AlloantibodyDependent Pathways in Allograft Rejection

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Alloantibody-Mediated Rejection Complement and
non-Complement Dependent Mechanisms
B.A. Wasowska
JHMI
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Specific recognition of MHC Antibodies
Complement independent
Macrophage
cytokines
FcR
Endothelial cells
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Specific recognition of MHC Antibodies
Complement dependent
Complement independent
cytokines
Macrophage
Macrophage
cytokines
CRs
FcR
C4
C3
Endothelial cells
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Experimental Design
Heart donors Recipients Incompatibilities
B10.A C57BL/6 H-2, H-9 IgKO
(m-/m-) passively transferred with mAbs C
activation IgG2b yes IgG1 no IgG2bIgG1 yes

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Passive Transfer of IgG1 and IgG2b to IgKO
Recipients of Cardiac Allografts
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Passive Transfer of IgG1 and IgG2b to IgKO
Recipients of Cardiac Allografts
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Pro-Inflammatory Effects of anti-MHC Antibodies
in the Absence of Complement
Anti-MHC Ab
Endothelial Cells
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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).

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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
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Cytokine Array on Culture Supernatants
Endothelial Cells Stimulated by IgG1 IgG2b
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What Effects Do Antibodies Have on Endothelial
Cells and Leukocytes in the Absence of Complement?
?
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Cytokine Array on Culture Supernatants
Endothelial Cells Stimulated by IgG1 IgG2b
Peritoneal and Endothelial Cells Stimulated by
IgG1 IgG2b
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ELISA for MCP-1 and IL-6 in Cultures of
Endothelial Cells Peritoneal Cells
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ELISA for MCP-1 and IL-6 in Cultures of
Endothelial Cells Alloantigen-primed SPL from
Heart Recipient
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What Effects Do Antibodies Have on FcgR Cells
in the Absence of Complement ?
Macrophages
?
FcgR
?
Endothelial Cells
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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 transplant were
co-cultured with mouse endothelial cells in the
presence of IgG1 Ab-s.
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The Affinity of Mouse FcgRs to Bind IgG Subclasses
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ELISA for IL-6 in Cultures of Endothelial Cells
IgG1 Lymph Node Cells from WT and FcgRIII KO
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ELISA for MCP-1 in Cultures of Endothelial Cells
IgG1 Lymph Node Cells from WT and FcgRIII KO
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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.

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What Effects Do Antibodies Have on Human
Endothelial Cells and Human FcgRI Cells?
U 937
Anti-MHC Ab W6/32 (IgG2a)
?
FcgRI
BMEC
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The affinity of human FcgRs to bind mouse IgG
subclasses
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Expression of CD 64 (FcgRI) on U-937 Macrophages
after Stimulation with IFN-g
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ELISA for MCP-1 and IL-6 in Cultures of Human
BMEC anti-HLA Ab (IgG2a) IFN-g-Stimulated
U-937 Macrophages
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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.

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Specific recognition of MHC Antibodies
Complement independent
Macrophage
cytokines
FcR
Endothelial cells
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Specific recognition of MHC Antibodies
Complement dependent
Complement independent
cytokines
Macrophages
Macrophages
cytokines
CRs
FcR
C4
C3
Endothelial cells
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Complement Receptors for C4 and C3
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Possible Interactions of iC3b and C3b with CR3 in
IgG1IgG2b-Mediated Cell Activation in the
Presence of Complement
WT and CR3 KO C57BL/6 peritoneal macrophages were
co-cultured with mouse endothelial cells in the
presence of IgG1Ig2b mAb-s Complement.
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ELISA for MCP-1 and IL-6 in Cultures of
Endothelial Cells IgG1IgG2b C Macrophages
from WT and CR3 KO
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Other Complement Receptors
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Complement and non-Complement Dependent
Mechanisms Contribute to Alloantibody-Mediated
Activation of Endothelial Cells and Macrophages
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ACKNOWLEDGEMENTS

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