SECTION TWO The adversaries – host defences

67

9

The body has both ‘innate’ and ‘adaptive’ immune defences When an organism infects the body, the defence systems

already in place may well be adequate to prevent replica-

tion and spread of the infectious agent, thereby prevent-

ing development of disease. These established mechanisms

are referred to as constituting the ‘innate’ immune system.

However, should innate immunity be insufficient to parry

the invasion by the infectious agent, the so-called ‘adaptive’

immune system then comes into action, although it takes

time to reach its maximum efficiency ( Fig. 9.1 ). When it does

take effect, it generally eliminates the infective organism,

allowing recovery from disease.

The main feature distinguishing the adaptive response

from the innate mechanism is that specific memory of infec-

tion is imprinted on the adaptive immune system, so that

should there be a subsequent infection by the same agent, a

particularly effective response comes into play with remark-

able speed. It is worth emphasizing, however, that there is

close synergy between the two systems, with the adaptive

mechanism greatly improving the efficiency of the innate

response.

The contrasts between these two systems are set out

in Table 9.1 . On the one hand, the soluble factors such as

lysozyme and complement, together with the phagocytic

cells, contribute to the innate system, while on the other

the lymphocyte-based mechanisms that produce antibody

and T lymphocytes are the main elements of the adap-

tive immune system. Not only do these lymphocytes pro-

vide improved resistance by repeated contact with a given

infectious agent, but the memory with which they become

endowed shows very considerable specificity to that infec-

tion. For instance, infection with measles virus will induce

a memory to that microorganism alone and not to another

virus such as rubella.

DEFENCES AGAINST ENTRY INTO THE BODY

A variety of biochemical and physical barriers operate at the body surfaces Before an infectious agent can penetrate the body, it must

overcome biochemical and physical barriers that operate at

the body surfaces. One of the most important of these is the

skin, which is normally impermeable to the majority of infec-

tious agents. Many bacteria fail to survive for long on the

skin because of the direct inhibitory effects of lactic acid and

fatty acids present in sweat and sebaceous secretions and the

lower pH to which they give rise ( Fig. 9.2 ). However, should

there be skin loss, as can occur in burns, for example, infec-

tion becomes a major problem.

The membranes lining the inner surfaces of the body

secrete mucus, which acts as a protective barrier, inhibit-

ing the adherence of bacteria to the epithelial cells, thereby

preventing them from gaining access to the body. Microbial

and other foreign particles trapped within this adhesive

mucus may be removed by mechanical means such as cili-

ary action, coughing and sneezing. The flushing actions of

tears, saliva and urine are other mechanical strategies that

help to protect the epithelial surfaces. In addition, many

of the secreted body fluids contain microbicidal factors,

Introduction

In the preceding chapters, we have outlined some of the fundamental characteristics of the myriad types of microparasites and macroparasites that may infect the body. We now turn to consider the ways in which the body seeks to defend itself against infection by these organisms.

The innate defences of the body 9

infection re-infection

innateimmunity

adaptive immunity

specificimmunologic

memory

disease recovery no disease

1 2

3

4 6

5

Figure 9.1 Innate and adaptive immunity. An infectious agent first encounters elements of the innate immune system. These may be sufficient (1) to prevent disease but if not, disease may result (2). The adaptive immune system is then activated (3) to produce recovery (4) and a specific immunologic memory (5). Following re-infection with the same agent, no disease results (6) and the individual has acquired immunity to the infectious agent.

68

CHAPTERCHAPTER9 The innate defences of the body

e.g. the acid in gastric juice, spermine and zinc in semen,

lactoperoxidase in milk, and lysozyme in tears, nasal secre-

tions and saliva.

The phenomenon of microbial antagonism is associ-

ated with the normal bacterial flora of the body. These

commensal organisms suppress the growth of many

potentially pathogenic bacteria and fungi at superficial

sites, first by virtue of their physical advantage of previ-

ous occupancy, especially on epithelial surfaces, second by

competing for essential nutrients, or third by producing

inhibitory substances such as acid or colicins. The latter are

a class of bactericidins that bind to the negatively charged

surface of susceptible bacteria and form a voltage-depen-

dent channel in the membrane, which kills by destroying

the cell's energy potential.

DEFENCES ONCE THE MICROORGANISM PENETRATES THE BODY Despite the general effectiveness of the various barriers, micro-

organisms successfully penetrate the body on many occasions.

When this occurs, two main defensive strategies come into

play, based on:

• the mechanism of phagocytosis, involving engulfment

and killing of microorganisms by specialized cells, the

‘professional phagocytes’

• the destructive effect of soluble chemical factors, such as

bactericidal enzymes.

Two types of professional phagocyte Perhaps because of the belief that professionals do a better

job than amateurs, the cells that shoulder the main burden

of our phagocytic defences have been labelled ‘professional

phagocytes’. These consist of two major cell families, as orig-

inally defined by Elie Metchnikoff, the Russian zoologist

( Box 9.1 ; Fig. 9.3 ):

• the large macrophages

• the smaller polymorphonuclear granulocytes, which

are generally referred to as polymorphs or neutrophils

because their cytoplasmic granules do not stain with

haematoxylin and eosin.

As a very crude generalization, it may be said that the poly-

morphs provide the major defence against pyogenic (pus-

forming) bacteria, while the macrophages are thought to be

at their best in combating organisms capable of living within

the cells of the host.

