5a. Inflammation II_Hazen-Martin_NOTES.pdf

Inflammation 2 Debra Hazen-Martin, PhD Office: 792-2906 Email: [email protected] Inflammation 2: Chemical Mediators of Inflammation Outline: I. Chemical Mediators of Acute Inflammation A. Definition and Classification II. Systemic Mediators and Associated Activities A. Hepatic Plasma Proteases 1....

Inflammation 2 Debra Hazen-Martin, PhD Office: 792-2906 Email: [email protected] Inflammation 2: Chemical Mediators of Inflammation Outline: I. Chemical Mediators of Acute Inflammation A. Definition and Classification II. Systemic Mediators and Associated Activities A. Hepatic Plasma Proteases 1. Kinin Cascade 2. Coagulation Cascade 3. Fibrinolytic Cascade 4. Complement Cascade III. Local Mediators A. Cellular Origins B. Preformed Mediators 1. Vasoactive Amines 2. Lysosomal Enzymes C. Newly Synthesized Mediators 1. Products of Membrane Phospholipids a) Arachidonic Acid Metabolites b) Platelet-activating Factor 2. Cytokines a) Inflammatory TNF and IL1 b) Chemokines 3. Free Radicals a) Nitric Oxide 4. Others a) Neuropeptides IV. Termination of Acute Inflammation Suggested Reading: Robbins Basic Pathology by Kumar, Abbas and Aster, Chapter 3 (70-77) Objectives: 1) Define the term “chemical mediator.” 2) Explain the origin or precursors of local and systemic chemical mediators. 3) Identify the cascades activated by Factor XII. 1 Inflammation 2 4) Describe the product(s) of each of the cascades and the mechanism by which each acts to mediate inflammation. 5) Identify the target(s) for the main chemical mediators of inflammation? 6) Describe and identify the two pathways of the arachidonic acid cascade. 7) Identify the important chemical mediators derived from each pathway of arachidonic acid metabolism and how the specific metabolites function in an inflammatory response. 8) Describe the origin of arachidonic acid. 9) Describe the opposing functions of eicosanoids and the significance of this when choosing drugs that inhibit production of various enzymes that metabolize arachidonic acid. 10) Describe the role of NO in inflammatory events in various tissues. 11) Describe the role of alpha-1 antitrypsin in the event of acute inflammation. I. Chemical Mediators of Acute Inflammation Def A. Types(2) Definition and Classification - Chemical mediators are substances from many different sources that direct the vascular and cellular events in acute inflammation. Chemical mediators may be systemic meaning that they are circulating in the plasma after synthesis and secretion by the liver. Others are produced locally by many cells found at the site of acute inflammation and/or injury. 2 Inflammation 2 II. Systemic Mediators and Associated Activities A. Hepatic Plasma Proteases: Ex(3) Systemic mediators include proteins in the complement, kinin, and coagulation cascades. The proteins are produced in the liver and normally circulate as inactive precursors that require proteolytic cleavage for activation. Xter(2) All 3 cascades produce proteins that mediate many of the events occurring during acute inflammation. The cascades have a common activator, the Hageman factor (Factor XII) which is part of the coagulation cascade. Table Factor XII activation factors(3) Fig In this overview slide you can see that Factor XII is inactive until it encounters collagen, basement membrane, or activated platelets in the presence of a cofactor (high molecular weight kininogen). It then undergoes conformational change (exposing serine) to become active form (Factor XIIa). Active Factor XII cleaves a number of substrates. Products of the three cascades are inter-regulated and form substances that either directly or indirectly influence inflammation. 3 Inflammation 2 Flow diagram This diagram illustrates the components of the cascades and how they interact. We will start with the Kinin Cascade and proceed with the others. Bradykinin fxn(3) 1. Kinin Cascade -This cascade produces bradykinin, a potent vasoactive mediator that increases vascular permeability, arteriole dilation, and bronchial smooth muscle contraction. The first step in the cascade is the conversion of prekallikrein to the active kallikrein by active Factor XII. Kallikrein then converts an inactive high molecular weight kininogen to active bradykinin. Note: Active bradykinin is quickly degraded by kinases in tissues and plasma. The intermediate kallikrein acts to amplify this cascade and others by activating more Factor XII. 4 Inflammation 2 The Coagulation Cascade is highlighted on the right. You will study this cascade in much more detail in upcoming lectures. The overall purpose of the cascade is the production of fibrin Purpose(1) to help form a permanent clot in hemostasis. /def 2. Coagulation Cascade: Active Factor XII initiates a series of inactive-to-active conversions of clotting proteins finally resulting in the conversion of thrombin from its inactive precursor prothrombin. In turn, thrombin converts a soluble fibrinogen into an insoluble fibrin clot. Note: The important point in today’s lecture is that intermediates of this cascade are chemical mediators of inflammation. Factor Xa increases vascular permeability and leukocyte emigration. Thrombin enhances leukocyte adhesion to endothelial cells and cleaves C5. The by-products of fibrinogen cleavage, fibrinopeptides, increase vascular permeability and are chemotactic for leukocytes. 