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Cytokines are a class of protein signaling compounds that are essential for both innate and adaptive immune responses. Cytokines mediate a broad sequence of cellular responses, including cell migration, DNA replication, cell turnover, and immunocyte proliferation (Table 2-5). When functioning locally at the site of injury and infection, cytokines mediate the eradication of invading microorganisms and also promote wound healing. However, an exaggerated proinflammatory cytokine response to inflammatory stimuli may result in hemodynamic instability (i.e., septic shock) and metabolic derangements (i.e., muscle wasting). Anti-inflammatory cytokines also are released, at least in part, as an opposing influence to the proinflammatory cascade. These anti-inflammatory mediators may also result in immunocyte dysfunction and host immunosuppression. Cytokine signaling after an inflammatory stimulus can best be represented as a finely tuned balance of opposing influences and should not be oversimplified as a “black and white” proinflammatory/anti-inflammatory response. A brief discussion of the important cytokine molecules is included.
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Tumor Necrosis Factor-α
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TNF-α is a cytokine that is rapidly mobilized in response to stressors such as injury and infection and is a potent mediator of the subsequent inflammatory response. TNF is primarily synthesized by immune cells, such as macrophages, dendritic cells, and T lymphocytes, but nonimmune cells have also been reported to secrete low amounts of the cytokine.
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TNF is generated in a precursor form called transmembrane TNF that is expressed as a trimer on the surface of activated cells. After being processed by the metalloproteinase TNF-α–converting enzyme (TACE; also known as ADAM-17), a smaller, soluble form of TNF is released, which mediates its biologic activities through type 1 and 2 TNF receptors (TNFR1; TNFR2).66 Transmembrane TNF-α also binds to TNFR1 and TNFR2, but its biologic activities are likely mediated through TNFR2. While the two receptors share homology in their ligand binding regions, there are distinct differences that regulate their biologic function. For example, TNFR1 is expressed by a wide variety of cells but is typically sequestered in the Golgi complex. Following appropriate cell signaling, TNFR1 is mobilized to the cell surface, where it sensitizes cells to TNF, or it can be cleaved from the surface in the form of a soluble receptor that can neutralize TNF.67 In contrast, TNFR2 expression is confined principally to immune cells where it resides in the plasma membrane. Both TNF receptors are capable of binding intracellular adaptor proteins that lead to activation of complex signaling processes and mediate the effects of TNF.
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Although the circulating half-life of soluble TNF is brief, it acts upon almost every differentiated cell type, eliciting a wide range of important cellular responses. In particular, TNF elicits many metabolic and immunomodulatory activities. It stimulates muscle breakdown and cachexia through increased catabolism, insulin resistance, and redistribution of amino acids to hepatic circulation as fuel substrates. TNF also mediates coagulation activation, cell migration, and macrophage phagocytosis, and enhances the expression of adhesion molecules, prostaglandin E2, platelet-activating factor, glucocorticoids, and eicosanoids. Recent studies indicate that a significant early TNF response after trauma may be associated with improved survival in these patients.68
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IL-1α and IL-1β, which are encoded by two distinct IL-1 genes, were the first described members of the IL-1 cytokine family. Currently, the family has expanded to 11 members, with the three major forms being IL-1α, IL-1β, and IL-1 receptor antagonist (IL-1Rα). IL-1α and IL-1β share similar biologic functions, but have limited sequence homology. They use the same cell surface receptor, termed IL-1 receptor type 1 (IL-1R1), which is present on nearly all cells. Although IL-1Rα is synthesized and released in response to the same stimuli that lead to IL-1 production, it lacks the necessary domain to form a bioactive complex with the IL-1 receptor when bound. Thus, IL-1Rα serves as a competitive antagonist for the receptor. IL-1R activation initiates signaling events, which result in the synthesis and release of a variety of inflammatory mediators.