Macrophages are widespread throughout the tissues Macrophages originate as bone marrow promonocytes,

which develop into circulating blood monocytes ( Fig. 9.4 )

and finally become the mature macrophages, which are

widespread throughout the tissues and collectively termed

the ‘mononuclear phagocyte system’ ( Fig. 9.5 ). These

macrophages are present throughout the connective tissue

Innate immune system Adaptive immune system

Major elements

Soluble factors Lysozyme, complement, acute phase proteins, e.g. C-reactive protein, interferon

Antibody

Cells Phagocytes Natural killer cells

T lymphocytes

Response to microbial infection

First contact + + +

Second contact + + + + +

Non-specific; no memory Resistance not improved by repeated contact

Specific; memory Resistance improved by repeated contact

Table 9.1 Comparison of innate and adaptive effector immune systems

Innate immunity is sometimes referred to as ‘natural’, and adaptive as ‘acquired’. There is considerable interaction between the two systems. ‘Humoral’ immunity due to soluble factors contrasts with immunity mediated by cells. Primary contact with antigen produces both adaptive and innate responses, but if the same antigen persists or is encountered a second time the specific adaptive response to that antigen is much enhanced.

lysozymein most

tears, nasal secretions and

saliva

sebaceousgland

secretions

commensalorganisms in

gut and vagina

spermine insemen

mucus

cilia liningtrachea

skin

acid instomach

biochemical chemical and physical

Figure 9.2 Exterior defences. Most of the infectious agents encountered by an individual are prevented from entering the body by a variety of biochemical and physical barriers. The body tolerates a variety of commensal organisms, which compete effectively with many potential pathogens.

69

SECTION TWO The adversaries – host defences

and are associated with the basement membrane of small

blood vessels. They are particularly concentrated in the

lung (alveolar macrophages), liver (Kupffer cells) and the

lining of lymph node medullary sinuses and splenic sinu-

soids ( Fig. 9.6 ), where they are well placed to filter off

foreign material ( Fig. 9.7 ). Other examples are the brain

microglia, kidney mesangial cells, synovial A cells and

osteoclasts in bone. In general, these are long-lived cells that

depend upon mitochondria for their metabolic energy and

show elements of rough-surfaced endoplasmic reticulum

( Fig. 9.8 ) related to the formidable array of different secre-

tory proteins that these cells generate.

Polymorphs possess a variety of enzyme-containing granules The polymorph is the dominant white cell in the blood-

stream and, like the macrophage, shares a common haemo-

poietic stem cell precursor with the other formed elements

of the blood. It has no mitochondria, but uses its abundant

cytoplasmic glycogen stores for its energy requirements;

therefore, glycolysis enables these cells to function under

anaerobic conditions, such as those in an inflammatory

focus. The polymorph is a non-dividing, short-lived cell,

with a segmented nucleus; the cytoplasm is characterized by

an array of granules, which are illustrated in Figure 9.9 .

A B

Figure 9.4 Phagocytic cells. (A) Blood monocyte and (B) polymorphonuclear neutrophil, both derived from bone marrow stem cells. (Courtesy of P.M. Lydyard.)

4. alveolarmacrophages

7. splenicmacrophages

1. bloodmonocytes

8. lymph noderesident andrecirculating

macrophages

precursors inbone marrow

6. brainmicroglial cells

2. liverKupffer cells

3. kidneymesangialphagocytes

synovialA cells

osteoclasts

5. connectivetissuehistiocytes

Figure 9.5 The mononuclear phagocyte system. Tissue macrophages are derived from blood monocytes, which are manufactured in the bone marrow. (The numbers relate to those in Fig. 9.6 .)

Elie Metchnikoff (1845–1916)

This perceptive Russian zoologist can legitimately be regarded as the father of the concept of cellular immunity, in which it is recognized that certain specialized cells mediate the defence against microbial infections. He was intrigued by the motile cells of transparent starfish larvae and made the critical observation that a few hours after introducing a rose thorn into the larvae, the rose thorn became surrounded by the motile cells. He extended his investigations to mammalian leukocytes, showing their ability to engulf microorganisms, a process that he termed ‘phagocytosis’ (literally, eating by cells).

Because he found this process to be even more effective in animals recovering from an infection, he came to the conclusion that phagocytosis provided the main defence against infection. He defined the existence of two types of circulating phagocytes: the polymorphonuclear leukocyte, which he termed a ‘microphage’, and the larger ‘macrophage’.

Although Metchnikoff held the somewhat polarized view that cellular immunity based upon phagocytosis provided the main, if not the only, defence mechanism against infectious microorganisms, we now know that the efficiency of the phagocytic system is enormously enhanced through cooperation with humoral factors, in particular antibody and complement.

Figure 9.3 Elie Metchnikoff (1845–1916). (Courtesy of the Wellcome Institute Library, London.)

Box 9.1 Lessons in Microbiology

70

CHAPTERCHAPTER9 The innate defences of the body

capillaryendothelial cells

sinusoidal space

hepatocyte

Kupffer cell

endothelial cell

basementmembrane

mesangialmacrophage

endotheliumpodocyte

pneumocytetype II

macrophage

pneumocytetype I

air space

basementmembrane

capillary

capillary

mesotheliumbasement membrane

reticular fibres

macrophage

capillary

microglial cell

ependyma

nerve cell basementmembrane

macrophage

sinus endothelium

erythrocytesmacrophages

endothelialcell

circulatingblood monocyte

1Kupffer cellsin the liver

intraglomerularmesangial cells of the kidney

alveolar macrophagesin the lung

connective tissuehistiocytes

brain microglia spleen sinusmacrophages

lymph node sinusmacrophages

2 3 4

8765

Figure 9.6 Tissue location of mononuclear phagocytes.

V

NORMAL INJECTED

L

SG

Figure 9.7 Localization of intravenously injected particles in the mononuclear phagocyte system. ( Right ) A mouse was injected with fine carbon particles and killed 5 min later. Carbon accumulates in organs rich in mononuclear phagocytes: lungs (L), liver (V), spleen (S) and areas of the gut wall (G). ( Left ) Normal organ colour shown in an uninjected control mouse. (Courtesy of P.M. Lydyard.)

LM

E

PN

Figure 9.8 Monocyte ( × 8000), with ‘horseshoe’ nucleus (N). Phagocytic and pinocytic vesicles (P), lysosomal granules (L), mitochondria (M) and isolated profiles of rough-surfaced endoplasmic reticulum (E) are evident. (Courtesy of B. Nichols; © Rockefeller University Press.)

