Quantification of interleukin-2 mRNA may have a role in individualization of immunosuppressant drugs

Enas Ali1, Salim Fredericks2, Mohamed Attia3, Tim Rutherford4, Atholl Johnston1 , David W Holt2 . 1 Clinical Pharmacology, William Harvey Research Institute, Barts and The London, Charterhouse Square, London, EC1M 6BQ2The Analytical Unit, Cardiac and Vascular Science, 3Department Cellular and Molecular Medicine, 4Medical Biomics Centre,; St George’s Hospital Medical School. London, UK.

The calcineurin inhibitors (CNI), ciclosporin and tacrolimus are potent immunosuppressant drugs used mainly following organ transplantation to prevent rejection by inhibiting lymphocyte proliferation. Their pharmacokinetic monitoring does not accurately reflect their biological status. Methods of pharmacodynamic monitoring are presently not well established. A major effect of the calcineurin inhibitors is to decrease the production of interleukin 2 (IL-2). In this study we attempted to demonstrate interindividual variation by assessing cytokine IL-2 production at the cellular and extra cellular levels using an in vitro technique.

Whole blood samples from healthy volunteers (n=13) were incubated for 24 hours in culture medium, in the presence of anti CD3 and anti CD28 antibodies to stimulate in vitro lymphocyte proliferation. Each experimental sample was split into three and pre-incubated for 2 hours either with tacrolimus (25µg/l), ciclosporin (1000µg/l) or without any drug. Non-stimulated control incubates were also included without the addition of the two antibodies. Following the culturing period total RNA was isolated from 16 ml of incubate mixture. Quantitative reverse transcriptase PCR (RT-PCR), using TaqMan™ probes, was employed to quantify IL-2 mRNA expression. mRNA expression of the housekeeping gene GAPDH, which is constitutively does not change with lymphocyte cell cycling, was also quantified, and IL-2 mRNA copy number was reported according to an absolute standard curve. Supernatants of the culture were also assayed for IL-2 protein concentration using ELISA.

All incubates demonstrated an increase in IL-2 mRNA production, as compared to non-stimulated baseline control incubates. As expected, the relative amounts of IL-2 mRNA were suppressed by the presence of either tacrolimus or ciclosporin in most cases. Nine of the thirteen samples treated with tacrolimus and seven of the thirteen samples treated with ciclosporin demonstrated attenuation of mRNA expression of more than 50% relative IL-2 mRNA as compared to incubates that were not exposed to either Tacrolimus or ciclosporin. A paradoxical increase, ranging between 100% and 150% was observed with the rest of samples. Only one sample of the thirteen showed up regulation of IL-2 mRNA when treated with tacrolimus and down-regulation when treated with ciclosporin. All the stimulated samples showed increase in the IL-2 protein concentration which was attenuated on adding tacrolimus or ciclosporin (p=0.03, p=0.02 respectively).

Although there was relatively small number of samples, the frequency of the occurrence of paradoxical response to calcineurin inhibitor drugs in this type of in vitro experiment reflected previously published reports. Quantitative measurement of cytokine IL-2 regulated gene expression may represent a method to assess the efficacy of CNI drugs.