BACKGROUND Siglec-8 is a CD33 subfamily cell-surface receptor selectively expressed on human eosinophils. After cytokine priming, Siglec-8 mAb or glycan ligand binding causes eosinophil apoptosis associated with reactive oxygen species (ROS) production. Most CD33-related Siglecs function as inhibitory receptors, but the ability of Siglec-8 to stimulate eosinophil ROS production and apoptosis suggests that Siglec-8 might instead function as an activating receptor. OBJECTIVE We sought to determine the role of IL-5 priming and identify the signaling molecules involved in Siglec-8 function for human eosinophils. METHODS We used an mAb and/or a multimeric synthetic sulfated sialoglycan ligand recognizing Siglec-8 in combination with integrin blocking antibodies, pharmacologic inhibitors, phosphoproteomics, and Western blot analysis to define the necessity of various proteins involved in Siglec-8 function for human eosinophils. RESULTS Cytokine priming was required to elicit the unanticipated finding that Siglec-8 engagement promotes rapid β2-integrin-dependent eosinophil adhesion. Also novel was the finding that this adhesion was necessary for subsequent ROS production and apoptosis. Siglec-8-mediated ROS was generated through reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation because pretreatment of eosinophils with catalase (an extracellular superoxide scavenger) or NSC 23766 (a Rac GTPase inhibitor) completely inhibited Siglec-8-mediated eosinophil apoptosis. Finally, engagement of Siglec-8 on IL-5-primed eosinophils resulted in increased phosphorylation of Akt, p38, and c-Jun N-terminal kinase 1 that was also β2-integrin dependent; pharmacologic inhibition of these kinases completely prevented Siglec-8-mediated eosinophil apoptosis. CONCLUSIONS These data demonstrate that Siglec-8 functions uniquely as an activating receptor on IL-5-primed eosinophils through a novel pathway involving regulation of β2-integrin-dependent adhesion, NADPH oxidase, and a subset of protein kinases.

中文翻译：

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结合唾液酸的免疫球蛋白样凝集素8（Siglec-8）是激活受体，介导人类嗜酸性粒细胞的β2-整联蛋白依赖性功能。

背景技术Siglec-8是在人类嗜酸性粒细胞上选择性表达的CD33亚家族细胞表面受体。启动细胞因子后，Siglec-8 mAb或聚糖配体结合会导致与活性氧（ROS）产生相关的嗜酸性粒细胞凋亡。大多数与CD33相关的Siglecs均起抑制受体的作用，但是Siglec-8刺激嗜酸性粒细胞ROS产生和凋亡的能力表明，Siglec-8可能起激活受体的作用。目的我们试图确定IL-5引发的作用，并鉴定参与人类嗜酸性粒细胞的Siglec-8功能的信号分子。方法我们将识别Siglec-8的mAb和/或多聚体合成的硫酸化唾液酸聚糖配体与整联蛋白阻断抗体，药理抑制剂，磷酸化蛋白质组学，免疫印迹和蛋白质印迹分析来确定与人类嗜酸性粒细胞Siglec-8功能有关的各种蛋白质的必要性。结果需要细胞因子引发来引发意想不到的发现，即Siglec-8参与促进快速的β2-整合素依赖性嗜酸性粒细胞粘附。同样新颖的发现是该粘附对于随后的ROS产生和细胞凋亡是必需的。Siglec-8介导的ROS是通过减少烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶的活化而产生的，因为用过氧化氢酶（一种细胞外超氧化物清除剂）或NSC 23766（一种Rac GTPase抑制剂）预处理嗜酸性粒细胞会完全抑制Siglec-8介导的嗜酸性粒细胞凋亡。最后，Siglec-8与IL-5引发的嗜酸性粒细胞结合导致Akt，p38，和c-Jun N-末端激酶1，其也是β2-整联蛋白依赖性的；这些激酶的药理学抑制作用完全阻止了Siglec-8介导的嗜酸性粒细胞凋亡。结论这些数据表明，Siglec-8通过涉及调节β2-整合素依赖性粘附，NADPH氧化酶和一部分蛋白激酶的新型途径，在IL-5引发的嗜酸性粒细胞中作为激活受体发挥独特作用。