3

THE INNATE IMMUNE

RESPONSE

When tissue damage occurs as a result of trauma or infection, a complex series

of cellular and biochemical events occur, which are designed to limit the spread
of infection/degree of tissue damage, eliminate any microorganisms and repair
the damaged tissue. Initially, these activities are limited to the innate immune
response, i.e. they do not require specific identification of the precipitating
organism but often a successful outcome requires the combined activity of both
the innate and specific immune responses. In this chapter, we are going to look
at the cellular and molecular events that occur early in a response to tissue
damage – the innate immune response.
As a result of trauma and/or infection, cells in your tissues are damaged and
release their contents into the lymph, which bathes the tissues. One of the
earliest consequences of tissue damage or infection is the release of cytokines
from activated tissue macrophages and mast cells, in particular the colony-
stimulating factors (CSF) G(ranulocyte)-CSF and G(ranulocyte)M(onocyte)-
CSF. This causes the release of granulocytes and monocytes from the bone
marrow providing a ready reserve of these front-line cells. Other cytokines
released include those responsible for the increased expression of adhesion
molecules on the endothelial cells lining the blood vessels and on leukocytes in
the vicinity. The interaction of these adhesion molecules allows cells to attach to
the endothelium. Other factors released at the site of damage/infection affect the
vascular tone and the integrity of the endothelial layer, giving attached cells the
opportunity to escape into the underlying tissues. This they do in response to
certain cytokines known as chemokines. Once at the site of infection/damage,
the cells may actively phagocytose material recognised either through a group of
molecules known as pattern recognition receptors or as a result of binding
antibody or proteins of the complement pathways which encourage phago-
cytosis (i.e. they act as opsonins). As a result of phagocytosis, the organism may
be destroyed by the action of defensins or antimicrobial enzymes found in the
cellular granules. Alternatively, stimulation of the oxidative (respiratory) burst

Immunology for Life Scientists, Second Edition. Lesley-Jane Eales.

& 2003 John Wiley & Sons, Ltd: ISBN 0 470 84523 6 (HB); 0 470 84524 4 (PB)
 THE INNATE IMMUNE RESPONSE 75

may lead to the production of reactive oxygen radicals, which can destroy the
microorganism. This chemical and cellular response involves a number of other
cells and molecules, which collectively are known as the inflammatory response.
It is intimately associated with the kinin system (responsible for the recognition
of pain), the clotting cascade and the fibrinolytic pathway. The following sections
describe some of these key mediators/functions in detail.

3.1 THE COMPLEMENT CASCADES

Complement is a term used to describe a group of serum and cell surface

proteins, which have a number of important functions including lysis of cells
and microorganisms, opsonisation of microorganisms (a mechanism which
increases phagocytosis) and regulation of inflammatory and immune responses.
Owing to these wide-ranging and often dramatic effects, the complement
components are present as inactive precursors in the blood. There are three
major pathways by which complement may be activated. These are the classical,
alternative and lectin pathways.
The components of the complement system interact with each other in a
sequential manner such that the product of one reaction forms the enzyme for
the next. This leads to the formation of an enzyme complex, which binds and
cleaves C3 – the third component of the complement cascade. This component
is central to the complement activation pathways and forms the point at which
they converge. Subsequent components and complexes act to form the
membrane attack complex (MAC), which ultimately causes lysis of the cell/
microorganism on which it forms.
Cleavage fragments of complement molecules are identified by an adjunct.
Classically, the small fragments have been denoted by a (e.g. C3a, C5a) and the
large fragments by b (e.g. C3b, C5b). Unfortunately, the fragments of C2 do not
follow this notation and the larger fragment is designated ‘‘a’’ and the smaller
fragment is ‘‘b’’.

3.1.1 THE CLASSICAL COMPLEMENT PATHWAY

The classical complement pathway is normally activated by immune complexes

(antibody bound to specific antigen), containing IgG or IgM, which bind to the
first component, C1. In addition, the acute phase protein – C reactive
protein – endotoxin (a component of some bacterial cell walls) and certain
viruses may directly activate the classical pathway.
76 IMMUNOLOGY FOR LIFE SCIENTISTS

Figure 3.1 Representation of the structure of the first component of the complement
cascade
C1q comprises six subunits with globular heads and associates with two molecules each of C1s and
C1r.

Table 3.1 Effectiveness of different classes of antibody in

classical pathway activation

Class Subclass Classical pathway

IgM ++++
IgG
IgG1 ++++
IgG2 +
IgG3 +++
IgG4 7
IgA 7
IgE 7
IgD 7

C1

C1 comprises three different types of molecule – C1q, C1r and C1s – which, in
the presence of calcium, are held together (Figure 3.1). The Fc portion of an
antibody in an immune complex binds to the globular heads of the six subunits,
which comprise C1q. The affinity of C1q for immunoglobulin alone is very
weak but when several antibodies are closely associated (as in an immune
complex), the affinity of the binding is greatly increased. Not all forms of
immunoglobulin bind to C1 with the same affinity; some classes are better than
others at activating the classical complement pathway (Table 3.1).
 THE INNATE IMMUNE RESPONSE 77

The enzymatic activity of C1 resides in the C1r and C1s chains. Each molecule
of C1 comprises one molecule of C1q and two molecules each of C1r and C1s.
Binding of antibody to C1q leads to the cleavage of the two chains of C1r,
which in turn cleaves the C1s chains into long and short fragments. This results
in the appearance of an enzymatic site on C1s which acts on the next
component in the pathway, C4.

C4

C4 has three chains, the largest of which – the alpha chain – is cleaved by C1s
causing the release of a small fragment, C4a. The larger fragment – C4b –
binds to the target antigen (an infected cell or a microorganism) via the
formation of covalent amide or ester bonds. In the presence of magnesium ions,
C4b bound to the target antigen is capable of interacting with, and binding, the
next component of complement, C2.

C2

C2 is a single chain molecule, which binds to C4b and, in the presence of C1s, is
cleaved. The larger fragment – C2a – contains the enzymatic site of the C2
molecule and remains in the complex whilst the smaller fragment is released.
This new complex – C4b2a – acquires the ability to activate the next
component in the classical pathway, C3. This complex is also known as the
classical pathway C3 convertase1 (Figure 3.2).
Both C4b and C2a have labile active sites and most of the molecules formed
lose their binding sites before achieving association with membranes or with one
another, diffusing away as inactive reaction products. In addition, C4b2a is
unstable and quickly loses the C2 peptide, which becomes inactive upon
dissociation. C4b bound to an activator can accept another molecule of C2 and,
in the presence of active C1, will form an active enzyme capable of continuing
the complement cascade.

C3

C3 has two disulphide-linked chains (a and b) and like C4 has an internal

thiolester bond in the a chain. When this bond is cleaved, the molecule
undergoes a conformational change, which leads to an alteration in its
biochemical properties.

1
You may see this referred to as C4b2b in some texts that do not use the classical notation.
78 IMMUNOLOGY FOR LIFE SCIENTISTS

Figure 3.2 Formation of the classical pathway C3 convertase

C1 is activated by immune complexes and splits C4 and C2. C4b binds the activator surface and
C2a to form the C3 convertase. The C4a and C2b are soluble fragments with other biological
properties.

When C4b2a acts on C3, a small peptide is cleaved from the a chain, C3a.
This exposes a thioester bond in the remaining fragment – C3b – which will
interact with any suitable acceptor in the environment (e.g. molecules with
exposed reactive hydroxyl or amino groups). If this thiolester bond does not
form a covalent bond with an appropriate acceptor, it is hydrolysed through
interaction with water in the tissues. The majority of cleaved C3 molecules fail
to bind to an activator.
The attachment of C3b to membranes leads to the formation of C4b2a3b,
which is covalently bound to the antigen (via the C3 thiolester linkage) and
forms the classical pathway C5 convertase (Figure 3.3). This cleaves C5 into two
fragments – C5a and C5b – the larger of which, C5b, associates with the
convertase and can interact with subsequent components of the complement
cascade leading to lysis of membranes and microorganisms.
The smaller fragments released by the actions of the enzymes of the classical
complement pathway – i.e. C3a, C4a and C5a – have a number of potent
biological effects which are important in inflammation and will be discussed
later.
 THE INNATE IMMUNE RESPONSE 79

Figure 3.3 Formation of the classical pathway C5 convertase

The C3 convertase attached to the activator splits C3, the larger fragment of which (C3b) attaches
to the activator leading to the formation of C4b2a3b which is covalently bound to the antigen and
forms the classical pathway C5 convertase.