71

SECTION TWO The adversaries – host defences

Azurophil granules Specific granules

0.5 µ m 0.2 µ m1500/cell 3000/cell

Lysozyme Lysozyme

Myeloperoxidase Cytochrome b 558

Elastase Alkaline phosphatase

Cathepsin G Lactoferrin

Acid hydrolases

Defensins Vitamin B12 binding protein

BPI (bactericidal permeability increasing protein)

Phagocytosis and killing Phagocytes recognize pathogen-associated molecular patterns (PAMPs) The first event in the uptake and digestion of a microorgan-

ism by the professional phagocyte involves the attachment

of the microbe to the surface of the cell through the recog-

nition of repeating pathogen-associated molecular patterns

(PAMPs) on the microbe by pattern recognition receptors

(PRRs) on the phagocyte surface ( Fig. 9.10 ). A major sub-

set of these PRRs belongs to the class of so-called ‘Toll-

like receptors’ (TLRs) because of their similarity to the Toll

receptor in the fruit fly, Drosophila , which, in the adult, trig-

gers an intracellular cascade generating the expression of

antimicrobial peptides in response to microbial infection.

A series of cell surface TLRs acting as sensors for extra-

cellular infections have been identified ( Fig. 9.11 ) which

are activated by microbial elements such as peptidogly-

can, lipoproteins, mycobacterial lipoarabinomannan, yeast

zymosan and flagellin. Other PRRs displayed by phago-

cytes include the cell bound ‘C-type (calcium-dependent)

lectins’, of which the macrophage mannose receptor is an

example, and ‘scavenger receptors’, which recognize a vari-

ety of anionic polymers and acetylated low density proteins.

Examples of intracellular PAMPs are the unmethylated

guanosine-cytosine (CpG) sequences of bacterial DNA and

double-stranded RNA from RNA viruses.

The phagocyte is activated through PAMP recognition The attached microbe may then signal through the phago-

cyte receptors to initiate the ingestion phase by activating

an actin-myosin contractile system, which sends arms of

cytoplasm around the particle until it is completely enclosed

within a vacuole (phagosome; Fig. 9.12 ; see Fig. 9.10 ). Shortly

afterwards, the cytoplasmic granules fuse with a phago-

some and discharge their contents around the incarcerated

microorganism.

The internalized microbe is the target for a fearsome array of killing mechanisms As phagocytosis is initiated, the attached microbes also signal

through one of the PRRs to engineer an appropriate defensive

response to the different types of infection through a number of

NF κ B-mediated responses. This activation of a unique plasmamembrane reduced nicotinamide adenine dinucleotide phos-

phate (NADPH) oxidase reduces oxygen to a series of power-

ful microbicidal agents, namely superoxide anion, hydrogen

Figure 9.9 Neutrophil. The multi-lobed nucleus and primary azurophilic, secondary specific and tertiary lysosomal granules are well displayed. In some granules there is an overlap in the contents between azurophilic and secondary granules. Typical conventional lysosomes with acid hydrolase are also seen. (Courtesy of D. McLaren.)

attachment by patternrecognition receptors

pseudopodia forminga phagosome

granule fusionand killing

release ofmicrobial products

phagosomeforming

granule damage anddigestion

phagocyte

receptor

PAMP

phagolysosome

complete formationof the phagolysosome

A B

C D

Figure 9.10 Phagocytosis. (A) Phagocytes attach to microorganisms (blue icon) via their cell surface receptors which recognize pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide. (B) If the membrane now becomes activated by the attached infectious agent, the pathogen is taken into a phagosome by pseudopodia, which extend around it. (C) Once inside the cell, the various granules fuse with the phagosome to form a phagolysosome. (D) The infectious agent is then killed by a battery of microbicidal degradation mechanisms, and the microbial products are released.

72

CHAPTERCHAPTER9 The innate defences of the body

peroxide, singlet oxygen and hydroxyl radicals ( Box 9.2 ;

see also Ch. 14 ). Subsequently, the peroxide, in association with

myeloperoxidase, generates a potent halogenating system from

halide ions, which is capable of killing both bacteria and viruses.

As superoxide anion is formed, the enzyme superoxide

dismutase acts to convert it to molecular oxygen and

hydrogen peroxide, but in the process consumes hydrogen

ions. Therefore initially there is a small increase in pH,

which facilitates the antibacterial function of the families

of cationic proteins derived from the phagocytic granules.

These molecules damage microbial membranes by the

proteolytic action of cathepsin G and by direct adherence

to the microbial surface. The defensins have an amphipa-

thic structure which allows them to insert into microbial

membranes to form destabilizing voltage-regulated ion

channels. These antibiotic peptides reach extraordinarily

high concentrations within the phagosome and act as dis-

infectants against a wide spectrum of bacteria, fungi and

enveloped viruses. Other important factors are:

• lactoferrin, which complexes iron to deprive bacteria of

essential growth elements

• lysozyme, which splits the proteoglycan cell wall of

bacteria

• nitric oxide, which can lead not only to iron seclusion

but, together with its derivative, the peroxynitrite

radical, can also be directly microbicidal.

The pH now falls so that the dead or dying microorganisms

are extensively degraded by acid hydrolytic enzymes, and

the degradation products released to the exterior.

NF κ B activation can also lead to the release of proinflam-matory mediators. These include the antiviral interferons,

PAMPsPRRs TLR1 TLR2 TLR4 TLR5 TLR6 TLR10 TLR11

BacterialLipopeptidesLipoproteins Bacterial LPS

Bacterialflagellin

TLR3

Viral dsRNA

TLR7

Viral ssRNANucleotide analogues

TLR8 TLR9

Bacterialunmethylated

CpG DNA

BacterialLipopeptidesLipoproteins Unknown

Plasmamembrane

Endosome

Toxoplasma gondiiProfilin

NF!B IRF-5

Transcription

Chromosome

IRF-3

IRF-7 NFκB IRF-5

Figure 9.11 Recognition of PAMPs by a subset of pattern recognition receptors (PRRs) termed Toll-like receptors (TLRs). TLRs reside within plasma membrane or endosomal membrane compartments, as shown. All TLRs have multiple N-terminal leucine-rich repeats forming a horseshoe-shaped structure which acts as the PAMP-binding domain. Upon engagement of the TLR ectodomain with an appropriate PAMP (some examples are shown), signals are propagated into the cell that activate the nuclear factor kB (NFkB) and/or interferon regulated factor (IRF) transcription factors, as shown. NFkB and IRF transcription factors then direct the expression of numerous antimicrobial gene products such as cytokines and chemokines, as well as proteins that are involved in altering the activation state of the cell.

