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

2. Specific Specific recognition of MHCAntibodies Complementindependent FcR cytokines Endothelial cells Macrophage

3. Specific Specific recognition of MHCAntibodies Complementdependent Complementindependent FcR CRs cytokines Endothelial cells C4 C3 Macrophage Macrophage

4. Heart donors Recipients Incompatibilities B10.A C57BL/6 H-2, H-9 IgKO (  -/  -) passively transferred with: mAbsC activation IgG2byes IgG1 no IgG2b+IgG1yes Experimental Design

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

6. mAb

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

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

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

11. Endothelial Cells Macrophages Anti-MHC Ab MHC 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-primed SPL from Heart Recipient

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

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

17. Receptor The affinity of binding Fc  RI IgG2a 10 7 -10 8 M -1 for IgG2a>>IgG1>IgG2b Fc  RIII IgG1 Ig2a>IgG2b The Affinity of Mouse Fc  Rs to Bind IgG Subclasses

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

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

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

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

22. Receptor The affinity of binding Fc  RI IgG2a 10 8 -10 9 M -1 for IgG2a=IgG3>>>IgG1, IgG2b Fc  RIII IgG2a 10 7 M -1 for IgG3>IgG2a> IgG2b >> IgG1 The affinity of human Fc  Rs to bind mouse IgG subclasses

23. Expression of CD 64 (Fc  RI) on U-937 Macrophages after Stimulation with IFN- 

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

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

26. Specific Specific recognition of MHCAntibodies Complementindependent FcR cytokines Endothelial cells Macrophage

27. Specific Specific recognition of MHCAntibodies Complementdependent Complementindependent FcR CRs Macrophages cytokines Endothelial cells C4 C3 Macrophages

28. Complement Receptors for C4 and C3 ReceptorLigands Cell expression CR1C3b>C4b>iC3b Monocytes, Macrophages, PMN, B, T cells, RBC, FDC CR2C3d/C3dg>iC3b B cells, FDC CR3 (Mac-1) iC3b>C3b, ICAM Monocytes, Macrophages, PMN, NK cells

29. Possible Interactions of iC3b and C3b with CR3 in IgG1+IgG2b-Mediated Cell Activation in the Presence of Complement CR3 KO +Complement. WT and CR3 KO C57BL/6 peritoneal macrophages were 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 ReceptorLigands Cell expression CR1C3b>C4b>iC3b Monocytes, Macrophages, PMN, B, T cells, RBC, FDC CR3 (Mac-1) iC3b>C3b, ICAM Monocytes, Macrophages, PMN, NK cells C5aRC5a Monocyte, Macrophage. T cells, Endothelial Cells C3aRC3a

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

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