Effect Of Interleukin-35on Apoptosis Of The Activated T Cells And Its Mechanism In Mice

Objective:The present study was performed to investigate:(1) the effect of the interleukin-35(IL-35) on the apoptosis of the activated T cells and its potential mechanism in mice,(2) the time/dose dependent manner of the effect of IL-35stimulation on the apoptosis of the activated T cells,(3) the possible role of the protein B-cell lymphoma2(Bcl-2) in the process of IL-35-mediated apoptosis of the activated T cells, and (4) the effect of treatment with IL-35on the secretion of the cytokines such as interleukin-10(TL-10), transforming growth factor-β (TGF-β), interleukin-4(IL-4), as well as interferon-γ (IFN-γ) of the activated T cells.MetAod:CD4+T cells isolated from mice in vitro were suspended in RPMI-1640with10%FBS at1×106cell/ml on12-well cell culture plates. In the current experiment of part Ⅰ, CD4+T cells which were activated by Con A (1μg/ml) were stimulated with IL-35at different time points of12h,24h,48h, and different doses in2,10,50ng/ml, respectively. Apoptosis rates of the CD4+T cells stimulated by IL-35were tested by the method of Annexin-FITC using flow cytometer. Then, the time/dose effect of IL-35stimulation on the apoptosis of CD4+T cells were analysed. In the part Ⅱ:by use of both RT-PCR and Western blot, changes in Bcl-2mRNA expression and protein level were determined at various groups. The possible role of Bcl-2in the regulation of IL-35-mediated apoptosis of activated CD4+T calls were also evaluated. In the part Ⅲ: with the method of ELISA, the secretion of cytokines including IL-10, IL-4, TGF-β, as well as IFN-y in the supernatants of CD4+T cells stimulated by IL-35at different doses for24hours were measured.Result:1Treatment with IL-35could markedly enhance the apoptosis of the activated CD4+T cells. Comparing with the control group, it could be noted that in the IL-35treatment group that the apoptosis rates of the activated CD4+T cells increased evidently at24h and48h with the doses of10ng/ml and50ng/ml (P<0.01). However, there were no significant difference between the control and treatment groups in the dose of2ng/ml IL-35. In the does of10ng/ml, IL-35apparently increased the apoptosis rates of the activated CD4+T cells at24h following stimulation (P<0.01), and the response reached its peak at48h (P<0.01). In the dose of50ng/ml, the effect of IL-35on the apoptosis of the activated CD4+T cells peaked at24h after stimulation (P<0.01). Nevertheless, the apoptosis rates were markedly decreased along with the elevation of rate of the dead cells at48h after IL-35treatment.2The whole cell protein and RNA were extracted from the activated CD4+T cells after24h stimulated by the IL-35of2ng/ml,10ng/ml and50ng/ml, respectively. By means of Western bolt and RT-PCR, changes in Bcl-2were determined in the level of both protein and mRNA expression. The result showed that as the increase in the doses of IL-35, the expression of Bcl-2molecule varied in both protein and mRNA levels (P<0.01).3. With the method of ELISA, the secretion levels of the cytokines including IL-10, IL-4, TGF-β, as well as IFN-y in the supernatants of CD4+T cells stimulated by IL-35in the doses of10ng/ml and50ng/ml for24hours were measured. It was found that along with the increased doses in IL-35stimulation, levels of Th2class cytokines such as IL-10and TGF-β were significantly elevated (P<0.01). There were no marked difference in the srelease of IL-4in the control and treatment groups. In addition, secretion of the IFN-y was decreased only in the10ng/ml IL-35treatment group.Conclusion:1. IL-35could induce apoptosis of the activated CD4+T cells in a time/dose dependent manner, which might be one of the important mechanisms leading to the immune dysfunction in septic response.2. IL-35could markedly influence the expression of the Bcl-2both in the protein and mRNA level, and this effect was enhanced along with the increases in dosages of IL-35. The results suggested that IL-35-mediated apoptosis might be related to the mitochondrion pathway, in which Bcl-2appeared to be involved in the process.3. IL-35affected the secretory function of the CD4+T cells. Under the treatment with IL-35, the secretion of negative-regulation cytokines such as IL-10, TGF-β was markedly increased without significant changes in IL-4release. These data indicated that IL-35might be critically involved in Th2-cell differentiation accompanied with the apoptosis of CD4~+T cell-mediated immunesuppressive activity.