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| TEXT BOOK of MEDICINE |
Section VII Principles of Immunology and Inflammation
| 45 MECHANISMS OF INFLAMMATION AND TISSUE REPAIR Gary S. Firestein • |
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THE INFLAMMATORY RESPONSE
Host defense mechanisms have evolved to rapidly recognize pathogens, render them harmless, and repair the damaged tissue. This complex and highly regulated sequence of events can also be triggered by environmental stimuli such as noxious mechanical and chemical agents. The cascade is characterized clinically by the classically described signs and symptoms of “rubor et tumor cum calore et dolore,” or redness and swelling with heat and pain. Under normal circumstances, tightly controlled responses protect against further injury and clear damaged tissue. In disease states, however, pathologic inflammation can lead to marked destruction of the extracellular matrix (ECM) and organ dysfunction.
Initiation of the Inflammatory Response
Toll-like Receptors and Innate Immunity
When normal tissue encounters a pathogen, resident cells are stimulated by engagement of pattern recognition receptors expressed on their cell membranes. These receptors include the toll-like receptor (TLR) family of proteins, which bind molecular structures on microbial pathogens that normally are not found in mammalian cells. Of the TLRs identified to date, perhaps the best studied is TLR2, which is activated primarily by bacterial peptidoglycan and lipoproteins, and TLR4, which is activated by lipopolysaccharide (LPS, or endotoxin) (Table 45-1). In addition, TLR9 is activated by unmethylated bacterial sequences that are enriched for CpG motifs, and TLR3 is important for antiviral defense because it binds double-stranded viral RNA. The range of recognized microbial stimulants is increased through the structural diversity of the TLR intracytoplasmic domains, their ability to heterodimerize, and their associations with tissue-specific accessory molecules. In addition to exogenous molecules, some endogenous structures can bind to TLRs, including heat shock proteins and oxidized low-density lipoproteins (oxLDLs). The latter might be especially important in the pathogenesis of atherosclerosis, where TLR4 is activated by LDL within vascular plaques. Local endothelial cell–and macrophage-derived chemotactic factors can then recruit activated T cells into the atheroma.
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This primitive pattern recognition system is known as innate immunity. The ability to recognize specific foreign molecular structures is encoded in the host genome and provides intergenerational immunologic memory. In contrast, the adaptive immune response involving T and B cells requires a complex system of somatic gene mutations and rearrangements to develop diversity and requires constant monitoring and deletion of autoreactive clones. Each host must develop a unique repertoire of pathogen recognition molecules in adaptive immunity. After encountering a new antigen, generation of the T-cell response can take considerable time (days to weeks) and is fraught with peril if autoreactive clones escape. In contrast, innate immunity mediated by the TLRs leads to rapid deployment (minutes to hours) of host defenses.
TLR engagement initiates a stereotypic response that includes transcription of a customized array of pro-inflammatory genes. Signaling by TLRs often progresses through adaptor proteins and converges on a kinase known as MyD88, which orchestrates several downstream cascades. By directing the phosphorylation of IKB kinase-β (IKKβ), MyD88 activates nuclear factor-KB (NF-KB), a master switch for inflammatory genes. Translocation of NF-KB to the cell nucleus stimulates the production of cytokines (e.g., interleukin-6 [IL-6], IL-8, and tumor necrosis factor-α [TNF-α]), the machinery for prostaglandin release (e.g., cyclooxygenase 2 [COX2]), and genes that regulate the ECM (e.g., metalloproteinases). This rapid response is normally transient, although it can persist in pathogenic states. MyD88-indpendent pathways that stimulate innate immunity also exist. For instance, TLR3 stimulation by RNA viruses uses a separate pathway involving IKKε and interferon regulating factor-3 (IRF-3). IRF-3, in combination with several other transcription factors, induces the expression of genes such as interferon-β (IFN-β) to establish an antiviral state. The mitogen-activated protein (MAP) kinases, such as p38, extracellular signal–regulating kinase (ERK), and c-Jun N-terminal kinase (JNK), are also phosphorylated after ligation of many TLRs and can enhance production of cytokines and proteases.
Activation of transcription factors and signaling cascades has an element of peril for the host. The genes expressed primarily offer protection against pathogens by initiating key defense mechanisms. However, these same pathways can create a hazardous milieu that is toxic to normal cells through the production of oxygen radicals, nitric oxide, and other reactive intermediaries. These molecules can damage DNA and harm bystander cells, or even lead to neoplasia (Table 45-2). For instance, long-standing inflammation in the colon, as in ulcerative colitis, is associated with adenocarcinoma. Increased COX2 expression as a result of NF-KB translocation is another mechanism that also contributes to the development of tumors at inflammatory sites. An unanticipated finding is that NF-KB itself can also directly augment carcinogenesis by serving as a survival signal for damaged cells that would normally be deleted by apoptosis.
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The TLR signal transduction mechanisms, initiated by broad categories of nonmammalian structures, integrate the environmental stimuli and generate a broadly antipathogen response. Fine-tuning of host defenses against unique pathogen structures to provide long-lived immunity requires the slower, more precise adaptive immune system. Although it is more cumbersome and primitive, innate immunity provides signals that activate adaptive responses. For instance, TLRs can stimulate dendritic cells (DCs), which have internalized and processed antigen, to migrate from peripheral tissues to central lymphoid organs. The dendritic cells can also produce cytokines and, after maturation, present antigens to T cells in the context of class II major histocompatibility molecules and surface costimulatory proteins. The activated T cells can then migrate to the tissue to enhance and amplify the host response. T cells also provide help to B cells, thereby stimulating antibody production and activating other components of innate immunity (e.g., the complement system).
