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

Specific Specific recognition of MHCAntibodies Complementindependent FcR cytokines Endothelial cells Macrophage

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

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

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

mAb

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

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).

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

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

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

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

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

ELISA for MCP-1 and IL-6 in Cultures of Endothelial Cells + Alloantigen-primed SPL from Heart Recipient

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

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.

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

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

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

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

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

Receptor The affinity of binding Fc  RI IgG2a 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

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

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

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.

Specific Specific recognition of MHCAntibodies Complementindependent FcR cytokines Endothelial cells Macrophage

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

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

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.

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

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

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

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