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

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Abstract

Background.

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).

Methods.

For in vitro experiments, peripheral blood from healthy volunteers was collected and treated either with 1 μM cyclosporin A (CsA; n=6), 10 μM 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.

Results.

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.

Conclusion.

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.

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