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Acs0826 Molecular And Cellular Mediators Of The Inflammatory Response
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Acs0826 Molecular And Cellular Mediators Of The Inflammatory Response


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  • 1. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice 8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 1 26 MOLECULAR AND CELLULAR MEDIATORS OF THE INFLAMMATORY RESPONSE Vivienne M. Gough, M.B., CH.B., Constantinos Kyriakides, M.D., and Herbert B. Hechtman, M.D. The Inflammatory Response MECHANISM OF NEUTROPHIL-MEDIATED INJURY The inflammatory response to injury is complex, involving not Once it was established that neutrophils were invariably pres- only the area of injury but also systemic tissues. The four cardi- ent during an acute inflammatory reaction, efforts were nal signs of acute inflammation—calor (heat), rubor (redness), launched to explore their role in mediating this response. In the dolor (pain), and tumor (swelling)—were first described by 1960s and 1970s it became clear that during inflammation, neu- Celsus in the first century A.D. Subsequent descriptions added a trophils actively secreted products that were injurious3,4; these fifth sign, functio laesa (loss of function). Although the descrip- products included reactive oxygen metabolites (ROMs) as well tive features of acute inflammation have long been known, there as proteases such as elastase and collagenase.The importance of is still no single satisfactory definition of this phenomenon. It is the neutrophil in mediating inflammation was further under- commonly considered to be the reaction of living tissues to all scored by the finding that the increased permeability resulting forms of injury and involves vascular, neurogenic, humoral, and from an injection of the eicosanoid leukotriene B4 (LTB4), the cellular responses. complement fragment C5a, or the formylated bacterial peptide The vascular events associated with inflammation typically formyl-methionyl-leucyl-phenylalanine (fMLP) into the skin involve changes in blood flow and alterations in permeability. was prevented by previous neutrophil depletion.That neutrophil In 1924, Lewis first described the so-called triple response, in depletion did not affect the release of serotonin, histamine, and which drawing a blunt instrument across the skin induced a bradykinin from mast cells in response to these stimuli suggest- flush, a flare, and a wheal.Thus, directly after injury, local arte- ed that these mast cell–derived agents did not play a major role riolar dilatation, preceded by a fleeting interval of vasocon- in mediating permeability.5 striction, is the dominant vascular response. Precapillary In the early 1980s, it was shown that neutrophils stimulated sphincters dilate, leading to increased flow in already active with phorbol myristate acetate damaged endothelial cells via a capillaries as well as to the opening of inactive capillary beds. mechanism that could be interrupted by catalase, a hydrogen At the same time, postcapillary venules dilate to cope with the peroxide scavenger.6 Subsequent investigations demonstrated increased flow of blood. The rise in hydrostatic pressure is fol- that neutrophil-mediated endothelial damage was also caused lowed by fluid exudation through the vascular bed, leading to by elastase.7 The mechanism of injury required further clarifi- edema (wheal). As the edema increases, the wheal turns pale as cation, given that normal plasma was known to contain potent a result of the increased interstitial pressure compressing the protease inhibitors and superoxide dismutase (SOD), which capillaries. The changes observed in the skin after mechanical inactivate circulating proteases and ROMs, respectively. trauma are typical of those seen in other organs after various Furthermore, a better understanding of how neutrophils were injuries. selectively activated was needed, given that indiscriminate In what follows, we focus on humoral and cellular responses release of proteases and ROMs would clearly be both inefficient to injury, making use of several types of clinically important and dangerous. events (e.g., ischemia-reperfusion and sepsis) to illustrate impor- The mechanism by which the neutrophil is currently thought tant inflammatory pathways. to produce its injurious effects on target cells involves the for- mation of a protected environment in which there is close con- tact between cells. This concept, though not proved, has some Neutrophil evidence to support it. Neutrophils lyse substrate protein only That leukocytes are involved in inflammation was first when in close contact.8 Phorbol myristate acetate–induced lung demonstrated in 1887 by Metchnikoff, who found that rose injury occurs only when neutrophils are in direct contact with thorns inserted into the body of starfish resulted in the local the endothelium.9 When neutrophils are stimulated with fMLP,7 accumulation of leukocytes within hours.1 Metchnikoff also doc- C5a, or endotoxin,10 they must adhere to the endothelium to umented the existence of different types of leukocytes and the cause vascular injury, which is governed by molecules expressed ability of these cells to kill and digest bacteria, and he was the on the surfaces of both cell types. first to suggest that products released by white blood cells could The normal immune action of the neutrophil is to phagocytose damage vessel walls. In 1960, Marchesi and Florey, using elec- its target cell. This process results in the creation within the neu- tron microscopy to follow the movement of neutrophils across trophil of a protected microenvironment known as a phagosome, blood vessel walls in rat mesentery, found that neutrophils pen- which contains the targets for destruction [see Figure 1].When the etrated vascular endothelium at or near cell junctions and that phagosome has been established, the neutrophil releases large after their diapedesis, no sign of a passageway could be seen.2 quantities of ROMs and proteases into it. Any plasma protease Furthermore, they demonstrated that the site at which such dia- inhibitors gaining access to this restricted space are themselves pedesis occurred was the postcapillary venule. inhibited by the neutrophil secretory products hydrogen peroxide
  • 2. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice 8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 2 Circulating energy phosphates; in contrast, such depolarization was limited NEUTROPHIL during hindlimb reperfusion in neutropenic animals.16 Microbe Lysosome Neutrophils have also been implicated as mediators of the remote lung injury observed to occur as a consequence of skele- Proteases tal muscle ischemia. Investigators documented leukosequestra- tion and increased lung permeability during reperfusion after Phagolysosome hindlimb tourniquet ischemia.17 Neutropenia, however, was protective in this setting, maintaining normal microvascular per- MPO meability and lung histology. O2 H2O2 Circulating Antiproteases Adhesion Molecules ROMs To patrol the body effectively for infectious organisms and to PSGL-1 CD18 Serum repair injuries in disparate regions of the body, neutrophils must Antioxidants be able both to circulate as nonadherent particles in blood and lymph and to adhere to and migrate through specific tissues sites P/E ICAM where they are needed. Evidence from the past 20 years indi- Selectin Endothelial Damage cates that inflammatory sites emit signals that activate the adja- cent endothelium and circulating phagocytes. Inflammatory events also trigger release of circulating mediators such as Chemoattractants cytokines, which produce more generalized activation of Endothelium (Chemokines, C5a, LTB4, PAF) endothelial cells and leukocytes, thereby rendering one or both cell types more adhesive.18 Circulating phagocytes then adhere to endothelium; in response to a chemical chemoattractant gra- Figure 1 Proteases and ROMs are stored in intracellular lyso- somes. These fuse with microbe-containing phagosomes, forming dient across the microvessel, they undergo diapedesis between phagolysosmes that initiate destruction of the microbes. Anti- the junctions of endothelial cells, migrate through the suben- proteases that gain entry to this area, such as serum α1-anti- dothelial matrix, and participate in the parenchymal inflamma- trypsin, are inhibited by hydrogen peroxide and myeloperoxidase tory reaction. This phagocyte adherence, though essential to the secreted by neutrophils. The same neutrophil products that cause immune response and the repair of injury, may induce patho- microbial destruction may also damage surrounding endothelial logic consequences. tissue. Tissue damage is somewhat reduced by natural protective For the first few hours of the inflammatory response, the neu- mechanisms, such as ROM scavenging by SOD, catalase, and α1- trophil is the predominant leukocyte at inflammatory sites.19 antitrypsin. After 12 to 24 hours, mononuclear phagocytes predominate.20 When injury is extensive and the inflammatory response robust, and myeloperoxidase, either of which is capable of neutralizing this second phase may be accompanied by immunomodulation α1-antitrypsin, the major protease inhibitor.11,12 Furthermore, and increased susceptibility to infection. myeloperoxidase activates other neutrophil proteases, such as The importance of leukocyte adhesion to the endothelium in collagenase and gelatinase.4 Thus, ROMs and proteases act in maintaining host defenses against infection is illustrated by the synergy to digest phagocytosed matter. example of patients with a congenital deficiency of CD11/CD18, In cases of strong neutrophil activation, the cells may release a family of adhesion-promoting leukocyte surface proteins.21 This their toxic products in concentrations that overwhelm the body’s condition, known as leukocyte adhesion deficiency I (LAD I), regulatory defenses, causing substantial damage to surrounding results in a profound defect in phagocyte accumulation because tissues through excess vascular permeability and parenchymal the leukocytes, though stimulated, cannot adhere and migrate to injury. extravascular sites.7 Patients with LAD I have little or no pus at bacterial infection sites and suffer recurrent life-threatening bac- ROLE IN REPERFUSION INJURY terial infections.21 On the other hand, unmoderated leukocyte The importance of neutrophils in the generation of reperfu- adhesion to the endothelium can also have pathologic conse- sion injury was first demonstrated in the context of myocardial quences, such as lung failure (i.e., the acute respiratory distress ischemia.13 Histologically, the injury was characterized by an syndrome [ARDS]) or organ transplant rejection.22,23 Thus, a bet- extensive early accumulation of extravasated neutrophils, thus ter understanding of the mechanism of adhesion may lead to bet- resembling an acute inflammatory process. Depletion of neu- ter therapy for a number of disease states. Such therapy will have trophils by means of an antiserum led to a significant reduction to balance the benefit of reducing tissue damage caused by leuko- in the size of myocardial infarction after temporary occlusion of cyte adhesion against the possible increased risk of infection. the left circumflex coronary artery.13 Restoration of blood flow Adhesion molecules on endothelial cells and neutrophils may to ischemic skeletal muscle was also associated with significant be classified into three groups: selectins, integrins, and leukocyte infiltration,14 whereas leukocyte depletion attenuated immunoglobulins [see Table 1]. the associated increase in microvascular permeability and vascu- SELECTINS lar resistance.15 Besides enhancing permeability, neutrophils were shown to The selectins are expressed only on cells within the vascula- cause skeletal myocyte injury after ischemia, as evidenced by ture or the lymphatic system. To date, three selectins have been membrane dysfunction. Such dysfunction was indicated by identified: L-selectin, P-selectin, and E-selectin, or endothelial depolarization of resting skeletal muscle that persisted after leukocyte adhesion molecule–1 (ELAM-1).These molecules are restoration of blood flow despite repletion of intracellular high- also collectively referred to as lectin cell adhesion molecules
  • 3. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice 8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 3 Table 1—Adhesion Molecules Involved in INTEGRINS Adherence of Leukocytes to Endothelium The integrin family comprises a vast number of adhesion mol- ecules that mediate intercellular recognition and cellular binding Family Members to the extracellular matrix. These molecules are heterodimeric transmembrane receptors composed of α and β subunits.The β2 L-selectin (LAM-1, Mel-14, Leu-8, LECCAM-1) subfamily, collectively known as the leukocyte integrins or leuko- Selectins E-selectin (ELAM-1) cyte cell adhesion molecules (LeuCAMs), is expressed exclu- P-selectin (CD62, GMP-140, PADGEM) sively by leukocytes. This subfamily comprises three related het- LFA-1 (CD11a/CD18) erodimers, each made up of a common β subunit (CD18) com- Integrins Mac-1 (CD11b/CD18, Mo-1, CR3) plexed with a unique α subunit (CD11a, CD11b, or CD11c). gp 150,95 (CD11c/CD18) CD11a/CD18, also known as lymphocyte function–associat- ed antigen–1 (LFA-1), is expressed on all leukocytes and medi- ICAM-1 Immunoglobulins ates the attachment of unstimulated neutrophils to the vascular ICAM-2 endothelium through a specific interaction with ICAM-1 and VCAM-1 ICAM-2. CD11b/CD18, previously referred to as complement receptor type 3 (CR3) because of its ability to bind the comple- (LEC-CAMs). In general, the selectins are responsible for the ment fragment iC3b but now more generally known as Mac-1, earliest interaction of circulating cells, setting the stage for inte- is constitutively expressed on the surface of neutrophils, mono- grin-mediated firm adherence. All three help regulate the initial cytes, and macrophages. It binds to endothelial ICAM-1 at a site leukocyte rolling on activated endothelium. Without selectins, distinct from the LFA-1 binding domain.34 In addition to the leukocytes interact poorly with endothelium. Leukocyte adhe- Mac-1 expressed on the cell surface, an intracellular pool of pre- sion deficiency II (LAD II), a genetic deficiency of glucosyl- formed Mac-1 is stored within specific granules in neutrophils. transferase, prevents formation of sialyl Lewisx (sLex), the key Translocation of Mac-1 from intracellular pools to the neu- selectin counterreceptor, thereby increasing the risk of infection. trophil surface via granule fusion with the plasma membrane is L-selectin, the smallest member of the selectin family, is pres- stimulated by chemotaxins, including C5a, LTB4, IL-8, and ent on the surface of neutrophils, monocytes, and lymphocytes platelet-activating factor (PAF).35 Unlike the adhesive interac- and binds both constitutive and inducible carbohydrate ligands tions between LFA-1 and ICAM-1, which can be demonstrated on the endothelial cell surface.The migration of neutrophils into in both stimulated and unstimulated cells, Mac-1–mediated certain