5 Inflammation 2 Def The fibrinolytic cascade occurs when products of the kinin cascade participate in plasmin activation. Plasmin degrades fibrin produced in the coagulation cascade to limit the size of the fibrin clot. 3. The Fibrinolytic Cascade: This cascade counter-regulates the clotting process with the formation of plasmin, an active protease that cleaves the fibrin clot. Plasmin is produced by cleavage of an inactive precursor plasminogen. This cleavage is performed by plasminogen activator (produced by endothelial cells and leukocytes) and kallikrein from the kinin cascade. Cleavage of fibrin produces fibrin degradation products. Note: Fibrin degradation products increase vascular permeability. Plasmin participates in cleavage of C3 in the complement cascade (this will lead to increased vasodilation and vascular permeability) and also activates Fig additional Factor XII to amplify the cascades. 6 Inflammation 2 4. Complement Cascade: (The complement cascade will be well described in HRR lectures.) This cascade involves a group of plasma proteins that function to form holes on microbe membranes (MAC, membrane attack complex). Along the pathway, complement components are formed that participate in inflammatory activities we have discussed and we will concentrate on these activities in today’s lecture. Cleavage products of the complement cascade 1) enable phagocytosis of pathogens by opsonization, 2) increase vascular permeability, or 3) activate leukocytes and increase their adherence and chemotaxis. cascade components fxn(3) Briefly, the nomenclature of this pathway names 9 inactive proteins - C1 through C9. When these proteins are cleaved they are activated and the products of cleavage are designated as a (small fragment, ex: C3a) or b (the larger fragment, ex: C3b). Cleavage of C3 is the most important step in activating the biological function of complement. C3 is cleaved into C3a and C3b by C3 convertases formed by complement components in the classic pathway or alternate pathways. What other roles do specific complement components have in inflammation? Vascular effects: C3a and C5a (anaphylatoxins) induce mast cell release of histamine that contributes to vasodilation and increased vascular permeability. Fxns C5a participates in arachidonic acid metabolism which will produce mediators, increases the leukocyte integrin affinity for endothelial adhesion molecules, and is chemotactic for leukocytes. C3b acts as an opsonin on microbial surfaces with specific binding to C3b receptors on macrophages and neutrophils. 7 Inflammation 2 III. Local Mediators A. Cellular Origins: Cells at the site of inflammation produce local chemical mediators. The cells include macrophages, mast cells, endothelial cells, and leukocytes. Specific types of chemical mediators may be in preformed granules (vasoactive amines and lysosomal enzymes) and are therefore ready for quick release. Others require de novo synthesis (arachidonic acid metabolites, cytokines and free radicals). Whether preformed or requiring synthesis, a stimulus is required for the production and/or release. B. Preformed Mediators 1. Vasoactive amines Histamine - This mediator is widely distributed and is present in the preformed granules of mast cells near vessels, basophils and platelets. Release of histamine-containing granules results in arteriolar dilation and endothelial contraction to form intercellular gaps in venules. These actions explain its role as the principal mediator in immediate increased vascular permeability. The effect of histamine is short-lived due to the rapid degradation of histamine by histaminase. The stimuli for release of histamine are many and include: physical injury or trauma (remember the triple response?), immune reactions involving IgE, complement C3a and C5a (anaphylactic responses), leukocyte derived histamine releasing proteins, neuropeptides, and cytokines (IL-1, IL8). 8 Inflammation 2 Serotonin - This mediator is found in the dense core granules of platelets which also contain histamine, ADP, and calcium. Stimulation for release is by platelet aggregation. Serotonin also modulates vasoconstriction at high concentrations. 