2 Inflammation

2 InflammationHedwig S. Murphy

Inflammation is the response to injury of a tissueand its microcirculation and is characterized by elab-oration of inflammatory mediators as well as move-ment of fluid and leukocytes from the blood into ex-travascular tissues. Inflammation localizes andeliminates microorganisms, damaged cells, and foreignparticles, paving the way for a return to normal struc-ture and function.

The clinical signs of inflammation, recognized inEgyptian medical texts before 1000 BC, were codifiedas the four cardinal signs of inflammation: rubor(redness), calor (heat), tumor (swelling), and dolor(pain) by the Roman encyclopedist Aulus Celsus in

the second century AD. These features correspond toinflammatory events of vasodilation, edema, and tis-sue damage. A fifth sign functio laesa (loss of func-tion) was added in the 19th century by RudolfVirchow, who recognized inflammation as a responseto tissue injury.

Overview Of Inflammation

Inflammation is best viewed as an ongoing process that canbe divided into phases.

• Initiation results in a stereotypic, immediate responsetermed acute inflammation. The acute response is

2323

Overview Of InflammationAcute Inflammation: Vascular EventsPlasma-Derived Mediators Of Inflammation

Hageman Factor Kinins Complement System and the Membrane AttackComplex (MAC)

Cell-Derived Mediators Of InflammationArachidonic Acid and Platelet-Activating Factor Prostanoids, Leukotrienes, and Lipoxins Cytokines Reactive Oxygen Species (ROS)

Cells Of InflammationNeutrophilsEndothelial Cells Monocyte/Macrophages Mast Cells and Basophils Eosinophils Platelets

Leukocyte Recruitment In Acute InflammationLeukocyte Adhesion Chemotactic Molecules Leukocytes Traverse the Endothelial Cell Barrier to GainAccess to the Tissue

Leukoctye Functions In Acute InflammationPhagocytosisNeutrophil Enzymes Oxidative and Nonoxidative Bactericidal Activity

Outcomes Of Acute InflammationChronic Inflammation

Cells From Both the Circulation and Affected TissuePlay a Role in Chronic Inflammation

Injury And Repair In Chronic InflammationGranulomatous InflammationSystemic Manifestations Of Inflammation

Acute Phase Response Fever Shock

FPO FPO FPO FPO

characterized by the rapid flooding of the injured tissuewith fluid, coagulation factors, cytokines, chemokines,platelets and inflammatory cells, and neutrophils in par-ticular (Fig. 2-1).

• Amplification depends upon the extent of injury andactivation of mediators such as kinins and complementcomponents. Additional leukocytes and macrophagesare recruited to the area.

• Destruction of the inciting agent by phagocytosis andenzymatic or nonenzymatic processes reduces or elimi-nates foreign material or infectious organisms. At thesame time, damaged tissue components are also re-moved, paving the way for repair to begin (see Chapter 3).

• Termination of the inflammatory response is mediatedby intrinsic anti-inflammatory mechanisms that limittissue damage and allow for either restoration of tis-sue, with return to normal physiological function, orrepair and the development of scar in place of normaltissue.

Certain types of injury trigger a sustained inflammatoryresponse associated with the inability to clear injured tissueand foreign agents. Such a persistent response (which oftenhas an immune component) is termed chronic inflamma-tion. Chronic inflammatory infiltrates are composed largelyof lymphocytes, plasma cells, and macrophages and oftenhave an immune component (Fig. 2-2). Acute and chronic in-flammatory infiltrates often coexist.

Acute Inflammation: Vascular Events

Among the earliest responses to tissue injury are alterationsin the anatomy and function of the microvasculature,which may promote edema (see Figs. 2-3 and 2-4). These re-sponses include:

1. Transient vasoconstriction of arterioles at the site ofinjury is the earliest vascular response to mild skin in-jury. This process is mediated by both neurogenic andchemical mediator systems and usually resolves withinseconds to minutes.

2. Vasodilation of precapillary arterioles then increasesblood flow to the tissue, a condition known as hyper-emia. Vasodilation is caused by release of specific medi-ators and is responsible for redness and warmth at sitesof tissue injury.

3. An increase in endothelial cell barrier permeabilityresults in edema. Loss of fluid from intravascular com-partments as blood passes through capillary venulesleads to local stasis and plugging of dilated small vesselswith erythrocytes. These changes are reversible followingmild injury: within several minutes to hours, the ex-travascular fluid is cleared through lymphatics.

The vascular response to injury is a dynamic event thatinvolves sequential physiological and pathological changes.

24 Essentials of Rubin’s Pathology

Acute inflammation with densely packed polymor-phonuclear neutrophils (PMNs) with multilobed nu-

clei (arrows).

FIGURE 2-1. Chronic inflammation. Lymphocytes, plasma cells(arrows), and a few macrophages are present.FIGURE 2-2.

Vasoactive mediators, originating from both plasma andcellular sources, are generated at sites of tissue injury (seeFig. 2-4). These mediators bind to specific receptors on vas-cular endothelial and smooth muscle cells, causing vasocon-striction or vasodilation. Proximal to capillaries, vasodilationof arterioles increases blood flow and can exacerbate fluidleakage into the tissue. Distally, vasoconstriction of postcap-illary venules increases capillary bed hydrostatic pressure, po-tentiating edema formation. By contrast, vasodilation ofvenules decreases capillary hydrostatic pressure and inhibitsmovement of fluid into extravascular spaces.

After injury, vasoactive mediators bind specific receptorson endothelial cells, causing endothelial cell contractionand gap formation, a reversible process (see Fig. 2-3B). Thisbreak in the endothelial barrier leads to extravasation (leak-age) of intravascular fluids into the extravascular space.

Mild direct injury to the endothelium results in a biphasicresponse: an early change in permeability occurs within 30 minutes after injury, followed by a second increase invascular permeability after 3 to 5 hours. When damage is se-vere, exudation of intravascular fluid into the extravascularcompartment increases progressively, peaking 3 to 4 hoursafter injury.

Severe direct injury to the endothelium, such as iscaused by burns or caustic chemicals, may result in irre-versible damage. In such cases, the endothelium separatesfrom the basement membrane, resulting in cell blebbing(blisters or bubbles between the endothelium and thebasement membrane). This leaves areas of basementmembrane naked (see Fig. 2-3C), thereby disrupting thebarrier between the intravascular and extravascularspaces.

C H A P T E R 2 : INFLAMMATION 25

Time course of changein permeability

0 1 2 3 4 5C

hang

e in

perm

eabi

lity

Hours

Time course of changein permeability

0 1 2 3 4 5

Cha

nge

inpe

rmea

bilit

y

Hours

Mild

Severe

NORMAL VENULE

VASOACTIVE MEDIATOR-INDUCED INJURY

DIRECT INJURY TO ENDOTHELIUM

Gap formation

Basementmembrane

Endothelialcell

Tight junction

Endothelialretractionand gapformation

Electrolytes,fluid, protein

Denudedbasementmembrane

Blebbing

A

B

C

Responses of the microvasculature to injury. A. The wall of the normal venule is sealed by tight junctionsbetween adjacent endothelial cells. B. During mild vasoactive mediator-induced injury, the endothelial cells

separate and permit the passage of the fluid constituents of the blood. C. With severe direct injury, the endothelial cells form blebs(b) and separate from the underlying basement membrane. Areas of denuded basement membrane (arrows) allow a prolongedescape of fluid elements from the microvasculature.

FIGURE 2-3.

Several definitions are important for understanding thevascular components of inflammation:

• Edema is accumulation of fluid within the extravascularcompartment and interstitial tissues.

• A transudate is edema fluid with low protein content(specific gravity <1.015). Transudates tend to occur innoninflammatory conditions, where the endothelial bar-rier remains intact and prevents the loss of large mole-cules from the vasculature.

• An exudate is edema fluid with a high protein concen-tration (specific gravity >1.015), which frequently con-tains inflammatory cells. Exudates are observed early inacute inflammatory reactions and are produced by mildinjuries, such as sunburn or traumatic blisters.

• A fibrinous exudate contains large amounts of fibrin asa result of activation of the coagulation system. When afibrinous exudate occurs on a serosal surface, such as thepleura or pericardium, it is referred to as “fibrinous pleu-ritis” or “fibrinous pericarditis.”

• A purulent exudate or effusion contains prominent cel-lular components. It is frequently associated with patho-logical conditions such as pyogenic bacterial infections,

in which the predominant cell type is the polymor-phonuclear neutrophil (PMN).

Plasma-Derived Mediators Of Inflammation

Numerous chemical mediators are integral to initiation,amplification, and termination of inflammatory processes(Fig. 2-4). Cell- and plasma-derived mediators work in con-cert to activate cells by (1) binding specific receptors, (2) re-cruiting cells to sites of injury, and (3) stimulating the re-lease of additional soluble mediators. These mediatorsthemselves are relatively short-lived, or are inhibited by in-trinsic mechanisms, effectively turning off the response andallowing the process to resolve. Cell-derived mediators areconsidered below.