Immune Complexes and Complement
The complement system is another ancient defense mechanism that links innate immunity and the humoral arm of adaptive immunity (see Chapter 47). Both the classical complement pathway, activated by immunoglobulin G (IgG)- and IgM-containing immune complexes, and the alternative pathway, activated by bacterial products, converge at the third component of complement, C3, with proteolytic release of fragments that amplify the inflammatory response and mediate tissue injury. The anaphylotoxins C3a and C5a directly increase vascular permeability and contraction of smooth muscle. C3a and C3a desArg also induce TNF-α and interleukin-1β (IL-1β) production by peripheral blood mononuclear cells. C5a induces mast cell release of histamine, thereby indirectly mediating increased vascular permeability. C5a also activates leukocytes and enhances their chemotaxis, adhesion, and degranulation, with release of proteases and toxic metabolites. C5b attaches to the surface of cells and microorganisms and is the first component in the assembly of the C5b–9 membrane attack complex.
Complement plays a critical role in immune regulation, and individuals with genetic or acquired deficiencies have increased susceptibility to many diseases. Individuals with abnormalities of the early complement components, especially C1q, C2, and C4, usually have a minimally increased incidence of infection but demonstrate an enhanced risk of developing autoimmune diseases such as systemic lupus erythematosus (SLE). The mechanism of increased disease susceptibility is probably related to inefficient clearance of immune complexes. Enhanced activation and consumption of complement proteins can also occur in SLE accompanied by low plasma C3 and C4 levels, especially in association with disease exacerbations. C3 or C5 deficiency causes increased susceptibility to bacterial infections, whereas defects in the late components that form the membrane attack complex result in an increase incidence of Neisseria bacteremia.
Environmental Stress and Tissue Damage
Tissue injury due to direct trauma or noxious stimulus also initiates an inflammatory response and is associated with microvascular damage, extravasation of leukocytes through vascular walls, and leakage of plasma and proteins into the tissue. Hemostasis at the site of damaged vessels quickly ensues to stem the flux. Platelets then release fibrinogen, fibronectin, thrombospondin, and von Willebrand's factor, which permit homotypic aggregation as well as adherence to collagen. The resulting thrombus not only serves as a mechanical plug but also begins the inflammatory cascade through the release of vasoactive amines (e.g., serotonin), release of lysosomal proteases, and formation of eicosanoid products. The platelets can also later regulate healing with release of growth factors such as platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β).
Second Wave of the Inflammatory Response
Activation of innate immunity quickly leads to the robust influx of inflammatory cells. Resident cells, such as vascular endothelial cells (ECs), mast cells, dendritic cells, and interstitial fibroblasts, respond by releasing soluble mediators, including the eicosanoids and pro-inflammatory cytokines (Table 45-3). These mediators amplify the inflammatory response and recruit additional leukocytes. Locally stimulated cells, along with the newly arrived inflammatory cells, release toxic reactive intermediates of nitrogen and oxygen as well as a myriad of proteases, principally matrix metalloproteinases (MMPs), serine proteases, and cysteine proteases. These molecules are designed to help destroy infectious agents and remove damaged cells, thus clearing the injured site for tissue repair. Although these processes are carefully balanced and controlled under ordinary circumstances, prolonged stimulation of acute inflammatory mechanisms can cause severe tissue destruction. However, in the vast majority of situations, the normal physiologic response is an exquisitely coordinated program that utilizes proteolytic enzymes to remodel the ECM and promote a supportive environment for wound healing rather than tissue damage.
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Cellular Response
Inflammatory cell infiltration at the site of initial tissue damage typically progresses in an orderly fashion. The process begins with release of chemokines and soluble mediators from resident cells, including interstitial fibroblasts, mast cells, and vascular endothelial cells. Signaling from these events alters the local adhesion molecule profile and creates a chemotactic gradient that recruits cells from the blood stream. Mast cells, in particular, play an important role by releasing vasoactive amines. In fact, some immune complex animal models of inflammation, such as the passive K/BxN arthritis model in mice, have an absolute requirement for mast cells. This model involves activation of complement by autoantibody complexes with glucose-6-phosphate isomerase and is dependent on the alternative complement pathway. Rapid mast cell activation by these autoantibodies increases vascular permeability, enhancing articular immune complex deposition and inflammatory cell ingress into the synovium. In most acute responses, polymorphonuclear leukocytes (PMNs) are the first inflammatory cells to extravasate from the circulation and arrive at the site of injury, followed later by mononuclear cells under the influence of separate signals.
Most resident tissue fibroblasts and vascular endothelial cells are quiescent before migration of PMNs into the tissue. However, these resident cells can be triggered to proliferate and migrate toward the site of injury as well as to synthesize cytokines, proteases, and ECM components. Growth factors are released, such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), stimulating new blood vessel formation. Together with granulocyte-macrophage colony-stimulating factor (GM-CSF), these locally released growth factors contribute to cellular proliferation and amplification of the inflammatory response and also induce maturation of dendritic cells that process antigens. In addition, fibroblasts and endothelial cells secrete new ECM proteins, MMPs, and other ECM-digesting enzymes. The balance of protease and ECM production varies as tissue is remodeled during the course of inflammation. Initially, the response favors proteolytic activity to clear damaged infrastructure. This is followed by a shift to increased production of new ECM to allow tissue repair and wound healing.