inflammatory sites is significantly attenuated by L- adherence to endothelial ICAM-1 requires chemotactic activa- selectin blockade24; however, other molecules play a greater role tion.36 CD11c/CD18, also referred to as gp150,95, has been in neutrophil adhesion than L-selectin does. Where L-selectin identified on the surfaces of neutrophils and monocytes and is plays a dominant role in cell trafficking is in the lymphocyte: L- believed to play its primary role in monocyte adhesion. selectin–deficient knockout mice have small, poorly formed lymph nodes and defective T cell function.25 IMMUNOGLOBULINS P-selectin, the largest member of the family, plays a key part Leukocyte integrins bind to endothelial surface receptors in leukocyte rolling.26,27 It is constitutively associated with the α- belonging to the immunoglobulin supergene family. Members of granules of platelets28 and the cytoplasmic Weibel-Palade bodies this superfamily, which includes major histocompatibility com- of endothelial cells29 and is rapidly translocated to the endothe- plex (MHC) antigens I and II, T cell receptors, ICAM-1, and lial cell surface after stimulation by histamine or thrombin.29 ICAM-2, share an immunoglobulin domain composed of 90 to Expression of P-selectin may also be induced by exposure of the 100 amino acids arranged in two sheets of antiparallel β strands vascular endothelium to free radicals (particularly superoxide),30 stabilized at the center with a disulfide bond. Although constitu- C5a, the complement membrane attack complex (C5b-C9),31 tively expressed, copies of ICAM-1 are increased after stimula- and tumor necrosis factor–α (TNF-α).32 The extracellular N- tion by TNF-α, IL-1, LPS, lymphotoxin, or interferon gamma terminal domain of the molecule binds to its carbohydrate li- (IFN-γ). IL-1–induced upregulation can be observed 30 min- gand, sLex. An additional P-selectin mechanism of neutrophil- utes after incubation, after which time it increases steadily, endothelial interaction involves platelets. By adhering to reaching a peak after 24 hours.37 ICAM-1 is the counterreceptor endothelial surfaces, platelets provide a rich surface of exposed for CD11a and CD11b. P-selectin to which neutrophils can bind. Platelet-endothelial binding may occur via fibrinogen attachment to endothelial STUDIES USING ANTIBODIES TO ADHESION MOLECULES intercellular adhesion molecule–1 (ICAM-1) and to platelet The development of monoclonal antibodies (mAbs) that bind GPIIb/IIIa. Platelet endothelial cell adhesion molecule–1 to the functional epitopes of adhesion molecules has yielded use- (PECAM-1) can also mediate attachment. ful data on adhesion mechanisms. In addition, a number of stud- E-selectin, the third member of the selectin family, is a glyco- ies have aroused clinical interest. protein found exclusively on the surface of endothelial cells. In two trials, survival was significantly increased in rabbits and Although not expressed on resting endothelium, E-selectin is baboons treated with a CD18 mAb after hemorrhagic shock.38,39 markedly upregulated in response to the cytokines interleukin-1 In a canine model of myocardial ischemia-reperfusion, treatment (IL-1) and TNF-α as well as to lipopolysaccharide (LPS). E- with a CD11b mAb led to reduced infarct size and neutrophil selectin plays an important role in slightly later leukocyte adhe- sequestration.40 In a murine model of liver cell injury induced by sion to the microvascular endothelium.33 LPS and galactosamine, a CD11b mAb reduced liver cell injury For all three selectins, P-selectin glycoprotein ligand–1 by more than 90% (as determined by assessment of alanine (PSGL-1), which is rich in sLex moieties, is the principal coun- aminotransferase levels) but reduced neutrophil sequestration by terreceptor; it is located on the surface of circulating leukocytes only 40%.41 In this setting, it is likely that neutrophils were and participates in the earliest inflammatory responses. bound to a large extent by CD11a, which may explain the dis-
  • 4. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice 8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 4 crepancy between the degree of protection afforded by the anti- Table 2—General Classification of Cytokines body and the reduction in neutrophil sequestration. The benefi- by Primary Activity cial effects of CD11b antibodies may well be related more to the inhibition of neutrophil signaling and function than to seques- Proinflammatory Cytokines Anti-inflammatory Cytokines tration per se. In another study, treatment with a mAb to ICAM-1 led to a TNF IL-10 reduction in phorbol myristate acetate–stimulated neutrophil IL-1 IL-4 accumulation in rabbit lungs.42 This mAb also reduced lympho- Chemokines IL-13 cyte sequestration and acute graft rejection after renal transplan- Interferons TGF-β tation in primates.23 Attempts to modify acute allograft rejection IL-2 in humans have met with little success. It must be kept in mind, CSFs however, that our understanding of the effects of antibodies to IL-6 adhesion molecules on the immune system is still in its infancy. This rapidly developing field promises to offer powerful tools that may well prove applicable to the control of inflammatory discovered in a variety of different settings and consequently and immunologic reactions. bear several different names associated with specific functions. For example, TNF, initially named for its ability to cause tumor necrosis, is also known as cachectin because of its association Cytokines with cachexia. Current cytokine nomenclature follows a more The term cytokine refers to a diverse group of polypeptides consistent naming system, in which the term interleukin (IL), and glycoproteins that are important mediators of inflammation. referring to a substance that acts between leukocytes, is used in They are produced by numerous cell types but primarily by conjunction with a number (e.g., IL-10); however, many of the leukocytes. Cytokines are potent intercellular messengers that cytokines are still commonly referred to by their historical (and exert most of their actions on immune and inflammatory cells. frequently misleading) names. Taken separately, they have a wide range of effects; considered as The cytokines we address in the following discussion do not a group, their principal functions are to coordinate the immune constitute an exhaustive list. Such a list would be beyond the response to foreign antigens by acting as growth factors and cel- scope of this chapter; furthermore, given the rate at which new lular activators and also to moderate this response via various cytokines are being found and classified, it would be out of date feedback mechanisms. before it was published. Accordingly, we focus on the better- Cytokines are not stored intracellularly: upon antigenic stim- known and more thoroughly studied cytokines. ulus of the cell, they are synthesized rapidly by novel mRNA To facilitate understanding of cytokine function, it may be transcription, then quickly released. For example, LPS from helpful to classify them on the basis of their primary actions. bacterial cell walls causes macrophage activation and induces Various classification systems have been proposed; however, TNF release. The mRNA encoded during this process tends to none have proved entirely satisfactory, because these agents, be unstable, which ensures that cytokine synthesis after cell being pleiotropic by nature, often have conflicting actions. In our activation will be brief and self-limiting. Cytokines exert their view, the most useful approach is to categorize the cytokines effects through very high-affinity binding to various receptors according to whether they exert primarily proinflammatory or on target cells. Thus, as a rule, very small amounts of cytokines primarily anti-inflammatory effects [see Table 2]. Admittedly, the suffice to produce their biologic effects. External signals boundary between the two categories is frequently blurred. increase the number of receptors on a cell, thereby enhancing PROINFLAMMATORY CYTOKINES the cellular response to a particular cytokine. For example, antigenic stimulation of lymphocytes leads to increased The general distinction between proinflammatory and anti- cytokine receptor expression. The cytokine itself can also stim- inflammatory cytokines is based on the observation that certain ulate cytokine receptor upregulation via a positive feedback cytokines—principally TNF, IL-1, and IL-8—promote the mechanism. inflammatory response by upregulating expression of genes Cytokines usually act on cells locally in an autocrine and encoding phospholipase A2, cyclooxygenase-2 (COX-2), or paracrine fashion, thereby ensuring that the inflammatory inducible nitric oxide (NO) synthase, thereby increasing pro- response is evoked at the site of infection or injury. When they duction of the corresponding proinflammatory mediators (PAF, are produced in very large quantities, they can enter the circula- eicosanoids, and NO, respectively). In addition, proinflamma- tion and exert significant systemic effects. On one hand, a single tory cytokines mediate neutrophil action, acting as chemoat- cytokine may act on more than one cell type, causing a number tractants, upregulating expression of leukocyte-endothelial of different consequences (pleiotropism); on the other, several adhesion molecules, causing cell migration into the tissues, and different cytokines may produce the same functional effects activating neutrophil degranulation—all of which lead to tissue (redundancy). This relative nonspecificity of action limits the damage. clinical usefulness of exogenously administered cytokines: the likelihood is high that such agents will either have unwanted side Tumor Necrosis Factor and Interleukin-1 effects or be functionally useless as a result of compensation by Although TNF and IL-1 are structurally dissimilar, their func- other cytokines.43 Another common feature of cytokines is that tions overlap considerably, and they are known to act synergisti- they often act in a cascade, with one cytokine leading to the pro- cally. Originally discovered as a substance causing ischemic duction of another. They may also act synergistically or may necrosis of tumors in rabbits treated with endotoxin,TNF is pro- antagonize the actions of other cytokines. duced principally by monocytes and macrophages but also by Discussion of cytokines is complicated by the often confusing various other immune and endothelial cells. The most potent nomenclature. Several of the earliest identified cytokines were stimulus for its production is LPS (endotoxin) from gram-nega-
  • 5. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice 8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 5 bomodulin; and profound hypoglycemia results from tissue overutilization of glucose and impaired liver function.43 Table 3 Systemic Actions of TNF Like TNF, IL-1 is produced primarily by macrophages and to a lesser extent by other leukocytes and endothelial cells. IL-1 is Fever a primary responder in the inflammatory cascade, and its actions Acute-phase protein synthesis are very similar to those of TNF. It mediates local inflammatory Cachexia responses at low concentrations and exerts adverse systemic Myocardial depression effects at higher concentrations. Unlike TNF, it cannot induce Disseminated intravascular coagulation (DIC) apoptosis. IL-1 also minimizes the perception of posttraumatic Hypoglycemia pain by causing the release of β-endorphins from the pituitary gland and increasing the number of central opioid receptors. TNF and IL-1 act synergistically to produce the acute innate immune response to invading microorganisms, manifested by tive bacteria, but other stressors (e.g., infection, trauma, neutrophil activation and release of other cytokines that promote ischemia, and toxins) can also induce its release. further immune activity. In small concentrations, TNF acts in an autocrine and paracrine fashion, causing increased expression of endothelial E- Chemokines selectin and leukocyte integrins and promoting leukocyte adhe- The chemokines are a group of cytokines that regulate migra- sion to the endothelium.TNF causes neutrophils to be attracted tion of leukocytes from blood to other tissues. Chemokines bind first, followed by monocytes and lymphocytes, thereby helping to heparan sulfate proteoglycans on endothelium, thus present- to regulate the inflammatory response. It also stimulates ing themselves to white blood cells that have become loosely endothelial cells to produce a cytokine subset known as the attached to endothelial cells via the action of selectins and that chemokines (e.g., IL-8), which promote leukocyte migration roll along the endothelium. When a white cell interacts with a into the tissues, and induces mononuclear phagocytes to pro- chemokine, its shape changes rapidly, becoming flatter and more duce IL-1. Finally, TNF can cause apoptosis in some cell types, motile. At the same time, surface integrins are exposed, allowing an event that is of unknown physiologic significance.43 firmer attachment to the endothelium. Chemokines also induce In large concentrations, TNF mediates a number of physio- rhythmic contraction of cellular actin filaments, causing cellular logic effects that are well-recognized components of infection. It migration from the blood vessel at an interendothelial cell junc- acts on the hypothalamus via prostaglandin synthesis to cause tion across a chemokine concentration gradient to the region of fever [see Table 3]. It also induces hepatocyte production of some tissue injury [see Figure 2]. of the acute-phase proteins, the best known of which are C-reac- For the purposes of this discussion, chemokines may be clas- tive protein, fibrinogen, serum amyloid A, and α1-antitrypsin. sified into two main subsets, CC and CXC, depending on Prolonged TNF production also leads to cachexia, probably by whether their two terminal cysteine residues are adjacent (CC) reducing appetite. Massive doses of TNF produce the classic or separated (CXC). The CXC group, which includes IL-8 symptoms of septic shock. Hypotension results from reduced (probably the best known of the chemokines), acts mainly on vascular smooth muscle tone and myocardial depression; dis- neutrophils, whereas the CC chemokines act on monocytes, seminated intravascular coagulation (DIC) results from eosinophils, and lymphocytes. Both subsets are produced by endothelial expression of tissue factor and inhibition of throm- leukocytes, endothelial cells, and fibroblasts in response to Leukocyte Endothelium Ligand (e.g., sLeX) Leukocyte Chemokine Displayed Integrin on Endothelium Loose Binding Diapedesis Leukocyte Firm Adherence Rolling Endothelial Integrin Ligand Chemokines Selectin (P or E) (ICAM-1/VCAM-1) in Tissues Figure 2 Leukocytes bind loosely to the endothelium via low-affinity interaction between selectins and their li- gands. Once loosely bound, they begin rolling slowly along the endothelium. Chemokines cause a conformational change in the leukocyte, exposing the leukocyte integrins that bind to endothelial ICAM-1 and VCAM-1 and thus inducing firm adherence. The leukocyte then undergoes diapedesis into tissues along the chemokine gradient.