Plasma contains the elements of three major enzyme cas-cades, each composed of a series of proteases. Sequential ac-tivation of proteases results in release of important chemi-cal mediators. These interrelated systems include (1) thecoagulation cascade and fibrinolytic system, (2) kinin


CELL-DERIVED

PLASMA-DERIVED

• Hageman factor activation

• Complement system activation

Clotting/fibrinolyticsystem

Kallikrein-kininsystem

Fibrin splitproducts

SOURCE MEDIATOR

Kinins(bradykinin)

C3a, C5a

• Mast cell/basophil degranulation

• Platelets

• Inflammatory cells

Histamine

Serotonin

Increasedvascularpermeability

EDEMA

• Platelet- activating factor• Prostaglandins• Leukotrienes

• Endothelium• Nitric oxide• Platelet- activating factor• Prostaglandins

Inflammatory mediators of increased vascular permeability.FIGURE 2-4.

generation, and (3) the complement system (Fig. 2-5). Thecoagulation cascade is discussed in Chapters 10 and 20; thekinin and complement systems are presented here.

Hageman Factor is a Key Source of VasoactiveMediators

Hageman factor (clotting factor XII) is generated within theplasma and is activated by exposure to negatively chargedsurfaces such as basement membranes, proteolytic enzymes,bacterial lipopolysaccharides, and foreign materials. Thiskey component triggers activation of additional plasma pro-tease systems important in inflammation including (1) the“intrinsic” coagulation cascade, (2) fibrinolysis with the con-comitant elaboration of plasmin and plasmin-derived bioac-tive peptides, (3) generation of kallikrein and subsequentproduction of kinins, and (4) activation of the alternatecomplement pathway (see Fig. 2-5).

Kinins Amplify the Inflammatory Response

Kinins are potent inflammatory agents formed in plasmaand tissue by the action of serine protease kallikreins onspecific plasma glycoproteins termed kininogens.Bradykinin and related peptides regulate multiple physio-logical processes, including blood pressure, contraction

and relaxation of smooth muscle, plasma extravasation, cellmigration, inflammatory cell activation, and inflammatory-mediated pain responses. Kinins amplify the inflammatoryresponse by stimulating local tissue cells and inflamma-tory cells to generate additional mediators, includingprostanoids, cytokines (especially tumor necrosis factor-α[TNF-α] and interleukins), and nitric oxide (NO•). Kininsare rapidly degraded to inactive products by kininases and,therefore, have rapid and short-lived functions.

Complement is Activated Through ThreePathways to Form the Membrane Attack Complex (MAC)

The complement system is a group of proteins found inplasma and on cell surfaces, whose primary function is de-fense against microbes. The physiological activities of thecomplement system include (1) defense against pyogenicbacterial infection by opsonization, chemotaxis, activation ofleukocytes and lysis of bacteria and cells; (2) bridging innateand adaptive immunity for defense against microbial agentsby augmenting antibody responses and enhancing immuno-logical memory; and (3) disposal of immune products andproducts of inflammatory injury by clearance of immunecomplexes from tissues and removal of apoptotic cells.


ACTIVATION OF HAGEMAN FACTOR (XII)

Plasminogen

Plasmin

FibrinolysisComplement

activation

Activation ofkallikrein

Activation ofcoagulation system

CLOTFORMATION

KININGENERATION

FIBRIN DEGRADATIONPRODUCTS

• Negatively charged surfaces (e.g., basement membrane, collagen, elastin, glycosaminoglycans)• Bacterial lipopolysaccharide• Sodium urate crystals• Enzymes (e.g., trypsin, plasmin)

AGENTS ASSOCIATED WITH INJURY

Hageman factor activation and inflammatory mediator production. Hageman factor activation is a keyevent leading to conversion of plasminogen to plasmin, resulting in generation of fibrin split products and ac-

tive complement products. Activation of kallikrein produces kinins, and activation of the coagulation system results in clot forma-

FIGURE 2-5.

The endpoint of complement activation is formation ofthe MAC and cell lysis. The cleavage products generated ateach step of the way catalyze the next step in the cascadeand have additional properties that render them importantinflammatory molecules (Fig. 2-6):

• Anaphylatoxins (C3a, C4a, C5a): These proinflamma-tory molecules mediate smooth-muscle contraction andincrease vascular permeability.

• Opsonins (C3b, iC3b): Bacterial opsonization is theprocess by which a specific molecule (e.g., IgG or C3b)binds to the surface of the bacterium. The process en-hances phagocytosis by enabling receptors on phago-cytic cell membranes (e.g., Fc receptor or C3b receptor)to recognize and bind the opsonized bacterium. Viruses,parasites, and transformed cells also activate complement

by similar mechanisms, an effect that leads to their inac-tivation or death.

• Proinflammatory molecules (MAC, C5a): These chemo-tactic factors also activate leukocytes and tissue cells togenerate oxidants and cytokines and induce degranula-tion of mast cells and basophils.

The complement system is activated by three convergentpathways termed classical, mannose-binding lectin(MBL), and alternative pathways (see Fig. 2-6).

The Classical PathwayActivators of the classical pathway include antigen-antibody(Ag-Ab) complexes, products of bacteria and viruses, pro-teases, urate crystals, apoptotic cells, and polyanions(polynucleotides). The proteins of this pathway are C1through C9, the nomenclature following the historical or-der of discovery. Ag-Ab complexes activate C1, initiating acascade that leads to formation of the MAC, which pro-ceeds as follows (see Fig. 2-6).

The Mannose-Binding PathwayThe mannose- or lectin-binding pathway has some compo-nents in common with the classical pathway. It is initiatedby the binding of microbes bearing terminal mannosegroups to mannose-binding lectin, a member of the fam-ily of calcium-dependent lectins, termed the collectins.This multifunctional acute-phase protein has propertiessimilar to those of immunoglobulin M (IgM) antibody (itbinds to a wide range of oligosaccharide structures), IgG (itinteracts with phagocytic receptors), and C1q. This lastproperty enables it to interact with either C1r-C1s or with aserine protease called MASP (MBL-associated serine pro-tease) to activate complement (see Fig. 2-6).

Alternative PathwayThe alternative pathway is initiated by derivative productsof microorganisms, such as endotoxin (from bacterial cellsurfaces), zymosan (yeast cell walls), polysaccharides ,viruses, tumor cells, and foreign materials. Proteins of thealternative pathway are called “factors,” followed by a letter.Activation of the alternative pathway occurs at the level ofC3 activation to produce small amounts of C3b. which be-come covalently bound to carbohydrates and proteins onmicrobial cell surfaces (see Fig. 2-6).

The Complement System and DiseaseThe importance of an intact and appropriately regulatedcomplement system is exemplified in persons who have ac-quired or congenital deficiencies of specific complementcomponents or regulatory proteins. Such patients have anincreased susceptibility to infectious agents, and in somecases, a propensity for autoimmune diseases associatedwith circulating immune complexes.


AlternativePathway

ClassicalPathway

Mannose-BindingPathway

Bacteria,activation surfaces

Antigen-antibodycomplexes

Mannose on microbesurfaces

bind C3b Plasma C3

C3bBb(C3 convertase)

C3bBb3b(C5 convertase)

C4b,2a(C3 convertase)

C3aANAPHYLATOXIN

C5aCHEMOTAXISANAPHYLATOXIN

C5b-9MAC (Membrane Attack Complex)

C4b,2a,3b(C5 convertase)

bind C1 bind MBL

C4

C1 Activation

C2

C3

C5

C5b

C6,7,8,9

C3b

Factors D and B

C1r/C1s-MBLor

MBL-MASP

CELL LYSIS

C3b, iC3bOPSONIZATION

Complement activation. The alternative, classical,and mannose-binding pathways lead to generation

of the complement cascade of inflammatory mediators and cell lysis bythe membrane attack complex (MAC). MBL, mannose-binding lectin;MBL-MASP, MBL-associated serine protease.

FIGURE 2-6.

Cell-Derived Mediators Of Inflammation

Circulating platelets, basophils, PMNs, endothelial cells,monocyte/macrophages, tissue mast cells, and the injuredtissue itself are all potential cellular sources of vasoactivemediators. In general, these mediators are (1) derived frommetabolism of phospholipids and arachidonic acid (e.g.,prostaglandins, thromboxanes, leukotrienes, lipoxins,platelet-activating factor [PAF]), (2) preformed and storedin cytoplasmic granules (e.g., histamine, serotonin, lysoso-mal hydrolases), or (3) derived from altered production ofnormal regulators of vascular function (e.g., NO•).

Arachidonic Acid and Platelet-Activating Factorare Derived from Membrane Phospholipids

Phospholipids and fatty acid derivatives released fromplasma membranes are metabolized into mediators andhomeostatic regulators by inflammatory cells and injuredtissues. As part of a complex regulatory network, prostanoids,leukotrienes and lipoxin, (derivatives of arachidonic acid)both promote and inhibit inflammation (Table 2-1).

Arachidonic AcidDepending on the specific inflammatory cell and the natureof the stimulus, activated cells generate arachidonic acid byone of two pathways, involving either stimulus-induced ac-tivation of phospholipase A2 (PLA2) or phospholipase C.Once generated, arachidonic acid is further metabolizedthrough two pathways: (1) cyclooxygenation, with subse-quent production of prostaglandins and thromboxanes;and (2) lipoxygenation, to form leukotrienes and lipoxins(Fig. 2-7).

Corticosteroids are widely used to suppress tissue de-struction associated with many inflammatory diseases.These drugs induce synthesis of an inhibitor of PLA2 andblock release of arachidonic acid in inflammatory cells.Although corticosteroids (e.g., prednisone) are widely usedto suppress inflammatory responses, their prolonged ad-ministration can have significant harmful effects, includingincreased risk of infection, damage to connective tissue,and adrenal gland atrophy.