Lt

Lt

Lt

Lt

Lt

Figure 9.12 Electron micrographic study of phagocytosis. These two micrographs show human phagocytes engulfing latex particles (Lt). (A) × 3000; (B) × 4500. (Courtesy of C.H.W. Horne.)

73

SECTION TWO The adversaries – host defences

the small protein cytokines interleukin-1 β (IL-1 β ), IL-6, IL-12and TNF (TNF α ), which activate other cells through bindingto specific receptors, and chemokines such as IL-8, which rep-

resent a subset of chemoattractant cytokines.

Microbial nucleotide breakdown products of infectious

agents that have succeeded in gaining access to the interior

of a cell can be recognized by the so-called NOD proteins

and the typical CpG DNA motif which binds to the endo-

somal TLR9. Other endosomal Toll-like receptors, TLR3 and

TLR7/8, are responsive to intracellular viral RNA sequences

and engender production of antiviral interferon.

Phagocytes are mobilized and targeted onto the microorganism by chemotaxis Phagocytosis cannot occur unless the bacterium first

attaches to the surface of the phagocyte, and clearly this

cannot happen unless both have become physically close

to each other. There is therefore a need for a mechanism

that mobilizes phagocytes from afar and targets them

onto the bacterium. Many bacteria produce chemical

substances, such as formyl methionyl peptides, which

directionally attract leukocytes, a process known as ‘che-

motaxis’. However, this is a relatively weak signalling

system, and evolution has provided the body with a far

more effective ‘magnet’ that uses a complex series of pro-

teins collectively termed ‘complement’.

Activation of the complement system Complement resembles blood clotting, fibrinolysis and kinin

formation in being a major triggered enzyme cascade sys-

tem. Such systems are characterized by their ability to pro-

duce a rapid, highly amplified response to a trigger stimulus

mediated by a cascade phenomenon in which the product

of one reaction is the enzymic catalyst of the next. The most

abundant and most central component is C3 (complement

components are designated by the letter ‘C’ followed by a

number), and the cleavage of this molecule is at the heart of

all complement-mediated phenomena.

Oxygen-independent antimicrobial mechanisms

Cathepsin G and elastase

Damage to microbial membranesLow molecular weight defensins High molecular weight cationic proteins Bactericidal permeability-increasing protein Lactoferrin Complex with iron Lysozyme Splits proteoglycanAcid hydrolases Degrade dead microbes

Oxygen-dependent antimicrobial mechanisms

Reaction Sequence Generated by NADPH Oxidase:

Glucose + NADP + pentose phosphate + NADPH

} O 2 burst plus generation of superoxide anionNADPH + O 2 −++NADP 2O − ++22O 2H +

12 2 2H O O Spontaneous formation

of further microbial agents

H 2 O 2 + CI − Myeloperoxidase generation

of microbicidal moleculesOCI − + H 2 O 2

O 2 + H 2 O 2 Protective mechanisms used by host and many microbes

2H 2 O 2 2H 2 O + O 2

Nitric Oxide Reaction Sequence

O 2 + L –arginine NO· Reactive species −+⋅ ⋅ 2 NO O ·ONOO −

NO· + Fe/RSH Fe(RS) 2 (NO) 2 Complexes iron

Microbicidal species in bold letters. Fe/RSH, a complex of iron with a general sulfhydryl molecule; Fe (RS) 2 , oxidized Fe/RSH; 2O− ,

superoxide anion; 1 O 2 , singlet (activated) oxygen; OH, hydroxyl free radical; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NADP + , oxidized NADPH; H 2 O 2 , hydrogen peroxide; OCI

– , hypochlorite anion; NO; nitric oxide; ONOO – , peroxynitrite radical.

}

Hexose monophosphate shunt

NADPH oxidase>

Spontaneous dismutation > }

− +2 2 2O H O > �−+ +1 2OH OOH

Myeloperoxidase>

− + 2H OOCI }> −+ +1 2O 2CI H O

22O 2H− ++ Superoxide dismutase

> }Catalase >NO synthase

> }>>

Box 9.2 Antimicrobial Mechanisms in Phagocytic Vacuoles

74

CHAPTERCHAPTER9 The innate defences of the body In normal plasma, C3 undergoes spontaneous activation

at a very slow rate to generate the split product C3b. This is

able to complex with another complement component, fac-

tor B, which is then acted upon by a normal plasma enzyme,

factor D, to produce the C3-splitting enzyme C3bBb. ThisC3 convertase can then split new molecules of C3 to give C3a

(a small fragment) and further C3b. This represents a posi-

tive feedback circuit with potential for runaway amplifica-

tion; however, the overall process is restricted to a tick-over

level by powerful regulatory mechanisms, which break the

unstable soluble-phase C3 convertase into inactive cleavage

products ( Fig. 9.13 ).

In the presence of certain molecules, such as the carbohy-

drates on the surface of many bacteria, the C3 convertase can

become attached and stabilized against breakdown. Under

these circumstances, there is active generation of new C3

convertase molecules, and what is known as the ‘alternative’

complement pathway can swing into full tempo (see Ch. 10 ).

Complement synergizes with phagocytic cells to produce an acute inflammatory response Activation of the alternative complement pathway with

the consequent splitting of very large numbers of C3 mole-

cules has important consequences for the orchestration of an

integrated antimicrobial defense strategy ( Fig. 9.14 ). Large

numbers of C3b produced in the immediate vicinity of the

microbial membrane bind covalently to that surface and act

as opsonins (molecules that make the particle they coat more

susceptible to engulfment by phagocytic cells; see below).

This C3b, together with the C3 convertase, acts on the next

component in the sequence, C5, to produce a small fragment,

C5a which, together with C3a, has a direct effect on mast

cells to cause their degranulation ( Fig. 9.15 ). This results in

the release not only of mediators of vascular permeability,

but also of factors chemotactic for polymorphs ( Table 9.2 ).