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The IL-1α precursor is constitutively expressed and stored in a variety of healthy cells, including epithelium, endothelium, and platelets. Both the precursor and mature forms of IL-1α are active. With appropriate signals, IL-1α moves to the cell membrane where it can act on adjacent cells bearing the IL-1 receptor. It can also be released directly from injured cells. In this way, IL-1α is believed to function as a DAMP, which promotes the synthesis of inflammatory mediators, such as chemokines and eicosanoids. These mediators attract neutrophils to the injured site, facilitate their exit from the vasculature, and promote their activation. Once they have reached their target, neutrophil lifespan is extended by the presence of IL-1α.69
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IL-1β, a multifunctional proinflammatory cytokine, is not detectable in healthy cells. Rather, its expression and synthesis occur in a more limited number of cells, such as monocytes, tissue macrophages, and dendritic cells, following their activation. IL-1β expression is tightly regulated at multiple levels (e.g., transcription, translation, and secretion), although the rate-limiting step is its transcription. IL-1β is synthesized and released in response to inflammatory stimuli, including cytokines (TNF, IL-18) and foreign pathogens. IL-1α or IL-1β itself can also induce IL-1β transcription. In contrast to IL-1α, IL-1β is synthesized as an inactive precursor molecule. The formation of mature IL-1β requires the assembly of the inflammasome complex by the cell and the activation of caspase 1, which is required for the processing of stored pro-IL-1β. Mature IL-1β is then released from the cell via an unconventional secretory pathway. IL-1β has a spectrum of proinflammatory effects that are largely similar to those induced by TNF, and injection of IL-1β alone is sufficient to induce inflammation. High doses of either IL-1β or TNF are associated with profound hemodynamic compromise. Interestingly, low doses of both IL-1β and TNF combined elicit hemodynamic events similar to those elicited by high doses of either mediator, which suggests a synergistic effect.
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There are two primary receptor types for IL-1: IL-1R1 and IL-1R2. IL-1R1 is widely expressed and mediates inflammatory signaling on ligand binding. IL-1R2 is proteolytically cleaved from the membrane surface to soluble form on activation and thus serves as another mechanism for competition and regulation of IL-1 activity. IL-1α or IL-1β binds first to IL-1R1. This is followed by recruitment of a transmembrane coreceptor, termed the IL-1R accessory protein (IL-1RAcP). A complex is formed of IL-1R1 plus IL-1 plus the coreceptor. The signal is initiated with recruitment of the adaptor protein MyD88 to the toll–IL-1 receptor (TIR) domains of the receptor complex and signal transduction then occurs via intermediates, which are homologous to the signal cascade initiated by TLRs. These events culminate in the activation of NF-κB and its nuclear translocation.70
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IL-2 is a multifunctional cytokine produced primarily by CD4+ T cells after antigen activation, which plays a pivotal role in the immune response. Other cellular sources for IL-2 include CD8+ and NK T cells, mast cells, and activated dendritic cells. Discovered as a T-cell growth factor, IL-2 also promotes CD8+ T-cell and NK cell cytolytic activity and modulates T-cell differentiation programs in response to antigen. Thus, IL-2 promotes naïve CD4+ T-cell differentiation into T helper 1 (Th1) and T helper 2 (Th2) cells while inhibiting T helper 17 (Th17) and T follicular helper (Tfh) cell differentiation. Moreover, IL-2 is essential for the development and maintenance of T regulatory (Treg) cells and for activation-induced cell death, thereby mediating tolerance and limiting inappropriate immune reactions. The upregulation of IL-2 requires calcium as well as protein kinase C signaling, which leads to the activation of transcription factors such as nuclear factor of activated T cells (NFAT) and NF-κB. MicroRNAs also play a role in the regulation of IL-2 expression.71