  • 6. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice 8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 6 Naive Interferon Gamma Antigen- Presenting Helper T Cell IFN-γ is produced by T cells and NK cells in response to anti- Cell gen, an event greatly enhanced by IL-12. It activates macro- phages, inducing them to kill microbes by activating enzymes that trigger synthesis of ROMs and NO. In addition, by increas- ing IL-12 release from the macrophages it activates, IFN-γ increases differentiation of T helper cells into the Th1 cell subset that produces it while inhibiting proliferation of the Th2 subset, which produces anti-inflammatory cytokines [see Figure 3]. IL-12 IL-4 Interleukin-6 It is probably most accurate to think of IL-6 as a mixed proin- Activated flammatory/anti-inflammatory cytokine. IL-6 is produced by Macrophage mononuclear phagocytes as well as by endothelial cells and fibroblasts. It acts in a proinflammatory manner by providing a potent stimulus for hepatocyte synthesis of acute-phase proteins. Th1 Th2 It is also involved in promoting T cell differentiation and matu- Cell Cell ration and immunoglobulin production and in enhancing NK cell activity.44 However, IL-6 also has important anti-inflamma- tory effects. It has been shown to inhibit production of TNF and IL-1 by mononuclear cells and to reduce release of TNF sec- Positive Positive ondary to endotoxin challenge.45 Furthermore, IL-6 leads to Cytokine Production Feedback Feedback reduced plasma concentrations of soluble TNF receptors and IL-1 receptor antagonist (IL-1Ra), which are naturally occurring anticytokines.46 Proinflammatory Anti-inflammatory Cytokines Cytokines Interferon Alfa and Interferon Beta (Type I Interferons) TNF IL-4, IL-5, IFN-α and IFN-β are two functionally similar cytokines that IL-2 IL-10, IL-13 are often jointly referred to as the type I interferons; they are also IFN-γ known as the antiviral interferons because they are produced by Figure 3 The function of T helper cells is to secrete cytokines in antigen-stimulated T cells in response to viral infection.They act response to antigenic stimulation; these cytokines then act to reg- in a paracrine fashion to inhibit viral replication, preventing ulate an appropriate immune response by triggering proliferation infection of neighboring cells and putting cells into an antiviral and stimulation of T cells, B cells, and other leukocytes. There are state. IFN-α and IFN-β also increase the efficiency of cytotoxic two distinct subsets of T helper cells—Th1 and Th2—which are T cell–mediated cell lysis by upregulating expression of MHC descended from the same precursors, namely, naive CD4+ T cells. class 1 molecules on infected cells. They are frequently used in The development of Th1 and Th2 subsets occurs in response to the clinical setting: IFN-α seems to be effective therapy for viral naïve T cell stimulation by different cytokines. In turn, each sub- hepatitis, and INF-β has been used, with some success, to treat set secretes a distinct set of cytokines. IL-12 is the major stimu- multiple sclerosis. lant for Th1 subset development. This subset assists in phagocyte- mediated responses, with IL-12 being produced by phagocytes in Interleukin-2 response to a variety of antigens (most of which are intracellu- lar). Th1 cells secrete the proinflammatory cytokines, mainly Unlike the cytokines already mentioned, which exert most of IFN-γ, IL-2, and TNF. IL-2 acts in an autocrine fashion, stimulat- their effects via the innate immune system, IL-2 mediates ing further growth of this subset. Th2 cell growth is stimulated by acquired immunity—that is, the proliferation and differentiation the anti-inflammatory cytokine IL-4. This subset produces the of lymphocytes after antigen recognition. Originally known as T anti-inflammatory cytokines IL-4, Il-5, IL-13, and IL-10, with IL- cell growth factor, IL-2 acts in an autocrine manner on the T 4 acting as an autocrine growth factor through a positive feed- cells that produce it (i.e., T helper cells), upregulating IL-2 back mechanism. These cytokines also mediate the immune receptors and thereby causing preferential proliferation of T cells response to helminths and allergens. specific for the stimulating antigen.43 It also promotes growth and differentiation of NK cells. stimulation by either antigens or other cytokines (e.g., TNF IL-2 has an immunomodulatory function as well, in that it and IL-1). Some are also produced constitutionally by lym- potentiates apoptosis of antigen-stimulated T cells. This action phocytes to regulate the flow of these cells through the lym- appears to limit immune responses. It is noteworthy that IL-2 phatic system. knockout mice experience overproduction of lymphocytes with hyperplasia of lymphoid organs and the development of autoim- Interleukin-12 mune diseases (most strikingly, autoimmune hemolytic anemia When activated by intracellular microbes (e.g., viruses, Liste- and inflammatory bowel disease). These events are probably ria, and mycobacteria), mononuclear phagocytes and dendritic attributable to loss of the regulatory apoptosis function. cells (professional antigen-presenting cells [APCs] located in lymph nodes) produce IL-12, the essential action of which is to Hematopoietic Cytokines mediate the early immune response. The most important single The hematopoietic cytokines—including the colony-stimulat- action of IL-12 is to stimulate synthesis and release of IFN-γ by ing factors (CSFs), IL-7, and IL-3—are, strictly speaking, proin- T cells and natural killer (NK) cells (see below). flammatory, in that they induce the growth and differentiation of
  • 7. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice 8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 7 bone marrow progenitor cells; however, this is a normal compo- knockout mice acquire multifocal inflammatory disease, partic- nent of bone marrow hematopoiesis. CSFs are produced by ularly involving the heart and the lungs.51 bone marrow stromal cells and act to stimulate the formation of THERAPEUTIC IMPLICATIONS bone marrow cell colonies. There are several different varieties, named according to the type of cell line they promote (e.g., gran- Interleukin-1 Receptor Antagonist ulocytes [G-CSF], monocytes [M-CSF], or both [GM-CSF]). A naturally occurring cytokine inhibitor is the endogenously IL-7 is synthesized by fibroblasts and bone marrow cells and secreted protein IL-1Ra, which competitively binds the IL-1 induces the differentiation of B and T cells. IL-3 is also known receptor without signaling. IL-1Ra is secreted by mononuclear as multilineage colony-stimulating factor (multi-CSF) because phagocytes and has nearly the same affinity for the IL-1 recep- its actions promote the differentiation of bone marrow precur- tor as IL-1 itself does.52 IL-1Ra is present in high concentrations sors into all known mature cell types. in the serum of patients with a variety of diseases, often in much ANTI-INFLAMMATORY CYTOKINES higher concentrations than IL-1.53 In patients with sepsis, juve- nile rheumatoid arthritis, and inflammatory bowel disease, IL- The anti-inflammatory cytokines exert their effects by inhibit- 1Ra levels have been found to correlate with disease severity; ing the production of proinflammatory cytokines, countering presumably, IL-1Ra acts as a natural negative feedback mecha- their actions, or both. In so doing, they reduce gene expression nism in inflammatory states. Unfortunately, the results of clini- of agents such as TNF, IL-1, and the chemokines, thereby miti- cal trials evaluating recombinant IL-1Ra in the setting of septic gating or preventing their subsequent inflammatory effects. shock have been largely disappointing.54 Interleukin-10 TNF Antagonists IL-10 is important in the homeostatic control of both innate There are two cell surface receptors for TNF, p55 and p75. and acquired immunity. It is produced by activated macro- These are normally shed after activation and are found in the cir- phages and is a potent inhibitor of these cells, regulating them culation. In the presence of disease and in response to endotox- via negative feedback; it is also produced by Th2 cells and B in challenge, they circulate in much higher concentrations.55 It is cells. IL-10 antagonizes a broad range of immune functions: it thought that p55 and p75 act as carriers for TNF, thereby reduc- inhibits IL-12 production by activated macrophages, thereby ing the amount of TNF available to act on cells. TNF itself reducing IFN-γ release and, consequently, decreasing macro- induces the production of its soluble receptors. Elevated levels of phage activation. It also inhibits Th1 cells, thereby reducing pro- TNF receptors have been recorded in cancer patients, and these duction of proinflammatory cytokines (e.g., TNF, IFN-γ, and levels correlate well with tumor burden and the extent of metas- IL-2). However, IL-10 also has proinflammatory effects, stimu- tasis.56 Elevated TNF receptor levels in blood or joint fluid have lating B cell function and enhancing the development of cyto- also been recorded in patients with a variety of autoimmune and toxic T cells.47 inflammatory diseases. The importance of IL-10 in normal regulation of the immune In response to such observations, commercial anti-TNF drugs response is demonstrated by studies of IL-10 knockout mice, began to be developed. The first such drug to be approved was which experience fatal inflammatory bowel disease and show etanercerpt, in 1998.This agent, a recombinant TNF p75 recep- markedly increased levels of TNF in response to endotoxin chal- tor, has proved to be effective in treating rheumatoid arthritis lenge.48 Administration of recombinant IL-10 before lethal doses and spondyloarthropathies. Infliximab, approved in 1999, is a of endotoxin reduces TNF release and prevents death.49 In genetically engineered anti-TNF mAb that is capable of binding humans, it prevents fever, proinflammatory cytokine release, and to both cell-bound and soluble TNF. It causes lysis of TNF-pro- clotting cascade activation during endotoxin challenge. ducing cells via complement- and antibody-dependant cytotox- Interleukin-4 and Interleukin-13 icity.57 Infliximab is approved for use in patients with Crohn dis- ease, in whom it can induce and maintain remissions, bring IL-4 and IL-13 are structurally similar, and both are produced about endoscopically and histologically demonstrable mucosal by Th2 cells as well as by mast cells and basophils.They are con- healing, reduce the number of perianal fistulae, and decrease sidered anti-inflammatory cytokines by virtue of their ability to inflammation in ileoanal pouches.58 Infliximab also shows great antagonize INF-γ and thereby reduce macrophage activation. promise in the treatment of rheumatoid arthritis, ankylosing Because INF-γ is the major promoter of Th1 cells, these two spondylitis, and psoriatic arthritis. agents indirectly suppress cytokine production by Th1 cells in much the same way that IL-10 does. Modulation of Cytokine Activity in Sepsis, SIRS, and CARS IL-4 is the principal growth factor for Th2 cells, which, in addi- Key terms and concepts associated with the systemic activa- tion to their immune suppressor functions, mediate the immune tion of the body’s inflammatory mechanisms—systemic inflam- response to helminths and are responsible for allergic reactions. It matory response syndrome (SIRS), sepsis, septic shock, and is also the major stimulus for production of IgE by B cells. multiple organ dysfunction syndrome (MODS)—are defined and discussed more fully elsewhere [see VI:10 Clinical and Transforming Growth Factor–β Laboratory Diagnosis of Infection,VI:11 Blood Cultures and Infection Transforming growth factor–β (TGF-β) is produced by a in the Patient with the Septic Response, and VII:7 Multiple Organ large variety of cells, including activated T cells, phagocytes, and Dysfunction Syndrome].59 The general understanding is that sys- endothelial cells. It has potent anti-inflammatory effects, inhibit- temic inflammatory activity (i.e., SIRS), which may occur in ing T cell differentiation and proliferation and macrophage acti- response to either infectious or noninfectious stimuli, is the fun- vation. It also counteracts the effects of proinflammatory damental phenomenon; in this view, sepsis may be understood cytokines, causing downregulation of endothelial intercellular as SIRS occurring specifically in response to microbial invasion. adhesion molecules,50 downregulation of TNF endothelial This whole-body inflammatory response can lead ultimately to receptor expression, and inhibition of chemokine action. TGF-β MODS, which is associated with mortalities higher than 50%.
  • 8. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice 8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 8 The role of cytokines in the pathophysiology of SIRS and sep- Vascular Endothelium sis was first appreciated when experimental studies demonstrat- ed that antibodies to TNF reduced mortality in animals to which endotoxin had been administered.60,61 Further research revealed Selectin- that the proinflammatory cytokines TNF and IL-1 were both Mediated able to evoke a clinical picture mimicking that of sepsis (i.e., Rolling Clotting hypotension and fever). In addition, TNF activated the coagula- Cascade tion cascade.54 Accordingly, it was postulated that SIRS and sep- VIIIa sis were attributable to an overwhelming proinflammatory Neutrophil immune response mediated by TNF and other cytokines. A great deal of research on sepsis has now been carried out in animal Activated and clinical trials, mostly using TNF mAbs and soluble TNF Protein C Va receptors. The initial results were positive, in that TNF amelio- ration led to increased survival in endotoxin-sensitized animals. Subsequently, however, these findings were countered by the Thrombin observation that TNF had no significant effect in animal sepsis models of cecal ligation and puncture (which would be more rel- evant to clinical application in humans). Large-scale clinical tri- Monocyte Cytokine Production als employing TNF mAbs and receptors or IL-1 ablation with IL-1Ra have met with little success.62 IL-1, TNF, IL-6 The disappointing clinical results from proinflammatory cytokine ablation have brought into question the thesis that sep- Figure 4 Activated protein C has both anti-inflammatory and antithrombotic actions that make it a valuable antisepsis drug. It sis is entirely understandable in terms of persistent and uncon- reduces cytokine release by mononuclear phagocytes, reduces trolled inflammation. Another view—which is not incompatible leukocyte adhesion to the vascular endothelium, and inhibits with the basic concept of sepsis as a systemic inflammatory thrombin formation. response—is that the body also mounts a countering anti- inflammatory reaction to an insult, which then can lead to what is referred to as the compensatory anti-inflammatory response by presumably combat CARS. In one, administration of G-CSF syndrome (CARS).63 According to this hypothesis, when the to patients with community-acquired pneumonia resulted in proinflammatory response to an insult predominates, SIRS and reduced rates of systemic complications and sepsis.62 INF-γ was shock result, and when the anti-inflammatory response predom- employed in a small clinical study of patients with sepsis and inates, CARS, immunosupression, and increased susceptibility later in a multicenter study of burn patients,66 the rationale being to infection ensue. Homeostasis and return to health reflect a that monocyte function had been found to be depressed in state in which the two responses are in balance. It is likely that in severely ill patients. In these settings, the goal of restoring mono- cases of trauma or infection, the body’s first response is proin- cyte activity, as defined by HLA-DR expression, was achieved, flammatory, and the resulting SIRS is responsible for early mor- though no clinical benefit was noted with regard to mortality or tality. This initial response is followed by an anti-inflammatory incidence of infectious complications. response, and the resulting CARS is responsible for late mortal- It is clear that the role of cytokines in sepsis is a complex one: ity secondary to immunodepression.62 both hypoinflammation and hyperinflammation occur, and Numerous studies have measured cytokine concentrations in both are major determinants of clinical outcome. To date, no the peripheral vasculature in an effort to identify markers of sep- therapy has been found that reliably regulates cytokine activity sis, predictors of mortality, and possible clinical interventions. and reduces sepsis-related mortality. It may be that future treat- Perhaps surprisingly, levels of the proinflammatory cytokines ments will be chosen and given on a patient-to-patient basis, (TNF, IL-1, IL-12, and IFN-γ) are not consistently correlated with markers of immune competence (e.g., HLA-DR, IL-6, and with either disease severity or mortality in patients with septic TNF) indicating whether a proinflammatory approach or an shock. This finding provides some evidence for the CARS anti-inflammatory approach is preferable in a specific instance. hypothesis. It must be remembered, however, that systemic blood sampling is not indicative of tissue cytokine dynamics. In patients with pneumonia, for example, concentrations of TNF, Activated Protein C IL-1, and IL-12 were found to be markedly higher in bronchial It has long been recognized that the clotting system plays a lavage samples than in serum.64 The cytokine for which periph- major role in inflammation and in the response to invading organ- eral blood levels seem to correlate best with disease severity is isms; DIC is one of the cardinal signs of sepsis. The proinflam- IL-6; it is unclear why this is so, given that IL-6 certainly does matory cytokines TNF, IL-1, and IL-6 are known to trigger acti- not produce shock. A 1994 study found that circulating levels of vation of the clotting cascade by stimulating the release of tissue the anti-inflammatory cytokine IL-10, which are normally factor from monocytes and endothelium, leading to thrombin for- unmeasurable, were detectable in more than 80% of patients mation and a fibrin clot.These cytokines also inhibit endogenous with severe sepsis.65 A so-called refractory immunologic state fibrinolytic mechanisms by stimulating the release of plasmino- developing after infection or trauma has also been described,47 gen-activator inhibitor–1 (PAI-1) from platelets and endothelium. characterized by reduced proinflammatory cytokine production At the same time, thrombin stimulates many inflammatory path- by mononuclear cells but increased synthesis of IL-1Ra (which ways and suppresses natural anticoagulant response by activating correlates with CARS). thrombin-activatable fibrinolysis inhibitor (TAFI).67 This proco- Several studies have examined therapies aimed at promoting agulant response leads to microvascular thrombosis and is impli- cytokines that enhance the proinflammatory response and there- cated in the multiple organ failure associated with sepsis.