The circulating equivalent of the tissue mast cell, the baso-

phil, is shown in Figure 9.16 .

The vascular permeability mediators increase the perme-

ability of capillaries by modifying the intercellular forces

between the endothelial cells of the vessel wall. This allows

the exudation of fluid and plasma components, including

more complement, to the site of the infection. These media-

tors ( Table 9.2 ) also up-regulate molecules such as intercel-

lular adhesion molecule-1 (ICAM-1) and endothelial cell

leukocyte adhesion molecule-1 (ELAM-1), which bind to

specific complementary molecules on the polymorphs and

encourage them to stick to the walls of the capillaries, a pro-

cess termed ‘margination’.

The chemotactic factors, on the other hand, provide a

chemical gradient which attracts marginated polymorpho-

nuclear leukocytes from their intravascular location, through

the walls of the blood vessels, and eventually leads them to

the site of the C3b-coated bacteria that initiated the whole

activation process. Polymorphs have a well-defined recep-

tor for C3b on their surface, and as a result, the opsonized

bacteria adhere very firmly to the surface of these newly

arrived cells.

The processes of capillary dilation (erythema), exudation

of plasma proteins and of fluid (oedema) due to hydro-

static and osmotic pressure changes, and the accumulation

of neutrophils are collectively termed the ‘acute inflam-

matory response’, and result in a highly effective way of

focusing phagocytic cells onto complement-coated micro-

bial targets.

It also seems clear that the macrophage can be stimu-

lated by certain bacterial toxins such as the lipopolysaccha-

rides (LPS), by the action of C5a, and by the phagocytosis

of C3b-coated bacteria, to secrete other potent mediators of

acute inflammation, independently of the mast cell-directed

pathway ( Fig. 9.17 ).

C9 molecules form the ‘membrane attack complex’, which is involved in cell lysis We have already introduced the idea that following the

activation of C3 the next component to be cleaved is C5;

the larger C5b fragment that results becomes membrane

bound. This subsequently binds components C6, C7 and

C8, which form a complex capable of inducing a criti-

cal conformational change in the terminal component C9.

The unfolded C9 molecules become inserted into the lipid

bilayer and polymerize to form an annular ‘membrane

attack complex’ (MAC) ( Figs 9.18, 9.19 ). This behaves as a

transmembrane channel that is fully permeable to electro-

lytes and water; because of the high internal colloid osmotic

pressure of cells, there is a net influx of sodium (Na + ) and

this frequently leads to lysis.

microbialpolysaccharide

properdin

C3 convertasestabilization

C3bB

factor BC3b

C3a

C3

C3bBb

iC3b

C3dC3c +(inactive products)

proteases

factor D

factors H and I

C3b regulation

(C3 convertase)

feedbackloop

Figure 9.13 Activation of complement by microorganisms. C3b is formed by the spontaneous breakdown of C3 complexes with factor B to form C3bB which is split by factor D to produce a C3 convertase C3bBb, capable of further cleaving C3. The convertase is heavily regulated by factors H and I but can be stabilized on the surface of microbes and properdin. The horizontal bar indicates an enzymically active complex. iC3b, inactive C3b.

75

SECTION TWO The adversaries – host defences

Acute phase proteins Certain proteins in the plasma, collectively termed ‘acute

phase proteins’, increase in concentration in response to

early ‘alarm’ mediators such as the cytokines interleukin-1

(IL-1), IL-6 and tumour necrosis factor (TNF), released as

a result of infection or tissue injury. Many acute phase

reactants such as mannose binding lectin and C-reactive

protein (CRP) increase dramatically during inflamma-

tion ( Fig. 9.20 ). Like the professional phagocytes, both

use pattern recognition receptors to bind to molecular

patterns on the pathogen (PAMPs), to generate defen-

sive effector functions ( Fig. 9.21 ). Other acute phase reac-

tants show more moderate rises, usually less than fivefold

(see Table 9.3 ). In general, these proteins are thought to

have defensive roles.

capillary

C3bBb

C3b C3

1

8

93

4

2

4

57

7

6

C5

C5a/C3a

vascularpermeability

mediators

chemotacticfactors

exudation

polymorph

C3b receptor

C3b

bacterium initiation

MC

Figure 9.14 The defensive strategy of the acute inflammatory reaction initiated by bacterial activation of the alternative complement pathway. Activation of the C3bBb C3 convertase by the bacterium (1) leads to the generation of C3b (2) (which binds to the bacterium (3)), C3a and C5a (4), which recruit mast cell (MC) mediators. These in turn cause capillary dilation (5), exudation of plasma proteins (6), and chemotactic attraction (7) and adherence of polymorphs to the C3b-coated bacterium (8). Note that C5a itself is also chemotactic. The polymorphs are then activated for phagocytosis and the final kill (9).

Figure 9.15 Electron micrographs of rat peritoneal mast cells. These show (A) the resting cell with its electron-dense granules ( × 6000) and (B) a granule in the process of exocytosis ( × 30 000). (Courtesy of T.S.C. Orr.)

A

B

76

CHAPTERCHAPTER9 The innate defences of the bodyInfl ammatory mediators

Mediator Main source Actions

Histamine Mast cells, basophils Increased vascular permeability, smooth muscle contraction, chemokinesis

5-hydroxytryptamine (5HT – serotonin)

Platelets, mast cells (rodent) Increased vascular permeability, smooth muscle contraction

Platelet activating factor (PAF) Basophils, neutrophils, macrophages Mediator release from platelets, increased vascular permeability, smooth muscle contraction, neutrophil activation

IL-8 (CXCL8) Mast cells, endothelium, monocytes and lymphocytes

Polymorph and monocyte localization

C3a Complement C3 Mast cell degranulation, smooth muscle contraction

C5a Complement C5 Mast cell degranulation, neutrophil and macrophage chemotaxis, neutrophil activation, smooth muscle contraction, increased capillary permeability

Bradykinin Kinin system (kininogen) Vasodilation, smooth muscle contraction, increased capillary permeability, pain

