Complement Material in this document is given for internal use only and exclusively for teaching purposes Jules Bordet Discovery in 1890s due to the “complement activity” of a heat-labile component of normal plasma that augments the opsonization and killing of bacteria by antibodies. Nobel Prize was awarded in 1919 for his discoveries relating to immunity, particularly complement-mediated lysis Complement originally evolved as part of innate immune system providing protection early in infection Definition Composed of more than 30 different plasma proteins produced mainly by the liver. In absence of infection, proteins circulate in inactive form: proteases of the complement are synthesized as inactive pro-enzymes, the zymogens. Zymogens become enzymatically active only after proteolytic cleavage, usually by another complement protein, triggering a proteolytic cascade. Final effector complement involved in the removal of the pathogen Small number of pathogen produces a rapid response greatly amplified at each step. Nomenclature Three pathways to activate: classical, alternative, lectin First proteins discovered for the classical pathways: C1, C2, … Named in the order of discovery rather than sequence of reactions Cleavage of fragments (C3) produces a small fragment called a (C3a) and a bigger fragment called b (C3b) except for C2 because the larger fragment C2a was enzymatically active and this name has survived Nomenclature Another exception to the rule: C1q, C1r and C1s which are not cleavage products but distinct proteins together comprising C1 Proteins of alternative pathway were discovered later and designed by capital letters, for anaphylatoxins example factor B and D Cleavage products also called Bb the larger fragment and Ba the small fragment. Pathways of complement activation CRP, SAP, PTX3 MBL, ficolins anaphylatoxin anaphylatoxin Three main functions: Opsonization and phagocytosis Stimulation of inflammation Complement-mediated cytolysis The complement system recognizes features of microbial surfaces and marks them for destruction by the deposition of C3b Complement is a system of soluble pattern- recognition receptors and effector molecules that detect and destroy microorganisms. Anaphylatoxins: C3a, C4a, C5a Stimulation of Opsonization and Cytolysis inflammation phagocytosis Complement pathways The alternative pathway can be activated by spontaneous cleavage of C3 2 different ways of activation: Tickover of C3 (cleavage of 1-2% total plasma C3/hour) C3b produced from lectin or classical pathway Alternative pathway can act as an amplification loop to increase C3b production The alternative pathway Anaphylatoxins: C3a The alternative pathway can be activated by spontaneous cleavage of C3 C3 convertase: C3bBb Properdin stabilizes the alternative pathway C3 convertase on pathogen surfaces Properdin-deficient patients are particularly susceptible to infections with Neisseria meningitidis The alternative pathway Anaphylatoxins: C3a C5a Complement pathways Mannose-binding lectin (MBL) and Ficolins form complexes with serine proteases and recognize particular carbohydrates on microbial surfaces Lectin pathway triggered by 4 different pattern recognition molecules in blood and extracellular fluids Human ficolins: MBL ü M-ficolin (1) produced in secreted by liver and activated circulate in macrophages the blood in tissues ü L-ficolin (2) synhesized by liver and circulate in the blood MASP: MASP-3 MASP-3 ü H-ficolin (3) MBL-associated synhesized by serine protease liver and circulate in the blood The actions of C3 convertase result in the binding of large numbers of C3b molecules to the pathogen surface Individuals deficient with MBL or MASP-2 experience substantially more respiratory infections by common extracellular bacteria during early childhood Complement activation Only antibodies bound to antigens can initiate complement activation CH3 CH2 CRP, SAP, PTX3 can The classical also bind to C1q pathway Anaphylatoxins: C4a C2 is cleaved in small soluble fragment and larger fragment For historical reasons: large fragment C3a is named C2a and is a serine protease, active enzyme of C3 convertase C5a C4a: anaphylatoxin of the classical and lectin pathways MAC formation Pathways leading to potent inflammatory and destructive effects must be tightly regulated One important safeguard is that key activated components are rapidly inactivated unless they bind pathogen surface Complement can be activated by dying cells (sites of ischemic injury, apoptosis). Complement coating helps phagocytes to dispose of the dead and dying cells limiting the risk of cell contents being released and triggering an autoimmune response Presence of regulatory proteins to prevent activation of complement on the surfaces of healthy host cells Complement activation is regulated by a series of proteins that serve to protect host cells from accidental damage Complement activation is regulated by a series of proteins that serve to protect host cells from accidental damage DAF: Decay Accelerating Factor / C4BP: C4 Binding Protein / CR1: Complement Receptor 1/ MCP: Membrane Cofactor Protein Complement activation spares host cells which are protected by complement regulatory proteins C3bBb DAF: Decay Accelerating Factor CR1: Complement Receptor 1 MCP: Membrane Cofactor Protein Factor H Factor I = Protease I Complement activation is regulated by a series of proteins that serve to protect host cells from accidental damage Complement activation is regulated by a series of proteins that serve to protect host cells from accidental damage Protectina =CD59 Complement activation spares host cells which are protected by complement regulatory proteins Complement components C4a C4b Ingestion of complement-tagged pathogens by phagocytes is mediated by receptors for the bound complement proteins Integrins: The cleavage products of C3b are recognized by different complement receptors iC3b: CR2, CR3, CR4 C3dg, C3d: CR2 The anaphylatoxin C5a can enhance the phagocytosis of microorganisms opsonized in innate immune response Local inflammatory responses can be induced by small complement fragments C5a > C3a > C4a Functions of complement 4.Clearance of immune complexes 5. B cell activation through binding of fragments to CR2 (co- receptor), 6. selection of high- affinity B cells within germinal centers by exposure of Ag through CR1, CR2, and CR3 Close evolutionary relationship Difetti delle proteine di controllo del complemento sono associati con diverse malattie Deficit in proteine regolatorie del complemento causa malattie patologia Angioedema ereditario (HAE) HAe = hereditary angioedema Edema angioneurotico ereditario o angioedema ereditario C1-INH si lega anche a MASP2 attivata - L’origine dell’Angioedema Ereditario è quindi un difetto genetico, responsabile di una insufficiente quantità o di un deficit funzionale del C1 esterasi-inibitore (C1-INH) - Malattia autosomica dominante, incidenza 1:50.000 caratterizzata da angioedemi ricorrenti Edema angioneurotico ereditario o angioedema ereditario Trattamento con C1 INH ricombinante Deficit in proteine regolatorie del complemento causa malattie patologia CD55 emoglobinuria parossistica notturna Deficit di DAF e CD59: Emoglobinuria parossistica notturna (PNH: Paroxysmal Nocturnal Hemoglobinuria) Anemia emolitica accompagnata CD55 = DAF dall’emissione ricorrente di urine scure CD59 = protectin (eliminazione di emoglobina per via renale), trombosi venosa Deficit di DAF (CD55) e CD59: Emoglobinuria parossistica notturna (PNH: Paroxysmal Nocturnal Hemoglobinuria) CD55 = DAF CD59 = protectina Deficit in proteine regolatorie del complemento causa malattie patologia Sindrome uremica emolitica (HUS, Hemolytic-Uremic Syndrome ) Condizione caratterizzata da: - un danno alle piastrine e globuli rossi - glomerulonefrite provocata da inadeguata eliminazione degli immunocomplessi che tendono a depositarsi a livello renale I difetti dei componenti del complemento determinano una deficienza nella funzione dell’immunità umorale 12.