Apoptosis-inducing factor of a cytotoxic T cell line:: involvement of a secretory phospholipase A2
Apoptosis-inducing factor of a cytotoxic T cell line:: involvement of a secretory phospholipase A2
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DOI：
10.1007/s00441-005-0095-y
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发表时间：
2006-05-01
影响因子：
3.6
通讯作者：
Persechini, P
中科院分区：
文献类型：
--
作者：
Costa-Junior, HM;Hamaty, FC;Persechini, P
自然杀伤（NK）细胞和细胞毒性T淋巴细胞（CTL）通过颗粒胞吐途径和通过属于肿瘤坏死因子家族的分子的接合来杀伤靶细胞。分泌型磷脂酶A（2）（sPLA（2））参与NK细胞的细胞毒性过程。然而，其分子身份和细胞内定位仍然未知，其作用机制知之甚少。在这里，我们通过研究CTL细胞系的全细胞提取物的细胞毒活性来重新解决这个问题。我们观察到，在37摄氏度在1 mM Ca 2+的存在下，穿孔素-颗粒酶途径的失活增强了CTL提取物诱导细胞凋亡的能力。这种细胞死亡的增强作用是Ca 2+依赖性的、耐热的，并被4-溴苯甲酰甲基溴和scalaradial（sPLA的两种抑制剂）抑制（2）。通过用抗sPLA（2）多克隆抗体阻断Ca 2+处理的细胞提取物的促凋亡活性，证实了sPLA（2）的参与。通过细胞分级分析，我们发现促凋亡sPLA（2）定位于细胞质部分，而不是富含穿孔素的颗粒或质膜部分。Western印迹分析显示存在四个不同的条带，分别为56、29.5、21和15 kDa。最高分子量条带与第III组sPLA的表达一致（2）。综上所述，这些数据表明诱导细胞毒性的sPLA（2）在CTL细胞系的胞质溶胶中表达，并表明其在CTL介导的细胞毒性中起效应子作用。
Natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) kill target cells by the granule-exocytosis pathway and by the engagement of molecules belonging to the tumor necrosis factor family. The involvement of secretory phospholipase A(2) (sPLA(2)) in the cytotoxic process has been proposed in NK cells. However, its molecular identity and intracellular localization remain unknown, and its mechanism of action is poorly understood. Here, we have readdressed this issue by studying the cytotoxic activity of whole cell extracts of a CTL line. We observed that inactivation of the perforin-granzyme pathway at 37 degrees C in the presence of 1 mM Ca2+ enhanced the ability of CTL extracts to induce apoptosis. This potentiation of cell death was Ca2+-dependent, thermoresistant, and inhibited by 4-bromophenacyl bromide and scalaradial ( two inhibitors of sPLA(2)). The involvement of an sPLA(2) was confirmed by blocking the pro-apoptotic activity of the Ca2+-treated cell extract with an anti-sPLA(2) polyclonal antibody. By cell fractionation assays, we showed that the proapoptotic sPLA(2) was localized in the cytoplasmic fraction but not in perforin-rich granules or plasma membrane fractions. Western blotting analysis revealed the presence of four distinct bands of 56, 29.5, 21, and 15 kDa. The highest molecular weight band was consistent with the expression of a group III sPLA(2). Taken together, these data indicate that an apoptosis-inducing sPLA(2) is expressed in the cytosol of a CTL cell line and suggest that it plays an effector role in CTL-mediated cytotoxicity.