Inflammatory Response

1. Inflammatory Response

2. Cytokines • Cytokines are small proteins or ____________ secreted for the purpose of altering the function of target cells in an _____________, ____________, or ________ fashion. • Cytokines are produced by cells individually (e.g., lymphocytes or macrophages) or as components of a tissue (e.g., the intestinal epithelium). • Many cytokines are_____________; these cytokines are capable of inducing many different biologic effects, depending on the target cell types involved and the presence or absence of other modulating factors. • Redundancy is another characteristic feature of cytokines; that is, several different cytokines can exert very similar biologic effects. glycoproteins paracrine autocrine endocrine pleiotropic

3. Cytokines • Another way that cytokines can be categorized is on the basis of structure. • Type I cytokines • are a large group of proteins that share a characteristic tertiary structure consisting of a bundle of four a helices. • The receptors for type I cytokines also share structural similarities and are referred to as type I cytokine receptors. • Type I cytokines include the following proteins: interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-13, IL-15, and granulocyte colony-stimulating factor (G-CSF). • Type II cytokines • including interferon-α (IFN-α), IFN-β, IFN-γ, and IL-10, are a second structurally related group of proteins.

4. Cytokines • Yet another way of grouping cytokines is based on the recognition that naive CD4+ T cells (TH0 cells) can differentiate into either of two T helper (TH) subsets called TH1 and TH2. • TH1 cells, responsible for directing the ___________ immune responses necessary for the eradication of intracellular pathogens, favor macrophage activation. • TH2 cells have been implicated in the pathogenesis of atopy and allergic inflammation and favor B-cell growth and differentiation. cell-mediated

5. Cytokines • TH1 cells produce IL-2, as well as the potent proinflammatory cytokines IFN-γ and lymphotoxin-α (LT-α). • TH2 cells produce IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13. • TH1 cytokines are often viewed as being proinflammatory, whereas TH2 cytokines are thought of as being anti-inflammatory. What cytokine predominantly drives TH1 differentiation? IL-12 What cytokine predominantly induces TH2 differentiation? IL-4

6. Cytokines Chemokines • _____________ are a special family of cytokines that consist of small proteins with molecular weights in the range of 8 to 11 kd and have as their primary biologic activity the ability to act as chemoattractants for leukocytes or fibroblasts. • Another cytokine subclass is a group of proteins that act primarily to stimulate the growth or differentiation (or both) of hematopoietic progenitor cells; these mediators are collectively referred to as ________________________. • Other growth and differentiation factors, including the various platelet-derived growth factors, epidermal growth factor, and keratinocyte growth factor, also fit into the broad category of cytokines. colony-stimulating factors.

7. Moving along.. • The immune response to infection has two broad components. • Innate responses. • ????? responses which occur early and are not antigen specific, depend largely on proper functioning of natural killer (NK) cells and phagocytic cells, such as monocytes, macrophages, and neutrophils. • ??????? responses • Acquired responses which develop later after antigen processing and the clonal expansion of T- and B-cell subsets, are antigen specific. A number of cytokines, including transforming growth factor-β (TGF-β), tumor necrosis factor (TNF), IL-1, IL-6, IL-10, IL-12, and IL-18, are synthesized by cells of the innate immune system and contribute to the ability of the host to mount an early, innate immune response to an infectious challenge. Another group of cytokines, the interferons, are also key components of the innate immune system

8. Interferon • The interferons, named for their ability to interfere with viral infection, were initially discovered in the 1950s as soluble factors secreted by leukocytes. • The type 1 interferons, IFN-α and IFN-β, are primarily involved as mediators of innate immune responses to viral infection. • IFN-γ, though also important in the immune response to viral infection, has much broader activity as a proinflammatory mediator.

9. IFN-γ • IFN-γ is produced by three types of cells: • CD4+ TH1 cells, • CD8+ TH1 cells • and NK cells. • IFN-γ, along with two other cytokines, IL-12 and IL-18, plays a critical role in promoting the differentiation of CD4+ T cells to the TH1 phenotype. • Because TH1 cells also produce IFN-γ, the potential exists for a positive feedback loop.