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IL-2 binds to IL-2 receptors (IL-2R), which are expressed on leukocytes. IL-2Rs are formed from various combinations of three receptor subunits: IL-2Rα, IL-2Rβ, and IL-2Rγ; these form low-, medium-, and high-affinity forms of the receptor depending on the subunit combination. IL-2Rγ has been renamed the common cytokine receptor γ chain (γc), which is now known to be shared by IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Constitutive IL-2 receptor expression is low and is inducible by T-cell receptor ligation and cytokine stimulation. Importantly, the transcription of each receptor subunit is individually regulated via a complex process to effect tight control of surface expression. Once the receptor is ligated, the major IL-2 signaling pathways that are engaged include Janus kinase (JAK) signal transducer and activator of transcription (STAT), Shc-Ras-MAPK, and phosphoinositol-3-kinase (PI3K)-AKT. Partly due to its short half-life of <10 minutes, IL-2 is not readily detectable after acute injury. IL-2 receptor blockade induces immunosuppressive effects and can be pharmacologically used for organ transplantation. Attenuated IL-2 expression observed during major injury or blood transfusion may contribute to the relatively immunosuppressed state of the surgical patient.72
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Following burn or traumatic injury, DAMPs from damaged or dying cells stimulate TLRs to produce IL-6, a pleiotropic cytokine that plays a central role in host defense. IL-6 levels in the circulation are detectable by 60 minutes, peak between 4 and 6 hours, and can persist for as long as 10 days. Further, plasma levels of IL-6 are proportional to the degree of injury. In the liver, IL-6 strongly induces a broad spectrum of acute-phase proteins such as CRP and fibrinogen, among others, whereas it reduces expression of albumin, cytochrome P450, and transferrin. In lymphocytes, IL-6 induces B-cell maturation into immunoglobulin-producing cells and regulates Th17/Treg balance. IL-6 modulates T-cell behavior by inducing the development of Th17 cells and inhibiting Treg cell differentiation in conjunction with transforming growth factor-β. IL-6 also promotes angiogenesis and increased vascular permeability, which are associated with local inflammatory responses. To date, 10 IL-6 family cytokines have been identified, including IL-6, oncostatin M, neuropoietin, IL-11, IL-27, and IL-31, all of which use trans signaling.73
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The IL-6 receptor (IL-6R, gp80) is expressed on hepatocytes, monocytes, B cells, and neutrophils in humans. However, many other cells respond to IL-6 through a process known as trans signaling.74 In this case, soluble IL-6Rs (sIL-6R) exist in the serum and bind to IL-6, forming an IL-6/sIL-6R complex. The soluble receptor is produced by proteolytic cleavage from the surface of neutrophils in a process that is stimulated by CRP, complement factors, and leukotrienes. The IL-6/sIL-6R complex can then bind to the gp130 receptor, which is expressed ubiquitously on cells. Upon IL-6 stimulation, gp130 transduces two major signaling pathways: the JAK-STAT3 pathway and the SHP2-Gab-Ras-Erk-MAPK pathway, which is regulated by cytoplasmic suppressor of cytokine signaling (SOCS3). These signaling events can lead to increased expression of adhesion molecules as well as proinflammatory chemokines and cytokines. High plasma IL-6 levels have been associated with mortality during intra-abdominal sepsis.75
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We have talked almost exclusively about the factors that initiate the inflammatory response following cellular stress or injury. The re-establishment of immune homeostasis following these events requires the resolution of inflammation and the initiation of tissue repair processes. IL-10 plays a central role in this anti-inflammatory response by regulating the duration and magnitude of inflammation in the host.
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The IL-10 family currently has six members including IL-10, IL-19, IL-20, IL-22, IL-24, and IL-26. IL-10 is produced by a variety of immune cells of both myeloid and lymphoid origin. Its synthesis is upregulated during times of stress and systemic inflammation; however, each cell type that produces IL-10 does so in response to different stimuli, allowing for tight control of its expression. IL-10 exerts effects by binding to the IL-10 receptor (IL-10R), which is a tetramer formed from two distinct subunits, IL-10R1 and IL-10R2. Specifically, IL-10 binds first to the IL-10R1 subunit, which then recruits IL-10R2, allowing the receptor complex to form. Whereas IL-10R2 is widely expressed, IL-10R1 expression is confined to leukocytes so that this differential expression of the receptor confines the effects of IL-10 to the immune system. Once receptor ligation occurs, signaling proceeds by the activation of JAK1 and STAT3. In particular, STAT3 in conjunction with IL-10 is absolutely required for the transcription of genes responsible for the anti-inflammatory response. IL-10 inhibits the secretion of proinflammatory cytokines, including TNF and IL-1, partly through the downregulation of NF-κB, and thereby functions as a negative