Chapter 19: The Inflammatory Response

The sentinel clinical features of acute inflammation—rubor, calor, tumor, and dolor—have been recognized for at least 5000 years.¹ Dr. John Hunter, the renowned late 18th-century Scottish surgeon, observed that the inflammatory response is not a disease per se but rather a nonspecific and salutary response to a variety of insults. Through his microscopic examinations of transparent vital membrane preparations, German pathologist Julius Cohnheim concluded that the inflammatory response is fundamentally a vascular phenomenon. Phagocytosis was described late in the 19th century by Elie Metchnikoff and his colleagues at the Pasteur Institute. Morphologic studies, using both live animals and fixed histologic preparations, transformed our understanding of inflammation and led to the currently held concepts of inflammation-associated hemodynamic alterations, acute inflammation and chronic inflammation.^1,2 During the past 5 decades, the modern techniques of biochemistry, tissue culture, monoclonal antibody production, recombinant DNA technology, and the genetic manipulation of isolated cells and whole animals have enabled a more detailed understanding of the cellular and molecular mechanisms which underpin the inflammatory response, the active resolution of inflammation, and the transition to tissue repair and restitution. These studies, in concert with “experiments of nature,” such as chronic granulomatous disease (Chap. 66) and the leukocyte adhesion deficiency disorders (Chap. 66), have permitted the formulation of complex, yet elegant, models of acute and chronic inflammation and led to the promise of incisive therapeutic approaches. A large array of human diseases is marked by either defects in the development of the inflammatory response or the deleterious effects of the inflammatory response itself.

It is useful to consider inflammation as an acute or chronic (persistent) process. Acute inflammation lasts from minutes to several days and is characterized by pronounced local hemodynamic and microvascular changes and leukocyte accumulation.^2,3 The acute inflammatory response is consistently marked by microvascular leakage and the accumulation of neutrophils. The four cardinal signs of acute inflammation, alluded to above, can be accounted for within the physiologic parameters of inflammation. The systemic effects of inflammation, particularly acute inflammation, account for the familiar clinical findings of fever, acute-phase response and altered sensorium. In turn, sepsis is a systemic inflammatory response syndrome that occurs in response to infection; severe sepsis is sepsis complicated by acute organ dysfunction; and septic shock is sepsis complicated by either fluid resuscitation-resistant hypotension or by hyperlactatemia.⁴

The chronic inflammatory response, which lasts much longer and is more varied in its effects, is marked by growth of new capillaries and proliferation of resident fibroblasts.^2,3 Cellular infiltrates typically include monocytes and lymphocytes, but there are many variations in the cellular composition, anatomic distribution and tempo of development of chronic inflammatory lesions. Chronic inflammatory processes can be classified according to these variations. For example, granulomatous inflammation is a chronic process marked by nodular aggregates of mononuclear phagocytes that have become “transformed” into epithelioid histiocytes, so-called because of their similar appearance to epithelial cells.² Granulomas may be distributed along blood vessels (e.g., angiocentric), along upper airways (e.g., bronchocentric), or randomly throughout the interstitium or parenchyma of an organ. Granulomas may vary morphologically. Tuberculous granulomas often contain areas of caseous necrosis while sarcoidosis-associated granulomas are often cellular and exhibit fibrosis but usually without areas of necrosis.² Other chronic inflammatory processes are marked by a preponderance of eosinophils or plasma cells. In contrast to the more stereotyped appearance of an acute inflammatory lesion, the particular appearance of a chronic inflammatory lesion can sometimes provide insight into its cause (e.g., caseating granulomas in tuberculosis, eosinophil-rich infiltrates in a parasitic infection and plasma cell-rich infiltrates in viral hepatitis).

Superimposed upon acute and chronic inflammatory responses is repair.^2,5 Resolution or termination of the inflammatory response is an important step in the pathway to repair; it occurs through a complex set of regulated processes.⁵ Repair, which may entail the regeneration of parenchymal cells damaged as the result of an insult per se or as “bystanders” to the inflammatory response, is characterized by the growth of new capillaries (angiogenesis) and the activation of fibroblasts which produce extracellular matrix molecules (e.g., scar tissue). In some circumstances an inflammatory response is self-limited (e.g., sunburn), whereas in other situations the response may persist for many years (e.g., tuberculous granulomas). The elimination or persistence of an insult has a major influence on outcome–whether ongoing chronic inflammation, complete regeneration or scar formation. There is great complexity in terms of the networks of proinflammatory and antiinflammatory soluble mediators (e.g., cytokines) and the phenotypes, as well as the functional and regulatory roles of both indigenous cells and recruited inflammatory cells in inflammation, resolution and repair.^2,3,5,6,7

This chapter first addresses acute inflammation, which encompasses localized changes in blood flow, alterations in microvascular permeability and neutrophil exudation.^2,3 In addition to hemodynamic changes, inflammation encompasses endothelial cell activation, low-affinity leukocyte–endothelial adhesion, high-affinity or stationary leukocyte–endothelial adhesive interactions, leukocyte emigration, leukocyte activation, and the subsequent dampening and resolution of the inflammatory response. The highly regulated migration of leukocytes from the vasculature into sites of inflammation and of lymphocytes through secondary lymphoid tissues and, in turn, into sites of microbial invasion, are pivotal to host defense in the contexts of inflammation and immunity.^2,3,6 This extraordinary complexity of regulatory processes that control inflammation is exemplified by, but not limited to, proinflammatory cytokines (e.g., tumor necrosis factor [TNF]-α, interleukin [IL]-1β, IL-6) that drive inflammation, countered by rises in antiinflammatory cytokines (e.g., IL-4, IL-10, IL-11, IL-13, transforming growth factor [TGF]-β, IL-1ra and soluble cytokine receptors) that dampen inflammatory responses (see “Cytokines and Chemolines”).^2,7 Both the termination of an inflammatory process and the transition from an active inflammatory milieu to a wound-healing, tissue-remodeling environment are actively regulated processes. The concept of “active” termination of the inflammatory response, including the roles of chemokine depletion, neutrophil apoptosis, resolvins and protectins, and the shift from interferon (IFN)-γ–driven “classical M1” macrophages to IL-4/IL-13–driven “alternative M2” macrophages, is introduced at the end of the first section of this chapter (M1 and M2 Macrophages). The section “Regulators of the Inflammatory Response” of this chapter introduces (and where appropriate, reiterates) the vast array of soluble and surface-active mediators that regulate both acute and chronic inflammatory responses, as well as some aspects of resolution. These mediators include substances that range from short-lived reactive oxygen and nitrogen intermediates to entire regulatory systems (e.g., complement and coagulation). Many mediators of inflammation have become targets for therapeutic interruption strategies. See section “Chronic Inflammation and Repair.” This chapter provides a framework for understanding the basic processes of inflammation while promoting an appreciation for the highly complex and integrated nature of the regulated inflammatory response.

HEMODYNAMIC CHANGES

The hemodynamic changes that occur early in acute inflammation include arteriolar vasodilatation and localized increases in microvascular permeability (Fig. 19–1). In many circumstances, arteriolar vasodilation follows a rapid and transient period of vasoconstriction.^2,3 Arteriolar vasodilation results in increased blood flow, thus explaining the familiar redness and warmth that characterize a site of acute inflammation. The increase in blood flow, coupled with increases in microvascular permeability, results in hemoconcentration and increased local viscosity. These hemodynamic changes are critical to subsequent leukocyte emigration because selectin-mediated low-affinity rolling leukocyte–endothelial adhesive interactions occur only under such conditions of low shear force. Experimental studies using in vitro flow chambers and transparent vital membrane preparations in live animals indicate that selectin-mediated leukocyte–endothelial rolling adhesive interactions cannot occur in the face of the shear forces that exist under conditions of normal blood flow velocity. Increased microvascular permeability leads initially to protein-poor transudation followed by protein-rich plasma exudation, another characteristic of acute inflammation.² Microvascular leakage occurs through a variety of temporally regulated mechanisms, including rapid, reversible, and short-lived venular endothelial cell contraction attended by widening of intercellular junctions; so-called endothelial cell retraction, which is less-well understood but involves long-lived cytokine-mediated cytoskeletal changes; direct endothelial injury and disruption by physical trauma; leukocyte-mediated endothelial cell injury; and leakage via new capillaries that do not yet possess completely “closed” intercellular junctions.² Increases in rate of transcytosis by which plasma constituents cross endothelial cells in vesicles or vacuoles (vesiculovacuolar organelles) occur in neoplastic blood vessels and may play a role in inflammation.^2,8 Alterations in local blood flow occur at the level of arterioles, the key to vascular resistance and regulated largely by the autonomic nervous system, nitric oxide (NO) (formerly called endothelium-derived relaxing factor), vasoactive peptides, and eicosanoids. A variety of soluble mediators can induce increases in microvascular permeability through several of the above-mentioned mechanisms.

