Complement

1. Complement

2. Objectives • Discuss the role of complement in the immune system. • Discuss complement regarding its: • Components • Activation pathways • Biological activities

3. Complement • System comprised of more than 25 glycoproteins • Make up about 10% of total serum proteins • Components formed mainly in the liver • C1 forms in intestinal epithelial cells • Factor D forms in adipose tissues • Components form a cascade, with each step triggering (and often amplifying) the next step

4. Complement • In plasma – the components are in inactive form • Once activated, each component splits • The smaller “a” fragment serves to stimulate the immune system • The larger “b” fragment further activates the cascade • The exception to this is C2… C2a is the larger molecule that promotes the cascade • Activated components are written with a line over the letter or number. For example: ____ • C4b2a

5. 3 Functions • Cell lysis – Cell swells and bursts • Opsonization - Neutrophils and macrophages have receptors for C4b or C3b, which promotes phagocytosis

6. 3 Functions 3. Regulate immune and inflammatory response • Immune adherence – enhanced response to an antigen. Receptors for complement found on Red Blood Cells (RBCs), platelets, B lymphocytes, endothelial and epithelial cells • Anaphylatoxins - chemicals that increase vascular permeability, contract smooth muscle, and cause the release of histamine from basophils and mast cells • C3a, C4a, and C5a are anaphylatoxins • Chemotaxins – signal leukocytes to migrate to an affected area • C5a is also a chemotaxin • C5b67 promotes monocyte and neutrophil adherence to blood vessel endothelium, and extravasation

7. 3 Activation Pathways • Classical • Alternative • Lectin

8. Classical Pathway • Begins with antibody sensitization of antigen on cell • 2 Fc pieces in close proximity • IgM better than IgG (1 IgM vs. 800 IgG) • IgG1 & IgG3 better at activation than IgG2 or IgG4 • C1 = Recognition unit • C1q • C1r • C1s Y C1 Y

9. Recognition Unit Y • C1q combines with the Fc piece • 6 globular heads attached to collagen-like tails. • C1r that acts as a protease on C1s • C1r and C1s intertwine with the tails. • 2 C1q heads must interact with Fc pieces for complement to be activated. Y

10. Classical - Activation Unit C4a C4 C2 C4b2a Y C1 C1s Y C2b • C4, C2, and C3 components participate • Amplification of cascade • 1 molecule of C1s activates approximately 30 C4 molecules.

11. Activation Unit • C2 is active only if it binds to C4b before being cleaved by C1s • This reaction is enhanced if C4b binds to the antigen rather than being free in serum • Once C4b2a is formed, antibody is no longer necessary to ensure cell lysis

12. Activation Unit C4a C3a C4 C2 C4b2a C3 Y C1 C1s Y C3b C4b2a3b C2b • C4b2a = C3 convertase • One C4b2a complex converts about 200 C3 molecules • Requires Mg+

13. Activation Unit • C3 most abundant complement component • C3 common to all pathways • C3b on a cell enhances opsonization • C3b combines with C4b2a to form C4b2a3b = C5 convertase

14. Classical – MAC C4a C3a C5a C4 C2 C9 C8 C7 C6 C4b2a C3 C1s Y C5 C1 C3b C4b2a3b Y C2b C5b • Membrane Attack Complex = C5, C6, C7, C8, C9

15. MAC • C5b binds to C6 and C7 in serum • This complex will bind to any nearby cell membrane, not just the cell that originally triggered the cascade. (The “innocent by-stander” effect) • May also form a micelle – free floating sphere- which has antiviral properties • C5b678 form a surface on the cell membrane for polymerization of C9 • A pore forms in the cell, allowing an influx of water • The cell swells and lyses

16. Hemolysin • An antibody that can activate complement, resulting in lysis of an RBC is termed a hemolysin. C1 Y Y

17. Alternative (Properdin) Pathway Properdin C5a C5 C3b C3bBb C3bBbP C9 C8 C7 C6 Factor B Factor D C5b • Activated by bacteria, fungus, yeast, viruses, parasites, and tumor cells • Oldest pathway • Relies on the natural splitting of C3 into C3a and C3b (exact process unclear)

18. Alternative • C3b and Factor B need Mg+ to combine • Factor D splits Factor B that has been bound to C3b • Properdin stabilizes the C3bBb complex • C3bBbP is a C5 convertase • Once C5b formed, cascade continues as in the classical pathway

19. Lectin Pathway • Mannose is a sugar commonly found in bacterial cell walls • This pathway can be activated by bacteria, yeast, viruses and protozoa C4a C3a C4 C2 MBL C4b2a C3 C5a C2b C9 C8 C7 C6 C3b C4b2a3b C5 C5b

20. Lectin Pathway • MBL= Mannose Binding Lectin equivalent to C1q of classical pathway • MBL also increases opsonization • MASP-1 and MASP-2 function similarly to C1r and C1s respectively • MASP = Mannose Associated Serine Proteases • Once MBL activates the cascade, the cascade continues as in the classical pathway

21. Inhibitors of Complement • Activated enzymes decay quickly. • Half life of activated components ranges from a fraction of a second to approximately 30 minutes. • If Ca+ and Mg+ are not present, the cascade can NOT be activated. • Carboxypeptidase N inactivates anaphylatoxins. • Heating serum at 56oC for 30 minutes destroys complement components.

22. Specific Inhibitors • C1-INH irreversibly binds to the active sites of C1r and C1s. • Factor I degrades C4b and C3b • Membrane cofactor protein –cofactor for Factor I • C4-binding protein – prevents C4 and C2 from joining (cofactor for Factor I)

23. Specific Inhibitors • Complement Receptor 1 (CR1) • Found as a surface protein on most WBCs and follicular dendritic cells • Binds C3b allowing degradation by Factor I • Increases clearance of C3b coated cells via macrophages of liver and spleen • Also increases the length of time antigen remains near germinal centers of lymph nodes (May assist with B cell differentiation)

24. Specific Inhibitors • Sialic acid of cell membrane inactivates C3b bound to cells • Decay Accelerating Factor (DAF) – dissociates C2 from C3 • S protein – prevents C5b67 complex from binding to cell • MIRL – Binds to C8 • MIRL = Membrane Inhibitor of Reactive Lysis • Protectin – Prevents binding of C5b678 to cell, so no polymerization of C9 is allowed

25. Inhibitors - Alternative Properdin C5a C5 C3b C3bBb C3bBbP C9 C8 C7 C6 Factor B C5b • DAF & Factor H – compete with Factor B for binding with C3b

26. Complement Deficiencies • Complement deficiencies rare • C2 deficiency most common (1 in 10,000) • C3 deficiency most problematic as it participates in all pathways • Deficiencies in complement components can lead to: • Increased susceptibility to infection • Accumulation of immune complexes • Autoimmune disease

27. Pathologic Conditions • Complement is harmful if: • Activated systemically • Activated by tissue necrosis • Allows build up of immune complexes • Immune complexes play a role in Goodpasture’s syndrome, SLE, MS, Guillain-Barre’ syndrome and other autoimmune diseases

28. Pathologic Conditions • Paroxysmal Nocturnal Hemoglobinuria (PNH) • Abnormal DAF on RBCs • Cells more susceptible to lysis • C1-INH deficiency – Hereditary angioedema • C2b accumulates, but does not participate in the complement cascade • Increases vascular permeability

29. The End Can you list the complement components in order of activation?