Coordinated with the changes induced in the ECM through proteolysis, alterations in endothelial morphology affect the barrier function of the ECM. Increased vascular permeability, caused by disruption of endothelial cell tight junctions, allows blood-borne proteins such as fibrinogen, fibronectin, and vitronectin to extravasate into the perivascular ECM. Interaction with preexisting ECM allows the assembly of new ligands for a subset of adhesion molecules (e.g., integrins α5β1 and αvβ3). This increased vascular permeability and change in the profiles of adhesion molecules and ligands, in conjunction with release of chemoattractant molecules, leads to the recruitment of leukocytes to sites of inflammation with subsequent retention. Some of the chemokines involved are IL-8 (for neutrophils), RANTES (regulated on activation, T-cell expressed and secreted; for monocytes and eosinophils), and IL-16 (for CD4+ T cells).
The precise combination of chemokines and adhesion molecules present in an inflammatory lesion determines the specificity of time and event for the recruitment of subsets of inflammatory cells. For instance, in the synovial lining and microvasculature of patients with rheumatoid arthritis, induction of the adhesion molecule intercellular adhesion molecule 1 (ICAM-1) on endothelial cells and fibroblasts, in combination with chemokines such as IL-8 and monocyte chemoattractant protein 1 (MCP-1), serves to recruit neutrophils and monocytes using the β2 integrins. Similarly, vascular cell adhesion molecule 1 (VCAM-1) recruits T cells and monocytes that express α4β1 integrin. PMNs lack α4β1 and therefore are not recruited by expression of VCAM-1 by vascular endothelium. Thus, the selective expression of integrins and other adhesion molecules regulates the time course of migration of various cell lineages into inflamed tissue. Ligation of these integrins on leukocytes also prolongs cell survival once they have moved into the tissue, by preventing apoptosis. The central role of certain specific adhesion molecule–ligand pairs has been confirmed in human diseases. For instance, α4β1 plays a key role in the recruitment of lymphocytes to the central nervous system in multiple sclerosis, and blocking this interaction suppresses disease activity. Eosinophils also use the same adhesion receptors to migrate into the lung in allergen-induced asthma.
Increased expression of ICAM-1 and VCAM-1, as well as increased chemokine expression, is also evident in other cell types, such as the airway epithelium after allergen challenge in asthma. Rapid and transient influx of neutrophils occurs in allergic airway disease, along with activation of the local T cells and mast cells. These neutrophils produce lipid mediators, reactive oxygen intermediates, and proteases such as elastase, which may contribute to airflow obstruction, epithelial damage, and remodeling. Neutrophil elastase, together with chemokines released by both recruited and allergen-activated T cells and mast cells, serves to recruit eosinophils.
Soluble Mediators
In addition to the activation of local cells and the recruitment of leukocytes, the inflammatory response leads to the production of soluble mediators. In some cases, as with cytokines, these products serve as a primary communication system between cells, orchestrating subsequent infiltration and activation. Other molecules, such as reactive oxygen intermediates, act as effectors that directly kill pathogens. However, damage to normal tissues can be a byproduct of these events.
Pro-inflammatory Cytokines
Pro-inflammatory cytokines, often derived from macrophages and fibroblasts, are primary mediators that activate the immune system. Scores of factors have been identified, but IL-1 and TNF-α are among the most important. The pro-inflammatory members of the IL-1 family (IL-1α, IL-1β, and IL-18) and TNF-α have pleiotropic activities and can enhance adhesion molecule expression on endothelial cells, induce proliferation of endogenous cells, and stimulate antigen presentation. IL-1 and TNF-α also increase expression of matrix-degrading enzymes, such as collagenase and stromelysin. In addition, they stimulate synthesis of inflammatory mediators such as prostaglandin E2 (PGE2) from fibroblasts. Direct injection of these cytokines into animals induces local inflammatory responses, and IL-1 or TNF-α blockade abrogates tissue damage in many animal models of inflammation. Inhibitors of TNF-α are effective therapeutic agents in inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease, and IL-1 inhibitors are beneficial in genetic diseases such as Muckle-Wells syndrome and familial cold autoinflammatory syndrome.
IL-1 and TNF-α comprise only a small fraction of the acute cytokine response. Many other factors also participate, including IL-6 and its related cytokines (IL-11, osteopontin, and leukemia inhibitory factor), which can both induce acute phase reactants and bias an immune response toward a helper T type 1 (TH1) or TH2 phenotype (see Chapter 44). GM-CSF can regulate dendritic cell maturation, increase expression of human leukocyte antigen (HLA-DR) on these cells, and enhance antigen-specific responses, in addition to increasing bone marrow production of myeloid cells. Specialized chemoattractant cytokines called chemokines recruit specific cell types to the site of injury. Through G protein–coupled receptors, some of these molecules act to activate neutrophil effector functions directly. The T-cell lymphokine IFN-γ, although generally considered part of the secondary wave that ensues after T-cell activation, can also induce expression of HLA-DR, increase expression of endothelial cell adhesion molecules, and inhibit collagen production. IL-15, IL-17 (a T cell–derived pro-inflammatory factor that is stimulated by the production of macrophage-derived IL-23), and many other mediators participate in this complex cytokine network.