10. IFN-γ • IFN-γ inhibits the differentiation of lymphocytes into cells with the TH2 phenotype. • TH2 cells secrete the counter-regulatory cytokines: • IL-4 and IL-10 • Effect of IFN-γ to down-regulate the production of these cytokines by TH2 cells promotes the development of an inflammatory response to an invading pathogen.

11. IFN-γ • When responsive target cells are exposed to IFN-γ, a number of genes are activated within minutes • IFN-γ–induced signal transduction occurs through the activation of a protein tyrosine phosphorylation cascade known as the JAK-STAT pathway • JAK initially stood for _________________ because the biologic role of these proteins was not established when they were initially discovered. • Because these receptor-associated kinases look both outside and inside the cell, JAK has now come to stand for Janus kinases, after _______________________ just another kinase the two-faced Roman god.

12. IFN-γ • Prophylactic treatment with recombinant IFN-γ has been shown to markedly reduce the frequency of infections in patients with chronic granulomatous disease,a life-threatening condition caused by an inherited defect in _______________, the enzyme complex responsible for generating reactive ________ species in phagocytes. • Three major clinical trials of prophylactic IFN-γ treatment were conducted in patients with multiple trauma or major thermal injury. Unfortunately, in all three studies, the incidence of infection and mortality was similar in cytokine- and placebo-treated patients. NADPH oxidase oxygen

13. INTERLEUKIN-1 AND TUMOR NECROSIS FACTOR • IL-1 and TNF are structurally dissimilar pluripotent cytokines. • Although these compounds bind to different cellular receptors, their multiple biologic activities overlap considerably • Through their ability to potentiate the activation of helper T cells, IL-1 and TNF can promote nearly all types of humoral and cellular immune responses • Both these cytokines are capable of activating neutrophils and macrophages and inducing the expression of many other cytokines and inflammatory mediators. + + + + + + + + + - - + - + + + + +

14. Interleukin-1 IL-1 was first described as a lymphocyte-activating factor produced by stimulated macrophages. IL-1 is not a single compound, but rather a family of three distinct proteins: Name Them!!!!! IL-1α, IL-1β, and IL-1 receptor antagonist (IL-RA) , which are products of different genes located close to one another on the long arm of human chromosome 2. IL-1RA, the third member of the IL-1 family of proteins, is biologically inactive but competes with IL-1α and IL-1β for binding to IL-1 receptors on cells and thereby functions as a competitive inhibitor to limit IL-1–mediated effects.

15. IL-1 • IL-1 is synthesized by a wide variety of cell types, including : • Monocytes • Macrophages • B lymphocytes • T lymphocytes • NK cells, • Keratinocytes • dendritic cells • Fibroblasts • Neutrophil • endothelial cells • enterocytes. Name One !!!!!

16. IL-1 • Compounds that can trigger the production of by monocytes, macrophages, or other cell types include microbial cell wall products, such as : • lipopolysaccharide (LPS) • lipoteichoic acid • zymosan gram-negative bacteria gram-positive bacteria yeast

17. IL-1RA • In some tissues, such as intestinal epithelium, the formation of intracellular IL-1RA may serve a counter-regulatory function to limit inflammation and thereby confer mucosal protection. • Moreover, an imbalance between the production of IL-1 and IL-1RA may promote the development of chronic inflammation in certain pathologic conditions such as Crohn's disease

18. IL-1 receptors There are two distinct IL-1 receptors, IL-1RI and IL-1RII • IL-1RI is an 80-kd transmembrane protein with a long cytoplasmic tail. • IL-1RI is present on a wide variety of cell types, including T cells, endothelial cells, hepatocytes, and fibroblasts. • IL-1RII is actually a decoy receptor that serves a counter-regulatory role by competing with IL-1RI, the fully functional IL-1 receptor, for IL-1 in the extracellular space. • IL-1RII is the predominant IL-1 receptor found on B cells, monocytes, and neutrophils. The extracellular domains of IL-1RI and IL-1RII are shed by activated neutrophils and monocytes. The shed receptors can act as a sink for secreted IL-1 and, thus, along with IL-1RA, represent an important counter-regulatory component of the inflammatory response.

