inflam1.pdf

Chapter 9

Inflammation and fever

9.1 Inflammation

9.1.1 Principles of inflammation

A human or animal must defend itself against mul-titude of different pathogens including viruses, bac-teria, fungi, and protozoan and metazoan parasitesas well as tumours and a number of various harmfulagents which are capable to derange its homeostasis.For this, a plenty of effector mechanisms capable ofdefending the body against such antigens and agentshave developed and these can be mediated by sol-uble molecules or by cells. If infection occur as aconsequence of the tissue damage, the innate and,later, the adaptive immune systems are triggered todestroy the infectious agent.Inflammation is a complex stereotypical reaction

of the body expressing the response to damage of itscells and vascularized tissues. In avascular tissues,e.g. in normal cornea, the true inflammation doesnot occure.The discovery of the detailed processes of inflam-

mation has revealed a close relationship between in-flammation and the immune response.The five basic symptoms of inflammation - red-

ness (rubor), swelling (tumour), heat (calor), pain(dolor) and deranged function (functio laesa) havebeen known since the ancient Greek and Roman era.These signs are due to extravasation of plasma andinfiltration of leukocytes into the site of inflamma-tion. Early investigators considered inflammationa primary host defence system. From this point of

view inflammation is the key reaction of the innateimmune response but in fact, inflammation is morethan this, since it can lead to death, as in anaphy-lactic shock, or debilitating diseases, as in arthritisand gout.

According to different criteria, inflammatory re-sponses can be divided into several categories. Thecriteria include:

1. time hyperacute (peracute), acute, subacute,and chronic inflammation;

2. the main inflammatory manifestation - alter-ation, exudation, proliferation;

3. the degree of tissue damage - superficial, pro-found (bordered, not bordered);

4. characteristic picture - nonspecific, specific;

5. immunopathological mechanisms

allergic (reaginic) inflammation, inflammation mediated by cytotoxic anti-

bodies,

inflammation mediaded by immune com-plexes,

delayed-type hypersensitivity reactions.

9.1.1.1 The response to injury and infection

Inflammation is the bodys reaction to invasion byan infectious agent, antigen challenge or even justphysical, chemical or traumatic damage.

The mechanism for triggering the response thebody to injury is extremely sensitive. Responses areto tissue damage that might not normally be thoughtof as injury, for example when the skin is strokedquite firmly or if some pressure is applied to a tissue.

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578 Chapter 9. Inflammation and fever ( I.Huln, M. Ferenck, V. Stvrtinova, J. Jakubovsky)

In addition, the body has the capacity to respondto both minor injuries such as bruising, scratching,cuts, and abrasions, as well as to major injuries suchas severe burns and amputation of limbs.Depending on the severity of the tissue damage

resulting from an injury, the integrity of the skin orinternal surfaces may be breached and damage to theunderlying connective tissue and muscle, as well asblood vessels can occur. In this situation infectioncan, and frequently does result because the normalbarrier to the entry of harmful organisms has beenbroken. It is obviously most important that the bodycan respond to injury by healing and repairing thedamaged tissue, as well as by eliminating the infec-tious agents that may have entered the wound andtheir toxins. It is also important that the appropri-ate response to the tissue damage and infection canbe made: it is no use bringing all of the bodys de-fences into action to repair a minor scratch, just asone would not expect a single mechanism to be ableto deal with the sudden loss of a limb or a majorinfection.The inflammatory reaction is phylogenetically and

ontogeneticaly the oldest defence mechanism. Thecells of the immune system are widely distributedthroughout the body, but if an infection or tissuedamage occurs it is necessary to concentrate themand their products at the site of damage. Three ma-jor events occur during this response :

1. An increased blood supply to the tissue in dan-ger. It is performed by vasodilation. The in-flamed tissue looks like containing greater num-ber of vessels.

2. Increased capillary permeability caused by re-traction of the endothelial cells. This permitlarger molecules than usual to escape from thecapillaries, and thus allows the soluble media-tors of immunity to reach the site of inflamma-tion.

3. Leukocytes migrate out of the capillaries intothe surrounding tissues. In the earliest stagesof inflammation, neutrophils are particularlyprevalent, but later monocytes and lymphocytesalso migrate towards the site of infection.

For the possibility of surrounding tissue damage,inflammatory responses must be well ordered andcontrolled. The body must be able to act quickly

in some situations, for example to reduce or stopthe lost of blood, whereas tissue repair and recon-struction can begin a little later. Therefore, a widevariety of interconnected cellular and humoral (sol-uble) mechanisms are activated when tissue damageand infection occur. On the other hand if the injuryis negligible, the body must have mechanisms whichare able to stop the tissue damage when the injuryagent was removed.

The development of inflammatory reactions is con-trolled by cytokines, by products of the plasma en-zyme systems (complement, the coagulation cloth-ing, kinin and fibrinolytic pathways), by lipid medi-ators (prostaglandins and leukotrienes) released fromdifferent cells, and by vasoactive mediators releasedfrom mast cells, basophils and platelets. These in-flammatory mediators controlling different types ofinflammatory reaction differ. Fast-acting media-tors, such as vasoactive amines and the productsof the kinin system, modulate the immediate re-sponse. Later, newly synthesized mediators such asleukotrienes are involved in the accumulation andactivation of other cells. Once leukocytes have ar-rived at a site of inflammation, they release media-tors which control the later accumulation and acti-vation of other cells.

However, in inflammatory reactions initiated bythe immune system, the ultimate control is exertedby the antigen itself, in the same way as it controlsthe immune response itself. For this reason, the cel-lular accumulation at the site of chronic infection,or in autoimmune reactions (where the antigen can-not ultimately be eradicated), is quite different fromthat at sites where the antigenic stimulus is rapidlycleared.

The nervous system can also participate in thecontrol of inflammation, especially axon reflexes, butinflammation may be realized in denervated tissuesas well.

Inflammation can become chronic. In certain set-tings the acute process, characterized by neutrophilinfiltration and edema, gives way to a predomi-nance of mononuclear phagocytes and lymphocytes.This probably occurs to some degree with the nor-mal healing process but becomes exaggerated andchronic when there is ineffective elimination of for-eign materials as in certain infections (e.g. tuber-culosis) or following introduction of foreign bodies(e.g. asbestos) or deposition of crystals (e.g. urate

9.1. Inflammation 579

crystals). Chronic inflammation is often associatedwith fusion of mononuclear cells to form multinu-cleated gigant cells, which eventually become granu-loma. Chronic inflammation is seen under conditionsof delayed hypersensitivity.Main humoral and cellular components involved in

the amplification and propagation of both acute andchronic inflammation are showed in Table 9.1.

9.1.1.2 Factors involved in cell damage

There are two categories of factors capable to inducethe damage of cells and tissues - endogenous and ex-ogenous. Endogenous damaging factors include im-munopathological reactions, and some neurologicaland genetical disorders. Exogenous factors can bedivided into:

mechanical (traumatic injury),

physical (extremely low or high temperature,ionising irradiation, microwaves),

chemical (caustic agents, poisons, venoms, geno-toxic and proteotoxic compounds),

nutritive (deficiency of oxygen, vitamins and ba-sic nutrients),

biological (viruses, microorganisms, protozoanand metazoan parasites).

Immunopathological reactions may be also trig-gered by exogenous antigens. Genetically caused al-terations leading to inflammation are manifested bydestruction of membrane structures, by derangementof transport mechanisms, or by defective activity ofsome enzymes and mediators. Cell damage also oc-curs during ageing. It is very complicated processin which genetic, metabolic, immunologic, neurolog-ical and other factors are involed. In ageing cells,probably metabolic intermediates such as differentfree radicals, aldehydes, ketones, and their reactionproducts, or on the contrary non-degradable com-pounds are accumulated. This results in a seriousdefect in the integrity and physiological homeostasisof cells and tissue.It seems that aging cells are losing their multi-

plication capacity at a particular generation. For in-stance, cultivated fibroblasts lose their ability to mul-tiply between 40 and 60 generations. The cell aging

may result as a tissue atrophy. In other cases, hy-pertrophy or hypoplasia is the compensatory mecha-nism for this situation. The altered cellular activitiesmay lead to metaplasia, dysplasia, or neoplasia be-cause aging cells are more susceptible to destructionof their DNA, RNA and vital proteins.

Extremly low temperature is able to form crystalsinside the cell. Mild decrease in temperature causesparalysis of vasomotors and an increase in perme-ability of vessels. Blood viscosity rises proportion-ally with the lowering temperature and cells are de-stroyed by hypoxia. Low temperature acting for alonger time provokes the destruction of myelin in ex-posed area. Microthrombi are produced in vesselsand they are the cause of gangrene.

High temperature increases the permeability ofcell membranes. Very high temperature is respon-sible for the coagulation of vessels and denaturationof vital biopolymers, especially proteins.

According to the dose and the way of expo-sition ionizing irradiation may primarily damagehaematopoietic, gastrointestinal or neural tissues.Whole-body irradiation produces nonspecific im-munosupression which is the cause of increased sensi-tivity to infection. The infection is developed mainlydue to leukopenia and the loss of physical integrityof mucosal membranes especially in the gastroin-testinal tract. Whole-body irradiation eliminatesmost of the mature lymphocytes of the immune sys-tem while preserving the more radiation-resistant el-ements such as the thymic epithelium. Ionizing ra-diation is also used for the treatment of patient withcancer and sometimes in the form of local graft irra-diation. An alternative form of radiation therapy istotal lymphoid irradiation e.g. for the treatment ofHodgkins disease. Lethally irradiated persons canbe given immature bone marrow cells to reconstitutethe immune systems.

