- Email: [email protected]
Inflammation in Horses
IMMUNOLOGY
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INFLAMMATION IN HORSES Robert
J.
MacKay, BVSc, PhD
Inflammation is vital to the survival of all complex organisms and has a profound central role in health and disease. Normally, inflammation is a subclinical localized response that destroys, dilutes, or walls off injurious agents or damaged tissues. In some cases, the inflammatory response is of such vigor as to cause the classic signs of dolor (p,ain), calor (heat), rubor (redness), tumor (swelling), and functio laesa (loss of function).27 Such inflammation usually has a salutary effect. With activation of the "counter-regulatory" (anti-inflammatory) systems there is resolution of disease and restoration of homeostasis. Occasionally, however, the inflammatory or counter-regulatory responses go awry, resulting in potentially lethal inflammatory or immunosuppressive disorders. 7 The processes of inflammation increasingly are being revealed as complex overlapping networks of cellular and humoral events with net effects dependent upon spatial, temporal, and contextual considerations. The following description is a consensus sequence of inflammatory events applicable to most inflammatory processes involving the innate immune system. After inflammation is initiated by detection of antigen, plasma components and activated leukocytes are concentrated at the inflammatory site. Cellular and chemical effectors of inflammation are focused on the offending antigen, usually resulting in its destruction and elimination. Activation of endogenous counter-regulatory systems damps down the inflammatory process and is the first stage of repair. In addition to local effects, the inflammatory focus may initiate a continuum of systemic acute-phase responses ranging from the systemic in-
From the Department of Large Animal Clinical Sciences, Large Animal Medicine Service, College of Veterinary Medicine, University of Florida, Gainesville, Florida
VETERINARY CLINICS OF NORTH AMERICA: EQUINE PRACTICE VOLUME 16 • NUMBER 1 • APRIL 2000
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flammatory response syndrome (SIRS) to generalized immunosuppression. This schema of inflammation is discussed herein in more detail. INFLAMMATION IS INITIATED BY THE DETECTION OF ANTIGEN
Antigen is material recognized as nonself by the innate or adaptive immune systems of the body. Frequentl~ antigen is in the form of pathogenic microbes, but host molecules may appear foreign in autoimmune conditions or after aseptic tissue necrosis. Detection of antigen typically is by resident mononuclear phagocytes (usually macrophages) patrolling in serosal or interstitial spaces or by plasma-derived proteins (Table 1). In some cases, phagocytes crawl toward the microbe up a concentration gradient of microbial chemoattractant. A prototypic chemoattractant of this type is N-formylmethionyl-leucyl-phenylalanine (fMLP) at the amino terminus of bacterial polypeptides; however, equine neutrophils appear unable to respond chemotactically to this peptide. 6 Once the phagocyte has contacted particulate antigen, attachment is mediated by cell membrane receptors. Carbohydrate molecules on the surface of most microbes are ligands for unlinked macrophage cellsurface lectins such as the scavenger, mannose, and asialoglycoprotein receptors. 17, 33, 36 Toxic microbial lipids often are detected by transmembrane receptors with connection to the interior of the macrophage. An example is CD14, which binds to the lipid A portion of gram-negative bacterial lipopolysaccharide and to lipotechoic acid of gram-positive bacteria. 19 Microbes also may be recognized by the low concentrations of plasma proteins present in normal tissues. In this way, phagocytes become indirectly but firmly attached to antigen via receptors for opso-
Table 1. ANTIGEN-RECOGNITION MOLECULES OF THE INNATE AND ADAPTIVE IMMUNE SYSTEMS Molecular
Innate Scavenger receptor Mannose receptor Asialoglycoprotein receptor ~2 integrins CD14 LPS-binding protein Clq Mannose-binding protein C3b Adaptive Immunoglobulin T-cell receptor
Target
Polyanions on bacteria, LPS, LTA CHO on bacteria, yeast, viruses, parasites CHO on bacteria, viruses, senescent RBC LPS, gram-negative bacteria LPS, LTA LPS Ig, bacteria, viruses, protozoa, mycoplasma CHO on bacteria, yeast, viruses, parasites Low sialic acid surfaces of microbes Specific antigen Specific antigen on APC
LPS = lipopolysaccharide; LTA = lipotechoic acid; CHO = carbohydrate; Ig = immunoglobulin; APC = antigen-presenting cells.
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nins such as C3b (via CD35, CD11c/CD18), and IgG (CD16, CD32, CD64). As a result of receptor engagement by microbes or their products (e.g., lipopolysaccharide), mononuclear phagocytes are stimulated to a higher level of respiratory and metabolic activity. Usually this results in phagocytosis of the antigenic particle and release of proinflammatory mediators. Among these mediators are reactive oxygen species (ROS), matrix metalloproteinases (MMPs )13, 14 and other hydrolases, cytokines, chemokines, and prostanoids. Some of these substances signal adjacent endothelium and begin the processes of recruitment of leukocytes and plasma proteins to the inflammatory site. Processed foreign antigen may be displayed on the surface of macrophages in association with histocompatability antigens. In this form, antigen can be recognized by antigen-specific receptors of T lymphocytes. The interactions of resident tissue macrophages. and plasma-derived proteins with antigen can be considered as the nucleating events in a rapidly escalating process of inflammation. ACTIVATED LEUKOCYTES ARE RECRUITED TO THE INFLAMMATORY SITE
Trafficking of leukocytes to inflamed tissues is dependent upon a sequential series of interactions between leukocyte and endothelium. 4, 5, 11 In acute inflammation, this begins within seconds of an inflammatory stimulus and results initially in the emigration of neutrophils. In response to inflammatory mediators such as platelet-activating factor (PAF), C3a, tumor necrosis factor (TNF), and thromboxane A 2 (TXA2 ), and chemoattractants such as C5a, leukotriene B4 (LTB 4 ), a (CC) chemokines, and ~ (CXC) chemokines, endothelial cells express P- and Eselectins and other adhesion molecules. 35 These adhesion molecules IItether" leukocytes via specific cell-membrane ligands (including Lselectin/ CD62) and a series of transient interactions between ligands and receptors allows leukocytes to roll along the endothelial surface (Fig. 1). Leukocyte capture is most efficient in areas of low shear force such as the walls of postcapillary venules. During rolling, leukocytes are activated or triggered" by selectins, chemokines, and PAF expressed on endothelial cells. Tethered leukocytes may be guided over the endothelial surface by chemotactic gradients, haptotactic (adhesion molecule) gradients, or both. 12 The firm attachment or arrest step of the cascade is mediated by the avid interaction of leukocyte integrins with adhesion molecules of the immunoglobulin superfamily expressed on endothelial cells. During firm attachment, the activated leukocyte spreads out and then squeezes between the intercellular junctions of adjacent endothelial cells. Leukocyte transmigration is dependent upon homophilic interaction between CD31 expressed both on leukocytes and on the intercellular surfaces of endothelial cells. More than 108 neutrophils/h may enter an inflamed joint by this process. II
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Figure 1. Adhesion of neutrophils to endothelium at sites of inflammation. Neutrophils are tethered, activated, and firmly attached via sequential but overlapping interactions with selectins, chemoattractants, and integrins.