Platelet-Activating Factor (PAF)Another potent inflammatory mediator derived frommembrane phospholipids is PAF, synthesized by virtuallyall activated inflammatory cells, endothelial cells, and in-jured tissue cells. PAF is derived from membrane phos-pholipids by the PLA2 pathway. During inflammatory andallergic responses, PAF stimulates platelets, neutrophils,monocyte/macrophages, endothelial cells, and vascular

smooth muscle cells. PAF induces platelet aggregation anddegranulation at sites of tissue injury and enhances releaseof serotonin, thereby causing changes in vascular perme-ability. The molecule is also an extremely potent vasodila-tor, augmenting permeability of microvasculature at sitesof tissue injury.

Prostanoids, Leukotrienes, and Lipoxins are Biologically Active Metabolites of Arachidonic Acid

ProstanoidsArachidonic acid is further metabolized by cyclooxygenases 1and 2 (COX-1, COX-2) to generate prostanoids (see Fig. 2-7).COX-1 is constitutively expressed by most cells and in-creases upon cell activation. It is a key enzyme in the syn-thesis of prostaglandins, which in turn (1) protect the gas-trointestinal mucosal lining, (2) regulate water/electrolytebalance, (3) stimulate platelet aggregation to maintain nor-mal hemostasis, and (4) maintain resistance to thrombosison vascular endothelial cell surfaces. COX-2 expression isgenerally low or undetectable but takes over as the majorsource of prostanoids as inflammation progresses. BothCOX isoforms generate prostaglandin H (PGH2), which isthen the substrate for production of prostacyclins (PGI2),PGD2, PGE2, PGF2, α and TXA2 (thromboxane). The profileof prostaglandin production (i.e., the quantity and varietyproduced during inflammation) depends in part on thecells present and their activation state (see Table 2-1).


TABLE 2–1Biological Activities of Arachidonic AcidMetabolites

Metabolite Biological ActivityPGE2, PGD2 Induce vasodilation,

bronchodilation, inhibitinflammatory cell function

PGI2 Induces vasodilation,bronchodilation, inhibitsinflammatory cell function

PGF2α Induces vasodilation,bronchoconstriction

TXA2 Induces vasoconstriction,bronchoconstriction, enhancesinflammatory cell functions(especially platelets)

LTB4 Chemotactic for phagocyticcells, stimulates phagocyticcell adherence, enhancesmicrovascular permeability

LTC4, LTD4, LTE4 Induce smooth musclecontraction, constrict pulmonaryairways, increase microvascularpermeability

PG, prostaglandin; TXA2, thromboxane A2; LT, leukotriene.

Inhibition of COX is one mechanism by which nonsteroidal anti-in-flammatory drugs (NSAIDs), including aspirin, indomethacin,and ibuprofen, exert their potent analgesic and anti-inflammatoryeffects. NSAIDS block COX-2–induced formation ofprostaglandins, thereby mitigating pain and inflammation.However, they also inhibit COX-1 and lead to adverse ef-fects on the stomach and kidneys. This complication led todevelopment of COX-2–specific inhibitors (see Fig. 2-7).

LeukotrienesSlow-reacting substance of anaphylaxis has long been recog-nized as a smooth- muscle stimulant and mediator of hyper-sensitivity reactions. It is, in fact, a mixture of leukotrienes,the second major family of derivatives of arachidonic acid(see Fig. 2-7 and Table 2-1). Leukotriene A4 (LTA4) serves asa precursor to several other leukotrienes. LTB4 is a majorproduct of neutrophils as well as certain macrophage popu-lations and has potent chemotactic activity for neutrophils,monocytes, and macrophages. In other cell types, especiallymast cells, basophils and macrophages, LTC4, LTD4, andLTE4 are produced. These three cysteinyl-leukotrienes (1)stimulate smooth-muscle contraction, (2) enhance vascularpermeability, and (3) are responsible for the development ofmany of the clinical symptoms associated with allergic-typereactions, notably asthma. Leukotrienes exert their actionthrough high-affinity specific receptors that may prove to beimportant targets of drug therapy.

LipoxinsLipoxins, the third class of proinflammatory products ofarachidonic acid, are synthesized by platelets and neu-trophils within the vascular lumen in a manner dependent

on cell� cell interactions (see Fig. 2-7). Neutrophil LTA 4

serves as a source for platelet-dependent synthesis of lipox-ins. Monocytes, eosinophils, and airway epithelial cells gen-erate 15S-hydroxyeicosatetraenoic acid (15S-HETE), whichis taken up by neutrophils and converted to lipoxins.

Cytokines are Cell-Derived InflammatoryHormones

Cytokines constitute a group of low-molecular-weight hor-mone-like proteins secreted by cells. Many cytokines are pro-duced at sites of inflammation, including interleukins,growth factors, colony-stimulating factors, interferons, andchemokines (Fig. 2-8). Cytokines produced at sites of tissueinjury regulate inflammatory responses, ranging from initialchanges in vascular permeability to resolution and restora-tion of tissue integrity. These molecules are inflammatoryhormones that exhibit autocrine (affecting themselves),paracrine (affecting nearby cells), and endocrine (affectingcells in other tissues) functions. Through production of cy-tokines, macrophages are pivotal in orchestrating tissue inflamma-tory responses. Lipopolysaccharide (LPS), a molecule derivedfrom the outer cell membrane of gram-negative bacteria, isone of the most potent activators of macrophages, as well asof endothelial cells and leukocytes (Fig. 2-9). LPS activatescells via specific receptors, either directly or after binding aserum LPS-binding protein (LBP). It is a potent stimulus forproduction of TNF-α and interleukins (IL-1, IL-6, IL-8, IL-12, and others). Macrophage-derived cytokines modulateendothelial cell� leukocyte adhesion (TNF- α), leukocyte re-cruitment (IL-8), the acute phase response (IL-6, IL-1), andimmune functions (IL-1, IL-6, IL-12).


CYCLOOXYGENASE PATHWAY LIPOXYGENASE PATHWAY

ARACHIDONIC ACID

COX-1, COX-2 (NSAIDs)

5-LOX

5-LOX

12-LOX

15R HETE 15S-HETE HpETE

HETE

Aspirin PGH2

LXA4LXB4

15-epi LXPGI2PGD2PGE2PGF2α

TXA2

PROSTAGLANDINS THROMBOXANE LIPOXINS LEUKOTRIENES

LTA4

LTC4LTD4LTE4

LTB4

T

Biologically active arachidonic acid metabolites. The cyclo-oxygenase pathway of arachidonic acid me-tabolism generates prostaglandins (PG) and thromboxane (TXA2). The lipoxygenase pathway forms lipoxins

(LX) and leukotrienes (LT); COX, cyclooxygenase; HETE, hydroxyeicosatetraenoic acid; HpETE, 5-hydroperoxyeicosatetraenoicacid; NSAIDs, nonsteroidal anti-inflammatory drugs.

FIGURE 2-7.

InterleukinsIL-1 and TNF-α, produced by macrophages, as well as othercells, are central to the development and amplification ofinflammatory responses. These cytokines activate endothe-lial cells to express adhesion molecules and release cy-tokines, chemokines, and reactive oxygen species (ROS) (seebelow). TNF-α induces priming and aggregation of neu-trophils. IL-1 and TNF-α are also among the mediators offever, catabolism of muscle, shifts in protein synthesis, andhemodynamic effects associated with inflammatory states(see Fig. 2-9). IFN-γ, another potent stimulus formacrophage activation and cytokine production, is pro-duced by a subset of T lymphocytes as part of the immuneresponse (see Chapter 4).

Chemokine Structure and FunctionChemokines direct cell migration (a process termedchemotaxis), The accumulation of inflammatory cells atsites of tissue injury requires their migration from thevascular space into extravascular tissue. Chemokines area large class of cytokines (over 50 known members) thatregulate leukocyte trafficking in inflammation and immunity. For example, chemokines are importantchemotactic factors for PMNs in acute inflammation (seelater).

Chemokines are small molecules that interact with G-protein� coupled receptors on target cells. These secretedproteins are produced by a variety of cell types, either con-stitutively or after induction, and differ widely in biologicalaction. This diversity is based on specific cell types targeted,

specific receptor activation, and differences in intracellularsignaling.

Two functional classes of chemokines have been distin-guished, namely inflammatory chemokines and homingchemokines. Inflammatory chemokines are produced inresponse to bacterial toxins and inflammatory cytokines(especially, IL-1, TNF-α and IFN-γ) by a variety of tissuecells, as well as leukocytes themselves. Homing chemokinesare constitutively expressed and upregulated during diseasestates and direct trafficking and homing of lymphocytesand dendritic cells to lymphoid tissues during an immuneresponse (see Chapter 4).

Chemokines function as immobilized or soluble mole-cules that generate a chemotactic gradient by binding toproteoglycans of the extracellular matrix or to cell surfaces.As a result, high concentrations of chemokines persist atsites of tissue injury. Specific receptors on the surface of themigrating leukocytes bind the matrix-bound chemokinesand associated adhesion molecules, which tend to movecells along the chemotactic gradient to the site of injury.This process of responding to a matrix-bound chemoattrac-tant is termed haptotaxis. As soluble molecules,chemokines control leukocyte motility and localizationwithin extravascular tissues by establishing a chemotacticgradient. The multiplicity and combination of chemokinereceptors on cells allows an extensive variety in biologicalfunction. Neutrophils, monocytes, eosinophils, and ba-sophils share some receptors but express other receptors ex-clusively. Thus, specific chemokine combinations can re-cruit selective cell populations.


