Effects of cyclosporine A and methotrexate on induction of tumour necrosis factor-alpha in rat cardiac allografts

Abstract

Experimental and clinical studies have yet to determine th extent to which methotrexate (MTX) or cyclosporine A (CSA) treatment alone affects the expression in vivo of tumour necrosis factor-alpha (TNFα), a cytokine produced primarily by macrophages and believed to be directly involved in the pathogenesis of cardiac allograft rejection. In light of previously published findings from this laboratory examining the effects of combination CSA/MTX treatment, these studies were designed to examined the individual effects of CSA and MTX upon TNFα gene expression post-transplant (post-tx) using an accessory cervical heart transplant model in the rat. These studies have focused on a higly selective method with which to detect changes in gene expression, reverse transcriptase-polymerase chain reaction (RT-PCR) methodology and enzyme-linked immunosorbant assay (ELISA) assessment of transplant TNFα protein levels. Both techniques consistently demonstrated biphasic TNFα expression in cardiac transplant tissue obtained from untreated allograft recipients during the first week post-tx in contrast to isograft recipients. Previously demonstrated in combination to prolong cardiac allograft survival, low-dose MTX and low-dose CSA were each evaluated alone in the course of these studies to determine their impact on TNFα gene expression. While TNFα levels were up-regulated during untreated allograft rejection, both TNFα RNA and protein were significantly diminished with low-dose combinations CSA/MTX treatment, with CSA alone, but not significantly with MTX treatment alone. In conclusion, TNFα gene expression in untreated allografts is consistent with the hypothesis that TNFα may play a role in events leading to allograft rejection. Results of these studies indicate that TNFα levels are significantly regulated in vivo by CSA but not by MTX treatment. These studies further implicate a role for low-dose MTX in mediating statistically significant immunosuppressive effects in conjunction with low-dose CSA.