Depending upon the array of trafficking molecules expressed, endothelial cells of different tissues may be relatively selective for leukocyte types. For example, neutrophils followed by monocytes typically are recruited to foci of acute bacterial infection, whereas lymphocytes emigrate in high numbers through the high endothelial vessels of lymph nodes and Peyer's patches. 34 As neutrophils enter into an inflammatory focus, "respiratory burst" activity is stimulated and partial degranulation occurs. Such activity is heightened by exposure to inflammatory mediators, phagocytosis, or both of particulate antigen. The NADPH-oxidase-dependent respiratory burst generates short-lived ROS including superoxide anion (O~), singlet oxygen C02), hydrogen peroxide (H20 2 ), and hydroxyl radical (-OH).30 In the presence of granule myeloperoxidase, highly toxic hypohalous acids (usually hypochlorous acid) are formed that are corrosive to both pathogen and host. 25 The broad-spectrum antimicrobial effect of systemic iodide treatment is thought to relate to upregulated hypoiodous acid formation by this pathway. Other neutrophil granule contents include antimicrobial proteins such as lysozyme, lactoferrin, bactericidal/ permeability-increasing protein and defensins, digestive hydrolases, and matrix proteases such as elastase and collagenase that facilitate passage of neutrophils across the basement membrane and through tissues. I8 During initial passage of neutrophils into inflamed tissues there also is rapid processing of membrane phospholipids into lipid or peptidolipid mediators such as PAF and LTB 4. Freshly emigrated monocytes also are stimulated to produce ROS and prostanoids. In certain tissues (including joints in horses),9 early effects of inflammation are amplified by the proinflammatory effect of neuropeptides released from sensory nerve endings. This class of mediators,
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exemplified by substance P but also including vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), and somatostatin, cause vasodilatation, influx and activation of leukocytes, and upregulation of cytokine synthesis. 2o,32 This early phase of inflammation is characterized by an exponential increase in numbers of inflammatory cells and results in an environment rich in mediators and effector molecules. These may include (1) microbial products including exotoxins, degradative proteases, endotoxin, and lipotechoic acid; (2) products of leukocyte and endothelial cell membrane phospholipid metabolism including leukotrienes, thromboxanes, prostaglandins, lipoxins, and PAF; (3) ROS and hypohalous acid; and (4) granule contents of neutrophils (and other granulocytes). Over minutes to hours after the initiation of inflammation there is de novo synthesis and secretion by macrophages and other cells of proinflammatory cytokines such as interleukin 1(3 (IL-1(3), TNF, and IL-6 and chemokines such as IL-8. 3, 16, 24 During this period there also is induction, principally in macrophages and endothelial cells, of three important enzymes that in comparison with constitutive homologues generate massive amounts of product: (1) cyclooxygenase II (COX-II), which produces prostanoids such as thromboxane, prostacyclin (PGI2) and PGE226; (2) inducible nitric oxide synthase (iNOS), which catalyzes formation of NO from arginine21,22; and (3) heme oxygenase 1 (HO-1), which processes heme to bile pigments and CO.38 These products have potent regulatory effects. PLASMA COMPONENTS ENTER THE INFLAMMATORY SITE
Beginning early in inflammation, plasma exudes into affected tissues through permeable capillaries and postcapillary venules. Exposure of plasma components to basement membrane, other extracellular structures, or microbes results in activation of the kinin-kininogen, contact coagulation, extrinsic coagulation, and complement cascades, resulting in the liberation of numerous active mediators and effector molecules. The contact system of plasma begins with the activation of coagulation factor XII (Hageman factor) and proceeds down dichotomous pathways to bradykinin and fibrin formation. 1, 23 Factor XII is activated to factor Xlla by contact with macromolecular complexes exposed or formed during inflammation. Factor Xlla in turn liberates the active protease factor Xla from factor XI and initiates the intrinsic coagulation cascade. Factor Xlla also converts plasma prekallikrein to kallikrein; kallikrein then digests high-molecular-weight kininogen to liberate bradykinin, a potent vasoactive peptide. During inflammation, all of the components of the contact system are thought to be located on the surface of endothelial cells. Bradykinin also is generated by a tissue pathway. Tissue kallikrein and plasma amino-peptidase present in inflammatory exudate digest low-molecular-weight kininogen to release
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bradykinin. Bradykinin acts on cellular B2 receptors to cause venular dilatation, increased vascular permeability, and activation of phospholipase A 2 to begin arachidonic acid metabolism. In the absence of inflammation, the antithrombotic phenotype of endothelial cells is maintained by expression of thrombomodulin and heparan sulfate proteoglycan species, presence of low amounts of PGI2 and NO, and receptor-mediated. engagement of protein C and tissue plasminogen activator. With stimulation, endothelium supports thrombosis by loss of expression of antithrombotic molecules and upregulation of plasminogen activator inhibitor 1 (PAl-I) and procoagulant tissue factor. Procoagulant activity on endothelial cells and phagocytes activates the extrinsic pathway of coagulation; thrombin formation on endothelial surfaces promotes platelet adherence and platelet activation is stimulated by surface-bound PAF. Complement proteins from plasma can recognize and eliminate pathogens and altered host cells via any to all of three different pathways-namely, classic mannose-binding lectin (MBL), and alternative. Is The classic pathway is initiated by the binding of Clq to immunoglobulin multimers (Le., IgM or 2::2 IgG) or to numerous bacteria and viruses. Mannose-binding lectin is activated by binding to high-mannose polysaccharides found on numerous microbes but not on host cells. To initiate the alternative pathwa)T, C3b generated by autodigestion binds covalently to microbial surfaces where it is protected from the degradative actions of regulatory factors Hand 1. Each of the three recognition/ activation pathways culminates in the production of a C3 convertase. These serine proteases mediate cleavage and activation of large numbers of C3 molecules, thereby generating C3b and enormously amplifying complement activation at this crucial central stage of the reaction sequence. Complexes of C3-associated proteins on cell surfaces initiate formation of the hydrophobic transmembrane pore structure known as the membrane attack complex (MAC). Initially, C5 is cleaved to form C5b and then C5b-8 is assembled nonenzymatically into the cell membrane. Finall)T, 12 to 18 molecules of C9 polymerize onto each C5b-8 complex. In addition to the MAC, which has been shown to be cytolytic against some bacteria, activation of complement generates numerous active peptides with potent regulatory roles in inflammation. Several complement proteins, including Clq, MBL, C3b, iC3b, C4b, and iC4b bind to foreign surfaces and target them for ingestion and destruction by phagocytic cells. Neutrophils, monocytes, and macrophages bind to these opsonins via surface receptors including CD35, CDllb / CDI8, and CDllc/ CDI8. Opsonization by complement leads to phagocytosis if the phagocytic cell is exposed to a second activating signal (e.g., PAF, cytokines, Ig). The anaphylatoxins C3a, C4a, and C5a increase vascular permeability, cause vasodilatation, and have indirect proinflammatory action via induction of mast cell degranulation and activation of phospholipase A 2 • C5a also has potent chemotactic and activating activities for neutrophils, monocytes, and macrophages.
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Finally, plasma immunoglobulins are the recognition molecules of the humoral arm of adaptive immune system. They mediate epitopespecific opsonization and phagocytosis, mast cell degranulation, complement activation (by the classic pathway), phagocyte activation, and antibody-dependent cellular cytotoxicity. The role of immunoglobulins and the adaptive immune response are discussed in more detail elsewhere in this issue. PROINFLAMMATORY CELLS AND MEDIATORS ARE ACTIVE IN THE INFLAMMATORY SITE
As previously described, during the amplification stage of inflammation, there is activation of endothelial cells and recruitment and stimulation of large numbers of inflammatory leukocytes (mainly neutrophils and macrophages). In most cases, this leads to internalization of microbes or other material (e.g., immune complexes). Phagosomes containing internalized microbes fuse with cytoplasmic organelles to form phagoendosomes or phagolysosomes wherein microbes are killed by the oxygen-dependent and independent means previously described.' Some microbes escape destruction by preventing phagosome-organellar fusion or acidification of the phagosome or by escaping into the cytosol. Other microbes subvert oxygen-dependent mechanisms by deploying scavenger systems such as superoxide dismutase. As the inflammatory site matures, phagocytes, particularly macrophages, acquire new killing capabilities that may dispatch even these resistant organisms. Interferon ~ (IFN-~) production by natural killer (NK) cells in the inflammatory site is stimulated by small amounts of IL-12 and IL-18 secreted by stimulated macrophages. IFN-~ in combination with other cytokines or microbial products activate macrophages to produce NO (via action of iNOS) and reactive nitrogen species (RNS), molecules with potent microbicidal effects. Adaptive immune responses either may greatly upregulate macrophage function in this way (THI responses) or may suppress such responses (TH2 responses). In addition to their salutary defensive function, inflammatory cells and mediators often have adverse local effects on host tissues. Many vasoactive mediators are produced at inflammatory sites (Table 2) but the predominant early effect is vasodilatation, evident clinically as redness and heat. In some circumstances, the same mediator can cause relaxation of arterioles and constriction of venules. 37 This phase of the inflammatory response correlates closely with the induction of COX-II in numerous cell types by numerous inflammatory mediators, suggesting that products of this enzyme, such as PGE 2 and PGI2t are particularly important vasodilators. Leakiness of capillaries and venules is induced by many mediators (e.g., cytokines such as IFN-~, as well as bradykinin, PAF, histamine, anaphylatoxin, and thrombin) in combination with infiltration of leukocytes and causes swelling and edema of tissues. Extracellular matrix is liquefied by MMP produced by stimulated
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Table 2. VASOACTIVE AGENTS GENERATED DURING INFLAMMATION Vasodilators
PGE 2 PGI 2 Bradykinin
NO
Vasoconstrictors TXA2 PGF 2a
Angiotensin II Endothelin-l
CO Anaphylatoxins Histamine Substance P Lipoxins Serotonin Acetylcholine ADP PGE z = Prostaglandin Ez; PGlz = prostacyclin; NO = nitric oxide; CO adenosinephosphate; TXA z = thromboxane Az; PGF za = prostaglandin Fza .