LEUKOCYTES

The recruitment of leukocytes into a site of inflammation is a fundamental characteristic of the inflammatory response.^2,3,9 The orchestrated recruitment of particular types of leukocytes into specific tissues, whether sites of acute inflammation, in the course of physiologic lymphocyte recirculation through lymph nodes or in the cellular immune response to microbial invasion, is referred to as homing.³ The general mechanisms of leukocyte homing are similar, but the leukocytes and particular mediator molecules vary. For example, neutrophils bind and traverse postcapillary venules in acute inflammation, naïve T lymphocytes bind and traverse lymph node high-endothelial venules (HEVs) in lymphocyte recirculation, and effector and memory T lymphocytes bind and traverse postcapillary endothelial cells in sites of chronic infection.³ The importance of white blood cells in host defense is highlighted in patients with either numerical leukocyte deficiencies or functional defects. Leukocytes are critical because of their central role in the phagocytosis and killing or containment of microbes and in the digestion of necrotic tissue debris. Leukocyte-derived products, such as proteolytic enzymes and reactive oxygen intermediates, contribute to tissue injury.

Leukocyte Adhesion and Transmigration

Vascular stasis that results from the hemodynamic changes of early acute inflammation leads to displacement of leukocytes from the central axial column of circulating blood cells to positions along the endothelial surface. This process, margination, is enhanced under conditions of slow blood flow.^2,3 Leukocytes adhere transiently and weakly to the endothelial surface. Vital membrane preparations and flow chamber studies using endothelial cell monolayers and suspensions of purified leukocytes have revealed that cells “tumble and roll” along the endothelial surface.¹⁰ Transient, weak, rolling neutrophil–endothelial adhesive interactions occur within minutes of initiation of an acute inflammatory response and can, depending upon the time point within the evolution of an inflammatory response, involve neutrophils, lymphocytes, monocytes, basophils, or eosinophils. Leukocyte–endothelial cell-rolling adhesive interaction is a specific and necessary step that precedes high-affinity, or so-called stationary adhesion and emigration.^9,10,11 Early rolling adhesive interactions are mediated largely by selectins and their carbohydrate-rich counterreceptors.^2,3,9,10 In turn, the cell-surface expression of selectins (and other intercellular adhesion molecules) is regulated by locally produced proinflammatory mediators.^9,10,11

Selectins contain an extracellular N-terminal carbohydrate-binding region that is homologous to mammalian lectins, an epidermal growth factor-like domain, a series of complement regulatory domains, and a lipophilic transmembrane domain (Table 19–1).^2,3,9,10 P-selectin is expressed by endothelial cells and platelets, E-selectin by endothelial cells, and L-selectin by most white blood cells. P-selectin is constitutively synthesized and stored in endothelial intracytoplasmic granules (Weibel-Palade bodies).⁹ When endothelial cells are exposed to histamine, thrombin, platelet-activating factor (PAF) or tissue factor, preformed P-selectin is rapidly (within minutes) translocated to the endothelial surface where it engages marginated leukocytes via carbohydrate moieties that contain sialic acid residues (e.g., P-selectin glycoprotein ligand-1 [PSGL-1]).^2,3,10,12 This transient, low-affinity, binding interaction accounts in part for the early rolling interactions (Fig. 19–2). The development of single knockout mice (i.e., lacking individual selectins [P−/−; E−/− or L−/−], double knockout mice (e.g., E−/− and P−/−), and even triple knockout mice, has confirmed that leukocyte rolling can be almost completely accounted for by selectins.^13,14 Exposure of endothelial cells to TNF-α or IL-1β results in new protein synthesis–dependent expression of E-selectin, a response that occurs within 1 to 2 hours and peaks at 4 to 6 hours.^2,3,9 As in the case of P-selectin–mediated leukocyte adhesion, E-selectin–mediated adhesion occurs via a series of sialylated and fucosylated carbohydrate moieties related to the sialyl Lewis X and sialyl Lewis A blood group antigens, but found on leukocytes (Table 19–1).^9,10 Selectin counterreceptors consist of several different mucin-like glycoproteins coated with various sialyl moieties. L-selectin is constitutively expressed by leukocytes, participates in neutrophil and monocyte binding to activated endothelium in inflammatory sites, lymphocyte–endothelial cell homing (e.g., lymphocyte homing to lymph nodes via HEVs), leukocyte–leukocyte adhesive interactions via sulfur-containing mucin-like glycoproteins, and is cleaved from cells by means of “sheddase” enzymes such as a disintegrin and metalloproteinase (ADAM)-17 (TNF-α converting enzyme [TACE]) when the leukocyte is activated (see Table 19–1).^3,15 The relevant mucin-like glycoprotein counter-receptors on HEVs are collectively referred to as peripheral node addressins (PNAds) and include mucosal addressin cell adhesion molecule (MadCAM)-1, GlyCAM-1, and CD34.^3,15 L-selectin shedding facilitates leukocyte emigration by allowing the weakly adherent white blood cell to detach from the endothelium. Low-affinity rolling adhesive interactions set the stage for β-integrin– and immunoglobulin superfamily mediated high-affinity adhesive interactions and leukocyte transmigration.^2,3,9

Weak selectin-mediated rolling and high-affinity stationary adhesive interactions are not temporally or mechanistically completely discrete. For example, TNF-α and IL-1β both induce E-selectin, which is not expressed by quiescent cells, and both increase endothelial expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1, which are constitutively expressed in low cell-surface densities.^2,3 ICAM-1 is involved in the recruitment of all types of leukocytes and VCAM-1 is involved in the recruitment of chronic inflammatory leukocytes (lymphocytes, monocytes, eosinophils, and basophils).^2,3,9 ICAM-1 binds to β₂ (leukocyte) integrins, which are heterodimeric structures that contain one species of α chain (e.g., CD11a, CD11b, CD11c, CD11d) and a common β chain (CD18).¹⁶ VCAM-1 binds to β₁ integrins (e.g., very-late antigen [VLA]-4/α₄β₁) (see Table 19–1).^2,3 Activated endothelial cells secrete PAF and CXCL8 (IL-8), which activate overlying selectin-bound leukocytes.^2,3,9,16 Individual leukocyte CD11a/CD18 (LFA-1) heterodimers undergo a transient conformational change and groups of CD11a/CD18 molecules form multimolecular clusters.^2,3,9 Both the conformational change in CD11a/CD18 and the clustering contribute to increases in binding affinity to endothelial ICAM-1.^3,16 CD11b/CD18 (Mac-1) binds ICAM-1, ICAM-2, and iC3b (see section “Complement”), the latter of which opsonizes complement-coated particulates. CD11c/CD18 also binds to iC3b and initiates phagocytosis, but plays a lesser role in neutrophil adhesion than do CD11a/CD18 and CD11b/CD18. Intercellular adhesion molecules are found on a variety of cell types other than endothelial cells. The roles of CD11c/CD18, CD11d/CD18, and ICAM-3 in leukocyte–endothelial adhesion are less-well established. β₁-Integrins, notably VLA-4, are found primarily on chronic inflammatory leukocytes (e.g., lymphocytes, monocytes, basophils, and eosinophils) and mediate leukocyte binding via VCAM-1.^2,3,9 β₁-Integrin–mediated adhesive interactions occur via arginine-glycine-aspartic acid peptide sequences (RGDs) displayed by VCAM-1, as well as on exposed surfaces of matrix molecules (e.g., fibronectin). β₂-Integrin–ICAM-1 and β₁-integrin–VCAM-1–mediated adhesive interactions occur later (hours to days) in the inflammatory response than do selectin-mediated interactions.