feedback regulator of the inflammatory cascade.76 In macrophages, IL-10 suppresses the transcription of 20% of all lipopolysaccharide (LPS)-induced genes. Further, experimental models of inflammation have shown that neutralization of IL-10 increases TNF production and mortality, whereas restitution of circulating IL-10 reduces TNF levels and subsequent deleterious effects. Increased plasma levels of IL-10 also have been associated with mortality and disease severity after traumatic injury. IL-10 may significantly contribute to the underlying immunosuppressed state during sepsis through the inhibition and subsequent anergy of immunocytes. For example, IL-10 produced by Th2 cells directly suppresses Th1 cells and can feedback to suppress Th2 cell activity.77
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IL-12 is unique among the cytokines in being the only heterodimeric cytokine. This family, which includes IL-12, IL-23, IL-27, and IL-35, consists of an α-chain that is structurally similar to the IL-6 cytokine and a β-chain that is similar to the class I receptor for cytokines. The individual IL-12 family members are formed from various combinations of the α and β subunits. Despite the sharing of individual subunits and the similarities of their receptors, the IL-12 cytokines have different biologic functions. IL-12 and IL-23 are considered proinflammatory, stimulatory cytokines with key roles in the development of Th1 and Th17 subsets of helper T cells. In contrast, both IL-27 and IL-35 appear to have immunoregulatory functions that are associated with cytokine inhibition in specific Treg cell populations, particularly the Th17 cells.78 The effects of these cytokines require specific receptor chains that are also shared among the cytokines. The complexity of signaling is evidenced by the fact that these receptor chains can function both as dimers and as monomers. Ligation of the IL-12 receptors initiates signaling events mediated by the JAK-STAT pathway. IL-12 synthesis and release are increased during endotoxemia and sepsis.79 IL-12 stimulates lymphocytes to increase secretion of IFN-γ with the costimulus of IL-18 and also stimulates NK cell cytotoxicity and helper T-cell differentiation in this setting. IL-12 release is inhibited by IL-10. IL-12 deficiency inhibits phagocytosis in neutrophils. In experimental models of inflammatory stress, IL-12 neutralization conferred a mortality benefit in mice during endotoxemia. However, in a cecal ligation and puncture model of intraperitoneal sepsis, IL-12 blockade was associated with increased mortality. Furthermore, later studies of intraperitoneal sepsis observed no difference in mortality with IL-12 administration; however, IL-12 knockout mice exhibited increased bacterial counts and inflammatory cytokine release, which suggests that IL-12 may contribute to an antibacterial response. IL-12 administration in chimpanzees is capable of stimulating the release of proinflammatory mediators such as IFN-γ and also anti-inflammatory mediators, including IL-10, soluble TNFR, and IL-1 receptor antagonists. In addition, IL-12 enhances coagulation as well as fibrinolysis.
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IL-18 is a member of the IL-1 superfamily of cytokines. First noted as an IFN-γ–inducing factor produced by LPS-stimulated macrophages, IL-18 expression is found both in immune cells and nonimmune cells at low to intermediate levels. However, activated macrophages and Kupffer cells produce large amounts of mature IL-18. Similar to IL-1β, IL-18 is synthesized and stored as an inactive precursor form (pro-IL-18), and activation requires processing by caspase 1 in response to the appropriate signaling. It then exits the cell through a nontraditional secretory pathway. The IL-18 receptor (IL-18R) is composed of two subunits, IL-18Rα and IL-18Rβ, and is a member of the IL-1R superfamily, which is structurally similar in its cytoplasmic domains to the TLR.
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One unique biologic property of IL-18 is the potential, in conjunction with IL-12, to promote the Th1 response to bacterial infection. At the same time, exogenous IL-18 can also enhance the Th2 response and Ig-mediated humoral immunity, as well as augment neutrophil function. Recent studies suggest that IL-18 therapy may hold promise as effective therapy in promoting immune recovery after severe surgical stress.80
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Interferons were first recognized as soluble mediators that inhibited viral replication through the activation of specific antiviral genes in infected cells. Interferons are categorized into three types based on receptor specificity and sequence homology. The two major types, type I and type II, are discussed here.