=
carbon dioxide; ADP
=
leukocytes. This facilitates migration of leukocytes but also may irreversibly damage host tissues. Important examples are the destruction of cartilage matrix that occurs in arthritic jointslO and laminitic hooves. 29 Inflamed tissues are damaged in a more general way by exposure to the microbicidal products of stimulated leukocytes, particularly ROS and RNS. Deposition of fibrin serves to localize inflammatory lesions but also may be the first step in adhesion formation or intravascular coagulation. ACTIVATION OF ENDOGENOUS COUNTERREGULATORY SYSTEMS DAMPS DOWN THE INFLAMMATORY PROCESS AND IS THE FIRST STAGE OF REPAIR
The same stimuli that generate proinflammatory responses also evoke, with slightly delayed kinetics, anti-inflammatory or regulatory molecules that serve to rein in the inflammatory response, contain it locally, and restore homeostasis. Macrophages are exposed to deactivating signals including PGE 2I IL-IO, cortisol, and TGF-~. Furthermore, macrophages become tolerant to the actions of proinflammatory mediators. TNF activity is neutralized by soluble receptor, and IL-l receptor antagonist competes with active IL-l for receptor sites. In the yin and the yang of inflammation, all of the proinflammatory cascades of plasma proteins induce parallel cascades of regulatory molecules. Reactive oxygen and nitrogen species and degradative enzymes are consumed on host macromolecules and acute-phase proteins. Even intracellularly proinflammatory pathways are turned off by the actions of inflammationinduced heat-shock proteins. Within hours of extravasation, neutrophils undergo apoptosis in response to signals in the inflammatory milieu such as Fas ligand and TNF. Apoptotic neutrophils undergo cell death in an orderly manner so that lysosomal enzymes are not released extra-
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cellularly. These cells usually undergo phagocytosis via the scavenger receptors of macrophages. 31 Phagocytosis of apoptotic neutrophils is evident microscopically in inflammatory exudates as leukophagocytosis. IN ADDITION TO LOCAL EFFECTS, THE INFLAMMATORY FOCUS MAY INITIATE A CONTINUUM OF SYSTEMIC RESPONSES
Most inflammatory sites that are clinically evident elicit a systemic acute-phase response. Broadly interpreted, the acute-phase response includes all global processes initiated by the inflammatory site. These may include fever, leukocytosis, anemia of chronic disease, production by the liver of acute-phase proteins, increase in circulating concentration of copper and reduced iron and zinc concentration,2 and stimulation of the neuroendocrine system. In most cases, the acute-phase response acts to combat inflammation and restore systemic homeostasis. Extreme examples of the acute-phase response are the systemic inflammatory response syndrome or generalized immunosuppression. Acute-phase response proteins are normal plasma constituents whose rate of synthesis by the liver is affected positively or negatively by inflammation. The principal signal for acute-phase response protein synthesis by hepatocytes is IL-6, which originates from the inflammatory site and circulates to the liver. Glucocorticoids and other cytokines including TNF, IL-IB, IFN-')', and transforming growth factor B (TGF-B) may influence the pattern of acute-phase response proteins induced by IL-6. The function of acute-phase response proteins generally is to facilitate local inflammatory processes but minimize harmful side effects; thus, fibrinogen is a substrate for thrombin action; complement proteins C3 and C4 are important in microbial recognition and elimination; ceruloplasmin and hemopexin are antioxidants; plasminogen, tissue plasminogen activator, PAl-I, and protein S are fibrinolytics and anticoagulants; and numerous acute-phase response proteins are protease inhibitors (including
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be a common terminal phase characterized by malignant global activation of proinflammatory pathways known as the systemic inflammatory response syndrome (SIRS). The cause of SIRS in hypovolemic shock is unclear but several mechanisms have been proposed. (1) Ischemic damage to endothelial cells exposes blood to procoagulant/proinflammatory substrates. (2) Prolonged splanchnic ischemia allows for translocation of microbes and/ or microbial products from the gut lumen into blood, causing secondary generalized septic shock. (3) Vigorous clinical resuscitation efforts are tantamount to whole body ischemia-reperfusion. The endothelial cells of certain organs are rich in xanthine dehydrogenase (XD), an enzyme that is degraded permanently to xanthine oxidase during ischemia. -Upon reperfusion, xanthine oxidase converts O 2 to superoxide anion, a proinflammatory oxygen radical. Once the SIRS is set in motion by LPS or other stimuli, all the proinflammatory events described for local inflammation are activated within the cardiovascular system, resulting in a bewildering array of circulating harmful mediators. These include cytokines (e.g., TNF, IL1~, IL-8), lipids (thromboxanes, prostaglandins, lipoxins), glycolipids (platelet-activating factor), peptidolipids (leukotrienes), anaphylatoxins (C3a), vasoactive proteins (bradykinin, endothelin), chemotactic proteins (C5a), ROS, and vasoactive gases (NO, CO). Because the proximate stimuli for these mediators frequently overlap and multiple mediators have the same activity (redundancy), it is readily apparent that neutralization of any single mediator is unlikely to be the "magic bullet" treatment for SIRS. The net effect of SIRS in horses usually is increased pulmonary vascular resistance (an effect of thromboxane A 2 ), systemic vasodilatation (prostacyclin, bradykinin, NO?, CO?), reduced cardiac contractility, capillary leakiness (LTB 4I bradykinin, C3a), and extravasation of neutrophils. As a consequence, endothelial surfaces are activated and damaged and become substrate for tissue factor expression and activation of the extrinsic coagulation pathway. These insults-namely, intravascular coagulation plus reduced perfusion pressure because of plasma leakage, systemic vasodilatation, and reduced myocardial contractility-combine to deprive tissues of oxygen. Organ dysfunction ensues comparable to that seen with hypovolemic/hemorrhagic shock. The effect of tissue hypoperfusion is compounded by an endotoxin-induced defect in oxygen extraction by tissues. Anaerobic glycolysis combined with direct inhibition of pyruvate dehydrogenase generate intense local lactic acidosis. SIRS may progress into a syndrome of irreversible shock. The discussion to this point has dealt with the SIRS phenomenon that dominates the acute clinical presentation of horses in septic shock. As previously described, there also is a counter-regulatory response mounted by the host that is designed to check the inflammatory response? This includes production of macrophage deactivators (IL-4, IL10, PGE 2I cortisol), soluble cytokine receptors that can neutralize IL-1 or TNF, receptor antagonists that can prevent binding of cytokines to cells, and intracellular responses (heat shock proteins) that protect cells from
INFLAMMATION IN HORSES Normal Response
Pro-inflammatory
Counter-regulatory
Net Effect
SIRS
Immunosuppression
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Figure 2. Net inflammatory responses during normal inflammation, systemic inflammatory response syndrome (SIRS), and immunosuppression. Each panel depicts the intensity of the inflammatory response over time. A positive response indicates proinflammatory influences, whereas a negative response indicates activation of the counter-regulatory system. During a normal response (left panels), there is brief proinflammatory environment (bottom left panel), then homeostasis is restored. In contrast, there is persistent inflammation in SIRS (central panels) and persistent immunosuppression when the counter-regulatory system is overinduced (right panels).
