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Alloantibody-Mediated Rejection: Complement and non-Complement Dependent Mechanisms

Alloantibody-Mediated Rejection: Complement and non-Complement Dependent Mechanisms. B.A. Wasowska. JHMI. Specific recognition of MHC Antibodies. Complement independent. Macrophage. cytokines. FcR. Endothelial cells. Specific recognition of MHC Antibodies. Complement dependent.

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Alloantibody-Mediated Rejection: Complement and non-Complement Dependent Mechanisms

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  1. Alloantibody-Mediated Rejection: Complement and non-Complement Dependent Mechanisms B.A. Wasowska JHMI

  2. Specific recognition of MHC Antibodies Complement independent Macrophage cytokines FcR Endothelial cells

  3. Specific recognition of MHC Antibodies Complement dependent Complement independent cytokines Macrophage Macrophage cytokines CRs FcR C4 C3 Endothelial cells

  4. Experimental Design Heart donors Recipients Incompatibilities B10.A C57BL/6 H-2, H-9 IgKO (m-/m-) passively transferred with: mAbsC activation IgG2b yes IgG1 no IgG2b+IgG1 yes

  5. Passive Transfer of IgG1 and IgG2b to IgKO Recipients of Cardiac Allografts

  6. mAb Passive Transfer of IgG1 and IgG2b to IgKO Recipients of Cardiac Allografts

  7. Pro-Inflammatory Effects of anti-MHC Antibodies in the Absence of Complement Anti-MHC Ab Endothelial Cells

  8. Contribution of Anti-MHC antibodies to graft rejection and activation of EC Ligation of MHC class I molecules with anti-MHC antibodies on EC and smooth muscle results in: • Increased phosphorylation of intracellualr proteins and induction of fibroblast growth factor receptor on EC. (Bian, Reed. JI, 1999, 163: 1010). • Increased phosphorylation of Src, Paxillin and Focal Adhesion Kinase and enhanced proliferation to fibroblast growth factor. (Jin,…Reed. JI, 2002, 168: 5415). • Activation of P13K/Akt signaling pathway and regulation of apoptotic genes (Bad, Bcl-2 and Bcl-xL). (Jin, Fishbein,...Reed, Human Immunol. 2004, 65: 291).

  9. Experimental Design:EC Activation in vitro Methods: Addition of different IgG subclasses to mouse endothelial cell line SVEC4-10 Measurement: cytokine production by EC in 48 hr cultures

  10. Cytokine Array on Culture Supernatants Endothelial Cells Stimulated by IgG1 + IgG2b

  11. Macrophages Anti-MHC Ab MHC Endothelial Cells What Effects Do Antibodies Have on Endothelial Cells and Leukocytes in the Absence of Complement? ?

  12. Cytokine Array on Culture Supernatants Endothelial Cells Stimulated by IgG1 + IgG2b Peritoneal and Endothelial Cells Stimulated by IgG1 + IgG2b

  13. ELISA for MCP-1 and IL-6 in Cultures of Endothelial Cells+ Peritoneal Cells

  14. ELISA for MCP-1 and IL-6 in Cultures of Endothelial Cells + Alloantigen-primedSPL from Heart Recipient

  15. What Effects Do Antibodies Have on FcgR+ Cells in the Absence of Complement ? Macrophages ? FcgR ? Endothelial Cells

  16. Possible Involvement of FcgR III in IgG1-Mediated Cell Activation in the Absence of Complement WT and FcgR III KO C57BL/6 splenocytes and lymph node lymphocytes primed in vivo by 10-day exposure to B10.A skin transplantwere co-cultured with mouse endothelial cells inthe presence of IgG1 Ab-s.

  17. The Affinity of Mouse FcgRsto Bind IgG Subclasses

  18. ELISA for IL-6 in Cultures of Endothelial Cells + IgG1 + Lymph Node Cells from WT and FcgRIII KO

  19. ELISA for MCP-1 in Cultures of Endothelial Cells + IgG1 + Lymph Node Cells from WT and FcgRIII KO

  20. SUMMARY • There is effective interaction between mouse endothelial cells, alloantibodies and alloantigen-primed effector cells. • FcgR III participates in IgG1-mediated activation of mouse effector cells.

  21. MHC What Effects Do Antibodies Have on Human Endothelial Cells and HumanFcgRI+Cells? U 937 Anti-MHC Ab W6/32 (IgG2a) ? FcgRI BMEC

  22. The affinity of human FcgRs to bind mouse IgG subclasses

  23. Expression of CD 64 (FcgRI) on U-937 Macrophages after Stimulation with IFN-g

  24. ELISA for MCP-1 and IL-6 in Cultures of Human BMEC + anti-HLA Ab (IgG2a) + IFN-g-Stimulated U-937 Macrophages

  25. SUMMARY • Two lines of evidence, in mouse and human models of EC sensitized with anti-MHC antibodies, suggest that: • FcgR participate in IgG-mediated activation of EC.

  26. Specific recognition of MHC Antibodies Complement independent Macrophage cytokines FcR Endothelial cells

  27. Specific recognition of MHC Antibodies Complement dependent Complement independent cytokines Macrophages Macrophages cytokines CRs FcR C4 C3 Endothelial cells

  28. Complement Receptors for C4 and C3

  29. Possible Interactions of iC3b and C3b with CR3 in IgG1+IgG2b-Mediated Cell Activation in the Presence of Complement WT and CR3 KO C57BL/6 peritoneal macrophageswere co-cultured with mouse endothelial cells in the presence of IgG1+Ig2b mAb-s+Complement.

  30. ELISA for MCP-1 and IL-6 in Cultures of Endothelial Cells + IgG1+IgG2b +C+ Macrophages from WT and CR3 KO

  31. Other Complement Receptors

  32. Complement and non-Complement Dependent Mechanisms Contribute to Alloantibody-Mediated Activation of Endothelial Cells and Macrophages

  33. Zhiping Qian, M.D.Wink Baldwin, M.D., Ph.D. Salma RahimiMillie Samaniego, M.D. Mike Coonfield Justin Garyu Jinhuan Liu Karen Fox Miriam Reynolds ACKNOWLEDGEMENTS

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