Inflammation, phagocytes, complement (Milan Číž) Inflammation Body is against injury/invasion of microorganisms protected by physiological and physical barriers. Skin – keratinocytes, sweat (washing, fatty acids) GI tract – low pH in stomach, fatty acids, bile acids, natural microflora in intestines Respiratory tract – cilia, mucosal coating Nasopharynx and eye – mucosal coating, saliva, lysozyme in tears (peptidoglycan hydrolysis) Inflammation If these barriers are overcome → inflammation, immune reaction. Definition: Complex of immune and physological reactions to a disruption of organism integrity, which lead to an injury localisation, protection of damaged site and its healing. Formation of inflammation: • Antigenic stimulus (infection microorganisms) • Chemical or physical injury • Ischemia of organs and tissues Typical signs of local inflammation: • Rubor = redness • Tumor = swelling • Dolor = pain • Calor = increased temperature Types of inflammation: • Acute – physiological defense reaction; passes without consequences and injured tissue is completely recovered • Chronic = usually pathologic; tissue is to some extent replaced by connective tissue • Sterile = without microorganisms Inflammation First signals for inflammatory response development give by: 1. Degranulated tissue mast cells 2. Phagocytes 3. Mediators released from injured cells and extracellular matrix Inflammatory response process Processes accompanying inflammation • Increased vascular permeability, leak of plasma into extravascular space and swelling. • In the case of injury the first response is hemocoagulation – cessation of blood loss. Factors released from damaged cells activate coagulation. Major principle is conversion of soluble protein fibrinogen to insoluble fibrin. • Activation of fibrinolytic system – prevention of blood clotting spreading outside the injury site. During fibrinolysis, enzyme plasmin is generated from inactive plasminogen. Plasmin cleaves fibrin to fibrin degradation products and reduces compactness of blood clot, eventually partially dissolves th blood clot. • Activation of kinin system – activated coagulation factor XII acts on prekallikrein, kallikrein is created, followed by bradykinin, which increases capillary permeability. • Increased expression of adhesion molecules on endothelium – attachment of phagocytes and later of lymphocytes. • Activation of complement. • Activation of phagocytes. • Influencing of local nerve endings (pain). • Increased synthesis of arachidonic acid metabolites (TX, LT, PG). • Changes in temperature regulation (fever). It is induced by stimulation of hypotalamic center by pro-inflammatory cytokines. This is followed by activation of tissue metabolism via mobilization of hypothalamus-hypophysisadrenal axis (adrenal cortex– steroida, stress hormone cortisol). Increased temperature increase metabolism of immunocompetent cells. Expression of heat shock proteins (Hsp) is induced. They function as chaperones – they bind newly synthesised polypeptide chains and intracellular denatured proteins and help them to assemble into native conformations. • Liver scavenge trace elements important for bacterial growth. • Cytokines produced in inflammatory site stimulate production of acute phase proteins (C-reactive protein, CRP; complement components C3 and C4; serum amyloid P, SAP) in liver. Processes accompanying inflammation Coagulation cascade Intrinsic pathwayExtrinsic pathway Factor XII Plasma prekallikrein Plasma kallikrein Bradykinin High molecular weight kininogen Vasodilation Increased capillary permeability Mechanisms of protective inflammation Phagocytes and their mediators Complement system Prostanoids Blood coagulation Kinins Vascular endothelial reactions Protective inflammation Specific mechanisms of inflammation Ag fragments bound to MHC are presented to lymphocytes Phagocytes History I. Metchnikoff – Phagocytosis and innate immunity – Nobel prize in 1908 • Term „phagocyte“ used for the first time • Phagocytes of starfish larvae engulfed a splinter; phagocytosis and digestion of bacteria by macrophages and PMNs • From greek words „phagein“ = eat, „cytos“ = cell http://fb.ru/article/214500/kratkaya- biografiya-ili-ilicha-mechnikova-istoriya- jizni-otkryitiya-dostijeniya-i-osobennosti- deyatelnosti Cells of the immune system Pluripotent stem cell Lymphoid stem cell Myeloid stem cell NK cells Thymus Megakaryocytes Erythroblasts ErythrocytesPlatelets T-lymphocytes B-lymphocytes Dendritic c. Neutrophil Eosinophil Basophil Mast cell Monocytes Macrophages Polymorphonuclear leukocytes (PMNs) • 40-65% of all leukocytes (3-5 x 103 /µl of blood) • short-living, abundant in blood, absent in healthy tissues • characteristic nucleus • granules and CD66 membrane marker • the first line of defense against pathogenic microorganisms • chemotaxis • phagocytosis, intracellular killing • generation of ROS and RNS • degranulation • inflammation, tissue damage Polymorphonuclear leukocytes (PMNs) • Primary granules – azurophilic – typical for young neutrophils – neutral proteases - cathepsin G, elastase, proteinase 3 – lysozyme, defensins, phospholipase A2, myeloperoxidase • Secondary granules – specific for mature neutrophils – lysozyme, NADPH oxidase, lactoferrin, elastase, colagenase • Terciary granules – Gelatinase granules – in front end of migrating phagocytes – gelatinase (membrane destruction) • Secretory vesicles – Reservoir of membrane components Eosinophils Their role in an organism is still widely discussed and reassessed. Originally – defense against parasites (worms) • NADPH oxidase (similarly like in