On the cell level, irradiation destroy importantbiopolymers (DNA, proteins) and biological mem-branes. At first, the degenerative changes of nu-cleus and chromosomal aberrations can be seen. Theincreased membrane permeability and activation ofhydrolytic lysosomal enzymes disrupt cell structuresand compartments. Irreversible damage of irradiatedcells causes their complete destruction, necrosis.

Some chemicals, namely caustic agents and min-eral acids are able to damage tissues directly, othersuch as heavy metals, poisons and venoms mainly


Process Effector cells and molecules

Antigen recognition:

Specific T lymphocytes, antibodies (immunoglobulins)

Nonspecific Professional phagocytes (neutrophils, eosinophils, monocytes and tissuemacrophages), alternative complement pathway, Hageman factor (coagulationcascade)

Amplification Complement system, arachidonate products, mast cell products, platelet-activating factor (PAF), bradykinin, serotonin, coagulation cascade, cytokines(IFN-, TNF-, IL-1, IL-6, IL-8, IL-11, chemokines, growth factors), lysosomalcontents of neutrophils

Antigen destruction Neutrophils, eosinophils, macrophages, cytotoxic lymphocytes, terminal com-plement components and other perforins, reactive oxygen and nitrogen inter-mediates.

Table 9.1: Components of inflammation

derange important enzymatic reactions. Metabolichomeostasis of cells and tissues is also disturbed bythe action of genotoxic and proteotoxic agents. Tothe often observed defects belong: destruction of cellmembranes, decrease of intracellular pH, release oflysosomal enzymes and changes similar as in hypoxia(decrease of oxidative phosphorylation). Lysosomalenzymes and free radicals derived from oxygen (re-active oxygen intermediates - ROI) or from nitrogen(reactive nitrogen intermediates - RNI) have an es-sential role in the damage of cell structures especiallyduring the injuring inflammation. These substancesmay be also activated by the action of many am-phiphilic detergents that are components of differentcleaning and washing preparations and tooth pastes.They are dangerous if they reach inside the bodyin the inappropriate amount or in the inappropriateway.The oxygen deficiency is manifested in 35 min-

utes. In mitochondria, oxidative phosphorylation isvery quickly impaired and insufficient production ofATP appears. Deficiency of ATP activates anaerobicmetabolism in which ATP is formed from glycogen.But the reserves of glycogen are again quickly de-pleted. Because of persistent ATP insufficiency the

sodium-potassium pump loses its operating capac-ity. This leads to the intracellular accumulation ofsodium and the leakage of potassium from cells. Ac-cumulation of sodium induces the transfer of ions andwater into cell. It is the reason of endoplasmic retic-ulum dilatation. The dilatation provides completedamage to ribosomes and blocks proteosynthesis.

If the hypoxia continues, the whole cell is overfilledwith water, sodium, and chlorides. This state is stillreversible, after the renewing of oxygen transport,the cell should recover. In the others cases, vacuolesin the cytoplasm and the damage of mitochondrialmembrane appear. Now, it is the irreversible pro-cess. Because of the membrane damage, the extra-cellular calcium may enter the cell and accumulate inmitochondria. The production of ATP is completelyterminated that is thought to be the real death ofcell. The cell or tissue death is performed as necro-sis.

Cell damage may be also caused by differentgasses, especially by nitrogen oxides, sulphur dioxide,carbon monoxide, formaldehyde, chlorine, etc. Car-bon monoxide is bound by hemoglobin with 300timeshigher affinity than oxygen. Therefore the exposure


to CO develops the secondary oxygen deficiency dueto the termination of oxygen transport to cells.Infections are often involved in cell damage. Viru-

lence of microorganisms and the induction of inflam-mation depend on their ability to replicate in humanor animal body and to destroy cellular structures.During growth and multiplication, microorganismscan produce and release different exotoxins whichare potent injuring agents. Other microorganisms,after destruction or lysis, release from phospholipidand lipopolysaccharide envelops toxins known as en-dotoxins. The term endotoxin is generally usedto refer to the thermostable polysaccharide toxin,firmly bound to the bacterial cell, in contrast tothe thermolabile protein exotoxin, secreted intothe external environment. Endotoxin (lipolysaccha-ride, LPS) is responsible for many pathophysiologicalsymptoms observed during gram-negative bacterialinfections. They include pyrogenicity (the ability tocause an increase in body temperature), changes inthe number of circulating leukocytes (leukocytope-nia, leukocytosis), complement activation, activationof macrophages, aggregation of platelets, increaseof capillary permeability and others. In addition,LPS induces an immune response. Administrationor release of a higher dose of endotoxin may pro-duce lethal shock. All these biological activities aremediated through the endogenous mediator tumornecrosis factor- (TNF-).Viruses do not produce exotoxins or endotoxins.

They are typical intracellular parasites and use cellsfor their own replication. During this, damage of cellstructures leading to the death of cell is observed. Inaddition viruses may be responsible for the tumoroustransformation of cells.During the immune responses, the cells may be

damaged by effector cells and molecules participatingin immune mechanisms. From this point of view theyare thought to be the immunopathological responses.They include:

1. Immediated allergic anaphylactic reactions me-diated by IgE antibodies (reagines).

2. Cytotoxic reactions during which complementis activated by IgG or IgM antibodies reactingwith antigens of self cells and structures (au-toantigens) which immediately damage the tar-get cells and surrounding tissues.

3. Reactions of the immune complex type. Thecomplement system is also activated by the im-mune complexes. During the activation, chemo-tactic factors are formed which attract granulo-cytes to the inflammation area. Neutrophils de-stroy target cells by released lysosomal enzymes,especially by proteinases, and free radicals ofoxygen.

4. Reactions of delayed or cell-mediated hypersen-sitivity. Specific subpopulation of T lympho-cytes and several cytokines are involed in theseprocesses.

5. Cytotoxic reactions influencing the function ofcell receptors. They are also mediated by au-toantibodies that may have a function of ago-nists or antagonists. Hence, the autoantibodiescan pathologically stimulate and/or block thetransfer of specific signal through the receptor.

It follows that cell damage following the inflamma-tory reaction may be useful or harmful. The usefulactivities include:

1. destruction of injuring and infectious agents andtheir elimination from the inflammatory site;

2. limitation of spreading of injuring factors;

3. stimulation of the specific immune response;

4. help in the healing process.

To the harmful inflammatory reactions belong au-toimmune and other immunopathological processes.

9.1.1.3 The phases of inflammation

The main purpose of inflammation, this immenselycomplex response seems to be to bring fluid, proteins,and cells from the blood into the damaged tissues. Itshould be remembered that the tissues are normallybathed in a watery fluid (extracellular lymph) thatlacks most of the proteins and cells that are presentin blood, since the majority of proteins are too largeto cross the blood vessel endothelium. Thus therehave to be mechanisms that allow cells and proteinsto gain access to extravascular sites where and whenthey are needed if damage and infection has occured.

The main features of the inflammatory responseare, therefore: vasodilation, i.e. widening of theblood vessels to increase the blood flow to the in-fected area; increased vascular permeability, which


allows diffusible components to enter the site; cellu-lar infiltration by chemotaxis, or the directed move-ment of inflammatory cells through the walls of bloodvessels into the site of injury; changes in biosynthetic,metabolic, and catabolic profiles of many organs;and activation of cells of the immune system as wellas of complex enzymatic systems of blood plasma.Of course, the degree to which these occur is nor-mally proportional to the severity of the injury andthe extent of infection.Inflammation can be divided into several phases.

The earliest, gross event of an inflammatory re-sponse is temporary vasoconstriction, i.e. narrowingof blood vessels caused by contraction of smoothmuscle in the vessel walls, which can be seen asblanching (whitening) of the skin. This is followedby several phases that occur over minutes, hours anddays later, outlined below.

1. The acute vascular response follows within sec-onds of the tissue injury and last for some min-utes. This results from vasodilation and in-creased capillary permeability due to alterationsin the vascular endothelium, which leads to in-creased blood flow (hyperaemia) that causesredness (erythema) and the entry of fluid intothe tissues (oedema). This phase of the in-flammatory response can be demonstrated byscratching the skin with a finger-nail. Thewheal and flare reaction that occurs is com-posed of (a) initial blanching of the skin due tovasoconstriction, (b) the subsequent rapid ap-pearance of a thin red line when the capillariesdilate; (c) a flush in the immediate area, gener-ally within a minute, as the arterioles dilate; and(d) a wheal, or swollen area that appears withina few minutes as fluid leaks from the capillar-ies. It is usually terminates after several tensminutes.

2. If there has been sufficient damage to the tis-sues, or if infection has occured, the acute cellu-lar response takes place over the next few hours.The hallmark of this phase is the appearanceof granulocytes, particularly neutrophils, in thetissues. These cells first attach themselves tothe endothelial cells within the blood vessels(margination) and then cross into the surround-ing tissue (diapedesis). During this phase ery-throcytes may also leak into the tissues and ahaemorrhage can occur (e.g. a blood blister). If

the vessel is damage, fibrinogen and fibronectinare deposited at the site of injury, platelets ag-gregate and become activated, and the red cellsstack together in what are called rouleau tohelp stop bleeding and aid clot formation. Thedead and dying cells contribute to pus forma-tion.

3. If the damage is sufficiently severe, a chroniccellular response may follow over the next fewdays. A characteristic of this phase of inflamma-tion is the appearance of a mononuclear cell in-filtrate composed of macrophages and lympho-cytes. The macrophages are involved in micro-bial killing, in clearing up cellular and tissue de-bris, and they also seem to be very important inremodelling the tissues.

4. Over the next few weeks, resolution may occur,meaning that the normal tissue architecture isrestored. Blood clots are removed by fibrinoly-sis, and if it is not possible to return the tissue toits original form, scarring results from in-fillingwith fibroblasts, collagen, and new endothelialcells. Generally, by this time, any infection willhave been overcome. However, if it has not beenpossible to destroy the infectious agents or to re-move all of the products that have accumulatedat the site completely, they are walled off fromthe surrounding tissue in granulomatous tissue.A granuloma is formed when macrophages andlymphocytes accumulate around material thathas not been eliminated, together with epith-eloid cells and gigant cells (perhaps derived frommacrophages) that appear later, to form a ballof cell.