  • 9. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice 8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 9 Protein C is an endogenous anticoagulant that, when activat- Table 4—Reactive Oxygen Metabolites ed by thrombin bound to thrombomodulin, reduces the genera- and Other Free Radicals tion of thrombin by inactivating clotting factors Va and VIIIa. It also exerts an anti-inflammatory effect by inhibiting monocyte Chemical production of TNF, IL-1, and IL-6 and limiting leukocyte adhe- Radicals and Metabolites Symbol Comments sion to endothelium by binding selectins68 [see Figure 4]. Activation of protein C is impaired during the inflammatory Oxygen-centered free response because endothelial injury results in reduced levels of radicals thrombomodulin. Decreased circulating levels of activated pro- Molecular oxygen 3 tein C are usually present in sepsis and are associated with Ground state (triplet) O2 Constitutes 20% of the oxygen atmosphere increased mortality.69 1 Singlet oxygen O2 Much more reactive These observations have led to the investigation of activated than 3O2 protein C as a potential treatment for severe sepsis. A multicen- Superoxide radical O2• – Initial product of ter, placebo-controlled phase III clinical trial comprising 1,690 respiratory burst patients documented a 6.1% reduction in total mortality in of neutrophils patients treated with drotrecogin alpha (the generic name for Hydroxyl radical OH• Highly unstable; extremely toxic recombinant human activated protein C).67 The only major side Alkoxy radical RO• Important toxic effect noted with this agent was an increased risk of serious bleed- intermediate of free ing: 3.5%, compared with 2% in the placebo group. Drotrecogin radical chain reactions is thus the first therapy to have shown a real clinical benefit in Peroxy radical ROO• Important mediator of DNA damage sepsis trials, and high hopes are held for its future therapeutic use. Lipoxy radical LOO• Important toxic The United States Food and Drug Administration has recently metabolite of lipid approved this agent for treament of sepsis, and further clinical tri- peroxidation als are planned. It is marketed under the brand name Xigris. Non–oxygen-centered free radicals Reactive Oxygen Metabolites Nitrogen centered Nitric oxide NO• Neurotransmitter Oxygen and its metabolites [see Table 4] are often very toxic, Peroxynitrite OONO• Important endogenous and this toxicity seems to have influenced the early evolution of toxin Carbon centered metabolic pathways to detoxify oxygen and make life possible in Lipid radical L• Important toxic an aerobic environment.70 Although aerobic organisms are capa- metabolite of lipid ble of exploiting oxygen’s high reactivity for cellular energy, this peroxidation reactivity also poses a continual assault on living tissues. Sulfur centered Whenever the rate of endogenous oxidant generation exceeds Thiyl R-S• Metabolites of cysteine the body’s endogenous antioxidant capacity, tissue injury ensues. and D-penicillamine This occurrence is known as oxidative stress. Hydrogen centered Free radicals and other ROMs have been implicated in a Hydrogen atom H• — diverse array of human disease processes, including reperfusion Iron centered injury, ischemic hepatitis and pancreatitis, inflammatory bowel Perferryl radical Fe3+-O2-Fe2+ Important final mediator of tissue injury disease, and ARDS. The common pathway for the tissue injury (along with OH•) in these illnesses is the action of free radicals. Non–free radical reactive CHEMICAL STRUCTURE metabolites Ozone O3 Major air pollutant The high reactivity of free radicals is a consequence of their Hydroperoxides chemical structure. Free radicals are molecules that contain one Hydrogen peroxide H2O2 Important endogenous or more unpaired electrons in their outer orbits. As a result, they tissue toxin possess higher energy levels than molecules with full outer orbits Lipid peroxide LOOH Intermediate lipid and, accordingly, are less stable and more reactive. peroxidation The most abundant radical in the biologic system is molecular Hypochlorous acid HOCl Important mediator of oxygen itself (O2), which has two unpaired electrons in its outer neutrophil toxicity orbit. In this ground (i.e., low-energy) state, the two electrons have Chloramines R′RNCl Important final mediator of neutrophil toxicity parallel spins, rendering the molecule relatively unreactive. When the electrons have antiparallel spins, the radical—termed singlet oxygen (1O2)—is much more reactive.Various oxygen-centered free radicals and ROMs are believed to play important roles in tissue The hydroperoxides constitute an important class of toxic – injury.The superoxide anion (O2• ) and the hydroxyl radical (OH•) ROMs, with hydrogen peroxide (H2O2) playing a particularly each have one unpaired electron in the outer orbit.The latter is par- important part. Much of the superoxide-initiated tissue damage ticularly unstable: it reacts within 10-6 seconds of its formation and caused by neutrophils and macrophages can be attributed to within a distance of 15 nm. Consequently, the hydroxyl radical is halogenated oxidants (e.g., hypochlorous acid [HOCl]) and not only highly toxic but also very difficult to scavenge effectively. chloramines, which are formed by the secondary reaction of Some ROMs, technically speaking, are not true radicals but halogenated radical species with nitrogen compounds. This pro- are still quite toxic. For example, much of superoxide’s tissue duction of toxic ROMs as redox products substantially amplifies toxicity is actually mediated by its oxidant reduction products. the toxicity of free radicals.
  • 10. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice 8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 10 Mitochondrion ELECTRON TRANSPORT CHAIN Cytochrome Cytochrome Cytochrome Complex Complex Complex Ubiquinone Cyt.C 2 NADH 4 e- 4 e- 4 e- 4 e- 2 ATP 2 ATP 2 ATP 2 NAD+ Oxygen Water e- Hydroxyl e- H2O Superoxide Radical O2 Hydroperoxyl Hydrogen Anion e- Radical e- Peroxide OH• O2• Catalase/ HO2• H2O2 Peroxidase Superoxide Dismutase UNIVALENT LEAK Figure 5 The mitochondrial cytochrome oxidase complex reduces molecular oxygen by the essentially simulta- neous addition of four electrons, producing water and generating adenosine triphosphate (ATP) without the pro- duction of free reactive oxygen intermediates. However, 1% to 5% of oxygen leaks from this pathway and under- goes stepwise, univalent reduction. The one-electron reduction of molecular oxygen generates the superoxide free radical (superoxide anion). This can subsequently be reduced to hydrogen peroxide (H2O2), most commonly in a dismutation reaction catalyzed by SOD. Hydrogen peroxide can be directly reduced to water (by a catalase- or peroxidase-catalyzed reduction), or it can be reduced to the hydroxyl radical (OH·) (usually in an iron-catalyzed reaction). The highly reactive hydroxyl radical can subsequently abstract a hydrogen atom to reduce itself to water. Normally, protective biochemical mechanisms detoxify the toxic intermediates generated by this so-called univalent leak. When increased concentrations of oxygen generate excessive amounts of reactive oxygen metabo- lites, however, protective mechanisms may be overwhelmed and tissue injury may result. FORMATION and peroxidases) can generate ROMs.71 The arachidonic acid cas- All cells continuously generate ROMs as normal by-products cade, which produces eicosanoids [see Eicosanoids, below], also of metabolism. During oxidative phosphorylation, mitochondrial generates peroxy compounds and hydroxyl radicals as by-products. cytochromes couple the production of adenosine triphosphate In inflammatory cells, ROMs are the primary product of a (ATP) to the tetravalent reduction of molecular oxygen to water highly specialized NADPH-dependent oxidant system. When [see Figure 5]. In the course of this process, all four electrons are activated, they cause reduction of oxygen to the superoxide effectively added to the oxygen molecule at once. No intermedi- anion.This process, known as the respiratory burst, may account aries are formed, and thus no free radicals are generated. for more than 90% of the oxygen consumption of an activated However, about 1% to 5% of the oxygen reduced to water with- neutrophil [see Figure 6].72 It is followed by formation of the in the mitochondria leaks from this pathway and undergoes a major toxic product, HOCl, catalyzed by an enzyme (myeloper- stepwise, noncatalyzed reduction—the so-called univalent leak— oxidase [MPO]) that is unique to phagocytes. that produces a series of toxic intermediaries.These ROMs must then be detoxified by endogenous antioxidants to prevent tissue MECHANISM OF TISSUE DAMAGE injury. All biochemical cell components are potentially subject to ROMs are also formed in many other circumstances. Certain attack by free radicals; however, lipids, proteins, and nucleic cellular oxidases (e.g., cytochrome P-450, mixed-function oxidase, acids are particularly vulnerable. It should also be noted that the
  • 11. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice 8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 11 toxic and mutagenic effects of ionizing radiation and those of 2. Protein denaturation. Both structural and enzymatic proteins many toxic chemicals (e.g., carbon tetrachloride [CCl4]) are in are vulnerable to free radical–mediated denaturation. This fact mediated by the formation of ROMs. ROMs exert their process can lead to loss of membrane fluidity and structural inflammatory effects in five major ways. disintegration, which can cause the destruction of organelles or even entire cells. Calcium adenosine triphosphatase 1. Lipid peroxidation. Because ROMs are so reactive, they gen- (ATPase) is particularly susceptible to oxidative damage; erally react with the first molecule encountered, which is denaturation of this enzyme results in increased intracellular often a cell membrane lipid. By removing a hydrogen atom calcium concentration with subsequent swelling and injury. from the carbon chain of a polyunsaturated fatty acid, a ROM 3. Nucleic acid denaturation. Free radicals can attack nucleic can form a fatty acid radical (LOO•), which is then able to acids directly, causing base hydroxylation, cross-linking, or react with neighboring lipids, proteins, or nucleic acids, in a scission. Such attacks may be facilitated by the presence of chain reaction.Thus, a single free radical can cause the break- DNA-bound iron acting as a catalyst. Depending on the down of several polyunsaturated fatty acids, generating dam- extent of DNA damage, the effect may be mutagenic or aged and reactive products along its path. The reaction prod- lethal. Ionizing radiation exerts most of its damaging effect by ucts include aldehydes, hydrocarbon gases (e.g., pentane, this mechanism. methane, and ethane) and various chemical residues; these 4. Cell matrix injury. Free radicals also attack extracellular pro- agents cause cell edema, influence vascular permeability, give teins and glycoproteins (e.g., collagen and hyaluronic acid). rise to inflammation and chemotaxis, and alter the activity of When these molecules lose their structural integrity, damage cell enzymes, including phospholipase. This destructive to the connective tissue basement membrane and the extra- sequence is terminated only when two of these radicals react cellular matrix may ensue, which can affect vascular perme- together or when the radicals are scavenged by an electron ability and epithelial membrane integrity and can even result acceptor. in petechiae.73 This process may be the cause of certain con- HOCl Cl- H2O2 O2 H2O - • O2 O2 Myeloperoxidase e- Plasma Membrane NADPH Oxidase Neutrophil Cytoplasm NADP NADPH Lysosome Hexose Monophosphate Shunt Figure 6 Neutrophils and other phagocytes generate the superoxide radical via the membrane-integrated enzyme NADPH oxidase. On stimulation, more than 90% of the oxygen used by the neutrophil is consumed in this respiratory burst. Although the superoxide radical itself is toxic to many invading microorganisms, the sec- ondary generation of HOCl from hydrogen peroxide and chloride ions, catalyzed by the neutrophil-generated enzyme myeloperoxidase, substantially increases the bactericidal potential of the neutrophil.
