Walsh G M, Williamson M L, Symon F A, Willars G B, Wardlaw A J
Department of Respiratory Medicine, Leicester University School of Medicine, Glenfield Hospital, UK.
Blood. 1996 Apr 1;87(7):2815-21.
Peripheral blood (PB) eosinophils rapidly undergo apoptosis and cell death in vitro unless cultured in the presence of cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) in which their survival is prolonged for up to 10 days. CD69 is a type II membrane antigen expressed by cytokine-activated, but not freshly isolated, PB human eosinophils. We have examined the effect of ligation of CD69 by specific monoclonal antibody (MoAb) on the viability of human eosinophils cultured with recombinant human (rh)GM-CSF. Eosinophils were purified by immunomagnetic selection and cultured with GM-CSF (10(-10) mol/L). Eighteen hours after the start of culture, a panel of CD69 MoAb or controls (anti-CR3 or isotype-matched control MoAb) were added. Viability was assessed by trypan blue exclusion and apoptosis by morphologic assessment, DNA laddering, and flow cytometric analysis of eosinophil red autofluorescence. Up to 50% of the eosinophils had undergone apoptosis 48 hours after addition of anti-CD69 MoAb compared with less than 10% apoptosis for CR3 or the isotype matched control. The majority of apoptotic eosinophils excluded trypan blue at 48 hours post CD69 ligation. More apoptotic eosinophils were observed at later time-points and this was associated with loss of viability. At 120 hours post-addition of the anti-CD69 MoAb MLR3, 24% +/- 10.6% eosinophils were viable compared with 84% +/- 3.4% for the CR3 control (P < .001). A F(ab)2 fragment of CD69 MoAb P8, also induced apoptosis in GM-CSF cultured eosinophils. A more rapid induction of eosinophil apoptosis was obtained with CD69 MoAb immobilized via their Fc portions on protein-A coated plastic 96 well plates. Ligation of CD69 or CR3 resulted in the release of comparable quantities of eosinophil peroxidase at 48 hours post-ligation. These levels of EPO were consistent with the viability of these cells at 48 hours as assessed by exclusion of trypan blue. Finally, a neutralizing MoAb to TGF beta 1 had no effect on CD69-dependent apoptosis induction nor were there detectable quantities of TGF beta 1 in supernatants from GM-CSF--cultured eosinophils ligated with CD69 or control MoAb. These results suggest that eosinophils cultured with GM-CSF can be induced to undergo apoptosis as a result of cell signalling mediated by perturbation of CD69. This may represent an important physiologic mechanism for eosinophil removal in vivo.
外周血（PB）嗜酸性粒细胞在体外会迅速发生凋亡和细胞死亡，除非在细胞因子如粒细胞-巨噬细胞集落刺激因子（GM-CSF）存在的情况下培养，此时它们的存活时间可延长至10天。CD69是一种II型膜抗原，由细胞因子激活的PB人嗜酸性粒细胞表达，但新鲜分离的细胞不表达。我们研究了用特异性单克隆抗体（MoAb）连接CD69对用重组人（rh）GM-CSF培养的人嗜酸性粒细胞活力的影响。嗜酸性粒细胞通过免疫磁选纯化，并与GM-CSF（10⁻¹⁰ mol/L）一起培养。培养开始18小时后，加入一组CD69 MoAb或对照（抗CR3或同型匹配对照MoAb）。通过台盼蓝拒染法评估活力，通过形态学评估、DNA梯状条带分析和嗜酸性粒细胞红色自发荧光的流式细胞术分析评估凋亡情况。加入抗CD69 MoAb后48小时，高达50%的嗜酸性粒细胞发生凋亡，而CR3或同型匹配对照的凋亡率不到10%。大多数凋亡的嗜酸性粒细胞在CD69连接后48小时拒染台盼蓝。在随后的时间点观察到更多凋亡的嗜酸性粒细胞，这与活力丧失有关。加入抗CD69 MoAb MLR3后120小时，24%±10.6%的嗜酸性粒细胞仍存活，而CR3对照为84%±3.4%（P <.001）。CD69 MoAb P8的F(ab)2片段也能诱导GM-CSF培养的嗜酸性粒细胞凋亡。通过将CD69 MoAb的Fc部分固定在蛋白A包被的塑料96孔板上，能更快速地诱导嗜酸性粒细胞凋亡。连接CD69或CR3后48小时，释放的嗜酸性粒细胞过氧化物酶量相当。这些EPO水平与通过台盼蓝拒染法评估的这些细胞在48小时的活力一致。最后，抗TGFβ1中和MoAb对CD69依赖性凋亡诱导没有影响，在用CD69或对照MoAb连接的GM-CSF培养的嗜酸性粒细胞上清液中也未检测到TGFβ1的量。这些结果表明，用GM-CSF培养的嗜酸性粒细胞可因CD69扰动介导的细胞信号传导而被诱导发生凋亡。这可能代表了体内嗜酸性粒细胞清除的一种重要生理机制。