Inflammation is often considered in terms of acuteinflammation that includes all the events of the acutevascular and acute cellular response (1 and 2 above),and chronic inflammation that includes the eventsduring the chronic cellular response and resolutionor scarring (3 and 4).

In addition, a large number of more distant ef-fects occur during inflammation. These include: theproduction of acute phase proteins, including com-plement components, by the liver; fever, caused bypyrogens acting on the hypotalamus in the brain;and systemic immunity, resulting in part from lym-phocyte activation in peripheral lymphoid tissues.


9.1.2 Exudation and swelling

9.1.2.1 Fluid exudate

In acute inflammation, the pressure in postcapil-lary venules may overcome the osmotic pressure ofplasma proteins. Therefore fluid and low molecularsubstances have the tendency to penetrate into thesurrounding area. The vascular permeability for pro-teins and some smaller molecules differs from tissueto tissue. For example, the brain and thymus vesselsare less permeable. The sinusoids in liver and sinusesin spleen are highly open vessels even at normal con-ditions.The increased capillary permeability for plasma

proteins is the key factor for the production of in-flammatory exudate. In the interstitial area, high-molecular proteins may be split into smaller frag-ments that participate in the raising of osmotic pres-sure of interstitial fluid. In addition, the alterationof general matrix is observed. It becomes more fluidwhich helps to make easier the diffusion of exudate.On the other hand, a sudden increase of pressure intissue is thus prevented.There are two phases of inflammatory infiltration.

The immediate temporary phase with a peak be-tween 8 and 10 min and duration about 30 min. Itis developed by the release of fluid from venules me-diated by histamine. This is followed by immediateprolonged phase which is similar, only the time ofduration is greater a few days. The second delayedphase needs a few hours for its development. Thedamage to capillaries and venules is observed.In the fluid exudate, all components of plasma,

including fibrinogen, kinins, complement, im-munoglobulins etc., are present. Fibrinogen is impor-tant for clot formation and the prevention of furtherloss of blood. Fibrin, which is originated from fib-rinogen, acts as the beginning of a scaffold on whichtissues may subsequently be repaired and on whichnew capillaries can be constructed, a process knownas angiogenesis. Although the rapid response of thecoagulation pathway is essential, the extent of bloodclothing must be limited so that it does not progressto undamaged vessels. In addition, the clots must ul-timately be removed from the area of damage. Thisis controlled by fibrinolysis (fibrin breakdown) dueto the enzyme plasmin.The kinins are important mediators of inflam-

matory responses. For kinin generation to pro-

ceed efficiently, activated Hageman factor activatesprekallikrein via a series of prekallikrein activators,resulting in the production of kallikrein. The gener-ation of kallikrein triggers kinin production, includ-ing the formation of bradykinin, which is responsiblefor induction pain, increasing vascular permeability,and causing vasodilation. Kallikrein also activatesthe fibrinolytic pathway, leading to the removal ofblood clots.

The complement cascade, as a part of the innateimmune response, may be activated via the alter-native and/or collectin (lectin) pathway to destroysome invading microorganisms. In addition, duringactivation of complement, important opsonins (C3b),chemotactic factors for neutrophils and mononuclearphagocytes (C5a), and anaphylatoxins (C5a, C3a)are formed. They all participate in inflammationduring phagocytosis or immediate allergic reactions.

Immunoglobulins may act as specific or nonspe-cific opsonins facilitating thus the process of phago-cytosis, or may participate in antibody-dependentcell-mediated cytotoxicity (ADCC) by which targetcells are destroyed by killer cells.

In the fluid infiltrate, all components of plasma,including administered drugs, are present. There-fore it is important to administer effective antibioticor other chemotherapy as soon as possible in orderto reach the inflammatory area in the concentrationsimilar to that in plasma.

Exudative infiltrate contributes to the generalsigns of inflammation. It is responsible for edema(swelling, tumour). The increased pressure in tissuemay participate in the production of pain (dolor).Actually, the pain is observed before the occurrenceof greater edema, since also other factors such asthe acidic pH of exudate, the accumulation of potas-sium ions and the presence of bradykinin, serotoninor other mediators take part in this process.

9.1.2.2 Cellular exudate

Cellular exudate is formed during the second and thethird phase of inflammation acute and chronic cel-lular response. During the former, neutrophils areprevalent, whereas mononuclear cells (macrophagesand lymphocytes) overcome later. Cell compositionof exudate differ not only depending on the phaseof inflammation but also on the type of inflamedtissue and factors triggering inflammatory process.Central effector and regulatory functions in acute in-


flammation posses neutrophils. They are also dom-inant when a pyogenic bacterial infection or localdeposition of immune complexes containing IgG arethe cause of inflammation. Mononuclear phagocytesrepresent the main infiltrating cells in subacute andchronic phase of the majority of inflammatory reac-tions, and in the case of infection with intracellularlyparasitizing microorganisms as well. Eosinophils andbasophils are predominant when inflammation hasbeen initiated by immediate alergic reactions or byparasites.

So, a number of different cell types are recruitedinto the area where damage has occured, and theseare responsible for inactivation and removing of theinvading infectious agents, for removing the damagedtissues, for inducing the formation of new tissue, andreconstructing the damaged cell matrix, includingbasement membranes and connective tissue. A newblood supply to the area is also established duringthe repair process.

Professional phagocytes (neutrophils, eosinophils,monocytes and tissue macrophages) are essentialperforming phagocytosis, lymphocytes are involvedin the specific immune responses, endothelial cellin the regulation of leukocyte emigration from theblood into inflamed tissue and platelets with mastcells in the production od early phase mediators.

The accumulation of leukocytes in inflamed tis-sue results from adhesive interactions between leuko-cytes and endothelial cells within the microcircu-lation. These adhesive interactions and the exces-sive filtration of fluid and protein that accompa-nies an inflammatory response are largely confinedto one region of the microvasculature postcap-illary venules. The nature and magnitude of theleukocyte-endothelial cell adhesive interactions thattake place within postcapillary venules are deter-mined by a variety of factors, including expression ofadhesion molecules on leukocytes and/or endothelialcells, products of leukocyte (superoxide and otherROI) and endothelial cell (nitric oxide) activation,and the physical forces generated by the movementof blood along the vessel wall. The contribution ofdifferent adhesion malecules to leukocyte rolling, ad-herence, and emigration in venules will be discussedlater.

This process is similar for granulocytes, mono-cytes, and lyphocytes only different chemotactic fac-tors and cytokines may be involved in its initiation

and control. The white blood cells leave the postcap-illarly venule by extending pseudopodia between ap-posing endothelial cells and pulling themselves intothe subendothelial space and the adjacent interstitialcompartment. This complex event, which is oftentermed leukocyte extravasation, emigration, or dia-pedesis, is dependent not only on an array of cellu-lar processes including adhesion molecule expressionand activation, but also on cytoskeletal reorganiza-tion, and alteration in membrane fluidity.

9.1.3 Cells participating in inflamma-tion

9.1.3.1 Mast cells and basophils

Mast cells and basophils play a central role in in-flammatory and immediate allergic reactions. Theyare able to release potent inflammatory mediators,such as histamine, proteases, chemotactic factors, cy-tokines and metabolites of arachidonic acid that acton the vasculature, smooth muscle, connective tis-sue, mucous glands and inflammatory cells.

Mast cells settle in connective tissues and usuallydo not circulate in the blood stream.

Basophils are the smallest circulating granulocyteswith relatively the least known function. They arisein the bone marrow, and following maturation anddifferentiation, are released into the blood circula-tion. If they are adequately stimulated they maysettle in the tissues.

Both mast cells and basophils contain special cy-toplasmic granules which store mediators of inflam-mation. The extracellular release of the mediators isknown as degranulation and may be induced by:

(a) physical destruction, such as high temperature,mechanical trauma, ionising irradiation, etc.;

(b) chemical substances, such as toxins, venoms,proteases;

(c) endogenous mediators, including tissue prote-ases, cationic proteins derived from eosinophilsand neutrophils;

(d) immune mechanisms which may be IgE-dependent or IgE-independent. The former iselicited by aggregation of IgE bound to high-afinity receptors (FcRI) on the surface of thesecells. Specific antigen (allergen) is responsiblefor the IgE aggregation. In the IgE-independent


way, the anafylatoxins C5a, C3a and C4a areformed during activation of complement. Then,the degranulation is triggered through C5a-receptors on the surface of mast cells and ba-sophils.

