Thiopurine-induced peripheral T cells apoptosis and drug response in patients with Crohn's disease

Background and Aim: in Crohn's disease(CD), both azathioprine(AZA)and 6-mercaptopurine( 6-MP) showed proven efficacy in induction of remission. The response increases after 17 weeks, thus suggesting that there is a minimum length of time for trial of thioupurine therapy. Up to 20% of patients treated with thiopurines need to discontinue therapy due to the occurrence of adverse events. Therefore, besides their proven efficacy, the relatively long period necessary to obtain the full pharmacological effect and the possibility of adverse reactions, do not encourage the use of thioupurine as early therapeutic option. The development of tests to predict responsiveness to thiopurine represents a major attempt in the clinical management of CD patients. It has been demonstrated that AZA is able to induce apoptosis of T cells. Aim of the present study was to analyze the “In Vitro” thiopurineinduced peripheral T cells apoptosis in a group of CD patients with known response to a previous treatment with either AZA or 6-MP. Results were then analyzed according to the response to previous treatment. Methods: A heparinised peripheral blood sample was obtained from a total of 11 CD patients previously treated with thiopurines during a scheduled visit. CD4+ T cells were obtained from density-gradient isolated PBMC by immunomagnetic selection. Cells were stimulated “In Vitro” for 4-5 days with aCD3/28 Abs in complete medium + 20U/ml of hrIL-2 in the presence or absence of AZA or 6-MP or 6-thioguanine at 5μM concentration, and apoptosis was assessed using Annexin V staining and analyzed by cytofluorimeter. IFN-γ production was evaluated in supernatants by ELISA. Results: among the 11 CD patients enrolled, 6 were not responsive to a previous treatment with thiopurines. The % of apoptosis after aCD3/aCD28 stimulation was not different between responders patients (R) and not responders patients (NR) (R: 16.7 (10.8-38.8); NR: 15.6 (8.1-58.3) median (range). All the patients showed an increase in the % of apoptotic cells after aCD3/aCD28 stimulation in the presence of AZA (p<0.05 by Wilcoxon test). To quantify the ability of AZA to increase the apoptosis, we conveyed the data as apoptosis stimulation index (ASI). NR patients showed a significant reduced ASI when compared to R patients (NR: 1.62 (1.37-1.93); R: 2.45 (2.19-2.65) median (range) p=0.004 by Mann Whitney test). Incubation with AZA reduced the IFN-γ production in R to a greater extent when compared to NR. Conclusions: evaluation of apoptosis stimulation index of peripheral CD4+T cell after incubation with AZA might represent a parameter useful for a proper selection of CD patients candidate to thiopurine treatment.