Table 12-1. Specificity of Innate and Adaptive Immunity
Innate immunity
Adaptive immunity
Specificity
For structures shared by classes of microbes ("molecular patterns")
For structural detail of microbial molecules
(antigens); may recognize nonmicrobial antigens
Receptors
Encoded in germline; limited diversity
Encoded by genes produced by somatic
recombination of gene segments; greater diversity
Distribution of Nonclonal: identical receptors on all cells of the same lineage
receptors
Clonal: clones of lymphocytes with distinct
specificities express different receptors
Discrimination Yes; host cells are not recognized or they may express molecules
of self and
that prevent innate immune reactions
nonself
Yes; based on selection against self-reactive
lymphocytes; may be imperfect (giving rise to
autoimmunity)
Table 12-2. Examples of Molecular Patterns of Microbes and Pattern
Recognition Receptors of Innate Immunity
Molecular pattern of Source
microbe
Pattern recognition
receptor of innate
immunity
Principal innate
immune response
dsRNA
Replicating viruses
Toll-like receptor?
Type I interferon
production by infected
cells
LPS
Gram-negative
bacterial cell wall
Toll-like receptor/CD14
Macrophage activation
Unmethylated CpG
nucleotides
Bacterial DNA
Toll-like receptor
Macrophage activation
N-formylmethionyl
peptides
Bacterial proteins
N-formylmethionyl peptide
receptors
Neutrophil and
macrophage activation
Mannose-rich glycans
Microbial
glycoproteins or
glycolipids
1. Macrophage mannose
receptor
2. Plasma mannose-binding
lectin
1. Phagocytosis
2. Opsonization,
complement activation
Phosphorylcholine and
related molecules
Microbial membranes Plasma C-reactive protein
Abbreviations: dsRNA, double-stranded RNA; LPS, lipopolysaccharide.
Opsonization,
complement activation
Table 12-3. Components of Innate Immunity
Components
Principal functions
Barriers
Epithelial layers
Prevent microbial entry
Defensins
Microbial killing
Intraepithelial lymphocytes
Microbial killing
Circulating effector cells
Neutrophils
Early phagocytosis and killing of microbes
Macrophages
Efficient phagocytosis and killing of microbes, secretion of cytokines that
stimulate inflammation
NK cells
Lysis of infected cells, activation of macrophages
Circulating effector proteins
Complement
Killing of microbes, opsonization of microbes, activation of leukocytes
Mannose-binding lectin
(collectin)
Opsonization of microbes, activation of complement (lectin pathway)
C-reactive protein (pentraxin)
Opsonization of microbes, activation of complement
Coagulation factors
Walling off infected tissues
Cytokines
TNF, IL-1, chemokines
Inflammation
IFN-α,-β
Resistance to viral infection
IFN-γ
Macrophage activation
IL-12, IL-18, IL-23
IFN-γproduction by NK cells and T cells
IL-15
Proliferation of NK cells
IL-10, TGF-β
Control of inflammation
BOX 12-1 Toll-like Receptors
The Toll-like receptors (TLRs) are a family of membrane proteins that serve as
pattern recognition receptors for a variety of microbe-derived molecules and
stimulate innate immune responses to the microbes expressing these
molecules. The first protein to be identified in this family was the Drosophila
Toll protein, which is involved in establishing the dorsal-ventral axis during
embryogenesis of the fly as well as mediating antimicrobial responses. Ten
different mammalian TLRs have so far been identified on the basis of sequence
homology to Drosophila Toll, and they are named TLR 1 to 10, but more
members of the family may exist. All these receptors contain leucine-rich
repeats flanked by characteristic cysteine-rich motifs in their extracellular
regions and a Toll/IL-1 receptor (TIR) homology domain in their cytoplasmic
region, which is essential for signaling. TIR domains are also found in the
cytoplasmic tails of the IL-1 and IL-18 receptors, and similar signaling pathways
are engaged by TLRs, IL-1, and IL-18. The TLRs are expressed on many
different cell types that are important components of the innate immune
system, including macrophages, dendritic cells, neutrophils, mucosal epithelial
cells, and endothelial cells.