There are two categories of inflammatory (ana-phylactic) mediators in mast cells and basophils.Preformed mediators, stored in secretory granulesand secreted upon cell activation, include a bio-genic amine, typically histamine, proteoglycans, ei-ther heparin, over-sulphate chondroitin sulphates orboth, and a spectrum of neutral proteases. Re-leased histamine acts at H1, H2 and H3 receptorson cells and tissues, and is rapidly metabolized ex-tracellularly. The proteoglycan, which imparts themetachromatic staining characteristic of mast cellswhen exposed to certain basic dyes such as toluidineblue, has two functions: it may package histamineand basic proteins into secretory granules, and inhuman mast cells it appears to regulate the stabilityof the protease called tryptase. Neutral proteases,which account for the vast majority of the granuleprotein, serve as markers of mast cells and of differ-ent types of mast cells.Newly generated mediators, often absent in the

resting mast cells, are typically produced during IgE-mediated activation, and consist of arachidonic acidmetabolites, principally leukotriene C4 (LTC4) andprostaglandin D2 (PGD2) and cytokines. Of partic-ular interest in humans is the production of tumournecrosis factor (TNF-), IL-4, IL-5 and IL-6. In thecytoplasma of both mastocytes and macrophages arespecial organelles lipid bodies where metabolismof arachidonic acid occur and where their products,including leukotrienes, may be stored.Mast cells are heterogeneous two types of them,

mucosal and connective tissue, were reported in ro-dent tissue back in the 1960s on the basis of his-tochemical and fixation characteristics that reflect,in part, whether heparin proteoglycan was presentin secretory granules. Neutral proteases better re-flect the heterogeneity or plasticity of mast cells invivo and in vitro, particularly in humans where his-tochemical heterogeneity is less apparent (Table 9.2).In murine mast cells, five chymases ( mouse mast

cell protease MMCP-1, -2, -3, -4 and -5), one mastcell carboxypeptidase and two tryptases (MMCP-6and -7) have been reported.In human mast cells, genes encoding two chy-

motryptic enzymes (chymase and cathepsin G-likeprotease) and one mast cell carboxypeptidase en-zyme, and at least two genes encoding tryptase pep-tides have been detected. The gene encoding chy-mase resides on chromosome 14, closely linked tothe gene encoding cathepsin G, an enzyme appar-ently expressed in mast cells and various myelomono-cytic cells, and to the genes encoding granzymes,which are expressed in cytotoxic T lymphocytes andnatural killer cells. Two types of mast cells havebeen found by immunohistochemical analyses. TheMCTCtype contains tryptase, chymase, cathepsin Glike protease and mast-cell carboxypeptidase, andpredominates in normal skin and intestinal submu-cosa, whereas the MCTtype contain only tryptase,and predominates in normal intestinal mucosa andlung alveolar wall. Nearly equivalent concentrationsof each type are found in nasal mucosa. In MCTCcells, tryptase, chymase and mast-cell carboxypepti-dase reside in macromolecular complexes with pro-teoglycan, but interestingly, tryptase reside in a sep-arate complex from that in which chymase and mast-cell carboxypeptidase are found.

The biological function of mast cell neutral pro-teases, like mast cells themselves, remain to be fullyclarified. In serum, elevated levels of tryptase are de-tected in systemic mast-cell disorders, such as ana-phylaxis and mastocytosis. Ongoing mast-cell ac-tivation in asthma appear to be a charakteristic ofthis chronic inflammatory disease. It is detected byelevated levels of tryptase and PGD2 in bronchoalve-olar lavage fluid, higher spontaneous release of his-tamine by mast cell obtained from the bronchoalve-olar lavage fluid of asthmatics than non asthmat-ics, and ultrastructural analysis of mast cell in pul-monary tissue.

The number of basophils and mast cells increaseat sites of inflammation. To reach these areas, ba-sophils must migrate from the blood into tissue sites.A crucial step in this process is the adherence ofcells to the endothelium. Cell adherence is medi-ated by several families of adhesion molecules andadhesion receptors in the surface of basophils andmast cells that can mediate binding to other celland to the extracellular matrix (ECM) glycoproteins.Upon stimulation, basophils and mast cells releasecytokines, including TNF- and IL-4, that can mod-ulate adhesion molecules on endothelial cells. Acti-vated endothelial cells express the intercellular adhe-


Mast cell type Biogenic amine Neutral protease Proteoglycan

Mouse:

Mucosal H MMCP-1,-2 Chondroitin sulphate E

Connective tissue H+5-HT MMCP-3,-4,-5,-6, HeparinCarboxypeptidase

Human:

MCTcells H Tryptase Heparin, chondroitin sulphate

MCTCcells H Tryptase, chymase, Heparin, chondroitin sulphatecathepsin G-like protease,carboxypeptidase

H: histamine; 5-HT: serotonin; MMCP: mouse mast cell protease

Table 9.2: Predominant granule mediators of mast cells

sion molecule (ICAM-1), endothelial-leukocyte ad-hesion molecule (ELAM-1) and vascular cell adhe-sion molecule (VCAM-1) on their cell surface. Hu-man basophils express integrins as receptors for thesemolecules.

Until recently, the effects of adherence on cell func-tion were believed to result only from changes in cellshape and cytoskeletal organization. However, in ad-dition to cell spreading, aggregated adhesion recep-tors transduce a variety of intracellular signals thatregulate cell function. These signals include proteintyrosine phosphorylation, phosphoinositide hydroly-sis, changes in intracellular pH or calcium concentra-tion and the expression of several genes. The adhe-sion properties of basophils and mast cells regulatetheir migration, localization, proliferation and phe-notype.

Different mechanisms could contribute to the in-crease in the number of mast cells at sites of tissueinjury: mast cells or their progenitors could migrateto these sites; or resident mast-cell precursors couldproliferate. Adhesion receptors and their ligands alsoplay a role in the localization and migration of mastcells in normal tissues. ECM proteins that are theligands for adhesion receptors are chemotactics for

mast cells. Adherence of mast cells to fibroblasts,other cells or to ECM proteins can transduce signalsthat affect cell growth and differentiation.

The increase in the number of mast cells and ba-sophils, and the enhanced secretion at sites of in-flammation, can accelerate the elimination of thecause of tissue injury or, paradoxically, may lead to achronic inflammatory response. Thus, manipulatingmast-cell and basophil adhesion may be an impor-tant strategy for controlling the outcome of allergicand inflammatory responses.

9.1.3.2 Eosinophils

The eosinophil is a terminally differentiated, end-stage leukocyte that resides predominantly in sub-mucosal tissue and is recruited to sites of specificimmune reactions, including allergic diseases. Themean generation time for eosinophils in the bonemarrow is approximately 2-6 days. They mainly set-tle in the tissue where their number is about onehundred times higher than in the blood. Like othergranulocytes, they posses a polymorphous nucleus,although with only two lobes and no nucleolus. Theeosinophil cytoplasm contains large ellipsoid granuleswith an electron-dense crystalline nucleus and par-


tially permeable matrix. In addition to these largeprimary crystalloid granules, there is another granuletype that is smaller and lacks the crystalline nucleus.These large specific granules are the principal in-

dentifying feature of eosinophils. They contain fourdistinct cationic proteins which exert a range of bi-ological effects on host cells and microbial targets:major basic protein (MBP), oesinophil cationic pro-tein (ECP), eosinophil derived neurotoxin (EDN),and eosinophil peroxidase (EPO). Basophils containabout one fourth as much MBP as did eosinophilsand detectable amounts of EDN, ECP and EPO.Small amounts of EDN and ECP were also foundin neutrophils.These proteins have major effects not only on the

potential role of eosinophils in host defence againsthelminthic parasites, but also in contributing to tis-sue dysfunction and damage in eosinophil related in-flammatory and allergic diseases. As MBP lack en-zymatic activity, one mechanism whereby this highlycationic polypeptide may exert its toxic activitiesis by interactions with lipid membranes leading totheir derangement. Both MBP and EPO have beenshown to act as selective allosteric inhibitors of ago-nist binding to M2 muscarinic receptors. Thus, theseproteins may contribute to M2 receptor dysfunctionand enhance vagally mediated bronchoconstrictionin asthma. EDN specifically damage the myelin coatof neurons.In addition, histaminase and a variety of hy-

drolytic lysosomal enzymes are also present in thelarge specific granules.Among the typical small granule enzymes are aryl

sulphatase, acid phosphatase and a 92 kDa metallo-proteinase, a gelatinase.Only recently has it been recognized that

eosinophils are capable of elaborating cytokineswhich include those with potential autocrine growth-factor activities for eosinophils and those with po-tential roles in acute and chronic inflammatory re-sponses. Three cytokines have growth-factor activi-ties for eosinophils: granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3 and IL-5. Othercytokines produced by human eosinophils that mayhave activities in acute and chronic inflammatory re-sponses include IL-1, IL-6, IL-8, TNF- and bothtransforming growth factors, TGF- and TGF-.Eosinophils also participate in hypersensitivity re-

actions, especially through two lipid inflammatory

mediators, leukotriene C4 (LTC4) and platelet acti-vating factor (PAF). Both mediators contract airwaysmooth muscle, promote the secretion of mucus, altervascular permeability and elicit eosinophil and neu-trophil infiltration. In addition to the direct activ-ities of these eosinophil-derived mediators, MBP bya non-cytotoxic mechanism can stimulate the releaseof histamine from basophils and mast cells, and EPOfrom mast cells. Thus, once stimulated, eosinophilscan serve as a local source of specific lipid mediatorsas well as induce the release of mediators from mastcells and basophils.

The processes that lead to the accumulation ofeosinophils within tissue sites of specific inflam-mation, as for other leukocytes, involve numeroussequential interactions that enable eosinophils toadhere to and then transmigrate through the en-dothelium and to respond to local chemoattractants.The adhesion of eosinophils to endothelium includeCD18-dependent pathways, interaction between E-selectin and P-selectin and adherence to VCAM bymeans of very late antigen 4 (VLA-4) expressed onthe eosinophil.

The eosinophil granule content is released follow-ing similar stimuli to neutrophil granules (e.g. duringphagocytosis of opsonized particles and by chemotac-tic factors). However, whereas the neutrophil lyso-somal enzymes act primarily on material engulfed inphagolysosomes, the eosinophil granule content actmainly on extracellular target structure such as par-asites and inflammatory mediators.

The eosinophil functional activity, like the im-mune response in general, may be beneficial or harm-ful for the organism. Compared to neutrophils,eosinophils have limited phagocytic activity which ismainly aimed at killing multicellular parasites. An-other beneficial activity is the inactivation of me-diators of anaphylaxis. Thus, for example, acyl-sulphatase B may inactivate the slow-reacting sub-stance of anaphylaxis (SRS-A, a mixture of LTC4,LTD4 and LTE4), phospholipase D destroys theplatelet lytic factor, histaminase degrades histamineand lysophospholipase (phospholipase B) may inac-tivate the membrane-active lysophosphatides.