3.1.2 THE ALTERNATIVE COMPLEMENT PATHWAY

In the alternative complement pathway, C3 exists in two molecular forms: the

native form, which circulates in the serum, and a conformationally altered form
in which the thiolester bond has been hydrolysed. This altered C3 can bind
another factor in the presence of magnesium ions – Factor B of the alternative
pathway. In the presence of Factor D (a serine protease), Factor B may be
cleaved giving rise to the fragment Bb. These proteins are analogous to the C4b,
C2 and C1 of the classical pathway respectively. Together they form the
alternative pathway C3 convertase – C3bBb (Figure 3.4). Like the convertase of
the classical pathway, it catalyses the breakdown of C3 to C3a and C3b. The
C3b so formed may either continue the alternative pathway activation by
binding Factor B or it may bind the C3 convertase to form the alternative
pathway C5 convertase – (C3b)nBb. This compound is extremely unstable
under normal physiological conditions and is stabilised by another serum
protein – properdin (C3bnPBb).
Activation of the alternative pathway may be achieved through the
presence of surfaces to which the conformationally altered C3 may attach,
e.g. rabbit erythrocytes, Gram-negative bacteria, aggregates of IgA and certain
80 IMMUNOLOGY FOR LIFE SCIENTISTS

Figure 3.4 Formation of the alternative pathway C3 convertase

C3 can naturally exist in an altered form which can bind Factor B of the alternative pathway. In the
presence of Factor D (a serine protease), Factor B may be cleaved ^ Bb. These proteins are
analogous to the C4b, C2 and C1 of the classical pathway respectively. Together they form the
alternative pathway C3 convertase ^ C3bBb.

B-lymphocytes. Once attached, the alternative pathway C3 convertase becomes

highly active and is able to act on large numbers of C3 molecules to produce
C3a and C3b. Like the classical pathway, the alternative pathway may be
activated by immune complexes, IgA-containing complexes being the most
efficient (Table 3.2).

Table 3.2 Effectiveness of different classes of antibody

in alternative pathway activation

Class Subclass Classical pathway

IgM ++
IgG
IgG1 ++
IgG2 ++
IgG3 ++
IgG4 ++
IgA +++
IgE ++
IgD ++
 THE INNATE IMMUNE RESPONSE 81

3.1.3 THE LECTIN PATHWAY

The third complement activation pathway is the lectin pathway (Figure 3.5).
Lectins are carbohydrate-binding proteins, which are able to bind to a wide
range of microorganisms. There are two major types found in serum: the
ficollins (consisting of a fibrinogen- and a collagen-like domain) and the
mannose-binding lectin (MBL). Both of these lectins can activate complement.
Like the classical pathway, which involves the activation of the serine proteases
C1r and C1s, the lectin pathway is activated by the binding of carbohydrates on
pathogens followed by the activation of two novel serine proteases (MBL-
associated serine proteases – MASPs), which are responsible for the subsequent
activation of the lectin pathway. The structural similarities between the initial
activating complexes of the classical and lectin pathways suggest that the former
may have evolved from the latter to take advantage of the evolution of the
specific immune response and the presence of immune complexes.
MBL is a member of a family of proteins containing a collagen-like domain
and a carbohydrate recognition domain (CRD) as is seen in C1q. These proteins
are called collectins. Structurally, MBL consists of three identical chains but
these structures can cross-link giving a final structure similar to that of C1q.
MBL binds certain carbohydrates such as mannose and N-acetyl glucosamine
(GlcNAc) on bacterial surfaces. It associates with MASPs, the whole structure

Figure 3.5 Lectin pathway of complement activation

Carbohydrates on pathogens bind to mannose-binding lectin (MBL) or ficollins found in the serum.
This causes the activation of MASPs (MBL-associated serine proteases ^ MASPs), which activate
the lectin pathway in a manner analogous to C1q.
82 IMMUNOLOGY FOR LIFE SCIENTISTS

forming the first active enzyme in the complement cascade. MASP 1 cleaves C3
and C2 while MASP 2 cleaves C4 and C2.
MASPs also associate with ficollins. Ficollins possess a fibrinogen-like
domain responsible for binding to carbohydrates. They do not bind mannose
but bind GlcNAc in proximity to galactose and elastin. Thus, these lectins bind
a distinct range of carbohydrates compared to MBL. In addition, ficollins are
found expressed on cell membranes such as monocytes and aid in the
phagocytosis of organisms such as E. coli.

3.1.4 THE MEMBRANE ATTACK COMPLEX

The membrane attack complex (MAC) is formed by complement components

C5 to C9. Upon attachment to its convertase (derived from either the classical
or alternative pathways), C5 is cleaved into C5a and C5b. The latter binds to its
ligand, C6. Failure to do so results in its swift inactivation. The C5b6 complex
so formed binds C7. The resulting complex, being relatively hydrophobic,
interacts with lipids present in the membranes surrounding the immunogen
(Figure 3.6).
The C7 molecule, when bound to the C5b6 complex, can insert in the
membrane and may bind a single molecule of C8. The resulting small, highly
charged channel is stabilised by the incorporation of several molecules of C9
giving rise to a cylindrical, pore-like structure which spans the membrane
(Figure 3.7). The stability of this membrane attack complex – C5b678(9)n –

Figure 3.6 Formation of the C5b67 complex

The C5 convertase splits C5, allowing the fragment C5b to attach to the antigen. This binds to C6
and the C5b6 complex so formed binds C7. This complex is relatively hydrophobic and interacts
with lipids present in the membranes surrounding the antigen.
 THE INNATE IMMUNE RESPONSE 83

Figure 3.7 Formation of the membrane attack complex

The C7 molecule, when bound to the C5b6 complex, can insert in the membrane and may bind a
single molecule of C8.The resulting small, highly charged channel is stabilised by the incorporation
of several molecules of C9 giving rise to a cylindrical, pore-like structure which causes lysis of the
cell.

arises from the association between the hydrophobic exterior of the MAC with
membrane lipids. The hydrophilic interior of the channel allows the loss of
water and small ions from the target cell, thus eliminating its osmotic and
chemical balance and resulting in lysis.

3.1.5 REGULATION OF COMPLEMENT ACTIVATION

Since the formation of the MAC is antigen-independent and may attach to cell
surfaces in the vicinity and cause lysis of nearby host cells, it is vital to maintain
strict control of the complement system. It has powerful lytic and inflammatory
activities and if uncontrolled may lead to serious tissue damage.
Neither the classical, nor the alternative pathway can be activated by
antibody alone; it must be bound to antigen to be effective. In addition, a single
antibody molecule bound to antigen is ineffectual; more than one must coat the
antigen before complement can be activated. This limitation is probably an
important method of regulation since relatively large amounts of specific
antibody must be formed before complement activation can occur. Infections,
which are controlled quickly and effectively by other mechanisms, may only
stimulate a small antibody response. If the organism has already been
eliminated, activation of complement would probably lead to pathology
(tissue damage). By contrast, a poorly controlled infection with increasing
microorganismal load will stimulate a strong antibody response leading to
complement activation, which may help to eliminate the infecting agent.
84 IMMUNOLOGY FOR LIFE SCIENTISTS

However, complement may be activated in the absence of specific antibody

and so regulation at different stages of activation is achieved through the
involvement of other proteins.

C1 INHIBITOR (C1INH)

This prevents the function of activated C1s and C1r by binding to their active
sites. It also inhibits activated Hageman factor (one of the components of the
clotting cascade) and all the systems activated by Hageman factor fragments.
Thus, C1INH regulates enzymes of the kinin-generating system (chemicals
which stimulate the sensation of pain), the clotting system and the fibrinolytic
system (molecules involved in the regulation of blood clotting and wound
repair). The importance of this regulatory protein in prevention of pathological
damage is evidenced by the immunodeficiency disease hereditary angioedema
(HAE) where sufferers are unable to produce normal levels of functional
C1INH. This is discussed further in Section 5.3.

REGULATORS OF COMPLEMENT ACTIVATION (RCA) FAMILY

These proteins downregulate the activity of C3 convertases in the classical and

alternative pathways, either by causing their decay and/or by acting as cofactors
for the serine protease Factor I in the breakdown of C3b and C4b.
One member of the RCA family is the C4-binding protein (C4BP). As its name
suggests, it binds C4b which may be cleaved (and therefore inactivated) by the
protease Factor I. The latter, in the presence of another member of the RCA
family, Factor H, may cleave the a chain of hydrolysed C3 or C3b to form a
partially degraded molecule, iC3b. This molecule does not play a part in the
complement cascade but is capable of promoting phagocytosis. In addition,
under appropriate conditions, Factor I can degrade iC3b further to C3dg.
Regulation by Factors I and H is dependent upon the surface to which C3b is
bound. If the surface is that of a microorganism, the C3b is protected from
Factors H and I, which are unable to bind, and the complement cascade
proceeds to its termination with the formation of the MAC. By contrast, when
C3b is bound to host cell membranes, Factors H and I are able to interact with
it causing its degradation and preventing the continuation of the pathway. This
difference in ability to bind to certain surfaces appears to be related to the
presence of charged carbohydrates such as sialic acid on mammalian cells,
which promote the binding of Factor H. Other members of the RCA family are
shown in Table 3.3.
 THE INNATE IMMUNE RESPONSE 85

Table 3.3 Some members of the RCA family and their activities

RCA component Activity

CR1 (CD35) Unknown

CR2 (CD21) Expressed on B cells and is a receptor for breakdown products
of C3 including C3dg
CD46 (membrane cofactor protein) Found on a wide variety of human cells and binds C3/C4
CD55 (decay-accelerating factor) Promotes breakdown of the two C3 convertases and is
expressed on many human cells

REGULATORS OF THE MEMBRANE ATTACK COMPLEX

Regulation of the formation of the membrane attack complex also occurs.