β₁- and β₂-integrins are clearly important in leukocyte recruitment into sites of acute and chronic inflammation. Eighteen different integrin α subunits, eight integrin β subunits, and 24 in vivo heterodimer combinations have been identified in mammals.¹⁷ In addition to leukocyte–endothelial cell adhesion interactions, integrins function in a variety of other cell–cell and cell–extracellular matrix interactions. A large number of integrin-targeted small molecule, peptide, and designer antibodies have been developed for therapeutic applications.¹⁷ Inflammatory–immunologic diseases treated with integrin antagonists include, among others, multiple sclerosis, Crohn disease, and age-related macular degeneration.

High-affinity stationary adhesive interactions precede leukocyte transmigration across the endothelium into the subjacent interstitium. The functional importance of complementary leukocyte–endothelial adhesive interactions has been clarified by in vitro binding studies and in vivo studies that have employed neutralizing antibodies directed against adhesion molecules, pharmacologic antagonists of adhesion molecules, and knockout mice.^2,3 The functional importance of leukocyte integrins (CD11a/CD18, CD11b/CD18, CD11c/CD18) has also been highlighted by clinical and experimental observations in patients with rare genetic leukocyte adhesion deficiencies (Chap. 66).

β₂-Integrin ICAM-1 and ICAM-2, as well a β₁-integrin (VLA-4) VCAM-1, induce adhesive interactions that lead to cytoskeletal reorganization in leukocytes that flatten and spread out on the endothelial surface, extend pseudopodia between endothelial cells, and migrate along extravascular chemotactic gradients. Most leukocytes exit the vascular space between adjacent endothelial cells (paracellular transmigration). Paracellular transmigration depends not only on integrin-ligand interactions but also on CD31 (PECAM-1 [platelet-endothelial cell adhesion molecule-1]) expressed on both leukocytes and endothelial cells, and transient reversible disassembly of tight interendothelial vascular endothelial (VE)–cadherin junctional complexes.¹⁸ There is evidence that leukocytes can also exit the vascular space via a less-well-characterized transcellular pathway.

Leukocyte Chemotaxis and Activation

Leukocytes bound tightly to endothelium emigrate from the vascular space into the interstitium by extending pseudopods between intercellular junctions (see Fig. 19–2).¹⁸ Secreted neutral proteases such as elastase, cathepsin G, and proteinase 3, play a role in the passage or “invasion” of leukocytes through the subendothelial extracellular matrix. Collagenases are particularly important in leukocyte transmigration through basement membranes. A variety of matrix metalloproteinases, produced by several cell types, participate in leukocyte migration, and also play roles in resolution of inflammation and tissue remodeling. Leukocyte emigration and subsequent movement through the interstitium follow chemical concentration gradients; processes facilitated by binding interactions between leukocyte integrins and complementary sites on extracellular matrix molecules (e.g., fibronectin).⁹ A wide variety of soluble mediators can provide the motive trigger for this process.^2,3,19 Chemotactic factors for neutrophils include peptides derived from bacteria (e.g., N-formyl peptides), complement-derived peptides (e.g., C5a), cell membrane-derived chemotactic lipids (e.g., PAF), and cytokines and chemokines produced by a variety of cell types (e.g., CXCL8 [IL-8] from endothelial cells).^2,3,19 Chemotactic factors vary with respect to their specificity for different types of leukocytes. For example, C5a and N-formyl peptides both induce neutrophil and monocyte chemotaxis, and CXCL8 [IL-8] induces neutrophil chemotaxis, whereas CCL2 (monocyte chemoattractant protein [MCP]-1) induces chemotactic responses in monocytes and a specific subset of memory T lymphocytes.³ The Th17 subset of CD4 T-helper lymphocytes secretes IL-17 and IL-22 which participate in the recruitment of neutrophils.³ (There are several species of IL-17, including homodimers IL-17A–IL-17F as well as heterodimeric species.²⁰) Each of these chemotactic factors activates “target” cells by engaging specific cell surface receptors, which, in turn, are linked to the contractile cell motility apparatus.^3,19

In addition to chemotaxis, soluble and cell-surface mediators induce leukocyte activation manifested by a wide array of changes in cellular function (e.g., leukocyte integrin upregulation and increased binding affinity [e.g., CD11b/CD18], selectin shedding [e.g., L-selectin], lysosome degranulation, and initiation of the respiratory burst). There have been great advances in understanding of the biochemical pathways involved in chemotaxis, cell activation, and degranulation.²¹ Although there are many nuances in the signal transduction pathways involved in these processes, several themes have emerged. Cell surface receptors are activated by specific ligands (e.g., C5a, leukotriene B₄ [LTB₄], CXCL8 [IL-8] and receptor activation is transduced via specific G proteins and membrane-associated phospholipases, which leads to mobilization of intracellular calcium, influx of extracellular calcium and phosphorylation of series of cytosolic proteins.¹⁹ Rare genetic diseases linked to receptor and effector defects (e.g., IFN-γ receptor defects and nicotinamide adenine dinucleotide phosphate [reduced form] [NADPH] oxidase defects) have provided insight into leukocyte function and the importance of such specific activities in host defense (Chap. 66).

A principal result of neutrophil and monocyte recruitment is provision of large numbers of activated leukocytes that can release lytic substances and reactive oxygen and nitrogen intermediates needed to destroy foreign invaders, and a vehicle to contain foreign particulates through phagocytosis. Some recruited monocytes differentiate into macrophages and recruited effector and memory lymphocytes play pivotal roles in the adaptive immune response.^3,6 The products and functions of activated inflammatory cells are at once salutary because they contain and destroy invaders and deleterious because they cause tissue damage. The roles of neutrophil apoptosis (programmed cell death) in the termination of acute inflammatory responses and IFN-γ–driven M1 macrophages and IL-4/IL-13–driven M2 macrophages in the transition of an inflammatory milieu to a wound-healing or tissue-remodeling milieu are discussed in “M1 and M2 Macrophages”.

Leukocyte activation, especially of neutrophils and mononuclear phagocytes, results in the secretion of microbicidal peptides (e.g., defensins, bactericidal permeability-increasing protein [BPI], cationic antimicrobial protein [e.g., CAP37]) and lytic enzymes (e.g., myeloperoxidase, elastase, cathepsin G).^6,21,22 The release of such granular constituents is accompanied by the generation of reactive oxygen and nitrogen intermediates (e.g., O₂⁻, H₂O₂, NO), the generation of arachidonate metabolites (e.g., leukotrienes and prostaglandins) and the production of other proinflammatory mediators (see section “Regulators of the Inflammatory Response”).^21,22 In some circumstances these mediators are released into phagolysosomes where they contribute to the destruction of engulfed microbes, while in other circumstances they are secreted into the extracellular milieu where they amplify the inflammatory response and cause tissue damage. The various types of neutrophil granules (primary azurophilic, secondary specific, tertiary gelatinase-containing, and secretory vesicles) are released in a differentially coordinated fashion.^21,22

An important recently formulated concept is that of the “inflammasome,” a cytosolic multiprotein complex that is formed in a variety of leukocytes and leads to the generation of IL-1β via activated caspase-1–mediated cleavage of pro–IL-1β.²³ Inflammasome formation can be induced by a wide variety of microbial products (e.g., lipopolysaccharide) as well as by other proinflammatory molecules (e.g., urate crystals, damage-associated molecular patterns also known as “alarmins”).²³ Pathogens can activate immune cells via several classes of pattern-recognition receptors, including C-type lectin receptors, toll-like receptors (TLRs), retinoic acid–inducible gene (RIG) 1-like cytosolic receptor and nucleotide-binding oligomerization domain (NOD)-like receptors.^23,24