Mammalian TLRs are involved in responses to widely divergent types of
molecules that are commonly expressed by microbial but not mammalian cells
(see Figure). The innate immune response to one species of microbe may
reflect an integration of the responses of several TLRs to different molecules
produced by the microbe. Some of the microbial products that stimulate TLR
signals include gram-negative bacterial lipopolysaccharide (LPS), grampositive bacterial peptidoglycan, bacterial lipoproteins, lipoteichoic acid,
lipoarabinomannan, zymosan, the bacterial flagellar protein flagellin, heat
shock protein 60, respiratory syncytial virus fusion protein, unmethylated CpG
motifs, and double-stranded RNA. In many cases, a single TLR is responsible
for stimulating inflammatory responses to a particular microbial ligand, such as
TLR9-mediated responses to CpG. However, the repertoire of specificities of
the TLR system is apparently extended by the ability of TLRs to heterodimerize
with one another. For example, dimers of TLR2 and TLR6 are required for
responses to peptidoglycan. In some cases, one of the TLRs of a heterodimer
may confer ligand-binding specificity, and the other TLR may be required for
signaling. Specificities of the TLRs are also influenced by various non-TLR
accessory molecules. This is most thoroughly understood for TLR4 and its
ligand LPS. LPS first binds to soluble LPS-binding protein (LBP) in the blood or
extracellular fluid, and this complex serves to facilitate LPS binding to CD14,
which exists as both a soluble plasma protein and a glycophosphatidylinositollinked membrane protein on most cells except endothelium. Once LPS binds to
CD14, LBP dissociates, and the LPS-CD14 complex physically associates with
TLR4. An additional extracellular accessory protein called MD2 also binds to
the complex with CD14. LPS, CD14, and MD2 are all required for efficient LPSinduced signaling, but it is not yet clear if direct physical interaction of LPS with
TLR4 is necessary. Different combinations of accessory molecules in TLR
complexes may serve to broaden the range of microbial products that can
induce innate immune responses. For example, both CD14 and MD2 are
associated with complexes of other TLRs (e.g., TLR2), and TLR4 may form
complexes with other accessory molecules, such as MD1 and RP105, in
certain cell types, such as B lymphocytes.
The predominant signaling pathway used by TLRs results in the activation of
NF-κB (see Figure). In this pathway, ligand binding to the TLR at the cell
surface leads to recruitment of several cytoplasmic signaling molecules through
specific domain-domain interactions. The first protein to be recruited is a
cytoplasmic adapter protein that probably binds to the TIR domain of the TLR.
The adapter protein that binds to most TLRs is called MyD88, but some TLRs
may recruit other proteins and trigger MyD88-independent signaling pathways.
The second protein to be recruited into the signaling complex is called IL-1
receptor-associated kinase (IRAK). IRAK contains a domain that mediates
interactions with MyD88 and a serine/threonine protein kinase domain. On
recruitment, IRAK undergoes autophosphorylation, dissociates from MyD88,
and activates TNF-R-associated factor-6 (TRAF-6). TRAF-6 then activates the
IκB kinase cascade, leading to NF-κB activation. This same pathway is
involved in IL-1-and IL-18-induced activation of NF-κB. In some cell types,
certain TLRs also engage other signaling pathways, such as the MAP kinase
cascade, leading to activation of the AP-1 transcription factor. The relative
importance of these additional pathways of TLR signaling and the way the
"choice" of pathways is made are not well understood.
The genes that are expressed in response to TLR signaling encode proteins
important in many different components of innate immune responses. These
include inflammatory cytokines (TNF-α, IL-1, and IL-12), endothelial adhesion
molecules (E-selectin), and proteins involved in microbial killing mechanisms
(inducible nitric oxide synthase). The particular genes expressed will depend
on the responding cell type.
Killing of Phagocytosed Microbes
BOX 12-2 Physiologic and Pathologic Responses to Bacterial
Lipopolysaccharide
Bacterial lipopolysaccharide (LPS or endotoxin) is a product of gram-negative
bacteria, and a potent stimulator of innate immune responses that enhance
killing of the bacteria, but it may also cause significant pathologic changes in
the host. LPS is a mixture of fragments of the outer cell walls of gram-negative
bacteria and contains both lipid components and polysaccharide moieties. The
polysaccharide groups can be highly variable and are the major antigens of
gram-negative bacteria recognized by the adaptive immune system. The lipid
moiety, by contrast, is highly conserved and is an example of a molecular
pattern recognized by the innate immune system.