inflammatory damage. This response may be activated systemically in a way analogous to the SIRS. The SIRS and counter-regulatory systems ideally cancel each other out (Fig. 2), allowing repair systems to begin and homeostasis to be restored. If proinflammatory responses predominate, the auto-cannibalistic" events of septic shock ensue; if counterregulatory systems persist, a state of dangerous generalized immunosuppression may exist-such is the case in human patients with severe burns and is likely in some horses after large intestinal surgery or enterocolitis. /I
References 1. Auer DE, Ng JC, Reilly JS, et al: Assessment of histamine, bradykinin, prostaglandins E1 and E2 and carrageenin as vascular permeability agents in the horse. J Vet Pharmacol Ther 14:61-69, 1991 2. Auer DE, Ng JC, Thompson HL, et al: Acute phase response in horses: Changes in plasma cation concentrations after localised tissue injury. Vet Rec 124:235-239, 1989 3. Bazzoni F, Beutler B: The tumor necrosis factor ligand and receptor families. N Engl J Med 334:1717-1725, 1996 4. Bevilacqua M~ Nelson RM, Mannori G, et al: Endothelial-leukocyte adhesion molecules in human disease. Annu Rev Med 45:361-378, 1994 5. Bochsler PN, Slauson DO, Neilsen NR: Modulation of an adhesion-related surface antigen on equine neutrophils by bacterial lipopolysaccharide and antiinflammatory drugs. J Leukoc BioI 48:306-315, 1990 6. Bochsler PN, Slauson DO, Neilsen NR: Secretory activity of equine polymorphonUclear leukocytes: stimulus specificity and priming effects of bacterial lipopolysaccharide. Vet Immunol Immunopathol 31:241-253, 1992
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7. Bone RC: Immunologic dissonance: A continuing evolution in our understanding of the systemic inflammatory response syndrome (SIRS) and the multiple organ dysfunction syndrome (MODS) [see comments]. Ann Intern Med 125:680-687, 1996 8. Bone RC: The sepsis syndrome: Definition and general approach to management. Clin Chest Med 17:175-181, 1996 9. Bowker RM, Sonea 1M, Vex KB, et al: Substance P innervation of equine synovial membranes: Joint differences and neural and nonneural receptor localizations. Neurosci Lett 164:76-80, 1993 10. Brama PA, TeKoppele JM, Beekman B, et al: Matrix metalloproteinase activity in equine synovial fluid: Influence of age, osteoarthritis, and osteochondrosis. Ann Rheum Dis 57:697-699, 1998 11. Carlos TM, Harlan JM: Leukocyte-endothelial adhesion molecules. Blood 84:20682101, 1994 12. Carter SB: Haptotaxis and the mechanism of cell motility. Nature 213:256-260, 1997 13. Clegg PD, Burke RM, Coughlan AR, et al: Characterisation of equine matrix metalloproteinase 2 and 9; and identification of the cellular sources of these enzymes in joints [see comments]. Equine Vet J 29:335-342, 1997 14. Clegg PD, Coughlan AR, Carter SD: Equine TIMP-1 and TIMP-2: Identification, activity and cellular sources. Equine Vet J 30:416-423, 1998 15. Cooper NR: Biology of the complement system. In Gallin JI, Snyderman R (eds): Inflammation: Basic Principles and Clinical Correlates, ed 3. Philadelphia, LippincottWilliams & Wilkins, 1999 16. Dinarello CA: Biologic basis for interleukin-1 in disease. Blood 87:2095, 1996 17. Elomaa 0, Kangas M, Sahlberg C, et al: Cloning of a novel bacteria-binding receptor structurally related to scavenger receptors and expressed in a subset of macrophages. Cell 80:603, 1995 18. Elsbach ~ Weiss J, Levy 0: Oxygen-independent antimicrobial systems of phagocytes. In Gallin JI, Snyderman R (eds): Inflammation: Basic Principles and Clinical Correlates, ed 3. Philadelphia, Lippincott-Williams & Wilkins, 1999 19. Grunwald U, Fan X, Jack RS, et al: Monocytes can phagocytose gram-negative bacteria by a CD14-dependent mechanism. J Immunol 157:4119--4125, 1996 20. Gutwald J, Goebeler M, Sorg C: Neuropeptides enhance irritant and allergic contact dermatitis. J Invest Dermatol 96:695-698, 1991 21. Hammond RA, Hannon R, Frean SP, et al: Endotoxin induction of nitric oxide synthase and cydooxygenase-2 in equine alveolar macrophages. Am J Vet Res 60:426--431, 1999 22. Hobbs AJ, Moncada S: Inducible nitric oxide synthesis and inflammation. In Willoughby DA, Tomlinson A (eds): Inducible enzymes in the inflammatory response. Basel, Birkhauser Verlag, 1999 23. Kaplan A~ Kusumam J, Shibayama ~ et al: Bradykinin formation: Plasma and tissue pathways and cellular interactions. In Gallin JI, Snyderman R (eds): Inflammation: Basic Principles and Clinical Correlates, ed 3. Philadelphia, Lipincott Williams & Wilkins, 1999, p 347 24. Kaplan D: Autocrine secretion and the physiological concentration of cytokines. Immunol Today 17:303-304, 1996 25. Klebanoff SJ: Myeloperoxidase: Occurrence and biological function. In Everse J, Everse KE, Grisham MB (eds): Peroxidases in Chemistry and Biology. Boca Raton, CRC Press, 1991 26. Loll PJ, Garavito RM: The isoforms of cyclooxygenase: Structure and function. Current Opinions in Investigational Drugs 3:1171-1180, 1994 27. Majno G: The Healing Hand: Man and Wound in the Ancient World. Cambridge, MA, Harvard University Press, 1975 28. Pepys MB, Baltz ML, Tennent GA, et al: Serum amyloid A protein (SAA) in horses: Objective measurement of the acute phase response. Equine Vet J 21:106-109, 1989 29. Pollitt CC, Pass MA, Pollitt S: Batimastat (BB-94) inhibits matrix metalloproteinases of equine laminitis. Equine Vet J Suppl 119-124, 1998 30. Rossi F: The 02-forming NADPH oxidase of the phagocytes: Nature, mechanisms of activation and function. Biochim Biophys Acta 853:65-89, 1986 31. Savill J: Macrophage recognition of senescent neutrophils. Clin Sci 83:649-655, 1992
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32. Smith CH, Barker IN, Morris RW, et al: Neuropeptides induce rapid expression of endothelial cell adhesion molecules and elicit granulocytic infiltration in human skin. J Immunol151:3274-3282, 1993 33. Speiss M: The asialoglycoprotein receptor: A model for endocytic transport receptors. Biochemistry 29:10009-10018, 1990 34. Springer TA: Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu Rev Physiol 57:827-872, 1995 35. Springer TA: Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. In Paul LC, Issekutz TB (eds): Adhesion Molecules in Health and Disease. New York, Marcel Dekker, 1999 36. Tenner AJ, Robinson SL, Ezekowitz RA: Mannose binding protein (MBP) enhances mononuclear phagocyte function via a receptor that contains the 126,000 M(r) component of the C1q receptor. Immunity 3:485--493, 1995 37. Venugopalan CS, Moore RM, Holmes EP, et al: Biphasic responses of equine colonic vessel rings to vasoactive inflammatory mediators. J Auton Pharmacol18:231-237, 1998 38. Willis D: Overview of HO-1 in inflammatory pathologies. In Willoughby DA, Tomlinson A (eds): Inducible Enzymes in the Inflammatory Response. Basel, Birkhauser Verlag, 1999, p 91 39. Yamashita K, Fujinaga T, Okumura M, et al: Serum C-reactive protein (CRP) in horses: The effect of aging, sex, delivery and inflammations on its concentration. J Vet Med Sci 53:1019-1024, 1991
Address reprint requests to Robert J. MacKa)T, BVSc, PhD Department of Large Animal Clinical Sciences College of Veterinary Medicine University of Florida PO Box 100136, JHMHSC Gainesville, FL 32610 e-mail: [email protected]£1.edu
0749-0739/00 $15.00
+ .00
INFLAMMATION IN HORSES Robert
J.