neutrophils) • Eosinophil peroxidase • Major basic protein and other granular proteins Monocytes / Macrophages • phagocytosis, killing • tissue renewal • antigen presentation for specific immune response • characteristic nucleus and CD14 membrane marker • adher to plastic and glass • activated by cytokines • produce cytokines • also eliminate malignant and altered self structures Activation of phagocytes in inflammation • SOS signals – N-fMLP – PGs, LTs, PAF – Complement – Pro-inflammatory cytokines • Response of phagocytes – chemotaxis – adherence – diapedesis – activation – peptides of hemocoagulation cascade – phagocytosis and killing Tissue Receptors on phagocytes • complement receptors • Fc receptors • Toll-like receptors • chemotactic receptors (fMLP) • mannose receptors recognizing sugar structures on bacterial and viral surfaces • scavenger receptors – recognize acetylated LDL Killing mechanisms of phagocytes NADPH oxidase activation Neutrophil extracellular trap (NETs) generation Proper functioning of phagocytes is important for the organism Deficiency in phagocyte functions = severe course of trivial infections Example: CGD – defective NADPH oxidase On the other hand, overactivated phagocytes – problems Damage of neighbouring cells and tissues by reactive metabolites and proteolytic enzymes Complement Komplement: historie Discovered in 1894 – 1899 Jules Bordet (1870-1961) Worked in Metchnikoff laboratory (Pasteur Institute, Paris) Complement fixation test • composed from various plasma proteins (30) • individual components interact • pathogen opsonization • induction of inflammatory response Complement system • C-activation: alteration of complement proteins and their interactions with other components • C-fixation: complement utilization of Ag-Ab complexes • Hemolytic activity: complement-mediated lysis of erythrocytes coated by antibody • C-inactivation: heat denaturation Complement – basic terms Nomenclature: • C1complex(qrs), C2, C3, C4, C5, C6, C7, C8, C9 • factors B, D, H and I, properdin (P) • mannose-binding lectin (MBL), MBL-associated serine proteases (MASP-1 MASP-2) Proteins of complement system Activated products of complement proteins (nomenclature) Activated components are usually overlined: eg. C1qrs After being enzymatically cleaved, a larger fragment usually binds to activation complex or membrane and smaller peptide is released into surroundings. Letter “b” is usually used for larger peptide and letter “a” for smaller peptide, eg. C3b/C3a, C4b/C4a, C5b/C5a. Several complement proteins are proteases, which are themselves activated by proteolytic cleavage. These enzymes are called zymogens. General biochemical principle of complement activation cascade Ways of complement activation Complement cascade can be activated by one out of three ways: Classical pathway – initiated by C1q binding to: • Ab-Ag complex • Surface components of bacteria (proteins, polyanionic structures) • C-reactive protein (acute phase protein which binds to phosphocholin residues of bacterial polysaccharides) Lectin pathway – initiated by binding of MBL (mannose-binding lectin) = serum protein, concentration of which increases during acute phase of immune response, to surface structures of bacteria and viruses, which contain mannose Alternative pathway – initiated by binding of spontaneously activated C3 to the surface of pathogens Ways of complement activation Classical pathway Alternative pathway LYTIC pathway Lectin pathway C3 activation Generation of C5 convertasey C5 activation Antibody-dependent Antibody-independent Classical pathway activation C1q is a complex with 6 heads = binding site of C1q is associated with 2 molecules of C1r and C1s. Binding leads to activation of C1r, then of C1s and cleavage of C4. Lectin pathway activation MBL (mannose binding lectin) resembles C1 complex. 6 heads with affinity to saccharide structures of pathogen surface are associated with 2 molecules of MBLassociated serine proteases (MASP-1 a MASP-2). Binding leads to activation of MASP and cleavage of C4. Classical (and lectin) pathway activation Alterntive pathway activation C3 is spontaneously cleaved – formation of functionally active C3b. C3b binds factor B, which cleaved by factor D. Soluble C3 convertase is formed, which cleaves more C3 to C3a and C3b. C3b binds to the surface of own cells or pathogens. Rapidly inactivated on the surface of own cells – formation of iC3b. C3bBb on the surface of pathogen – no regulátory proteins present. Binding of factor P (properdin), which stabilizes C3bBb convertase aktivity. C3bBb is an equivalent of C4b2b of classical pathway. 40 Figure 2-30 C4b2b3b from classical (lectin) pathway or its equivalent C3b2Bb from alternative pathway are C5 convertases 41 Video (The Immune response – Complement Systém) Results of complement activation 43 44 Biological effects of complement activation products • C3a (anaphylatoxin) - degranulation of mast cells; increased vascular permeability; anaphylaxis • C3b (opsonin) - opsonization; phagocyte activation • C4a – anaphylaxis • C5a - anaphylaxis similarly to C3, but more intensive; attractant and activator of neutrophils, elicits neutrophil aggregation, stimulation of oxidative metabolism and release of leukotrienes, degranulation of mast cells • MAC – cytolysis Opsonization and phagocytosis Complement – summary of effets • useful: • opsonization and facilitated phagocytosis • chemoattraction and phagocyte activation • Lysis of bacteria and infected cells • regulation of antibody response • clearance of immune complexes • clearance of apoptotic cells • harmful: • inflammation, anaphylaxis