Selective rejection of porcine islet xenografts by macrophages.

BACKGROUND: Our previous study has shown that porcine antigen-primed and CD4+ T cell-activated macrophages are capable of recognition and rejection of porcine xenografts after adoptive transfer. However, whether this is an absolute xenograft specific rejection remains to be confirmed. METHODS: Mouse islet allografts and neonatal porcine islet cell cluster (NICC) xenografts were admixed and transplanted under the left kidney capsule, and NICC xenografts alone were transplanted under the right kidney capsule of strepotozotocin-induced diabetic NOD-SCID mice. After achievement of normoglycemia, the NOD-SCID recipients were transferred with macrophages purified from NICC transplant NOD-SCID mice reconstituted with CD4+ T cells. Five weeks after macrophage transfer the left kidney with the admixed grafts were removed. Graft survival and function following macrophage transfer was assessed by blood glucose measurement and immunohistochemistry. RESULTS AND CONCLUSIONS: Adoptive transfer with activated macrophages did not affect the normalized blood glucose levels in NOD-SCID recipients of admixed grafts until left nephrectomy 5 weeks post-macrophage transfer. Insulin-positive and porcine C-peptide-negative mouse islets were detected in the admixed grafts. The surviving mouse islets in the admixed grafts were surrounded but not infiltrated by macrophages. The nephrectomized recipients demonstrated sustained hyperglycemia and completely destroyed NICC xenografts in their remaining right kidneys 8 weeks after macrophage transfer. Taken together, these data provide direct evidence of porcine islet xenograft specific rejection by activated macrophages.

Requirement of MyD88 for macrophage-mediated islet xenograft rejection after adoptive transfer.

BACKGROUND: Porcine antigen primed and CD4+ T-cell activated macrophages are able to migrate to and destroy porcine xenografts. However, the specific signaling mechanisms involved remain to be identified. METHODS: In this study macrophages which lack the universal toll-like receptor (TLR) adaptor MyD88 were used to investigate the role of TLR in the recognition and activation of macrophages in islet xenograft rejection. Macrophages were isolated from rejecting MyD88(-/-) and wild-type C57BL/6 mice that were recipients of neonatal porcine pancreatic cell cluster (NPCC) xenografts, and were transferred to NPCC recipient NOD-SCID mice. RESULTS: Both wild-type C57BL/6 and MyD88(-/-) mice rejected NPCC xenografts 8 and 10 days, respectively after transplantation, and the grafts were heavily infiltrated with CD4+ T cells and macrophages. However, graft infiltrating macrophages from rejecting MyD88(-/-) recipients demonstrated impaired up-regulation of TLR expression and impaired activation phenotype, when compared to those from rejecting C57BL/6 recipients. Transfer of NOD-SCID recipients with macrophages from rejecting C57BL/6 mice resulted in NPCC xenograft rejection along with massively infiltrated macrophages 8 days after transfer, whereas NPCC xenografts in NOD-SCID mice transferred with macrophages from rejecting MyD88(-/-) mice remained intact until the end of this study, 90 days after transfer, with insulin-positive islets and no infiltration by macrophages. CONCLUSION: This study demonstrates that deletion of MyD88 causes impaired macrophage activation after pig islet xenotransplantation. However, graft survival is not prolonged and xenografts are rejected rapidly by alternate mechanisms.

Involvement of CCR5 signaling in macrophage recruitment to porcine islet xenografts.

BACKGROUND: Porcine antigen primed and CD4+ T-cell-activated macrophages are capable of both recognition and rejection of porcine xenografts. However, the specific signaling mechanisms involved remains to be addressed. The aim of this study was to examine the role of chemokine receptor and CD40 signaling in macrophage recruitment and graft destruction. METHODS: Macrophages were isolated from rejecting CCR2, CCR5, CD40 and control C57BL/6 mice that were recipients of neonatal porcine pancreatic cell cluster (NPCC) xenografts and were transferred to NPCC recipient NOD-SCID mice. RESULTS: Macrophages isolated from rejecting NPCC xenografts in CD40 and wildtype C57BL/6 mice demonstrated upregulated expression of macrophage activation markers as well as CCR5 and CCR2 genes, and caused pig islet xenograft destruction 8 days after transfer to NOD-SCID recipients. Graft infiltrating macrophages from rejecting CCR2 mice showed a similar activation phenotype and destroyed NPCC xenografts 10 days after transfer to NOD-SCID mice. Blockade of MCP-1 by anti-MCP-1 mAb did not prolong graft survival in CD4+ T cell reconstituted NPCC recipient NOD-SCID mice. By contrast, the graft infiltrating macrophages from rejecting CCR5 recipients showed impaired macrophage activation when compared to control C57BL/6 recipients, and transfer of these macrophages did not result in xenograft destruction in NOD-SCID recipients until day 16 after transfer. Analysis of graft infiltrating macrophages from these rejecting NOD-SCID mice showed an impaired activation phenotype. CONCLUSION: These results demonstrate that CCR5 is involved in both the activation and recruitment of macrophages to rejecting islet xenografts but other pathways are involved.