School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510641, China.
Cell Res. 2009 Dec;19(12):1350-62. doi: 10.1038/cr.2009.114. Epub 2009 Sep 29.
Cytotoxic lymphocytes are key players in the orchestration of immune response and elimination of defective cells. We have previously reported that natural killer (NK) cells enter target tumor cells, leading to either target cell death or self-destruction within tumor cells. However, it has remained elusive as to the fate of NK cells after internalization and whether the heterotypic cell-in-cell process is different from that of the homotypic cell-in-cell event recently named entosis. Here, we show that NK cells undergo a cell-in-cell process with the ultimate fate of apoptosis within tumor cells and reveal that the internalization process requires the actin cytoskeletal regulator, ezrin. To visualize how NK cells enter into tumor cells, we carried out real-time dual color imaging analyses of NK cell internalization into tumor cells. Surprisingly, most NK cells commit to programmed cell death after their entry into tumor cells, which is distinctively different from entosis observed in the homotypic cell-in-cell process. The apoptotic cell death of the internalized NK cells was evident by activation of caspase 3 and DNA fragmentation. Furthermore, NK cell death after internalization is attenuated by the caspase inhibitor, Z-VAD-FMK, confirming apoptosis as the mode of NK cell death within tumor cells. To determine protein factors essential for the entry of NK cells into tumor cells, we carried out siRNA-based knockdown analysis and discovered a critical role of ezrin in NK cell internalization. Importantly, PKA-mediated phosphorylation of ezrin promotes the NK cell internalization process. Our findings suggest a novel regulatory mechanism by which ezrin governs NK cell internalization into tumor cells.
细胞毒性淋巴细胞是免疫反应协调和缺陷细胞消除的关键参与者。我们之前曾报道过，自然杀伤 (NK) 细胞进入靶肿瘤细胞，导致靶细胞死亡或肿瘤细胞内的自我破坏。然而，NK 细胞内化后的命运以及异型细胞内细胞过程是否与最近命名的胞吞作用的同源细胞内事件不同，仍然难以捉摸。在这里，我们表明 NK 细胞经历了一个细胞内过程，最终在肿瘤细胞内凋亡，并揭示内化过程需要肌动蛋白细胞骨架调节剂 ezrin。为了可视化 NK 细胞如何进入肿瘤细胞，我们对 NK 细胞内化进入肿瘤细胞进行了实时双色成像分析。令人惊讶的是，大多数 NK 细胞在进入肿瘤细胞后都会选择程序性细胞死亡，这与同源细胞内细胞过程中观察到的胞吞作用明显不同。内化的 NK 细胞的凋亡细胞死亡通过 caspase 3 的激活和 DNA 片段化来证明。此外，内化后 NK 细胞死亡被 caspase 抑制剂 Z-VAD-FMK 减弱，证实凋亡是 NK 细胞在肿瘤细胞内死亡的方式。为了确定 NK 细胞进入肿瘤细胞所必需的蛋白因子，我们进行了 siRNA 基敲低分析，发现 ezrin 在 NK 细胞内化中起着关键作用。重要的是，PKA 介导的 ezrin 磷酸化促进了 NK 细胞的内化过程。我们的研究结果表明了 ezrin 控制 NK 细胞内化进入肿瘤细胞的新的调节机制。