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Type I interferons, of which there are 20, include IFN-α, IFN-β, and IFN-ω, which are structurally related and bind to a common receptor, IFN-α receptor. They are likely produced by most cell types and tissues in response to appropriate pathogens or DAMP signaling. Type I interferons are expressed in response to many stimuli, including viral antigens, double-stranded DNA, bacteria, tumor cells, and LPS. Type I interferons influence adaptive immune responses by inducing the maturation of dendritic cells and by stimulating class I major histocompatibility complex (MHC) expression. IFN-α and IFN-β also enhance immune responses by increasing the cytotoxicity of NK cells both in culture and in vivo. Further, they have been implicated in the enhancement of chemokine synthesis, particularly those that recruit myeloid cells and lymphoid cells. Thus, IFN/STAT signaling has important effects on the mobilization, tissue recruitment, and activation of immune cells that compose the inflammatory infiltrate. In contrast, IFN-I appears to inhibit inflammasome activity, possibly via IL-10.81
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Many of the physiologic effects observed with increased levels of IL-12 and IL-18 are mediated through IFN-γ. IFN-γ is a type II interferon that is secreted by various T cells, NK cells, and antigen-presenting cells in response to bacterial antigens, IL-2, IL-12, and IL-18. IFN-γ stimulates the release of IL-12 and IL-18. Negative regulators of IFN-γ include IL-4, IL-10, and glucocorticoids. IFN-γ binding with a cognate receptor activates the JAK-STAT pathway, leading to subsequent induction of biologic responses. Macrophages stimulated by IFN-γ demonstrate enhanced phagocytosis and microbial killing and increased release of oxygen radicals, partly through an NADP-dependent phagocyte oxidase. IFN-γ mediates macrophage stimulation and thus may contribute to acute lung injury after major surgery or trauma. Diminished IFN-γ level, as seen in knockout mice, is associated with increased susceptibility to both viral and bacterial pathogens. In addition, IFN-γ promotes differentiation of T cells to the helper T-cell subtype 1 and also enhances B-cell isotype switching to immunoglobulin G.82
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Receptors of all IFN subtypes belong to the class II of cytokine receptors and use the JAK-STAT signaling pathway for nuclear signaling, although different STAT activation (e.g., STAT1 and STAT2) is favored by individual receptors.
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Granulocyte-Macrophage Colony-Stimulating Factor/Interleukin-3/Interleukin-5
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GM-CSF, IL-3, and IL-5 compose a small family of cytokines that regulate the growth and activation of immune cells. They are largely the products of activated T cells, which when released stimulate the behavior of myeloid cells by inducing cytokine expression and antigen presentation. In this way, GM-CSF, IL-3, and IL-5 are able to link the innate and acquired immune responses. With the exception of eosinophils, GM-CSF, IL-3, and IL-5 are not essential for constitutive hematopoietic cell function. Rather, they play an important role when the host is stressed, by serving to increase the numbers of activated and sensitized cells required to bolster host defense.83 Currently, GM-CSF is in clinical trials for administration to children with an Injury Severity Score >10 following blunt or penetrating trauma. The goal of the study is to provide evidence of the effectiveness of GM-CSF as an agent that can ameliorate posttraumatic immune suppression.
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Receptors for the GM-CSF/IL-3/IL-5 family of cytokines are expressed at very low levels on hematopoietic cells. Similar to the other cytokine receptors discussed, they are heterodimers composed of a cytokine-specific α subunit and a common β subunit (βc), which is shared by all three receptors and is required for high-affinity signal transduction. The binding of cytokine to its receptor activates JAK2-STAT–, MAPK-, and PI3K-mediated signaling events to regulate a variety of important cell behaviors including effector function in mature cells.
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Omega-6 Polyunsaturated Fat Metabolites: Arachidonic Acid
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Eicosanoids are derived primarily by oxidation of the membrane phospholipid, arachidonic acid [all-cis-5,8,11,14-eicosatetraenoic acid; 20:4(ω-6) eicosatetraenoic acid], which is relatively abundant in the membrane lipids of inflammatory cells. They are composed of three families, which include prostaglandins, thromboxanes, and leukotrienes. Arachidonic acid is not stored free in the cell but in an esterified form in phospholipids and neutral lipids. When a cell senses the proper stimulus, arachidonic acid is released from phospholipids or diacylglycerols by the enzymatic activation of phospholipase A2 (Fig. 2-6A). Prostanoids, which include all of the prostaglandins and the thromboxanes, result from the sequential action of the cyclooxygenase (COX) enzyme and terminal synthetases on arachidonic acid. In contrast, arachidonic acid may be oxidized along the lipoxygenase pathway via the central enzyme 5-lipoxygenase, to produce several classes of leukotrienes and lipoxins. In general, the effects of eicosanoids are mediated via specific receptors, which are members of a superfamily of G-protein–coupled receptors.
