Leibson P J, Midthun D E, Windebank K P, Abraham R T
Department of Immunology, Mayo Clinic, Rochester, MN 55905.
J Immunol. 1990 Sep 1;145(5):1498-504.
NK cells can mediate either FcR-dependent cytotoxicity against antibody-coated target cells or direct cytotoxicity against a variety of tumor cells. We used homogeneous, cloned populations of CD16+/CD3- human NK cells to characterize and compare the transmembrane signaling mechanisms used during these alternative forms of cytotoxicity. Cross-linkage of NK cell FcR with anti-FcR (anti-CD16) mAb or direct binding to NK-sensitive tumor targets resulted in a rapid release of inositol phosphates and increases in [Ca2+]i. The receptor-dependent [Ca2+]i increase (as monitored in indo-1 loaded NK cells by flow cytometry) consisted of an initial release of calcium from intracellular stores, followed by a sustained influx of calcium across the plasma membrane. To assess the potential regulatory feedback role of protein kinase C (PKC) activation in these proximal signaling events, NK cells were pretreated with either PKC-activating phorbol esters, nonactivating phorbol ester homologs, or synthetic diacylglycerols. Brief pretreatment with activating phorbol esters rapidly inhibited, in a concentration-dependent manner, both phosphoinositide hydrolysis and increases in [Ca2+]i induced by FcR ligation, whereas pretreatment with an inactive phorbol ester had no effect. This acute inhibitory effect was not explained by FcR down-regulation, which occurred with more prolonged exposure to phorbol esters. In contrast, the phosphoinositide turnover and [Ca2+]i increase in NK cells stimulated with NK-sensitive tumor targets were not affected by prior exposure to PKC-activating phorbol esters. This differential regulatory effect of phorbol ester on proximal signaling was paralleled by a corresponding effect on cytotoxicity, i.e., phorbol ester-induced activation of PKC inhibited FcR-dependent cytotoxicity, but did not alter direct cytotoxicity against NK-sensitive tumor cells. These results indicate that PKC activation can differentially regulate alternative forms of NK cell-mediated cytotoxicity by rapidly and specifically desensitizing the FcR.
自然杀伤（NK）细胞可介导针对抗体包被靶细胞的FcR依赖性细胞毒性，或针对多种肿瘤细胞的直接细胞毒性。我们使用CD16⁺/CD3⁻人NK细胞的均一克隆群体来表征和比较这些不同形式细胞毒性过程中所使用的跨膜信号传导机制。用抗FcR（抗CD16）单克隆抗体使NK细胞FcR交联，或直接与NK敏感肿瘤靶标结合，会导致肌醇磷酸迅速释放，细胞内钙离子浓度（[Ca²⁺]i）升高。受体依赖性[Ca²⁺]i升高（通过流式细胞术在负载indo-1的NK细胞中监测）包括细胞内钙库的初始钙释放，随后是钙持续流入细胞膜。为了评估蛋白激酶C（PKC）激活在这些近端信号事件中的潜在调节反馈作用，NK细胞用PKC激活佛波酯、非激活佛波酯类似物或合成二酰基甘油进行预处理。用激活佛波酯进行短暂预处理以浓度依赖性方式迅速抑制FcR连接诱导的磷酸肌醇水解和[Ca²⁺]i升高，而用无活性佛波酯预处理则无影响。这种急性抑制作用不能用FcR下调来解释，FcR下调发生在更长时间暴露于佛波酯时。相反，用NK敏感肿瘤靶标刺激的NK细胞中磷酸肌醇周转和[Ca²⁺]i升高不受先前暴露于PKC激活佛波酯的影响。佛波酯对近端信号的这种差异调节作用与对细胞毒性的相应作用平行，即佛波酯诱导的PKC激活抑制FcR依赖性细胞毒性，但不改变对NK敏感肿瘤细胞的直接细胞毒性。这些结果表明，PKC激活可通过快速且特异性地使FcR脱敏来差异调节NK细胞介导的不同形式细胞毒性。
