1. Humoral Immune Effector Mechanisms

2. Ig of Different Isotypes How do the functional differences impact the immune response ? The focus will be on Ig and its interactions with immune effector systems and receptors

3. Complement - an important effector system has important functions related to immune defense 1.Lysis of cells. This is the original function identified and causes hypotonic cell death by making hole. It is not effective against organisms with cell walls such as fungi and Gram positive bacteria

4. Complement - an important effector system has important functions related to immune defense 2.Opsonization. Macrophage and PMNs have FcRs and at least two different kinds of complement receptors that aid in phagocytosis. C3b, a cleavage product formed during activation is the major player. Antigen coated with C3b binds to cells bearing complement receptors and if the cell is a phagocyte the antigen will be phagocytosed.

5. Complement - an important effector system has important functions related to immune defense 3.Inflammation. Peptides generated during activation play a role in inflammation. The anaphylatoxins of which C5a is the most potent bind receptors on mast cells and basophils and cause degranulation with the release of pharmacologically active mediators which induce smooth- muscle contraction and increases in vascular permeability. C3a, C5a and C5b67 act as chemoattractants and induce monocytes and neutrophils to adhere to vascular endothelial cells, extravasate through the endothelial lining of the capillaries and migrate to the site of complement activation in the tissue.

6. Complement - an important effector system has important functions related to immune defense 4. Immune clearance. Removes immune complexes from the circulation and deposits them in the liver where they are degraded. C3b facilitates immune complex binding to CR1 on RBCs. In the liver and spleen the complexes are stripped from the RBC and phagocytosed. Complement also helps to solubilize immune complexes.

7. Complement - an important effector system has important functions related to immune defense 5.Enhanced immune response. CD21, part of the co- receptor on the B cell, binds cleaved C3. Recently it has also been shown that C3 is required for optimal expansion of T cells during a systemic viral infection.

8. Complement - an important effector system has important functions related to immune defense 6.Virus neutralization. Complement mediates viral neutralization by facilitating viral aggregation and by coating the viral surface.

10. Classical pathway Requires Ab Mannose binding lectin pathway Alternative pathway

12. IgM The first isotype made during the immune response Exists in pentameric/hexameric forms One consequence of polymerization is higher avidity The higher avidity helps to compensate in part for the low affinity of IgM made early in the immune response before somatic mutation has taken place. Also each molecule has ten/twelve C1q binding sites In contrast to IgM, complement activation by IgG requires aggregation. Monomeric IgG has an exposed C1q binding site but it is of low affinity; adjacent sites are required in order to achieve an interaction of sufficient avidity to trigger the cascade. IgG

13. IgM is a very rigid molecule Binding of Ag by IgM leads to a conformational change exposing the C1q binding sites. There are multiple sites in each IgM molecule so one IgM can bind C1q and activate the complement cascade

14. C1 C1 q binding siteC1 q binding site

16. MBL - resembles C1q in structure except specificity is for carbohydrate

18. C3a and C5a are anaphylatoxins C3a - 77 amino acid peptide that causes smooth muscle contraction, increases vascular permeability and mast cell and basophil degranulation C5a- a77 amino acid peptide is also a chemoattractant and activator of WBC

19. C3a and C5a are anaphylatoxins Also amplify the inflammatory response by inducing the synthesis of pro-inflammatory cytokines. Their receptors are present on many cell types including leukocytes, mast cells, macrophages, endothelial cells, astrocytes and microglial cells

20. C3a and C5a are anaphylatoxins Anaphylatoxin triggered cascade of events that contribute to the pathogenesis of a number of disease and conditions including hypersensitivity reactions, endotoxin shock, multiples organ failure and respiratory distress syndrome. Neutralization of the effects of anaphylatoxin is therefore of substantial clinical significance

21. C3a and C5a are anaphylatoxins Neutralization of the effects of anaphylatoxin is therefore of substantial clinical significance It has now been shown that C3a and C5a bind to immunoglobulin molecules through the constant region of the Fab. (Nature Medicine, published online March 3, 2003).

22. Intravenous immunoglobulin (IVIG) and F(ab)’ 2 prepared from it inhibit C3a and C5a induced histamine release

23. Binding is to F(ab)’ 2 and not Fc and is not inhibited by binding to Ag

24. Cleavage products of C3b are opsonins

26. Complement receptors (CD21 = CR2) potentiate the immune response

28. Fc  receptors are important humoral effector molecules

34. FcRs function for the phagocytosis of Ab coated particles by macrophages and killing by NK cells.

35. Phagocytic Index (PI) = (percentage of cells containing at least on particle)X(mean number of particles per positive cell)

36. For the Arthus reaction Ab is injected into the skin followed by intravenous Ag It had been thought to be complement mediated but it can be virtually absent in  chain knock-out mice However, there are strain differences and some strains show more complement dependence than others

39. In this experiment the mice were treated with suboptimal amounts of antibody

40. Activated macrophages treated with IFN-  and LPS are biased to IFN-  production when stimulated by Ag alone, but produce IL-4 when stimulated by immune complexes. This bias is stable and is seen when CD4 + cells were stimulated 7 days later with OVA alone. Intracellular cytokine expression reflects what is seen in the secretions (J. Immunol 168:3701) Immune complexes can determine whether macrophages bias to TH1 or TH2

41. Immune complexes facilitate DC priming of CTLs and induce DC maturation in the absence of T cell help J. Immunol. 168:2240-2246, 2002

42. There is increased binding and uptake of Ag present in ICs J. Immunol. 168:2240-2246, 2002

43. FcRn plays a major role in determining serum persistence

48. CD89 - Fc  RI receptor for IgA Found on cells of the myeloid lineage including neutrophils, eosinophils, most monocytes, interstitial dendritic cells, and Kuppfer cells. Can be expressed associated with FcR .

49. Uptake of FITC-labeled E. coli opsonized with IgA into Kupffer cells of transgenic mice expressing CD89

50. When the CD89 is occupied, it inhibits the phagocytosis of E. coli opsonized with IgG Anti-CD89 Fab + opsonized E. coli Buffer + Unopsonized E. coli Buffer + opsonized E. coli Opsonized E. coli + irrelevant Fab In this case an anti-CD89 was used in place of IgA to mimic its effects

51. A rat mast cell line was engineered to express Fc  RI Addition of IgA but not of IgG inhibited IgE mediated granule release. However, extensive crosslinking with F(ab’)2 and rabbit anti-mouse resulted in granule release. Therefore a model emerges of FcaRI acting as an inhibitory receptor at low occupany, and as a inflammatory receptor when extensively crosslinked. Both activities required FcR  with a functional ITAM.