1. THE COMPLEMENT SYSTEM

2. The complement system is a set of plasma proteins that act in a cascade to attack and kill extracellular pathogens. Approximately 30 components: - activating molecules - regulator factors - complement receptors - membranproteins wich inhibit the lysis of host cells Most of the complement proteins and glycoproteins are produced in the liver in an inactive form. Activation is induced by proteolitic cleavage. Complement system

3. (i) complement activation, (ii) C3-convertase  C3a, C3b, (iii) C3a  platelet activation, (iv) C3b  C5-convertase  C5a, C5b, (v) C5a  tissue factor, PAI-1 expression, (vi) C5b  MAC,…(ix) contact activation (intrinsic pathway) (prekallikrein, factor XII), (x) extrinsic pathway (TF+factor VII), (xi) factor X, (xii)  from protrombin (II) to trombin (IIa), (xiii) trombin  fibrinpolimerisation (fibrin), (xiv) trombin  C3 and C5 breakdown Markiewski MM 2007 Enzyme cascades of the plasma: complement and coagulation

4. A plazma enzim rendszerei MAC Enzyme cascades of the plasma Coagulation cascade Kallikrein-kinin system Complement cascade Alternative pathway MB-lectin pathway Classical pathway

5. AMPLIFICATION OF THE COMPLEMENT CASCADE inactive precursors limited proteolysis activating surface enzyme Activating sutface needed!

6. ACTIVATION OF THE COMPLEMENT SYSTEM COMPLEMENT SYSTEM CLASSICAL PATHWAY MB-LECTIN PATHWAY ALTERNATIVE PATHWAY COMPLEMENT ACTIVATION RECRUITMENT OF INFLAMMATORY CELLS OPSONIZATION OF PATHOGENS KILLING OF PATHOGENS

7. Antigen-antibody complex MannosePathogen surface C1q, C1r, C1s Serin protease C4, C2 MBL MASP-1/MASP-2 Serin protease C4, C2 C3 B, D COMPLEMENT SYSTEM CLASSICAL PATHWAYMB-LECTIN PATHWAYALTERNATIVE PATHWAY C3 CONVERTASE C4a* C3a, C5a Inflammatory peptid mediators Phagocyte recruitment C3b Opsonization Binding to phagocyte CR Immune complex removal Terminal C5b – C9 MAC Pathogen/cell lysis

8. THE C1 COMPLEX C1 is always present in serum but it can operate on an activating surface in normal case Low affinity binding to the C-terminal of antibody - Multiple interaction with immune complexes Only in classical pathway! Classical pathway Collagen „legs” Gobular „heads”

9. Immunoglobulin Fragments: Structure/Function Relationships antigen binding complement binding site placental transfer binding to Fc receptors C1 component Association between native and adaptive immunity Only the antigen-linked antibodies are able to associate to complement. Why?

10. The classical pathway of complement activation is initiated by binding of C1q to antibody on a bacterial surface

11. Eukariotic cells Glucoseamine Mannose Galactose Neuraminic acid (sialic acid) GLYCOSYLATION OF PROTEINS IS DIFFERENT IN VARIOUS SPECIES Mannose Prokariotic cells

12. MANNAN-BINDING LEKTIN ACTIVATES THE COMPLEMENT SYSTEM

13. THE CENTRAL COMPONENT OF THE COMPLEMENT SYSTEM C3CGEQ One of the proteins present at the highest concentration in serum 1.2mg/ml Is it a lot??? 3 900.000.000.000.000 molecules/ml CLEAVAGE SITE

14. ACTIVATION OF C3 InflammationBinding C3aC3b CGEQ C3CGEQ Active thioester ROH ROH RO CGEQ Bacterium CGEQ ROH ROHROH RO Cell

15. Membrane attack complex (MAC) The membrane attack complex affects to the bacterial cell wall, but complement fragments can be attached to the body’s cell surface also Complement-mediated lysis of the cells is blocked by cell surface and soluble inhibitory factors Certain bacteria can activate the C3 complement component directly (ALTERNATIVE PATHWAY) Complement-mediated lysis of bacteria opsonized by antibodies takes place in the absence of alternative pathway also

16. MAC in the cell membrane live and dead bacteria The membrane-attack complex assembles to generate a pore in the lipid bilayer membrane

18. Homologue components of classical and alternative pathways

19. Complement receptors NameLigandExpression CR1 CD35 C3b>C4b, iC3bRBC, Mo/MØ, Gr, B Act-T, FDC CR2 CD21, CD21L C3d, C3dg, iC3b EBV, IFN , CD23 B, activated T, FDC CR3 CD11b/CD18 iC3b> C3dg, C3d ICAM-1, LPS, fibrinogen Mo/MØ, Gr, NK CR4 CD11c/CD18 iC3b, C3dg, C3d Fibriogen Mo/MØ, Gr, NK C3aR C3aM, B, Gr, Mo/MØ, Trombocites, simaizom, Neur C5aR C5a,, des-Arg-C5aM, B, Mo/MØ, Trombocites, SMC, Neur C1qR C1q collagen partB, NGr, Mo/MØ, endothel C1qRp C1qFagocyte

20. The role of complement system in in vivo Lectin and alternative pathway classical pathway C3 C3b opsonization phagocytosis C3b C5a C4aC3a MAC lysis