Effective phagocytosis involves three distinct steps: (1) recognition and attachment, (2) engulfment, and (3) degradation (killing in the case of microbes) of the ingested material.^21,22 Phagocytosis is greatly enhanced when particles (e.g., bacteria) are coated with opsonins, which, in turn, function as ligands for leukocyte surface receptors. The major opsonins include the Fc domains of immunoglobulin (Ig) G and IgM and the complement-derived fragments C3b and iC3b, which are generated via activation of the complement cascades and covalently bound to the surfaces of nearby large molecules and particles (e.g., microbes). There are a variety of Fc receptors (FcγRI, FcγRII, FcγRIIIB, etc.) and complement receptors (e.g., CR1, CR3, CR4) that specifically engage their respective opsonins when the latter coat foreign particulates.⁶ In addition to facilitating receptor-mediated phagocytosis of opsonized particles, Fc receptors trigger cell activation with the attendant release of granular constituents and the generation of reactive oxygen intermediates.⁶ Other important recognition molecules expressed by leukocytes include integrins, the C1q receptor, mannose receptors, scavenger receptors and TLRs.^6,24,25 Mannose receptors bind to mannose and fucose moieties, which are present on some microbes but not mammalian cells; scavenger receptors bind to a variety of microbes, as well as to oxidized and acetylated low-density lipoproteins; and TLRs bind to a variety of microbial moieties including endotoxins (lipopolysaccharide) and prokaryotic nucleic acids (e.g., double-stranded RNA).^6,24,25 Humans express at least nine species of TLR, some of which are expressed on external cell surfaces and others on the inner surfaces of endosomes.^24,25 Some enhanced phagocytic reactions occur independently of opsonins. The engulfment, degranulation, and oxidative burst triggered as the result of engagement of FcR is enhanced by the concurrent engagement of complement receptors. In some circumstances, engulfment is enhanced by the simultaneous binding of the leukocyte to specific extracellular matrix molecules (e.g., fibronectin) or soluble cytokines. Engulfment results in the formation of phagosomes, which fuse with lysosomes to form phagolysosomes in which foreign particles are oxidized and degraded. Numerous mechanisms for killing and/or degradation of microbes have been elucidated (Table 19–2). Although these mechanisms are classified as either oxygen-dependent or oxygen-independent, both types of processes may be involved in the destruction of a given microorganism, and a given microorganism may vary greatly in its susceptibility to various mechanisms of destruction.^6,21,22 Extracellular release of reactive oxygen and nitrogen intermediates, lysosomal enzymes, lipid mediators, and cationic proteins can all contribute to inflammation-related tissue injury.

As noted above, acute inflammation may be followed by chronic inflammation and a superimposed series of reparative processes that can result in resolution or scar formation. Resolution of inflammation was long viewed to be a passive process that included a poorly understood decline in concentrations of proinflammatory mediators and cells.⁵ It has become clear that the balance between a proinflammatory and an antiinflammatory milieu is regulated by networks of proinflammatory mediators (e.g., proinflammatory cytokines: TNF-α, IL-1β, IL-6) and antiinflammatory mediators (e.g., antiinflammatory cytokines: IL-4, IL-10, IL-11, IL-13, TGFβ, IL-1ra, and soluble cytokine receptors). Clearance of inflammatory cells and mediators is an active process that encompasses leukocyte apoptosis, inactivation and sequestration of proinflammatory chemokines, and egress of leukocytes from sites of inflammation.⁵ Detailed serial biochemical analyses of inflammatory exudates by liquid chromatography-mass spectroscopy have, along with structure–function analyses and in vivo animal studies, elucidated the identities of “resolvins” and “protectins,” lipids derived from omega-3 polyunsaturated fatty acids that facilitate resolution of inflammation.^5,26,27 The recognition of functionally distinguishable macrophages as “classical, IFN-γ–driven M1” phenotype and “alternative, IL-4/IL-13–driven M2” phenotype has provided insight into the transition of an active inflammatory milieu to a wound-healing or tissue-remodeling milieu.^5,28,29 It has become clear that there are several different macrophage phenotypes, a reflection of the great complexity of the transition from active inflammation to resolution and remodeling. Insight into actively regulated termination of inflammation as well as the transition from active inflammation to resolution and remodeling has provided for new therapeutic strategies.

ACUTE-PHASE RESPONSE

The acute-phase response is a stereotyped host response to insults that include trauma, tissue damage, and infection.³ TNF-α, IL-1β, and IL-6 are consistently produced regardless of trigger. As discussed throughout, these soluble cytokines mediate several proinflammatory processes. (Proinflammatory cytokines TNF-α, IL-1β, IL-6, and antiinflammatory cytokines are further discussed in “Cytokines and Chemokines”.) TNF-α, IL-1β and IL-6 act on the hypothalamus to increase the body temperature set point, resulting in fever. Because they are produced endogenously and induce fever in the context of a systemic host response, these mediators have sometimes been referred to as “endogenous pyrogens.” Exogenous pyrogens (e.g., endotoxin or lipopolysaccharide) originate from outside the host but induce TNF-α, IL-1β, and IL-6. The downstream effects of these cytokines are responsible for several familiar clinical manifestations of infection, including fever, altered sensorium, and the production of fibrinogen, serum amyloid protein, C3, C4, and C-reactive protein by hepatocytes and other cell types. The biochemical fingerprints of an acute-phase response are revealed through several widely available laboratory tests (C-reactive protein measurement, serum protein electrophoresis). Severe, overwhelming infections attended by high serum concentrations of endotoxin can induce very high concentrations of TNF-α, IL-1β, and IL-6.⁴ High concentrations of TNF-α are directly responsible for several of the key manifestations of severe sepsis and septic shock (e.g., cardiac suppression, intravascular thrombosis, capillary leakage and insulin resistance).^3,4

Interruption of TNF-α has been a therapeutic strategy in localized inflammatory conditions like the joint inflammation (and destruction) of rheumatoid arthritis and bowel wall inflammation in Crohn disease and ulcerative colitis, as well as in severe sepsis and septic shock.³⁰ Anti–TNF-α therapy (via engineered monoclonal antibodies [infliximab and adalimumab] that neutralize TNF-α as well as inhibitory soluble TNF receptors [etanercept]) has been effective in conditions such as rheumatoid arthritis and inflammatory bowel disease, but not in severe sepsis or septic shock in humans.^4,30 Major adverse events associated with anti–TNF-α therapies include an increased risk of mycobacterial infection, development of autoantibodies (but not autoimmune disease), and injection site inflammation.³⁰ There have been isolated reports of cytopenias, skin cancer and worsened congestive heart failure.³⁰

Since the advent of morphologic examinations of tissue from inflammatory lesions, it has been recognized that resolution of acute inflammation is marked by the disappearance of neutrophils and the engulfment of cellular debris by recruited monocytes and tissue macrophages. It has been relatively recent that resolution of inflammation has been understood to be an active process.⁵ Key aspects of resolution include: (1) cessation of neutrophil influx effected by chemokine inactivation and sequestration; (2) neutrophil apoptosis; (3) functional polarization or switching of macrophages from a proinflammatory (M1) to a wound-healing and tissue-remodeling (M2) phenotype; and (4) the rapid, localized generation of proresolution lipid mediators that include lipoxins, resolvins, and protectins.⁵

INACTIVATION AND SEQUESTRATION OF CHEMOKINES

As described in the preceding section, neutrophil influx into an inflammatory site is in part mediated by locally generated chemokines. The actions of proinflammatory chemokines are terminated as a result of their cleavage into inactive fragments and through sequestration or removal from participation by binding to indigenous nonfunctional decoy receptors or to locally generated decoy receptors. Examples include the cleavage of neutrophil-directed CXC-chemokines within a critical ELR motif by macrophage-derived matrix metalloproteinase 12 and matrix metalloproteinase cleavage of the monocyte-directed CC-chemokine, CCL7.^5,31 Cleaved CCL7 can still bind its cognate receptors, CCR1, CCR2, and CCR3, but these cells are not mobilized by the truncated version of CCL7.⁵

Chemokine receptors that lack an intact highly-conserved DRY motif, which normally links the receptor to key signal transduction molecules, function as decoy receptors. The best understood of these are the “Duffy antigen receptor for chemokines” (DARC) and D6.⁵ DARC is expressed by endothelial cells at sites of leukocyte egress and binds both CC and CXC chemokines. Functional studies reveal that disruption of DARC leads to increased neutrophil recruitment into sites of inflammation. D6 binds several different CC chemokines, thus rendering them ineffective. Chemokine decoy receptors can also be derived in situ from previously active receptors in sites of inflammation. IL-10 facilitates maintenance of CCR1, CCR2, and CCR5 expression, but induces the functional inactivity of these receptors thus hastening the resolution of inflammation.