Fibrinopeptides and fibrin breakdown products

Clotting system Increased vascular permeability, neutrophil and macrophage chemotaxis

Prostaglandin E 2 (PGE 2 ) Cyclo-oxygenase pathway, mast cells Vasodilation, potentiates increased vascular permeability produced by histamine and bradykinin

Leukotriene B 4 (LTB 4 ) Lipoxygenase pathway, mast cells Neutrophil chemotaxis, synergizes with PGE 2 in increasing vascular permeability

Leukotriene D 4 (LTD 4 ) Lipoxygenase pathway Smooth muscle contraction, increasing vascular permeability

Table 9.2 The major inflammatory mediators that control blood supply and vascular permeability or modulate cell movement

Other mediators are generated from the coagulation process. Chemotaxis refers to directed migration of granulocytes up the concentration gradient of the mediator, whereas chemokinesis describes randomly increased motility of these cells. (Reproduced from Male D, Brostoff J, Roth DB, Roitt I. Immunology , 7th edition, 2006. Mosby Elsevier, with permission.)

Figure 9.16 Morphology of the basophil. (A) This blood smear shows a typical basophil with its deep violet-blue granules. Wright's stain ( × 1500). (B) Electron micrograph showing the ultrastructure of the basophil. Basophils in guinea pig skin showing the nuclei (N) and characteristic randomly distributed granules (G) ( × 6000). (Courtesy of D. McLaren.)

A

N

G

B

77

SECTION TWO The adversaries – host defences

Other extracellular antimicrobial factors There are many microbicidal agents that operate at short range

within phagocytic cells, but also appear in various body fluids

in sufficient concentration to have direct inhibitory effects on

infectious agents. For example, lysozyme is present in fluids

such as tears and saliva in amounts capable of acting against

the proteoglycan wall of susceptible bacteria. Similarly, lacto-

ferrin may appear in the blood in sufficient concentration to

complex iron and deprive bacteria of this important growth

factor. Whether agents that normally act over a short range,

such as reactive oxygen metabolites or TNF (a cytotoxic mol-

ecule produced by macrophages and other cell types), can

reach concentrations in the body fluids that are adequate to

allow them to act at a distance from the cell producing them

will be discussed in Chapter 14 , particularly when consider-

ing the mechanisms by which the blood-borne forms of para-

sites such as malaria are attacked.

Interferons are a family of broad spectrum antiviral molecules Interferons (IFNs) are widespread throughout the animal

kingdom and are again discussed further in Chapter 14 .

They were first recognized by the phenomenon of viral inter-

ference, in which a cell infected with one virus is found to

be resistant to superinfection by a second unrelated virus.

Leukocytes produce many different α -interferons (IFN α ),

Acute phase reactant Function

Dramatic increases in concentration

C-reactive protein Fixes complement, opsonizes

Mannose binding lectin Fixes complement, opsonizes

α 1 acid glycoprotein Transports proteinSerum amyloid A protein

Complexes chondroitin sulphate

Moderate increases in concentration

α 1 proteinase inhibitors Inhibit bacterial proteases α 1 anti-chymotrypsin Inhibits bacterial proteasesSurfactant protein A Binds influenza virus

haemagglutinin

C3, C9, factor B Increase complement function

Ceruloplasmin O 2 scavenger

Fibrinogen Coagulation

Angiotensin Blood pressure

Haptoglobin Binds haemoglobin

Fibronectin Cell attachment

Table 9.3 Acute phase proteins produced in response to infection in the human

ICAM-1

bacterial toxin C5a (?C3a)

microbe

PGE2 , LTB4 IL-1, TNFα NAP-2

induce adhesion molecules

ELAM-1

PMNactivation andchemotaxis

neutrophil

NAP-1

Mø

C3b

basementmembrane

endothelial cell

IL-8

increasevascular

permeability

Figure 9.17 A role for the macrophage (Mø) in the initiation of acute inflammation. Stimulation induces macrophage secretion of mediators. Blood neutrophils stick to the adhesion molecules on the endothelial cell and use them to provide traction as they force their way between the cells, through the basement membrane (with the help of secreted elastase) and up the chemotactic gradient. During this process they become progressively activated by neutrophil activating peptide-2 (NAP-2). PGE 2 , prostaglandin E 2 ; LTB 4 , leukotriene B 4 ; IL-1, interleukin-1; PMN, polymorphonuclear neutrophil; TNF α , tumour necrosis factor alpha; ELAM-1, endothelial cell leukocyte adhesion molecule-1; ICAM-1, intercellular adhesion molecule-1.

78

CHAPTERCHAPTER9 The innate defences of the body

while fibroblasts and probably all cell types synthesize IFN β . A third type (IFN γ ) is not a component of the innate immunesystem and will be discussed in Chapter 10 as a member of

the important cytokine family.

When cells are infected by a virus, they synthesize and

secrete IFNs α and β , which bind to specific receptors onnearby uninfected cells. The bound IFN exerts its antivi-

ral effect by facilitating the synthesis of two new enzymes,

which interfere with the machinery used by the virus for its

own replication. The mechanism of action of IFN is discussed

more fully in Chapter 14; the net result is to set up a cordon

of infection-resistant cells around the site of virus infection, so

restraining its spread ( Fig. 9.22 ). IFN is highly effective in vivo,

as supported by experiments in which mice injected with an

antiserum to murine IFN were found to be killed by several

hundred times less virus than was needed to kill the controls.

It should be emphasized, however, that IFN seems to play a

significant role in recovery from, rather than prevention of,

viral infections.

Extracellular killing Natural killer cells attach to virally infected cells, allowing them to be differentiated from normal cells There is a widely held view that viruses represent fragments

of the genome of multicellular organisms that have achieved

the ability to exist in an extracellular state. The small num-

ber of genes present in the viral genome, however, does not

C5b

C6

C7 C8

C5b678

C9C9

membraneattack

complex

C5aC3bBb

C6 C7 C8

solutes

C9 C9

C5b678

C3b C5b C3b C5b C3bC5b

etc.

membrane

MAC

etc.