Cytokines play a key role in the establishment and perpetuation of inflammatory diseases. In rheumatoid arthritis, autocrine and paracrine cytokine networks play a critical role in the perpetuation of inflammation. Effects of IL-1 and TNF-α are often central to continued synovitis, and there is increasing evidence that IL-15 participates by enhancing TNF-α production. IL-17 contributes to synoviocyte activation, and IL-18 can trigger T-cell differentiation toward a TH1 phenotype. Factors such as MCP-1 recruit and activate macrophages into atheromas containing oxLDLs and foam cells. In allergic asthma, IL-13 is emerging as a central inflammatory cytokine. IL-13 functions through binding to cell surface IL-4 receptors, and IL-4Rα–deficient mice are relatively resistant to the development of asthma. In addition, overexpression of IL-13 in the murine lung causes inflammation, mucus hypersecretion, subepithelial fibrosis, and production of chemokines for eosinophils. Administration of IL-13 directly into the airways of mice induces hyperreactivity, eosinophilia, and increased production of IgE. Finally, the administration of soluble IL-13 receptors leads to an antagonism of these responses, reducing bronchial hyperreactivity and mucus production in the mouse model of asthma.
Eicosanoids
In addition to cytokines and immune complexes, local inflammatory responses lead to the release of eicosanoids, which are lipid-derived molecules. Because lipids are present in the cell membrane, they are readily available substrates for the synthesis of mediators. These molecules are functional immediately adjacent to sites of synthesis, and their half-lives range from seconds to minutes. Eicosanoids are not stored but are produced de novo from membranes when cell activation by mechanical trauma, cytokines, growth factors, or other stimuli leads to release of arachidonic acid (AA). Cytosolic phospolipase A2 (cPLA2) is the key enzyme in eicosanoid production. Cell-specific and agonist-dependent events coordinate the translocation of cPLA2 to the nuclear envelope, endoplasmic reticulum, and Golgi apparatus, where interaction with cyclooxygenase (in the case of prostaglandin synthesis) or 5-lipoxygenase (in the case of leukotriene synthesis) can occur. The temporal sequence of events in acute inflammation may be governed by eicosanoid profile switching. This shift can be mediated, in part, by the induction of COX2 in macrophages and fibroblasts after exposure to IL-1 or TNF-α.
Prostaglandins
Prostanoids are produced when AA is released from the plasma membrane of injured cells by phospholipases and metabolized by cyclooxygenases and specific isomerases. These molecules act both at peripheral sensory neurons and at central sites within the spinal cord and brain to evoke pain and hyperalgesia. Their production is increased in most acute inflammatory conditions, including arthritis and inflammatory bowel disease. In response to exogenous and endogenous pyrogens, PGE2 derived from COX2 mediates a central febrile response. In addition, prostaglandins synergize with bradykinin (BK) and histamine to enhance vascular permeability and edema. The levels of prostaglandins are usually very low in normal tissues and increase rapidly with acute inflammation, well before leukocyte recruitment. Levels increase further with cellular infiltration and local cytokine production. COX2 induction with inflammatory stimuli most likely accounts for the high levels of prostanoids in chronic inflammation.
COX2 also plays a key role in platelet–endothelial cell interactions by increasing the production of prostacyclin (PGI2) in endothelial cells. Increased risk of myocardial infarction associated with the use of selective COX2 inhibitors might be related to unopposed production of thromboxane A2 by COX1 in platelets. Of interest, prostacyclin production also protects against atherosclerosis in female mice, and COX2 blockade abrogates this beneficial effect. Increased appreciation of the inflammatory nature of atherosclerosis and recent prospective studies evaluating cardiovascular events suggest that COX inhibitors might have a long-term stimulatory effect on thrombotic events and the progression of atherosclerosis.
Leukotrienes
In addition to prostaglandins, a distinct set of enzymes direct AA metabolites towards the synthesis of leukotrienes. Their relative importance depends on the specific target organ of an inflammatory response. For instance, leukotriene receptor antagonists have demonstrated efficacy in asthma, whereas similar approaches have been less impressive in rheumatoid arthritis. Unlike prostaglandins, leukotrienes are primarily produced by inflammatory cells such as neutrophils, macrophages, and mast cells. 5-Lipoxygenase (5-LO) is the key enzyme in this cascade, transforming released AA to the epoxide leukotriene A4 (LTA4) in concert with 5-lipoxygenase–activating protein (FLAP). LTA4 can be hydrolyzed by cytosolic LTA4 hydrolase to LTB4, a potent neutrophil chemoattractant and stimulator of leukocyte adhesion to endothelial cells. LTA4 can also conjugate with glutathione to form LTC4 by LTC4 synthase at the nuclear envelope. LTC4 migrates out of the cell, using transporters such as the multidrug resistance–associated protein, and can be metabolized extracellularly to LTD4 and LTE4. These three cysteinyl leukotrienes comprise the “slow-reacting substance of anaphylaxis” for their slow and sustained smooth muscle–contracting abilities. They promote plasma leakage from postcapillary venules, upregulation of expression of cell surface adhesion molecules, and bronchoconstriction.