19. Moving along…… • Three major anticoagulant pathways exist and all can be inhibited by the inflammatory cascade: • antithrombin, • protein C system, • TF pathway inhibitor. • Antithrombin is a serine protease inhibitor that antagonizes thrombin and factor Xa. • During severe inflammatory responses, antithrombin levels are markedly decreased as the result of consumption, impaired synthesis (negative acute phase response), and degradation by elastase from activated neutrophils.

20. Protein C • Protein C is activated by thrombin bound to thrombomodulin. • During systemic inflammation, protein C levels are reduced because of impaired synthesis and degradation by neutrophil elastase. • The protein C system is inhibited by decreased expression of thrombomodulin induced by TNF and IL-1β. • In addition to its role in regulating coagulation, the protein C system also modulates the inflammatory response. • Activated protein C binds to the endothelial protein C receptor. • Activation of this signaling pathway inhibits inhibits secretion of TNF, IL-1β, IL-6, and IL-8 by monocytes.

21. Circulating levels of protein C decrease in patients with severe sepsis or septic shock, and a marked deficiency of protein C in these patients is a prognostic indicator for an unfavorable outcome. • Various strategies to inhibit excessive activation of the coagulation system were extensively evaluated both in animal models of endotoxemia and sepsis and in clinical trials. • One of these approaches, the administration of recombinant human activated protein C, also called drotrecogin alfa (activated), was shown in a large multicentric randomized clinical trial to significantly improve survival in patients with severe sepsis. (Understandably, bleeding complications can occur with the use of drotrecogin alfa (activated). Furthermore, this agent was not beneficial for septic patients with an Acute Physiology and Chronic Health Evaluation II (APACHE II) score lower than 25, postoperative patients with single–organ system dysfunction,or pediatric patients with severe sepsis.)

22. INTERLEUKIN-6 • IL-6 is a pluripotent cytokine intimately associated with the inflammatory response to injury or infection. • IL-6 can be produced not only by immunocytes (e.g., monocytes, macrophages, and lymphocytes) but also by many other cell types, including endothelial cells and intestinal epithelial cells. • Factors known to induce expression of IL-6 include IL-1, TNF, platelet-activating factor, LPS, and reactive oxygen metabolites.

23. IL-6 • The cellular and physiologic effects of IL-6 are diverse and include • induction of fever, • promotion of B-cell maturation and differentiation • stimulation of T-cell proliferation and differentiation • promotion of differentiation of nerve cells • stimulation of the hypothalamic-pituitary-adrenal axis, • induction of the synthesis of acute phase proteins (e.g., C-reactive protein) by hepatocytes. Name One !!!!!

24. IL-6 • Circulating concentrations of IL-6 increase dramatically after tissue injury. • Elevated plasma levels of IL-6 are consistently observed in patients with sepsis or septic shock. • The degree to which circulating IL-6 levels are elevated after tissue trauma or during sepsis has been shown to correlate with the risk for postinjury complications or death.

25. INTERLEUKIN-11 • IL-11 is expressed in a variety of cell types, including neurons, fibroblasts, and epithelial cells. • Expression of IL-11 can be up-regulated by IL-1, TGF-β, and other cytokines or growth factors. • From a functional standpoint, IL-11 is a hematopoietic growth factorwith particular activity as a stimulator of megakaryocytopoiesis and thrombopoiesis.

26. IL-11 • Circulating levels of IL-11 increase in patients with DIC and sepsis. • IV or oral administration of recombinant IL-11 improves survival in neutropenic rodents with sepsis, possibly by preserving the integrity of the intestinal mucosal barrier

27. INTERLEUKIN-8 AND OTHER CHEMOKINES Chemotaxis • ____________ is the term used to denote the directed migration of cells toward increasing concentrations of an activating substance. • The ability to recruit leukocytes to an inflammatory focus by promoting chemotaxis is the primary biologic activity of a special group of cytokines called __________. chemokines

28. Chemokines • Four chemokine subgroups have been described. The subgroups are defined by the degree of separation of the first two NH2-terminal cysteine residues. • CXC or α-chemokines, the first two cysteine moieties are separated by a single nonconserved amino acid residue, • CC or β-chemokines, the NH2-terminal cysteines are directly adjacent to each other. • C chemokine subgroup is characterized by the presence of only a single NH2-terminal cysteine moiety. • CX3C subgroup has only one member (fractalkine); in this chemokine, the NH2-terminal cysteine residues are separated by three intervening amino acids. Name them !!!!!!!