Vitronectin (S protein) binds to C5b67 complexes and prevents their binding to
cell membranes. Although C8 and C9 can still bind to the complex in the fluid
phase it cannot insert into membranes and cause lysis.

KEY POINTS FOR REVIEW

. Complement is a group of serum proteins that are in an inactive state but when
appropriately stimulated, form an enzyme cascade designed to stimulate
inflammation and kill infectious agents.
. There are three main complement activation pathways: the classical, alternative
and lectin pathways.
. The classical pathway of complement is activated through C1q binding to
immune complexes.
. The classical pathway involves the activity of C1–C5.
. The alternative pathway is activated by a shift in the normal metabolic ‘tick-
over’ of C3 by partially hydrolysed C3 binding to the surface of micro-
organisms.
. The alternative pathway involves the activity of components designated as
factors.
. The lectin pathway is activated by microorganisms.
. The membrane attack complex can directly lyse microorganisms.
. Byproducts of the activation of complement such as C3a and C5a are
anaphylatoxins which cause inflammation.
. Byproducts also act as chemoattractant for immunologically active cells.
86 IMMUNOLOGY FOR LIFE SCIENTISTS

. The complement pathways are highly damaging and therefore are tightly
regulated to prevent unnecessary host cell damage.

3.2 PHAGOCYTOSIS

The process of phagocytosis may be divided into a number of sequential stages:

recognition, ingestion and digestion; we shall discuss each of these in turn.
Although tissue macrophages are often the first cells to encounter and ingest an
invading microorganism, they are not very efficient at killing. This is best
achieved by neutrophils, which may be attracted to the site of infection as a
result of mediators released by macrophages (chemokines). Neutrophils are by
far the most efficient of the professional phagocytes. However, newly recruited
macrophages (derived from blood monocytes attracted to the site of infection)
also show the ability to kill phagocytosed organisms. Eosinophils, which are
also efficient killers, are geared to killing extracellular pathogens that are too
large to phagocytose such as parasites like Schistosoma spp.

3.2.1 PATTERN RECOGNITION RECEPTORS

Once at the site of inflammation, phagocytes have to recognise the causative

agent. They have a number of cell surface receptors, which help to identify the
agent as foreign and attach to them. These receptors are known as pattern
recognition receptors and bind to groups of molecules known as pathogen-
associated molecular patterns (PAMPs). These PAMPs have three chief
characteristics: (i) they are found on microorganisms and not on host cells;
(ii) their structure is relatively constant within a given group of organisms; (iii)
they are essential for microbial survival. PAMPs are the ligands for pattern
recognition receptors (PRR) on host cells, which allow the innate system to
discriminate between ‘‘self’’ and ‘‘non-self’’ in a general manner. These pattern
recognition receptors may be secreted (e.g. lipopolysaccharide-binding protein,
mannose-binding protein), membrane bound (e.g. CR3, CD14, Toll-like
receptors (TLRs), macrophage scavenger receptor, mannose receptor, surfac-
tant protein A receptor) or intracellular. PRRs are found on the surface of those
cells recruited early in an immune response, i.e. neutrophils, mononuclear
phagocytes and NK cells. These cells rapidly respond to microbial invaders
owing to recognition of non-self via pattern recognition receptors. Some of
these will be described below.
 THE INNATE IMMUNE RESPONSE 87

COMPLEMENT RECEPTOR 3 (CR3)

CR3 (CD11b/CD18) is a member of a group of molecules known as the alpha2

integrins. The members of this family share a common molecule – CD18 – and
are expressed only on white blood cells. Other major members of the family
include leukocyte function antigen-1 (LFA-1 or CD11a/CD18) and CR4
(CD11c/CD18). LFA-1 promotes migration of neutrophils and T cells across
the endothelium and acts in concert with CR3 to promote migration of
monocytes. CR3 provides a critical link between cells and the extracellular
matrix. It exists in both inactive and active states. When activated, it promotes
phagocytosis by binding complement opsonised microorganisms. In addition to
its ability to bind to the extracellular matrix, C3bi and its cellular counter-
receptors (intercellular adhesion molecules 1 and 2), CR3 is also able to bind to
a range of molecules found on the surface of microbes including lipopoly-
saccharide (LPS), polysaccharides from Mycobacterium tuberculosis and
zymosan from yeast.

CD14

CD14 is expressed on monocytes, macrophages and on neutrophils. It binds to

lipopolysaccharide (either alone or in conjunction with the soluble LPS-binding
protein). It is also known to bind to the exotoxin Staphylococcus aureus protein
A and the lipoarabinomannan from mycobacteria. Recent studies suggest that
CD14 acts in cooperation with members of the Toll family to discriminate
between ligands, CD14 acting as the signalling unit directing the cellular
response to the ligand.

TOLL-LIKE RECEPTORS

Toll receptors were first identified in the fruitfly Drosophila. Structurally similar
molecules were subsequently identified in mammals and the group of molecules
have been defined both structurally and functionally. These TLRs are able to
discriminate between different PAMPs leading to the stimulation of an
appropriate immune response. The binding affinities of the Toll receptors are
shown in Table 3.4.
TLR4 has been demonstrated on the surface of macrophages and dendritic
cells, its activation causing upregulation of co-stimulatory molecules, increased
antigen presentation, secretion of proinflammatory cytokines and microbicidal
activity.
Most TLRs show limitations in the range of PAMPs that they recognise.
TLR2 does not appear to do so. However, it has been suggested that perhaps
TLRs are able to associate with each other to form heterodimers with distinct
ligands, thus explaining the apparent promiscuity of TLR2.
88 IMMUNOLOGY FOR LIFE SCIENTISTS

Table 3.4 Binding specificities of humanToll-like receptors

TLR Function

TLR2 Lipotechoic acid, peptidoglycan, lipoarabinomannan, lipopeptide, atypical LPS

TLR3 Viral double-stranded RNA
TLR4 Lipopolysaccharide (LPS)
TLR5 Bacterial flagellin
TLR9 CpG DNA

The binding of a ligand to a Toll receptor results in the production of

cytokines such as interleukin 1 and tumour necrosis factor. This is achieved
through the interaction of a series of cytoplasmic molecules that transduce the
signal from the receptor to the nucleus resulting in the transcription of specific
genes (see Figure 3.8).

MACROPHAGE SCAVENGER RECEPTORS

There are two classes of scavenger receptors, SR-A and SR-B. The class A
macrophage scavenger receptor (SR-A) was identified as one of the main
receptors mediating the endocytosis of lipids by macrophages. However, as
more functions have been attributed to these receptors (including macrophage
growth and maintenance, adhesion to the substratum, cell–cell interactions,
phagocytosis and host defence), it is thought that their earliest evolutionary
roles involved their scavenger function related to innate immunity but that they
have evolved to play a vital role in cellular transport of fatty acid and the
regulation of angiogenesis.
SR-A: The class A macrophage scavenger receptors bind to modified low-
density lipoproteins (LDLs) such as oxidised LDL and acetylated LDL. Three
class A scavenger receptors have been identified. SR-AI and SR-AII are
encoded by the same gene and the third receptor – MARCO (macrophage
receptor with collagenous structure) – has a structure related to SR-AI.
MARCO is expressed on a subgroup of macrophages. These cells are found
in distinct tissue locations and are thought to be important in scavenging
foreign material and debris. By contrast, SR-A is found on all macrophages and
on some endothelial cells.
SR-B: The class B scavenger receptor family includes the molecule CD36,
which is expressed on the endothelium of the microvasculature, dendritic cells,
platelets, monocytes and macrophages. Initially, the chief role of CD36 was
thought to be facilitation of cell–cell (platelets and monocytes) and tumour cell–
extracellular matrix interactions but recent work has demonstrated its role in
cellular lipid transport.
 THE INNATE IMMUNE RESPONSE 89

Figure 3.8 Toll receptor signalling

Upon activation theToll receptor binds the adapter molecule MyD88 which has a modular structure:
the C-terminal domain binds the TollR whilst the N-terminal portion is a ‘‘death-domain’’ module,
which recruits the IL-1R-associated kinase (IRAK) to the signalling complex. Downstream, IRAK
interacts with the adapter molecule tumour necrosis factor receptor-associated factor 6 (TRAF6)
that binds them to the protein kinase NF-kB-inducing kinase (NIK). Activated IKK phosphorylates
IkBa in the NFkB signalsome leading to recognition by the specific ubiquitin^protein ligase
complex (SCF^ubiquitin^Slimb) resulting in the association of ubiquitin with the signalsome. This
leads to the rapid degradation of IkB, exposing the active NFkB which translocates to the nucleus
and initiates transcription of the target genes.