  • 12. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice 8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 12 ATP XANTHINE DEHYDROGENASE ADP Proteolysis Oxidation Ca2+ ISCHEMIA AMP Adenosine Inosine Hypoxanthine Xanthine Oxidase Xanthine Oxidase Xanthine Urate SOD O2 O2 • H2O2 REPERFUSION Fe2+ NH3 OONO – NO2 Neutrophil RCINO3 OH • HOCl Peroxynitrite Nitric Chloramines Oxide Endothelial Cells Reactive Adhesion/ Oxidants Activation TISSUE INJURY Neutrophils Figure 7 Xanthine oxidase may serve as the initial source of free radical generation in postischemic reperfusion injury. With the onset of ischemia, ATP is degraded to its purine bases (e.g., hypoxanthine), the concentration of which increases. Simultaneously, xanthine dehydrogenase is converted by ischemia to xanthine oxidase. Although the concentrations of substrate and enzyme are high during ischemia, the absence of oxygen prevents purine oxi- dation until reperfusion. At reperfusion, oxygen becomes available, suddenly and in excess, and the oxidation of hypoxanthine (and xanthine) proceeds rapidly, generating a burst of superoxide radicals as a by-product, proba- bly at or near the endothelial cell surface. nective tissue diseases (e.g., osteoarthritis and rheumatoid can occur immediately after formation—a critical point, in view arthritis), with inflammatory cells serving as the source of of the short radii of the antioxidants’ activity. For example, SOD ROMs. is located almost exclusively in the mitochondrial matrix, where 5. Apoptosis. Apoptosis, a genetic mechanism that regulates cell it scavenges superoxide radicals formed via the univalent leak, numbers, is characterized by cell shrinkage (pyknosis), in and vitamins A and E are found in cell membranes, where they contrast with the cell swelling and rupture typical of necrosis. inhibit lipid peroxidation. Pyknotic cells are cleared by macrophages with minimal ROLE IN REPERFUSION INJURY inflammation. Apoptosis is induced by ROMs, acting via diverse mechanisms. Postischemic reperfusion disease provides the classic example of free radical–mediated tissue injury. This phenomenon was ANTIOXIDANT DEFENSE MECHANISMS first demonstrated in 1981 in the feline small intestine, where Various endogenous mechanisms have evolved to limit or administration of SOD near the end of the ischemic period but counteract the tissue damage caused by ROMs. An example is before reperfusion largely prevented the microvascular and the production and release of enzymatic and nonenzymatic epithelial injury seen after periods of partial ischemia.74 Since antioxidants such as vitamin E, vitamin C, provitamin A, glu- that initial study, SOD has been shown to attenuate reperfusion tathione, superoxide dismutase (SOD), and glutathione peroxi- injury in many organs, including skeletal muscle75 and the dase, which act by scavenging ROMs. The enzymatic antioxi- heart.76 In similar models, injury was ameliorated by (1) scav- dants require trace metals as cofactors for maximal effect: sele- enging of hydrogen peroxide by catalase,77 (2) scavenging of nium for glutathione peroxidase; copper, zinc, or manganese for hydroxyl radicals by dimethyl sulfoxide or mannitol,78 or (3) pre- SOD; and iron for catalase. vention of radical formation by chelating iron with transferrin or These endogenous antioxidants are generally present at sites deferoxamine.79 Equivalent protection was provided by inhibit- where free radicals are produced, which ensures that scavenging ing generation of superoxide by xanthine oxidase through
  • 13. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice 8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 13 administration of allopurinol, pterin aldehyde, or tungsten.80 dizing agent. Experimental studies using NO• donors and antag- These studies suggest that xanthine oxidase, which is known to onists have not yielded sufficiently consistent results to permit a be present in high concentrations in the intestinal mucosa, is the clear definition of the role of NO• in mediating or moderating initial trigger of free radical generation at reperfusion. inflammatory events. In nonischemic tissue, the dehydrogenase form of xanthine oxidoreductase predominates. Ischemia appears to mediate the conversion of xanthine dehydrogenase to xanthine oxidase via a Complement mechanism that remains to be defined. At the same time, catab- Complement was discovered in 1889 by Bordet while he was olism of ATP during ischemia leads to an increased concentra- studying the bactericidal action of serum.86 Bordet demonstrat- tion of the purine metabolites, xanthine and hypoxanthine. At ed that if fresh serum containing an antibacterial antibody was reperfusion, oxygen is suddenly reintroduced in excess.The sub- added to the bacteria at physiologic temperature, the bacteria sequent generation of superoxide by xanthine oxidase triggers a were lysed, but if the serum was heated to a temperature of 56º free radical chain reaction and subsequent tissue injury [see C or higher, the bacteria were not lysed. Reasoning that the Figure 7]. That either scavenging the hydroxyl radical or block- serum’s loss of lytic capacity could not have been caused by ing its generation by an iron-catalyzed reaction has the same decay of antibody activity—because antibodies were known to protective effect as more proximal blockage of the cascade sug- be heat stable, and even heated serum remained capable of gests that the hydroxyl radical, or one of its metabolites, is the agglutinating the bacteria—Bordet concluded that serum must final agent of injury.77 contain another heat-labile component that assisted or comple- Reperfusion injury is also known to be mediated by neu- mented the lytic function of antibodies. trophils, which would seem to suggest that the injury is entirely COMPLEMENT CASCADE accounted for by neutrophil superoxide generation.This thesis is unlikely to be true, however: a more plausible thesis is that neu- The complement cascade consists of the classical and alter- trophils act in concert with ROMs. Thus, in some models of native pathways, each of which comprises a series of plasma pro- injury, neutrophil depletion fails to ameliorate injuries that are teins that are known to mediate several aspects of the inflamma- blocked by SOD and catalase. It has been observed that free rad- tory response.This cascade has four principal biologic functions: ical scavengers prevent neutrophil infiltration into postischemic tissue,81 which has led to the hypothesis that ROMs act primar- 1. Osmotic cell lysis, effective in the killing of foreign organisms. ily as neutrophil chemoattractants. 2. Binding of complement proteins to the surfaces of foreign organisms or particles. These opsonins bind to specific coun- Clinical Trials terreceptors on macrophages and leukocytes, facilitating In view of the success achieved in moderating reperfusion phagocytosis. injury with antioxidants in animal models, it is perhaps surpris- 3. Augmentation, potentiation, and promotion of the inflam- ing that similar approaches have not met with comparable suc- matory response. Complement fragments bring about cess in humans. A 1989 study using human recombinant SOD chemotaxis of inflammatory cells. In addition, C3a and C5a to treat myocardial ischemia-reperfusion injury after angioplasty stimulate a neutrophil oxidative burst and mast cell degranu- and renal ischemia failed to demonstrate any benefit.82 lation, which potentiate the inflammatory response. However, a renal transplant study in which SOD was infused 4. Clearance of immune complexes from the circulation. immediately before reperfusion reported reduced rates of acute renal failure in graft recipients who underwent cold ischemia for The classical pathway and the alternative pathway have differ- longer than 30 hours83; graft survival was longer as well. ent activating mechanisms; however, both result in the formation In general, ROM scavengers are partially effective at best. of the membrane attack complex C5b-C9, which leads to the Despite the enormous amount of data that has been collected, creation of pores in the cell membrane and consequently to the precise relationship between ROM generation and tissue abnormal ion transport, impaired signaling, and potential lysis, injury remains unclear. particularly of nonnucleated cells. Activation of either pathway leads to production of the biologically active anaphylatoxins C3a and C5a. These peptides are generated via cleavage of native C3 Nitric Oxide and C5 complement components by C3 convertase and C5 con- NO• is produced in a variety of tissues (e.g., endothelial cells vertase, which are formed at intermediate steps in the process of and platelets) by either a constitutive or an inducible form of complement activation. C3a and C5a exert a variety of physio- NO• synthase. In the setting of inflammation, the inducible logic effects, including vasodilatation and increased vascular per- enzyme is activated by cytokines such as TNF. NO• is a potent meability. The effects on neutrophils include chemotaxis, dia- inhibitor of leukocyte and platelet adhesion, exerting its effects pedesis, degranulation, and free radical production as well as via activation of soluble guanylcyclase and generation of cyclic translocation of intracellular complement receptors to the neu- guanosine monophosphate (cGMP).84 With regard to platelets, trophil-plasma membrane. In addition, the complement frag- NO• has been shown to inhibit the binding of fibrinogen to ments C3b and C4b serve as opsonins, thus facilitating phago- GPIIb/IIIa and to limit the expression of P-selectin.85 Another cytosis of particles to which they have attached. Inactive C3b important anti-inflammatory role of NO• is its ability to scav- (iC3b) is a counterreceptor for the neutrophil CD11b/CD18 enge superoxide at least as fast as SOD does. Finally, inhaled (Mac-1) receptor and thus has the ability to enhance neutrophil NO• moderately reduces the pulmonary hypertension seen with binding and signaling in response to complement-coated mole- ARDS; however, it does not increase cardiac output or reduce cules, thereby facilitating their phagocytosis. mortality in these patients. The complement cascade has specific features that make it an Moreover, NO• acts as a source of potent free radicals when important effector mechanism in the inflammatory response. it interacts with superoxide to form peroxynitrite, a strong oxi- Many of its components, once activated, possess enzymatic
  • 14. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice 8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 14 activity. As the cascade proceeds, these activated components seems actually to have evolved before the classical pathway. Both cause substantial amplifications at each step. In addition to this pathways require the production of C3 and C5 convertases to important positive feedback loop, there are multiple levels at generate the terminal sequence leading to generation of the which inhibitory proteins can intervene in the cascade to prevent membrane attack complex; however, each uses a different series its accidental or inappropriate generalized activation. Another of activators and proteins to produce these convertases. important property of the complement cascade is that it has both The alternative pathway is in a constant state of low-level acti- specific and nonspecific activators. Once a specific antibody and vation as a result of spontaneous hydrolysis of the internal antigen have reacted, the classical pathway can be activated by thioester bond in the α chain of C3 (the so-called tickover of C3), several closely spaced IgG molecules or by a single IgM pen- a process that is believed to be ongoing at a low intensity in the cir- tameter.The alternative pathway is triggered by direct binding of culation at all times.This hydrolysis leads to the unstable interme- components to the surface of foreign organisms. In this way, the diate C3b. (The classical pathway may also initiate the alternative alternative pathway participates in nonspecific immunity. pathway by serving as the source of the first C3b molecules.) C3b is usually hydrolysed by water, but sometimes it reacts with amino Classical Pathway or hydroxyl groups of cell surface proteins or polysaccharides to The classical pathway [see Figure 8] is usually initiated by inter- form stable amide or ester bonds. The cell-bound C3b then action between C1 and an antigen-antibody complex. It may also attaches to factor B, which in turn is proteolytically cleaved by fac- be activated by infectious organisms (e.g., Mycoplasma) or certain tor D to form C3bBb, the solid-phase alternative pathway C3 con- molecules (e.g., DNA, heparin, and chondroitin sulfate).87 vertase, which requires properdin for stabilization. C1 does not interact with IgG or IgM until the antibody has The C3 convertase then acts on additional C3b to form the bound its specific antigen. The subunit of C1 that binds is C1q. C3bBbC3b complex, which is the C5 convertase of the alterna- C1q attaches either to the CH3 domain of IgM or to the CH2 tive pathway. This C5 convertase initiates hydrolysis of C5, domain of IgG, both of which reside in the Fc portion of the which, as in the classical pathway, eventually results in the for- antibody. A single C1q molecule must bind to at least two Fc mation of the membrane attack complex. portions of an immunoglobulin. Because IgM is secreted as a pentameter containing five separate Fc portions, a single mole- Membrane Attack Complex cule is an efficient activator of complement. Because IgG is Both of the C5 convertases cleave C5 into C5a and C5b. The secreted as a monomer, C1q must bind to two or more adjacent C5b component binds to C6 and C7, and the resulting C5b67 IgG molecules to be activated. C1q is uniquely able to bind these complex is then inserted into the lipid bilayer of the plasma different Fc regions of immunoglobulins because it consists of membrane. Once in place in the membrane, this molecule six identical subunits, each of which has a projecting head. becomes a receptor for and binds to C8, at which point pore for- Once C1q is bound to an immune complex, the other com- mation, ionic movement, and cell lysis may begin. The C5b678 ponents of C1—C1r and C1s—become operative. A tetramer complex undergoes hydrophilic association within the plasma consisting of two C1r and two C1s molecules is formed and membrane, weakening it and causing the formation of trans- bonded with C1q. This process results in a serum esterase–type membrane channels. This process may lead to cell death by enzyme that acts on the next two components in the cascade, C4 osmotic lysis because of the cell’s inability to maintain critical and C2. C4, like several other complement proteins, has an intra- and extracellular concentration gradients. Because of the internal thioester bond. C4 is split into two parts, C4a and C4b, amplification mechanisms involved in both pathways, as many as with the former diffusing away. Generation of C4b results in a 12 to 15 C9 molecules may be added to the C5b678 complex, reactive molecule called metastable C4b, which possesses an resulting in the formation of C5b-C9, the membrane attack internal thioester. Many C4b molecules react with water at the complex. Scanning electron microscopy suggests that the mem- site of the internal thioester, thereby inactivating the C4 com- brane attack complex acts as a pore within the cell membrane, plex; alternatively, some of the C4b thioesters may esterify to cell allowing the collapse of ionic and osmotic gradients. surface proteins or carbohydrates, thereby stabilizing the C4 complex. Regulation In the presence of magnesium, C2 binds to C4b molecules To prevent inappropriate activation and damage to the host, that in turn are bound to cell surface proteins or carbohydrates. the complement cascade must be tightly regulated. There are a A C1s molecule splits C2, generating C2a and C2b. The latter number of circulating soluble factors that inactivate various may diffuse away, whereas the C2a fragment remains complexed aspects of the complement cascade, including C1 inhibitor, C4- with C4b, resulting in C4b2a, which is the C3 convertase of the binding protein, factor H, and factor I. Molecules that inhibit classical pathway [see Figure 8]. In the presence of calcium and membrane attack complex formation include homologous magnesium, this C3 convertase binds C3 and cleaves it into C3a restriction factor and the membrane inhibitor of reactive lysis, and C3b. Like C4b, C3b has an internal thioester, and this both of which are thought to play a key role in protecting normal thioester may form a covalent bond with C4b2a or be inactivat- surrounding cells in adjacent areas where complement activation ed by reacting with water. The addition of C3b to the preexist- and cell lysis are taking place. ing C4b2a complex yields C4b2a3b, which is the C5 convertase In addition, there are several regulatory proteins that are of the classical pathway. This C5 convertase cleaves C5 and ini- found within the membranes of most cells of the myeloid line as tiates the common terminal sequence of the complement cas- well as those of platelets and mast cells; these include comple- cade, which results in the formation of C5b-C9, the membrane ment receptor type 1 (CR1), membrane cofactor protein, and attack complex. decay-accelerating factor. CR1 is present on erythrocytes and in smaller quantities on neutrophils, monocytes, macrophages, Alternative Pathway eosinophils, and lymphocytes. It has at least three important The alternative pathway [see Figure 8] was so named because functions: (1) it can inhibit C3 convertase activity, (2) it it was discovered after the classical pathway was described, but it enhances the ability of leukocytes to ingest C3b- or C4b-coated
