In Vitro Effects of Clinical Immunosuppressive Regimens on Human Islet Function and Apoptosis Are Dependent on Doses (2012)

Background: Despite increasing success for clinical islet transplantation, immunosuppressive therapy still is a two-edged sword that on one hand helps avoiding allograft rejection and on the other it hampers islet survival and function by its toxicity. To improve islet transplantation therapy, the impact of immunosuppressive regimens on islet function in terms of doses, agents, and combinations are of importance. The purpose of this study was to evaluate the impact of clinically used immunosuppressive regimens on human islet function and apoptosis in vitro. Methods: Human islets were isolated using a modified automated method from independent donors (n=8) and aliquots of purified islets were cultured in supplemented CMRL-1066 with or without selected immunosuppressive agents. Islets were either exposed to a therapeutic or high dose of Tacrolimus (T)(12nM or 37nM), Sirolimus (S)(11nM or 33nM), Mycophenolic Acid (MPA)(9nM or 28nM) or Methylprednisolon (MP)(100ng/ml or 1000ng/ml) alone or in combinations as indicated, (T+S), (T+S+MP), (T+MPA), (T+MPA+MP) for 24 hours. We evaluated the viability by Cell Death EIA, induction of apoptosis by Caspase 3/7 activity, the expression of apoptosis related genes (Bcl-2 and Bax) and the pro-inflammatory gene, IL-8. Islets function was assessed by glucose-stimulated insulin secretion presented as insulin stimulation index (SI) and glucose-stimulated oxygen consumption rate (OCR) analysed by an XF24 bioenergetic assay (Seahorse Bioscience). Results: Human islets exposed to therapeutic doses of the immunosuppressive agents alone or in combination, showed the same rate of apoptosis and no effects on viability (n=8, p=0.4, p=0.2 by one-way ANOVA, respectively) compared to untreated islets. Moreover, no adverse effects on islet function (no change of SI or glucose-stimulated OCR) were observed of the immunosuppressive agents alone, whereas the combination T+S, T+S+MP, T+MPA, T+MPA+MP tended to reduce SI compared to untreated islets. However, when islets were exposed to high doses of the immunosuppressive agents alone, OCR and SI were significantly reduced and totally abolished when islets were exposed to the combinations (p< 0,001). In addition, we found a significantly increased apoptosis in islets exposed to immunosuppressive agents alone compared to untreated cells (p< 0.05). The Bcl-2/Bax mRNA ratio was unchanged. When the strong immunoprotecting agent MP was added to the combinations we observed reduced cell death and expression of IL-8 (p< 0.05). Conclusions: Therapeutic doses of immunosuppressive agents, either alone or in clinically used combinations, have no hampering effect on islet survival in vitro, whereas acute effects of high doses of the immunosuppressive drugs have deleterious effects on islets, especially on glucose-stimulated insulin secretion. However, when adding MP to the combinations, both cell death and inflammation were reduced. Overall, our results show that the toxicity of clinical immunosuppressive regimens on islet function and survival is dependent on the doses. This is important in clinical intraportal islet transplantation where islets probably are exposed to higher concentrations of immunosuppressants than measured in the peripheral bloodstream, supporting the use of low-dose immunosuppressive drug protocols in clinical islet transplantation.