Histamine
One of the hallmarks of allergic inflammation is the activation of mast cells with release of histamine. This mediator is a vasoactive amine produced by basophils and mast cells that markedly increases capillary leakage. In basophils, histamine is released in response to bacterial formylmethionyl-leucyl-phenylalanine (f-MLP) sequences, complement fragments C3a and C5a, and IgE. The resultant edema can be readily observed clinically in urticaria and allergic rhinitis. Despite the production of histamine in asthma and in acute synovitis, histamine blockers have minimal therapeutic effect in these conditions. The stimulus for release of histamine from mast cell granules is the same as in basophils, except for the absence of f-MLP receptors in this cell type. Histamine can also synergize with locally produced LTB4 and LTC4. In addition, histamine enhances leukocyte rolling and firm adhesion, and induces gaps in the endothelial cell lining, enhancing leukocyte extravasation.
Kinins
Pain plays a key role in host responses as a mechanism to protect damaged sites from subsequent trauma by modulating behavior. Although these pathways are quite complex, the kinins are known to participate in vasodilation, edema, and smooth muscle contraction, as well as in pain and hyperalgesia through stimulation of C fibers. They are formed from high- and low-molecular-weight kininogens by the action of serine protease kallikreins in plasma and peripheral tissues. The primary products of kininogen digestion are bradykinin and lysyl-bradykinin. These products have high affinity for the B2 receptor, which is widely expressed and is responsible for the most common effects of kinins. The peptides desArg-BK and Lys-desArg-BK are generated by carboxypeptidases and bind the kinin B1 receptor subtype, which is not expressed in normal tissues but is rapidly upregulated by LPS and cytokines. The kinin B2 receptor is internalized rapidly and desensitized, whereas the B1 receptor remains highly responsive. Both receptors belong to the G protein–coupled receptor superfamily; they signal through phospholipase C with activation of protein kinase C and subsequent flux of intracellular calcium. Kinin actions are associated with the secondary production of other mediators of inflammation, including nitric oxide, mast cell–derived products, and the pro-inflammatory cytokines IL-6 and IL-8. In addition, kinins can increase IL-1α production through initial stimulation of TNF-α, and can increase prostanoid production through activation of phospholipase A2 and release of AA.
Mechanisms of Tissue Damage in Inflammation
Reactive Oxygen and Nitrogen
Macrophages, neutrophils, and other phagocytic cells can generate large amounts of highly toxic reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs) that can directly kill pathogens. These molecules can damage DNA, oxidize membrane lipids, and nitrosylate proteins. Hence, their roles in inflammatory responses caused by infections are obvious. The ability of ROIs or RNIs to serve as critical signal transduction molecules that regulate expression of inflammatory genes is equally important.
Uncontrolled production of ROIs and RNIs can also lead to tissue damage. Release of reactive intermediates can be initiated by microbial products such as LPS and lipoproteins, by cytokines such as IFN-γ or IL-8, or by engagement of Fc receptors by IgG. These events cause translocation of several cytosolic proteins, including Rac2 and Rho-family guanosine triphosphatase (GTPase) to the membrane-bound complex carrying cytochrome c, with subsequent activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The reaction catalyzed by NADPH oxidase leads primarily to production of superoxide, which can be converted to hydrogen peroxide, hydroxyl radicals and anions, hypochlorous acid, and chloramines.
ROIs are critically important to the antimicrobial activity of neutrophils, but it is not clear to what extent other phagocytes utilize ROIs for control of intracellular bacteria in vivo. In some cases, ROIs can contribute directly to the initiation of chronic disease. Atherosclerosis is an especially important example. Lipid oxidation produces aldehydes that substitute lysine residues in apolipoprotein B-100. This altered moiety either binds to TLR2 to induce cytokine production or is internalized by macrophages, leading to the production of foam cells and fatty streaks, the primary lesions of atherosclerosis. Subsequently, altered epitopes in damaged host proteins can be presented to T cells to initiate an adaptive immune response that amplifies the inflammatory vascular lesion.
Nitric oxide synthases (NOS) convert l-arginine and molecular oxygen to l-citrulline and nitric oxide (NO). There are three known isoforms of NOS: neuronal NOS (ncNOS or NOS1) and endothelial cell NOS (ecNOS or NOS3) are both constitutively expressed, whereas macrophage NOS (macNOS, iNOS, or NOS2) is induced by inflammatory cytokines such as TNF-α and IFN-γ. The expression of NOS2 is suppressed by TGF-β. Products of viruses, bacteria, protozoa, and fungi, as well as low oxygen tension and low environmental pH, enhance NOS2 gene transcription.
Together with prostaglandins, the production of NO by NOS2 and ROIs by NADPH oxidase are key mechanisms by which macrophages paradoxically impair T-cell proliferation in response to mitogens or antigens. This may serve to control inflammatory processes or to delete autoreactive T cells, but it at least partially accounts for the immunosuppressed state seen in certain infections, malignancies, and graft-versus-host reactions. In addition, the intracellular balance of ROIs and RNIs may help to govern cell survival at a site of inflammation. For example, hydrogen peroxide promotes apoptosis in natural killer (NK) cells, but it contributes to macrophage resistance to NO-mediated apoptosis (presumably via scavenging of NO). Even within the lineage of a single cell, ROIs and RNIs may have both pro-apoptotic and anti-apoptotic effects. NO can prevent activity of caspases, a family of enzymes that can initiate apoptosis. Increased NADPH oxidase activity in neutrophils suppresses caspase activity but promotes externalization of phosphtidylserine, which signals the presence of an apoptotic cell to macrophages. Thus ROIs, like endogenous NO, may inhibit caspase function and simultaneously mediate neutrophil clearance through translocation of phosphotidlyserine and stimulation of cell death.