  • 15. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice 8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 15 CLASSICAL PATHWAY ALTERNATIVE PATHWAY examining therapeutic use of sCR1 in animals documented sig- Spontaneous nificant reductions in myocardial infarct size and dramatic A–Ab Complex Hydrolysis reductions in the number of neutrophils sequestered in the Covalently myocardium.90 Moreover, treatment with sCR1 did not appear Bound C1 C3 to impair healing of the infarcted area. Immunoperoxidase stain- C4 Bound to ing for the membrane attack complex demonstrated little or no Microbial myocardial deposition of C5b-C9 in the sCR1-treated group in C4a Cell comparison with untreated animals. Surface C4b C3b Several studies have examined the role of complement in skeletal muscle ischemia-reperfusion injury. In one, concentra- C2 Factor B Factor D tions of C3a and C5a were elevated in patients with ischemic limbs.91 Levels of these anaphylatoxins returned to normal after C3 C3 amputation, which suggested that complement was activated by Convertase C4b2a C3bBb Convertase acute limb ischemia. In another study, there was a significant C3 C3 dose-dependent reduction in muscle permeability with sCR1 treatment after hindlimb ischemia-reperfusion.92 Subsequently, it was found that during reperfusion, skeletal muscle permeabil- C3a ity was dependent on the presence of IgM natural antibody and C5 C5 on activation of the classical complement pathway. These inves- Convertase C4b2a3b Anaphylatoxins C3bBbC3b Convertase tigations lend strong support to the thesis that complement is a C5a significant mediator of skeletal muscle injury after ischemia- reperfusion. C5 C5b C5 In further studies, it was postulated that ischemic injury led to expression or unmasking of a neoantigen. Immunohistochem- C6 istry demonstrated colocalization of IgM and C3, indicating the C7 formation of an immune complex in conjunction with comple- ment activation (shown by other techniques to involve the clas- sical pathway). Animals genetically depleted of immunoglobu- C5b67 lins—specifically, IgM—were protected from reperfusion injury. C8 Mast Cell C5b678 Although mast cells were first identified more than a century ago, only comparatively recently have major advances been made C9 in understanding their biology. All mast cells are derived from progenitors present in the bone marrow, which migrate to C5b6789 peripheral tissues as immature cells and undergo differentiation in situ.Thus, unlike neutrophils, mature mast cells are not found Membrane Attack Complex in the circulation but are located throughout the body near blood vessels and nerves, beneath epithelia, and in lymphoid organs. Figure 8 Shown are the classical and alternative pathways of the complement cascade. STRUCTURE AND TYPOLOGY Mast cells have (1) a mononuclear nucleus, (2) numerous cytoplasmic granules containing mediators (including peptides, particles, and (3) it facilitates clearance of immune complexes proteins, proteoglycans, and amines, and (3) a plicated cell from the circulation. CR1 also inhibits the activity of C4b and membrane. The cell membrane contains a number of receptors thus is capable of preventing activation of the classical pathway. for important regulatory compounds, such as growth factors, This powerful inhibitor of both complement pathways is cur- activators of secretion (e.g., receptors for the Fc portion of the rently the subject of much attention among researchers. A solu- IgE molecule), and receptors for adhesive molecules that enable ble form of CR1 that lacks the cytoplasmic and transmembrane the mast cell to attach to certain sites. domains has been engineered from human erythrocytes. This Mast cells can be differentiated into two types, those found in human soluble complement receptor type 1 (sCR1) retains the mucosa and those found in connective tissue. These two pheno- C3b- and C4b-binding activities of membrane-associated CR1. types are relatively easy to distinguish in rodents because they stain differently, differ in size, and are found in different tissues. ROLE IN REPERFUSION INJURY GI mucosal mast cells have chondroitin sulfate as their major The first evidence that complement might play a role in the granule proteoglycan and contain little histamine. In contrast, pathogenesis of ischemia-reperfusion injury was the finding that connective tissue mast cells (found in the lungs and in the serosa myocardial ischemia induced complement activation.88 It was of body cavities) have heparin as their major granule proteogly- also reported that mitochondrial fragments in cardiac lymph can and produce large quantities of histamine. activated both classical and alternative complement pathways. Two techniques have enabled researchers to distinguish Later studies using cobra venom factor to deplete complement between the two types of mast cells in humans. One uses elec- by triggering its activation demonstrated reductions in myocar- tron microscopy to examine the completeness of the scroll or dial necrosis after coronary artery occlusion.89 Researchers whorl structure of the granule (only connective tissue mast cells
  • 16. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice 8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 16 have a complete scroll). The other uses mAbs to identify mast lar mast cells were exposed to hypoxia, they released their gran- cell subtypes on the basis of their constituent protease enzymes. ules, thereby causing lung injury.93 Perivascular mast cells from All human mast cells contain tryptase, but only connective tissue the hypoxic lungs showed none of the morphologic changes mast cells contain chymase. Furthermore, there are a number of associated with IgE-mediated mast cell degranulation, such as chymase isotypes that are unique to particular organs, which granule swelling, fusion, and exocytosis. suggests that the pattern of mediators produced by mast cells In a murine model of hindlimb ischemia-reperfusion that might vary with anatomic location. made use of two congenic animal strains, one mast cell–suffi- cient and the other mast cell–deficient, it was demonstrated that ACTIVATION pulmonary mast cells mediated neutrophil sequestration and In diseases associated with mast cell activation, increases in pulmonary edema, probably through the production and release the total number of mast cells and variations in the relative abun- of leukotrienes.94 Vagal efferent nerves appeared to play an dance of mast cell phenotypes occur at different tissue sites. The important role in the induction of remote lung injury after best understood immunologic stimulus of mast cell activation is hindlimb ischemia; in this setting, bilateral cervical vagotomy an immune IgE complex. Other important immunologic triggers limited mast cell degranulation and associated lung injury.95 are cytokines and various histamine-releasing factors generated Vagal mechanisms also appear to play a key role in mast cell by neutrophils, platelets, mononuclear leukocytes, and macro- activation in a murine model of acid aspiration.96 Blocking sub- phages as a consequence of the immune response. stance P, a neuropeptide released at the end of C-fibers located Numerous nonimmunologic stimuli are also important in near mast cells, modified the lung injury; the C-fibers travel with mast cell activation associated with wheal-and-flare reactions. vagal nerves. A similar benefit was obtained by using the chy- Such stimuli include the complement fragments C3a, C4a, and mase antagonist chymostatin, which blocked the effect of a key C5a; various neuropeptides, including calcitonin gene–related mast cell secretory product. The reduction in injury achieved peptide; substance P; neurotransmitters; and drugs. These non- with these two approaches was the same as the injury reduction immunologic activators are generally more effective on connec- observed in mast cell knockout mice.96 This similarity suggested tive tissue mast cells than on mucosal mast cells. that the final common pathway of lung damage in aspiration was When mast cells are activated, their granules move to the sur- activation of the alternative complement pathway. These data face, dissolve, and release their contents. Some of these products indicate that vagal stimulation with release of substance P leads (e.g., histamine and other low-molecular-weight components) to release of mast cell–derived chymase that triggers activation of are freely soluble; others (e.g., proteoglycans, enzymes, and larg- the alternative complement pathway, thereby causing aspiration er proteins) are poorly soluble. Differential release of these mate- lung injury. rials may have a bearing on the mast cell’s role in disease. ROLE IN ACUTE LUNG INJURY Eicosanoids The central role played by mast cells in the immediate hyper- Eicosanoids are vasoactive and immunomodulatory deriva- sensitivity reaction has long been known. In the lung, this reac- tives of membrane fatty acids that can be produced by virtually tion is typically manifested either by an asthma attack or by a all cells in the body. They may be divided into three major sub- more generalized systemic hypersensitivity reaction (i.e., ana- groups: prostaglandins, thromboxanes, and leukotrienes. These phylactic shock). agents are not stored but are rapidly synthesized in response to Asthma is characterized by paroxysms of bronchial constric- signaling stimuli. After release, they act in an autocrine and tion and increased production of thick mucus, which leads to paracrine fashion. They have very short half-lives (largely bronchial obstruction and exacerbation of respiratory distress. because of their susceptibility to spontaneous hydrolysis or active The smooth muscle in the airway is often hypertrophied and enzymatic conversion to inactive metabolites) and thus rarely act hyperreactive to constricting stimuli. Anaphylactic shock is char- as circulating hormones. acterized by vasodilation and exudation of plasma in vascular Prostaglandins were discovered in the 1930s by von Euler beds throughout the body, usually resulting from the presence of when a substance in human semen was found to cause myome- a triggering antigen that gains systemic access via injection, an trial contraction.The active agent, an acidic lipid, was thought to insect sting, or absorption across an epithelial surface (e.g., skin be a product of the prostate gland and hence was named or GI mucosa). The decrease in vascular tone and leakage of prostaglandin.The next major observation was the description of plasma lead to a fall in blood pressure that may be fatal.The car- the slow-reacting substance of anaphylaxis (SRS-A) in the early diovascular effects are complicated by constriction of the mus- 1940s. Thromboxanes were discovered in 1975 and leukotrienes culature surrounding the upper and lower airways, increased in 1979. In 1980, SRS-A was identified as a combination of the mucosal reactivity with outpouring of mucus in the gut and the three cysteinyl leukotrienes: LTC4, LTD4, and LTE4. respiratory tract, and urticarial lesions on the skin. FORMATION In both asthma and anaphylaxis, mast cell activation takes place in response to an immunologic trigger. Specifically, IgE antibody insertion on mast cell surface Fcε receptors leads to the Liberation of Arachidonic Acid production and release of inflammatory mediators. Mast Arachidonic acid (eicosatetraenoic acid), the major substrate cell–derived leukotrienes and PAF are thought to be major medi- for eicosanoid synthesis, is a long-chain polyunsaturated fatty acid ators of acute airway constriction. In addition, mast cell–derived that is incorporated into the phospholipid mammalian cell mem- cytokines (e.g., TNF-α, IL-4, and IL-5) are thought to be the brane. It is also present in plasma, but it is tightly bound to plas- major factors causing sustained airway inflammation via the ma proteins and therefore biologically unavailable. The first step recruitment of eosinophils and basophils. in eicosanoid synthesis is liberation of arachidonic acid from the Nonimmunologic mechanisms may also play a part in mast cell membrane. An agonist binds to the membrane receptor, caus- cell–mediated lung injury. In one study, when pulmonary alveo- ing activation of the enzyme phospholipase C, which generates
  • 17. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice 8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 17 Agonist Bound Phospholipase A2 Cell Receptor Arachidonic Membrane Acid Phospholipase C PIP2 IP3 DG Free Arachidonic Acid Free Ca2+ Free Arachidonic Acid Diacylglycerol and Monoacylglycerol Glycerol Lipases Intracellular Long-Chain Ca2+ Stores Fatty Acid NUCLEUS Figure 9 Shown is the production of free arachidonic acid in the cytosol. The binding of an agonist to a recep- tor activates phospholipase C, which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5- trisphosphate (IP3) and diacylglycerol (DG). From this point, free arachidonic acid can be generated in two ways. First, IP3, by increasing the intracellular calcium ion concentration, activates phospholipase A2, which then liberates arachidonic acid from the cell membrane. Second, DG is broken down enzymatically into sever- al products, one of which is arachidonic acid. free arachidonic acid via the production of the intracellular second verted to PGH2. PGH2 is then metabolized by different path- messengers inositol triphosphate and diacylglycerol. These mes- ways to prostaglandins, prostacyclin (PGI2), and thromboxanes sengers, in turn, activate phospholipase A2 [see Figure 9]. [see Figure 11]. Phospholipase activation is a crucial step in arachidonic acid Cells typically synthesize one or two COX products, depend- liberation; in fact, it serves as the rate-limiting step in all ing on the enzymes they express. Platelets, for example, produce eicosanoid production. Once freed, arachidonic acid is metabo- thromboxane A2 (TXA2) through the action of thromboxane lized via two pathways. The cyclooxygenase pathway produces synthetase, whereas endothelial cells primarily produce PGI2 prostaglandins and thromboxanes (collectively referred to as through the action of prostacyclin synthetase. prostanoids), and the lipoxygenase pathway, present in cells of Six cyclooxygenase products are currently in widespread use in myeloid origin, produces leukotrienes. clinical settings. Alprostadil (PGE1) is used for its smooth mus- cle–relaxing effects; misoprostol, a synthetic analogue of PGE1, is Cyclooxygenase Pathway used for gastric cytoprotection; PGE2 and latanoprost (PGF2α) All cells except nonnucleated red cells are capable of generat- are used in obstetrics and gynecology; latanoprost is used in oph- ing cyclooxygenase products. Two related but distinct cyclooxy- thalmology, and PGI2 is used for its properties as a potent genase isozymes have now been identified, COX-1 and COX-2. vasodilator and a moderate inhibitor of platelet aggregation. At COX-1 is constitutively present in cells, and its products are con- present, however, greater clinical importance is attached to inhi- sidered to have housekeeping functions (e.g., gastric mucosal bition of the synthesis of cyclooxygenase products by means of protection). COX-2 is an inducible enzyme that is rapidly upreg- nonsteroidal anti-inflammatory drugs (NSAIDs) and selective ulated in response to various immune and inflammatory media- COX-2 inhibitors, which act as anti-inflammatory and analgesic tors (e.g., cytokines, growth factors, and tumor promoters), and compounds [see Therapeutic Implications, Cyclooxygenase its products play a more inflammatory role [see Figure 10]. Inhibition with NSAIDs and COX-2 Antagonists, below]. Admittedly, this polarized view of the functions of COX-1 and COX-2 products is somewhat oversimplified; nevertheless, we Lipoxygenase Pathway believe it is useful for the purposes of general discussion. There are several differences between the cyclooxygenase and Both COX-1 and COX-2 convert arachidonic acid into an lipoxygenase pathways. Unlike cyclooxygenase, which is ubiqui- endoperoxide, prostaglandin G2 (PGG2), which in turn is con- tous, lipoxygenase is present only in inflammatory cells.