In addition to the acute release of protein, cytokineand lipid mediators of inflammation, eosinophilslikely contribute to chronic inflammation, includ-ing the development of fibrosis. Eosinophils arethe major source of the fibrosis-promoting cytokine


TGF- in nodular sclerosing Hodgkins disease. Ad-ditional roles for the eosinophil in modulating extra-cellular matrix deposition and remodeling are sug-gested by studies of normal wound healing. Duringdermal wound healing eosinophils infiltrate into thewound sites and sequentially express TGF- early,and TGF- later, during wound healing.

9.1.3.3 Neutrophils, central cells in acute in-flammation

Neutrophils, which are also known as polymorphonu-clear leukocytes (PMN), represent 50 to 60% of thetotal circulating leukocytes and constitute the firstline of defence against infectious agents or non-self substances that penetrate the bodys physicalbarriers. Once an inflammatory response is initiated,neutrophils are the first cells to be recruited to sitesof infection or injury. Their targets include bacteria,fungi, protozoa, viruses, virally infected cells and tu-mour cells. Their development in the bone marrowtakes about two weeks; during this period, they un-dergo proliferation and differentiation. During mat-uration, they pass trough six morphological stages:myeloblast, promyeloblast, myelocyte, metamyelo-cyte, non-segmented (band) neutrophil, segmentedneutrophil. The segmented neutrophil is a fully func-tionally active cell. It contains cytoplasmic granules(primary or azurophil and secondary or specific) anda lobulated chromatin-dense nucleus with no nucle-olus. The bone marrow of a normal healthy adultproduces more than 1011 neutrophils per day andmore than 1012 per day in settings of acute inflam-mation. Upon release from the bone marrow to thecirculation the cells are in a nonactivated state andhave a half-life of only 4 to 10 h before marginatingand entering tissue pools, where they survive for 1to 2 days. Cells of the circulating and marginatedpools can exchange with each other. Senescent neu-trophils are thought undergo apoptosis (programmedcell death) prior to removal by macrophages. Theviability is significantly shorter in individuals suffer-ing from infectious or acute inflammatory diseaseswhen the tissue requirement for newly recruited neu-trophils increases considerably.Subpopulations of neutrophils have been identi-

fied by various criteria. These cells exist not only indormant (resting) or activated states but also in var-ious intermediate stages. For, example, priming is amechanism whereby dormant neutrophils acquire a

state of preactivation that enable a more powerfulresponse to be generated once microbial activity isinitiated.

9.1.3.3.1 Neutrophil granules The neutrophilgranules are of major importance for neutrophilsfunction. When referring to phagocytes or leuko-cytes in general, the term granule is used more oftenthan lysosome. The terms are not fully equivalent;the term granules was originally derived from mor-phological observations whereas the term lysosomesis based on functional and biochemical characteris-tics of these cell organelles. Not all organelles thatlook like granules are necessarily typical lysosomes.The granules of neutrophils are generated during celldifferentiation; they are produced for storage ratherthan continually. On the basis of function and en-zyme content, human neutrophil granules can be di-vided into three main types - azurophil, specific andsmall storage granules. Their function is not justto provide enzymes for hydrolytic substrate degra-dation - as in classical lysosomes but also to killingested bacteria and, finally, to secrete their con-tents to regulate various physiological and patho-logical processes, including inflammation. Individualgranule populations can be characterized morpholog-ically (e.g. azurophil granules are larger and containmore electron-dense material than specific granules),or biochemically using enzyme markers or other sub-stances (Table 9.3).

Neutrophil granules contain antimicrobial or cy-totoxic substances, neutral proteinases, acid hydro-lases and a pool of cytoplasmic membrane receptors.Among azurophil granule constituents myeloperox-idase (MPO) is a critical enzyme in the conversionof hydrogen peroxide to hypochlorous acid. Togetherwith hydrogen peroxide and a halide cofactor it formsthe most effective microbicidal and cytotoxic mech-anism of leukocytes - the myeloperoxidase system.MPO is responsible for the characteristic green colorof pus.

Defensins, which constitute 30 to 50% ofazurophilic granule protein, are small (moleculeweight < 4 000) potent antimicrobial peptides thatare cytotoxic to a broad range of bacteria, fungi andsome viruses. Their toxicity may be due to mem-brane permeabilization of the target cell which issimilar to other channel-forming proteins (perforins).

Bacterial permeability-increasing (BPI) protein is


Constituents Granules

Azurophil Specific Small storage

Antimicrobial Myeloperoxidase LysozymeLysozyme LactoferrinDefensinsBPI

Neutral proteinases Elastase Collagenase GelatinaseCathepsin G Complement PlasminogenProteinase 3 activator activator

Acid hydrolases Cathepsin B Cathepsin BCathepsin D Cathepsin D-D-Glucuronidase -D-Glucuronidase-Mannosidase -MannosidasePhospholipase A2 Phospholipase A2

Cytoplasmic membrane CR3, CR4receptors FMLP receptors

Laminin receptors

Others Chondroitin Cytochrome b558 Cytochrome b558-4-sulphate Monocyte-chemotactic factor

HistaminaseVitamin B12binding protein

BPI: bactericidal permeability-increasing proteinFMLP: N-formylmethionyl-leucyl-phenylalanine

Table 9.3: Enzymes and other constituents of human neutrophil granules

also a member of perforins. It is highly toxic to gram-negative bacteria but not to gram-positive bacteriaor fungi and can also neutralize endotoxin, the toxiclipopolysaccharide component of gram-negative bac-terial cell envelope.

Lactoferrin sequesters free iron, thereby prevent-ing the growth of ingested microorganisms that sur-vive the killing process and increases bacterial per-meability to lysozyme.

Serine proteases such as elastase and cathepsin Ghydrolyze proteins in bacterial cell envelopes. Sub-strates of granulocyte elastase include collagen cross-linkages and proteoglycans, as well as elastin com-ponents of blood vessels, ligaments, and cartilage.Cathepsin D cleaves cartilage proteoglycans, whereasgranulocyte collagenases are active in cleaving type Iand, to a lesser degree, type III collagen from bone,cartilage, and tendon. Collagen breakdown prod-


ucts have chemotactic activity for neutrophils, mono-cytes, and fibroblasts.

Regulation of tissue destructive potential of lysoso-mal proteases is mediates by protease inhibitors suchas 2-macroglobulin and 1-antiprotease. These an-tiproteases are present in serum and synovial flu-ids. They are thought to function by binding toand covering the active sites of proteases. Protease-antiprotease imbalance is probably important in thepathogenesis of emphysema.

Azurophil granules function predominantly in theintracellular milieu (in the phagolysosomal vacuole),where they are involved in the killing and degrada-tion of microorganisms. On the other hand, neu-trophil specific granules are particularly susceptibleto release their contents extracellularly and appearto have an important role in initiating inflammation.Specific granules represent an intracellular reservoirof various plasma membrane components includingcytochrome b558 (component of NADPH oxidase, en-zyme responsible for the production of superoxide),receptors for complement fragment iC3b (CR3, CR4)for laminin, and formylmethionyl-peptide chemoat-tractants. In addition, there is also histaminase ca-pable for the degradation of histamine, vitamin B12binding protein, plasminogen activator (responsiblefor plasmin formation and cleavage of C5a from C5)and others.

The importance of neutrophil granules in inflam-mation is apparent from studies of several patientwith congenital abnormalities of the granules. Pa-tients with Chediak-Higashi syndrome have a pro-found abnormality in the rate of establishment ofan inflammatory response and have abnormally largelysosomal granules. The congenital syndrome of spe-cific granule deficiency is an exceedingly rare disordercharacterized by diminished inflammatory responsesand severe bacterial infections of skin and deep tis-sues.

9.1.3.3.2 Neutrophils in host defence Themajor role of neutrophils is to phagocytose and de-stroy infectious agents but they also limit the growthof some microbes, thereby buying time for adaptive(specific) immunological responses. With many mi-crobes, however, neutrophil defences are ineffectivein the absence of opsonins and various agents thatamplify the cytotoxic response.

Opsonization is a process, in which opsonins ad-sorb to the surface of bacteria or other particles andfacilitate their adherence to the phagocyte cytoplas-mic membrane through opsonin receptors. Specificbinding between the particle and phagocyte whichoccurs during immune phagocytosis is mediated byimmunoadherent receptors. There are two typesof immunoadherent receptors: Fc-receptors mainlyfor IgG antibodies (FcR) and complement receptors(CR1, CR3). It means that function of opsonins inthe first case is realized by antibodies and in the sec-ond case by iC3b. Specific binding between the par-ticle and phagocyte may be also performed by lectinsand lectin receptors (lectinophagocytosis).

To the phagocytosis itself chemotaxis of phago-cytes precede into the site where phagocytosable ma-terial occurs. This is regulated by chemotactic fac-tors generated by infectious agents themselves, aswell as those release as a result of their initial con-tact with phagocytes and other components of theimmune system.

Phagocytosis is a complex process composed ofseveral morphological and biochemical steps. Af-ter recognition and particle binding to the phagocytesurface, ingestion (engulfment), phagosome origina-tion, phagolysosome formation (fusion of phagosomewith lysosomes), killing and degradation of ingestedcells or other material proceed. Simultaneously withthe recognition and particle binding a dramatic in-crease in oxygen consumption (the respiratory burst)is observed. It is responsible for the production ofsuperoxide and other oxygen radicals, and also forthe secretion of a variety of enzymes and biologicallyactive substances controlling inflammatory and cy-totoxic reactions.