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Eicosanoids are not stored within cells but are instead generated rapidly in response to many stimuli, including hypoxic injury, direct tissue injury, endotoxin (lipopolysaccharide), NE, vasopressin, angiotensin II, bradykinin, serotonin, ACh, cytokines, and histamine. Eicosanoid pathway activation also leads to the formation of the anti-inflammatory compound lipoxin, which inhibits chemotaxis and NF-κB activation. Glucocorticoids, nonsteroidal anti-inflammatory drugs, and leukotriene inhibitors block the end products of eicosanoid pathways. Eicosanoids have a broad range of physiologic roles, including neurotransmission and vasomotor regulation. They are also involved in immune cell regulation (Table 2-6) by modulating the intensity and duration of inflammatory responses. The production of eicosanoids is cell- and stimulus-specific. Therefore, the signaling events that are initiated will depend on the concentrations and types of eicosanoids generated, as well as the unique complement of receptors expressed by their target cells. For example, prostaglandin E2 (PGE2) suppresses the effector function of macrophages (i.e., phagocytosis and intracellular pathogen killing) via a mechanism that is dependent on increased cAMP levels. PGE2 also modulates chemokine production and enhances local accumulation of regulatory T cells and myeloid-derived suppressor cells. Prostacyclin (PGI2) has an inhibitory effect on Th1- and Th2-mediated immune responses, while enhancing Th17 differentiation and cytokine production. Leukotrienes are potent mediators of capillary leakage as well as leukocyte adherence, neutrophil activation, bronchoconstriction, and vasoconstriction. Leukotriene B4 is synthesized from arachidonic acid in response to acute Ca2+ signaling induced by inflammatory mediators.84 High-affinity leukotriene receptors (BLT1) are expressed primarily in leukocytes, including granulocytes, eosinophils, macrophages, and differentiated T cells, whereas the low-affinity receptor is expressed in many cell types. Activation of BLT1 results in inhibition of adenylate cyclase and reduced production of cAMP. Not surprisingly, a role for leukotriene B4 signaling in abrogating the effects of prostaglandins on macrophage effector function has recently been shown.85
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Recent evidence supports a role for lipid droplets (LDs) as an important intracellular source of arachidonic acid. LDs are neutral lipid storage organelles ubiquitous to eukaryotic cells that are a rich source of esterified arachidonic acid especially in leukocytes. Accumulation of LDs in response to TLR signaling has been reported with an associated increase in the generation of eicosanoid metabolites.86
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While experimental models of sepsis have shown a benefit to inhibiting eicosanoid production, human sepsis trials have failed to show a mortality benefit.87 Eicosanoids also have several recognized metabolic effects. COX pathway products inhibit pancreatic β-cell release of insulin, whereas lipoxygenase pathway products stimulate β-cell activity. Prostaglandins such as PGE2 can inhibit gluconeogenesis through the binding of hepatic receptors and also can inhibit hormone-stimulated lipolysis.88
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Omega-3 Polyunsaturated Fat Metabolites: All-cis-5,8,11,14,17-Eicosapentaenoic Acid [20:5(ω-3) Eicosapentaenoic Acid]
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As noted earlier, polyunsaturated fatty acid (PUFA) metabolites of endogenous arachidonic acid function as inflammatory mediators and have significant roles in the inflammatory response. The major direct dietary source of arachidonic acid is from meat. However, a much larger quantity of ω-6 PUFAs is ingested as linoleic acid, which is found in many vegetable oils, including corn, sunflower, and soybean oils, and in products made from such oils, such as margarines. Linolenic acid is not synthesized in mammals; however, it can be converted to arachidonic acid through lengthening of the carbon chain and the addition of double bonds. The second major family of PUFAs is the ω-3 fatty acid. They can also be derived from shorter chain ω-3 fatty acids of plant origin such as α-linolenic acid, which can be converted after ingestion to eicosapentaenoic acid (EPA) and to docosahexaenoic acid (DHA). ω-3 fatty acids are found in cold water fish, especially tuna, salmon, mackerel, herring, and sardine, which can provide between 1.5 and 3.5 g of these long-chain ω-3 PUFAs per serving. EPA and DHA are also substrates for the COX and lipoxygenase (LOX) enzymes that produce eicosanoids, but the mediators produced have a different structure from the arachidonic acid–derived mediators, and this influences their potency (Fig. 2-6B). In addition, ω-3 fatty acids are reported to have specific anti-inflammatory effects, including inhibition of NF-κB activity, TNF release from hepatic Kupffer cells, and leukocyte adhesion and migration. These are achieved via two purported mechanisms: (a) by decreasing the production of arachidonic acid (ω-6)–derived proinflammatory mediators (by competition for the same enzymes) and (b) by generation of proresolving bioactive lipid mediators. In fact, key derivatives of ω-3 PUFAs, termed resolvins, have been identified and synthesized. Resolvins are now categorized as either E-series (from EPA) or D-series (from DHA). In a variety of model systems, resolvins have been shown to attenuate the inflammatory phenotypes of a number of immune cells.89
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The ratio of dietary ω-6 to ω-3 PUFAs is reflected in the membrane composition of various cells, including cells of the immune system, which has potential implications for the inflammatory response. For example, a diet that is rich in ω-6 PUFAs will result in cells whose membranes are “ω-6 PUFA rich.” When ω-6 PUFAs are the main plasma membrane lipid available for phospholipase activity, more proinflammatory PUFAs (i.e., two-series prostaglandins) are generated. Many lipid preparations are soy-based and thus primarily composed of ω-6 fatty acids. These are thought to be “inflammation enhancing.” Nutritional supplementation with ω-3 fatty acid has the potential to dampen inflammation by shifting the cell membrane composition in favor of ω-3 PUFAs.