Proteases
Production of enzymes that degrade the ECM represents a key mechanism of tissue turnover in inflammation. This process is generally considered detrimental in diseases marked by overproduction of proteases (e.g., cartilage in osteoarthritis, synovium in rheumatoid arthritis, alveoli in chronic obstructive lung disease, and colonic epithelium in inflammatory bowel disease). However, reconfiguring of the matrix plays an important role in the host response by remodeling damaged tissue, releasing matrix-bound growth factors and cytokines, preparing the tissue for the ingrowth of new blood vessels, and altering the local milieu to permit adherence and retention of newly recruited cells.
The MMPs are a family of more than 20 extracellular endopeptidases that participate in degradation and remodeling of the ECM matrix (Table 45-4). They are produced as pro-enzymes and require limited proteolysis or partial denaturation to expose the catalytic site. Their name is derived from their dependence on metal ions (zinc/metzincin superfamily) for activity and from their potent ability to degrade structural ECM proteins. MMPs can also cleave cell surface molecules and other pericellular nonmatrix proteins, thereby regulating cell behavior. For instance, MMPs can alter cell growth by digesting matrix proteins associated with growth factors. FGF and TGF-β have high affinities for matrix molecules that serve as depots for storage of these cytokines in their activation state. Matrix proteolysis releases some growth factors and can make them available to cell surface receptors. In addition, MMPs can directly cleave and activate growth factors, as in the processing of latent TGF-β into its active form by MMP-2 and MMP-9. MMP-2, -3, and -9 also change IL-1β from its biologically inactive precursor into its active mature form. MMPs affect cell migration by altering cell–matrix or cell–cell receptor sites. For instance, the adhesion molecule β4 integrin is cleaved by MMP-7. MMP-3 and MMP-7 digest E-cadherin and not only disrupt endothelial cell junctions but also stimulate cell migration.
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Degradation of the ECM is usually initiated by collagenases, which cleave native collagen. Denatured collagen is then recognized and further degraded by gelatinases and stromelysins. Unlike the collagenases, stromelysins demonstrate broad substrate specificity and act on many ECM proteins, such as proteoglycan, fibronectin, laminin, and many cartilage proteins. Stromelysins can also amplify the remodeling process by activating collagenase through limited proteolysis. MMP gene expression can be induced by many pro-inflammatory cytokines, including TNF-α, IL-1, IL-17, and IL-18, through MAP kinase signal transduction pathways. The MAP kinase pathway involving JNK is particularly important; it phosphorylates c-Jun, a component of the activator protein 1 (AP-1) transcription factor complex. The other two MAP kinase pathways, involving ERK and p38, can also activate MMP gene expression, depending on the specific cell type. NF-KB translocation can also enhance MMP production.
Several other classes of proteases contribute to matrix remodeling, including serine proteases and cysteine proteases. High levels of active serine proteases, such as trypsin, chymotrypsin, and elastase, are released by infiltrating PMNs at sites of inflammation and can directly digest the ECM or activate the pro-enzyme forms of secreted MMPs. The ADAM (a disintegrin and metalloproteinase) family can cleave the extracellular domain of cytokine receptors (e.g., ADAM-17 and cleavage of TNF-α). ADAM-related proteins and the ADAMT family and differ by the presence of multiple copies of thrombospondin 1–like repeats. These ECM proteases include two members of the aggrecanase family that are implicated in the degradation of cartilage in arthritis.
Tissue Repair and Resolution of Inflammation
Inflammation is a normal physiologic response, but it can cause serious host injury if it is allowed to persist. Hence, additional mechanisms are required to reestablish homeostasis once this response is initiated (Fig. 45-1). Suppression of acute inflammation by removal or deactivation of mediators and effector cells permits the host to repair damaged tissues through elaboration of appropriate growth factors and cytokines. The precise mechanisms controlling the switch from predominantly pro-inflammatory to anti-inflammatory pathways are not fully understood. However, as in the initial generation of an inflammatory response, components of resolution include a cellular response (apoptosis), formation of soluble mediators (such as anti-inflammatory cytokines and antioxidants), and production of direct effectors (such as protease inhibitors).
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| FIGURE 45-1 Anti-inflammatory me-chanisms that resolve inflammation and lead to repair of the extracellular matrix. IL = interleukin; SERPINs = serine protease inhibitors; TGF = transforming growth factor; TIMPs = tissue inhibitor of metalloproteinases; TNF = tumor necrosis factor. |
Deletion of Inflammatory Cells by Apoptosis
Apoptosis is a highly regulated process in eukaryotic cells that leads to cell death and marks the surface membrane for rapid removal by phagocytes. This clearance process does not elicit an inflammatory response, in contrast to cell death by necrosis, in which the release of intracellular contents into the microenvironment surrounding the dying cells promotes inflammation. Apoptosis is a normal process by which inflammatory cells are removed from healing sites, with the clearance of neutrophils representing a prominent example. PMN phagocytes have a very short half-life in the tissue, and the persistence or release of their contents into the microenvironment after death can be deleterious. In some pathologic conditions, such as leukocytoclastic vasculitis, abundant neutrophil apoptosis is readily apparent on histopathologic examination; in fact, it is one of the pathologic criteria for this diagnosis. Other cells, including T lymphocytes, undergo postactivation apoptosis to prevent their overwhelming of host responses. Defective apoptosis or even persistence of apoptotic cells that escape clearance may contribute to chronic inflammatory and autoimmune diseases. For instance, loss of tolerance to self-antigens might participate in autoimmune responses in SLE. Specific molecular defects in apoptosis pathways, such as the Fas–Fas ligand (FasL) system, induce lupus-like conditions in mice.