During phagocytosis, cytosolic granules (lyso-somes) fuse with the invaginating plasma membrane(around the engulfing microorganism) to form aphagolysosome into which they release their con-tents, thereby creating a higly toxic microenviron-ment. This step is of the first importance becauseduring it two categories of cytotoxic substances,present in the preformed state in azurophil and spe-cific granules and synthesized de novo during therespiratory burst, arrive at the same cell compart-ment. This degranulation normally prevents releaseof the toxic components into the extracellular milieu.However, some target may be too large to be fullyphagocytosed or they avoid engulfment, resulting in


frustrated phagocytosis in which no phagosome isformed. These may be killed extracellularly. How-ever, tissue damage occurs when neutrophil microbi-cidal products are released extracellularly to such anextent that host defences (antioxidant and antipro-tease screens) in the immediate vicinity are over-whelmed.The importance of neutrophils in fighting bacterial

and fungal infections is well recognized. Recently, ithas been shown that neutrophils are in abundancealso in virally induced lesions. Neutrophils bind toopsonized viruses and virally infected cells via anti-body (Fc) and complement (iC3b) receptors. Virusessuch as influenza can be inactivated by neutrophilstrough damage to viral proteins (e.g. hemagglutininand neuraminidase) mediated by the myeloperoxi-dase released during degranulation. In contrast tothese acute diseases, chronic influenza infections candiminish or exhaust the microbicidal potency of neu-trophils.

9.1.3.3.3 Neutrophils and host tissue dam-age Although neutrophils are essential to host de-fence, they have also been implicated in the pathol-ogy of many chronic inflammatory conditions andischemia-reperfussion injury. Hydrolytic enzymes ofneutrophil origin and oxidatively inactivated pro-tease inhibitors can be detected in fluid isolatedfrom inflammatory sites. Under normal conditions,neutrophils can migrate to sites of infection with-out damage host tissues. This damage may occurthrough several independent mechanisms. These in-clude premature activation during migration, extra-cellular release of toxic products during the killing ofsome microbes, removal of infected or damage hostcells and debris as a first step in tissue remodeling, orfailure to terminate acute inflammatory responses.Ischemia-reperfusion injury is associated with an

influx of neutrophils into the affected tissue and sub-sequent activation. This may be triggered by sub-stances released from damaged host cells or as a con-sequence of superoxide generation through xantineoxidase.Under normal conditions, blood may contain a

mixture of normal, primed, activated and spent neu-trophils. In the inflammatory site, mainly activatedand spent neutrophils are present. Activated neu-trophils have enhanced production of reactive oxygenintermediates (ROI). A subpopulation of neutrophils

with the anhanced respiratory burst has been de-tected in the blood of people with an acute bacte-rial infection and patients with the adult respira-tory distress syndrome (ARDS). This is a good ex-ample of the neutrophil paradox. Neutrophils havebeen implicated in the pathology of this conditionbecause of the large influx of these cells into thelung and the associated tissue damage caused byoxidants and hydrolytic enzymes released from ac-tivated neutrophils. The impairment of neutrophilmicrobicidal activity that occurs as the ARDS wors-ens may be a protective response on the part of thehost, which is induced locally by inflammatory prod-ucts. This down-regulation of neutrophil functionmay explain why many of these patients eventuallydie from overwhelming pulmonary infections.

The acute phase of thermal injury is also associ-ated with neutrophil activation, and this is followedby a general impairment in various neutrophil func-tions. Activation of neutrophils by immune com-plexes in synovial fluid contributes to the pathologyof rheumatoid arthritis. Chronic activation of neu-trophils may also initiate tumour development be-cause some ROI generated by neutrophils damageDNA and proteases promote tumour cell migration.

In patient suffering from severe burns, a strongcorrelation has been established between the onsetof bacteremic infection and reduction in the propor-tion and absolute numbers of neutrophils positive forantibody and complement receptors.

Oxidants of neutrophil origin have also been shownto oxidize low-density lipoproteins (LDL) which arethen more effectively bound to the plasma membraneof macrophages through specific scavenger receptors.Uptake of these oxidized LDL by macrophages isthought to initiate atherosclerosis.

In addition, primed neutrophils have been foundin people with essential hypertension, Hodgkinsdisease, inflammatory bowel disease, psoriasis, sar-coidosis, and septicaemia, where priming corre-lates with high concentrations of circulating TNF-(cachectin).

Hydrolytic damage to host tissue and thereforechronic inflammatory conditions may occur onlywhen antioxidant and antiprotease screens are over-whelmed. Antiprotease deficiency is thought to beresponsible for the pathology of emphysema. Manyantiproteases are members of the serine protease in-hibitor (SERPIN) family. Although the circulation is


rich in antiproteases, these large proteins may be se-lectively excluded at sites of inflammation becauseneutrophils adhere hightly to their targets. Ox-idative stress may initate tissue damage by reduc-ing the concentration of extracellular antiproteasesto below the level required to inhibit released pro-teases. Chlorinated oxidants and H2O2 can inacti-vate antiproteases such as 1-protease inhibitor and2-macroglobulin (which are endogenous inhibitorsof elastase) but, surprisingly, simultaneously acti-vate latent metalloproteases such as collagenases andgelatinase, which contribute to the further inactiva-tion of antiproteases.Cytoplasmic constituents of neutrophils may also

be a cause of formation of specific anti-neutrophil cy-toplasmic antibodies (ANCA) which are closely re-lated to the development of systemic vasculitis andglomerulonephritis. ANCA are antibodies directedagainst enzymes that are found mainly within theazurophil or primary granules of neutrophils. Thereare three types of ANCA that can be distinguishedby the patterns they produce by indirect immunoflu-orescence when tested on normal ethanol-fixed neu-trophils. Diffuse fine granular cytoplasmic fluores-cence (cANCA) is typically found inWegeners gran-ulomatosis, in some cases of microscopic polyarteri-tis and Churg Strauss syndrome, and in some casesof crescentic and segmental necrotising glomeru-lonephritis, but is rare in other conditions. The tar-get antigen is usually proteinase 3. Perinuclear fluo-rescence (pANCA) is found in many cases of micro-scopic polyarteritis and glomerulonephritis. Theseantibodies are often directed against myeloperoxi-dase but other targets include elastase, cathepsin G,lactoferrin, lysozyme and -D-glucuronidase. Thethird group designated atypical ANCA includesneutrophil nuclear fluorescence and some unusual cy-toplasmic patterns and while a few of the target anti-gens are shared with pANCA, the others have notbeen identified yet.pANCA are also found in a third of patients with

Crohns disease. The reported incidence of ANCAin rheumatoid arthritis and SLE varies considerablybut the patterns are predominantly pANCA andatypical ANCA.

9.1.3.3.4 Free radicals produced by neu-trophils Two types of free radicals are producedby neutrophils, macrophages, endothelial and other

cells. The first type is represented by reactive oxy-gen intermediates which are formed in neutrophils bythe activity of NADPH oxidase, the enzyme of therespiratory burst. The second type includes reactivenitrogen intermediates, the first member of them, ni-tric oxide being produced by nitric oxide synthase.

Reactive oxygen intermediates (ROI)

Upon activation neutrophils and mononuclearphagocytes have increased oxygen consumption, aprocess known as the respiratory burst. During this,oxygen is univalently reduced by NADPH oxidase tosuperoxide anion or its protonated form, perhydroxylradical, which then is catalytically converted by ac-tion of superoxide dismutase to hydrogen peroxide:

O2 + e + H+ HO2 = O2 + H+O2 + O2 + 2H+ O2 + H2O2

NADPH oxidase is an electron transport chainfound in the wall of the endocytic vacuole of pro-fessional phagocytes and in B and T lymphocytes.It is so called because NADPH is used as an elec-tron donor to reduce oxygen to superoxide and hy-drogen peroxide. NADPH oxidase is a complex en-zyme composed at least of five members. Two ofthem are p21phox and gp91phox subunits of a very un-usual flavocytochrome b558 in the cytoplasmic mem-brane. Two cytosolic proteins (p47phox, p67phox), aqiunone, and a Rac-related GTP-binding protein arethought to be the other functional components of thiselectron transport system. (phox meas phagocyteoxidase, p protein, and gp glycoprotein). TheNADPH oxidase system is dissociated and thus in-active in dormant neutrophils. While some com-ponents are membrane bound, others are stored inthe cytosol. Upon activation, the cytosolic compo-nents translocate to the plasma membrane to assem-ble the active oxidase. The absence of, or an ab-normality in, any one of these components result inchronic granulomatous disease (CGD) characterizedby the absence of respiratory burst from neutrophilsand monocytes of these patients. The children suf-fer from repeated infections that respond poorly toconventional therapy and almost invariably lead toearly death.

Superoxide anion (O2 is both a one-electron re-ductant and a one-electron oxidant that can passthrough cell membrane via anion channels. It apears


that superoxide does not have direct toxic effects ontargets but, rather exerts its toxicity by penetrationto important sites where it subsequently is convertedto other ROI. Hydrogen peroxide (H2O2), hydroxylradical (OH) and singlet oxygen are of the first im-portance of them.Hydrogen peroxide interacts with myeloperoxi-

dase (MPO), contained in neutrophil azurophil gran-ules to produce hypochlorous acid, which is metabo-lized to hypochlorite (bleach) and chlorine:

H2O2 + Cl +H+ MPO

H2O+HOCl

HOCl H+ +OCl

HOCl + Cl Cl2 +OH

Hydroxyl radical (OH) is formed by several waysfrom which decomposition of H2O2 catalyzed byFe2+ is the most important:

Fe2+ + H2O2 Fe3+ +OH + OH

This reaction is supposed to be involved, for in-stace, in asbestosis because asbestos contains highconcentrations of iron. The toxicity of OH is be-lieved to result from the ability of OH to serve as apowerful one-electron oxidant capable of abstractingelectrons from a large variety of compounds with theformation of a new radical, which can oxidize othersubstances:

OH +R OH + R

Hydroxyl radical and hypochlorite are the mostpowerfull substances involved in microbicidal and cy-totoxic reactions. HOCl is 100 to 1000 times more ef-fective than H2O2. Furthermore, HOCl-induced celldeath occurs very rapidly in comparison to that me-diated by H2O2.Singlet oxygen (1O2) is an oxygen form whose elec-

trons are excited at a higher energy level compared tothe normal (ground) triplet oxygen. When returningto the ground state they emit light (chemiluminis-cence) which may have antimicrobial and cytotoxiceffects.These oxidants also promote the margination of

neutrophils by triggering the expression of adhesionmolecules on endothelial cells.ROI are involved in a variety of pathological con-

ditions. For example pulmonary diseases in whichoxygen radicals are thought to be involved includeARDS, hyperoxia, asbestosis, silicosis, paraquat tox-icity, bleomycin toxicity, cigarette smoking, ionizingradiation and others.