Interleukins GrowthFactors

Chemokines Interferons Pro-Inflammatory

Cytokines

IL-1IL-6IL-8

IL-13IL-10

GM-CSFM-CSF

CCCXCXC

CX3C

IFNα

IFNβ

IFNγ

TNFα

• Inflammatory cell activation

• Macrophage• Bactericidal activity

• NK and dendritic cell function

• Leukocyte chemotaxis

• Leukocyte activation

• Antiviral• Leukocyte activation

• Fever• Anorexia• Shock• Cytotoxicity• Cytokine induction• Activation of endothelial cells and tissue cells

Cytokines important in inflammation. GM-CSF, granulocyte macrophage-colony stimulating factor; IL, in-terleukin; NK, natural killer; IFN, interferon; TFN, tumor necrosis factor.

FIGURE 2-8.

Reactive Oxygen Species are Signal-Transducing,Bactericidal, and Cytotoxic Molecules

ROS are chemically reactive molecules derived from molec-ular oxygen. Normally, they are rapidly inactivated, butwhen generated inappropriately, they can be cytotoxic (seeChapter 1). ROS create oxidative stress by activating signal-transduction pathways and combining with proteins,lipids, and DNA. Leukocyte-derived ROS, released withinphagosomes, are bactericidal. ROS important in inflamma-tion include superoxide (O2•, O2

–), NO•, hydrogen peroxide(H2O2), and hydroxyl radical (•OH) (Fig. 2-10) (see belowand Chapter 1).

Cells Of Inflammation

Leukocytes are the major cellular components of the in-flammatory response and include neutrophils, T and Blymphocytes, monocytes, macrophages, eosinophils, mastcells, and basophils. Specific functions are associated witheach of these cell types, but such functions overlap and varyas inflammation progresses. In addition, local tissue cellsinteract with one another and with inflammatory cells, in acontinuous response to injury and infection.

Neutrophils are the Major Cellular Participant inAcute Inflammation

The PMN is the major cellular participant in acute inflam-mation. It has granulated cytoplasm and a nucleus withtwo to four lobes. PMNs are stored in the bone marrow, cir-culate in the blood, and rapidly accumulate at sites of in-jury or infection (Fig. 2-11A). They are activated in responseto phagocytic stimuli, cytokines, chemotactic mediators orantigen�–antibody complexes, which bind specific receptorson their surface membrane. In tissues, PMNs phagocytoseinvading microbes and dead tissue (see below). Once theyare recruited into tissue, they do not re-enter the circula-tion.

Endothelial Cells Line Blood Vessels

Endothelial cells comprise a monolayer of cells lining bloodvessels and help to separate intra- and extravascular spaces.They produce agents that maintain blood vessel patency andalso vasodilators and vasoconstrictors that regulate vasculartone. Injury to a vessel wall interrupts the endothelial barrierand exposes a local procoagulant signal (Fig. 2-11B).

Endothelial cells are gatekeepers in inflammatory cell re-cruitment: they can promote or inhibit tissue perfusion andthe influx of inflammatory cells. Inflammatory agents, suchas bradykinin and histamine, endotoxin and cytokines, in-


Gram negativebacteria

LPS IFNγMacrophage

T cells

TNFα, IL-1

ENDOTHELIALCELLS

Adhesion molecules

Cytokines

Eicosanoids

Chemokines

Oxygen radicals

Aggregation

Priming

Fever

Anorexia

Hypotension

Increased heart rate

Corticosteroid andACTH release

NEUTROPHILS

ACUTE PHASERESPONSE

Central role of interleukin (IL)-1 and tumor necrosisfactor (TNF)-a in inflammation. Lipopolysaccharide

(LPS) and IFN-γ activate macrophages to release inflammatory cytokines,principally IL-1 and TNF-α, responsible for directing local and systemic in-flammatory responses. ACTH, adrenocorticotropic hormone.

FIGURE 2-9.

Neutrophil

Respiratoryburst

O2

O2-SOD

Activateneutrophilgranules

myeloper-oxidase

Fe2+

Phagocytosedbacteria

Degradativeenzymes

NADPHoxidase

H2O2

HOCl (OCl-) OH

Neutrophilgranules

Generation of reactive oxygen species in neu-trophils as a result of phagocytosis of bacteria. Fe2+,

ferrous iron; H2O2, hydrogen peroxide; HOCl, hypochlorous acid;NADPH, nicotinamide adenine dinucleotide phosphate; OCl–, hypochlo-rite radical; •OH, hydroxyl radical; SOD,superoxide dismutase.

FIGURE 2-10.

duce endothelial cells to reveal adhesion molecules that (1)anchor and activate leukocytes, (2) present major histocom-patibility complex (MHC) class I and II molecules, and (3)generate cytokines and important vasoactive and inflam-matory mediators.

Monocyte/Macrophages are Important in Acute and Chronic Inflammation

Circulating monocytes (Fig. 2-11C) have a single lobed orkidney-shaped nucleus. They are derived from the bonemarrow and can exit the circulation to migrate into tissueand become resident macrophages. In response to inflam-matory mediators, they accumulate at sites of acute in-flammation where they ingest and process microbes.

Monocyte/macrophages produce potent vasoactive medi-ators, including prostaglandins and leukotrienes, PAF,and inflammatory cytokines. These cells are especiallyimportant for maintaining chronic inflammation.

Mast Cells and Basophils are Important in Allergic Hypersensitivity Reactions

Mast cell products play an important role in regulating vas-cular permeability and bronchial smooth muscle tone, espe-cially in allergic hypersensitivity reactions (see Chapter 4).Granulated mast cells and basophils (Fig. 2-11D) containcell surface receptors for IgE. Mast cells are found in theconnective tissues and are especially prevalent along lungand gastrointestinal mucosal surfaces, the dermis, and the


Granules(lysosomes)

Secondary granule

Primary granule

CHARACTERISTICS AND FUNCTIONS

POLYMORPHONUCLEAR LEUKOCYTES

• Central to acute inflammation• Phagocytosis of microorganisms and tissue debris• Mediates tissue injury

PRIMARY INFLAMMATORY MEDIATORS• Reactive oxygen metabolites• Lysosomal granule contents Primary granules Myeloperoxidase Lysozyme Defensins Bactericidal/permeability increasing protein Elastase Cathepsins Protease 3 Glucuronidase Mannosidase Phospholipase A2

Secondary granules Lysozyme Lactoferrin Collagenase Complement activator Phospholipase A2 CD11b/CD18 CD11c/CD18 Laminin Tertiary granules Gelatinase Plasminogen activator Cathepsins Glucuronidase Mannosidase

Capillary lumen

CHARACTERISTICS AND FUNCTIONS• Maintains vascular integrity• Regulates platelet aggregation• Regulates vascular contraction and relaxation• Mediates leukocyte recruitment in inflammation

PRIMARY INFLAMMATORY MEDIATORS• von Willebrand factor• Nitric oxide• Endothelins• Prostanoids

ENDOTHELIAL CELLS

A

B

Cells of inflammation: morphology and function. A. Neutrophil. B. Endothelial cell. Cells of inflammation:morphology and function.

FIGURE 2-11.

microvasculature. Basophils circulate in small numbers andcan migrate into tissue.

When IgE-sensitized mast cells or basophils are stimu-lated by antigens; physical agonists such as cold andtrauma, or cationic proteins, inflammatory mediators inthe dense cytoplasmic granules are secreted into extracellu-lar tissues. These bodies contain acid mucopolysaccharides(including heparin), serine proteases, chemotactic media-tors for neutrophils and eosinophils, and histamine, a pri-mary mediator of early increased vascular permeability.Histamine binds specific H1 receptors in the vascular wall,thereby inducing endothelial cell contraction, gap forma-tion, and edema, an effect that can be inhibited pharmaco-logically by H1-receptor antagonists. Stimulation of mastcells and basophils also leads to the release of products ofarachidonic acid metabolism and cytokines, such as TNF-αand IL-4.

Eosinophils are Important in Defense Against Parasites

Eosinophils circulate in the blood and are recruited totissue in a manner similar to that of PMNs. They are

characteristic of IgE-mediated reactions, such as hyper-sensitivity, allergic, and asthmatic responses (Fig. 2-12A).Eosinophils contain leukotrienes and PAF, as well as acidphosphatase and peroxidase. They express IgA receptorsand exhibit large granules that contain eosinophil majorbasic protein, both of which are involved in defenseagainst parasites.

Platelets Play a Role in Normal Hemostasis

Platelets play a primary role in normal hemostasis and ininitiating and regulating clot formation (see Chapter 20).They are sources of inflammatory mediators, including po-tent vasoactive substances and growth factors that modu-late mesenchymal cell proliferation (Fig. 2-12B). Theplatelet is small (2 mm in diameter), lacks a nucleus, andcontains three distinct kinds of inclusions:

• dense granules, rich in serotonin, histamine, calciumand adenosine diphosphate

• α granules, containing fibrinogen, coagulation proteins,platelet-derived growth factor, and other peptides andproteins

• lysosomes, which sequester acid hydrolases


Lysosome

Phagocyticvacuole

CHARACTERISTICS AND FUNCTIONS• Regulates inflammatory response• Regulates coagulation/fibrinolytic pathway• Regulates immune response (see Chapt. 4)

PRIMARY INFLAMMATORY MEDIATORS• cytokines - IL-1 - TNF-α - IL-6 - Chemokines (e.g. IL-8, MCP-1)• lysosomal enzymes - acid hydrolases - serine proteases - metalloproteases (e.g. collagenase)• cationic proteins• prostaglandins/leukotrienes• plasminogen activator• procoagulant activity• oxygen metabolite formation

MONOCYTE/MACROPHAGE

C

Continued. CC.. Monocyte/macrophage. DD.. Mast cell. IL, interleukin; MCP-1, monocyte chemoattractant pro-tein-1; TNF-, tumor necrosis factor-.

