Graves S S, Bramhall J, Bonavida B
J Immunol. 1986 Sep 15;137(6):1977-84.
Previous studies in our laboratory on the natural killer (NK) lytic mechanism demonstrated that following interaction of target cell with effector cell, the effector cell releases NK cytotoxic factors (NKCF) that can then bind to and lyse the target cell. This study investigates the mechanism by which the target cell signals the effector cell to release NKCF. Studies on other cell systems with secretory functions have indicated that receptor-induced transmembrane signaling leads to the metabolism of phosphatidylinositol and activation of protein kinase C (PKC) by increased cytosolic Ca++ and diacylglycerol (DAG). We tested the hypothesis that a similar sequence of activation events occurs in human NK cells by examining the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), and the calcium ionophores A23187 and ionomycin in their ability to induce release of NKCF. The amount of NKCF released was determined in a 20-hr 51Cr release assay against an NK-sensitive target cell. A23187, ionomycin, or TPA alone did not induce release of NKCF. However, ionophores (200 mM) in conjunction with TPA (20 ng/ml) induced release of NKCF. Several properties of the induced NKCF by TPA and ionophores were concordant with those of the NK cell-mediated cytotoxicity (CMC) reaction. The kinetics of release were faster (less than 1 hr) than when either Con A or target cells were used to stimulate NKCF. Only NK-sensitive target cells were killed by NKCF. Pretreatment of effector cells with interferon enhanced release of NKCF from effector cells. Several lines of evidence suggested that the pathway of activation takes place through phosphatidyl inositol metabolism. Activation of PKC was indicated because TPA and A23187 enhanced protein phosphorylation in the LGL-enriched fraction. Experiments that made use of oleoyl acetyl glycerol, a synthetic DAG, showed release of NKCF in the absence of A23187 but was augmented by the ionophore. The above studies suggest that NKCF is released from NK effector cells within a period of time consistent with NK CMC, and the release of NKCF results either directly or indirectly from protein phosphorylation by PKC.
我们实验室之前关于自然杀伤（NK）细胞溶解机制的研究表明，靶细胞与效应细胞相互作用后，效应细胞会释放NK细胞毒性因子（NKCF），该因子随后可结合并裂解靶细胞。本研究旨在探究靶细胞向效应细胞发出释放NKCF信号的机制。对其他具有分泌功能的细胞系统的研究表明，受体诱导的跨膜信号传导会导致磷脂酰肌醇代谢，并通过增加胞质Ca++和二酰基甘油（DAG）来激活蛋白激酶C（PKC）。我们通过检测佛波酯12-O-十四酰佛波醇-13-乙酸酯（TPA）以及钙离子载体A23187和离子霉素诱导NKCF释放的能力，来验证人类NK细胞中是否发生类似的激活事件序列。在针对NK敏感靶细胞的20小时51Cr释放试验中测定释放的NKCF量。单独使用A23187、离子霉素或TPA均未诱导NKCF释放。然而，离子载体（200 mM）与TPA（20 ng/ml）联合使用可诱导NKCF释放。TPA和离子载体诱导产生的NKCF的几个特性与NK细胞介导的细胞毒性（CMC）反应的特性一致。释放动力学比使用Con A或靶细胞刺激NKCF时更快（不到1小时）。只有NK敏感靶细胞会被NKCF杀死。用干扰素预处理效应细胞可增强效应细胞释放NKCF。几条证据表明激活途径是通过磷脂酰肌醇代谢发生的。表明PKC被激活是因为TPA和A23187增强了富含大颗粒淋巴细胞（LGL）的组分中的蛋白质磷酸化。利用合成DAG油酰乙酰甘油进行的实验表明，在没有A23187的情况下会释放NKCF，但离子载体可增强其释放。上述研究表明，NKCF在与NK CMC一致的时间段内从NK效应细胞中释放出来，并且NKCF的释放直接或间接源于PKC介导的蛋白质磷酸化。
