1. THE COMPLEMENT SYSTEM Sections from chapter 2 and 11 in Parham’s book

2. The complement system is a set of about 30 soluble proteins, constitutively produced by the liver, that are found in the blood, lymph and extracellular fluids, and act against extracellular pathogens. Complement activation proceeds by a cascade of enzymatic reactions (proteases), in which each protease cleaves and activates the next enzyme in the pathway. Complement system

4. THE CENTRAL COMPONENT OF THE COMPLEMENT SYSTEM C3CGEQ One of the proteins present at the highest concentration in serum 1.2mg/ml CLEAVAGE SITE (3 900 000 000 000 000 molecules/ml)

5. Inflammation C3aC3b CGEQC3CGEQ ROH Bacterium CGEQROH RO ROH ROCGEQROHROH Cell Binding

6. THE CENTRAL COMPONENT OF THE COMPLEMENT SYSTEM

7. Complement fixation- Covalent binding of C3b to the pathogens’ surface Covalent binding of C3b to the pathogens’ surface

9. The alternative pathway The alternative soluble C3 convertase

10. The alternative C3 convertase  C3bBb

11. AMPLIFICATION OF THE COMPLEMENT CASCADE inactive precursors limited proteolysis activating surface enzyme

12. Regulation of the complement system Positive regulation Negative regulation (Inhibits both alternative and classical convertases)

13. Negative regulatory proteins on human cells protecting them from complement-mediated attack MCP binds to sialic acid on the surface of human cells and prevents the complement activation DAF and MCP

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

16. The classical pathway Collagen „legs” Gobular „heads” THE C1 COMPLEX C1qR binding by Phagocytes Cleavage of C4 and C2 components Binding the Fc part of an antibody

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

18. Low affinity binding to the C-terminal of an antibody Multiple interactions with immune complexes

19. The classical C3 convertase  C4bC2a

20. CRP binds to phosphocholine component of the lipopolisaccharides in bacterial and fungal cell wall but not to phosphocholine component of phospholipids on human cell membranes!

21. ACUTE-PHASE RESPONSE INCREASES THE SUPPLY OF INNATE IMMUNITY MOLECULES  C-reactive protein CRP levels can increase up to 1000-fold during an acute-phase response!

22. 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

24. The Mannose-binding Lectin pathway Binds Mannose-containing carbohydrates of bacteria, fungi, protozoans and viruses Similar to C1q protein in triggering a complement cascade MASP-1 and 2 have common gene ancestors with C1r and C1s A member of the Collectin family

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

26. ACUTE-PHASE RESPONSE INCREASES THE SUPPLY OF INNATE IMMUNITY MOLECULES  Mannose-binding lectin MBL levels can increase up to 1000-fold during an acute-phase response! * SP-A and SP-D belong to the collectin family as well, opsonyzing pathogens in the lung

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

30. Bacterium complement receptors macrophage Opsonization C3b Ex:CR1, CR3, CR4

31. 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, Trombocytes, SMC, Neur C1qR C1q collagen partB, NGr, Mo/MØ, endothel C1qRp C1qPhagocytes

33. Membrane attack complex (MAC) C3bBbC3b = alternative C5 convertase Or C4bC2aC3 = classical C5 convertase

34. The membrane-attack complex assembles to generate a pore in the lipid bilayer membrane

35. MAC in the cell membrane

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

38. Diseases caused by deficiencies in the complement pathways Complement protein Effects of deficiency C1, C2, C4Immune-complex diseases C3Susceptibility to a wide range of pyogenic infections C5-C9Susceptibility to Neisseria Factor D, ProperdinSusceptibility to capsulated bacteria and Neisseria but no Immune-complex disease Factor ISimilar to C3 deficiency DAF, CD59Autoimmune-like conditions including paroxysmal nocturnal hemoglobinuria (PNH) C1INHHereditery angioneurotic edema (HANE)

39. Immune complex diseases Early components of the classical pathway (C1-C4) are necessary for the elimination of immune complexes! Attachment of the complement components to the soluble immune complexes allows them to be transported, or ingested and degraded by CR-bearing cells. Deficiencies in these components lead to the accumulation of immune complexes in the blood, lymph and extracellular fluid and their deposition in tissues. Damage is caused by the deposition itself and by the activation of phagocytes causing inflammation. These may include: Pyogenic infections Systemic Lupus Erythematosus  Vasculitis Glomerulonephritis

40. Paroxysmal Nocturnal Hemoglobinuria (PNH) Acqired clonal mutation of PIG-A gene  no GPI enchor proteins on RBCs  No expression of the complement regulatory proteins CD59 and DAF on these RBCs  episodes of complement-mediated RBCs lysis  hemolytic anemia Symptoms include:Anemia (tiredness, shortness of breath, palpitations) Hemoglobin in the urine 40% develope thrombosis Therapy include: Anti-C5-Mab, transfusion, immunosuppression and BM transplantation.

41. Hereditary Angioneuretic Adema (HANE) Deficiency in C1INH complement regulatory protein. The C1INH is a serine protease inhibitor that regulates the C1 complex and complement activation as well as inhibiting proteins in the coagulation cascade. Symptoms include: swellings of skin, gut and respiratory tracts serious acute abdomenal pain, vomiting Therapy include: C1INH from donor blood, Androgens and other bradykinin inactivators

42. Supplementary materials

43. C1Inh: C1-inhibitor (serine-protease inhibitor, can affect in many steps) Factor H: inhibits C3-convertase of alternative pathway, co-factor of factor I, cleaves C4b and C3b Properdin: stabilizes 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

44. 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

45. 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, Trombocytes, SMC, Neur C1qR C1q collagen partB, NGr, Mo/MØ, endothel C1qRp C1qPhagocytes

46. 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

47. Deficiencies of complement system – cascade molecules

48. Deficiencies of complement system – regulatory molecules, receptors

49. Hereditary angioneurotic edema (HANE) (hereditary C1INH defect) 17-year old boy - severe abdominal pain (frequent sharp spasms, vomiting) appendectomia  normal appendix similar symptoms occured repeatedly earlier in his life with watery diarrhea 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 became avaible by the time Main symptoms: swellings of skin, guts, respiratory tracts serious acute abdomenal pain, vomiting larynx swelling – may cause death Treatment: iv C1INH kallikrein and bradykinin receptor antagonists Child with symptomes of HANE

50. 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

51. 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 is no 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 cleavage by activated C1.

52. 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.

53. Acqired clonal mutation of PIG-A gene – no GPI-enchored proteins in the 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 pics). Eleveted levels of TF derived from complement-damaged leukocytes Paroxysmal nocturnal hemoglobinuria (PNH)

54. Paroxysmal nocturnal hemoglobinuria (PNH) symptoms and therapy Haemolytic anaemia and associated symptoms Haemoglobin and its products in the urine Thrombosis: in brain veins, mesentheric veins, vv. hepaticae (Budd-Chiari- syndrome) transformation to acut myelogenous leukemia (AML), aplastic anaemia, myelodisplastic syndrome (MDS) Specific th.: eculizumab (Soliris - anti-C5 monoclonal antibody) Curative th.: bone marrow transplantation Alternative th.: steroids (general immunosuppression) Anticoagulants: sc. heparin  p.o. kumarin Iron replacement Transfusion (filtered-irradiated blood)