Chapter 20 Cellular and Humoral Mediators of Inflammation

1. Chapter 20 Cellular and Humoral Mediators of Inflammation WuJianmin

2. The“cardinal signs”of inflammation include redness，swelling，heat，pain，and loss of function．Inflammation represents an orderly series of events which protect the host by destroying foreign invaders，eliminating the debris，and repairing any damage to host tissue．

3. NONSPECIFIC CELLULAR RESPONSE TO INFLAMMATION

4. Granulocytes Polymorphonuclear neutrophils (PMNs) are the largest population of leukocytes in the peripheral blood. These phagocytic cells contain distinct primary granules composed of proteases.

5. Mononuclear Phagocytes Blood and bone marrow monocytes and tissue macrophages are mononuclear phagocytes that reside in every tissue and organ, known as the mononuclear phagocyte system. Their functions include removing debris such as old or injured erythrocytes, leukocytes, platelets, bacteria, antigen-antibody complexes, and degenerated or damaged cell membranes.

6. Tissue macrophages include Kűpffer cells of the liver; alveolar macrophages; splenic macrophages; histiocytes of the lymph nodes, peritoneum, and other areas; bone osteoclasts, nervous system microglial cells, andsynovial A and C cells.

7. Natural Killer Cells Natural killer (NK) cell-mediated cytotoxicity, although not MHC-restricted, now is known to be regulated by the expression of class I MHC molecules expressed on target cells. Different subsets of NK cells express receptors that recognize MHC molecules, delivering inhibitory signals to NK cells to avoid eliminating normal self cells. Other receptors on NK cells trigger NK cells to eliminate allogeneic or autologous cells expressing "abnormal" MHC-molecules. Since NK cells also kill MHC-negative target cells, certain receptors might recognize non-MHC ligands.

9. Cytokines and Cytokine Receptors (CR) Cytokines are small polypeptide or glycopeptide molecules (mol. wt. 5 to 70,000 daltons) whose primary functions are to mediate infectious and inflammatory processes. Cytokines act at very lowconcentrations as pleiotropic hormones, overlapping suppressive functions and stimulatory events.

10. The inflammatory cytokines (IL-la, IL-1b, TNF-a, IFN-a, -b, -r) are involved in the pathogenesis of many infectious and autoimmune diseases. The antiinflammatory cytokine (IL-10) profoundly suppresses macrophages, inhibiting the synthesis of proinflammatory cytokines. Cytokines can be categorized loosely as interleukins, interferons, cytotoxins, chemokines, and growth and stimulatory factors.

15. Cytokine receptors (CyR) are noncovalently associated with intracellular tyrosine kinases. CyRs can be classified based on their subunit composition (1) homodimers where two receptor molecules bind one molecule of ligand (e.g., hGH-R, EPO-R, G-CSF-R); (2) two different subunits (α and β) where the α subunit functions primarily in ligand binding while the β subunit functions as a signaling subunit (e.g., IL-6R, GM-CSF-R, IL-3R, IL-5R); and (3) three different subunits (α and β and γ) where one of these subunits is common to several different receptors (e.g., IL-2R, IL-4R, IL-7R, IL-9R, IL-13R, and IL-15R).

16. Interleukins and Interleukin Receptors Most interleukins are synthesized by activated T-lymphocytes, however IL-1 and several other interleukins are also secreted by monocytes/macrophages. Many other cell types produce interleukins including fibroblasts, endothelial Cells, PMNs, NK cells, B cells, hepatocytes, and others. To date, 17 biologically potent signaling molecules have been designated as interleukins: IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 (NKCF), IL-13, IL-14, IL-15. Several of these molecules share biologic activities.

17. Interferons and Interferon Receptors Interferons originally were described as antiviral substances, but are now known to be important regulators of the immune response in general. IFN-γ (type II IFN), in conjunction with TNF and in the presence of bacteria or virus, activates macrophages stimulating the production of IL-12 and IFN-α and -β (type I IFNs). Type I IFNs, by increasing the expression of MHC class I antigens, enhance NK cell activity to virus-infected cells.

18. Cytotoxins and Receptors TNF activity was first described as a monokine that could cause tumor cell death (necrosis). Subsequently, TNF was shown to be homologous to cachectin, the mediator of cachexia accompanying parasitic infections. Some biologic properties of TNF are quite similar to those of IL-1; i.e., pyrogenic, induces shock, stimulates hepatocytes to synthesize acute phase proteins, induces prostaglandin E2 and collagen synthesis.