NEUTROPHIL APOPTOSIS

Contrary to a long-held belief that extravascular neutrophil life spans are fixed, recent studies suggest that neutrophil life spans can be modulated by local mediators.⁵ Low concentrations of macrophage-derived TNF-α and Fas-ligand prolong neutrophil life span, whereas high concentrations of the same ligands lead to shortened life spans.⁵ The latter results in neutrophil apoptosis via phosphoinositide 3-kinase–triggered oxygen metabolite generation and Btk-NADPH–modulated pathways. Other local regulators of neutrophil apoptosis include oxygen tension-modulated hypoxia-inducible factor 1α and granulocyte-macrophage colony-stimulating factor (GM-CSF).⁵

In turn, apoptotic neutrophils attenuate inflammation via the secretion of annexin A1, which inhibits the recruitment of additional neutrophils as well enhances neutrophil apoptosis and phagocytosis by macrophages.⁵ Lactoferrin, a neutrophil secondary granule protein, also inhibits neutrophil recruitment and induces apoptosis following its release into the inflammatory milieu. (The effect of lactoferrin on neutrophil survival is influenced by its degree of iron saturation.) Macrophage ingestion of apoptotic neutrophils is called “efferocytosis,” a process directed by distinct “find me” and “eat me” signals.⁵ “Find me” signals include the nucleotides ATP and uridine triphosphate (UTP), fractalkine (CX₃CL1), lysophosphatidylcholine and sphingosine-1-phosphate (S-1-P). The corresponding counterreceptors for these “find me” signals include P2Y2 receptors, CX₃CR1, G2A, and S-1-P_1–5 receptors, respectively. This set of “find me/eat me” ligand receptor pairs fits within a larger set of apoptotic cell-efferocyte interactions. In some cases, apoptotic cell “find me” molecules are expressed as a function of apoptosis per se, whereas in other cases, existing surface molecules are either modified or linked with mediators that facilitate recognition and ingestion by efferocytes.

M1 AND M2 MACROPHAGES

It was recognized during the 1980s and 1990s that various cytokines can differentially modulate macrophage function.⁵ From original specific observations of an IFN-γ–activated macrophage phenotype and an IL-4–activated macrophage phenotype, emerged the concept of “classical, IFN-γ–activated, M1” macrophages and “alternative, IL-4–activated, M2” macrophages.⁵ Such macrophages are sometimes described as being “polarized” by IFN-γ, IL-4, or other mediators. Several different activated macrophage “phenotype signatures” have been elucidated, a recognition that has led to a series of designations with “M1” and “M2” representing the most extreme differences.⁵ There are several M2 subtypes.⁵ IFN-γ–activated macrophages (M1) produce tissue-toxic radicals (e.g., NO) and proinflammatory cytokines (e.g., TNF-α), whereas M2 macrophages produce less NO and more IL-10 and TGF-β, the latter being important mediators of wound healing and/or tissue-remodeling.⁵ Insight into the role of tissue macrophages in the resolution of active inflammation and the transition to wound-healing and tissue-remodeling has helped foster the concept of inflammation as an actively regulated response.

LIPID REGULATORS: RESOLVINS AND PROTECTINS

The transition from peak acute inflammation, marked by maximum concentrations of neutrophils, toward resolution, is accompanied by marked changes not only in the local cytokine–chemokine milieu, but also in the lipid milieu. Specifically, there is a shift from high local concentrations of proinflammatory prostaglandins and leukotrienes to antiinflammatory lipoxins, resolvins, and protectins. Basic lipoxin biochemistry is outlined later in the section “Inflammatory Lipids”. Resolvins and protectins, derived from omega-3 polyunsaturated fatty acids, each encompass several classes of related molecules.^5,26,27 The synthesis of both resolvins and protectins occurs through enzymatic pathways; the mediators themselves exert their effects through specific receptors on neutrophils, macrophages and dendritic cells.^5,26,27 Antiinflammatory mechanisms exerted by resolvins and protectins are diverse and include enhancement of macrophage-mediated clearance of apoptotic neutrophils, upregulation of cell-surface CCR5 which sequesters proinflammatory chemokines, and the shift of biochemical pathways toward an antiinflammatory phenotype.^3,26,27 The identification and characterization of both resolvins and protectins has laid the groundwork for new therapeutic strategies to modulate inflammation.^5,26 Efficacy of several “proresolving lipids” has been reported in animal models of inflammation.⁵ Resolvin E1, a synthetic resolvin analogue (RX-100045), and LXA4-based compounds are under investigation in several human inflammatory diseases.⁵

The foregoing sections have provided a conceptual framework for the inflammatory response, specifically, the hemodynamic alterations, mechanisms of specific leukocyte–endothelial adhesive interactions, chemotaxis, leukocyte activation, phagocytosis, intracellular microbial killing mechanisms, active termination/resolution of the acute inflammatory response, and the contributions of M1 and M2 macrophages to inflammation and tissue repair. The many steps that constitute this paradigm are regulated by soluble mediators produced by endothelial cells and leukocytes at a site of inflammation, by other resident cells (e.g., tissue macrophages, fibroblasts, mast cells) and as byproducts of bloodborne proteins (e.g., complement system, coagulation cascade; Table 19–3). There are many examples of “crosstalk” among regulatory systems (e.g., proteinase-activated receptors), complex regulatory networks (e.g., proinflammatory and antiinflammatory cytokine balance), and pleiotropism exhibited by individual mediators (e.g., TNF-α and IL-1β).

REACTIVE OXYGEN INTERMEDIATES

Since the early 1970s it has been recognized that activated phagocytes exhibit a transient but marked increase in oxygen consumption and the mechanistically coupled generation of reduced oxygen metabolites.³² Although small quantities of reactive oxygen intermediates are produced as byproducts of several metabolic pathways, the chief source is the leukocyte cytosol and membrane-associated NADPH oxidase, an enzyme complex that is defective in most patients with chronic granulomatous disease (Chap. 66). Reactive oxygen intermediates include superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), hydroxyl radical (HO•), and singlet oxygen (¹O₂).³² These reduced oxygen products play a major role in intraphagolysosomal killing of microorganisms, and when released extracellularly, are directly or indirectly responsible for a variety of proinflammatory processes, including endothelial cell lysis, extracellular matrix degradation, activation of latent proteolytic enzymes (collagenase, gelatinase), inactivation of antiproteases, interaction with metabolites of l-arginine, and generation of chemotactic factors from arachidonic acid and the complement component, C5.³³ In addition to their role in endothelial cytotoxicity, reactive oxygen intermediates are cytotoxic to fibroblasts, erythrocytes, tumor cells and many types of parenchymal cells.³³ Implicated biochemical mechanisms include lipid peroxidation, formation of carbonyl moieties and nitrosylation products, intracellular enzyme inactivation, protein oxidation, and oxidant-mediated DNA damage. Reactive oxygen intermediates (e.g., O₂⁻) can also undergo reactions with reactive nitrogen intermediates (e.g., NO; see “Reactive Nitrogen Intermediates” below) to generate toxic NO derivatives.³³ Within limits, host cells are protected by antioxidant defense systems (e.g., superoxide dismutase, catalase, reduced glutathione).³³

REACTIVE NITROGEN INTERMEDIATES

Described in 1980 as “endothelium-derived relaxing factor,” NO is the soluble, gaseous, short-acting biosynthetic product of l-arginine, O₂, NADPH, and NO synthase (NOS).³⁴ As suggested by its original name, NO mediates vascular smooth muscle relaxation. NO binds to the heme moiety of guanylyl cyclase to trigger the generation of intracytoplasmic cyclic guanosine monophosphate (cGMP) and, through the activation of a series of kinases, induces smooth-muscle relaxation and vasodilation.³⁵ Three different forms of NOS have been characterized: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS).³⁵ Nitric oxide can be produced either constitutively (eNOS, nNOS) or induced (iNOS) in a wide variety of cell types (e.g., endothelial cells, neurons, macrophages, respectively). Nitric oxide produced by eNOS plays a particularly important role in the localized regulation of vascular tone, whereas NO derived from nNOS is important in neuronal signal transduction. NO also plays important roles in the inhibition of smooth-muscle proliferation and in inflammation. The roles of NO in inflammation include inhibition of cell-mediated inflammation, reduction in platelet aggregation and adhesion, and as a regulator of leukocyte recruitment. Specifically, NO produced by cytokine-iNOS reduces leukocyte recruitment into sites of inflammation. NO can react with reactive oxygen intermediates to form both reactive oxygen and nitrogen species (e.g., NO + O₂⁻ → NO₂⁻ + HO•); it can inhibit DNA synthesis; it can directly kill microbes and tumor cells; and it can inactivate cytosolic glutathione and other sulfhydryl enzymes. NO and its generating enzymes, eNOS, nNOS, and iNOS, represent a regulatory system that has varied effects on the inflammatory response depending upon location and setting.