Commitment of a cell to apoptosis can be initiated by a number of factors, including the ROIs in the cellular microenvironment, as well as signaling through several death receptor pathways (e.g., FasL/Fas and TNF-related apoptosis-inducing ligand [TRAIL]). The former can damage DNA, which is a common sequela of the genotoxic environment created by inflammation. In this context, the p53 tumor suppressor protein is induced, leading to p21-dependent cell cycle arrest. If DNA damage is excessive, repair by tightly regulated mismatch repair mechanisms is terminated, and programmed cell death begins. The burden of mutations induced by ROIs or RNIs in chronic inflammation can potentially accumulate over time and eventually lead to amino acid substitutions in key regulatory proteins. Ultimately, as has been observed in ulcerative colitis, neoplastic disease can ensue.
Removal of apoptotic bodies, or the remnants of packaged apoptotic cells, is rapid and can be accomplished by macrophages, fibroblasts, epithelial and endothelial cells, muscle cells, and dendritic cells. The surface receptors used in recognition and engulfment of apoptotic cells include integrins (e.g., αvβ3), lectins, scavenger receptors, adenosine triphosphate (ATP)-binding cassette transporter 1, LPS receptor, CD14, and complement receptors CR3 and CR4. However, some of these membrane molecules can be utilized in both pro-inflammatory and apoptotic pathways, the divergence of which may be based on differing ligands and accessory molecules. For example, in macrophages, the CD14-dependent pro-inflammatory process in clearance of LPS depends on LPS-binding protein, whereas CD14-dependent apoptotic cell clearance does not.
Apoptotic cells display a series of membrane-associated molecular patterns that interact with receptors on phagocytes. The details of interactions between apoptotic cells and phagocytes are only partially understood. A general feature of apoptotic cells is loss of phospholipid asymmetry, with external presentation of phosphotidylserine. Externalized phosphatidylserine may be sufficient to trigger phagocytosis, but other apoptotic cell surface structures exist, including sugars (e.g., mannose), ICAM-3 (on leukocytes), oxidized surface structures (including phospholipids), thrombospondin, complement components, and β2-glycoprotein I.
While some inflammatory and immune cells are being deleted, other cell lineages expand during the resolution phase. Mesenchymal cells, especially fibroblasts, proliferate and produce new matrix that can contract to form a fibrotic scar. Locally produced growth factors such as PDGF and TGF-β induce DNA synthesis of these stromal cells. In addition, mesenchymal stem cells that either reside in the tissue or migrate from the peripheral blood can differentiate into the appropriate organ-specific lineage. The pluripotential cells can, in the presence of the appropriate milieu, can become adipocytes, chondrocytes, bone cells, or other terminally differentiated stromal cells.
Soluble Mediators
Anti-Inflammatory Cytokines
Just as there are cytokines that initiate and induce the inflammatory response, an additional array of cytokines displays primarily anti-inflammatory activities. TGF-β and IL-10 are examples that are produced by macrophages, interstitial fibroblasts, or T cells. Their anti-inflammatory effects diminish the likelihood of an acquired immune response against apoptotic cell–derived antigens and deactivate other cells in the tissue. Some T-cell cytokines, including IL-4, IL-10, and IL-13, suppress the expression of MMP by cells stimulated by IL-1 or TNF-α. In addition increasing fibroblast proliferation, TGF-β suppresses collagenase production, increases collagen deposition, and decreases MMP activity by inducing production of the tissue inhibitors of metalloproteinases (TIMPs). Although resolution of inflammation clearly is necessary for normal wound healing, it is abnormal in diseases in which tissue fibrosis represents a major pathologic manifestation. For example, scleroderma is marked by diffuse fibrosis and is accompanied by high levels of TGF-β and increased production of ECM.
Cytokine decoy receptors can also downregulate the inflammatory response. These surface receptors recognize certain cytokines with high affinity and specificity but do not transduce intracellular signals. The receptors can also be shed from the cell surface after proteolytic cleavage and can absorb cytokines, thereby preventing them from ligating functional receptors on cell membranes. These cytokine inhibitors can be released as a coordinated attempt to prevent unregulated inflammation, as in septic shock, in which endotoxin induces production of soluble receptors after initial massive production of TNF-α and IL-1. Other types of cytokine-binding proteins are also produced as counter-regulatory mechanisms, including IL-18–binding protein (IL-18BP), which is an Ig superfamily-related receptor that captures IL-18. In bone remodeling, interactions of receptor activator of NF-KB (RANK) with RANK ligand are required for osteoclast-mediated resorption. The competitive antagonist osteoprotegerin (OPG) is a member of the TNF receptor family that binds to RANK ligand and inhibits osteoclast activation.