CD36 is the receptor for thrombospondin-1 (TSP-1) which is found in the extracellular
matrix and platelet a granules. TSP-1 is involved in cell–cell interactions, cellular
proliferation, TGFb activation and angiogenesis.
90 IMMUNOLOGY FOR LIFE SCIENTISTS

3.2.2 OPSONISATION

Once recognition has occurred, the attachment between cell and microbe may
be enhanced by the presence of opsonins.

An opsonin is something that coats a particle and makes it easier to phagocytose. For
example, complement activation at the site of inflammation causes C3b to be deposited on the
causative agent (which may be a microorganism or an inert particle). Both neutrophils and
macrophages have receptors for C3b, which allows them to bind the complement-coated
particles.

There are three main types of opsonin: IgG antibodies, fragments of the
complement protein C3 and the lectins described earlier. Each may act as a
bridge between the particle and the phagocytic cell. Acute phase proteins (such
as C reactive protein (CRP)) can bind to the surface of microorganisms and,
having a structure similar to C1, activate the classical complement pathway
leading to opsonisation by complement.
Molecules that bind IgG (Fc gamma receptors or FccR) and those binding
carbohydrates (lectins) are always in an active state and stimulate ingestion
immediately following ligand–receptor interaction. By contrast, the C3-
fragment receptors (Table 3.5) are inactive and require a further signal in the
form of fibronectin or an acute phase protein before activation may occur.
However, it has been shown that C3bi on the surface of bacteria or fungi may
bind to CR3 and trigger phagocytosis due to the interaction between
polysaccharides on the microbial surface and a lectin-like site on CR3.

3.2.3 INGESTION

The process of taking extracellular material inside a cell is known as

endocytosis. The active uptake of particulate material through the formation

Table 3.5 Complement receptors involved in opsonisation

Receptor Ligands

CR1 C3b
CR2 C3dg4C3d4iC3b4C3b
CR3 C3bi
 THE INNATE IMMUNE RESPONSE 91

Figure 3.9 Illustration of the different methods for uptake of extracellular material
The process of taking extracellular material inside a cell is known as endocytosis.The active uptake
of particulate material through the formation of pseudopodia is known as phagocytosis. Pinocytosis
is the process by which cells take up soluble material.

of finger-like projections of cell membrane and cytoplasm (pseudopodia) is

known as phagocytosis. Pinocytosis is the process by which cells take up soluble
material (Figure 3.9).
The process of phagocytosis involves alterations in the cell membrane caused
by changes in the structure of the cytoskeleton, the latter also affecting cell
motility. When a particle attaches to a phagocyte (either non-specifically
through chemical attraction or specifically via receptor–ligand interaction),
phagocytosis is only initiated if the cell surface signals are sufficient to activate
the process. Additional required signals include receptor clustering, receptor
activation through co-stimulation by cytokines, chemoattractants or other
microbial products, or cooperation with another type of receptor. Once the cell
has been activated, the cell membrane forms pseudopodia that surround the
particle. They ultimately fuse together, engulfing the particle in a membrane-
bound vesicle known as a phagosome. Once formed, the phagosome moves to
the interior of the cell where degradation of its contents occurs (Figure 3.10).
The process of phagocytosis is achieved by the transformation of the fluid
cytoplasm (cytosol) into a gel by interaction with actin-binding proteins located
on the inner surface of the cell membrane. This gelation results from the
polymerisation of actin in the cytoplasm to form filaments which connect with
each other and with myosin, giving a more rigid gel-like consistency, and is
stimulated by the activation signal received as a result of the occupancy of an
opsonin receptor. The activated actin transmits a signal to the myosin, which
contracts leading to the streaming of the cytosol pushing the plasma membrane
in one direction and thus forming the pseudopod (Figure 3.11). This
reorganisation of the cytoskeleton is regulated by a family of small GTPases
known as Rho (Rho, Rac and Cdc42). Members of this family have clearly
defined roles in the process. CR3-mediated phagocytosis requires the activity of
Rho and through sequential ligand–receptor binding (the zipper effect) results in
a tight-fitting phagosome. In contrast to this, FcR-mediated phagocytosis
depends on the activity of Rac and Cdc42 resulting in a more spacious
phagosome. These differences between CR3- and FcR-mediated phagocytosis
92 IMMUNOLOGY FOR LIFE SCIENTISTS

Figure 3.10 Phagocytosis

may also explain the lack of an oxidative burst and other proinflammatory
signals in the former but not the latter, since Rho is not involved in the
activation of NADPH oxidase.

3.2.4 DIGESTION

Digestion of the phagosomal contents is achieved by a variety of enzymes,

which are introduced into the phagosome when cytoplasmic granules
(polymorphs) and lysosomes (polymorphs and mononuclear phagocytes)
empty their contents into the phagosome. These membrane-bound organelles
fuse with the phagosomal membrane forming a larger phagolysosome. This
fusion may start before the phagosome is closed and thus, destructive enzymes
may be released outside the cell, resulting in the tissue damage associated with
some immunological reactions.
One of the first events to occur immediately after ingestion is the acidification of
the phagosome. This starts before the granule contents are released and is caused
 THE INNATE IMMUNE RESPONSE 93

Figure 3.11 Intracellular processes leading to pseudopodia formation

Pseudopodium formation is thought to occur due to the transformation of the fluid cytoplasm
(cytosol) into a gel by interaction with actin-binding proteins located on the inner surface of the
cell membrane. This gelation results from the polymerisation of actin in the cytoplasm to form
filaments which connect with each other and myosin, giving a more rigid gel-like consistency. The
activated actin transmits a signal to the myosin which contracts leading to the streaming of the
cytosol which pushes the plasma membrane in one direction thus forming the pseudopod.

by the accumulation of lactic acid and hydrogen ions produced by the respiratory
burst. The hydrogen ions are actively moved into the lysosomes by special
pumps, which derive their energy from adenosine triphosphate (ATP). The pH
rapidly reduces to about 4, assisted by the release of the acidic granule contents.
Few microorganisms can survive or multiply in an acid environment and the
lysosomal enzymes that bring about their destruction are most efficient at a low
pH.
Once the contents of the phagolysosome have been destroyed, the debris must
be eliminated. Some products of degradation may be re-used, such as amino
acids, nucleotides, sugars and lipids. Other, more indigestible parts, may be
exocytosed (although this is undesirable since tissue-damaging enzymes are
released at the same time), or may be stored within the cell until it dies and is
eliminated from the body, e.g. in the faeces or sputum.
94 IMMUNOLOGY FOR LIFE SCIENTISTS

3.2.5 THE RESPIRATORY BURST

Upon activation, the phagocyte undergoes a respiratory burst (also known as

the oxidative or metabolic burst), which is characterised by a rapid, marked
increase in the consumption of oxygen by the cell and results in the production
of toxic oxygen metabolites. Mitochondrial enzymes normally mediate
respiration. However, the respiratory burst involves the action of a group of
enzymes found in the cytoplasm and associated with the membrane of the
phagosome known as the nicotinamide adenine diphosphate oxidase (NADP-
oxidase). The principal effect of this group of enzymes is to convert molecular
oxygen to the superoxide anion – a highly reactive molecule, which has two
unpaired electrons each available for association with another electron.
Addition of one electron leads to the formation of superoxide; a second
electron converts it to peroxide (Figure 3.12). This conversion is mediated by the

Figure 3.12 Conversion of molecular oxygen to reactive intermediates

The respiratory burst involves the action of a group of enzymes known as the nicotinamide adenine
diphosphate oxidase (NADPH-oxidase). These enzymes convert molecular oxygen to the
superoxide anion ^ a highly reactive molecule which has two unpaired electrons each available for
association with another electron. Addition of one electron leads to the formation of superoxide; a
second electron converts it to peroxide.
 THE INNATE IMMUNE RESPONSE 95

Figure 3.13 The production of toxic oxygen metabolites and other microbicidal
compounds during the oxidative burst

reduced form of NADP (NADPH). Interaction between superoxide and

hydrogen peroxide leads to the formation of the hydroxyl anion or radical.
All molecules with unpaired electrons are called free radicals. They are highly
unstable and are capable of damaging proteins, lipids, DNA and cell
membranes. Thus, they may be responsible for the destruction of phagocytosed
microorganisms. Normally, endogenous, scavenger, enzymes (e.g. superoxide
dismutase, catalase, glutathione peroxidase) destroy free radicals and hydrogen
peroxide. However, in activated phagocytes they are produced in greater
quantities than can be destroyed allowing them to accumulate and indeed to be
secreted by the cells. Although the oxygen radicals are extremely short lived,
superoxide and H2O2 are released in the tissues; their persistence (and hence the
degree of resulting tissue damage) depends on the ability of the cells in the
locality to destroy them. In addition to these toxic oxygen metabolites, other
microbicidal compounds are generated during the oxidative burst including
hypohalous acids (Figure 3.13).

