1. Complement S. Barbour Suggested Reading: Janeway, Chapter 2, pp. 61-82 Chapter 9, pp. 406-412 Office hours by arrangement: Please contact me by email (sbarbour@vcu.edu) to schedule an appointment.

2. I. Overview of the Complement System

3. Hallmarks of Complement Sequential Activation Amplification Regulation

4. Overview of Complement Classical PathwayAlternative PathwayLectin Pathway Antibody binds to specific antigen on pathogen surface Mannose-binding protein binds pathogen surface Pathogen surface creates environment conducive to complement activation Complement Activation Formation of C3 and C5 convertases Membrane Attack Pathway Cytolysis of some pathogens Opsonization & phagocytosis of some pathogens Inflammatory responseClearance of Immune complexes Clearance of Apoptotic Cells

5. Complement Nomenclature Note: “i” denotes “inactive” fragments that do not support complement activation. However, these fragments can have other biological functions. (eg. iC3b or C3bi) 1. CX (X = 1-9); example: C5 2. Factor X (alternative pathway); example: Factor B (B) 3. complement fragments (usually a and b); example: C3 → C3a + C3b

6. II. Activation of the Complement System

7. Activation of the Classical Pathway Y Y Y Y Y C1 C3 C3b C3 convertase -Note: only antigen-antibodies complexes can activate the classical pathway! Antibody alone cannot -Classical pathway results in formation of a C3 convertase (C4b2a) that generates C3b -C3b is covalently attached to pathogen; triggers complement activation and biological activity Y Y IgM IgG (IgG1, IgG2, IgG3) Antigen (bacterial or yeast cell surface)

8. Activation of the Lectin Pathway C3 C3b C3 convertase MBL “activating surface” (bacterial or yeast cell surface) mannose sugars mannan binding lectin (MBL) -mannan binding lectin (MBL) recognizes mannose sugars on microbial cells -host mannose is hidden and is not accessible to MBL -lectin pathway results in the formation of a C3 convertase (C4b2a) that generates C3b

9. Activation of the Alternative Pathway (Sources of C3b) Y Y C1 C3 C3b Soluble C3 convertase “activating surface” (bacterial or yeast cell surface) C3 tickover -the alternative pathway is initiated by C3b binding to a bacterial or yeast cell surface -this C3b can come from the classical pathway or “C3 tickover” -C3b preferentially interacts with bacterial or yeast cells; host cells are spared -this is a primitive distinction of self versus non-self C3 convertase C3 C3b classical pathway

10. Activation of the Alternative Pathway (C3 Convertase) C3 C3b C3 C3b Soluble C3 convertase “activating surface” (bacterial or yeast cell surface) C3 tickover -alternative pathway results in the formation of a C3 convertase (C3bBb) that generates C3b -this is the same C3b that is produced by the classical pathway; two C3b molecules in alternative pathway -C3 tickover occurs constantly; regulatory proteins prevent C3b from binding host cells C3 convertase C3b

11. Please note this inconsistency in your text On pages 64-66 and 73, your text says the C3 convertase of the classical/ lectin pathway is C4b2a On page 72 (text and table) it calls the same enzyme C4b2b This discrepancy arises from a change in the nomenclature For the purposes of our discussion, we will call the C3 convertase of the classical/ lectin pathway C4b2a

12. C3 Convertase and C5 Convertase C3 convertases generate C3b C3b forms covalent attachment with pathogen surface Together with other components, attached C3b forms C5 convertase C5 convertase proteolyzes (activates) C5; last step in complement activation

13. II. Regulation of Complement Activation

14. Importance of C3 Activation of Classical, Lectin, and Alternative pathways (alternative pathway is constantly activated in serum!) Fragments have biological activity Opsonization / Phagocytosis B cell activation Inflammation Most abundant complement protein in serum ( > 1 mg/ml)

15. C3 Convertases C2aC4bBbC3b Classical / Lectin PathwayAlternative Pathway catalytic subunit receptor subunit catalytic subunit receptor subunit covalent association with pathogen surface P C3C3a + C3b -C3 convertases consist of two subunits; one with catalytic activity, one that binds C3 -subunits must be associated to have active C3 convertase -regulatory mechanisms are designed to disrupt this association C3 convertase

16. Regulation of C3 Convertases ProteinClassical/ Lectin AlternativeC4bC3b Factor H -+-+ C4bp +-+- DAF ++-- MCP --++ CR1 ++++ Dissociation Proteolysis (Factor I cofactor) In general, regulatory proteins are expressed on host cells, but not on bacteria. Therefore, host cells are spared from complement attack.

17. III. Effecter Functions of Complement

18. The Membrane Attack Pathway The MAC is especially important for the immune response against Neisseria spp. Membrane proteins (CD59, HRF) prevent MAC formation on host cells. MAC

19. Opsonization / Phagocytosis opsonized bacterium -both C3b and iC3b (fragment of C3b) are opsonins -CR3 and CR4 also mediate phagocytosis

20. Inflammation C5a—chemoattraction C3a, C4a----activation C3a, C4a---increased vascular permeability anaphylotoxins

21. Complement Proteins and Inflammation C4a, C3a, and C5a are pro-inflammatory “anaphylotoxins” C5a binds a G protein coupled receptor - neutrophils express high levels of the C5a receptor -C5a binding induces chemotaxis and activation / increased cytocidal activity C3a, C4a bind another G protein coupled receptor -C3a/4a receptor is expressed by phagocytes, endothelial cells -C3a/ C4a binding induces cell activation, increased cytocidal activity, increased vascular permeability

22. CR1 Clearance of Immune Complexes

23. B cell Activation or C3dg -microbial cell expresses antigens recognized by B cell receptor -microbial cell is coated with C3d or C3dg (fragments of C3b) -C3d/ C3dg is recognized by CR2 -simultaneous binding to B cell receptor and CR2 results in efficient B cell activation

24. Clearance of Apoptotic Cells iC3b phagocyte CR3, CR4 -Phagocyte recognizes iC3b deposited on the surface of apoptotic cell -Apoptotic cell is ingested and destroyed by phagocyte - This is an important mechanism for clearing self antigens and preventing autoimmune responses -Uptake of apoptotic cell also induces self tolerance, thereby prevents autoimmune response

25. Overview of Complement Classical PathwayAlternative PathwayLectin Pathway Antibody binds to specific antigen on pathogen surface Mannose-binding protein binds pathogen surface Pathogen surface creates environment conducive to complement activation Membrane Attack Pathway Cytolysis of some pathogens Opsonization & phagocytosis Of some pathogens Clearance of Immune complexes Inflammatory responseComplement Activation Formation of C3 and C5 convertases Clearance of Apoptotic Cells

26. C3d C3dg B cell activation Clearance of apoptotic cells