MacKay, BVSc, PhD
Inflammation is vital to the survival of all complex organisms and has a profound central role in health and disease. Normally, inflammation is a subclinical localized response that destroys, dilutes, or walls off injurious agents or damaged tissues. In some cases, the inflammatory response is of such vigor as to cause the classic signs of dolor (p,ain), calor (heat), rubor (redness), tumor (swelling), and functio laesa (loss of function).27 Such inflammation usually has a salutary effect. With activation of the "counter-regulatory" (anti-inflammatory) systems there is resolution of disease and restoration of homeostasis. Occasionally, however, the inflammatory or counter-regulatory responses go awry, resulting in potentially lethal inflammatory or immunosuppressive disorders. 7 The processes of inflammation increasingly are being revealed as complex overlapping networks of cellular and humoral events with net effects dependent upon spatial, temporal, and contextual considerations. The following description is a consensus sequence of inflammatory events applicable to most inflammatory processes involving the innate immune system. After inflammation is initiated by detection of antigen, plasma components and activated leukocytes are concentrated at the inflammatory site. Cellular and chemical effectors of inflammation are focused on the offending antigen, usually resulting in its destruction and elimination. Activation of endogenous counter-regulatory systems damps down the inflammatory process and is the first stage of repair. In addition to local effects, the inflammatory focus may initiate a continuum of systemic acute-phase responses ranging from the systemic in-
From the Department of Large Animal Clinical Sciences, Large Animal Medicine Service, College of Veterinary Medicine, University of Florida, Gainesville, Florida
VETERINARY CLINICS OF NORTH AMERICA: EQUINE PRACTICE VOLUME 16 • NUMBER 1 • APRIL 2000
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flammatory response syndrome (SIRS) to generalized immunosuppression. This schema of inflammation is discussed herein in more detail. INFLAMMATION IS INITIATED BY THE DETECTION OF ANTIGEN
Antigen is material recognized as nonself by the innate or adaptive immune systems of the body. Frequentl~ antigen is in the form of pathogenic microbes, but host molecules may appear foreign in autoimmune conditions or after aseptic tissue necrosis. Detection of antigen typically is by resident mononuclear phagocytes (usually macrophages) patrolling in serosal or interstitial spaces or by plasma-derived proteins (Table 1). In some cases, phagocytes crawl toward the microbe up a concentration gradient of microbial chemoattractant. A prototypic chemoattractant of this type is N-formylmethionyl-leucyl-phenylalanine (fMLP) at the amino terminus of bacterial polypeptides; however, equine neutrophils appear unable to respond chemotactically to this peptide. 6 Once the phagocyte has contacted particulate antigen, attachment is mediated by cell membrane receptors. Carbohydrate molecules on the surface of most microbes are ligands for unlinked macrophage cellsurface lectins such as the scavenger, mannose, and asialoglycoprotein receptors. 17, 33, 36 Toxic microbial lipids often are detected by transmembrane receptors with connection to the interior of the macrophage. An example is CD14, which binds to the lipid A portion of gram-negative bacterial lipopolysaccharide and to lipotechoic acid of gram-positive bacteria. 19 Microbes also may be recognized by the low concentrations of plasma proteins present in normal tissues. In this way, phagocytes become indirectly but firmly attached to antigen via receptors for opso-
Table 1. ANTIGEN-RECOGNITION MOLECULES OF THE INNATE AND ADAPTIVE IMMUNE SYSTEMS Molecular
Innate Scavenger receptor Mannose receptor Asialoglycoprotein receptor ~2 integrins CD14 LPS-binding protein Clq Mannose-binding protein C3b Adaptive Immunoglobulin T-cell receptor
Target
Polyanions on bacteria, LPS, LTA CHO on bacteria, yeast, viruses, parasites CHO on bacteria, viruses, senescent RBC LPS, gram-negative bacteria LPS, LTA LPS Ig, bacteria, viruses, protozoa, mycoplasma CHO on bacteria, yeast, viruses, parasites Low sialic acid surfaces of microbes Specific antigen Specific antigen on APC
LPS = lipopolysaccharide; LTA = lipotechoic acid; CHO = carbohydrate; Ig = immunoglobulin; APC = antigen-presenting cells.
INFLAMMATION IN HORSES
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nins such as C3b (via CD35, CD11c/CD18), and IgG (CD16, CD32, CD64). As a result of receptor engagement by microbes or their products (e.g., lipopolysaccharide), mononuclear phagocytes are stimulated to a higher level of respiratory and metabolic activity. Usually this results in phagocytosis of the antigenic particle and release of proinflammatory mediators. Among these mediators are reactive oxygen species (ROS), matrix metalloproteinases (MMPs )13, 14 and other hydrolases, cytokines, chemokines, and prostanoids. Some of these substances signal adjacent endothelium and begin the processes of recruitment of leukocytes and plasma proteins to the inflammatory site. Processed foreign antigen may be displayed on the surface of macrophages in association with histocompatability antigens. In this form, antigen can be recognized by antigen-specific receptors of T lymphocytes. The interactions of resident tissue macrophages. and plasma-derived proteins with antigen can be considered as the nucleating events in a rapidly escalating process of inflammation. ACTIVATED LEUKOCYTES ARE RECRUITED TO THE INFLAMMATORY SITE
Trafficking of leukocytes to inflamed tissues is dependent upon a sequential series of interactions between leukocyte and endothelium. 4, 5, 11 In acute inflammation, this begins within seconds of an inflammatory stimulus and results initially in the emigration of neutrophils. In response to inflammatory mediators such as platelet-activating factor (PAF), C3a, tumor necrosis factor (TNF), and thromboxane A 2 (TXA2 ), and chemoattractants such as C5a, leukotriene B4 (LTB 4 ), a (CC) chemokines, and ~ (CXC) chemokines, endothelial cells express P- and Eselectins and other adhesion molecules. 35 These adhesion molecules IItether" leukocytes via specific cell-membrane ligands (including Lselectin/ CD62) and a series of transient interactions between ligands and receptors allows leukocytes to roll along the endothelial surface (Fig. 1). Leukocyte capture is most efficient in areas of low shear force such as the walls of postcapillary venules. During rolling, leukocytes are activated or triggered" by selectins, chemokines, and PAF expressed on endothelial cells. Tethered leukocytes may be guided over the endothelial surface by chemotactic gradients, haptotactic (adhesion molecule) gradients, or both. 12 The firm attachment or arrest step of the cascade is mediated by the avid interaction of leukocyte integrins with adhesion molecules of the immunoglobulin superfamily expressed on endothelial cells. During firm attachment, the activated leukocyte spreads out and then squeezes between the intercellular junctions of adjacent endothelial cells. Leukocyte transmigration is dependent upon homophilic interaction between CD31 expressed both on leukocytes and on the intercellular surfaces of endothelial cells. More than 108 neutrophils/h may enter an inflamed joint by this process. II
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MACKAY
.~ Chemoattractants~~'${@ff.
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Figure 1. Adhesion of neutrophils to endothelium at sites of inflammation. Neutrophils are tethered, activated, and firmly attached via sequential but overlapping interactions with selectins, chemoattractants, and integrins.