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In experimental models of sepsis, ω-3 fatty acids inhibit inflammation, ameliorate weight loss, increase small-bowel perfusion, and may increase gut barrier protection. In human studies, ω-3 supplementation is associated with decreased production of TNF, IL-1β, and IL-6 by endotoxin-stimulated monocytes. In a study of surgical patients, preoperative supplementation with ω-3 fatty acid was associated with reduced need for mechanical ventilation, decreased hospital length of stay, and decreased mortality with a good safety profile.90
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Plasma Contact System
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Following traumatic injury, there is almost immediate activation of the complement system, which is a major effector mechanism of the innate immune system. The complement system was thought to act initially as the required “first line of defense” for the host against pathogens, by binding and clearing them from the circulation. Recent data indicate that complement also participates in the elimination of immune complexes as well as damaged and dead cells. In addition, complement is recognized as contributing to mobilization of hematopoietic stem/progenitor cells and lipid metabolism.91 Although complement activation is typically depicted as a linear process in which parallel pathways are activated, it actually functions more like a central node that is tightly networked with other systems. Then, depending on the activating signal, several initiation and regulatory events act in concert to heighten immune surveillance.
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Complement activation proceeds via three different pathways. Initiation of these pathways occurs by the binding and activation of the recognition unit of each pathway to its designated ligand. The classical pathway, which is often referred to as “antibody dependent,” is initiated by direct binding of C1q to its common ligands, which include immunoglobulin (Ig) M/IgG aggregates. Alternately C1q can activate complement signaling by binding to soluble pattern recognition molecules such as pentraxins (e.g., CRP). In a series of subsequent activation and amplification steps, the pathway ultimately leads to the assembly of the C3 convertase, which cleaves C3 into C3a and C3b. As C3b then complexes with C3 convertase, the C5 convertase is activated, cleaving C5 into C5a and C5b. C3a and C5a are potent anaphylatoxins. C3b acts as an opsonin, whereas C5b initiates the formation of the membrane attack complex. When C5b associates with C6 and C7, the complex becomes inserted into cell membrane and interacts with C8, inducing the binding of several units of C9 to form a lytic pore.
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The lectin pathway of complement activation is initiated by mannose-binding lectins or ficolins, which act as the soluble PRM by binding specific carbohydrate structures that are often present on pathogens. The alternative pathway also includes a PRM-based initiation mechanism that resembles those found in the lectin pathway but involves properdin. The latter recognizes several PAMPs and DAMPs on foreign and apoptotic cells. Once bound, it initiates and propagates the complement response by attracting fluid-phase C3b to recognized surfaces and by stabilizing C3 convertase complexes. Despite its name, the alternative pathway may account for up to 80% to 90% of total complement activation.92
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The major source of the circulating complement components is the liver. Complement proteins can also be produced locally where they have been implicated in the regulation of adaptive immune processes. Complement protein synthesis has been demonstrated in immune cells, including T cells, which when surface bound, interact with C3 and C4 receptors. Also, complement synergistically enhances TLR-induced production of proinflammatory cytokines through convergence of their signaling pathways.
