Granzyme B Short-Circuits the Need for Caspase 8 Activity during Granule-Mediated Cytotoxic T-Lymphocyte Killing by Directly Cleaving Bid

Cytotoxic T lymphocytes (CTL) can trigger an apoptotic signal through the Fas receptor or by the exocytosis of granzyme B and perforin. Caspase activation is an important component of both pathways. Granzyme B, a serine proteinase contained in granules, has been shown to proteolytically process and activate members of the caspase family in vitro. In order to gain an understanding of the contributions of caspases 8 and 3 during granule-induced apoptosis in intact cells, we have used target cells that either stably express the rabbitpox virus-encoded caspase inhibitor SPI-2 or are devoid of caspase 3. The overexpression of SPI-2 in target cells significantly inhibited DNA fragmentation, phosphatidylserine externalization, and mitochondrial disruption during Fas-mediated cell death. In contrast, SPI-2 expression in target cells provided no protection against granzyme-mediated apoptosis, mitochondrial collapse, or cytolysis, leading us to conclude that SPI-2-inhibited caspases are not an essential requirement for the granzyme pathway. Caspase 3-deficient MCF-7 cells were found to be resistant to CTL-mediated DNA fragmentation but not to CTL-mediated cytolysis and loss of the mitochondrial inner membrane potential. Furthermore, we demonstrate that granzyme B directly cleaves the proapoptotic molecule Bid, bypassing the need for caspase 8 activation of Bid. These results provide evidence for a two-pronged strategy for mediating target cell destruction and provide evidence of a direct link between granzyme B activity, Bid cleavage, and caspase 3 activation in whole cells.

细胞毒性T淋巴细胞（Cytotoxic T lymphocytes, CTL）可通过Fas受体（Fas receptor）触发凋亡信号，或通过颗粒酶B（granzyme B）与穿孔素（perforin）的胞吐作用诱导凋亡。半胱天冬蛋白酶（caspase）的激活是这两条凋亡通路的关键组成部分。颗粒酶B作为储存于胞浆颗粒中的丝氨酸蛋白酶，已被证实可在体外对半胱天冬蛋白酶家族成员进行蛋白水解加工并使其激活。为阐明完整细胞中caspase-8与caspase-3在颗粒酶介导的凋亡过程中的具体作用，我们采用了两类靶细胞：一类可稳定表达兔痘病毒（rabbitpox virus）编码的半胱天冬蛋白酶抑制剂SPI-2，另一类则缺失caspase-3。在Fas介导的细胞死亡过程中，靶细胞内过表达SPI-2可显著抑制DNA片段化、磷脂酰丝氨酸外翻（phosphatidylserine externalization）与线粒体破坏（mitochondrial disruption）。与之相反，靶细胞表达SPI-2无法为颗粒酶介导的凋亡、线粒体崩溃（mitochondrial collapse）与细胞溶解（cytolysis）提供保护，这表明受SPI-2抑制的半胱天冬蛋白酶并非颗粒酶通路的必需组分。研究发现，caspase-3缺陷型MCF-7细胞对CTL介导的DNA片段化具有抗性，但无法抵抗CTL介导的细胞溶解与线粒体内膜电位丧失。此外，我们证实颗粒酶B可直接剪切促凋亡蛋白Bid（proapoptotic molecule Bid），无需依赖caspase-8对Bid的激活。上述研究结果为靶细胞破坏的双管齐下策略提供了实验依据，同时证实了完整细胞中颗粒酶B活性、Bid剪切与caspase-3激活之间存在直接关联。