1.2. 3. 4.

Figure 9.18 Assembly of the C5b-9 membrane attack complex (MAC). (1) Recruitment of a further C3b into the C3bBb enzymic complex generates a C5 convertase which cleaves C5a from C5 and leaves the remaining C5b attached to the membrane. (2) Once C5b is membrane bound, C6 and C7 attach themselves to form the stable complex C5b67, which interacts with C8 to yield C5b678. (3) This unit has some effect in disrupting the membrane, but primarily causes the polymerization of C9 to form tubules traversing the membrane. The resulting tubule is referred to as a MAC. (4) Disruption of the membrane by this structure permits the free exchange of solutes, which are primarily responsible for cell lysis.

Figure 9.19 Electron micrograph of the MAC. The funnel-shaped lesion ( arrowed ) is due to a human C5b–9 complex that has been reincorporated into lecithin liposomal membranes ( × 234 000). (Courtesy of J. Tranum-Jensen and S. Bhakdi.)

2 4 6 8 10 12 140

10

100

1

days

acute phaseprotein titre

(arbitrary units)

infection disease recovery

C-reactive protein (CRP)

complement

opsonization

Ca2+

Figure 9.20 Acute phase proteins, here exemplified by C-reactive protein (CRP), are serum proteins that increase rapidly in concentration (sometimes up to 100-fold) following infection (graph). They are important in innate immunity to infection. CRP recognizes and binds in a calcium (Ca 2 + )-dependent fashion to molecular groups found on a wide variety of bacteria and fungi. In particular, it uses its pattern recognition to bind the phosphocholine moiety of pneumococci. The CRP acts as an opsonin and activates complement with all the associated sequelae. Mannose binding protein reacts not only with mannose but several other sugars, enabling it to bind to a wide variety of Gram-negative and -positive bacteria, yeasts, viruses and parasites, subsequently activating the complement system and phagocytic cells. The structurally related ficolins typically recognize PAMPs containing N -acetylglucosamine and can also activate the lectin complement pathway.

79

SECTION TWO The adversaries – host defences

include those required for viral replication. Accordingly, it is

essential for viruses to penetrate the cells of an infected host

in order to subvert the cells' replicative machinery towards

viral replication. Clearly, it is in the interests of the host to try

to kill such infected cells before the virus has had a chance

to reproduce. Natural killer (NK) cells are cytotoxic cells that

appear to have evolved to carry out just such a task. These

are large granular lymphocytes (LGLs) ( Fig. 9.23 ) that rec-

ognize virus-infected or stressed cells and allow them to be

differentiated from normal cells; this clever discrimination

is mediated by activating receptors on the NK cells such as

NKG2D that recognize ligands on the infected cell that are

related to MHC Class I molecules, and inhibitory receptors

which bind to MHC Class I molecules on normal cells,

generating signals that counteract those from the activating

receptors. Activation of the NK cell results in the extracel-

lular release of its granule contents into the space between

the target and effector cells. These contents include perfo-

rin molecules, which resemble C9 in many respects, espe-

cially in their ability to insert into the membrane of the target

cell and polymerize to form annular transmembrane pores,

like the MAC. This permits the entry of another granule pro-

tein, granzyme B, which leads to death of the target cell by

apoptosis (programmed cell death), a process mediated by

a cascade of proteolytic enzymes termed caspases, which

terminates with the ultimate fragmentation of DNA by a

Ca-dependent endonuclease ( Fig. 9.24 ).

Subsidiary mechanisms that can activate the caspase path-

way include engagement of Fas on the target cell by the NK

Fas ligand, and binding of tumour necrosis factor (TNF)

released from the NK granules to surface receptors. TNF

was first recognized as a product of activated macrophages

known to be capable of killing certain other cells, particu-

larly some tumour cells.

Yet a further mode of cytotoxicity can be turned on by the

activated macrophage, involving the direct ‘burning’ of the

surface of another cell by means of a stream of reactive oxy-

gen intermediates, produced at the macrophage membrane

by the respiratory oxygen burst, as discussed previously

(see Box 9.2 ).

Eosinophils act against large parasites It takes little imagination to realize that professional

phagocytes are far too small to be capable of physically

engulfing large parasites such as helminths. An alterna-

tive strategy, such as killing by an extracellular broad-

side of the type discussed above would seem to be a more

appropriate form of defence. Eosinophils appear to have

evolved to fulfil this role. These polymorphonuclear rela-

tives of the neutrophil have distinctive cytoplasmic gran-

ules, which stain strongly with acidic dyes ( Fig. 9.25 ) and

have a characteristic ultrastructural appearance. A major

microbe

PRR

transducer

effectorfunction:

complementactivation

phagocytosis

PAMP

receptor on solublemolecule or phagocyte

Figure 9.21 A major defensive strategy in which soluble factors, such as CRP (C reactive protein) and mannose binding protein, and professional phagocytes use their pattern recognition receptors (PRR) to bind to the pathogen-associated molecular patterns (PAMPs) on the microbial surface and signal through their transducer structures to initiate appropriate effector functions.

virus DNA mRNA

mRNA

IFNreceptor

protein synthesis

'antiviral' state

DNA

IFNsynthesis

Increased NK activityand expression of

MHC products

Figure 9.22 The action of interferon (IFN). Virus infecting a cell induces the production of IFN α / β . This is released and binds to IFN receptors on other cells. The IFN induces the production of antiviral proteins, which are activated if virus enters the second cell, and increased synthesis of surface MHC molecules which enhance susceptibility to cytotoxic T cells (cf. Ch. 10). NK, natural killer; MHC, major histocompatibility complex.

TC

NK

Figure 9.23 Electron micrograph of an NK cell killing a tumour cell (TC). NK cells bind to and kill IgG antibody-coated (see Fig. 10.13 ), and non-coated, tumour cells. It is essential for the membranes of the two cells to be closely apposed in order for the NK cell to deliver the ‘kiss of death’ ( × 4500). (Courtesy of P. Lydyard.)