ROI are highly toxic also for producing cells.Therefore neutrophils have to contain large reservesof endogenous antioxidants such as glutathione andascorbate. Their ability to maintain these antioxi-dants in the reduced state during phagocytosis mayprevent death from oxidative suicide.

Reactive nitrogen intermediates (RNI)

They are sometimes also called reactive oxynitro-gen intermediates (RONI). The pathway by whichthey are originated is an oxidative process in whichshort-lived nitric oxide (NO ) is derived from theguanidino nitrogen in the conversion of L-arginineto L-citrulline. This reaction is catalysed by NOsynthase and, like the respiratory burst, it involvesoxygen uptake.

Three distinct isoform of nitric oxide synthase(NOS) representing three distinct gene products havebeen isolated and purified. The three isoforms varyconsiderably in subcellular location, structure, ki-netics, regulation, and hence functional roles (Ta-ble 9.4).

Two of the enzymes are constantly present andtermed constitutive NOS (cNOS). The endothelialcNOS is mostly membrane bound and formed onlyin endothelial cells. The neuronal cNOS was identi-fied in the cytosol of central and peripheral neurons.NO derived from the cNOS isoform act as a physio-logic regulator by relaxing vascular smooth muscle orby functioning as a neurotransmitter. These isoformsproduce small amounts of NO for short periods ina calcium/calmodulin dependent manner upon stim-ulation. Endothelial cNOS with the endothelial cellacting as a signal transducer, releases NO contin-uously in varying amounts to regulate blood vesseltone and thus also the blood flow and pressure. Largeamounts of NO produced in a prolonged time maycause vasodilalation and hypotension, whereas insuf-ficient NO formation may be involved in hyperten-sion. It seems that NO plays a fundamental role inthe regulation of the cardiovascular system. The or-ganic nitrates used as vasodilatation drugs for manyyears spontaneously release or are biotransformed tothe active form which is NO Within the CNS, NO


Characteristic Endothelial cNOS Neuronal cNOS Inducible NOS

Depency on Ca2+, calmodulin Ca2+, calmodulin Independent

Molecular weight x103 150160 150160 132

Chromosomal location 7q3536 12q24.2 17q1112

Producing cells Endothelial Neurons Macrophages, monocytes,Kuppffers cells neutrophils,hepatocytes, myocytes,chondrocytes, smoothmuscle cells

Inductors of No No LPS, TNF, IL1,biosynthesis IFN, GMCSF

Inhibition by Yes Yes YesLarginine analogs

Inhibition by No No Yesglucocorticoids

Table 9.4: Isoforms of human NO synthase and their characteristics

is released in response to increases in intracellularCa2+ that follow stimulation of glutamate receptorsand may be classified as a mediator of slow synap-tic transmission. A second function for NO withinthe CNS may relate to the toxic effects because itsincreased release may lead to epileptic seizures andbrain damage.

The third isoform of NOS is not present in rest-ing cells but instead the cells must be induced toexpress the enzyme, thus the name inducible NOS(iNOS). Stimuli typically include cytokines and/orlipopolysaccharide (LPS), and once expressed theenzyme generates large amounts of NO. A numberof cytokines is involved in the production of iNOS.Among them IFN-, IL-1, IL-6, THF-, GM-CSF(granulocyte-macrophage colony stimulatory factor)and PAF (platelet activating factor) exert the stim-ulatory effect whereas the suppression has been

observed in the case of IL-4, IL-8, IL-10, TGF- (transforming growth factor), PDGF (platelet-derived growth factor) and MDF (macrophage de-activating factor).

NO may react with superoxide to form highlytoxic peroxynitrite anion:

NO + O2 ONOO

which may be transformed in an acid milieu toperoxynitrite acid and then to hydroxyl radical:

ONOO +H+ ONOOHONOOH OH +NO2 NO3 + H+

Independent pathways are involved in the sythe-sis of ROI and RNI. Dormant neutrophils produced


NO continuously but activation arrest this pathwayin favor of the oxidative burst. Thus, although theROI and RNI pathways are independent, they maycompete for common substrates such as NADPHand O2 and exert other modulating effects on eachother. The steady-state production of these speciesmay dictate the anti/proinflammatory balance. Mi-crobial killing appears to ROI dependent in normalneutrophils but RNI may play a role in cells withdeficiences in the NADPH oxidase/MPO pathways.Nitric oxide may also contribute to the microbicidalactivity of neutrophils by reacting with ROI to formsecondary cytotoxic species such as peroxynitrite.

The main role of neutrophil-derived NO may beto facilitate the migration of neutrophils from bloodvessels to surrounding tissue by causing vasodilata-tion. NO facilitates relaxation of vascular smoothmuscle, and ROI initiate vasoconstriction throughthe production of superoxide, which removes NO. Inaddition NO inhibits neutrophil adhesion to vascu-lar endothelium and this may prevent inflammatoryand ischemia-reperfusion injuries.

The basis of the functional activity of NO isits dual actions on some enzymes of target cells.The small amount of NO released by cNOS iso-forms is adequate to activate the known NO-sensitive enzymes (guanylate cyclase and ADP-ribosyl-transferase) and participate in NO signal-ing pathways. The larger amounts of NO gener-ated by iNOS may also activate the NO-sensitiveenzymes, but in many cell types the high out-put of NO also exceed the necessary concentra-tion threshold to inhibit the action of certain iron-containing enzymes, namely aconitase, NADPH-ubiquinone oxidoreductase, succinate-ubiquinone ox-idoreductase, ribonucleotide reductase, NADPH oxi-dase and glyceraldehyd-3-phosphate dehydrogenase.

Activation of soluble guanylate cyclase by NOleads to the synthesis of cGMP, which leads to re-laxantion of vascular smooth muscle cells, inhibitionof platelet adherence, aggregation, inhibition of neu-trophil chemotaxis, and signal transduction in thecentral and peripheral nervous system.

NO causes autoribosylation of glyceraldehyde-3-phosphate dehydrogenase, which inactivates this gly-colytic enzyme. NO also inhibits three mitochodrialenzymes: aconitase of the Krebs cycle and NADPHubiquinone oxidoreductase and succinate-ubiquinoneoxidoreductase of the electron transport chain.

Induced NO synthesis was reported in inflam-matory responses initiated by microbial productsor autoimmune reactions and also in the systemicinflammatory response, also referred to as sepsis.NO likely participates in the inflammatory reactionand subsequent joint destruction in some types ofarthritis. For instace synovial fluid from patientswith osteoarthritis exhibits elevated nitrate concen-trations (nitrate are end products of the L-arginine-NO synthase pathway). There is also evidence forchronic expression of iNOS in the smooth muscle inatherosclerotic aortic aneurysms, a disease in whichthere is progressive dilatation and destruction of theaortic wall leading often to fatal rupture.

9.1.3.3.5 Regulation of neutrophil functionUnder normal conditions, neutrophils roll along mi-crovascular walls via low affinity interaction of se-lectins with specific endothelial carbohydrate lig-ands. During the inflammatory response, chemotac-tic factors of different origin and proinflammatory cy-tokines signal the recruitment of neutrophils to sitesof infection and/or injury. This leads to the activa-tion of neutrophil 2-integrins and subsequent high-afinity binding to intercellular adhesion molecules onthe surface of activated endothelial cells in postcap-illary venules. Under the influence of a chemotac-tic gradient, generated locally and by diffusion ofchemoattractants from the infection site, neutrophilpenetrate the endothelial layer and migrate throughconnective tissue to sites of infection (diapedesis),where they finally congregate and adhere to extra-cellular matrix components such as laminin and fi-bronectin. A wide variety of adhesion molecules havebeen characterized on the surface of phagocytic cellsand will be shown later.

Cytokines are basic regulators of all neutrophilfunctions. Many of them including hematopoieticgrowth factors and pyrogens have shown to be potentneutrophil priming agents. Neutrophils also synthe-size and secrete small amounts of some cytokines in-cluding IL-1, IL-6, IL-8, TNF-, and GM-CSF; theymay act in an autocrine or paracrine manner. Thepyrogenic cytokines, IL-1, TNF-, and IL-6 all primevarious pathways that contribute to the activation ofNADPH oxidase. Pro-inflammatory cytokine IL-8,which is also known as neutrophil-activating factor,is also a potent chemoattractant; it synergizes withIFN-, TNF-, GM-CSF, and G-CSF to amplifyvarious neutrophil cytotoxic functions. Cytokines


also increase the microbiostatic and killing capac-ities of neutrophils against bacteria, protozoa andfungi. IFN- and GM-CSF independently amplifyneutrophil antibody-dependent cytotoxicity. Anti-inflammatory cytokines, IL-4 and IL-10 inhibit theproduction of IL-8 and the release of TNF- andIL-1 which reflects in the blockade of neutrophil ac-tivation.