  • 18. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice 8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 18 HOUSEKEEPING FUNCTIONS INFLAMMATORY FUNCTION Platelets Kidney Tissue Leukocytes Macrophages Free Stomach Arachidonic Acid Endothelium Endothelium COX-1 COX-2 TXA2 PGI2 Eicosanoid Products PGI2 PGE2 PGE2 GI Mucosal Integrity Inflammation Renal System Homeostasis Mitogenesis Platelet Aggregation Bone Formation Figure 10 The products of COX-1 tend to have so-called housekeeping functions. This enzyme is consti- tutively present in cells. In contrast, the COX-2 enzyme is induced in cells in response to inflammatory stimuli. The products of both enzymes tend to cause inflammation. Furthermore, whereas cyclooxygenase is always active, requiring reproductive tract; however, they also play important roles in only the presence of arachidonic acid for endoperoxidase pro- platelet regulation and in the nervous system.Their main biolog- duction, lipoxygenase is quiescent and must be activated for ic actions may be summarized as follows: leukotriene synthesis to occur. Activating stimuli for the lipoxy- genase pathway include GM-CSF, C5a, and fMLP. 1. Effects on the vasculature. PGI2 is a vasodilator that is syn- In the lipoxygenase pathway, liberated arachidonic acid is con- thesized by both smooth muscle and endothelial cells in verted to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) by 5- response to agents such as bradykinin, thrombin, serotonin, lipoxygenase (5-LO). This same enzyme then converts 5- platelet-derived growth factor, and IL-1. Its actions are essen- HPETE into LTA4, which then is either metabolized by LTA4 tially antagonistic to those of TXA2: it causes vasodilatation, hydrolase to LTB4 or conjugated by LTC4 synthetase with inhibits platelet aggregation, and is thought to control the reduced glutathione to form LTC4. LTC4, in turn, is converted release of tissue plasminogen activator. In contrast, PGF2α is into LTD4 and LTE4 [see Figure 11]. a potent vasoconstrictor. LTA4 may diffuse extracellularly and be taken up by cells that 2. Effects on the nervous system. PGE1 and PGE2 are responsi- possess LTA4 hydrolase and LTC4 synthetase (e.g., erythrocytes ble for the increase in body temperature associated with and endothelium), with the result that leukotrienes may be pro- inflammation.Their synthesis in the hypothalamus is promot- duced by nonmyeloid cell lines that do not possess 5-LO and ed by TNF and IL-1 (also known as endogenous pyrogen), thus do not ordinarily generate leukotrienes. This process can which are released by phagocytes in response to viruses, bac- amplify the inflammatory response. teria, fungi, endotoxin, and antigen-antibody complexes. PGE2 also exerts a strong hyperalgesic effect: it increases sen- PROSTAGLANDINS sitivity to a variety of mechanical and chemical pain stimuli, There are nine prostaglandin groups: A, B, C, D, E, F, G, H, with additive effects occurring over time and with increasing and I. With some prostaglandins (e.g., PGA2), the letter refers to concentration. At high concentrations, PGE2 can even stimu- the chemical structure of its five-member ring.With others, how- late nociperceptive nerve endings directly. ever, the letter means something else: for example, E originally 3. Effects on the renal system. PGE1, PGE2, and PGI2 increase stood for “ether” and F for “phosphate buffer,” which were the the glomerular filtration rate and moderate renal blood flow substances in which PGE and PGF were most readily soluble. by means of local vasodilatation. They also increase sodium Within a prostaglandin group, the subscript numerals 1, 2, and 3 and water excretion, though the mechanisms by which they are used to indicate the number of unsaturated double bonds do so are unclear. present in the two side chains. Finally, the subscript Greek letters 4. Effects on the GI tract. Prostaglandins play a central role in α and β are used to indicate the spatial orientation of the hydrox- gastric epithelial defense and repair and are produced in large yl groups on the side chains in relation to the ring. amounts by the gastric mucosa. Most of them induce con- Prostaglandins primarily exert their effects on the smooth traction of GI smooth muscle. Specifically, PGE2 and PGF2α muscle of the GI tract, the airways, the vasculature, and the contract longitudinal smooth muscle, whereas PGI2 and
  • 19. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice 8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 19 PGF2α contract circular smooth muscle. Administration of increase its synthesis and release from platelets or white blood these agents causes colicky abdominal pain. cells. The presence of thrombin or contact with collagen can 5. Effects on the respiratory tract. PGE1, PGE2, and PGI2 relax stimulate release of TXA2 from platelets, and cytokines, com- respiratory smooth muscle, and PGF2α contracts it. plement fragments, and PAF all can directly stimulate release of 6. Effects on the reproductive system. Prostaglandins have TXA2 from white blood cells. Thus, clinical situations associat- potent effects on the uterus and the cervix, moderating uterine ed with increased thromboxane production include vascular contraction during delivery. Although they were originally dis- injury; arteriosclerosis; conditions associated with complement covered in semen, their function there remains unknown. activation, such as sepsis, dialysis, and ischemia; and cytokine production. THROMBOXANES TXB2 is the metabolite of TXA2, produced via nonenzymatic TXA2, originally called rabbit aortic contracting substance, hydrolysis. Although it is only one tenth as potent as TXA2, it was identified in 1975 as an agent that contracted vascular does have some degree of biologic activity (e.g., enhancement of smooth muscle and caused platelet aggregation.97 In picogram endothelial permeability, activation of neutrophils, and promo- amounts, it exerts five major biologic effects. tion of neutrophil diapedesis). 1. Vasoconstriction. TXA2 is one of the most potent naturally LEUKOTRIENES occurring constrictors of smooth muscle. It acts on virtually Leukotrienes derive their name from their origin and their all vascular beds, but its effects are particularly prominent in chemical structure: they are products of leukocytes that have a small arteries and arterioles. conjugated triene as their common structural feature. They can 2. Bronchoconstriction. TXA2 exerts a marked spastic effect on be divided into two classes on the basis of differing biologic tracheal rings and pulmonary parenchymal strips and causes actions: (1) LTB4 and (2) the cysteinyl leukotrienes (LTC4, bronchoconstriction in intact animals. LTD4, and LTE4). The most important actions of the 3. Enhanced platelet aggregation. TXA2 activates platelets, leukotrienes have to do with their central role in allergic and inducing aggregation with subsequent stimulation to synthe- inflammatory diseases. size and release more TXA2. This autocrine action leads to The primary effects of LTB4 are neutrophil chemotaxis and further platelet activation. activation. Neutrophils activated by LTB4 exhibit increased 4. Increased membrane permeability. TXA2 increases vascular oxidative activity, granule release, and enhanced integrin expres- endothelial permeability, possibly via restructuring of the sion. LTB4 is also weakly chemotactic for eosinophils. The pri- endothelial cytoskeleton. It also causes the breakdown of actin mary effects of the cysteinyl leukotrienes are bronchoconstric- microfilaments, leading to increases in intercellular junction tion and stimulation of mucus secretion. LTC4 and LTD4 are size and loss of microvascular barrier function. 1,000 times more potent than histamine at causing smooth mus- 5. Neutrophil activation. TXA2 is both a chemoattractant and cle contraction in human bronchi. LTE4 also causes bron- chemoactivator of neutrophils; accordingly, it increases neu- choconstriction, but it is less potent. All of the cysteinyl trophil oxidative activity and diapedesis. leukotrienes are potent mucus secretagogues. They also con- TXA2 is usually produced at low baseline concentrations; tribute to plasma extravasation and tissue edema by increasing however, there are a number of triggers that can rapidly vascular permeability. ARACHIDONIC ACID Lipoxygenase Cyclooxygenase 5-HPETE PGG2 LTA4 PGH2 LTC4 LTA4 Synthetase Hydrolase Glutathione Thromboxane Prostacyclin Prostaglandin Synthetase Synthetase Synthetase LTC4 LTB4 LTD4 TXA2 PGI2 PGE2 PGD2 PGF2α LTE4 TXB2 6-Keto-PGF1α Figure 11 Arachidonic acid is metabolized by two different enzymes, cyclooxygenase (including both COX-1 and COX-2 variants) and lipoxygenase. The products of cyclooxygenase are the prostaglandins and the thromboxanes; the products of lipoxyge- nase are the leukotrienes.