3.2.6 OTHER ANTIMICROBIAL ACTIVITIES OF LYSOSOMES

As we have discovered in the previous section, lysosomes contain a wide variety

of enzymes and chemicals involved in the destruction of microorganisms. One
96 IMMUNOLOGY FOR LIFE SCIENTISTS

of the major granule constituents in neutrophils is a group of proteins that have

broad-spectrum antimicrobial activity against bacteria, fungi, mycobacteria and
enveloped viruses. These proteins, which are found in a wide range of other
cells, are highly conserved between species and are known as defensins.

DEFENSINS

There are two main types of defensin, the a defensins found in neutrophils and the
Paneth cells of the small intestine and the b defensins found in a range of other
tissues including the skin, salivary glands and airway epithelial cells.
Defensins are usually linear polypeptides that are folded, their conformation
being maintained by intra-chain disulphide bonds. These molecules are
positively charged and therefore interact with negatively charged PAMPs from
microbial membranes such as lipopolysaccharide from Gram-negative bacteria
and lipotechoic acid from Gram-positive bacteria. Thus, defensins are
electrostatically specific for microbial cells. The production of defensins is
positively regulated by the interaction between PRRs and PAMPs.
It has been suggested that defensins aggregate to form pores in the cytoplasmic
membranes of Gram-positive bacteria. In Gram-negative bacteria, the defensins,
which have a high affinity for LPS, are thought to displace the stabilising Ca2+
and Mg2+ ions from the outer membrane, leading to disruption.
Defensins can also activate the classical complement pathway and, through
their ability to regulate cytokine production and adhesion molecule expression,
may influence the inflammatory response.
a-Defensins: These are preformed as pre-propetides that are processed
enzymatically and stored as the functional peptide in the granules of
neutrophils. They stimulate chemotaxis in monocytes, dendritic and T cells at
concentrations as low as 10710 M. In addition, a-defensins inhibit fibrinolysis by
modulating tissue-type plasminogen activator and plasminogen binding to
fibrin and endothelial cells. Thus, in pathological conditions, defensins released
in the circulation may adhere to the endothelium and contribute to the
inflammatory process.
b-Defensins: These are constitutively expressed in epithelial cells, their
expression being increased as a result of infection or inflammation. They may
influence the immune response by binding to the chemokine receptor CCR6 on
dendritic and memory T cells.

KEY POINTS FOR REVIEW

. Phagocytosis may be considered to have a number of phases, recognition and

attachment, ingestion and digestion.
 THE INNATE IMMUNE RESPONSE 97

. Pattern recognition receptors on phagocytes bind to pathogen-associated

molecular patterns, which are not found on host cells.
. CR3 is an alpha2 integrin providing a critical link between cells and the
extracellular matrix.
. CD14 is found on monocytes, macrophages and neutrophils and binds
lipopolysaccharide, Staphylococcus aureus enterotoxin A and lipoarabino-
mannan.
. Toll-like receptors discriminate between pathogen-associated molecular
patterns, binding of which produces lymphokines that influence the immune
response.
. Macrophage scavenger receptors play an important role in the transport of
fatty acids and regulation of angiogenesis.
. Opsonins such as antibody, complement fragments and acute phase proteins
enhance phagocytosis.
. Material taken up by phagocytes is broken down in acidified endosomes, which
fuse with lysosomes.
. The respiratory burst that may occur as a result of phagocytosis produces
reactive oxygen intermediates that are toxic to microorganisms.
. Other antimicrobial products of phagocytes include the defensins, which can
destroy bacteria and activate complement.

3.3 INFLAMMATION

Inflammation is the body’s reaction to an injury such as invasion by a

microorganism or mechanical or chemical damage and is characterised by five
cardinal signs: heat (calor), pain (dolor), redness (rubor), swelling (tumour) and
(in extreme cases) loss of function. The response may be initiated by the release
of chemicals from damaged tissue cells either as a direct response to trauma or
as a result of factors released from microorganisms such as toxins. However, it
is difficult to define precisely what triggers the inflammatory response since it
involves a large number of different cells and mediators. All the events
occurring during inflammation are geared towards increasing the local blood
flow (caused by dilation of the blood vessels) and the permeability of the
vasculature (blood vessels). This allows cells and serum components increased
access to the area of tissue damage in order to limit the spread of infection and
tissue damage and to promote healing.
The inflammatory process involves the concerted action of the immune, kinin,
fibrinolytic and clotting systems which interact to maintain the integrity of the
vascular system and to limit the spread of infection/damage. Figure 3.14
illustrates the complexity of the interaction between the components of the
98 IMMUNOLOGY FOR LIFE SCIENTISTS

Figure 3.14 The cells and chemicals involved in inflammation

As a result of infection or tissue damage, complement is activated leading to the release of
complement fragments that can activate local mast cells leading to the release of vasoactive
chemicals such as leukotrienes, prostaglandins and histamine. These agents and a range of
cytokines are also released by activated tissue macrophages. They have an effect on the local
endothelium increasing the expression of a range of adhesion molecules (ICAM, P-selectin),
encouraging the tethering, rolling and immobilisation of leukocytes. Chemokines and chemotaxins
(such as C3a, C5a) released in the tissues encourage local extravasation of leukocytes where they
are able to remove damaged tissue and any causative agents and initiate repair. Endothelial damage
leads to platelet activation and the formation of fibrin, breakdown of which by plasmin results in the
formation of chemotactic fibrinopeptides.This ensures a constant supply of leukocytes at the site of
damage, ensuring repair of any tissue damage.

inflammatory response. The following sections will describe some of the

chemicals and cellular functions involved in inflammation.

3.3.1 INFLAMMATORY MEDIATORS

Toxins from microorganisms and enzymes from the lysosomes of polymorphs

cause or enhance tissue damage through the release of chemicals from a variety
 THE INNATE IMMUNE RESPONSE 99

of cells. Amongst these are the prostaglandins (PGs) and leukotrienes (LTs),
which belong to a family of unsaturated fatty acids derived from arachidonic
acid (a component of most cell membranes) by the phospholipase A2/
cyclooxygenase and 5-lipoxygenase pathways. Prostaglandins cause blood
vessel dilatation and enhance the effects of histamine and bradykinin on
vascular permeability. Leukotrienes stimulate the migration of leukocytes into
the tissues. Interestingly, metabolites of prostaglandins help resolve inflamma-
tion through the inhibition of key intracellular molecules involved in gene
transcription. Thus, the different molecules produced during inflammation have
different effects but overall are responsible for the induction of pain, fever,
vascular permeability and chemotaxis of polymorphonuclear leukocytes
(Table 3.6).
Although mast cells and basophils are the principle producers of these
inflammatory mediators, other cells such as eosinophils, neutrophils and
platelets are also capable of doing so. Substances released as a result of tissue
damage may activate mast cells. Their degranulation results in the release of
vasoactive amines such as histamine. In addition, basophils and platelets release
both histamine and serotonin (5-hydroxytryptamine). These mediators cause
increased capillary permeability. They are also important in the repair of tissue
damage as demonstrated in patients treated with large doses of anti-histamines.
Such individuals showed considerable impairment in the healing of surgical
wounds. Indeed, evidence suggests that histamine (and perhaps other
inflammatory mediators) may play a role in normal growth – especially foetal
growth.
When tissue injury occurs, enzymes are released and surfaces are exposed
which cause the activation of Hageman factor (Factor XII) of the clotting
cascade. This in turn activates, and is activated by, Factor XI of the clotting
cascade and kallikrein of the kinin system. The kinin system (a series of serum
peptides which are sequentially activated) produces bradykinin, which causes
pain, vasodilation and increased capillary permeability. This allows cells and
serum proteins such as components of the complement cascade to pass into the
tissues at the site of injury. If microorganisms are present at the site, the
complement cascade may be activated and fragments of complement,
particularly C3a and C5a, are released. These have dramatic, proinflammatory
effects, which are summarised in Table 3.7. Indeed, studies in complement-
deficient animal models have suggested that complement activation is vital for
the development of acute inflammation.

