts,
macrophages, and neurons and mediates adhesion of leukocytes expressing α4β1 integrins
(VLA-4) in response to inflammatory cytokines. VCAM-1–deficient embryos were not viable
and displayed severe defects in placental and heart development (Kwee et al., 1995).
VCAM-1 is a 715 amino acid (aa) type I transmembrane glycoprotein and displays an
extracellular region of 674 aa, a transmembrane region of 22 aa, and a 19 aa cytoplasmic tail
(Osborn et al., 1989). The proteolytic cleavage and release of transmembrane surface
proteins are important posttranslational mechanisms for the regulation of their functions
(Hooper et al., 1997). The ectodomain shedding occurs for proteins, such as growth factors,
cytokines, and adhesion molecules, and is mainly mediated by matrix metalloproteinases
and disintegrin metalloproteinases. TNF-–converting enzyme (TACE or ADAM 17) regulates
the phorbol 12-myristate 13-acetate(PMA)-induced release of sVCAM-1 by shedding of the
VCAM-1 expressed by murine endothelial cells (Garton et al., 2003).
In addition to the above-mentioned results, myeloid dendritic cells (mDCs) which are able to
take up antigens and initiate Th2-driven immune responses are also essential in asthma. In
vitro assays showed that circulating dendritic cells and bone marrow eosinophils collected
from P2Y2R-/- mice fail to migrate within an ATP gradient (Müller et al., 2010). Similar
observations have been made with neutrophils whose migratory response to ATP is
controlled by P2Y2R and (when metabolized to adenosine) A3 receptors (Chen et al., 2006 ;
Inoue et al., 2008). Additionally, mDCs and eosinophils from asthmatic subjects show a
higher expression of P2Y2R compared to healthy individuals (Müller et al., 2010).
In summary, first, the OVA challenge caused the accumulation of soluble VCAM-1 in the BAL
fluid, but to a lower level in P2Y2R-/- mice and also induced an up-regulation of membranebound VCAM-1 at the surface of lung endothelial cells, but to a lower extent in P2Y 2R-/- mice.
Second, P2Y2R expression is up-regulated on DCs and eosinophils and this is accompanied by
a stronger chemotactic response to ATP from both the cells. In addition, adhesion assays
confirmed that ATP promotes eosinophil migration and adhesion to the endothelial surface
through a P2Y2R-mediated increase in VCAM-1 surface expression. Finally, as a consequence
of the previous points, the P2Y2-/- mice exhibit a weaker capacity to recruit eosinophils in the
lungs in response to the experimentally OVA-induced asthma protocol than the wild-type
mice. Nevertheless, the ubiquitous expression of P2Y2R suggests that other inflammatory
defects in P2Y2-/- mice might contribute to the phenotype.
67
PART IV – DISCUSSION AND CONCLUSIONS
Collectively, these studies suggest that an amplification of the ATP-P2Y2R signalling in
asthmatic patients during an allergic reaction, contributes to the development of lung
eosinophilia. The proinflammatory action of ATP in the lung could occur through DC
activation, as well as through the regulation of endothelial and soluble VCAM-1 expression.
Accordingly, asthmatic patients may benefit from the intravenous injection of P2Y 2R
antagonists to suppress eosinophil recruitment to the airways.
Role of the P2Y2 receptor in the case of innate inflammatory disease
Lipopolysaccharide-induced acute lung injury model
To evaluate other contributions of P2Y2R in lung inflammation, we also performed an acute
lung injury (ALI) model induced by lipopolysaccharide (LPS) administration using P2Y2+/+ and
P2Y2-/- mice. This is a rapid model where leukocytes (mainly neutrophils and macrophages)
are recruited to the lung within 4 hours of LPS treatment. We observed comparable
infiltration of neutrophils and macrophages in the BALF of LPS-aerosolized P2Y2+/+ and P2Y2-/mice. The different data obtained with OVA and LPS models could be related to the much
lower level of ATP in the BALF of LPS-treated mice compared with OVA-treated mice
(Vanderstocken et al., 2010). Purinergic agonist ATPS (a nonhydrolized ATP analog) has
been shown to have a protective effect against LPS-induced model of ALI (Kolosova et al.,
2008). Indeed, the mice which received the ATPS exhibited about 50% less inflammatory
cells, cytokines and proteins in the BAL fluid, than the saline-treated LPS-exposed mice.
These data suggest that intravenous P2Y2 receptor agonists may reduce the airway
inflammatory responses and lung injury caused by airborne pathogens. In our LPS-induced
model of ALI it is intriguing that mice accumulate BALF neutrophils to the same extent as the
wild-type mice given the critical role of P2Y2R for their chemotaxis (Chen et al., 2006) as well
as ATPS seems to attenuate lung injury especially by reducing neutrophils accumulation.
