The activity of inducible nitric oxide synthase in rejected skin xenografts is selectively inhibited by a factor produced by grafted cells

BACKGROUND: Production of nitric oxide (NO) by graft infiltrating macrophages has been suggested as an important effector mechanism of allograft rejection. Expression of the gene for the inducible NO synthase (iNOS) and the production of NO in rejected graft has been demonstrated in various models of allotransplantation. However, whether NO plays a role in rejection of skin xenografts has not been documented. METHODS: Explants of rejected skin allografts or xenografts (rat to mouse) were cultivated in vitro and the production of NO, interleukin (IL)-2, IL-4, IL-10 and interferon-gamma (IFN-gamma) by graft infiltrating cells was determined by the Griess reaction or ELISA. Effects of supernatants from cultures of xenograft explants on the expression of gene for iNOS, accumulation of iNOS protein and NO production were determined by RT-PCR or Western blots. Molecular mass of the factor with the suppressive activity was characterized by filtration on chromatography Sephacryl S-200 Superfine column. In addition, the effects of 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), a selective iNOS inhibitor, on survival of skin xenografts were tested. RESULTS: While explants of rejected mouse skin allografts produced substantial amounts of NO, undetectable or only very low levels of NO were found in supernatants from cultured rat skin xenografts. Cocultivation of bacterial lipopolysaccharide (LPS)-stimulated mouse macrophages which produce high quantities of NO, with pieces of rejected xenografts, but not of syngeneic grafts, allografts or normal rat skin, completely inhibited production of NO. Production of IL-6 and IL-10 by LPS-stimulated macrophages was not inhibited under the same conditions. The inhibition of NO production was mediated by a factor which was produced by rejected rat xenograft and which was eluted from chromatography Sephacryl S-200 Superfine column in a fraction representing a molecular mass of 67 kDa. The factor did not inhibit the expression of the gene for iNOS, reduce the level of iNOS protein in stimulated macrophages, or function as a scavenger of NO. Rather, the factor inhibited the function of iNOS. The finding that NO does not play an important role during rejection of skin xenografts is supported by the observation that treatment of graft recipients with AMT, a specific iNOS inhibitor, did not enhance xenograft survival, while the same treatment resulted in prolongation of survival of skin allografts. CONCLUSION: The results thus demonstrate that a 67-kDa molecule produced by rejected rat skin xenografts selectively inhibits iNOS activity in graft infiltrating macrophages. We suggest that NO does not play a significant role in rejection of skin xenografts as it does in the case of allograft rejection.