3.3.2 CELLULAR RESPONSES IN INFLAMMATION

As a result of the action of inflammatory mediators, the flow of cells within the
blood vessels slows, the cells becoming located along the walls of the vessel. The
100 IMMUNOLOGY FOR LIFE SCIENTISTS

Table 3.6 Biochemical mediators of inflammation

Produced
Substance Chemistry by/from Characteristics/functions

Heparin Proteo- Degranulation It is an anti-coagulant, i.e. it prevents blood clotting. It

glycan of mast cells temporarily suspends blood clotting allowing
inflammatory cells to enter the area of tissue injury
Histamine Vasoactive Mast cells and Released on degranulation caused by cross-linking of
amine basophils. FcR. Acts on endothelial cells of blood vessels causing
Stored in them to become less tightly associated. This makes the
cytoplasmic blood vessels ‘‘leaky’’, allowing inflammatory cells and
granules serum proteins, such as Ab and complement to enter the
area of tissue damage. Also causes the contraction of
bronchiolar and vascular smooth muscle and increased
secretion by nasal and bronchial mucous glands. Effects
are maximal after 1^2 minutes and last for 10 minutes
Serotonin Derived Platelet A neurotransmitter in the central nervous system. Its
(5-hydroxy- from granules. ability to cause leakage from the blood vessels derives
tryptamine) tryptophan Released from its effect on the endothelial cells which become
during blood partially detached from each other
clotting
Kinins Small basic Kallikreins Affect the movement of smooth muscle, increase
peptides (arginine vascular permeability and vasodilation and induce pain.
esterases) Bradykinin is a 9 amino acid peptide derived from a
act on serum a2 macroglobulin precursor. It causes slow,
kininogens ^ sustained contraction of smooth muscles, including
large proteins those of the bronchi and vessels; increased vascular
in the plasma ^ permeability; increased secretion by mucous glands,
to give kinins including those of the bronchi, and stimulation of pain
fibres. It also activates phospholipase A2, which
stimulates arachidonic acid metabolism
Eosinophil Tetra- Mast cell Attract eosinophils to the site of inflammation and may
chemotactic peptides granules play a role in activation of the cells
factors
Prosta- Derived Lung mast cells. Lung mast cells preferentially form PGD2, a potent vaso-
glandins by cyclo- Neutrophils, dilator. From neutrophils and macrophages, this pathway
and oxygenase macrophages generates PGF2a , a bronchoconstrictor, and PGE1 and
thromb- metabolism PGE2, broncho- and vasodilators that regulate the tissue
oxanes of arachi- microenvironment. PGI2 causes disaggregation of plate-
donic acid lets; thromboxanes (TXA2 andTXB2) aggregate platelets
and regulate blood coagulation and homeostasis
Leukotrienes Metabolites Antigen^ Leukotrienes are spasmogenic and involved in the
of antibody continued bronchospasm of asthma. Also known as
arachidonic interactions; slow reacting substance of anaphylaxis (LTC4, LTD4,
acid neutrophils LTE4). 5-Lipoxygenase generates 5-hydroxyeico-
satetraenoic acid (5-HETE) which modulates cell motility
and possibly glucose transport and LTB4 which is a
chemotactic agent comparable to C5a
Platelet- Compounds IgE-containing Released from platelets, granulocytes, monocytes,
activating derived ICs and by macrophages, mast cells and endothelial cells.Very
factors from non-IgE potent, cause activation of neutrophils and monocytes
glycerol reactions from resulting in the release of inflammatory mediators;
basophils and induces lymphocyte proliferation and IL-2 secretion;
alveolar platelet aggregation and thromboxane release
macrophages
 THE INNATE IMMUNE RESPONSE 101

Table 3.7 The role of complement activation in inflammation

Product of
Proteins activation Activity

C3, 4, 5 C3a, 4a, 5a Anaphylatoxins which have intense effects on muscles and blood
flow. At its worst extreme, anaphylaxis may result in death. Cause
smooth muscle contraction, degranulation of mast cells and
basophils leading to release of histamine and other vasoactive
substances that induce capillary leakage. C5a is the most potent
anaphylatoxin
C3, 5 C3a, 5a C3a and C5a have important immunoregulatory effects onTcell
function, either stimulating or inhibiting aspects of cell-mediated
immunity
C5 C5a C5a is a potent chemotactic agent for neutrophils and monocytes. It
increases neutrophil adherence and causes aggregation; stimulates
neutrophil oxidative metabolism and the production of toxic oxygen
species; triggers lysosomal enzyme release from phagocytes

mediators also affect the expression of certain molecules on the endothelial cells
lining the capillaries. These adhesion molecules (Table 3.8) promote binding to
the endothelium of white blood cells, which express the corresponding ligands,
thus allowing them to move over the surface of the vessel wall in a process
known as rolling. The cells extend pseudopodia between the endothelial cells
and secrete chemicals to dissolve the basement membrane allowing them to
squeeze out of the capillaries into the tissues. This migration is known as
diapedesis.
Once in the tissues, cells detect certain molecules that have diffused away
from the site of infection/damage and move to that site by a process known as
chemotaxis. The diffusion of these chemotaxins follows the usual laws (the
molecules moving from a site of high concentration to one of low
concentration), thus establishing a gradient, which the cells are thought to
detect via surface receptors. One explanation proposed for the movement of
cells up the gradient suggests that engagement of these receptors on a particular
area of the cell surface stimulates pseudopod formation in that area and hence
movement. As the concentration increases and more receptors are engaged, it
is thought that their re-expression is downregulated, meaning that the
migratory signals cease and the movement of the cell ceases. A number of
molecules can act as chemotactic agents, including C3a and C5a. The latter
attracts both neutrophils and monocytes, but neutrophils predominate in acute
inflammation, due to their larger number in the circulation. In addition, a large
family of structurally and functionally proinflammatory cytokines known as
chemokines have been identified. Once cells have reached the site of tissue
injury, they are capable of phagocytosing any debris or pathogens that may be
present.
102 IMMUNOLOGY FOR LIFE SCIENTISTS

Table 3.8 Adhesion molecules, expression and function

Adhesion molecule pair Cellular expression Function in inflammation

Integrins Leukocytes. Different integrins Integrins are activated by chemo-

on different leukocytes kines (e.g. MCP-1, IL-8) allowing
providing specificity for binding to ICAMs. Causes flattening
binding to different types of of leukocytes on endothelium and
CAMs on endothelium subsequent extravasation
Intercellular adhesion Endothelial cells Corresponding ligands for integrins.
molecules (ICAM-1, 2, 3, Mediate cell binding and migration
VCAM-1, MadCAM)
Selectins (P-, L- and E-) L-selectin: leukocytes P&L-selectin mediate the initial
P-selectin: found in preformed interaction of leukocytes with
state in endothelial Weibel^ endothelium ^ ‘‘rolling’’of leuko-
Palade bodies and in alpha cytes. This is followed by stronger
granules of platelets. Mobilised interaction, involving E-selectin,
to cell surface in response to leading to extravasation to sites of
inflammatory signals inflammation
E-selectin: endothelium
GlyCAM-1, CD34, GlyCAM-1: peripheral and Ligands for L-selectin. Enable leuko-
MadCAM-1 mesenteric lymph node high cyte migration into lymphoid tissues
endothelial venules and inflammatory foci
CD34: variety of non-lymphoid
tissues; capillaries of peripheral
lymph nodes
MadCAM 1: mucosal lymph
node high endothelial venules
P-selectin glycoprotein Myeloid cells, neutrophils and Ligand for P&E-selectin. Mediates
ligand (PSGL) lymphocytes ‘‘rolling’’of leukocytes on
endothelium

3.3.3 CHEMOKINES

Chemokines are small proteins, which comprise the largest family of human
cytokines (Table 3.9). They are defined by the presence of four cysteine residues
and are classified according to the sequence of amino acids involving the first
two of these residues. Thus, there are two major families (CC and CXC) and
two minor subfamilies within this group.
Upon binding to their specific receptors, these molecules stimulate the
migration and activation of a variety of cells including neutrophils, monocytes,
lymphocytes and fibroblasts. Recently, it has been shown that Th1 and Th2
lymphocytes express a distinct range of chemokine receptors, suggesting that
recruitment in inflammation may be selective and influenced by the type of
chemokine produced (Table 3.10).
 THE INNATE IMMUNE RESPONSE 103