Depending upon the array of trafficking molecules expressed, endothelial cells of different tissues may be relatively selective for leukocyte types. For example, neutrophils followed by monocytes typically are recruited to foci of acute bacterial infection, whereas lymphocytes emigrate in high numbers through the high endothelial vessels of lymph nodes and Peyer's patches. 34 As neutrophils enter into an inflammatory focus, "respiratory burst" activity is stimulated and partial degranulation occurs. Such activity is heightened by exposure to inflammatory mediators, phagocytosis, or both of particulate antigen. The NADPH-oxidase-dependent respiratory burst generates short-lived ROS including superoxide anion (O~), singlet oxygen C02), hydrogen peroxide (H20 2 ), and hydroxyl radical (-OH).30 In the presence of granule myeloperoxidase, highly toxic hypohalous acids (usually hypochlorous acid) are formed that are corrosive to both pathogen and host. 25 The broad-spectrum antimicrobial effect of systemic iodide treatment is thought to relate to upregulated hypoiodous acid formation by this pathway. Other neutrophil granule contents include antimicrobial proteins such as lysozyme, lactoferrin, bactericidal/ permeability-increasing protein and defensins, digestive hydrolases, and matrix proteases such as elastase and collagenase that facilitate passage of neutrophils across the basement membrane and through tissues. I8 During initial passage of neutrophils into inflamed tissues there also is rapid processing of membrane phospholipids into lipid or peptidolipid mediators such as PAF and LTB 4. Freshly emigrated monocytes also are stimulated to produce ROS and prostanoids. In certain tissues (including joints in horses),9 early effects of inflammation are amplified by the proinflammatory effect of neuropeptides released from sensory nerve endings. This class of mediators,
INFLAMMATION IN HORSES
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exemplified by substance P but also including vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), and somatostatin, cause vasodilatation, influx and activation of leukocytes, and upregulation of cytokine synthesis. 2o,32 This early phase of inflammation is characterized by an exponential increase in numbers of inflammatory cells and results in an environment rich in mediators and effector molecules. These may include (1) microbial products including exotoxins, degradative proteases, endotoxin, and lipotechoic acid; (2) products of leukocyte and endothelial cell membrane phospholipid metabolism including leukotrienes, thromboxanes, prostaglandins, lipoxins, and PAF; (3) ROS and hypohalous acid; and (4) granule contents of neutrophils (and other granulocytes). Over minutes to hours after the initiation of inflammation there is de novo synthesis and secretion by macrophages and other cells of proinflammatory cytokines such as interleukin 1(3 (IL-1(3), TNF, and IL-6 and chemokines such as IL-8. 3, 16, 24 During this period there also is induction, principally in macrophages and endothelial cells, of three important enzymes that in comparison with constitutive homologues generate massive amounts of product: (1) cyclooxygenase II (COX-II), which produces prostanoids such as thromboxane, prostacyclin (PGI2) and PGE226; (2) inducible nitric oxide synthase (iNOS), which catalyzes formation of NO from arginine21,22; and (3) heme oxygenase 1 (HO-1), which processes heme to bile pigments and CO.38 These products have potent regulatory effects. PLASMA COMPONENTS ENTER THE INFLAMMATORY SITE
Beginning early in inflammation, plasma exudes into affected tissues through permeable capillaries and postcapillary venules. Exposure of plasma components to basement membrane, other extracellular structures, or microbes results in activation of the kinin-kininogen, contact coagulation, extrinsic coagulation, and complement cascades, resulting in the liberation of numerous active mediators and effector molecules. The contact system of plasma begins with the activation of coagulation factor XII (Hageman factor) and proceeds down dichotomous pathways to bradykinin and fibrin formation. 1, 23 Factor XII is activated to factor Xlla by contact with macromolecular complexes exposed or formed during inflammation. Factor Xlla in turn liberates the active protease factor Xla from factor XI and initiates the intrinsic coagulation cascade. Factor Xlla also converts plasma prekallikrein to kallikrein; kallikrein then digests high-molecular-weight kininogen to liberate bradykinin, a potent vasoactive peptide. During inflammation, all of the components of the contact system are thought to be located on the surface of endothelial cells. Bradykinin also is generated by a tissue pathway. Tissue kallikrein and plasma amino-peptidase present in inflammatory exudate digest low-molecular-weight kininogen to release
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bradykinin. Bradykinin acts on cellular B2 receptors to cause venular dilatation, increased vascular permeability, and activation of phospholipase A 2 to begin arachidonic acid metabolism. In the absence of inflammation, the antithrombotic phenotype of endothelial cells is maintained by expression of thrombomodulin and heparan sulfate proteoglycan species, presence of low amounts of PGI2 and NO, and receptor-mediated. engagement of protein C and tissue plasminogen activator. With stimulation, endothelium supports thrombosis by loss of expression of antithrombotic molecules and upregulation of plasminogen activator inhibitor 1 (PAl-I) and procoagulant tissue factor. Procoagulant activity on endothelial cells and phagocytes activates the extrinsic pathway of coagulation; thrombin formation on endothelial surfaces promotes platelet adherence and platelet activation is stimulated by surface-bound PAF. Complement proteins from plasma can recognize and eliminate pathogens and altered host cells via any to all of three different pathways-namely, classic mannose-binding lectin (MBL), and alternative. Is The classic pathway is initiated by the binding of Clq to immunoglobulin multimers (Le., IgM or 2::2 IgG) or to numerous bacteria and viruses. Mannose-binding lectin is activated by binding to high-mannose polysaccharides found on numerous microbes but not on host cells. To initiate the alternative pathwa)T, C3b generated by autodigestion binds covalently to microbial surfaces where it is protected from the degradative actions of regulatory factors Hand 1. Each of the three recognition/ activation pathways culminates in the production of a C3 convertase. These serine proteases mediate cleavage and activation of large numbers of C3 molecules, thereby generating C3b and enormously amplifying complement activation at this crucial central stage of the reaction sequence. Complexes of C3-associated proteins on cell surfaces initiate formation of the hydrophobic transmembrane pore structure known as the membrane attack complex (MAC). Initially, C5 is cleaved to form C5b and then C5b-8 is assembled nonenzymatically into the cell membrane. Finall)T, 12 to 18 molecules of C9 polymerize onto each C5b-8 complex. In addition to the MAC, which has been shown to be cytolytic against some bacteria, activation of complement generates numerous active peptides with potent regulatory roles in inflammation. Several complement proteins, including Clq, MBL, C3b, iC3b, C4b, and iC4b bind to foreign surfaces and target them for ingestion and destruction by phagocytic cells. Neutrophils, monocytes, and macrophages bind to these opsonins via surface receptors including CD35, CDllb / CDI8, and CDllc/ CDI8. Opsonization by complement leads to phagocytosis if the phagocytic cell is exposed to a second activating signal (e.g., PAF, cytokines, Ig). The anaphylatoxins C3a, C4a, and C5a increase vascular permeability, cause vasodilatation, and have indirect proinflammatory action via induction of mast cell degranulation and activation of phospholipase A 2 • C5a also has potent chemotactic and activating activities for neutrophils, monocytes, and macrophages.
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Finally, plasma immunoglobulins are the recognition molecules of the humoral arm of adaptive immune system. They mediate epitopespecific opsonization and phagocytosis, mast cell degranulation, complement activation (by the classic pathway), phagocyte activation, and antibody-dependent cellular cytotoxicity. The role of immunoglobulins and the adaptive immune response are discussed in more detail elsewhere in this issue. PROINFLAMMATORY CELLS AND MEDIATORS ARE ACTIVE IN THE INFLAMMATORY SITE
As previously described, during the amplification stage of inflammation, there is activation of endothelial cells and recruitment and stimulation of large numbers of inflammatory leukocytes (mainly neutrophils and macrophages). In most cases, this leads to internalization of microbes or other material (e.g., immune complexes). Phagosomes containing internalized microbes fuse with cytoplasmic organelles to form phagoendosomes or phagolysosomes wherein microbes are killed by the oxygen-dependent and independent means previously described.' Some microbes escape destruction by preventing phagosome-organellar fusion or acidification of the phagosome or by escaping into the cytosol. Other microbes subvert oxygen-dependent mechanisms by deploying scavenger systems such as superoxide dismutase. As the inflammatory site matures, phagocytes, particularly macrophages, acquire new killing capabilities that may dispatch even these resistant organisms. Interferon ~ (IFN-~) production by natural killer (NK) cells in the inflammatory site is stimulated by small amounts of IL-12 and IL-18 secreted by stimulated macrophages. IFN-~ in combination with other cytokines or microbial products activate macrophages to produce NO (via action of iNOS) and reactive nitrogen species (RNS), molecules with potent microbicidal effects. Adaptive immune responses either may greatly upregulate macrophage function in this way (THI responses) or may suppress such responses (TH2 responses). In addition to their salutary defensive function, inflammatory cells and mediators often have adverse local effects on host tissues. Many vasoactive mediators are produced at inflammatory sites (Table 2) but the predominant early effect is vasodilatation, evident clinically as redness and heat. In some circumstances, the same mediator can cause relaxation of arterioles and constriction of venules. 37 This phase of the inflammatory response correlates closely with the induction of COX-II in numerous cell types by numerous inflammatory mediators, suggesting that products of this enzyme, such as PGE 2 and PGI2t are particularly important vasodilators. Leakiness of capillaries and venules is induced by many mediators (e.g., cytokines such as IFN-~, as well as bradykinin, PAF, histamine, anaphylatoxin, and thrombin) in combination with infiltration of leukocytes and causes swelling and edema of tissues. Extracellular matrix is liquefied by MMP produced by stimulated
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Table 2. VASOACTIVE AGENTS GENERATED DURING INFLAMMATION Vasodilators
PGE 2 PGI 2 Bradykinin
NO
Vasoconstrictors TXA2 PGF 2a
Angiotensin II Endothelin-l
CO Anaphylatoxins Histamine Substance P Lipoxins Serotonin Acetylcholine ADP PGE z = Prostaglandin Ez; PGlz = prostacyclin; NO = nitric oxide; CO adenosinephosphate; TXA z = thromboxane Az; PGF za = prostaglandin Fza .