21. bacterium complement receptor macrophage OPSONIZATION C3b

22. Local inflammatory responses can be induced by the small complement fragments C3a, C4a, and especially C5a

23. DAF C1Inh Properdin positive feedback Factor I CR1MCP C4bp C-pept.ase N Factor I F act -H CR1MCP DAF CD59 HRF S-protein  -2macrogl LECTIN PATHWAY Regulation of complement system membrane protein soluble molecule

24. C1Inh: C1-inhibitor (serine-protease inhibitor, it can effect in many steps) Factor H: inhibits C3-konvertase of alternative pathway, co-factor of factor I, cleaves C4b and C3b Properdin: ballasts convertases of alternative pathway DAF: Decay Accelerating Factor MCP: Membrane Cofactor Protein CD59: inhibits the linking of C9 and C8 Major regulating factors of complement system

25. Regulation of C3 convertase f= FACTOR

26. One of the major function of C1 INHIBITOR C1q binds to IgM on bacterial surface C1q binds to at least two IgG molecules on bacterial surface Binding of C1q to Ig activates C1r, which cleaves and activates the serine protease C1s C1INH dissociates C1r and C1s from the active C1 complex Other functions are on the Figure 33.

27. Regulatory proteins on human cells protect them from complement-mediated attack

28. CD59 prevents assembly of terminal complement components into a membrane pore

29. Problem of xenotransplantation The ABO blood group antigens, cell surface carbohydrate components of endothel cells and the CD55/DAFand CD59 molecules are genus specific. Xenotransplantation from minipig – the complement regulators on pig cells can not protect from the attack of the recipient complement system. Transgenic (human CD55) animals – lower cytotoxic activity of human serum xenoreactive antibodies against xenotransplanted cells. (Transplant Proc. 2008 Mar;40(2):551-3 ) Cascalho M & Platt JL Nat Rev Immunol 2001

30. Deficiencies of complement system – cascade molecules

31. Deficiencies of complement system – regulatory molecules, receptors

32. Hereditary angioneurotic edema (HANO) (hereditary C1INH defect) 17-year old boy - severe abdominal pain (frequent sharp spasms, vomiting) appendectomia – normal appendix family history of prior illness immunologist’s suspicion: hereditary angioneurotic edema level of C1INH: 16% of the normal mean daily doses of Winstrol (stanozolol) – marked diminution in the frequency and severity of symptoms purified C1INH intravenously – the infusion relieves the symptoms within 25 minutes Main symptoms: swellings of skin, guts, respiratory tracts serious acut abdomenal pain, vomiting larynx swelling – may cause death Treating: IV C1INH kallikrein and bradykinin receptor antagonists

33. Pathogenesis of hereditary angioneurotic edema bradykinin and C2-kinin: enhance the permeability of postcapillar venules by contraction of endothel holes in the venule walls edema formation C1 is always active without activating surface because plasmine is always active Inhibition by C1INH in many steps activation of XII factor activation of kallikrein activation of proactivator cleveage of kininogen to generate bradykinin, vasoactive peptide activation of C1 cleveage of plasminogen to generate plasmin cleveage of C2 to generate C2a cleveage of C2a to generate C2-kinin, vasoactive peptide

34. Questions hereditary angioneurotic edema 1. Activation of complement system results in the release of histamine and chemokines, which normally produce pain, heat and itching. Why is the edema fluid in HANE free of cellular components, and why does the swelling not itch? - In HANE, C4b and C2b both generated free in plasma because plasmine always actives the C1 - There are not an activating surface, so C4b are not able to bind to a surface, so it is rapidly inactivated. The concentration of C4b and C2b are relatively low, no C3/C5 convertase is formed.  C3 and C5 are not cleaved and C3a and C5a are not generated.  After the complement activation histamine do not release which is caused by C3a  Without C5a there are not cell recruitment BUT there are C2a-kinin and bradykinin which cause edema. 2. Which complement component levels will be decreased? Why? C2 and C4, because of the continous cleveage by activated C1.

35. 4. What about the levels of the terminal components? The unregulated activation of the early components does not lead to the formation of the C3/C5 convertase, so the terminal components are not abnormally activated. 5. Despite the complement deficiency in patients with HANE, they are not unduly susceptible to infection. Why not? The alternative pathway of complement activation is intact and these are compensated for by the potent amplification step from the alternative pathway. 6. How might you decide the background of the laryngeal edema (HANO or anaphylactic reaction)? If the laryngeal edema is anaphylactic, it will respond to epinephrine. If it is due to HANO, it will not, C1INH needed. Questions hereditary angioneurotic edema 3. Would you expect the alternative pathway components to be low, normal or elevated? C1 plays no part in the alternative pathway. This pathway is not affected.

36. acqired clonal mutatio nof PIG-A gene – no GPI-enchored proteins in the cell membrane CD59 (upper pic) and CD55 complement regulatory proteins no CD59 and/or CD55: PNH patients are highly susceptible to complement-mediated lysis (lower pic). Eleveted levels of TF derived from complement-damaged leukocytes Treating PNH with a humanized anti- C5 antibody Paroxysmalis nocturnalis hemoglobinuria (PNH)

37. Paroxysmalis nocturnalis hemoglobinuria (PNH) symptoms and therapy haemolytic anaemia (9%) and associated symptoms (35%) haemoglobin and its products in the urine (26%) thrombosis (6%): in brain veins, mesentheric veins, vv. hepaticae (Budd-Chiari- syndrome) transformation to acut lymphoid leukemia (ALL) aplastic anaemia (13%) eculizumab (anti-C5 monoclonal antibody) steroids Iron replacement transfusion bone marrow transplantation