LYSOSOMAL GRANULE CONSTITUENTS

The activation of neutrophils, monocytes and macrophages results in the release, either through exocytosis or as the result of cell death, of a wide variety of proinflammatory mediators that have important roles in the inflammatory response. Neutrophils contain three major types of granules and also secretory vesicles (Chap. 60). Large, primary (azurophilic) granules contain myeloperoxidase, lysozyme, a variety of cationic proteins, defensins, phospholipase, acid hydrolases and neutral proteases (e.g., proteinase 3, collagenases, elastase). Smaller, secondary (specific) granules contain lactoferrin, lysozyme, type IV collagenase, subunits of NADPH oxidase and the β₂-integrin, CD11b/CD18. Tertiary granules contain gelatinase, subunits of NADPH oxidase and CD11b/CD18. Acid proteases function most efficiently within phagolysosomes where the pH is low, whereas neutral proteases can function efficiently within extracellular inflammatory exudates. Lysosomal granule constituents contribute to the inflammatory response and tissue injury through a wide array of mechanisms (e.g., degradation of extracellular matrix, proteolytic generation of chemotactic peptides and catalysis of reactive oxygen metabolite generation).

CYTOKINES AND CHEMOKINES

Cytokines are proteins that exhibit a variety of proinflammatory and antiinflammatory effects. They are produced by many cell types and modulate the function of other cell types. Individual cells may produce many different cytokines, and an individual cytokine may exert a wide variety of effects; they are pleiotropic.^2,3,6 In addition to their important roles in regulating various aspects of the immune response (e.g., lymphocyte activation, proliferation, and differentiation), many cytokines participate in innate immunity (e.g., TNF-α, IL-1β, IL-6, type I interferons), mediate the acute-phase response (TNF-α, IL1β, IL-6), activate inflammatory cells (e.g., IFN-γ) and participate in hematopoiesis (e.g., IL-3, granulocyte-monocyte colony-stimulating factor, granulocyte colony-stimulating factor, macrophage colony-stimulating factor).^2,3 Among the most thoroughly characterized cytokines are TNF-α and IL-1β, which are structurally dissimilar but share many biologic activities and can function as autocrine, paracrine and endocrine mediators of inflammation (Table 19–4).^2,3 TNF-α and IL-1β are produced by various cell types and are pleiotropic. Particularly important functions in inflammation include endothelial, leukocyte and fibroblast activation.

Elevated local (and sometimes systemic) concentrations of TNF-α, IL-1β, and IL-6 are consistently observed during the development of an inflammatory response. Based on their roles in the systemic acute-phase response and in the orchestration of important localized mechanistic steps in inflammation (e.g., induction of endothelial leukocyte adhesion molecules, phagocyte activation, procoagulant mediator induction), these mediators are prototypic “proinflammatory” cytokines. Their expression is regulated by nuclear factor κB (NFκB). NFκB is a transcription factor that exists as a heterodimer complexed with IκB (inhibitor κB) in the cytosol of many different cells types.^3,6 When cells are activated by various microbial products, viruses, reactive oxygen intermediates, cytokines, and chemotherapeutic agents, IκB is phosphorylated before it dissociates from NFκB heterodimers. Unbound NFκB translocates into the cell nucleus where it participates in the upregulation of as many as 200 different genes, including TNF-α, IL-1β and IL-6.

The proinflammatory cytokines and their activities are counterbalanced by a wide variety of “antiinflammatory” cytokines, including IL-4, IL-10, IL-11, IL-13, TGFβ, IL-1ra, and several soluble cytokine receptors.^2,3,7 IL-4, a 20-kDa peptide produced by CD4 Th2 cells, inhibits IL-1β synthesis and induces IL-1ra (IL-1 receptor antagonist). Soluble IL-1ra binds IL-1β (and IL-1α), preventing their binding to IL-1 receptors. IL-10 is also secreted by CD4 Th2 cells (and regulatory T cells, monocytes, and macrophages). Acting through its cognate receptor, IL-10 suppresses the expression of proinflammatory cytokines, adhesion molecules, chemokines, and cell-surface activation molecules of neutrophils, monocytes, macrophages, and T lymphocytes.⁷ IL-10 also induces the shedding of TNF-α receptors, which then function as soluble TNF-α antagonists. IL-11, IL-13, and TGFβ also each exert a set of activities that counter the proinflammatory actions of TNF-α, IL-1β, and IL-6. Recognition of the many counterbalancing actions between proinflammatory and antiinflammatory cytokines has led to the concept of “proinflammatory–antiinflammatory cytokine balance.” This concept is the basis for rational therapeutic strategies to manipulate or “reset” this balance.

TNF-α, IL-1β, and IL-6 are key proximate mediators of the “acute-phase response.” Stimuli such as bacterial endotoxin (lipopolysaccharide), exotoxins, immune complexes and physical stimuli (e.g., heat or trauma) can induce macrophages (and other cell types) to secrete TNF-α, IL-1β, and IL-6.^2,3,7 In turn, TNF-α, IL-1β, and IL-6 mediate fever, somnolence, increased production of proteins such as α₁-antitrypsin (α₁-antiprotease) and α₂-macroglobulin, and decreased production of proteins such as albumin and transferrin. As noted, the acute-phase response is a stereotyped host metabolic response to a wide variety of insults. In addition to the systemic acute-phase response, TNF-α and IL-1β induce endothelial activation marked by increases in leukocyte adherence and a procoagulant state, leukocyte activation marked by cytokine secretion, and fibroblast activation marked by proliferation, collagen synthesis, and collagenase production.^2,3,7 These actions are critical components of inflammation and wound healing; they exemplify the linkage between the inflammatory response and the coagulation system.

TNF-α, originally identified as “cachexin or cachectin” because of its role in the systemic wasting that accompanies some chronic infections and cancer, can induce cytokine production in a variety of cells.^2,3 TNF-α can induce neutrophil activation and the expression of adhesion molecules on endothelial cells. In contrast to IL-1β, TNF-α also possesses potent cytotoxic activities for some types of cells. Both IL-1β and TNF-α are produced in response to endotoxemia and both can mediate a systemic shock-like response.

IL-1β, which exhibits a wide variety of biologic activities, was initially termed endogenous pyrogen because of its ability to induce temperature elevation and the acute-phase response.^2,3,36 IL-1β is relevant to acute inflammation because of its ability to induce cytokine production in monocytes, macrophages, fibroblasts and endothelial cells. IL-1β can also induce NOS.³⁶ As noted previously, IL-1β can activate endothelial cells, resulting in the expression of adhesion molecules and a procoagulant phenotype.^2,3,36

IL-6 participates in the acute-phase response through the induction of proinflammatory mediators production by hepatocytes, via the differentiation of CD4 T lymphocytes that produce IL-17 and through the induction of marrow neutrophil production.^2,3 IL-6 is produced by a variety of cell types following activation by TNF-α, IL-1β, and pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharides (endotoxin), mannans, flagellin, and microbial nucleic acids.^3,6

Chemokines, or “intercrines,” are small proteins, which, in addition to many of the general properties of cytokines, exhibit prominent chemotactic activities.^3,6,37 Chemokines are grouped into four classes based on the amino acid sequence positions of conserved cysteine (C) residues in mature peptides.^6,37 The four classes include CC, CXC, XC, and CX₃C chemokines. There are four families of corresponding chemokine receptors: CCR, CXCR, XCR, and CX₃CR, respectively. Nomenclature of chemokines is based on the locations of N-terminal cysteine residues whereby “CC” indicates two adjacent residues, “CXC” indicates two cysteine residues separated by one intervening amino acid, and so on. Individual chemokines contain the letter “L” for ligand, followed by individual numbers (e.g., CCL1, CCL2, CCL3, etc.); more than 40 have been identified. The two most studied subfamilies include the alpha, or “CXC” chemokines, and the beta, or “CC” chemokines. Alpha chemokines, of which IL-8 (CXCL8) is the prototype, consistently exhibit neutrophil chemotactic activity, whereas the beta, or “CC” chemokines, of which MCP-1 (CCL2) is the prototype, exhibit monocyte chemotactic activity (Table 19–5).^37,38 Both in vitro and in vivo studies have provided insight into the roles of chemokines in inflammation. For example, MCP-1 knockout mice (MCP-1 −/−) exhibit reductions in monocyte influx into sites of experimentally induced peritonitis and delayed-type hypersensitivity.³⁹ Complementary studies using knockout mice devoid of the MCP-1 receptor CCR2 do not form typical granulomas.³⁹ These types of studies, as well as many that have employed specific chemokine-neutralizing antibodies or soluble chemokine receptor antagonists, have provided valuable insight into the pathophysiology of inflammation. Seemingly contradictory experimental results suggest that leukocyte recruitment mechanisms are multiple, overlapping or redundant, and not completely understood. Chemokine receptors noted above (CCR, CXCR, etc.) activate leukocytes through membrane receptors (sometimes called “serpentine” receptors) that contain seven transmembrane domains and are linked to cytosolic heterotrimeric G proteins.^3,6

