Effect of calcineurin inhibitor drugs, mTOR inhibitors and mycophenolate mophetil on the expression of mRNA IL-2

Enas A El-Safa1, Salim Fredericks2, Atholl Johnston1, David W Holt 2. Clinical Pharmacology, William Harvey Research Institute, Barts and The London, Charterhouse Square, London, EC1M 6BQ, UK. (2) The Analytical Unit, Division of Cardiac and Vascular Sciences; St George’s- University of London, London, SW17 0RE, UK.

Improved therapeutic strategies following transplantation have improved graft survival rates in recent years. The emergence of new immunosuppressant drugs challenging each other in relation to improved efficacy and reduced side effects has not been matched by improvements in methods of accurate monitoring. These drugs include: calcineurin inhibitors (CNIs) (ciclosporin and tacrolimus), mammalian target of rapamycin (mTOR) inhibitors (sirolimus and everolimus) and anti-proliferative drugs as mycophenolate mophetil (MMF). These different groups of immunosuppressants inhibit lymphocyte activation and proliferation through different mechanisms of action. Inhibition of the production of IL-2 is one of the main actions of CNIs. In this study we investigated the effect of different groups of immunosuppressant drugs on IL-2 mRNA expression. Whole blood samples from healthy volunteers (n =16) were cultured. Each sample of 200µl blood diluted in 1.8 mL of Iscove’s modified Dulbecco’s medium supplemented with penicillin (100000 units/L), streptomycin (100mg/L) and 10 mM/L glutamine. Samples were incubated for 24 hours in culture medium, in the presence of 1 mg/L anti CD3 and anti CD28 monoclonal antibodies (R&D Systems) to stimulate in vitro lymphocyte proliferation. Each experimental sample was split into three aliquots and pre-incubated for 2 hours either with tacrolimus (25μg/L), ciclosporin (1000μg/L) or without any drug. An identical procedure was performed for seven extra samples using blood pre-incubated for 2 hours with sirolimus (50 ng/ml), everolimus (10 ng/mL) and mycophenolic acid (150 μg/L). Following the culturing period total RNA was isolated and IL-2 mRNA was quantified using RT-PCR with reference to GAPDH mRNA. IL-2 protein concentration in the culture supernatant was also determined by ELISA, triplicate determination was made for each sample. The copy number of IL-2 mRNA was suppressed by the presence of either tacrolimus or ciclosporin in most cases. Eleven of the sixteen samples treated with tacrolimus and nine of the sixteen samples treated with ciclosporin demonstrated attenuation of IL-2 mRNA expression ranging from 27% to 83% for tacrolimus and from 76% to 82% for ciclosporin as compared to control. A paradoxical increase, ranging from 157% to 265% for tacrolimus and from 161% to 176% for ciclosporin was observed with the remaining samples. There was no significant change in IL-2 mRNA expression with either mTOR inhibitors or MPA. All the stimulated samples showed increase in the IL-2 protein concentration in the supernatant of the culture, which was attenuated in all samples containing tacrolimus or ciclosporin (p = 0.003, p = 0.001) respectively using the paired student t test.

We here described an up-regulation of IL-2 mRNA with some samples incubated with CNIs; these samples have not demonstrated any significant changes in IL-2 expression when incubated with mTOR inhibitors or MPA. Consequently, this paradoxical effect of CNI could be attributed to inter-individual variations based on genetic basis. These data demonstrate that individuals differ with respect to their sensitivity to CNIs.