80

CHAPTERCHAPTER9 The innate defences of the body

basic protein (MBP) has been identified in the core of the

granule, while the matrix has been shown to contain an

eosinophilic cationic protein, a peroxidase and a perfo-

rin-like molecule. Eosinophils have surface receptors for

C3b and when activated generate copious amounts of

active oxygen metabolites.

Many helminths can activate the alternative complement

pathway but, although resistant to C9 attack, their coat-

ing with C3b allows adherence to the eosinophils through

their C3b surface receptors. Once activated, the eosinophil

launches its extracellular ammunition, which includes the

release of major basic proteins and the cationic protein to

damage the parasite membrane, with a possibility of a fur-

ther ‘chemical burn’ from the oxygen metabolites and ‘leaky

pore’ formation by the perforins.

G

B

A

Figure 9.25 The eosinophil granulocyte is capable of extracellular killing of parasites (e.g. worms) by releasing its granule contents. (A) Morphology of the eosinophil. This blood smear enriched for granulocytes shows an eosinophil with its multilobed nucleus and heavily stained cytoplasmic granules. Leishman's stain ( × 1800). (Courtesy of P. Lydyard.) (B) Electron micrograph showing the ultrastructure of a guinea pig eosinophil. The mature eosinophil contains granules (G) with central crystalloids ( × 8000). (Courtesy of D. McLaren.)

NK cell

target cellnucleus

nucleus

granules

pore

virally induced structures

fluid phase perforin

NK cell receptor

membrane-bound perforin

granzyme B

TNFα

FasL

Fas

death signal

caspase cascade

Figure 9.24 Schematic model of lysis of virally infected target cell by a natural killer (NK) cell. As the NK cell receptors bind to the surface of the virally infected cell, and if signals from activation receptors exceed those from the inhibitory receptors that recognize normal MHC Class I molecules, there is exocytosis of granules and release of cytolytic mediators into the intercellular cleft. A calcium (Ca 2 + )-dependent conformational change in the perforin enables it to insert and polymerize within the membrane of the target cell to form a transmembrane pore, which allows entry of granzyme B into the target cell, where it causes programmed cell death (apoptosis). A back-up cytolytic system using engagement of the Fas receptor with its ligand (FasL), can also trigger apoptosis as can binding of granule-derived tumour necrosis factor alpha (TNF α ) to its receptor. Unlike the PRR-mediated activation of phagocytes by intracellular components – so-called danger-associated molecular patterns (DAMPs) – released on necrotic cell-death typically caused by tissue trauma, burns and other non-physiological stimuli, cells undergoing apoptotic death do not activate the immune system because they express surface molecules such as phosphatidyl serine which mark them out for phagocytic removal before they release their intracellular DAMPs.

81

SECTION TWO The adversaries – host defences

Mø

mast cell

acute phaseproteins

phagocytosis

acute inflammatory response

microbe mediators

PMN

margination

soluble factors

lysozymecomplement

interferon

transudation

mobilization

separation

endothelium

Figure 9.26 Mobilization of defensive components of innate immunity. Microbes, either through complement activation or through direct effects on macrophages, release mediators which increase capillary permeability to allow transudation of plasma bactericidal molecules, and chemotactically attract plasma polymorphs from the bloodstream to the infection site. PMN, polymorphonuclear neutrophil.

The innate system of immune defence consists of a formidable barrier to entry and second-line defence by phagocytes and circulating soluble factors. Colonization of the body by normally non-pathogenic (‘opportunistic’) microorganisms occurs whenever there is a hereditary or acquired deficiency in any of these functions.

The main phagocytic cells are polymorphonuclear neutrophils and macrophages. They adhere to the surface of the microbe by receptors which recognize pathogen-associated molecular patterns (PAMPs). This activates the engulfment process so that the organisms are taken inside the cell in a phagocytic vacuole which fuses with cytoplasmic granules. A formidable array of oxygen-dependent and oxygen-independent microbicidal mechanisms then comes into play.

The complement system, a multicomponent triggered enzyme cascade, is used to attract phagocytic cells to the microbes and engulf them.

The most abundant complement component, C3, is split by a convertase enzyme formed from its own cleavage product C3b and factor B and stabilized against breakdown caused by factors H and I through association with the microbial surface. As it is formed, C3b becomes covalently linked to the microorganism.

The next most abundant component, C5, is activated to yield a small peptide, C5a, while residual C5b binds to the surface of the microorganism and assembles the terminal components C6–9 into a membrane attack complex (MAC), which is freely permeable to solutes and can lead to osmotic lysis. In addition, C5a is a potent chemotactic agentfor polymorphs and greatly increases capillary permeability.

C3a and C5a act on mast cells, causing the release of further mediators such as histamine, LTB 4 and TNF α , with effects on capillary permeability and adhesiveness and

neutrophil chemotaxis. They also activate neutrophils, which bind to the C3b-coated microbes by their surface C3b receptors and then ingest them.

The influx of polymorphs and increase in vascular permeability constitute the potent antimicrobial acute inflammatory response.

Inflammation can also be initiated by tissue macrophages, which subserve a similar role to that of the mast cell since signalling by bacterial toxins C5a or by C3b-coated bacteria adhering to surface complement receptors on tissue macrophages causes the release of TNF α , LTB 4 , PGE 2 , the neutrophil chemotactic factor, IL-8, and a neutrophil-activating peptide.

Other humoral defences include the acute phase proteins such as CRP, and the IFNs, which can block viral replication.

Virally infected cells can be killed by NK cells, following increased recognition by activation receptors that overcomes inhibitory signals from normal MHC Class I recognition.

Extracellular killing can also be effected by C3b-bound eosinophils, which may be responsible for the failure of many large parasites to establish a foothold in potential hosts.

It is probably true to say that engulfment and killing by phagocytic cells is the mechanism used to dispose of the majority of microbes, and the mobilization and activation of these cells by orchestrated responses such as the acute inflammatory response ( Fig. 9.26 ) is a key feature of innate immunity. However, not every organism is readily susceptible to phagocytosis or even to killing by complement or lysozyme, and this brings us to the role of the adaptive immune response, which is explored in Chapter 10.

KEY FACTS