PROGRAMMED CELL DEATH IN LYMPHOCYTES

为了验证淋巴细胞程序性细胞死亡假说

In order to test the hypothesis programmed cell death in lymphocytes involves activation of an endogenous protease, we have tested the ability of several cysteine protease inhibitors to block the TcR-induced death of the CD4+ T cell hybridoma 2B4. Two chemically distinct families of inhibitors of calpains and cathepsins, the E-64 and leupeptin families, were found to rescue 2B4 cells from death measured by trypan blue or propidium iodide uptake after overnight culture on immobilized alpha-CD3 or alpha-TcR antibodies. DNA breakdown measured by flow microfluorimetry was also blocked. These drugs enhanced the TcR-induced IL-2 secretion by as much as 9-fold, showing that they do not act by interfering with signal transduction. Other programmed cell death systems in lymphocytes tested were the steroid-induced death of CD4+CD8+ thymocytes, which was blocked by leupeptin family inhibitors, and the CH31 B cell line, whose mIgM-induced death was unaffected by these inhibitors. We have also found that short-term-cultured lymphocytes can undergo programmed cell death after TcR cross-linking. After activation of purified resting mouse lymph node T cells (or PNA+ thymocytes) by immobilized alpha-CD3 or alpha-TcR antibodies, continuously dividing cells were be maintained in IL2 for periods of days to weeks. When re-exposed to surface-bound antibodies against TcR or CD3, these cells were found to undergo a decrease in cell division and mitochondrial metabolism, accompanied by substantial cell death. When purified populations of CD4+ and CD8+ cells were cultured this way, the cell death effect was principally seen in the CD4 population, with more modest effects in all responses in the CD8 population. In the CD4+ cells DNA breakdown was clearly seen to accompany cell death when analyzed by flow microfluorimetry. This effect was blocked by cyclosporin, co-culture with splenic macrophages, or cysteine protease inhibitors.