1) Bleomycin-Induced pulmonary fibrosis model
As mentioned previously, Idiopathic pulmonary fibrosis (IPF) is one other inflammatory lung
disease associated with elevated airway ATP concentrations (Riteau et al., 2010). The
development of fibrotic lesions is dependent on the release of chemokines, most notably
MCP-1/CCL2 or MCP-5/CCL12, and the recruitment of inflammatory cells such as monocytes,
lymphocytes and fibrocytes (Moore & Hogaboam, 2008). More recently, dendritic cells have
also been identifying as key proinflammatory cells potentially able to sustain pulmonary
inflammation and fibrosis in the bleomycin model (Bantsimba-Malanda et al., 2010).
Unfortunately, in our fibrosis model induced by bleomycin we did not succeed to observe
phenotypic differences between P2Y2+/+ and P2Y2-/- mice.
68
PART IV – DISCUSSION AND CONCLUSIONS
Role of the P2Y2 receptor in the case of Th1-related disease: Model
of viral pneumonia
After investigating the role of the P2Y2 receptor in the Th2 response in a model of asthma,
we have evaluated the consequences of P2Y2R loss in lung inflammation initiated after
pneumonia virus of mice (PVM) infection.
For this model we use the natural mouse pathogen PVM who recapitulates many of the
clinical and pathologic features of the most severe forms of RSV infection in human. Indeed,
the two viruses are closely related and provoke similar immune responses.
In our model, we demonstrate a protective role of P2Y2 receptor against lung infection by
PVM. As compared to P2Y2+/+ mice, P2Y2-/- mice displayed a higher morbidity and mortality
rate in response to PVM. Interestingly, this is not correlated with higher infiltration of
neutrophils and macrophages despite increased levels of KC/CXCL-1 and MIP-2/CXCL-2 in the
BALFs. Instead of a direct difference in the innate immune response, PVM seems to
unbalance the adaptive immune response by impeding Th1 immune responses. Indeed, we
observed a lower infiltration of DCs, CD4+ and CD8+ T cells in the BALFs of P2Y2-/- mice
compared to those of P2Y2+/+ mice. This lack of infiltration can be correlated to the data of
Müller and colleagues demonstrating that P2Y2R is involved in the recruitment of DCs in the
lungs (Müller et al., 2010). Moreover, IL-12 and BRAK/CXCL14 levels were significantly lower
in P2Y2-deficient mice. DCs are one primary producer of IL-12 which induces the proliferation
of NK, T cells, DCs and macrophages, the production of IFN- and increased cytotoxic activity
of these cells. IL-12 also promotes the polarization of CD4+ T cells to the Th1 phenotype
involved against viral infection. As for BRAK/CXCL14, it is a potent chemoattractant and
activator of DCs as well (Shurin et al., 2005). Finally, higher IL-6 level observed in P2Y2-/BALFs reflects a defective Th1 response in these mice. IL-6 is secreted by T cells and
macrophages and acts as both a pro- and anti-inflammatory cytokine. It was shown that IL-6
production by pulmonary dendritic cells impedes Th1 immune responses (Dodge et al.,
2003).
The analysis of PVM virus titers in P2Y2+/+ and P2Y2-/- mice could reveal a defective virus
clearance in P2Y2-/- mice. Indeed, both CD4+ and CD8+ T cells contribute to the clearance of
PVM from the lung (Frey et al., 2008). Genetically T-cell-deficient or T-cell-depleted mice
cannot eliminate PVM, therefore, increase morbidity and mortality in P2Y 2-/- mice could be
the consequence of the persisting virus titers due to defective Th1 response and the lack of
CD4+ and CD8+ T cell recruitment by DCs.
Our data support that P2Y2 receptor exerts a protective role during lung infection such as in
the Pseudomonas aeruginosa infection model (Geary et al., 2005). On the other hand,
respiratory syncytial virus inhibits fluid clearance by the bronchoalveolar epithelium through
a mechanism involving nucleotide release and P2Y receptor activation (Davis et al., 2004).
69
PART IV – DISCUSSION AND CONCLUSIONS
Finally, studies describes a role for ATP release and ATP-mediated responses in lymphocyte
functions which contributes to adaptive, as well as to innate immunity (Piccini et al., 2008,
Corriden et al., 2007, Kolachala et al., 2008).
Viral infections and allergies
As mentioned previously in the results chapter, respiratory viral infections in early childhood
may interact with the immune system and modify allergen sensitization and/or allergic
manifestations. In mice, RSV or PVM infection aggravates the effects of allergic sensitization
and challenge with ovalbumin, predominantly by increase Th2 cytokines in the lungs and
hyperreactivity of the airways (Barends et al., 2002; Peebles et al., 1999 ; Barends et al.,
2004). However, both enhancing and protective role have been proposed (Papadopoulos &
Psarras 2003).