=
carbon dioxide; ADP
=
leukocytes. This facilitates migration of leukocytes but also may irreversibly damage host tissues. Important examples are the destruction of cartilage matrix that occurs in arthritic jointslO and laminitic hooves. 29 Inflamed tissues are damaged in a more general way by exposure to the microbicidal products of stimulated leukocytes, particularly ROS and RNS. Deposition of fibrin serves to localize inflammatory lesions but also may be the first step in adhesion formation or intravascular coagulation. ACTIVATION OF ENDOGENOUS COUNTERREGULATORY SYSTEMS DAMPS DOWN THE INFLAMMATORY PROCESS AND IS THE FIRST STAGE OF REPAIR
The same stimuli that generate proinflammatory responses also evoke, with slightly delayed kinetics, anti-inflammatory or regulatory molecules that serve to rein in the inflammatory response, contain it locally, and restore homeostasis. Macrophages are exposed to deactivating signals including PGE 2I IL-IO, cortisol, and TGF-~. Furthermore, macrophages become tolerant to the actions of proinflammatory mediators. TNF activity is neutralized by soluble receptor, and IL-l receptor antagonist competes with active IL-l for receptor sites. In the yin and the yang of inflammation, all of the proinflammatory cascades of plasma proteins induce parallel cascades of regulatory molecules. Reactive oxygen and nitrogen species and degradative enzymes are consumed on host macromolecules and acute-phase proteins. Even intracellularly proinflammatory pathways are turned off by the actions of inflammationinduced heat-shock proteins. Within hours of extravasation, neutrophils undergo apoptosis in response to signals in the inflammatory milieu such as Fas ligand and TNF. Apoptotic neutrophils undergo cell death in an orderly manner so that lysosomal enzymes are not released extra-
INFLAMMATION IN HORSES
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cellularly. These cells usually undergo phagocytosis via the scavenger receptors of macrophages. 31 Phagocytosis of apoptotic neutrophils is evident microscopically in inflammatory exudates as leukophagocytosis. IN ADDITION TO LOCAL EFFECTS, THE INFLAMMATORY FOCUS MAY INITIATE A CONTINUUM OF SYSTEMIC RESPONSES
Most inflammatory sites that are clinically evident elicit a systemic acute-phase response. Broadly interpreted, the acute-phase response includes all global processes initiated by the inflammatory site. These may include fever, leukocytosis, anemia of chronic disease, production by the liver of acute-phase proteins, increase in circulating concentration of copper and reduced iron and zinc concentration,2 and stimulation of the neuroendocrine system. In most cases, the acute-phase response acts to combat inflammation and restore systemic homeostasis. Extreme examples of the acute-phase response are the systemic inflammatory response syndrome or generalized immunosuppression. Acute-phase response proteins are normal plasma constituents whose rate of synthesis by the liver is affected positively or negatively by inflammation. The principal signal for acute-phase response protein synthesis by hepatocytes is IL-6, which originates from the inflammatory site and circulates to the liver. Glucocorticoids and other cytokines including TNF, IL-IB, IFN-')', and transforming growth factor B (TGF-B) may influence the pattern of acute-phase response proteins induced by IL-6. The function of acute-phase response proteins generally is to facilitate local inflammatory processes but minimize harmful side effects; thus, fibrinogen is a substrate for thrombin action; complement proteins C3 and C4 are important in microbial recognition and elimination; ceruloplasmin and hemopexin are antioxidants; plasminogen, tissue plasminogen activator, PAl-I, and protein S are fibrinolytics and anticoagulants; and numerous acute-phase response proteins are protease inhibitors (including
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be a common terminal phase characterized by malignant global activation of proinflammatory pathways known as the systemic inflammatory response syndrome (SIRS). The cause of SIRS in hypovolemic shock is unclear but several mechanisms have been proposed. (1) Ischemic damage to endothelial cells exposes blood to procoagulant/proinflammatory substrates. (2) Prolonged splanchnic ischemia allows for translocation of microbes and/ or microbial products from the gut lumen into blood, causing secondary generalized septic shock. (3) Vigorous clinical resuscitation efforts are tantamount to whole body ischemia-reperfusion. The endothelial cells of certain organs are rich in xanthine dehydrogenase (XD), an enzyme that is degraded permanently to xanthine oxidase during ischemia. -Upon reperfusion, xanthine oxidase converts O 2 to superoxide anion, a proinflammatory oxygen radical. Once the SIRS is set in motion by LPS or other stimuli, all the proinflammatory events described for local inflammation are activated within the cardiovascular system, resulting in a bewildering array of circulating harmful mediators. These include cytokines (e.g., TNF, IL1~, IL-8), lipids (thromboxanes, prostaglandins, lipoxins), glycolipids (platelet-activating factor), peptidolipids (leukotrienes), anaphylatoxins (C3a), vasoactive proteins (bradykinin, endothelin), chemotactic proteins (C5a), ROS, and vasoactive gases (NO, CO). Because the proximate stimuli for these mediators frequently overlap and multiple mediators have the same activity (redundancy), it is readily apparent that neutralization of any single mediator is unlikely to be the "magic bullet" treatment for SIRS. The net effect of SIRS in horses usually is increased pulmonary vascular resistance (an effect of thromboxane A 2 ), systemic vasodilatation (prostacyclin, bradykinin, NO?, CO?), reduced cardiac contractility, capillary leakiness (LTB 4I bradykinin, C3a), and extravasation of neutrophils. As a consequence, endothelial surfaces are activated and damaged and become substrate for tissue factor expression and activation of the extrinsic coagulation pathway. These insults-namely, intravascular coagulation plus reduced perfusion pressure because of plasma leakage, systemic vasodilatation, and reduced myocardial contractility-combine to deprive tissues of oxygen. Organ dysfunction ensues comparable to that seen with hypovolemic/hemorrhagic shock. The effect of tissue hypoperfusion is compounded by an endotoxin-induced defect in oxygen extraction by tissues. Anaerobic glycolysis combined with direct inhibition of pyruvate dehydrogenase generate intense local lactic acidosis. SIRS may progress into a syndrome of irreversible shock. The discussion to this point has dealt with the SIRS phenomenon that dominates the acute clinical presentation of horses in septic shock. As previously described, there also is a counter-regulatory response mounted by the host that is designed to check the inflammatory response? This includes production of macrophage deactivators (IL-4, IL10, PGE 2I cortisol), soluble cytokine receptors that can neutralize IL-1 or TNF, receptor antagonists that can prevent binding of cytokines to cells, and intracellular responses (heat shock proteins) that protect cells from
INFLAMMATION IN HORSES Normal Response
Pro-inflammatory
Counter-regulatory
Net Effect
SIRS
Immunosuppression
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Figure 2. Net inflammatory responses during normal inflammation, systemic inflammatory response syndrome (SIRS), and immunosuppression. Each panel depicts the intensity of the inflammatory response over time. A positive response indicates proinflammatory influences, whereas a negative response indicates activation of the counter-regulatory system. During a normal response (left panels), there is brief proinflammatory environment (bottom left panel), then homeostasis is restored. In contrast, there is persistent inflammation in SIRS (central panels) and persistent immunosuppression when the counter-regulatory system is overinduced (right panels).