INFLAMMATORY LIPIDS

Lipid mediators of inflammation, commonly derived from cell membrane precursor molecules, can act either intracellularly or extracellularly, the latter in a short-lived, localized manner.⁴¹ Arachidonic acid, a 20-carbon polyunsaturated fatty acid (5,8,11,14-eicosatetraenoic acid) derived either from dietary sources or by conversion from linoleic acid, is maintained in cell membranes as an esterified phospholipid. Three families of inflammatory mediators derived from arachidonic acid are generated via the cyclooxygenase and lipoxygenase pathways. Arachidonic acid is released from membrane phospholipids via cellular phospholipases such as phospholipase A₂. Phospholipase activation is triggered by mechanical/physical or chemical stimuli. Arachidonic acid can be metabolized via the cyclooxygenase pathway to prostaglandins (e.g., PGG₂, PGH₂, PGD₂, PGE₂, PGF₂), prostacyclin (PGI₂) or thromboxane (TXA₂).⁴¹ Prostacyclin mediates vasodilation and the inhibition of platelet aggregation; thromboxane has the opposite effects; and PGD₂, PGE₂, and PGF₂ mediate vasodilation and edema. Activation of the lipoxygenase pathway results in the synthesis of 5-hydroperoxyeicosatetraenoic acid (5-HPETE), which is a potent chemoattractant of neutrophils and can be enzymatically modified to yield a series of other leukotrienes. LTB₄ induces neutrophil chemotaxis, aggregation, degranulation, and adherence, while LTC₄, LTD₄, and LTE₄ trigger smooth-muscle constriction, increases in vascular permeability and bronchoconstriction.⁴¹ Members of both of these families of lipid-derived mediators and their catabolites have been detected in inflammatory exudates. There are two important branches within the lipoxygenase pathway.^41,42 Lipoxins (A₄ [LXA₄] and B₄ [LXB₄]) are generated via the 12-lipooxygenase branch of the lipoxygenase pathway in conjunction with a unique transcellular biosynthetic pathway.⁴² Neutrophils generate LTA₄ via the 5-lipoxygenase pathway branch; in turn, lipoxins (LXA₄ and LXB₄) are generated through the action of platelet 12-lipooxygense on neutrophil LTA₄. Prevention of neutrophil-platelet binding interrupts this pathway. Lipoxins inhibit neutrophil chemotaxis and adhesion to endothelium.⁴² As noted above, resolvins and protectins each encompass several molecular species—all derived from omega-3 polyunsaturated fatty acids.^26,27

PAF is a potent proinflammatory lipid produced by a variety of cell types, including neutrophils, monocytes, endothelial cells and IgE-sensitized basophils.⁴³ Derived from the cell membrane constituent, choline phosphoglyceride, PAF is an acetyl glycerol ether phosphocholine that is synthesized following the activation of phospholipase A₂. PAF triggers platelet aggregation and degranulation, increases vascular permeability, and promotes leukocyte accumulation and activation. In vivo studies using specific PAF antagonists have suggested a role for PAF in a variety of acute inflammatory lesions.⁴³

KININS

The kinin system is activated by contact activation of clotting factor XII (Hageman factor) (Chaps. 113 and 114).⁴⁴ Activation of the kinin system results in the generation of bradykinin, a nine-amino-acid vasoactive peptide. Bradykinin possesses several activities, including the capacity to increase vascular permeability, induce smooth-muscle contraction, trigger vasodilation, and cause pain.⁴⁴ Activated Hageman factor (factor XIIa), also known as the prekallikrein activator, converts plasma prekallikrein to kallikrein. In turn, kallikrein cleaves high-molecular-weight kininogen to produce bradykinin. Models of septic shock reveal decreases in plasma kininogen that parallel decreases in peripheral arterial resistance.⁴⁴

VASOACTIVE AMINES

Histamine and serotonin (5-hydroxytryptamine) are low-molecular-weight vasoactive amines. Histamine is contained in mast cell and basophil granules, whereas platelets are a chief source of serotonin.⁴⁵ Localized release of histamine results in wheal formation as a consequence of increases in vascular permeability. Histamine induces the formation of reversible openings in endothelial tight junctions, triggers the formation of prostacyclin by endothelial cells and induces NO release from the endothelium. In addition, histamine, like thrombin, can induce the rapid upregulation of endothelial P-selectin.⁴⁵ Serotonin, acting through receptors on vascular smooth-muscle cells, is responsible for vasoconstriction, whereas interaction with endothelial receptors results in vasodilation (via release of NO) and increased permeability.² Release of histamine and serotonin from mast cells and platelets can be triggered by IgE-mediated type I hypersensitivity reactions, directly by C3a or C5a, and directly by neutrophil granule-derived cationic proteins.

COMPLEMENT

The complement system, including its soluble and cell membrane-associated regulators, consists of nearly two dozen plasma proteins that give rise to mediators of chemotaxis, increased vascular permeability, opsonic activity, phagocyte activation, and cytolysis.⁴⁶ In a manner analogous to coagulation, the complement system is activated through a cascade of proteolytic cleavage reactions. There are three convergent pathways (Fig. 19–3). The first of these, the “classical pathway,” is initiated primarily (but not exclusively) by complement-fixing immune complexes (IgG subclasses 1 to 3 and IgM), whereas the second, the “alternative pathway,” is triggered by a variety of substances that include IgA aggregates, endotoxin, cobra venom factor, and polysaccharide moieties found on some bacterial and fungal cell walls. The third pathway, the “mannan-binding” lectin (MBL) pathway, is activated when MBL binds to a microorganism coated with certain carbohydrate moieties (e.g., mannans). Upon binding, MBL activates MBL-associated serine proteases (e.g., MASP-1, MASP-2) which function in a manner analogous to C1r and C1s of the classical pathway. MBL recognizes carbohydrate moieties infrequently present in mammalian hosts, thus constituting a system for recognizing foreign particulates. As such, the alternative and MBL pathways are considered to be part of the innate system of host defense.⁶ The classical pathway is initiated by the fixation of C1 (C1qr₂s₂) by the Fc portion of surface-bound IgG or IgM immunoglobulins. Activated C1 (C1qr₂s₂) cleaves C2 and C4, which leads to the formation of the “classical pathway” C3 convertase, C4b2a. Activation of the alternative pathway results in the formation of an “alternative pathway” C3 convertase following direct cleavage of C3 and subsequent interactions of C3b with factors B and D in the presence of Mg²⁺. The resulting complex, C3bBb, is stabilized by properdin, leading to the stable C3 convertase, C3bBbP. C3 convertases generated via any of the three pathways efficiently cleave C3 to form C3a and C3b.

These enzymatic reactions exhibit high activity levels, thus serving to dramatically amplify the cascade. C3b can bind to either the classical or alternative pathway C3 convertase to form a C5 convertase, which cleaves C5 into C5a and C5b. C5a is released into the fluid phase, like C3a, whereas C5b combines first with C6 and then C7 to form C5b-7, which, in turn, binds with C8 and multiple C9 molecules to form C5b-9, the membrane attack complex. In addition to the cell-activating and cytolytic activities of C5b-9, individual complement cleavage products and complexes mediate a variety of specific and potent proinflammatory activities.⁴⁶ These functions, combined with the rapid amplification in numbers of complement-derived mediators, emphasize the vital role of complement in acute inflammation. The most important activation products of complement appear to be C5a, a major chemotactic factor, and the anaphylatoxins (C3a, C4a, C5a), of which C3a is the most abundant. C5b-9 is a major cytotoxic product, provided that this complex is assembled on the surface of a susceptible cell (e.g., bacterium).