Table 3.9 Some characteristics of chemokines

Major sources/
inducers/
Chemokine effects Description

Interleukin 8 Sources Produced by many cells including monocytes, lymphocytes, granulo-

cytes, fibroblasts, endothelial cells, hepatocytes, keratinocytes
Inducers IL-1and tumour necrosis factor
Effects Inflammatory cytokine which acts as a neutrophil chemoattractive and
activating factor. It also attracts basophils and a subpopulation of
lymphocytes. It is a potent angiogenic factor. It is a chemoattractive
and activating factor for Tcells
Macrophage Sources Monocytes/macrophages,Tcells and B cells
inflamma- Inducers Lipopolysaccharide (LPS; monocytes and macrophages), antibody to
tory proteins CD3 or phytohaemagglutinin (PHA) and phorbol myristate acetate
1a and1b (Tcells), S. aureus Cowan strain (B cells)
(MIP-1)
Effects In vitro, stimulates chemokinesis and H2O2 production by neutrophils
(human); acts as a prostaglandin-independent endogenous pyrogen
(rabbit); MIP-1a is a stem cell inhibitor (mouse), an activity that may be
antagonised by MIP-1b
Monocyte Sources PHA-stimulated peripheral blood mononuclear cells, fibroblasts,
chemo- endothelial cells, certain tumour cells and monocytes
attractant Inducers Some cells (e.g. certain tumour cells) express it constitutively,
protein endothelial cells produce it after IL-1,TNF or LPS stimulation. Conflict-
(MCP) ing evidence exists over the production by monocytes, i.e. whether it
is constitutive or induced
Effects Monocyte chemotaxis; enhances monocyte killing of certain tumour
cell lines
RANTES Sources T lymphocytes
Inducers May be constitutively expressed and enhanced on stimulation with Ag
or PHA
Effects Monocytic but not neutrophil chemoattractant. Also attractant for
Tcells. Shows subset specificity and migratingTcells are particularly
enriched for CD4+ cells bearing markers which are thought to identify
memory cells

Table 3.10 Chemokine receptor expression byTcell subsets

Chemokine
Tcell subset receptor Ligands

Th1 CXCR3 IP-10 and mIg

CCR5 RANTES, MIP-1a, 1b
Th2 CCR3 Eotaxin, RANTES, MCP-2, 3, 4
CCR4 TARC, RANTES, MCP-1, MIP-1a
104 IMMUNOLOGY FOR LIFE SCIENTISTS

KEY POINTS FOR REVIEW

. Inflammation is a complex series of biochemical and cellular events leading to

the five cardinal signs of heat, pain, swelling, redness and ultimately loss of
function.
. It involves the concerted action of the immune, clotting, fibrinolytic and kinin
systems.
. A range of chemicals such as prostaglandins and leukotrienes are released by
cells and affect immune cells and others contributing to a proinflammatory
state.
. Chemical mediators released affect vascular permeability, smooth muscle
contraction and the secretion of cytokines. This leads to upregulation of cell
surface antigens (adhesion molecules) encouraging migration of inflammatory
cells from the circulation to the periphery.
. Chemokines encourage the migration of cells into the tissues by interacting with
their receptors expressed on the surface of the cells.

3.4 HAEMOSTASIS AND THROMBOSIS

During inflammation, cells are stimulated leading to the production of

phospholipase A2 (PLA2), which converts the membrane lipid alkyl acyl
glycero-3-phosphocholine into arachidonic acid and the lyso-platelet-activating
factor (PAF). This precursor is acetylated converting it into active platelet
activating factor which has a diverse range of effects including the aggregation
and degranulation of platelets and neutrophils.
Usually, platelets do not adhere to each other or to normal endothelium but
do so when the basement membrane or extracellular matrix is exposed as a
result of endothelial damage. Platelet adhesion requires the secretion of von
Willebrand factor (VWF) (found in the vessel wall and in plasma), which binds
to the platelet surface receptor glycoprotein 1b. At the site of injury, platelets
develop pseudopodia and express a receptor (formed by the association of
glycoproteins IIb and IIIa), which binds fibrinogen and other adhesive proteins
resulting in platelet aggregation. Collagen and thrombin (formed as a result of
activation of the coagulation pathway) activate platelet phospholipase C,
leading to the hydrolysis of inositol phospholipids, activation of protein kinase
C, increased cytoplasmic calcium concentration and resulting in platelet
activation. Activated platelets express P-selectin, allowing them to adhere to
other cells such as monocytes, neutrophils and lymphocytes, thus providing a
bridge between these cells and the damaged endothelium. In addition, they
 THE INNATE IMMUNE RESPONSE 105

Figure 3.15 The fibrinolytic and coagulation pathways

The figure demonstrates the activation and inhibition of the fibrinolytic pathway and the extrinsic
and intrinsic coagulation pathways.

secrete adenosine diphosphate, which can activate adherent platelets and recruit
new platelets into the growing haemostatic plug. On the platelet surface, the
membrane reorganises to expose phospholipids. These, along with Factor V
secreted from platelet a granules, are required for the formation of enzyme/
cofactor complexes, which play a key role in the coagulation pathways
(Figure 3.15). Increased thrombin is generated, which converts fibrinogen to
fibrin. The latter forms strands that radiate from aggregated platelets to help
secure the haemostatic plug. Within the platelets, signals cause contraction of the
actomyosin elements of the cytoskeleton resulting in the compression and
consolidation of the haemostatic plug, securing it more tightly to the site of injury.

3.4.1 REGULATION OF COAGULATION

As a result of tissue damage and inflammation, blood coagulation occurs.

However, as with other immune-related pathways, the clotting cascades are
tightly regulated to prevent local thrombosis or disseminated intravascular
coagulation (DIC). Regulatory mechanisms include the neutralisation of the
106 IMMUNOLOGY FOR LIFE SCIENTISTS

enzymes and activated cofactors and clearance in the liver. In addition, there are
specific inhibitors, which can regulate coagulation. These include tissue factor
pathway inhibitor, antithrombin III (which inhibits thrombin, Factor Xa and
IXa), a2 macroglobulin, a1 antiprotease, heparin cofactor II, protein C (which
inhibits Factor VIIIa) and protein S (which inhibits Factor Va). Recently, the
therapeutic use of protein C has been established in persons with toxic shock.
Thrombin bound to thrombomodulin on endothelial cells cleaves a small peptide
from protein C, which becomes an active serine protease. The latter in the
presence of cofactors (protein S and procoagulant phospholipid) causes the
proteolysis of Factors VIIIa and Va, destroying their procoagulant activity.

3.4.2 THE FIBRINOLYTIC SYSTEM

When injury occurs to the vasculature, coagulation is initiated. However, whilst

it is important to block any leakage and ensure repair, it is vital to maintain
circulation. This is achieved by the fibrinolytic system. Fibrin deposition
activates this system and a balance is achieved between fibrin deposition and
lysis, which maintains and restructures the haemostatic seal during vessel repair.
When fibrinogen is converted to fibrin, lysine residues become available on
the molecule to which plasminogen can bind tightly by way of lysine-binding
sites. Breakdown of fibrin is initiated by plasmin, a proteolytic enzyme derived
from plasminogen as a result of the activity of plasminogen activators. Tissue
plasminogen activator (tPA) is secreted by endothelial cells (amongst others) and
when bound to fibrin in association with plasminogen is able to activate the
latter. Urokinase plasminogen activator (uPA) exists in two molecular forms.
The single-chain uPA is released by endothelial cells and activates plasminogen
bound to fibrin. The double-chain form (which can activate plasminogen in
solution as well as bound to fibrin) is produced when trace amounts of plasmin
cleave the single-chain uPA. Clearly, these powerful mediators require
regulation to ensure the desired balance between clot formation and
remodelling. Thus, plasma contains plasminogen activator inhibitors (e.g.
PAI-1 released from vascular endothelium and activated platelets) and plasmin
inhibitors that slow fibrinolysis. The primary plasmin inhibitor is a2
antiplasmin, which can rapidly inactivate free plasmin.
Thus, fibrinolysis is regulated by the rapid clearance and inactivation (by
PAI-1) of plasminogen activators.

KEY POINTS FOR REVIEW

. Platelet-activating factor promotes the aggregation and degranulation of

platelets and neutrophils.
 THE INNATE IMMUNE RESPONSE 107

. Platelet adhesion requires the secretion of von Willebrand factor that binds to
the surface glycoprotein 1b.
. Activated platelets bind to other cells and provide a bridge between them and
the damaged endothelium.
. Fibrinogen is converted to fibrin forming strands which bind to platelets to
secure the haemostatic plug.
. Coagulation must be tightly controlled to prevent thrombosis or disseminated
intravascular coagulation.
. Regulation is achieved by cofactors and specific inhibitors.
. The haemostatic plug is remodelled by a balance between fibrin deposition and
fibrinolysis.
. Fibrin is broken down by plasminogen, which is activated by the tissue
plasminogen activator or the urokinase plasminogen activator.
. Fibrinolysis is regulated by the clearance and inactivation of plasminogen
activators.