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Kallikrein-Kinin System
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The kallikrein-kinin system is a group of proteins that contribute to inflammation, blood pressure control, coagulation, and pain responses. Prekallikrein is synthesized in the liver and circulates in the plasma bound to high molecular weight kininogen (HK). A variety of stimuli lead to the binding of prekallikrein-HK complex to Hageman factor, (factor XII) followed by its activation, to produce the serine protease kallikrein, which plays a role in the coagulation cascade. HK, produced by the liver, is cleaved by kallikrein to form bradykinin (BK). The kinins (e.g., BK) mediate several physiologic processes, including vasodilation, increased capillary permeability, tissue edema, pain pathway activation, inhibition of gluconeogenesis, and increased bronchoconstriction. They also increase renal vasodilation and consequently reduce renal perfusion pressure. Kinin receptors are members of the rhodopsin family of G-protein–coupled receptors and are located on vascular endothelium and smooth muscle cells. Kinin receptors are rapidly upregulated following TLR4 signaling and in response to cytokines and appear to have important effects on both immune cell behavior and on immune mediators.93 For example, B1 activation results in increased neutrophil chemotaxis, while increased B2 receptor expression causes activation of arachidonic-prostaglandin pathways. Bradykinin and kallikrein levels are increased during gram-negative bacteremia, hypotension, hemorrhage, endotoxemia, and tissue injury. The degree of elevation in the levels of these mediators has been associated with the magnitude of injury and mortality. Clinical trials using bradykinin antagonists have shown some benefit in patients with gram-negative sepsis.94
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Serotonin is a monoamine neurotransmitter (5-hydroxytryptamine [5-HT]) derived from tryptophan. Serotonin is synthesized by neurons in the CNS as well as by intestinal enterochromaffin cells, which are the major source of plasma 5-HT. Once in the plasma, 5-HT is taken up rapidly into platelets via the serotonin transporter (SERT) where it is either stored in the dense granules in millimolar concentrations or targeted for degradation. It is interesting that the surface expression of SERT on platelets is sensitive to plasma 5-HT levels, which in turn modulates platelet 5-HT content. Receptors for serotonin are widely distributed in the periphery and are found in the gastrointestinal tract, cardiovascular system, and some immune cells.95 Serotonin is a potent vasoconstrictor and also modulates cardiac inotropy and chronotropy through nonadrenergic cAMP pathways. Serotonin is released at sites of injury, primarily by platelets. Recent work has demonstrated an important role for platelet 5-HT in the local inflammatory response to injury. Using mice that lack the nonneuronal isoform of tryptophan hydroxylase (Tph1), the rate-limiting step for 5-HT synthesis in the periphery, investigators demonstrated fewer neutrophils rolling on mesenteric venules.96 Tph1–/– mice, in response to an inflammatory stimulus, also showed decreased neutrophil extravasation. Finally, survival of lipopolysaccharide-induced endotoxic shock was reduced in Tph1–/– mice. Together, these data indicate an important role for nonneuronal 5-HT in neutrophil recruitment to sites inflammation and injury.
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Histamine is a short-acting endogenous amine that is widely distributed throughout the body. It is synthesized by histidine decarboxylase (HDC), which decarboxylates the amino acid histidine. Histamine is either rapidly released or stored in neurons, skin, gastric mucosa, mast cells, basophils, and platelets, and plasma levels are increased with hemorrhagic shock, trauma, thermal injury, and sepsis.97 Not surprisingly, circulating cytokines can increase immune cell expression of HDC to further contribute to histamine synthesis. There are four histamine receptor (HR) subtypes with varying physiologic roles, but they are all members of the rhodopsin family of G-protein–coupled receptors. H1R binding mediates vasodilation, bronchoconstriction, intestinal motility, and myocardial contractility. H1R knockout mice demonstrate significant immunologic defects, including impaired B- and T-cell responses. H2R binding is best described for its stimulation of gastric parietal cell acid secretion. However, H2R can also modulate a range of immune system activities, such as mast cell degranulation, antibody synthesis, Th1 cytokine production, and T-cell proliferation. H3R was initially classified as a presynaptic autoreceptor in the peripheral nervous system and CNS. However, data using H3R knockout mice demonstrate that it also participates in inflammation in the CNS. H3R knockout mice display increased severity of neuroinflammatory diseases, which correlates with dysregulation of blood-brain barrier permeability and increased expression of macrophage inflammatory protein 2, IFN-inducible protein 10, and CXCR3 by peripheral T cells. H4R is expressed primarily in bone marrow but has also been detected in leukocytes, including neutrophils, eosinophils, mast cells, dendritic cells, T cells, and basophils. H4R is emerging as an important modulator of chemoattraction and cytokine production in these cells. Thus, it is clear that cells of both the innate and adaptive immune response can be regulated by histamine, which is upregulated following injury.98