29. IL-8 • Increased circulating concentrations of IL-8 were detected in experimental animal models of infection or endotoxemia and in patients with sepsis. • Treatment of experimental animals with antibodies against IL-8improves survival or prevents pulmonary injury in models of sepsis or ischemia/reperfusion injury. • These observations support the view that IL-8–mediated activationof neutrophils plays an important role in the pathogenesis of organ system damage in these syndromes.

30. INTERLEUKIN-12 • produced primarily by antigen-presenting cells • IL-12 receptor is expressed on T cells and NK cells. • The most important biologic activity associated with IL-12 is to promote TH1 responses by helper T cells. • In this regard, IL-12 promotes the differentiation of naive T cells into TH1 cells capable of producing IFN-γ after activation

31. INTERLEUKIN-12 • IL-12 has also been implicated in the pathogenesis of IBD. T cells eluted from the lamina propria of intestinal resection specimens from patients with Crohn's disease secrete cytokines consistent with a TH1-like profile. • When antibodies to IL-12 are administered to mice with fecal peritonitis induced by cecal ligation and perforation, mortality is increased and clearance of the bacterial load is impaired. • Conversely, pretreatment or even post-treatment with recombinant IL-12 has been shown to improve survival in a murine model of bacterial peritonitis

32. INTERLEUKIN-18 • IL-18 is structurally related to IL-1β and is functionally a member of the TH1-inducing family of cytokines. • Like IL-1β, IL-18 is translated in the form of a precursor protein (pro–IL-18). • This precursor molecule requires cleavage by the same converting enzyme that activates IL-1β (i.e., ICE) to form biologically active IL-18. • The two cytokines, IL-1β and IL-18, are also similar with respect to the way that intracellular signaling occurs after association of the cytokine with its receptor on target cells.

33. INTERLEUKIN-18 • The main biologic activity of IL-18 is to induce production of IFN-γ by T cells and NK cells. • In this regard, IL-18 acts most potently as a co-stimulant in combination with IL-12. • IL-12–induced IFN-γ expression appears to depend on the presence of IL-18 • Circulating concentrations of IL-18 are higher in patients with sepsis than in those with just injuries, and high levels of this cytokine are associated with a fatal outcome in patients with postoperative sepsis

34. INTERLEUKIN-4, INTERLEUKIN-10, AND INTERLEUKIN-13 • Can all be regarded as inhibitory, anti-inflammatory, or counter-regulatory cytokines. • All three of these cytokines are produced by TH2 cells • serve to modulate the production and effects of proinflammatory cytokines such as TNF and IL-1.

35. IL-4 • IL-4, originally described as a B-cell growth factor, is a 15- to 20-kd glycoprotein that is synthesized by • TH2 cells • mast cells • basophils • eosinophils. • IL-4 has many biologic actions that promote expression of the TH2 phenotype; • down-regulation of proinflammatory and cell-mediated immune responses • up-regulation of humoral (B-cell–mediated) immune responses.

36. IL-4 • induces differentiation of CD4+ T cells into TH2 cells • conversely, down-regulates differentiation of CD4+ T cells into TH1 cells. • inhibits the production of TNF, IL-1, IL-8, and PGE2 by stimulated monocytes or macrophages • IL-4 acts as a comitogen for B cells and promotes expression of the class II major histocompatibility complex (MHC) on B cells.

37. IL-10 • originally called cytokine synthesis inhibitory factor, was first isolated from supernatants of cultures of activated T cells. • produced primarily by TH2 cells, but it is also released by activated monocytes • IL-10 acts to down-regulate the inflammatoryresponse through numerous mechanisms.

38. IL-10 • Administering exogenous IL-10 in an effort to blunt excessive inflammation has led to mixed results in experimental models of sepsis or septic shock.