LPS induces local and systemic inflammation, and many of the features of
tissue injury observed in infection by gram-negative bacteria can be mimicked
by administration of LPS. LPS is a potent activator of macrophages, and the
cellular response involves the LBP/CD14/mammalian Toll-like receptor 4
system, as described in Box 12-1. Macrophages, which synthesize and
express CD14, can respond to minute quantities of LPS, as little as 10 pg/mL,
and cells that lack CD14 are generally unresponsive to LPS. However, some
CD14 is shed and circulates as a plasma protein. Addition of soluble CD14 to
cells that are normally unresponsive to LPS, such as vascular endothelial cells,
allows such cells to respond to LPS, but usually at 100-to 1000-fold higher
concentrations than CD14+ macrophages. The genes in macrophages that are
induced by LPS encode cytokines and enzymes of the respiratory burst. The
functions of these proteins in innate immunity are described in the text.
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The systemic changes observed in patients who have disseminated bacterial
infections, sometimes called the systemic inflammatory response syndrome
(SIRS), are reactions to cytokines whose production is stimulated by LPS. In
mildly affected patients, the response consists of neutrophilia, fever, and a rise
in acute-phase reactants in the plasma. Neutrophilia is a response of the bone
marrow to circulating cytokines, especially G-CSF, resulting in increased
production and release of neutrophils to replace those consumed during
inflammation. An elevated circulating neutrophil count, especially one
accompanied by the presence of immature neutrophils prematurely released
from the bone marrow, is a clinical sign of infection. Fever is produced in
response to substances called pyrogens that act to elevate prostaglandin
synthesis in the vascular and perivascular cells of the hypothalamus. Bacterial
products such as LPS (called exogenous pyrogens) stimulate leukocytes to
release cytokines such as IL-1 and TNF (called endogenous pyrogens) that
increase the enzymes, especially cyclooxygenase-2, that convert arachidonic
acid into prostaglandins. Nonsteroidal anti-inflammatory drugs, including
aspirin, reduce fever by inhibiting cyclooxygenase-2 and thus blocking
prostaglandin synthesis. An elevated body temperature has been shown to
help amphibians ward off microbial infections, and it is assumed that fever does
the same for mammals, although the mechanism is unknown. One hypothesis
is that fever may induce heat shock proteins that are recognized by some
intraepithelial lymphocytes, promoting inflammation. Acute-phase reactants are
plasma proteins, mostly synthesized in the liver, whose plasma concentrations
increase as part of the response to LPS. Three of the best known examples of
these proteins are C-reactive protein, fibrinogen, and serum amyloid A protein.
Synthesis of these molecules by liver hepatocytes is up-regulated by cytokines,
especially IL-6 (for C-reactive protein and fibrinogen) and IL-1 or TNF (for
serum amyloid A protein). During the SIRS, serum amyloid A protein replaces
apolipoprotein A, a component of high-density lipoprotein particles. This may
alter the targeting of high-density lipoproteins from liver cells to macrophages,
which can use these particles as a source of energy-producing lipids. The rise
in fibrinogen causes erythrocytes to form stacks (rouleaux) that sediment more
rapidly at unit gravity than do individual erythrocytes. This is the basis for
measuring erythrocyte sedimentation rate as a simple test for the systemic
inflammatory response due to any number of stimuli including LPS.
When a severe bacterial infection leads to the presence of organisms and LPS
in the blood, a condition called sepsis, circulating cytokine levels increase and
the form of the host response changes. High levels of cytokines produced in
response to LPS can result in disseminated intravascular coagulation (DIC),
caused by mechanisms described below. Multiple organs show inflammation
and intravascular thrombosis, which can produce organ failure. Tissue injury in
response to LPS can also result from the activation of neutrophils before they
exit the vasculature, thus causing damage to endothelial cells and reduced
blood flow. The lungs and liver are particularly susceptible to injury by
neutrophils. Lung damage in the SIRS is commonly called the adult respiratory
distress syndrome (ARDS) and results when neutrophil-mediated endothelial
injury allows fluid to escape from the blood into the airspace. Liver injury and
impaired liver function result in a failure to maintain normal blood glucose levels
due to a lack of gluconeogenesis from stored glycogen. The kidney and the
bowel are also injured, largely as a result of reduced perfusion. Overproduction
of nitric oxide by cytokine-activated cardiac myocytes and vascular smooth
muscle cells leads to heart failure and loss of perfusion pressure, respectively,
resulting in hemodynamic shock. The clinical triad of DIC, hypoglycemia, and
cardiovascular failure is described as septic shock. This condition is often
fatal.
