The Influence of Immunosuppressive Drugs on T- and B-cell Apoptosis via p53-Mediated Pathway In Vitro and In Vivo

Abstract

This study was designed to assess the effects of calcineurin and inosine-5-monophosphate-dehydrogenase inhibitors on p53-mediated-apoptosis in T- and B-cells in vitro and in human heart-transplanted recipients (HTx-R). For in vitro experiments, peripheral blood from healthy volunteers was collected and treated either with 1 microM cyclosporin A (CsA; n = 6), 10 microM mycophenolic acid (MPA; n = 6) or 100 nM tacrolimus (TRL; n = 6). For the second part, peripheral blood was collected from HTx-R undergoing CsA-MPA (n = 11) or TRL-MPA (n = 11) therapy before (0 hr) and after (2 hr) acute drug application and from healthy volunteers (n = 11) without drug therapy. Whole blood (part 1+2) was stimulated (24 hr) with eight different concentrations of actinomycin-D (0-800 nM), an apoptosis inductor acting via p53-pathway. Apoptotic lymphocytes were measured by TUNEL and expression of Annexin-V using FACS. Drug effects were calculated by taking the effects of actinomycin-D as baseline values. In vitro drug treatment with CsA, MPA, and TRL significantly (P < 0.05) decreased the apoptotic effect of actinomycin-D in T-cells in a noncompetitive manner. In HTx-R undergoing drug therapy, there was a similar antiapoptotic effect observed in both T- and B-cells (P < 0.05). Differences between 0 hr and 2 hr after acute drug application did not exist. Apoptosis induced by actinomycin-D can be completely blocked by caspase-inhibitor zVAD-FMK. Our results suggest that, in vitro and in HTx-R, an inhibition of calcineurin and inosine-5-monophosphate-dehydrogenase by CsA, TRL, or MPA lead to an inhibition of T-and B-cell apoptosis via p53-pathway. This assay may be helpful to provide insights into mechanisms of immunosuppressive drugs in regulation of apoptosis in lymphocytes.