FIGURE 2-11.

MAST CELL (BASOPHILS)

CHARACTERISTICS AND FUNCTIONS• Binds IgE molecules• Contains electron-dense granules

PRIMARY INFLAMMATORY MEDIATORS• Histamine• Leukotrienes (LTC, LTD, LTE)• Platelet activating factor• Eosinophil chemotactic factors• Cytokines (e.g., TNF-α IL-4)

D

Platelets adhere, aggregate, and degranulate when theycontact fibrillar collagen (e.g., after vascular injury that ex-poses extracellular matrix [ECM] proteins) or thrombin (af-ter activation of the coagulation system).

Leukocyte Recruitment In AcuteInflammation

One of the essential features of acute inflammation is accu-mulation of leukocytes, particularly PMNs, in affected tis-sues. Leukocytes adhere to vascular endothelium, wherethey become activated. They then flatten and migrate fromthe vasculature through the endothelial cell layer into sur-rounding tissue. In the extravascular tissue, PMNs ingestforeign material, microbes, and dead tissue.

Leukocyte Adhesion to Endothelium Results from Interaction of Complementary Adhesion Molecules

Leukocyte recruitment to the postcapillary venules beginswith interaction of leukocytes with endothelial cell se-lectins, which are redistributed to endothelial cell surfacesduring activation. This interaction, called tethering, slowsleukocytes in the blood flow (Fig. 2-13). Leukocytes thenmove along the vascular endothelial cell surface with asaltatory movement, termed rolling. PMNs become acti-vated by proximity to the endothelium and by inflamma-tory mediators, and adhere strongly to intercellular adhe-sion molecules on the endothelium (leukocyte arrest). Asendothelial cells separate, leukocytes transmigrate throughthe vessel wall and, under the influence of chemotactic


EOSINOPHILS

Granules

CHARACTERISTICS AND FUNCTIONS• Associated with: - Allergic reactions - Parasite-associated inflammatory reactions - Chronic inflammation• Modulates mast cell-mediated reactions

PRIMARY INFLAMMATORY MEDIATORS• Reactive oxygen metabolites• Lysosomal granule enzymes (primary crystalloid granules) - Major basic protein - Eosinophil cationic protein - Eosinophil peroxidase - Acid phosphatase - β-glucuronidase - Arylsulfatase B - Histaminase• Phospholipase D• Prostaglandins of E series• Cytokines

A

PLATELETS

Vacuoles

Granules

PLATELETS

CHARACTERISTICS AND FUNCTIONS• Thrombosis; promotes clot formation• Regulates permeability• Regulates proliferative response of mesenchymal cellsPRIMARY INFLAMMATORY MEDIATORS• Dense granules -Serotonin -Ca2+

-ADP• α-granules -Cationic proteins -Fibrinogen and coagulation proteins -Platelet-derived growth factor (PDGF)• Lysosomes -Acid hydrolases•Thromboxane A2

Microtubules

More cells of inflammation: morphology and function. A. Eosinophil. B. Platelet. ADP, adenosine diphos-phate.

FIGURE 2-12.

B

factors, leukocytes migrate through extravascular tissue tothe site of injury.

The events involved in leukocyte recruitment are regu-lated as follows: (1) Inflammatory mediators stimulate resi-dent tissue cells, including vascular endothelial cells; (2)Adhesion molecules are expressed on vascular endothelialcell surfaces and bind to reciprocal molecules on the surfacesof circulating leukocytes; and (3) Chemotactic factors attractleukocytes along a chemical gradient to the site of injury.

Adhesion MoleculesFour molecular families of adhesion molecules are involvedin leukocyte recruitment: selectins, addressins, integrins,and immunoglobulins.

SelectinsThe selectin family (part of the C-type, calcium-dependentlectin group) includes P-selectin, E-selectin, and L-selectin,expressed on the surface of platelets, endothelial cells, andleukocytes. Selectins share a similar molecular structure,which includes a chain of transmembrane glycoproteinswith an extracellular carbohydrate-binding domain specificfor sialylated oligosaccharides. The last is the sialyl-Lewis X

moiety on addressins, the binding of which allows rapid at-tachment and rolling of cells.

P-selectin (CD62P, GMP-140, PADGEM) is preformedand stored within Weibel-Palade bodies of endothelial cellsand α-granules of platelets. On stimulation with histamine,thrombin, or specific inflammatory cytokines, P-selectin israpidly transported to the cell surface, where it binds tosialyl-Lewis X on leukocyte surfaces. Preformed P-selectincan be delivered quickly to the cell surface, allowing rapidadhesive interaction between endothelial cells and leuko-cytes.

E-selectin (CD62E, ELAM-1) is not normally expressedon endothelial cell surfaces but is induced by inflammatorymediators, such as cytokines or bacterial LPS. E-selectinmediates adhesion of neutrophils, monocytes, and certainlymphocytes via binding to molecules that contain Lewis X.

L-selectin (CD62L, LAM-1, Leu-8) is expressed on manytypes of leukocytes. It was originally defined as the “homingreceptor” for lymphocytes. It binds lymphocytes to high en-dothelial venules in lymphoid tissue, thereby regulating theirtrafficking through this tissue. L-selectin binds glycan-bear-ing cell adhesion molecule-1 (GlyCAM-1), mucosal addressincell adhesion molecule-1 (MadCAM-1), and CD34.


Blood FlowPMN

ENDOTHELIAL CELLS

ENDOTHELIALACTIVATION

TETHERING ROLLING FIRMADHESION

TRANSMIGRATION

EC: P-selectin

PMN: Sialyl-Lewis X

EC: E-selectin

PMN: Sialyl-Lewis X

EC: ICAM

PMN: β1, β2 integrins

EC: PECAM-1

PMN: elastase

Inflammatory mediators(Histamine, thrombin,

PAF, IL-1, TNF)

Inflammatorymediators

(Chemokines)

Neutrophil adhesion and extravasation. Inflammatory mediators activate endothelial cells to increase ex-pression of adhesion molecules. Sialyl Lewis X on neutrophil PSGL-1 and ESL-1 binds to P- and E-selectins

to facilitate tethering and rolling of neutrophils. Increased integrins on activated neutrophils bind to ICAM-1 on endothelial cells toform a firm attachment. Endothelial cell attachments to one another are released, and neutrophils then pass between separatedcells to enter the tissue. EC, endothelial cell; ICAM, intercellular adhesion molecule; IL, interleukin; PAF, platelet activating factor;PMN, polymorphonuclear neutrophil; TNF, tumor necrosis factor.

FIGURE 2-13.

AddressinsVascular addressins are mucin-like glycoproteins includingGlyCAM-1, P-selectin glycoprotein-1 (PSGL-1), E-selectinligand 1 (ESL-1), and CD34. They possess sialyl-Lewis X,which binds the lectin domain of selectins. Addressins areexpressed at leukocyte and endothelial surfaces. They regu-late localization of subpopulations of leukocytes and are in-volved in lymphocyte activation.

IntegrinsChemokines, lipid mediators, and proinflammatory mole-cules activate cells to express the integrin family of adhesionmolecules (see Chapter 3). Integrins have transmembrane αand β chains arranged as heterodimers. They participate incell–cell interactions and cell–ECM binding. Very late ac-tivation (VLA) molecules include VLA-4 (α4β1) on leuko-cytes and lymphocytes that bind VCAM-1 (an im-munoglobulin-domain-bearing molecule) on endothelialcells. The β2 integrins (CD18) form molecules by associa-tion with α integrin chains: α1β2 (also called CD11a/CD18or LFA-1) and αmβ2 (also termed CR3, CD11b/CD18 orMac-1) bind to both ICAM-1 and ICAM-2 (also members ofthe Ig domain-bearing family, see below). Leukocyte inte-grins exist in a low-affinity state, but are converted to a high-affinity state when these cells are activated.

Immunoglobulin SuperfamilyAdhesion molecules of the immunoglobulin (Ig) superfam-ily include ICAM-1, ICAM-2, and VCAM-1, all of which in-teract with integrins on leukocytes to mediate recruitment.They are expressed at the surfaces of cytokine-stimulated en-dothelial cells and some leukocytes, as well as certain ep-ithelial cells, such as pulmonary alveolar cells.

Recruitment of LeukocytesTethering, rolling, and firm adhesion are prerequisites forrecruitment of leukocytes from the circulation into tissues.For a rolling cell to adhere, there must first be a selectin-de-pendent reduction in rolling velocity. The early increase inrolling depends on P-selectin, whereas cytokine-induced E-selectin initiates early adhesion. Integrin family membersfunction cooperatively with selectins to facilitate rollingand subsequent firm adhesion of leukocytes. Leukocyte in-tegrin binding to the Ig superfamily of ligands expressed onvascular endothelium further retards leukocytes, increasingthe length of exposure of each leukocyte to endothelium. Atthe same time, engagement of adhesion molecules activatesintracellular signal transduction. As a result, leukocytes andvascular endothelial cells are further activated, with subse-quent upregulation of L-selectin and integrin binding. Thenet result is firm adhesion (see Fig. 2-13).