TNF produced by LPS-activated macrophages is a major mediator of LPSinduced injury. This is known because many of the effects of LPS can be
mimicked by TNF and in mice, anti-TNF antibodies or soluble TNF receptors
can attenuate or completely block responses to LPS. IL-12 and IFN-γ also
contribute to LPS-induced injury because IL-12 stimulates IFN-γ production by
NK cells and T cells, and IFN-γ increases TNF secretion by LPS-activated
macrophages and synergizes with TNF in effects on endothelium. Because
septic shock is the result of cytokine overproduction, many clinical trials have
been introduced to neutralize cytokines, such as TNF and IL-1, with antibodies,
soluble receptors, or natural antagonists. The results of these trials have been
disappointing, probably because of cytokine redundancy (i.e., the fact that
many cytokines contribute to the systemic response in fulminant sepsis). It
remains to be seen whether "cocktails" of multiple cytokine antagonists will be
more effective.
The mechanisms by which LPS induces tissue injury were extensively studied
in the rabbit initially by Robert Shwartzman, who had observed that when a
rabbit is given a low-dose (subinjurious) intravenous injection of LPS, it will
succumb to a second low-dose injection of LPS administered intravenously 24
hours later. The dead animal shows evidence of multiorgan injury and
widespread intravascular thrombus formation, a condition that mimics clinical
DIC. Thrombosis results from a simultaneous initiation of coagulation by LPSinduced tissue factor (TF) expression and from a down-regulation of natural
anticoagulation mechanisms, including a decrease in expression of tissue
factor pathway inhibitor (TFPI) and endothelial cell thrombomodulin. A localized
form of LPS-mediated tissue injury may be induced by injecting a small dose of
LPS into a skin site on a rabbit, followed 24 hours later by a second low dose of
LPS administered intravenously. This causes hemorrhagic necrosis of the skin
at the site of the first injection. On histologic examination, the tissue shows
intravascular thrombosis as well as neutrophil influx, endothelial damage, and
hemorrhage. In the local Shwartzman model of skin injury in the mouse, TNF
can replace the second dose of LPS, and TNF antagonists prevent tissue
injury, further supporting the central role of this cytokine in responses to LPS.
Interestingly, the first description of TNF (and the origin of its name) was as an
LPS-induced serum factor that mediated hemorrhagic necrosis of experimental
tumor implants. TNF-mediated necrosis of tumors histopathologically
resembles the local Shwartzman reaction. The current interpretation of these
data is that tumors release a factor or factors that act on local endothelium to
produce the same changes as the first injection of LPS does in the localized
Shwartzman reaction. Intravenous administration of TNF then replaces the
second intravenous injection of LPS, invoking a Shwartzman reaction with
vascular thrombosis in the tumor and subsequent tumor necrosis.
BOX 12-3 Inhibitory and Activating Receptors of NK Cells
NK cells recognize and kill infected or malignantly transformed cells, but they
do not usually harm normal cells. This ability to distinguish potentially
dangerous targets from healthy self is dependent on the expression of both
inhibitory and activating receptors. Inhibitory receptors on NK cells recognize
class I MHC molecules, which are constitutively expressed on most healthy
cells in the body but are often not expressed by cells infected with virus or
cancer cells. For the most part, activating receptors on NK cells recognize
structures that are present on both NK-susceptible target cells and normal
cells, but the influence of the inhibitory pathways dominates when class I MHC
is recognized. Some activating receptors recognize class I MHC-like molecules
that are expressed only on stressed or transformed cells. Different families of
NK cell receptors exist, and many of these receptors have evolved recently, as
indicated by their absence in rodents and their structural divergence between
chimpanzees and humans. The following discussion focuses on the properties
of human NK cell inhibitory and activating receptors, with only brief
consideration of murine NK receptors.
Numerous human NK inhibitory receptors have been identified, all of which
have immunoreceptor tyrosine inhibitory motifs (ITIMs) in their cytoplasmic tails
(see Figure). ITIMs recruit cytoplasmic protein tyrosine phosphatases SHP-1
and SHP-2, which dephosphorylate and thereby inactivate signaling
intermediates generated by activating receptors on the same cell. Inhibitory NK
receptors fall into three main families. The first family to be discovered is called
the killer Ig-like receptor (KIR) family because its members contain two or three
extracellular Ig-like domains. The KIRs recognize different alleles of HLA-A,-B,
and-C molecules. Structural and binding studies indicate that the sequence of
the peptides bound to the MHC molecules is important for KIR recognition of
MHC molecules. HLA molecule binding to KIRs is characterized by very fast
on-rates and off-rates, which would be consistent with the ability of NK cells to
rapidly "test" for the presence of MHC expression on many cells in a short time.