Furthermore, some cytokines prolong neutrophilsurvival. The acute inflammatory response maybe terminated by the secretion of macrophage in-flammatory protein-1 (MIP-1) from neutrophils;this protein may signal mononuclear cell recruit-ment and clear neutrophils from the affected tis-sue site. All these cytokines are produced by neu-trophils themselves and/or by lymphocytes, mono-cytes/macrophages or endothelial cells.

Along to cytokines other mediators, includingbioactive lipids, neuroendocrine hormones, his-tamine, and adenosine, are also involved in the reg-ulation of neutrophil activation.

Bioactive lipids originate mainly from arachi-donic acid which is an abundant constituent ofneutrophil membranes. Arachidonic acid is me-tabolized to prostaglandins, leukotrienes and lipox-ins. LTB4 is a strong neutrophil chemoattractantthat may play a role in the priming process. Va-soactive leukotrienes LTC4, LTD4 and LTE4 in-crease microvascular permeability and may con-tribute to ischemia-reperfusion injury. In contrastto leukotrienes, prostaglandins suppress most neu-trophil functions, possibly through their ability to el-evate intracellular cAMP. Lipoxins LXA4 and LXB4are potent inhibitors of neutrophil microbicidal ac-tivity.

In many inflammatory conditions, the level ofplatelet-activating factor (PAF) rise in the affectedtissues, but injury can be attenuated by PAF antag-onists. PAF directly primes superoxide generationand elastase release.

The major stress hormones are involved in theregulation of inflammation at both the systemicand, perhaps, local levels. The bidirectional in-teractions of cytokines and neurotransmitters withnervous and immune cells, respectively, provide ameans of indirect chemical communication betweenthe neuroendocrine and immune systems. Fromthe neuroendocrine hormones mainly growth hor-mone, prolactin, -endorphin, glucocorticoids and

catecholamines are involved in the neutrophil regula-tion. Growth hormone primes the oxidative burst ofhuman neutrophils. This is initiated by growth hor-mone to the prolactin (and not the growth hormone)receptor on neutrophils in a zinc-dependent process.The growth-promoting effects of growth hormone aremediated through insulin-like growth factor 1, whichis also a strong neutrophil-priming agent. Prolactin,which shares considerable functional and structuralsimilarities with growth hormone, is also a strongimmunopotentiating agent. Prolactin primes the ox-idative burst of neutrophils and macrophages to thesame intensity as that induced by growth hormone.

Although glucocorticoids and opioidsmay enhancesome immune responses at very low concentrations,they are generally considered to be immunosuppres-sive. These contrasting responses may be controlledby the presence of multiple receptors for the samemediator that are coupled to stimulatory and in-hibitory pathways. In fact, containment of the stressresponse may be the principal role of glucocorticoids.Glucocorticoids severely impair the phagocytic andcytotoxic activities of neutrophils and macrophages,their capacity to produce ROI and to induce iNOS,and secrete lysosomal enzymes in response to acti-vation. Oxidative burst of professional phagocytesis also inhibited with epinephrine and -endorphinwhich activity is mediated via nonopioid receptors.

Histamine is a potent inhibitor of neutrophil mi-crobicidal activity. Adenosine provides an interest-ing example of how a single mediators may playdual roles. Adenosine, a vasodilator, is a potentanti-inflammatory agent released from damaged hostcells. Neutrophil chemotaxis is activated by adeno-sine occupancy of A1 receptors and inhibition ofthe respiratory burst triggered through A2 receptors.Adenosine suppresses the respiratory burst only if itis added before the triggering agent, but it has noeffect on the initiation or progress of degranulation.

The interactions between platelets and neutrophilsare essential for both cell types. Activated plateletscan bind to neutrophils and stimulate the oxidativeburst while themselves synthesize vasoconstrictiveleukotrienes. Like prostaglandins, many immuno-suppressive mediators use cAMP as a second messen-ger. Increased intracellular cAMP in neutrophils isassociated with decreases in a number of microbicidalfunctions. Phagocyte priming and activation may, infact, be controlled by shifts in the intracellular ratio


of cGMP to cAMP, since cGMP is stimulatory.

9.1.3.4 Macrophages and monocytes

Originally, monocytes and macrophages were clas-sified as cells of the reticulo-endothelial system -RES (Aschoff, 1924). Van Furth et al. (1972) pro-posed the mononuclear phagocyte system MPS,and monocytes and macrophages became basic celltypes of this system. Their development takes inthe bone marrow and passes through the followingsteps : stem cell - committed stem cell - monoblast -promonocyte - monocyte (bone marrow) - monocyte(peripheral blood) - macrophage (tissues). Monocytedifferention in the bone marrow proceeds rapidly(1.5 to 3 days). During differentation, granules areformed in monocyte cytoplasma and these can be di-vided as in neutrophils into at least two types. How-ever, they are fewer and smaller than their neutrophilcounterparts (azurophil and specific granules). Theirenzyme content is similar.The process of haematopoiesis is controlled by

a group of at least 11 growth factors. Three ofthese glycoproteins initiate the differentiation ofmacrophages from uni- and bipotential progenitorcells in the bone marrow. The progression frompluripotential stem cell to myeloid-restricted pro-genitor is controlled by IL-3, which generates dif-ferentiated progeny of all myeloid lineages. AsIL-3-responsive progenitors differentiate, they be-came responsive to GM-CSF and M-CSF, the twogrowth factors giving rise to monocyte/macrophage-restricted progeny. After lineage commitment, cellsare completely dependent on these growth factors forcontinued proliferation and viability. More recently,TNF- has also been implicated in growth regulationfor macrophage precursors.The blood monocytes are young cells that al-

ready possess migratory, chemotactic, pinocytic andphagocytic activities, as well as receptors for IgG Fc-domains (FcR) and iC3b complement. Under mi-gration into tissues, monocytes undergo further dif-ferentiation (at least one day) to become multifunc-tional tissue macrophages. Monocytes are generally,therefore, considered to be immature macrophages.However, it can be argued that monocytes representthe circulating macrophage population and should beconsidered fully functional for their location, chang-ing phenotype in response to factors encountered inspecific tissue after migration.

Macrophages can be divided into normal andinflammatory macrophages. Normal macrophagesincludes macrophages in connective tissue (his-tiocytes), liver (Kupffers cells), lung (alveo-lar macrophages), lymph nodes (free and fixedmacrophages), spleen (free and fixed macrophages),bone marrow (fixed macrophages), serous fluids(pleural and peritoneal macrophages), skin (histio-cytes, Langerhanss cell) and in other tissues.

The macrophage population in a particular tis-sue may be maintained by three mechanisms: in-flux of monocytes from the circulating blood, localproliferation and biological turnover. Under nor-mal steady-state conditions, the renewal of tissuemacrophages occurs through local proliferation ofprogenitor cells and not via monocyte influx. Orig-inally, it was thought that tissue macrophages werelong-living cells. More recently, however, it has beenshown that depending on the type of tissue, theirviability ranges between 6 and 16 days.

Inflammatory macrophages are present in variousexudates. They may be characterized by various spe-cific markers, e.g. peroxidase activity, and since theyare derived exclusively from monocytes they sharesimilar properties. The term exudate macrophagesdesignates the developmental stage and not the func-tional state.

Macrophages are generally a population of ubiqui-tously distributed mononuclear phagocytes respon-sible for numerous homeostatic, immunological, andinflammatory processes. Their wide tissue distri-bution makes these cells well suited to provide animmediate defence against foreign elements prior toleukocyte immigration. Because macrophages par-ticipate in both specific immunity via antigen pre-sentation and IL-1 production and nonspecific im-munity against bacterial, viral, fungal, and neoplas-tic pathogens, it is not surprising that macrophagesdisplay a range of functional and morphological phe-notypes.

9.1.3.4.1 Heterogeneity and activation ofmacrophages Macrophage heterogeneity is awell-documented phenomenon, perhaps first ob-served by Metchnikoff, who described a progressionof infiltrating cell types in inflammatory exudates.It has also long been recognized that macrophagesisolated from different anatomical sites display adiversity of phenotypes and capabilities. Because


macrophage function is dependent in part on sig-nals received from the immediate microenvironment,it is suggested that macrophage heterogeneity mayarise from unique conditions within specific tissues.Obviously, the sterile, anaerobic environment of thespleen or peritoneum will impart different constraintson resident macrophages than does the aerobic en-vironment of the alveolar macrophage, which con-tains numerous external factors. Antibodies di-rected against specific membrane antigens have beenused to compare macrophage from different tissues.For instance, human breast milk macrophages ex-press an antigen not observed on monocytes, alve-olar macrophages, or peritoneal cells. Furthermore,human alveolar macrophage express high levels ofMHC class II antigen, whereas the opposite is foundfor peritoneal macrophages.

It has been just as quickly recognized thatmacrophages isolated from a given tissue display het-erogeneous function. For example, only a portionof peritoneal macrophages express low levels of 5-nucleotidase, and immune elicitation of peritonealmacrophages results in predominantly macrophageswith low 5-nucleotidase activity, presumably be-cause of an influx of monocytes. Thus, functionalheterogeneity results from the spectrum of matura-tional states in a given isolate because of the influxof monocytes and/or local proliferation.

Because macrophages are responsible for numer-ous inflammatory processes, it becomes impor-tant to distinguish between normal or steady-statehaematopoiesis and induced haematopoiesis associ-ated with immunological challenge. Production ofthe macrophage lineage from bone marrow progeni-tors is normally controlled by M-CSF, which is con-stitutively produced by many cell types. In re-sponse to invasive stimuli and inflammation, mono-cyte numbers increase dramatically, as do serum lev-els of M-CSF. In addition, GM-CSF appears in theserum. Although there appear to be a large overlapof macrophage progenitors able to respond to M-CSFor GM-CSF, the very different structures and signalt

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