  • 20. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice 8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 20 THERAPEUTIC IMPLICATIONS they are effective for inducing cervical softening before surgical termination of pregnancy. Antileukotrienes in Asthma It is now recognized that the pathogenesis of asthma is more Cyclooxygenase Inhibition with NSAIDs and COX-2 complicated than was previously realized: airway smooth muscle Antagonists contraction in response to an irritant is only part of a larger over- Management of inflammation Histologically, acute inflam- all picture. Asthma has been defined as “a chronic inflammatory mation is characterized by vasodilatation, edema, and early neu- disease of the airways,”98 in which many cells and cellular ele- trophil accumulation. Production of eicosanoids at the inflam- ments are involved, particularly mast cells, eosinophils, T cells, matory site by damaged vessels, leukocytes, and platelets leads to neutrophils, and epithelial cells. vasodilatation (caused by PGI2 and PGE2), increased vascular Leukotrienes have been shown to play a major role in asthma, permeability (caused by TXA2 and leukotrienes), and neutrophil producing the characteristic pathophysiologic signs (bron- recruitment and activation (caused by LTB4 and TXA2). In choconstriction, mucus, airway edema, and cellular infiltration). addition, LTB4 is a potent stimulus for production of the proin- The clinical manifestations of asthma can be reproduced by flammatory cytokines IL-1 and TNF, and PGE2 gives rise to administering leukotrienes. Furthermore, increases in bron- pain. Eicosanoids have been implicated in the pathophysiology choalveolar lavage and urinary leukotriene concentrations have of many inflammatory diseases, including rheumatoid arthritis, been documented in patients with asthma.99 ulcerative colitis, Crohn disease, and asthma. These findings have led to the development of a new class of The use of NSAIDs to treat the symptoms of inflammation is antiasthma drugs, the antileukotrienes. Two types of antileuko- a long-standing practice. NSAIDs counteract inflammation by trienes are now in clinical use. The first type, currently exempli- inhibiting cyclooxygenase, thereby decreasing the synthesis of all fied by zafirlukast and montelukast, acts through competitive prostanoids and exerting antipyretic and analgesic effects. antagonism of the cysteinyl leukotriene receptor, thereby block- NSAIDs are routinely used in the management of chronic ing the action of LTC4, LTD4, and LTE4. The second type, of inflammatory diseases such as rheumatoid arthritis and are which zileuton is the only example currently available, competi- among the most widely prescribed drugs for musculoskeletal tively inhibits 5-LO, thereby blocking all leukotriene synthesis. conditions and pain. By inhibiting cyclooxygenase, NSAIDs may Both types are highly effective at moderating asthma: their use also effectively redirect arachidonic acid into the lipoxygenase leads to improved airway function, less frequent nighttime awak- pathway, thus indirectly increasing leukotriene synthesis. This enings, fewer episodes of asthma exacerbation necessitating redirection is hypothesized to be the mechanism by which steroid therapy, and lower required dosages of inhaled steroids. aspirin can trigger asthma in susceptible individuals. An additional benefit of the antileukotrienes is their ease of use: they are orally administered, are well tolerated, and need be taken Regulation of blood flow Eicosanoids modulate blood only once or twice daily.100 Routine use of these agents is now rec- flow to organs and tissues by adjusting local balances between ommended as part of the multitiered pharmacologic approach to production of vasodilators and production of vasoconstricting asthma management. components. Loss of these controls, such as occurs when age, Whether the antileukotrienes might also be useful in the man- diabetes, or atherosclerosis decreases the capacity of the vascular agement of other inflammatory disease states is currently under endothelium for PGI2 production, may impair normal regulato- investigation. Preliminary reports suggest that they might some- ry mechanisms. For example, in occlusive vascular disease, day play a part in the management of rheumatoid arthritis, sys- ischemia may cause elevated TXA2 levels by stimulating the syn- temic lupus erythematosus, urticaria, and atopic dermatitis. thesis of TXA2 by neutrophils and platelets. This would explain why output of TXA2 is significantly elevated in the clinical set- Exogenous Prostaglandins in Obstetric and Gynecologic Settings tings of unstable angina and acute myocardial infarction and Prostaglandins are potent stimulators of myometrial smooth variably elevated after cerebrovascular accidents. Without muscle contractility, as are other prostanoids (e.g., TXA2 and enhanced PGI2 production, the resulting vasoconstriction and eicosatetraenoic acid). These mediators are thought to play an platelet aggregation may exacerbate ischemia in these settings. important—though not yet fully explained—role in normal fetal Aspirin, alone among the NSAIDs, causes irreversible meth- delivery. Concentrations of certain prostanoids and their ylation of COX-1 and COX-2. It is 50 to 100 times more potent metabolites are known to increase before the onset of sponta- at the COX-1 site than at the COX-2 site. Accordingly, it is capa- neous labor and continue to increase during birth. NSAIDs, ble of permanently eliminating platelets’ ability to produce TXA2 which inhibit prostanoid synthesis, are known to reduce myome- even when given only once daily in low doses; however, to antag- trial contractions and delay or even prevent labor.101 PGE2 and onize COX-2, which is largely responsible for inflammation, PGF2 also induce the cervical ripening that must occur before much higher and more frequent doses are necessary. Platelets, normal delivery. being nonnucleated, are unable to resynthesize COX-1 and Prostaglandin receptors are present on myometrial tissue COX-2. Because somewhat less than 10% of the platelet popu- throughout gestation, unlike many other receptors, such as those lation is turned over each day, it takes several days for platelet for oxytocin, which are only induced in the latter phase of preg- production of prostanoids to return to normal levels. Nucleated nancy. Consequently, therapy with prostaglandins is possible cells, on the other hand, rapidly resynthesize COX-1 and COX- throughout pregnancy. Gemeprost (the methyl ester of PGE1) 2 enzymes and thus can resume producing prostaglandins a few and dinoprostone (PGE2) are currently approved for cervical hours after aspirin administration. ripening and labor induction.When given vaginally, they are safe Aspirin, therefore, is antithrombotic when given in small and effective, posing no risk to the fetus even though they cross amounts and at wide intervals. When so used, it permits synthe- the placental barrier. These agents are safe in promoting first- sis of vasodilating, antithrombotic prostaglandins while inhibit- and second-trimester terminations, whether used alone or given ing platelet synthesis of thromboxane. Accordingly, aspirin has in combination with mifepristone or methotrexate; moreover, been widely applied to the management of atherosclerotic dis-
  • 21. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice 8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 21 ease. One large international trial reported that in patients who inhibition. Endogenous prostaglandins play a central role in took a single 162.5 mg tablet of aspirin daily, starting within 24 maintenance of the gastroduodenal mucosal defense barrier by hours of a suspected myocardial infarction and continuing for 5 regulating mucosal bicarbonate production, stimulating muco- weeks, mortality was reduced by 23%, the incidence of nonfatal sal blood flow, and modulating epithelial cell proliferation. Oral reinfarction by 49%, and the incidence of nonfatal stroke by therapy with misoprostol has proved effective in healing both 46%, with no increase in the incidence of either GI bleeding or gastric and duodenal ulcers. hemorrhagic stroke.102 It is now believed that long-term low- The coxibs, of which rofecoxib and celecoxib are the best- dose (75 to 180 mg/day) aspirin therapy may benefit all persons known examples, were developed specifically with the aim of who have unstable angina, have a history of myocardial infarc- overcoming the ulcerogenic side effects of COX-1 antagonists. tion, or are at risk for ischemic stroke, as well as many other per- The coxibs selectively inhibit COX-2 and thus should be capa- sons at high risk for atherosclerotic events. ble of exerting anti-inflammatory effects without interfering with Prostaglandins also play an essential role in regulating neona- the gastric protection afforded by COX-1 products. To date, tal circulatory changes. Very soon after birth, the fetal ductus they have been at least partially successful in achieving the arteriosus starts to contract, thereby reducing the size of the desired aim.The coxibs are as effective as conventional NSAIDs large vascular shunt present in the fetus, which diverts 90% of at treating rheumatoid arthritis and osteoarthritis and reducing right ventricular output away from the lungs and into the aorta. pain.106 In addition, patients receiving coxibs show significantly In approximately 20% of full-term infants, the ductus arteriosus lower rates of gastric ulceration than patients receiving conven- is functionally closed at 24 hours post partum, and in all, the tional NSAIDs.107 Use of coxibs is not, however, without draw- ductus is closed at 96 hours.The ductus is extremely sensitive to backs. These agents are associated with dyspepsia, though to a the vasodilatory action of PGE2. Normally, the high circulating lesser degree than conventional NSAIDs are. Moreover, they fetal concentration of this prostaglandin decreases after birth; exert the same delaying effect on ulcer healing that NSAIDs do; however, in preterm, low-birth-weight infants, this decrease does this is because both COX-1 products and COX-2 products are not occur, and thus the ductus does not close. In these instances, necessary for angiogenesis. administration of indomethacin is a safe and effective way of Another important side effect of NSAIDs is their nephrotox- promoting duct closure.103 icity, which results from interference with prostaglandin-mediat- There are, however, two groups of patients in whom mainte- ed control of local renal vascular flow. PGE2, PGI2, and TXA2 nance of a patent ductus arteriosus is necessary: (1) newborns play important roles in regulating the glomerular filtration rate, whose systemic blood flow depends on shunting from the pul- especially in disease states such as shock, cirrhosis, and conges- monary artery to the aorta (e.g., those who have an interrupted tive heart failure. In these settings, there is a compensatory aortic arch) and (2) newborns whose pulmonary blood flow increase in renal PGE2 production, leading to improved blood depends on shunting from the aorta to the pulmonary artery flow. Administration of NSAIDs may cause fluid retention. In (e.g., those who have pulmonary valve atresia). For both groups, addition, NSAIDs are responsible for 20% of cases of drug- infusions of PGE1 have proved highly beneficial, enabling resus- induced acute renal failure. To date, the coxibs have not been citation and stabilization even in the face of major respiratory shown to possess substantial advantages over NSAIDs in regard and electrolyte disturbances. to nephrotoxicity: they appear to produce the same degree of fluid retention as conventional NSAIDs do, though it is possible Prevention of tumors An exciting new potential thera- that their effect on the glomerular filtration rate is less damaging. peutic use of NSAIDs derives from epidemiologic studies that Because TXA2 is a COX-1 product, the coxibs do not inhib- document a reduced incidence of colorectal carcinoma in sub- it thromboxane-mediated platelet aggregation. Therefore, it is jects taking aspirin. Of the more than 90 rodent studies of car- unlikely that they will be able to reduce the thrombotic compli- cinogen-induced and genetically induced colorectal tumors car- cations of vascular disease, as NSAIDs do. Indeed, a recent clin- ried out to date, the majority showed definite decreases at all ical report indicated that COX-2 inhibitors were associated with stages of tumorigenesis with NSAID therapy.104 The results a slight but significant increase in myocardial infarction. from observational human studies suggest that NSAID therapy is associated with approximately a 40% relative risk reduction Dietary Fatty Acids with respect to the incidence of colorectal carcinoma. This issue The long-chain polyunsaturated fatty acids that are the pre- should be clarified further when the results of three ongoing cursors of eicosanoids cannot be synthesized in the human body phase III clinical trials of aspirin in colorectal carcinoma pre- and must therefore be supplied through diet. In North America vention become available. COX-2 antagonists have been less and western Europe, the primary dietary source for eicosanoid well studied, but there is some evidence suggesting that such synthesis is linoleic acid, the precursor of arachidonic acid. agents may modify the growth of bladder tumors. Linoleic acid is found predominantly in vegetable oils. It and its Precisely how NSAIDs exert their antitumor effects is not yet derivatives are termed omega-6 fatty acids because the last of fully understood. Colorectal carcinomas are known to overex- their four double bonds occurs at the sixth carbon atom from press COX-2 and PGE2,105 and it is possible that these agents the methyl end of the chain. α-Linolenic acid, found in marine act to promote cellular proliferation and inhibit cell apoptosis, fish and to a lesser extent in green vegetables, is the precursor of thereby giving rise to cancer. PGE2, the eicosanoid most preva- eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), lent in GI tissue, has immunosuppressive effects that may which are also eicosanoid precursors. These substances are reduce tumor surveillance. termed omega-3 fatty acids because their last double bond occurs at the third carbon atom from the methyl end of the Side effects of NSAIDs and development of COX-2 chain. inhibitors The ability of aspirin to cause gastric ulceration The relative prevalence of the various fatty acid precursors and bleeding, originally documented in the first half of the 20th has important clinical implications, in that higher proportions of century, is now understood to be attributable to prostaglandin one precursor or another will affect which eicosanoid products
  • 22. © 2002 WebMD Inc. All rights reserved. ACS Surgery: Principles and Practice 8 CRITICAL CARE 26 MOLECULAR AND CELLULAR MEDIATORS — 22 are generated. For example, when EPA is the substrate for is LTB5, which is less active than LTB4. Reduced synthesis of cyclooxygenase, TXA3, which is biologically inactive, is pro- LTB4 by neutrophils may underlie the observation that fish sup- duced; EPA also competitively binds cyclooxygenase, thereby plements reduce symptom severity in patients with rheumatoid reducing TXA2 synthesis. However, PGI3 synthesized from arthritis. It has also been noted that fewer low-birth-weight chil- omega-3 fatty acids is fully active. In this setting, therefore, with dren are born in northern European populations, who typically thromboxane activity reduced but prostacyclin activity main- consume substantial amounts of fish, than in non–fish-eating tained, the overall effects are to increase platelet survival, to populations. In animal studies, experimental subjects that either reduce platelet aggregation, and to increase bleeding time. are deficient in omega-6 fatty acids or have been fed diets high Interest in the possible clinical use of dietary omega-3 fatty in omega-3 fatty acid exhibit decreased prostaglandin synthesis acids came from the observation that Greenland Eskimos had a and longer gestation periods. low incidence of myocardial infarction despite eating a high-fat It is believed that early hominids evolved on a diet containing diet.108 Their plasma and platelet lipids were found to contain a roughly equal proportions of omega-3 and omega-6 fatty acids. high proportion of omega-3 long-chain polyunsaturated fatty In contrast, the typical diet in North America and western acids. Furthermore, they had hypoaggregable platelets and Europe today, which is high in dairy, meat, and fried foods, con- increased bleeding times. The same effects were noted in other tains 10 to 25 times more omega-6 than omega-3 fatty acids. populations who were fed dietary supplements containing This relative deficiency of omega-3 fatty acids may be a major omega-3 fatty acids. The potential long-term benefits of cause of the high rates of cardiac and inflammatory diseases increased omega-3 fatty acid intake were suggested by epidemi- recorded in the more developed areas of the world. ologic studies in the Netherlands and Japan that showed a reduced incidence of coronary artery disease in men whose diet contained a large amount of fish supplements.109,110 Subsequent Conclusion randomized, controlled clinical trials demonstrated that dietary Inflammation is a highly complex process that involves many supplementation with either fish or fish oil conferred significant interacting systems, which may complement each other, antago- benefits on men who had recently had myocardial infarctions, nize each other, or both. A degree of redundancy is built in, so reducing the risk of death and especially of sudden cardiac that antagonism of one pathway may lead to augmentation of death. In particular, increased dietary intake of omega-3 fatty another alternative inflammatory mechanism. Furthermore, acids had a potent antidysrhythmic effect, apparently because simultaneous or sequential production of both proinflammatory incorporation of these substances into the myocyte cell mem- and anti-inflammatory signals often occurs. Accordingly, the suc- brane increased the stability and lengthened the refractory peri- cess of a therapeutic intervention depends not only on the type od of these cells. of injury or insult present but also on the timing of the interven- Although the precise mechanisms underlying the protective tion. Unfortunately, the clinical trials done to date, evaluating a effect associated with dietary omega-3 fatty acids have not been host of anti-inflammatory agents of different classes, have been determined, it is likely that reduced TXA2 production is a major largely disappointing. For more successful therapies to be devel- contributor. Altered leukotriene biochemistry may also play a oped, much more information regarding the fundamental mech- role: the main leukotriene synthesized from omega-3 fatty acids anisms of the inflammatory response will be needed. References 1. Metchnikoff ME: Sur la des cellules de l’organ- meability to albumin of cultured endothelial leukocytes in reperfusion injury of skeletal muscle isme contre l’invasion des microbes. Ann Inst monolayers and isolated perfused lungs: role of after partial ischaemia. Am J Physiol 257:H1068, Pasteur 7:321, 1887 oxygen radicals and granulocyte adherence. Am 1989 2. 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