19. TNF is known to be a major cytokine involved in inflammatory reactions. IL-1 and TNF are induced during inflammatory reactions where they, in turn, increase the expression of adhesion molecules on endothelial cells. Vascular permeability is increased, facilitating cell and plasma protein extravasation. IL-1 and TNF then are directly involved in tissue damage, degrading cartilage, facilitating proteolysis of muscle cells, destroying bone at the site of the local inflammatory reaction.

20. Natural killer cytotoxic factor (NKCF) has now been designated as IL-12. This cytotoxin activates NK cells and LAK cells to enhance their lyric capacity.

21. Lymphotoxin is 27% to 32% homologous to TNF-β, the two substances compete for the same cellular receptor, and are functionally indistinguishable. Lymphotoxin, TNF-β, and IL-1 all can stimulate bone resorption. Lymphotoxin can be released from myeloma cells and may be involved in the hypercalcemia and osteolytic lesions associated with multiple myeloma.

22. There are many members of the TNF-R family, including TNF-R1, TNF-R2, CD27, CD30, CD40, Fas, and others. These receptors all have cysteine rich pseudorepeats, each containing about six cysteines and 40 amino acids. Two distinct TNF receptors, TNF-R1 (the primary TNF receptor on most cells) and R2 are representative of the TNF-R family.

23. Chemokines and Receptors Chemokines are very small (8 to 10 kd ) chemoattractant proteins with 20% to 70% amino-acid sequence homology. There are at least four families of chemokines, only two of which have been extensively studied to date: α-chemokines that have one amino acid separating the first two cysteine residues (CXC), and β-chemokines where the two cysteine molecules are adjacent (CC). IL-8 is an α-chemokine. Monocyte chemoattractant protein (MCP), RANTES (regulated on activation, normal T expressed and secreted), and macrophage inflammatory protein-1 (α and β) are β-chemokines.

24. Four human CXC chemokine receptors (CXCR1--CXCR4), eight human CC chemokine receptors (CCR1--CCR8) have been identified. CCR1 and CCR2 are constitutively expressed on monocytes, but CCR2 can be downregulated by lipopolysaccharide, making the cells unresponsive to MCP-1.

25. Growth and Stirnulatory Factors and Receptors Granulocyte / monocyte colony stimulating factor (GM-CSF) and IL-3 stimulate progenitor cells to produce hematopoietic cells of multiple lineages. Once IL-3 and GM-CSF stimulate the microenvironment, stroma cells and macrophages produce the other two CSFs (G-CSF and M-CSF) to support self-renewal and survival of stem cells. GM-CSF-R (CDw116) is a member of the IL-3, IL-5, GM-CSF receptor family. G-CSF-R is a member of the IL-6 receptor family.

26. Cytokine Antibodies Natural antibodies to cytokines in human sera have been reported to date against TNF-α, IL-1α, IL-lβ, IL-2, IL-6, IL-8, IL-10, IFN-α, IFN-β, IFN-γ, GM-CSF, chemokines, and nerve growth factor (NGF). These autoantibodies have been shown to bind to naturally occurring cytokines as well as recombinant cytokines. High avidity antibodies might interfere with the functions of their corresponding cytokines.

27. The following cautions have been identified: (1) cytokine autoantibodies can interfere with the quantitation of cytokines in biologic fluids, (2) existing autoantibodies could neutralize cytokine therapy, (3) monitoring of certain immunoinflammatory diseases by quantitation of specific cytokines might be efficacious, and (4) some pooled human IgG preparations contain high avidity cytokine autoantibodies.

28. Cytokines in Disease the role of cytokines in human disease is much more complicated. Inflammatory diseases (infections, sepsis, autoimmune diseases, allergic respiratory disease ) have been the target of most studies regarding the effects of cytokines in humans. IFN-α 2 and -ω have been shown in vitro to inhibit HIV replication. In vivo high serum levels of IFN appear to decrease viral burden during the acute stage of HIV infection, but late in the infection, these cytokines and sTNF-R are associated with disease progression.

29. Sepsis results from excessive systemic host inflammatory response to infection, largely mediated by cytokines. Cytokines that are elevated in human sepsis are TNF-α , IL-1 β, IL-6, andIL-8.

30. Inflammation, associated with abnormal cytokine production, is a prominent feature of several autoimmune diseases, e.g., joint disease in patients with RA, destruction of pancreatic islet cells in insulin-dependent diabetes, altered intestinal mucosa in inflammatory bowel disease, and myelin sheath destruction in multiple sclerosis.

31. NONSPECIFIC HUMORAL RESPONSE TO INFLAMMATION

32. Acute-Phase Response When the body is injured, it responds by increasing the hepatic synthesis of a number of plasma proteins. The systemic acute-phase response helps to ensure survival during the period immediately following injury. The systemic response must help to achieve the same goals as the localized inflammatory response, i.e., to contain or destroy infectious agents; to remove damaged tissue, and to repair the affected organ.