Chemotactic Molecules Direct Neutrophils to Sites of Injury

Leukocytes must be accurately positioned at sites of inflam-matory injury to carry out their biological functions. Forspecific subsets of leukocytes to arrive in a timely fashion,they must receive specific directions. Leukocytes are guidedthrough vascular and extravascular spaces by a complex interac-tion of attractants, repellants, and adhesion molecules.Chemotaxis is the dynamic and energy-dependent processof directed cell migration. Blood leukocytes are recruited bychemoattractants released by endothelial cells. They thenmigrate from the endothelium toward the target tissue,down a gradient of one chemoattractant in response to asecond more distal chemoattractant gradient. During mi-gration, the cell extends a pseudopod toward increasingchemokine concentration. At the leading front of thepseudopod, marked changes in levels of intracellular cal-cium are associated with assembly and contraction of cy-toskeleton proteins. This process draws the remaining tailof the cell along the chemical gradient. Neutrophils mustintegrate the various signals to arrive at the appropriate siteat the correct time to perform their assigned tasks. Themost important chemotactic factors for PMNs are:

• C5a, derived from complement• Bacterial and mitochondrial products, particularly

low-molecular-weight N-formylated peptides (such as N-formyl-methionyl-leucyl-phenylalanine)

• Products of arachidonic acid metabolism, especiallyLTB4

• Chemokines

Chemotactic factors for other cell types, including lym-phocytes, basophils, and eosinophils are also produced atsites of tissue injury and may be secreted by activated en-dothelial cells, tissue parenchymal cells, or other inflam-matory cells. They include PAF, transforming growth fac-tor-β (TGF-β), neutrophilic cationic proteins, andlymphokines. The cocktail of chemokines presented within a tis-sue largely determines the type of leukocyte attracted to the site.Cells arriving at their destination must then be able to stopin the target tissue. Contact guidance, regulated adhesion,or inhibitory signals may determine the final arrest of spe-cific cells in particular tissue locations.

Leukocytes Traverse the Endothelial Cell Barrier to Gain Access to the Tissue

Leukocytes adherent to the vascular endothelium emigrateby paracellular diapedesis, (i.e., passing between adjacentendothelial cells). Responding to chemokine gradients, neu-trophils extend pseudopods and insinuate themselves be-tween the cells and out of the vascular space. Vascular


endothelial cells are connected by tight junctions and ad-herens junctions. CD31 (platelet endothelial cell adhesionmolecule) is expressed on endothelial cell surfaces and bindsto itself to keep cells together. These junctions separate un-der the influence of inflammatory mediators, intracellularsignals generated by adhesion molecule engagement, andsignals from the adherent neutrophils. Neutrophils mobi-lize elastase to their pseudopod membranes, inducing en-dothelial cell retraction and separation at the advancingedge of the neutrophil. Neutrophils also induce increases inintracellular calcium in endothelial cells, to which the en-dothelial cells respond by pulling apart.

Neutrophils also migrate through endothelial cells bytranscellular diapedesis. Instead of inducing endothelialcell retraction, PMNs may squeeze through small circularpores in endothelial cell cytoplasm. In tissues that containfenestrated microvessels, such as gastrointestinal mucosaand secretory glands, PMNs may traverse thin regions ofendothelium, called fenestrae, without damaging endothe-lial cells. In nonfenestrated microvessels, PMNs may crossthe endothelium using endothelial cell caveolae or pinocy-totic vesicles, which form small, membrane-bound passage-ways across the cell.

Leukocyte Functions In AcuteInflammation

Leukocytes Phagocytose Microorganisms and Tissue Debris

Many inflammatory cells (including monocytes, tissuemacrophages, dendritic cells, and neutrophils) recognize,internalize and digest foreign material, microorganisms,or cellular debris by a process termed phagocytosis. Thisis now defined as ingestion by eukaryotic cells of large(usually > 0.5 µm) insoluble particles and microorganisms.The effector cells are phagocytes. The complex process in-volves a sequence of transmembrane and intracellular sig-naling events.

1. Recognition: Phagocytosis is initiated by recognition ofparticles by specific receptors on the surface of phago-cytic cells (Fig. 2-14). Phagocytosis of most biologicalagents is enhanced by, if not dependent on, their coating(opsonization) with plasma components (opsonins),particularly immunoglobulins or C3b. Phagocytic cellspossess specific opsonic receptors, including those forimmunoglobulin Fcγ and complement components.Many pathogens, however, have evolved mechanisms toevade phagocytosis by leukocytes. Polysaccharide cap-sules, protein A, protein M, or peptidoglycans aroundbacteria can prevent complement deposition or antigenrecognition and receptor binding.

2. Signaling: Clumping of opsonins on bacterial surfacescauses Fcγ receptors on phagocytes to cluster. Subsequentphosphorylation of immunoreceptor tyrosine-based acti-vation motifs, located in the cytosolic domain or γ sub-unit of the receptor, triggers intracellular signalingevents. Tyrosine kinases that associate with the Fcγ re-ceptor are required for signaling during phagocytosis.

3. Internalization: In the case of phagocytosis initiated viathe Fcγ receptor or the CR3 (CD11b/CD18 receptor),actin assembly occurs directly under the phagocytosedtarget. Polymerized actin filaments push the plasmamembrane forward. The plasma membrane remodels toincrease surface area and to form pseudopods surround-ing the foreign material. The resulting phagocytic cupengulfs the foreign agent. The membrane then “zippers”around the opsonized particle to enclose it in a cytoplas-mic vacuole called a phagosome (see Fig. 2-14).

4. Digestion: The phagosome that contains the foreignmaterial fuses with cytoplasmic lysosomes to form aphagolysosome, into which lysosomal enzymes are re-leased. The acid pH within the phagolysosome activatesthese hydrolytic enzymes, which then degrade thephagocytosed material. Some microorganisms haveevolved mechanisms for evading killing by neutrophilsby preventing lysosomal degranulation or inhibitingneutrophil enzymes.

Neutrophil Enzymes are Required for Antimicrobial Defense and Debridement

Although PMNs are critical for degrading microbes and celldebris, they also contribute to tissue injury. The release ofPMN granules at sites of injury is a double-edged sword.On the one hand, debridement of damaged tissue by prote-olytic breakdown is beneficial. On the other hand, degrada-tive enzymes can damage endothelial and epithelial cells, aswell degrade connective tissue.

Neutrophil GranulesThe armamentarium of enzymes required for degradationof microbes and tissue is generated and contained withinPMN cytoplasmic granules. Primary, secondary, and tertiarygranules in neutrophils are differentiated morphologicallyand biochemically: each granule has a unique spectrum ofenzymes (see Fig. 2-11A).

Inflammatory Cells Have Oxidative and Nonoxidative Bactericidal Activity

The bactericidal activity of PMNs and macrophages is mediated in part by production of ROS and in part byoxygen-independent mechanisms.


Bacterial Killing by Oxygen SpeciesPhagocytosis is accompanied by metabolic reactions in in-flammatory cells that lead to production of several oxygenmetabolites (see Chapter 1). These products are more reac-tive than oxygen itself and contribute to the killing of in-gested bacteria (see Fig. 2-14).

• Superoxide Anion (O2–): Phagocytosis activates a nicoti-

namide adenine dinucleotide phosphate (NADPH) oxidase in PMN cell membranes. NADPH oxidase is amulticomponent electron transport complex that re-duces molecular oxygen to O2

–. Activation of this enzymeis enhanced by prior exposure of cells to a chemotacticstimulus or LPS. NADPH oxidase activation increasesoxygen consumption and stimulates the hexosemonophosphate shunt. Together, these cell responses arereferred to as the respiratory burst.

• H2O2: O2– is rapidly converted to H2O2 by superoxide

dismutase at the cell surface and in phagolysosomes.H2O2 is stable and serves as a substrate for generatingadditional reactive oxidants.

• Hypochlorous Acid (HOCl): Myeloperoxidase (MPO),a neutrophil product with a strong cationic charge, is se-creted from granules during exocytosis. In the presenceof a halide, usually chlorine, MPO catalyzes conversionof H2O2 to HOCl. This powerful oxidant is a major bac-tericidal agent produced by phagocytic cells. HOCl alsoparticipates in activating neutrophil-derived collagenaseand gelatinase, both of which are secreted as latent en-zymes. At the same time, HOCl inactivates α1-antitrypsin.

• (•OH): Reduction of H2O2 occurs via the Haber-Weissreaction to form the highly reactive •OH. This reactionoccurs slowly at physiological pH, but in the presence offerrous iron (Fe2+), the Fenton reaction rapidly convertsH2O2 to •OH. Further reduction of •OH leads to for-mation of H2O (see Chapter 1).

• NO•: Phagocytic cells and vascular endothelial cells pro-duce NO• and its derivatives, which have diverse effects,both physiological and nonphysiological. NO• and otheroxygen-radical species interact with one another to bal-ance their cytotoxic and cytoprotective effects. NO• canreact with oxygen radicals to form toxic molecules suchas peroxynitrite and S-nitrosothiols. It can also scavengeO2

–, thereby reducing the amount of toxic radicals.

