Deem R L, Britvan L J, Targan S R
Department of Medicine, UCLA School of Medicine 90024.
Cell Immunol. 1987 Dec;110(2):253-64. doi: 10.1016/0008-8749(87)90121-3.
Phospholipase A2 (PA-2) is known to be involved in many calcium-dependent cellular processes and inhibitors of PA-2 have been shown to inhibit natural killer cell-mediated cytotoxicity (NK CMC). Since the trigger stage is calcium dependent, it was postulated that this effector cell-associated enzyme may play a role in early calcium-dependent processes. To define how PA-2 might be involved in NK lysis, the effect of both PA-2 inhibitors and exogenous PA-2 on the stages of NK lysis was examined. PA-2 inhibitors, quinacrine and p-bromophenacyl bromide, inhibited NK CMC at the effector cell level, but affected neither initial target-effector cell binding nor dissociated conjugates during the length of the NK assay, suggesting that they block post-binding lytic events. A calcium pulse assay showed that PA-2 inhibitors inhibit only moderately when added after calcium and only within the first 15 min, demonstrating that these inhibitors blocked very early post-binding lytic events. Because this very early post-binding inhibitory effect was consistent with effects upon the NK trigger mechanism, the effect of exogenous PA-2 on NK lysis was tested. Pretreatment of K562 target cells but not pretreatment of peripheral blood lymphocytes (PBL) with 20 units/ml PA-2 enhanced lysis by two to eight-fold (based upon lytic units), showing its enhancing effect to be at the target cell level. Single cell assays using effector cells purified by indirect panning with monoclonal antibody NKH-1 showed that only the number of killer cells was increased. Calcium pulse assays showed that enhancement of lysis was maximum 15 min after addition of calcium and decreased rapidly thereafter, demonstrating its effect at an early post binding stage. Additionally, PA-2 was shown to overcome inhibition by the monoclonal antibody 13.3, which has been shown to affect the trigger stage of NK lysis (post-binding but prior to calcium dependent events). Thus, it appears that an NK cell-associated PA-2 could function by modulating the target cell surface, revealing a structure which acts as a "secondary" trigger, subsequent to the 13.3 "trigger", requisite for activation of the NK lytic process.
已知磷脂酶A2（PA - 2）参与许多钙依赖性细胞过程，并且PA - 2抑制剂已被证明可抑制自然杀伤细胞介导的细胞毒性（NK CMC）。由于触发阶段是钙依赖性的，因此推测这种效应细胞相关酶可能在早期钙依赖性过程中发挥作用。为了确定PA - 2如何参与NK裂解，研究了PA - 2抑制剂和外源性PA - 2对NK裂解阶段的影响。PA - 2抑制剂奎纳克林和对溴苯甲酰溴在效应细胞水平上抑制NK CMC，但在NK测定期间既不影响初始靶细胞 - 效应细胞结合，也不影响解离的结合物，这表明它们阻断结合后裂解事件。钙脉冲试验表明，PA - 2抑制剂在钙加入后添加时仅适度抑制，且仅在最初15分钟内，这表明这些抑制剂阻断了结合后非常早期的裂解事件。因为这种结合后非常早期的抑制作用与对NK触发机制的影响一致，所以测试了外源性PA - 2对NK裂解的影响。用20单位/毫升PA - 2预处理K562靶细胞而非外周血淋巴细胞（PBL）可使裂解增强2至8倍（基于裂解单位），表明其增强作用在靶细胞水平。使用通过用单克隆抗体NKH - 1间接淘选纯化的效应细胞进行的单细胞试验表明，仅杀伤细胞数量增加。钙脉冲试验表明，裂解增强在添加钙后15分钟时最大，此后迅速下降，表明其在结合后早期阶段起作用。此外，PA - 2被证明可克服单克隆抗体13.3的抑制作用，该抗体已被证明影响NK裂解的触发阶段（结合后但在钙依赖性事件之前）。因此，似乎NK细胞相关的PA - 2可以通过调节靶细胞表面发挥作用，揭示一种结构，该结构作为13.3“触发”之后的“二级”触发，是激活NK裂解过程所必需的。