2. Lysosome enzymes: Lysosomes are preformed within leukocytes and monocytes which contain both neutral and acid proteases. During the process of phagocytosis, lysosomal enzymes are leaked into tissues either during cell death and necrosis or just by accident when the cells engulf the foreign material and the phagolysosome body is formed (Think of this as the way the phagocytic cell “burps”.) Fig diag Acid proteases require a pH optimum that is not present outside of the phagosome so they are not active in the extracellular tissue. Neutral proteases which include enzymes that degrade elastin, collagen, basement membrane, and other matrix proteins are fully active in the extracellular space and can cause a good bit of destruction. Deficiency in anti-proteases In addition, released neutral proteases may cleave complement C3 and C5 to generate anaphylatoxins outside of the normal complement cascade. They may also generate bradykinin peptides from kininogen. Each of these things can amplify the inflammatory process. Normally anti-proteases are present in tissue and serum to protect tissues from this nonspecific digestion (ex: alpha1 - antitrypsin, alpha2-macroglobulin). Individuals who are deficient in these protective enzymes are very susceptible to tissue damage during inflammation. 9 Inflammation 2 C. Newly Synthesized Mediators 1. Products of Membrane Phospholipids: Phospholipase is activated during inflammation and releases arachidonic acid (a precursor to the eicosanoids) and another potent phospholipid derived mediator, platelet activating factor (PAF) from membrane phospholipids. a) Arachidonic Acid (AA) Metabolites: Cellular phospholipases are released or activated in response to physical or chemical trauma, and/or release of C5a (complement mediator). These enzymes degrade cell membrane phospholipids releasing arachidonic acid (a 20Mech(4) carbon polyunsaturated fatty acid). Arachidonic acid is further metabolized by 1) cyclooxygenase, resulting in synthesis of prostaglandins and thromboxane or 2) lipoxygenase, resulting in leukotrienes and lipoxins. All of the metabolites of AA are called eicosanoids and they mediate inflammation. Cells that produce eicosanoids include leukocytes, mast cells, endothelial cells, and platelets. Cyclooxygenase pathway: This is the pathway that may be inhibited by common antiinflammatory drugs (aspirin, Cox-1 and Cox-2 inhibitors). Fig diag Different cells have specific enzymes in this pathway, leading to production of specific eicosanoids. Platelets produce thromboxane (TXA2) which promotes platelet aggregation and vasoconstriction while endothelial cells produce prostacyclin (PGI2) that opposes this action by vasodilating and inhibiting platelet aggregation. Mast cells produce PGD2 while the other prostaglandins PGE2 and PGF2 are widely distributed. All 3 cause vasodilation. PGE2 causes pain and, with the influence of additional cytokines, fever. 10 Inflammation 2 Lipoxygenase pathway: This pathway produces leukotrienes and lipoxin. The pathway is most active in leukocytes and influences leukocytes. LTB4 is the most potent chemotactic agent for neutrophils while the other downstream metabolites (also leukotrienes) serve as vasoconstrictors and increase vascular permeability. Lipoxins are also products of this pathway but their synthesis is more complex. LTA4 is a precursor for lipoxin, but the enzymes necessary for synthesis are not in platelets. LTA4 is transferred from neutrophils to platelets and then is processed by enzymes only in platelets to produce lipoxins. Lipoxins have opposing effects, promoting vasodilation and inhibiting neutrophil aggregation. So this represents another check and balance type of regulation. Lipoxins are extremely important in resolution of inflammation. Inhibiting the formation of Eicosanoids: Fig Steroids inhibit phospholipase and therefore the formation of AA removing the substrate for both of the downstream metabolic pathways. Long term administration of steroid has adverse outcomes as you will learn. 11 Inflammation 2 Aspirin and NSAID (nonsteroidal antiinflammatory drugs) act to more selective inhibit the cyclooxygenase pathway. However, it is important to remember that there are two isoforms of Cyclooxygenase , Cox 1 and Cox 2. Cox 1 in gastric mucosa has a beneficial role of protecting the mucosa from acid damage. Therefore nonselective inhibition of both isoforms may result in loss of Cox1 activity and development of stomach ulceration. It would seem that the logical next approach is to selectively block Cox 2. Cox 2 inhibitors were developed and marketed. The cyclooxygenase pathway leads to production of both prostacyclin (inhibits platelet aggregation) and prostaglandins (TXA2, promote platelet aggregation). Unfortunately the inhibition of prostacyclin generation was greater than the inhibition of prostaglandins. This leads to generation of a prothrombotic state and patients may experience increased acute coronary events. The message is: Drug development requires stringent testing and controls! 