39. IL-13 • IL-13 is 12-kd protein closely related to IL-4. • The two proteins have about 25% homology and share many structural characteristics. • IL-13 is produced by TH2 cells and also undifferentiated CD4+ T cells and CD8+ T cells. • Binding of either IL-4 or IL-13 to their respective receptors induces signaling by activating the same JAK kinases, JAK1 and Tyk2. • does not have any direct affects on T cells. • down-regulates the production of proinflammatory cytokines • increases the production of anti-inflammatory proteins

40. IL-13 • Additional anti-inflammatory properties of IL-13 include inhibition of induction of the enzyme COX-2 • required for the production of prostaglandins, and induction of an enzyme, 15-lipoxygenase, that catalyzes the formation of a lipid mediator (lipoxin A4) with anti-inflammatory properties.

41. EICOSANOIDS ?????????? ???? ??? ????

42. COX1/COX2 COX-2, however, is an inducible enzyme. Expression of COX-2 is induced by a number of stimuli, including various growth factors and proinflammatory cytokines. COX-1 is expressed constitutively in a variety of tissues, and mediators produced by this isoform are thought to be important in various homeostatic processes, such as regulating renal perfusion and salt and water handling, maintaining hemostasis by modulating platelet aggregation, and preserving gastrointestinal mucosal integrity.

43. identification of COX-2 as the so-called inflammatory isoform of cyclooxygenase led to intense efforts to develop drugs selective for the inducible enzyme. • Selective COX-2 inhibitors were initially widely prescribed by clinicians. • However, data from large multicentric trials of rofecoxib, one of the compounds in this class, showed that treatment with this agent was associated with an increased risk for death from cardiovascular complications. • As a result of these findings, rofecoxib was withdrawn from the market.

44. Many of the downstream actions of the proinflammatory cytokines occur as a result of increased expression of two key enzymes, iNOS and COX-2. • iNOS is one of three isoforms of an enzyme, nitric oxide synthase (NOS), that catalyzes conversion of the amino acid l-arginine to the free radical gas NO • produced by many different types of cells and serves as both a signaling and an effector molecule in mammalian biology. • NOS-1 (also called neuronal NOS or nNOS) and NOS-3 (also called endothelial or eNOS) tend to be expressed constitutively in various cell types, • iNOS is expressed for the most part only after stimulation of cells by proinflammatory cytokines (particularly IFN-γ, TNF, and IL-1) or LPS. B A

45. NO • NOS-1 and NOS-2 produce small puffs of NO· in response to transient changes in the intracellular ionized calcium concentration. • In contrast, iNOS, once induced, produces large quantities of NO· for a prolonged period • Many of the biologic actions of NO·, including vasodilation, induction of vascular hyperpermeability, and inhibition of platelet aggregation, are mediated through activation of the enzyme soluble guanylyl cyclase (sGC). • In addition, NO· reacts rapidly with another free radical, superoxide anion (O2-·), to form peroxynitrite anion (ONOO-), the conjugate base of the weak acid peroxynitrous acid (ONOOH • Being a potent oxidizing and nitrosating agent, ONOO-/ONOOH is thought to be responsible for many of the toxic effects of NO • ONOO-/ONOOH is capable of oxidizing sulfhydryl groups on various proteins at a rapid rate, peroxidizing membrane lipids, and inactivating mitochondrial aconitase

46. Heat Shock Proteins • The heat shock proteins were first identified as a family of proteins that are induced when cells or experimental animals are subjected to sublethal thermal stress. • These proteins are also induced by many other stimuli, such as inflammation, oxidative stress, and infection. • The primary role of heat shock proteins is to serve as molecular chaperones to facilitate the proper folding of nascent proteins.

47. Heat Shock Proteins • normally found inside cells, but under certain conditions these proteins can be detected in the extracellular milieu. • For example, elevated circulating levels of HSP70 have been found in trauma patients and patients in the immediate period after coronary artery bypass graft surgery. • these proteins may serve as endogenous danger signals and trigger activation of the inflammatoryresponse after damage to tissues.

48. The End