Furthermore, the inhibitory signals generated in an NK cell by KIR recognition
of an MHC molecule are not long-lived, and the same NK cell can quickly go on
to kill an MHC-negative target cell. Some members of the KIR family have short
cytoplasmic tails without ITIMs, and these function as activating receptors, as
discussed in more detail below. Mice do not express KIRs but instead use the
Ly49 family of proteins, which have similar class I MHC specificities and ITIMs
in their cytoplasmic tails.
A second family of inhibitory receptors consists of the Ig-like transcripts (ILTs),
which also contain Ig-like domains. One member of this family, ILT-2, has a
broad specificity for many class I MHC alleles and contains four ITIMs in its
cytoplasmic tail. Interestingly, cytomegalovirus encodes a molecule called
UL18 that is homologous to human class I MHC and that can bind to ILT-2.
This may represent a decoy mechanism by which the virus engages an
inhibitory receptor and protects its cellular host from NK cell-mediated killing.
The third NK inhibitory receptor family consists of heterodimers composed of
either of the C-type lectins NKG2A or NKG2B covalently bound to CD94.
NKG2A and NKG2B have two ITIMs in their cytoplasmic tails. The CD94/NKG2
receptors bind HLA-E, a nonclassical MHC molecule. Stable expression of
HLA-E on the surface of cells depends on the binding of signal peptides
derived from HLA-A,-B,-C, or-G. Therefore, the CD94/NKG2 inhibitory
receptors perform a surveillance function for the absence of HLA-E, classical
class I MHC, and HLA-G molecules. As is the case for the KIR receptors, some
CD49/NKG2 receptors do not have cytoplasmic ITIM motifs, and these function
as NK-activating receptors, as discussed below.
The activating receptors on NK cells include several structurally distinct groups
of molecules, and only some of the ligands they bind are known. A shared
feature of these receptors is their association with signaling molecules that
contain immunoreceptor tyrosine-based activation motifs (ITAMs), including
FcεRIγ, ζ, and DAP12 proteins. The activating NK receptors engage signaling
pathways involving protein tyrosine kinases Syk and ZAP-70 and adapter
molecules that are also part of the signaling pathways associated with
lymphocyte antigen receptors and Ig Fc receptors (see Chapter 8). CD16, one
of the first activating receptors identified on NK cells, is a low-affinity IgG Fc
receptor that associates with FcεRIγ and ζ proteins and is responsible for NK
cell-mediated antibody-dependent cellular cytotoxicity. A more recently
discovered group of human NK-activating receptors, called natural cytotoxicity
receptors, includes NKp46, NKp30, and NKp44. These are members of the Ig
superfamily; they associate with FcRγ and ζ proteins and are expressed
exclusively on NK cells. Although their ligands are not yet known, antibodyblocking studies suggest that they play a dominant role in NK-mediated killing
of various tumor target cells. Ligand binding to activating NK cell receptors
leads to cytokine production, enhanced migration to sites of infection, and
killing activity against the ligand-bearing target cells.
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As mentioned, some members of the KIR and CD94/NKG2 families of MHCspecific receptors do not contain ITIMs but rather associate with ITAM-bearing
accessory molecules (such as DAP12) and deliver activating signals to NK
cells. These include KIR2DS, CD94/NKG2C, CD94/NKG2E, and
CD94/NKG2H. All these receptors are known to recognize normal class I MHC
molecules, and it is not clear why these potentially dangerous receptors exist
on NK cells. These activating receptors bind class I MHC molecules with lower
affinities than the structurally related inhibitory receptors, and it is possible that
the activating receptors actually bind MHC-related molecules specifically
associated with pathological conditions. This is the case for NKG2D, which is
expressed on NK cells, as well as on T cells, and is distantly related to the
other NKG2 proteins. NKG2D associates with DAP10, a molecule homologous
to DAP12, which contains a PI-3 kinase-binding motif in its cytoplasmic tail
rather than an ITAM motif. This receptor recognizes the MICA, MICB, and
ULBP molecules in humans and the RAE-1 and H-60 molecules in mice, all of
which are encoded in the MHC, have domains homologous to class I MHC α
and β domains, but do not display peptides or associate with β2-microglobulin.
These NKG2 ligands are not abundantly expressed on normal cells but are upregulated by stress and are often found on tumor cells. Thus, NK cells may use
these receptors to eliminate stressed (injured) host cells and tumor cells.