12 Inflammation 2 b) Platelet-activating Factor: This factor is also a by-product of membrane phospholipid metabolism by phospholipase A2 . It is produced by a large number of cells including leukocytes, mast cells, endothelial cells, platelets and others. Although first thought to act only on platelets, it is now known to have broad inflammatory effects via G protein coupled receptors to increase vascular permeability and vasodilation up to 10,000X greater than histamine. PAF also aggregates leukocytes, is chemotactic, and stimulates other mediators. 2. Cytokines: Cytokines are polypeptides produced by many cell types (but primarily lymphocytes and macrophages). They modulate the activity of other cell types. Their secretion is targeted, wellregulated, and very transient. They are produced in immune and inflammatory events. They may be organized into 5 functional groups. Many have an IL label to indicate the function in interactions between leukocytes. Those most important to acute inflammatory events are TNF and IL-1 and chemokines. Interferon and IL-12 play Xter(2) Classes(5) a role in chronic inflammation. a) Tumor Necrosis Factor (TNF) and IL1: Both TNF and IL-1 are produced by macrophages, endothelial cells, and mast cells after stimulation by bacterial endotoxins, other toxins, injury, or other inflammatory mediators. The effects are local and systemic. In endothelial cells they induce increased expression of adhesion molecules, secretion of other chemical mediators including cytokines, growth factors, and promote production of eicosanoids and NO. TNF causes aggregation and activation of neutrophils. IL-1 causes proliferation of 13 Inflammation 2 fibroblasts contributing to matrix production. So they may help processes that remodel the site of inflammation. Acute Phase Reaction – The diagram above illustrates the contributions of IL 1 and TNF in local inflammation. In addition, these are the agents that initiate systemic changes that are typical of the acute-phase reactions. They along with IL-6 produce the symptoms of fever (by induced local PGE synthesis), lethargy, metabolic wasting (cachexia), and neutrophil release into the circulation. In addition, IL-6 will stimulate hepatic synthesis of coagulation proteins, complement, and fibrinogen. The increase fibrinogen will cause an elevation in the sedimentation rate of erythrocytes (SED) commonly seen after an inflammatory response. These inflammatory cytokines mediate the hypotension of shock and contribute to disseminated intravascular coagulation (DIC). b) Chemokines - The chemokines are very small, structurally related proteins that mediate chemotaxis of subsets of leukocytes. They are secreted by a variety of inflammatory cells and endothelium. Collections of chemokines attract specific types of white blood cells. Chemokines 14 Inflammation 2 bind G-protein coupled receptors to exert their effect on target cell populations. The take-home message is that chemotactic recruitment of each type of leukocyte population requires a specific grouping of chemokine proteins. 3. Free Radicals: a) Nitric Oxide (NO) is a free radical gas produced by macrophages to kill microbes and tumor cells, by endothelial cells to relax smooth muscle (vasodilation) and in the CNS where it regulates neurotransmitter release. Its half-life is so short, that the rate of synthesis by the enzyme nitric oxide synthase (NOS) efficiently regulates the impact of its presence. There are 3 isoforms of NOS. nNOS is found in neuronal tissues, iNOS has a broader distribution in macrophages, endothelial cells and smooth muscle, and many other cell types. eNOS is limited to endothelial cells. Each isoform responds to specific stimuli including calcium levels, chemical mediators, and stress. The production of NO in the macrophage can kill microbes. NO produced by endothelial cells results in vasodilation, reduction in platelet aggregation, and decreased leukocyte recruitment and adhesion. In the CNS, NO regulates neurotransmitter release and blood flow. 4. Others a) Neuropeptides: Substance P - This small peptide is localized in neurons particularly those of the lung and gut. Its effects are to initiate inflammation at these sites, transmit pain signals, regulate vessel tone, and modulate vascular permeability. It is suspected that they work through activation of NO synthesis. 15 Inflammation 2 IV. Termination of Acute Inflammation Ideally, acute inflammation ends as macrophages enter to begin the process of complete resolution of necrotic debris and prepare for healing and repair. 16