Monocytes, macrophages, and eosinophils also produceoxygen radicals, depending on their state of activation andthe stimulus to which they are exposed. Production of ROSby these cells contributes to their bactericidal and fungicidalactivity as well as their ability to kill certain parasites. Theimportance of oxygen-dependent mechanisms in bacterialkilling is exemplified in chronic granulomatous diseaseof childhood. In this hereditary deficiency of NADPH oxi-dase, failure to produce O2

– and H2O2 during phagocytosismakes these persons susceptible to recurrent infections, es-pecially with gram-positive cocci. Patients with a related ge-netic deficiency in MPO cannot produce HOCl and showincreased susceptibility to infections by the fungalpathogen Candida (Table 2-2).

Nonoxidative Bacterial KillingPhagocytes, particularly PMNs and monocytes/macrophages,have substantial antimicrobial activity, which is oxygen inde-pendent. This activity mainly involves preformed bactericidalproteins in cytoplasmic granules. These include lysosomalacid hydrolases and specialized noncatalytic proteins uniqueto inflammatory cells.

• Lysosomal hydrolases: Neutrophil primary and second-ary granules and lysosomes of mononuclear phagocytes


Fc

Fc receptor

C3b

C3b receptor

PMN

Bacterium

PHAGOSOME FORMATION

Phagolysosome

• Degranulation and NADPH oxidase activation• Bacterial killing and digestion

Primarygranule

Secondary granule

O2-O2

NADPHoxidase

Lysozyme,lactoferrin,PLA2

•OHFe2+

H2O2

H2O2

HOCl

MPO

Cationicproteins

Mechanisms of neutrophil bacterial phagocytosisand cell killing. Opsonins such as C3b coat the sur-face of microbes allowing recognition by the neu-

trophil C3b receptor. Receptor clustering triggers intracellular signallingand actin assembly within the neutrophil. Pseudopods form around themicrobe to enclose it within a phagosome. Lysosomal granules fuse withthe phagosome to form a phagolysosome into which the lysosomal en-zymes and oxygen radicals are released to kill and degrade the microbe.Fe2+, ferrous iron; HOCl, hypochlorous acid; MPO, myeloperoxidase;PLA2, phospholipase A2; PMN, polymorphonuclear neutrophil.

FIGURE 2-14.

contain hydrolases, including sulfatases, phosphatases,and other enzymes capable of digesting polysaccharidesand DNA.

• Bactericidal/permeability-increasing protein: Thiscationic protein in PMN primary granules can kill manygram-negative bacteria but is not toxic to gram-positivebacteria or to eukaryotic cells.

• Defensins: Primary granules of PMNs and lysosomes ofsome mononuclear phagocytes contain this family ofcationic proteins, which kill an extensive variety of gram-positive and gram-negative bacteria, fungi, and some en-veloped viruses.

• Lactoferrin: Lactoferrin is an iron-binding glycoproteinin the secondary granules of neutrophils and in most bodysecretory fluids. Its iron-chelating capacity allows it tocompete with bacteria for iron. It may also facilitate oxida-tive killing of bacteria by enhancing •OH formation.

• Lysozyme: This bactericidal enzyme is found in manytissues and body fluids, in primary and secondary gran-ules of neutrophils, and in lysosomes of mononuclearphagocytes. Peptidoglycans of gram-positive bacterialcell walls are exquisitely sensitive to degradation bylysozyme; gram-negative bacteria are usually resistantto it.

• Bactericidal Proteins of Eosinophils: Eosinophils con-tain several granule-bound cationic proteins, the most im-portant of which are major basic protein and eosinophiliccationic protein. Major basic protein accounts for abouthalf of the total protein of the eosinophil granule. Bothproteins are ineffective against bacteria but are potent cy-totoxic agents for many parasites.

Defects in Leukocyte FunctionThe importance of protection afforded by acute inflamma-tory cells is emphasized by the frequency and severity of in-fections when PMNs are greatly decreased or defective. Themost common such deficit is iatrogenic neutropenia resulting fromcancer chemotherapy. Functional impairment of phagocytesmay occur at any step in the sequence: adherence, emigra-tion, chemotaxis, or phagocytosis. These disorders may beacquired or congenital. Acquired diseases, such as leukemia,diabetes mellitus, malnutrition, viral infections, and sepsisare often accompanied by defects in inflammatory cell func-tion. Table 2-2 shows representative examples of congenitaldiseases linked to defective phagocytic function.

Outcomes Of Acute Inflammation

As a result of regulatory components and the short lifespan of neutrophils, acute inflammatory reactions are usu-ally self-limited and are followed by restoration of normaltissue architecture and physiological function (resolu-tion). Resolution involves removal of dead cells, clearanceof acute response cells, and re-establishment of the stroma.However, inflammatory responses can lead to other out-comes:

• Scar: If a tissue is irreversibly injured, the normal archi-tecture is often replaced by a scar, despite elimination ofthe initial pathological insult (see Chapter 3).

• Abscess: If the area of acute inflammation is walled offby inflammatory cells and fibrosis, PMN products de-stroy the tissue, forming an abscess.

• Lymphadenitis: Localized acute inflammation andchronic inflammation may cause secondary inflamma-tion of lymphatic channels (lymphangitis) and lymphnodes (lymphadenitis). The inflamed lymphatic chan-nels in the skin appear as red streaks, and the lymphnodes are enlarged and painful. Microscopically, thelymph nodes show hyperplasia of lymphoid follicles andproliferation of mononuclear phagocytes in the sinuses(sinus histiocytosis).

• Persistent inflammation: Failure to eliminate a patho-logical insult or inability to trigger resolution results ina persistent inflammatory reaction. This may be evidentas a prolonged acute response, with continued influx ofneutrophils and tissue destruction, or more commonlyas chronic inflammation.

Chronic Inflammation

When acute inflammation does not resolve or becomes dis-ordered, chronic inflammation occurs. Inflammatory cellspersist, stroma responds by becoming hyperplastic, and


TABLE 2–2Congenital Diseases of Defective PhagocyticCell Function Characterized by RecurrentBacterial Infections

Disease DefectLAD LAD-1 defective β2-integrin

expression or function(CD11/CD18) LAD-2 (defective fucosylation,selectin binding)

Hyper-IgE-recurrent infection, Poor chemotaxis(Job) syndrome

Chediak-Higashi syndrome Defective lysosomal granules,poor chemotaxis

Neutrophil-specific Absent neutrophil granulesgranule deficiency

Chronic granulomatous Deficient NADPH oxidase,disease with absent H2O2 production

Myeloperoxidase deficiency Deficient HOCl production

LAD, leukocyte adhesion deficiency; NADPH, nicotinamide adenine dinucleotidephosphate; H2O2, hydrogen peroxide; HOCl, hypochlorous acid; Ig, immunoglobulin.

tissue destruction and scarring lead to organ dysfunction.This process may be localized but more commonly pro-gresses to disabling diseases such as chronic lung disease,rheumatoid arthritis, asthma, ulcerative colitis, granuloma-tous diseases, autoimmune diseases, and chronic dermati-tis. Acute and chronic inflammation are ends of a dynamiccontinuum with overlapping morphological features: (1)Inflammation with continued recruitment of chronic inflammatory cells is followed by (2) tissue injury due toprolongation of the inflammatory response, and (3) an often-disordered attempt to restore tissue integrity. Theevents leading to an amplified inflammatory response resemble those of acute inflammation in a number of aspects:

• Specific triggers, microbial products or injury, initiatethe response.

• Chemical mediators direct recruitment, activation, andinteraction of inflammatory cells. Activation of coagula-tion and complement cascades generate small peptidesthat function to prolong the inflammatory response.Cytokines, specifically IL-6 and RANTES, regulate aswitch in chemokines, such that mononuclear cells aredirected to the site. Other cytokines (e.g., IFN-γ) thenpromote macrophage proliferation and activation.

• Inflammatory cells are recruited from the blood.Interactions between lymphocytes, macrophages, den-dritic cells, and fibroblasts generate antigen-specific re-sponses.

• Stromal cell activation and extracellular matrix re-modeling occur, both of which affect the cellular im-mune response. Varying degrees of fibrosis may result,depending on the extent of tissue injury and persistenceof the pathological stimulus and inflammatory response.

Chronic inflammation is not synonymous with chronicinfection, but if the inflammatory response to infectiousagents, including bacteria, viruses and notably parasites,cannot eliminate the organism, infection may persist.Chronic inflammation may also be associated with a varietyof noninfectious disease states including:

• Trauma: Extensive tissue damage releases mediators ca-pable of inducing an extended inflammatory response.

• Cancer: Chronic inflammatory cells, especiallymacrophages and T lymphocytes, may be the morpho-logical expression of an immune response to malignantcells. Chemotherapy may suppress normal inflammatoryresponses, increasing susceptibility to infection.

• Immune factors: Many autoimmune diseases includingrheumatoid arthritis, chronic thyroiditis, and primarybiliary cirrhosis are characterized by chronic inflamma-tory responses in affected tissues. This may be associatedwith activation of antibody-dependent and cell-mediated

immune mechanisms (see Chapter 4). Such autoimmuneresponses may account for injury in affected organs.

Cells from Both the Circulation and AffectedTissue Play a Role in Chronic Inflammation

Monocyte/macrophages, lymphocytes, and plasma cells(see Chapter 4) and cells discussed previously under AcuteInflammation recruited from circulation as well as cellsfrom the affected tissue including fibroblasts and vascularendothelial cells (see Chapter 3) play an active role inchronic inflammation.