inflammatory damage. This response may be activated systemically in a way analogous to the SIRS. The SIRS and counter-regulatory systems ideally cancel each other out (Fig. 2), allowing repair systems to begin and homeostasis to be restored. If proinflammatory responses predominate, the auto-cannibalistic" events of septic shock ensue; if counterregulatory systems persist, a state of dangerous generalized immunosuppression may exist-such is the case in human patients with severe burns and is likely in some horses after large intestinal surgery or enterocolitis. /I
References 1. Auer DE, Ng JC, Reilly JS, et al: Assessment of histamine, bradykinin, prostaglandins E1 and E2 and carrageenin as vascular permeability agents in the horse. J Vet Pharmacol Ther 14:61-69, 1991 2. Auer DE, Ng JC, Thompson HL, et al: Acute phase response in horses: Changes in plasma cation concentrations after localised tissue injury. Vet Rec 124:235-239, 1989 3. Bazzoni F, Beutler B: The tumor necrosis factor ligand and receptor families. N Engl J Med 334:1717-1725, 1996 4. Bevilacqua M~ Nelson RM, Mannori G, et al: Endothelial-leukocyte adhesion molecules in human disease. Annu Rev Med 45:361-378, 1994 5. Bochsler PN, Slauson DO, Neilsen NR: Modulation of an adhesion-related surface antigen on equine neutrophils by bacterial lipopolysaccharide and antiinflammatory drugs. J Leukoc BioI 48:306-315, 1990 6. Bochsler PN, Slauson DO, Neilsen NR: Secretory activity of equine polymorphonUclear leukocytes: stimulus specificity and priming effects of bacterial lipopolysaccharide. Vet Immunol Immunopathol 31:241-253, 1992
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7. Bone RC: Immunologic dissonance: A continuing evolution in our understanding of the systemic inflammatory response syndrome (SIRS) and the multiple organ dysfunction syndrome (MODS) [see comments]. Ann Intern Med 125:680-687, 1996 8. Bone RC: The sepsis syndrome: Definition and general approach to management. Clin Chest Med 17:175-181, 1996 9. Bowker RM, Sonea 1M, Vex KB, et al: Substance P innervation of equine synovial membranes: Joint differences and neural and nonneural receptor localizations. Neurosci Lett 164:76-80, 1993 10. Brama PA, TeKoppele JM, Beekman B, et al: Matrix metalloproteinase activity in equine synovial fluid: Influence of age, osteoarthritis, and osteochondrosis. Ann Rheum Dis 57:697-699, 1998 11. Carlos TM, Harlan JM: Leukocyte-endothelial adhesion molecules. Blood 84:20682101, 1994 12. Carter SB: Haptotaxis and the mechanism of cell motility. Nature 213:256-260, 1997 13. Clegg PD, Burke RM, Coughlan AR, et al: Characterisation of equine matrix metalloproteinase 2 and 9; and identification of the cellular sources of these enzymes in joints [see comments]. Equine Vet J 29:335-342, 1997 14. Clegg PD, Coughlan AR, Carter SD: Equine TIMP-1 and TIMP-2: Identification, activity and cellular sources. Equine Vet J 30:416-423, 1998 15. Cooper NR: Biology of the complement system. In Gallin JI, Snyderman R (eds): Inflammation: Basic Principles and Clinical Correlates, ed 3. Philadelphia, LippincottWilliams & Wilkins, 1999 16. Dinarello CA: Biologic basis for interleukin-1 in disease. Blood 87:2095, 1996 17. Elomaa 0, Kangas M, Sahlberg C, et al: Cloning of a novel bacteria-binding receptor structurally related to scavenger receptors and expressed in a subset of macrophages. Cell 80:603, 1995 18. Elsbach ~ Weiss J, Levy 0: Oxygen-independent antimicrobial systems of phagocytes. In Gallin JI, Snyderman R (eds): Inflammation: Basic Principles and Clinical Correlates, ed 3. Philadelphia, Lippincott-Williams & Wilkins, 1999 19. Grunwald U, Fan X, Jack RS, et al: Monocytes can phagocytose gram-negative bacteria by a CD14-dependent mechanism. J Immunol 157:4119--4125, 1996 20. Gutwald J, Goebeler M, Sorg C: Neuropeptides enhance irritant and allergic contact dermatitis. J Invest Dermatol 96:695-698, 1991 21. Hammond RA, Hannon R, Frean SP, et al: Endotoxin induction of nitric oxide synthase and cydooxygenase-2 in equine alveolar macrophages. Am J Vet Res 60:426--431, 1999 22. Hobbs AJ, Moncada S: Inducible nitric oxide synthesis and inflammation. In Willoughby DA, Tomlinson A (eds): Inducible enzymes in the inflammatory response. Basel, Birkhauser Verlag, 1999 23. Kaplan A~ Kusumam J, Shibayama ~ et al: Bradykinin formation: Plasma and tissue pathways and cellular interactions. In Gallin JI, Snyderman R (eds): Inflammation: Basic Principles and Clinical Correlates, ed 3. Philadelphia, Lipincott Williams & Wilkins, 1999, p 347 24. Kaplan D: Autocrine secretion and the physiological concentration of cytokines. Immunol Today 17:303-304, 1996 25. Klebanoff SJ: Myeloperoxidase: Occurrence and biological function. In Everse J, Everse KE, Grisham MB (eds): Peroxidases in Chemistry and Biology. Boca Raton, CRC Press, 1991 26. Loll PJ, Garavito RM: The isoforms of cyclooxygenase: Structure and function. Current Opinions in Investigational Drugs 3:1171-1180, 1994 27. Majno G: The Healing Hand: Man and Wound in the Ancient World. Cambridge, MA, Harvard University Press, 1975 28. Pepys MB, Baltz ML, Tennent GA, et al: Serum amyloid A protein (SAA) in horses: Objective measurement of the acute phase response. Equine Vet J 21:106-109, 1989 29. Pollitt CC, Pass MA, Pollitt S: Batimastat (BB-94) inhibits matrix metalloproteinases of equine laminitis. Equine Vet J Suppl 119-124, 1998 30. Rossi F: The 02-forming NADPH oxidase of the phagocytes: Nature, mechanisms of activation and function. Biochim Biophys Acta 853:65-89, 1986 31. Savill J: Macrophage recognition of senescent neutrophils. Clin Sci 83:649-655, 1992
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32. Smith CH, Barker IN, Morris RW, et al: Neuropeptides induce rapid expression of endothelial cell adhesion molecules and elicit granulocytic infiltration in human skin. J Immunol151:3274-3282, 1993 33. Speiss M: The asialoglycoprotein receptor: A model for endocytic transport receptors. Biochemistry 29:10009-10018, 1990 34. Springer TA: Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu Rev Physiol 57:827-872, 1995 35. Springer TA: Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. In Paul LC, Issekutz TB (eds): Adhesion Molecules in Health and Disease. New York, Marcel Dekker, 1999 36. Tenner AJ, Robinson SL, Ezekowitz RA: Mannose binding protein (MBP) enhances mononuclear phagocyte function via a receptor that contains the 126,000 M(r) component of the C1q receptor. Immunity 3:485--493, 1995 37. Venugopalan CS, Moore RM, Holmes EP, et al: Biphasic responses of equine colonic vessel rings to vasoactive inflammatory mediators. J Auton Pharmacol18:231-237, 1998 38. Willis D: Overview of HO-1 in inflammatory pathologies. In Willoughby DA, Tomlinson A (eds): Inducible Enzymes in the Inflammatory Response. Basel, Birkhauser Verlag, 1999, p 91 39. Yamashita K, Fujinaga T, Okumura M, et al: Serum C-reactive protein (CRP) in horses: The effect of aging, sex, delivery and inflammations on its concentration. J Vet Med Sci 53:1019-1024, 1991
Address reprint requests to Robert J. MacKa)T, BVSc, PhD Department of Large Animal Clinical Sciences College of Veterinary Medicine University of Florida PO Box 100136, JHMHSC Gainesville, FL 32610 e-mail: [email protected]£1.edu