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Over-expression of human HO-1 and A20 in porcine cells: Effects on susceptibility to cell-mediated lysis and inflammatory cytokines

Reinhard Schwinzer¹, Wiebke Baars¹, Katja Borns¹, Bjoern Petersen², Wilfried Kues², Heiner Niemann²

¹Transplant Laboratory, Hannover Medical School, Hannover; ²Department of Biotechnology, Friedrich-Loeffler-Institut, Mariensee; Germany

Objectives: Successful pig-to-human xenotransplantation will require multiple genetic modifications of the donor animal. In this study we assessed the effects of transgenic expression of human (h) A20 and HO-1 on the susceptibility of porcine cells to cellular cytotoxicity and inflammatory cytokines.

Methods: Fibroblasts and endothelial cells (EC) from transgenic (tg) pigs over-expressing hA20 or hHO-1 were used. The capacity of human NK cells, T-cells, and CD95L transfectants to lyse porcine cells was studied by 51Cr-release assays. The effects of cytokines were monitored by analyzing the expression of adhesion molecules and MHC class-II by flow cytometry.

Results: Human cytotoxic effector cells killed wild-type and hHO-1-tg cells with similar intensity suggesting that hHO-1 does not enhance resistance of porcine cells to cell-mediated lysis. However, TNF-α induced apoptosis and up-regulation of adhesion molecules (e.g. CD62E/E-selectin) was significantly reduced in hHO-1-tg EC. Furthermore, up-regulation of MHC class-II molecules in response to IFN-γ treatment was diminished in hHO-1-tg cells compared to controls. When cells from hA20-tg pigs were used as targets for human effector cells, reduced lysis was observed compared to wild-type targets (30 to 40% protection). Stimulation of wild-type EC with TNF-α resulted in strong up-regulation (10-15fold) of CD62E. In hA20-tg cells, however, TNF-α induced up-regulation of CD62E was significantly weaker (3-5fold).

Conclusion: Over-expression of hHO-1 enhances resistance of porcine cells to inflammatory stimuli. This can also be achieved by hA20 which in addition protects from cell-mediated lysis. Because of the large spectrum of protective effects, over-expression of hA20 might be an effective approach to diminish various types of immune responses to xenografts.

Supported by EU grant LSHB-CT-2006-037377, “Xenome”, and the Deutsche Forschungsgemeinschaft (FOR 535, “Xenotransplantation”).