A series of soluble and cell membrane-associated complement proteins play important roles in the regulation of the complement cascade.⁴⁶ The pivotal regulator of the proximal arm of classical pathway is C1 esterase inhibitor (C1E-INH), a serine protease inhibitor that covalently bonds to the activated esterase subunits of the C1qrs complex thus preventing activation of the downstream zymogen cascade.⁴⁶ Defects in C1E-INH, from genetic defects or those acquired (e.g., neutralizing antibodies against C1E-INH), can result in angioedema. Angioedema can manifest in a variety of ways including as life-threatening laryngeal soft tissue swelling.

COAGULATION SYSTEM

The coagulation system is reviewed in detail in Chaps. 113, 114, and 116. The interrelationships among the coagulation system and several inflammatory mediator systems are important in the context of host defense and the pathophysiology of septic shock.⁴ Activation of the clotting cascade results in the generation of fibrinopeptides which increase vascular permeability and are chemotactic for leukocytes. Thrombin and tissue factor induce endothelial expression of P-selectin, resulting in increased neutrophil adhesion.¹² In addition, plasmin is responsible for the activation of Hageman factor, which then can activate the kinin system and can cleave C3 into its active components.⁴⁴ It can also generate “fibrin-split” or “fibrin-degradation” products. The induction of tissue factor in endothelial cells exposed to TNF-α and IL-1β further links the coagulation system to the inflammatory response.

PROTEINASE-ACTIVATED RECEPTORS

Proteinase-activated receptors (PARs) define an important general mechanism that links several seemingly disparate regulatory systems involved in inflammation.⁴⁷ PARs subsume a G-protein–coupled receptor subfamily defined by a common activation mechanism.⁴⁷ Individual PARs include an N-terminal extracellular domain, seven transmembrane helices connected by three intracellular and three extracellular loops, and linkage to cytosolic G-protein–mediated signal transduction pathways.⁴⁷ PARs are activated when extracellular proteinases cleave the N-terminal extracellular domain at a specific site which results in the creation of a “tethered ligand.” The tethered ligand is the residual, now unmasked N-terminal portion of the PAR; it interacts with the nearby nontruncated extracellular PAR domain and activates the receptor. The PAR family possesses of four members: PAR₁, PAR₂, PAR₃, and PAR₄. The extracellular domain of each PAR possesses several potential cleavage sites. For example, the canonical PAR₁ tethered ligand sequence created after cleavage by thrombin is the amino acid sequence, SFLLRN.⁴⁷ A wide variety of proteinases that are pivotal in inflammation, thrombosis, hemostasis, and wound healing (as well as in development and cancer progression) can activate PARs. PAR₁, PAR₃, and PAR₄ are susceptible to cleavage by thrombin. Other coagulation system-related proteinases, such as factor Xa, activated protein C, plasmin, and kallikreins can also activate PAR₁. Likewise, PAR₁ can also be activated by matrix metalloproteinase-1, neutrophil elastase, and neutrophil proteinase-3. Various proteinases cleave the N-terminal extracellular domain of PARs at different, yet specific, sites. Examples of PAR activation relevant to inflammation include thrombin-induced CCL2 expression in osteoblasts and PAR₂ and PAR₄ activation in animal models of arthritis. A goal of rational therapeutic design is to target crosstalk interactions using paired drugs or bifunctional agents. Although no PAR-targeting compounds have yet come into clinical use, this is a promising area.⁴⁷

The chronic inflammatory response and repair processes are, like the acute inflammatory response, highly regulated. By definition, “chronic” inflammation connotes a process that lasts at least several days and more often, weeks to months, sometimes years. Chronic inflammation is characterized by the recruitment of mononuclear cells including lymphocytes, monocytes and plasma cells, as well as by the proliferation of new capillaries (angiogenesis) and increases in the deposition of extracellular matrix. Replacement of damaged tissue by new small blood vessels and extracellular matrix constitutes a fundamental aspect of chronic inflammation and, simultaneously, is an integral part of wound healing and repair. The recruitment of this wide variety of cell types is achieved by complex interactions among cytokines, chemokines and indigenous cells. Great advances in understanding of angiogenesis and extracellular matrix molecule metabolism have been made in recent years.

Chronic inflammation can be caused by persistent infections with a wide variety of microorganisms (e.g., Treponema pallidum, Mycobacterium tuberculosis). In contrast to highly virulent organisms that trigger acute pyogenic infections (e.g., Streptococcus pneumoniae, Haemophilus influenzae), organisms that induce chronic inflammation typically exhibit relatively low intrinsic toxicity, are poorly cleared and may provoke a delayed-type hypersensitivity reaction. Chronic inflammation is also triggered by long-term exposure to insoluble exogenous particles (e.g., carbon dust, silica).² The initiation of other chronic inflammatory processes such as atherosclerosis and autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus) is less-well understood, but it is clear that a variety of environmental factors (e.g., diet in atherosclerosis) and genetic factors (e.g., human leukocyte antigen [HLA]-linked susceptibility in rheumatoid arthritis) are important. The characteristics of individual chronic inflammatory responses are dependent on the location of the injury and the type of injurious agent. As noted throughout this chapter, the recruitment of mononuclear cells into an inflammatory lesion is governed by the same general paradigm that orchestrates the recruitment of neutrophils into sites of acute inflammation. Unlike most acute conditions, chronic inflammatory processes are sometimes marked by a relatively specific morphology (e.g., granuloma formation in tuberculosis, eosinophil infiltration in parasitic infections) and by the coexistence of tissue repair (i.e., angiogenesis and extracellular matrix production).

A key cell type in chronic inflammatory processes is the macrophage.^3,5 Tissue macrophages are derived primarily from circulating blood monocytes and can adopt relatively specific functions based on their differentiation in selected body sites (e.g., hepatic Kupffer cells, alveolar macrophages, central nervous system microglia). In the setting of chronic inflammation, tissue macrophages can be activated by immunologic means (IFN-γ secreted by antigen-activated CD4 T lymphocytes) and by nonimmunologic means (microbial endotoxin, extracellular matrix proteins and foreign particulates; Chap. 67). In turn, activated macrophages enlarge, become more metabolically active, exhibit enhanced phagocytosis and secrete a large array of mediators.^3,5 Mediators secreted by activated macrophages include proteases, reactive oxygen and nitrogen intermediates, coagulation factors, arachidonic acid-derived lipids and cytokines. These mediators, as detailed in preceding sections, participate in inflammation. Activated macrophages also secrete collagenases that participate in tissue remodeling, angiogenic factors (e.g., fibroblast growth factor) and profibrogenic growth factors (fibroblast growth factor, TGF-β, platelet-derived growth factor).^3,5 Consequently, activated tissue macrophages participate in inflammation per se, tissue remodeling, angiogenesis and fibrosis.

Although macrophages play a central role in all facets of chronic inflammation, other cell types are also important. Lymphocytes (both B and T cells) are recruited into chronic inflammatory lesions via leukocyte–endothelial adhesive interactions and via chemotactic mechanisms analogous to those involved in neutrophil recruitment. Antigen-activated CD4 Th1 lymphocytes produce IFN-γ, which, as discussed above, is an important activator of tissue macrophages.³ Activated CD4 Th2 lymphocytes produce a variety of proinflammatory mediators that are involved in lymphocyte activation (e.g., IL-2) and in immune regulation (e.g., IL-5 in IgE production).³

Eosinophils and mast cells also play important roles in some types of chronic inflammation. Mast cells, which tend to be distributed along small blood vessels, possess high-affinity FcεRI receptors for IgE.⁴⁸ Engagement of mast cell-bound IgE triggers degranulation that leads to histamine and arachidonic acid–derived lipid release (Chap. 63). Eosinophils are characteristically formed in IgE-mediated allergic reactions and in parasitic infections (Chap. 62). Eotaxin, a CC chemokine, binds to and activates eosinophils via CCR3.^3,48 Recruited eosinophils secrete various granule proteins that help kill parasites, but which can also cause tissue damage. As inferred above, the histopathologic appearance of many chronic inflammatory lesions can provide insight into their pathogenesis and cause. A variety of poorly degraded, intrinsically low toxicity agents can induce granulomatous inflammation (e.g., M. tuberculosis).⁴⁹ Many parasites induce an eosinophilic response (e.g., Toxocara canis). Finally, the induction of tissue remodeling, angiogenesis and fibrosis can contribute to both tissue damage and repair, and can also suggest underlying etiology (e.g., lung fibrosis associated with asbestos). The tremendous advances in understanding of the inflammatory response hold great promise for the future of both diagnostics and therapeutics.