BIBLIOGRAPHY

Anderson KV. (2000) Toll signaling pathways in the innate immune response. Curr Opin
Immunol, 12; 13–19.
Ehlers MRW. (2000) CR3: a general purpose adhesion-recognition receptor essential
for innate immunity. Microb Infect, 2; 289–294.
Febbraio M, Hajjar DP, Silverstein RL. (2001) CD36: a class B scavenger receptor
involved in angiogenesis, atherosclerosis, inflammation, and lipid metabolism. J Clin
Invest, 108; 785–791.
Fernandez EJ, Lolis E. (2002) Structure, function, and inhibition of chemokines. Ann
Rev Pharmacol Toxicol, 42; 469–499.
Greenberg S, Grinstein S. (2000) Phagocytosis and innate immunity. Curr Opin
Immunol, 14; 136–145.
Kraal G, van der Laan LJW, Outi Elomaa O, Tryggvason K. (2000) The macrophage
receptor MARCO. Microb Infect, 2; 313–316.
Lindahl G, Sjöbring U, Johnsson E. (2000) Human complement regulators: a major
target for pathogenic microorganisms. Curr Opin Immunol, 12; 44–51.
Matsushita M, Endo Y, Hamasaki N, Fujita T. (2001) Activation of the lectin
complement pathway by ficollins. Int Immunopharm, 1; 359–363.
Platt N, Haworth R, Darley L, Gordon S. (2002) The many roles of the class A
macrophage scavenger receptor. Int Rev Cytol, 212; 1–40.
Raj PA, Dentino AR. (2002) Current status of defensins and their role in innate and
adaptive immunity. FEMS Microbiol Lett, 206; 9–18.
Shirai H, Murakami T, Yamada Y, Doi T, Hamakubo T, Kodama T. (1999) Structure
and function of type I and II macrophage scavenger receptors. Mech Ageing Dev,
111; 107–121.
Teixeira MM, Almeida IC, Gazzinelli RT. (2002) Introduction: innate recognition of
bacteria and protozoan parasites. Microb Infect, 4; 883–886.
Turner MW. (1996) Mannose-binding lectin: the pluripotent molecules of the innate
immune system. Immunol Today, 17; 532–540.
108 IMMUNOLOGY FOR LIFE SCIENTISTS

NOW TEST YOURSELF!

1. Which of the following statements is FALSE? C1:

(a) Comprises three different types of molecule, C1q, C1r and C1s.
(b) Comprises one nonameric C1q molecule which binds the Fc portion of
antibody in an immune complex.
(c) Enzymatic activity does not reside in the C1q molecule.
(d) Comprises one molecule of C1q and two each of the other components.
(e) When activated possesses an enzymatic site that will act on the next
component in the pathway, C4.

2. C4:
(a) Has two chains, the largest of which is the beta chain.
(b) Has an alpha chain which is cleaved by C1q, causing the release of a small
fragment called C4a.
(c) Is cleaved to give a large fragment, C4b, which binds to the target antigen via
the formation of covalent amide or ester bonds.
(d) Is fragmented and, in the presence of calcium ions, C4b bound to the target
antigen interacts with the next component of complement, C2.
(e) Is fragmented to give C4b which, with C2b, forms the C5 convertase of the
classical pathway.

3. Which of the following statements concerning the alternative pathway of

complement activation is FALSE?
(a) In the alternative pathway, C3 exists in two molecular forms: the native form
which circulates in the serum, and a conformationally altered form in which
the thiolester bond has been hydrolysed.
(b) The hydrolysed form of C3 can bind Factor D in the presence of magnesium
ions.
(c) Factor D (a serine protease) cleaves Factor B to Bb.
(d) The alternative pathway C3 convertase is formed by the molecule C3bBb.
(e) The C5 convertase of the alternative pathway, (C3b)nBb, is stabilised by
properdin.

4. Which of the following components involved in the membrane attack complex

is the first to interact with the lipids present in the membrane surrounding the
immunogen?
(a) C5.
(b) C6.
(c) C7.
(d) C8.
(e) C9.
 THE INNATE IMMUNE RESPONSE 109

5. Which of the following statements relating to the regulation of complement

activation is FALSE?
(a) Neither the classical nor the alternative pathway can be activated by
antibody alone.
(b) Relatively large amounts of specific antibody must be formed before
complement activation can occur.
(c) C1 inhibitor inhibits the activity of activated C1s and C1r by binding to their
active sites.
(d) C4 binding protein regulates the activity of C4b in the presence of Factor H,
a protease which cleaves it.
(e) Factor I in the presence of Factor H cleaves the alpha chain of hydrolysed C3
or C3b to form iC3b.

6. Which of the following statements concerning phagocytes and phagocytosis is

CORRECT?
(a) Phagocytosis may be divided into a series of sequential stages that are in
order of occurrence, ingestion, opsonisation and digestion.
(b) Tissue macrophages are often the first cells to encounter and ingest a
microorganism and are highly efficient at killing.
(c) Neutrophils are the least efficient of the professional phagocytes.
(d) Neutrophils are attracted to the site of infection by mediators, which may be
released by macrophages.
(e) Basophils, which also phagocytose, are efficient killers but are geared to
killing extracellular pathogens.

7. Which of the following statements concerning opsonins and opsonisation is

INCORRECT?
(a) An opsonin is something which coats a particle and makes it easier to
phagocytose.
(b) C3b may act as an opsonin. Both neutrophils and macrophages have
receptors which bind C3b.
(c) IgG acts as an opsonin and facilitates phagocytosis by binding to Fc gamma
receptors on phagocytic cells.
(d) Certain lipopolysaccharides or LPS-binding proteins act as ‘‘go-betweens’’
linking the particle and the phagocyte, allowing attachment and enhanced
phagocytosis.
(e) Receptors for IgG are always in an active state, whilst receptors for
complement fragments are not. The former stimulate ingestion immediately.

8. Upon stimulation, the phagocyte undergoes a respiratory burst which:

(a) Is also known as the oxidative or metabolic burst and is characterised by a
gradual increase in the consumption of oxygen by the cell.
(b) Results in the production of non-toxic oxygen metabolites.
110 IMMUNOLOGY FOR LIFE SCIENTISTS

(c) Involves the action of a group of enzymes found in the cytoplasm and
associated with the membrane of the phagosome known as the nicotinamide
triphosphate oxidase.
(d) Results in the conversion of molecular oxygen to the superoxide anion, which
is highly reactive and has an unpaired electron.
(e) Results in the production of free radicals. Addition of an electron to the
superoxide anion produces superoxide; addition of a second electron converts
it to peroxide.

9. Defensins:
(a) Accumulate as multimers in the membrane of target cells, forming cyclic
peptides which form channels in the lipid bilayers.
(b) Are widely distributed, usually anionic and with a molecular mass of about
30–40 kDa.
(c) Are able to disrupt biological membranes, their richest source in humans
being the lysosomes of macrophages.
(d) Have a double-stranded structure, and those from humans trimerise in
crystalline form.
(e) Are a large number of small peptides which exhibit cytotoxicity to host cells.

10. The increased permeability of the blood vessels leads to the cardinal signs of
inflammation, which are:
(a) Heat, redness and pain.
(b) Heat, redness, pain and swelling.
(c) Heat, pain, swelling and loss of function.
(d) Heat, redness, pain and loss of function.
(e) Heat, redness, pain, swelling and loss of function.

11. Which of the following systems is NOT involved in the inflammatory

process?
(a) The immune system.
(b) The kallin system.
(c) The fibrinolytic system.
(d) The clotting system.
(e) The complement cascade.

12. Kinins are:

(a) Small, acidic peptides.
(b) Large, basic peptides.
(c) Derived from kininogens by the action of kallikreins, which are serine
esterases.
(d) Peptides which affect the movement of smooth muscle, preventing vasodila-
tion and causing pain.
 THE INNATE IMMUNE RESPONSE 111

(e) A group of peptides which include bradykinin, which is derived from a serum
macroglobulin precursor and causes the slow, sustained contraction of
smooth muscles.

13. Chemokines are:

(a) A large family of proinflammatory molecules which are not structurally
related.
(b) A group of proinflammatory molecules which stimulate the migration and
proliferation of a variety of cells.
(c) Molecules which stimulate cells such as neutrophils, monocytes, lymphocytes
and fibroblasts.
(d) May attract both neutrophils and monocytes but the latter predominate in
acute inflammation due to their capacity for replication.
(e) A small family of structurally related molecules which show distinct
functions.

14. Which of the following is part of the intrinsic coagulation pathway?

(a) Factor Xa.
(b) Plasminogen.
(c) Factor XI.
(d) Thrombin.
(e) Tissue Factor VII.

15. Which of the following statements is INCORRECT?

(a) Factor XII may be activated by surface exposed collagen.
(b) Antithrombin III may inhibit the activity of Factor XIa.
(c) Tissue Factor VII may activate Factor X.
(d) Plasmin degrades cross-linked fibrin.
(e) Thrombin converts fibrinogen to fibrin.