The need for tight control of the pro-inflammatory cytokine IL-1 is demonstrated by the existence of two separate mechanisms. An IL-1 decoy receptor, known as the type II IL-1R, has both cell membrane and soluble forms that neutralize IL-1 activity. In addition, a natural IL-1 antagonist, IL-1Ra, can bind to functional IL-1 receptors and compete with IL-1α or IL-1β. However, IL-1Ra does not transduce a signal to the cell and blocks the biologic functions of ambient IL-1. The balance of IL-1 and IL-1Ra production depends on many influences. For instance, monocytes produce more IL-1, whereas mature macrophages produce IL-1Ra, especially after engagement of Fc receptors by IgG.
Prostanoids/Cyclooxygenase
COX2 induced by pro-inflammatory mediators appears early and can contribute to inflammatory responses. However, COX2 expression late in the process has led to speculation that it also functions in the resolution of inflammation. This regulation might occur through formation of the cyclopentenone prostaglandins (CyPG). CyPG production is suppressed by COX inhibition and inhibits pro-inflammatory gene transcription. The prostanoids can serve as ligands for peroxisome proliferator-activated receptors (PPARs). There are three main classes PPAR receptors, PPARα, PPARβ/δ, and PPARγ, all of which bind to DNA as heterodimers in association with the retinoid X receptor. Activation of PPARγ by CyPG is associated with the suppression of AP-1 and signal transducer and activator of transcription (STAT) transcriptional pathways in macrophages. In addition, CyPG can directly inhibit IKKβ, thus preventing NF-KB activation. A variety of natural and synthetic PPAR agonists have demonstrated efficacy in models of ischemia-reperfusion injury, arthritis, and inflammatory airway disease.
Inhibitors of Direct Effectors
Antioxidants
An extensive array of antioxidant defenses exists to protect cells from the effects of ROIs and RNIs. In some cases, the damage induced by these reactive molecules can contribute to disease pathology. For instance, treatment of adjuvant arthritis in rats with antioxidants helps suppress joint swelling and destruction. Human diseases, such as atherosclerosis caused by oxLDL, appear to be more complex, and use of these agents has not met with universal success. Antioxidants can be divided into the antioxidant enzymes, chain-breaking antioxidants, and transition metal–binding proteins.
Antioxidant enzymes that can inactivate the toxic intermediates include catalase and superoxide dismutase. Catalase is a peroxisomal enzyme that catalyzes the conversion of hydrogen peroxide to water and oxygen. Most catalase activity is found in the liver and in erythrocytes. Superoxide dismutases (SOD) catalyze the dismutation of superoxide to hydrogen peroxide, which is then removed by catalase or glutathione peroxidase. Glutathione peroxidases and glutathione reductase are additional mechanisms for maintaining redox balance and removal of toxic metabolites. Insufficient production of intracellular antioxidants such as glutathione can suppress lymphocyte responses and could account for defective T-cell receptor signaling and blunted immunity in T cells derived from rheumatoid arthritis synovium.
Interactions of free radicals with surrounding molecules can generate secondary radical species in a self-propagating chain reaction. Chain-breaking antioxidants are small molecules that can receive or donate an electron and thereby form a stable byproduct with a radical. These antioxidant molecules are categorized as either aqueous phase (vitamin C, albumin, reduced glutathione) and lipid phase (vitamin E, ubiquinol-10, carotenoids, and flavonoids). In addition, transition metal–binding proteins (ceruloplasmin, ferritin, transferrin, and lactoferrin) can serve as antioxidants by sequestering cationic iron and copper and thereby inhibiting the propagation of hydroxyl radicals.
Protease Inhibitors
Mechanisms to protect the host and prevent tissue destruction using a complex system of protease inhibitors have evolved as part of the repair process. Protease inhibitors regulate the function of endogenous proteases and reduce the likelihood of collateral damage to tissues. These proteins form two functional classes, active site inhibitors and α2-macroglobulin (α2M). The latter class of protease inhibitors acts by covalently linking the protease to the α2M chain and thereby blocking access to substrates. α2M binds to all classes of proteases and, after forming a covalent bond, conveys them to cells through receptor-mediated endocytosis with subsequent enzymatic inactivation. The family of inhibitors of serine proteases (SERPINs) are the most abundant members of the former class of protease inhibitors and play a major role in regulation of blood clot resolution and inflammation, as indicated by many of their names: anti-thrombin III, plasminogen activator inhibitors 1 and 2, α2-antiplamsin, α1-antitrypsin, and kallistatin. In addition to direct inactivation via protease inhibitors, serine proteases can be inactivated by oxidation. In contrast, MMPs are activated by partial denaturation in the toxic environment.
A specialized mechanism for inhibiting MMP function has also evolved and can be induced during the reparative phase of inflammation. A family of TIMPs inhibits most members of the MMP family. The TIMPs bind to activated MMPs and irreversibly block their catalytic sites. Examples of disease states with an unfavorable balance between TIMPs and MMPs include loss of cartilage in arthritis and regulation of tumor metastasis. TIMP-MMP imbalance in destructive forms of arthritis appears be caused by the limited production capacity for protease inhibitors, which is overwhelmed by the prodigious expression of MMPs. Whereas IL-1 and TNF-α induce MMPs, IL-6, TGF-β, and several other growth factors suppress production of MMPs and increase levels TIMPs. TGF-β also increases the production of matrix proteins such as collagen. Therefore, the cytokine profile has a pivotal influence on the status of remodeling. When pro-inflammatory cytokines predominate, the balance favors matrix destruction; in the presence of pro-inflammatory cytokine inhibitors and growth factors, matrix protein production increases and MMPs are inhibited by TIMPs.
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