Influenza A virus infection is associated with asthmatic exacerbations in humans (Teichtahl
et al., 1997; Eriksson et al., 2000) and enhanced allergic sensitization in mice (Suzuki et al.,
1998; O’Donnell & Openshaw, 1998) but these results also appear in contrast to other
published data which demonstrate that influenza virus did not enhance pulmonary Th2
responses (Barends 2004). In our work, we infected P2Y2+/+ and P2Y2-/- mice with influenza A
virus (A/swine/Iowa/4/76 (H1N1)) without any observables differences between the two
group. Influenza virus is a member of the Othomyxoviridae family and induces inflammation
in humans throughout the respiratory tract as well in mice (with the mouse-adapted form)
(Murphy 1996). Two major differences exist between RSV/PVM and influenza and could
explain the absence of phenotype between wild-type and knock-out mice. First, the
difference in virus-structure (in particular in the attachment protein of the virus); and second
the site of virus replication in lung tissue (epithelial cells for RSV/PVM and alveolar
macrophages for influenza).
However, there is little doubt that a strong association exists between viral respiratory
infections and induction of wheezing illness and asthma exacerbations, but viral infections
may have multiple and contrasting effects on the development of allergy and asthma
(Xepapadaki 2007). A number of factors such as the type of agent, the severity of the
disease, the time of the infection and most importantly host predisposition, play a crucial
role.
Due to the potent demonstrated role of P2Y2 receptor in both PVM infection and asthma
and due to the strong synergy between viral infections and asthma, a combined model with
PVM on P2Y2-/--deficient asthmatic mice will be really relevant to further understand the
mechanisms of our receptor. A better understanding of this process will allow a long-term
strategy for targeting specific mediators or receptors that could alleviate progression of
acute disease episodes and chronic disease phenotypes mediated by RSV in human.
70
PART IV – DISCUSSION AND CONCLUSIONS
Therapeutic potential of P2Y2 receptor
The predominant P2 receptor in respiratory epithelium is the P2Y 2 receptor. Thus, release of
extracellular ATP/UTP influences the interactions between pathogens and their target cells
and regulates the lung immune system.
Since inflammatory lung diseases constitute a major public health problem with significant
economic impact, there is an increasing interest to study the lung immune system and the
mechanisms underlying lung inflammation, in order to identify candidate targets for the
development of new therapeutic drugs. A large number of existing therapeutic agents act
through various classes of G protein-coupled receptors (GPCRs), and concern all fields of
modern medicine. Recent progress in the development of selective agonists and antagonists
for P2Y receptors and study of knock-out mice have led to new drug candidates for cystic
fibrosis, dry eye disease, and thrombosis. On the horizon are novel treatments for
cardiovascular diseases, and neurodegeneration. More recently, another study showed that
extracellular ATP acts on P2Y2 receptors to facilitate HIV-1 infection (Séror et al., 2011).
These results reveal a novel signalling pathway involved in the early steps of HIV-1 infection
that may be targeted with new therapeutic approaches.
Through this work, we highlighted some possible roles of the P2Y2 receptor in lung diseases
such as in asthma and viral pneumonia. Numerous additional studies will be necessary to
understand fully the roles of P2Y2 in lung inflammation, and we can likely not transpose
directly to human our observations on animal models. Nevertheless, our data, the identities
between mouse and human sequence of P2Y2 receptor (85%), and the well-known role of
this receptor in disease such as cystic fibrosis, strongly suggest an interest of P2Y2 receptor
as a target for the development of therapeutic agents in the fields of inflammatory lung
diseases, which constitute life-threatening diseases. Pharmacologically, these therapies
should probably be based on UTP derivates rather than ATP due to their more stable form.
Conclusions
The aim of our work was to identify the potential roles of the P2Y 2 receptor in inflammatory
lung diseases. Using a set of models for various lung diseases in mouse, we have provided a
number of elements demonstrating interesting functions of this receptor at least in some
lung diseases which are of importance in human health.
First we demonstrated a function of P2Y2 receptor as a key mediator of Th2 immune
response through the ovalbumin-induced model of asthma. Indeed, via his effect on DCs
activation, as well as through the regulation of endothelial and soluble VCAM-1 expression,
the proinflammatory action of ATP provokes lung eosinophilia.
71
PART IV – DISCUSSION AND CONCLUSIONS
Second, our data support that P2Y2 receptor exerts a protective role during PVM infection
through his involvement in the initiation of Th1 immune response. Similar results were
previously highlighted in Pseudomonas aeruginosa infection model (Geary et al., 2005).
In conclusion, our study reveals that the purinergic P2Y2 receptor previously described as a
target in cystic fibrosis therapy, is a mediator of Th2 response in asthma, and is involved in
the initiation of Th1 response protecting mice against lung viral infection as well.
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PART V – REFERENCES
PART V – REFERENCES
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