Monocyte/MacrophagesActivated macrophages and their cytokines are central toinitiating inflammation and prolonging responses that leadto chronic inflammation. (see Fig. 2-11C). Macrophages pro-duce inflammatory and immunological mediators and reg-ulate reactions leading to chronic inflammation. They alsoregulate lymphocyte responses to antigens and secreteother mediators that modulate the proliferation and activ-ities of fibroblast and endothelial cells.

The mononuclear phagocyte system includes bloodmonocytes, different types of tissue histiocytes andmacrophages, particularly Kupffer cells of the liver. Underthe influence of chemotactic stimuli, IFN-γ and bacterialendotoxins, resident tissue macrophages are activated andproliferate, while circulating monocytes are recruited anddifferentiate into tissue macrophages (Fig. 2-15).


Activated T lymphocytesBacterial andtissue-derived

monocytechemotactic

factors Chemotacticfactors

Growthfactors

Tissue-derivedmitogen

Proliferation oftissue macrophages

Recruitment ofcirculating monocytes

INCREASEDMACROPHAGES

Long-livedtissue macrophages

Epithelioidcells

Multinucleatedgiant cells

CHRONIC INJURY

Accumulation of macrophages in chronic inflamma-tion.

FIGURE 2-15.

Within different tissues, resident macrophages differ intheir armamentarium of enzymes and can respond to localinflammatory signals. The activity of these enzymes is cen-tral to the tissue destruction in chronic inflammation. Inemphysema, for example, resident macrophages generateproteinases, particularly matrix metalloproteinases (MMPs)with elastolytic activity, which destroy alveolar walls and re-cruit blood monocytes into the lung. Other macrophageproducts include oxygen metabolites, chemotactic factors,cytokines, and growth factors

Lymphocytes and Plasma CellsLymphocytes and plasma cells play a central role in theadaptive immune response to pathogens and foreign agentsin damaged tissue and are discussed in detail in Chapter 4.

FibroblastsFibroblasts are long-lived, ubiquitous cells whose chieffunction is to produce components of the ECM (Fig. 2-16).They can also differentiate into other connective tissuecells, including chondrocytes, adipocytes, osteocytes, andsmooth muscle cells. Fibroblasts are the construction workersof the tissue, rebuilding the scaffold of ECM upon whichtissue is re-established.

Fibroblasts not only respond to immune signals thatinduce their proliferation and activation but are also ac-tive players in the immune response. They interact withinflammatory cells, particularly lymphocytes, via surfacemolecules and receptors on both cells. For example, whenCD40 on fibroblasts binds its ligand on lymphocytes,both cells are activated. Activated fibroblasts produce cy-tokines, chemokines, and prostanoids, creating a tissuemicroenvironment that further regulates the behavior of

inflammatory cells in the damaged tissue. Fibroblastsfunction in wound healing in combination with regener-ating vascular endothelial cells. Both are discussed morefully in Chapter 3.

Injury And Repair In ChronicInflammation

Chronic inflammation is mediated by both immunologicaland nonimmunological mechanisms and is frequently ob-served in conjunction with reparative responses, namely,granulation tissue and fibrosis. Neutrophil products, suchas proteases and ROS, protect the host by participating inantimicrobial defense and debridement of damaged tissue;however, these same products may prolong tissue damageand promote chronic inflammation if not appropriatelyregulated. Persistent tissue injury produced by inflamma-tory cells is important in the pathogenesis of several dis-eases, for instance, pulmonary emphysema, rheumatoidarthritis, certain immune complex diseases, gout, and adultrespiratory distress syndrome.

Granulomatous Inflammation

Granuloma formation is a protective response to chronic infection(fungal infections, tuberculosis, leprosy, schistosomiasis) or thepresence of foreign material (e.g., suture or talc). It prevents dis-semination and restricts inflammation due to exogenous sub-stances that are not effectively digested during the acute response,thereby protecting the host tissues. Some autoimmune diseases(e.g., rheumatoid arthritis, Crohn disease, and sarcoidosis


EndoplasmicReticuliun

Collagen fibrils

CHARACTERISTICS AND FUNCTIONS• Produce extracellular matrix proteins• Mediate chronic inflammation and

• PGE2• CD40 expression• Matricellular proteins• Extracellular proteins

PRIMARY INFLAMMATORY MEDIATORS• IL-6• IL-8• Cyclooxygenase-2• Hyaluronan

wound healing

Fibroblast: Morphology and function. IL, interleukin.FIGURE 2-16.

[a mysterious disease of unknown etiology]) are also associ-ated with granulomas.

The principal cells involved in granulomatous inflam-mation are macrophages and lymphocytes. Macrophagesare mobile cells that continuously migrate through the ex-travascular connective tissues. After amassing substancesthat they cannot digest, macrophages lose their motility,accumulate at the site of injury, and undergo transforma-tion into nodular collections of pale, epithelioid cells, creat-ing a granuloma (Fig. 2-17A, B). Multinucleated giant cellsare formed by the cytoplasmic fusion of macrophages.When the nuclei of such giant cells are arranged around theperiphery of the cell in a horseshoe pattern, the cell is calleda Langhans giant cell. If a foreign agent (e.g., silica or aHistoplasma spore) or other indigestible material is identifiedwithin the cytoplasm of a multinucleated giant cell, it istermed a foreign body giant cell. Granulomas are furtherclassified histopathologically by the presence or absence ofnecrosis. Certain infectious agents such as Mycobacterium tu-berculosis characteristically produce caseating granulomas,the necrotic centers of which are filled with an amorphousmixture of debris and dead microorganisms and cells.Other diseases such as sarcoidosis are characterized bygranulomas that lack necrosis.

Systemic Manifestations Of Inflammation

Under certain conditions, local injury may result in promi-nent systemic effects that can themselves be debilitating.For example, systemic effects are likely to result when apathogen enters the bloodstream, a condition known assepsis. The systemic symptoms associated with inflamma-tion, including fever, myalgia, arthralgia, anorexia and som-nolence, are attributable to cytokines, including IL-1α, IL-1β, TNF-α, IL-6, and interferons. The most prominentsystemic manifestations of inflammation, termed the sys-temic inflammatory response syndrome, are leukocytosisor the acute phase response, fever and shock.

The Acute Phase Response is a SystemicResponse to Elevated Levels of IL-1, IL-6, and TNF-αThe acute phase response is a regulated physiological re-action that occurs in inflammatory conditions in responseto elevated levels of IL-1, IL-6, and TNF-α. It is character-ized clinically by fever, leukocytosis, decreased appetite,and altered sleep patterns and notably by changes inplasma levels of certain acute phase proteins. These proteins(Table 2-3) are synthesized primarily by the liver and re-leased in elevated amounts into the circulation where they

may serve as markers for ongoing inflammation. For ex-ample, increases in acute phase proteins lead to the accel-erated erythrocyte sedimentation rate, a qualitative indexused clinically to monitor the activity of many inflamma-tory diseases.

Fever is a Clinical Hallmark of Inflammation

Fever is a clinical hallmark of inflammation. Release of py-rogens (molecules that cause fever) by bacteria, viruses, orinjured cells may directly affect hypothalamic thermoregu-lation. More importantly, they stimulate the production ofendogenous pyrogens, namely cytokines— including IL-1α, IL-1β, TNF-α, IL-6—, and interferons, which producelocal and systemic effects. IL-1 stimulates prostaglandinsynthesis in hypothalamic thermoregulatory centers,thereby altering the “thermostat” that controls body tem-perature. Inhibitors of cyclooxygenase (e.g., aspirin) blockthe fever response by inhibiting IL-1–stimulated synthesis


A

BGranulomatous inflammation. A. Section of lungfrom a patient with sarcoidosis reveals numerous dis-

crete granulomas. B. A higher-power photomicrograph of a single gran-uloma in a lymph node from the same patient depicts a multinucleatedgiant cell amid numerous pale epithelioid cells. A thin rim of fibrosis sep-arates the granuloma from the lymphoid cells of the node.

FIGURE 2-17.

of PGE2. Chills (the sensation of cold), rigor (profoundchills with shivering and piloerection), and sweats (to allowheat dissipation) are symptoms associated with fever.

Shock is Characterized by CardiacDecompensation

Under conditions of massive tissue injury or infection thatspreads to the blood (sepsis), significant quantities of cy-tokines, especially TNF-α and other chemical mediators ofinflammation, may be generated in the circulation. The sus-tained presence of these mediators induces cardiovasculardecompensation through its effects on the heart and on theperipheral vascular system, a process termed shock. Systemiceffects include generalized vasodilation, increased vascularpermeability, intravascular volume loss, myocardial depres-sion with decreased cardiac output, and potentially death(see Chapter 7). In severe cases, activation of coagulationpathways may generate microthrombi throughout the body,with consumption of clotting components and subsequentpredisposition to bleeding, a condition defined as dissemi-nated intravascular coagulation (see Chapter. 20). The netresult is multisystem organ dysfunction and death.


TABLE 2–3Acute Phase Proteins

Protein FunctionMannose binding protein Opsonization/complement

activation

C-reactive protein Opsonization

α1-Antitrypsin Serine protease inhibitor

Haptoglobin Binds hemoglobin

Ceruloplasmin Antioxidant, binds copper

Fibrinogen Coagulation

Serum amyloid A protein Apolipoprotein

α2-Macroglobulin Antiprotease

Cysteine protease inhibitor Antiprotease


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