Immunology 2008 Lecture 7 Complement 14 October

1. Immunology 2008 Lecture 7 Complement 14 October

2. TODAY Complement, Chapter 5, App. 6 Immunoglobulin Genetics, Chap. 6, App. 2,7

4. COMPLEMENT Three Pathways: CLASSICAL/ALTERNATE/MBLectin INFLAMMATION IMMUNE COMPLEXES I.C. DISEASE AUTOIMMUNITY COMPLEMENT FIXATION ASSAY

5. Cardinal Signs of Inflammation CALOR Heat[↑Blood flow, C3a, C5a] RUBOR Redness[↑Blood flow, C3a, C5a] TUMOR Swelling[Accumulation of fluid & cells, C3a, C5a, C5b67] DOLOR Pain[Swelling and tissue damage] …+Loss of function May be initiated by… Infection (bacteria, viruses et al.) Sunburn, allergens… Trauma… Mediated by immune & coagulation systems, etc.

6. Pfeiffer: …in the peritoneal cavity of immune guinea-pigs Cholera vibrios lost their motility, then disappeared completely. Lysis of Vibrio cholera by immune serum Fresh normal serum no lysis Fresh immune serum lysis Heated immune serum no lysis Heated immune serum + fresh normal serum LYSIS Normal serum “complements” the ability of antibodies in immune serum to lyse targets

7. How is complement-mediated damage targeted? Antibodies: Classical (& alternate) pathways Unique structures on microbes: Alternate & MBLectin Pathways

8. 1) Classical Pathway ●Initiated by AgAb complexes ●Protein/protein interactions ●Proteolytic cleavages and conformational changes… ●…which produce new enzymatic and biological activities

9. To bind C1qrs: • Ab must be bound to Ag • Two Fc's in close proximity (CH3) Structure of Mouse IgG2 Harris et al., Nature 360:369-72, 1992

10. “C3 convertase” Classical Pathway C1,4,2,3,5,6,7,8,9

11. specific & non-specific consequences

12. 2) Alternate Pathway ●Initiated by microbial cell walls et al... (& some IC’s)

14. 3) MBLectin Pathway ●Initiated by microbial carbohydrates… Importance of antibody-independent complement fixation (Alternate & MBLectin pathways): What do you do before the fire engine arrives?

15. Biological activities of Complement 1) Cytolysis; disruption of cell membrane ("hemolysis"). 2) Anaphylotoxin activity; ("vasoactive", "phlogistic"), increased capillary permeability, leakage of fluid. 3) Chemotaxis; attraction of PMN's. 4) Opsonization; phagocytosis by macrophages & PMN's. 5) Tissue damage; effects of lytic complex and accumulation of PMN's (Arthus reaction, Immune Complex Disease). heat, redness, swelling, pain, loss of function…

16. Biological Significance of Complement Activity ● Facilitation of Ab function. ● Inflammation - destruction and clearance of foreign material. ● Immune complex disease. ● NormalClearance of immune complexes.

17. IMMUNE COMPLEX DISEASE Circulating, pathological immune complexes may result from: Passive immunization (self-limiting) Drug reactions (penicillin, sulfa...) Autoimmune disease (autoantibodies) Chronic viral infection ●Immune complexes bind to basement membrane of kidney glomeruli and vasculature ●Complement is fixed ●Inflammation and tissue damage ensue

21. Initiated by a single dose of horse anti-diptheria toxin Transient Immune Complex Disease Passive immunization

22. Different Routes to Immune Complex Disease Intravenous: Drugs, anti-toxins & anti-venins Subcutaneous: Vaccination (tetanus) (classic Arthus reaction…) Inhaled: “Farmer’s Lung” (actinomycetes), etc. May also be autoimmune (e.g. SLE, RA)

23. COMPLEMENT DEFICIENCIES C5-9(MAC): Limitedsusceptibility to infections (!) P, B (Alternate pathway): Severe infections MBL: Mainly childhood infections C1,4: Increased risk of developing autoimmune disease Active in soluble phase, important in normal clearance of trace amounts of immune complexes. C4b receptors on RBC transport ICs to liver where they are removed.

25. Complement Fixation Assay Nurse: “Doctor, there’s an invisible man in the waiting room.” Doctor: “Tell him I can’t see him.” We want to be able to detect Abs by their ability to fix complement… …but like Ab/Ag binding, complement binding is invisible.

26. * *EA - visible indicator of the presence of complement Complement Fixation Assay  Standard Ab  Standard Complement "Sensitized" erythrocytes - EA Test unknown samples for antigen

27. 1:2 4 8 16 32 64 128 fresh guinea pig serum Titration of complement on sensitized RBCs

28. Ab/Ag + C Ab/Ag/C Presence of antigen is made visible by inhibition of lysis. Basis for Complement Fixation Assay Ab + C + EA Lysis Ab + Ag + C + EA No Lysis

29. Complement Fixation Assay: Example Tube No. Sample Result Interpretation 1) (saline alone + C) + EA lysis neg control 2) (Ab + saline + C) + EA lysis neg control 3) (Ab + VA + C) + EA no lysis pos control 4) (Ab + unk No. 1 + C) + EA lysis no VA present 5) (Ab + unk No. 2 + C) + EA no lysis VA present 6) ( unk No. 2 + C) + EA lysis neg control, OK ------------------------------------------------------------ 7) (Ab + unk No. 3 + C) + EA no lysis VA present?? 8) ( unk No. 3 + C) + EA no lysis anti-complement activity Core Notes, Appendix 6, p.174

31. Ag/Ab complexes are formed by Ab binding to… Antigen on a cell membrane: ● RBC – agglutination, hemolysis ● bacteria – opsonization/bacteriolysis Soluble Ag (e.g. in serum): ● small complexes (soluble) are removed via CR on RBCs ● larger complexes (precipitated) can be trapped in the vasculature, causing serum sickness

32. Fin