Division of Allergy and Immunology, Cincinnati Children's Hospital, and the Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.
J Allergy Clin Immunol. 2013 Aug;132(2):437-45. doi: 10.1016/j.jaci.2013.03.024. Epub 2013 May 16.
Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is expressed on human eosinophils, where its ligation induces cell death. Paradoxically, Siglec-8-mediated cell death is markedly enhanced by the presence of the activation and survival factor IL-5 and becomes independent of caspase activity.
In this report we investigate the mechanism of Siglec-8-mediated cell death in activated eosinophils.
Human peripheral blood eosinophils were treated with agonistic anti-Siglec-8 antibody and IL-5, and cell death was determined by using flow cytometry and morphology. Phosphorylation of mitogen-activated protein kinase (MAPK) was determined by using phosphoLuminex, flow cytometry, and Western blotting. Reactive oxygen species (ROS) accumulation was determined by using dihydrorhodamine fluorescence.
Costimulation with anti-Siglec-8 and IL-5 significantly increased the rate and proportion of cell death by means of necrosis accompanied by granule release compared with that seen after stimulation with anti-Siglec-8 alone, in which apoptosis predominated. Together with the caspase-independent mode of cell death in costimulated cells, these findings suggest the activation of a specific and distinct biochemical pathway of cell death during anti-Siglec-8/IL-5 costimulation. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and MAPK-ERK kinase (MEK) 1 was significantly enhanced and sustained in costimulated cells compared with that seen in cells stimulated with IL-5 alone; anti-Siglec-8 alone did not cause ERK1/2 phosphorylation. MEK1 inhibitors blocked anti-Siglec-8/IL-5-induced cell death. ROS accumulation was induced by Siglec-8 ligation in a MEK-independent manner. In contrast, an ROS inhibitor prevented the anti-Siglec-8/IL-5-induced enhancement of ERK phosphorylation and cell death. Exogenous ROS mimicked stimulation by anti-Siglec-8 and was sufficient to induce enhanced cell death in IL-5-treated cells. Collectively, these data suggest that the enhancement of ERK phosphorylation is downstream of ROS generation.
In activated eosinophils ligation of Siglec-8 leads to ROS-dependent enhancement of IL-5-induced ERK phosphorylation, which results in a novel mode of biochemically regulated eosinophil cell death.
唾液酸结合免疫球蛋白样凝集素（Siglec）-8 表达于人类嗜酸性粒细胞，其配体的结合可诱导细胞死亡。但矛盾的是，Siglec-8 介导的细胞死亡在激活和存活因子 IL-5 的存在下显著增强，并变得不依赖于半胱天冬酶活性。
本报告研究了 Siglec-8 介导的激活嗜酸性粒细胞细胞死亡的机制。
用激动性抗 Siglec-8 抗体和 IL-5 处理人外周血嗜酸性粒细胞，通过流式细胞术和形态学确定细胞死亡。通过磷酸化 Luminex、流式细胞术和 Western blot 确定丝裂原活化蛋白激酶（MAPK）的磷酸化。通过二氢罗丹明荧光测定活性氧（ROS）的积累。
与单独用抗 Siglec-8 刺激相比，抗 Siglec-8 和 IL-5 的共同刺激显著增加了坏死伴随颗粒释放的细胞死亡率和比例，而凋亡占主导地位。在共刺激细胞中，与细胞死亡的半胱天冬酶非依赖性模式一起，这些发现表明在抗 Siglec-8/IL-5 共刺激期间，细胞死亡的特定和不同生化途径被激活。与单独用 IL-5 刺激相比，共刺激细胞中 ERK1/2 和 MAPK-ERK 激酶（MEK）1 的磷酸化显著增强且持续，而单独用抗 Siglec-8 则不会引起 ERK1/2 磷酸化。MEK1 抑制剂阻断抗 Siglec-8/IL-5 诱导的细胞死亡。ROS 的积累是由 Siglec-8 配体以 MEK 非依赖性方式诱导的。相反，ROS 抑制剂可防止抗 Siglec-8/IL-5 诱导的 ERK 磷酸化和细胞死亡增强。外源性 ROS 模拟抗 Siglec-8 的刺激，足以诱导 IL-5 处理的细胞中增强的细胞死亡。总的来说，这些数据表明 ERK 磷酸化的增强是 ROS 产生的下游事件。
在激活的嗜酸性粒细胞中，Siglec-8 的结合导致 ROS 依赖性增强的 IL-5 诱导的 ERK 磷酸化，从而导致一种新型的嗜酸性粒细胞细胞死亡的生化调节模式。