33. Lysosomal Enzymes During phagocytosis and following the death of phagocytes, lysosomal contents of granules are released to enhance inflammation and provide microbicidal activity. Much of the tissue damage of inflammation is from destruction of "innocent bystander" cells by lysosomal enzymes released during the inflammatory process.

34. C-Reactive Protein C-reactive protein (CRP) was recognized in 1930 because of its ability to precipitate with the C-polysaccharide extract of pneumococcus. CRP can promote passive agglutination of red blood cells coated withbinding substrate. An example of the similarity between CRP and immunoglobulins is the initiation of the complement cascade through C1 activation by complexed CRP. Opsonization for ingestion by phagocytes can result from complement activation by CRP.

35. CRP is an especially useful marker of inflammation to monitor patients after surgery. In uncomplicated cases, the CRP levels peak between 48 and 72 hours and return to normal by day 7. If the postoperative course is complicated by inflammation or sepsis, the CRP levels remain elevated or increase. For CRP to be a valuable tool in assessing inflammatory complications, the test must be available 24 hours/day, 7 days/week. Recently, CRP has been proposed as predictor of coronary heart disease (CHD).

36. Complement The complement system consists of 16 components involved in three separate pathways of activation. Five proteins unique to the classical pathway include the trimolecular complex of C1 (C1q, C1r, C1s), C4 and C2. Three proteins unique to the alternative pathway include factor B, factor D and P (properdin). Two components are unique to the lectin pathway: mannose-binding lectin (MBL) and MBL-associated serine protease (MASP). Six components participate in all three pathways: C3, C5, C6, C7, C8 and C9. All three pathways can be divided into three units (recognition, activation, and membrane attack) to simplify the understanding of complement activation.

37. Measuring Complement in the Clinical Laboratory The complement components usually quantitated are C3 and C4; C3 because of its pivotal location and role in both the classical and alternative pathway, and C4 to distinguish classical from alternative pathway activation. All complement components can be quantitated, however, components other than C3 and C4 rarely are required. The only reason to measure any component other than C3 and C4 would be if a component deficiency is suspected. The screening method of choice for a component deficiency is the hemolytic assays.

38. Hemolytic complement assays are measured as CH50 (hemolytic complement 50% lysis point), which is a fluid phase assay, or CH100 (hemolytic complement 100% lysis), which is performed in agar. In both systems, either the classical pathway or the alternative pathway can be measured by varying the indicator cells.

39. Neither CH50 nor CH100 are useful to monitor disease activity because their endpoint (lysis) is very insensitive to changes in component levels and can occur even with significant depletion of most components. It takes very few C2 and C4 molecules to cause lysis and yield a "normal" CH50 level. Quantitation of C3 and C4 are much more sensitive measures of complement consumption in vivo than are the hemolytic assays. The patterns of complement consumption in various disease states are shown in table 20-1.

40. TABLE20-1 INTERPRETATION OF LABORATORY TESTS FOR COMPLEMENT ACTIVATION ↓CH50，↓C3，↓C4 　　↓CH50，↓C3，NormalC4 Classical Pathway ActivationAlternative Pathway Activation Active SLE Acute post-strep GN Serum sickness Membranoproliferative GN Immune complex disease Severe bacteremia Chronic active hepatitis C3 nephritic factor Subacute bacterial endocarditis C3, H, or I deficiency ↓to Normal CH50，NormaI C3，C4 ↓CH50，Normal C3．Normal C4 MildClassical Pathway ActivationDefective hemolytic function Cryoglobulinemia Improperly handled sera Vasculitis Coagulation-associated C1 Inh defiencv depletion C4 deficiency Component deficiency (e.g.，C2，C5 - C8)

41. Immune Complexes Immune complexes are formed whenever antigen and antibody combine．The dynamics of immune complex formation are continuously modified relative to the concentration of antigen or antibody present．The size of the immune complexes in circulation is determined by the relative ratio of antigen to antibody and the valence of the antigen and the antibody ．When antigen and antibody are present in more balanced ratios (equivalence)，they localize in vessels and mesangial areas．

42. Complement is activated while the immune complexes are in circulation，depositing C3b on their surface，which facilitates 1ocalization or deposition in blood vessels and renal glomeruli．C3b deposited on basement membranes provides the “activating surface” necessary to initiate the activation loop of the alternative complement pathway. Complement activation releases chemotactic factors that attract PMNs．During phagocytosis，the PMNs extrude the contents of their granules，Which are cytotoxic for endothelial cells．Thus immune complexes initiate as well as perpetuate the inflammatory process．

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