Department of Medicine, Division of Allergy and Clinical Immunology, Johns Hopkins University School of Medicine, Baltimore, MD 21224, USA.
Cytokine. 2012 Jan;57(1):169-74. doi: 10.1016/j.cyto.2011.10.007. Epub 2011 Nov 12.
IL-33 activates eosinophils directly via the ST2 receptor. Like IL-5, IL-33 induces eosinophilia and eosinophilic airway inflammation in mouse models and primes human eosinophil responses. Previously, we reported that IL-5 priming enhances Siglec-8 mediated mitochondrial and reactive oxygen species (ROS)-dependent eosinophilic apoptosis and eliminates caspase dependence of this cell death process. Whether IL-33, like IL-5, augments pro-apoptotic pathways involving receptors such as Siglec-8 and in a similar manner has not been explored. Annexin-V labeling was performed to detect apoptosis in human eosinophils pre-incubated with or without a range of concentrations of IL-33 and/or IL-5 in the presence or absence of Siglec-8 monoclonal antibody (mAb) 2C4 and inhibitors of caspases. Tetramethyl-rhodamine staining was used as a marker of mitochondrial membrane potential loss and injury. ROS production was determined by measuring the superoxide dismutase-inhibitable reduction of cytochrome c. Cleavage of poly(ADP-ribose) polymerase (PARP) was assessed using Western blotting. Eosinophils cultured alone or with mAb 2C4 underwent low levels of apoptosis at 24h. 2C4-induced eosinophil apoptosis was markedly and equally enhanced after culture for 24h with either IL-33 or IL-5, although IL-5 was more potent. Effects on apoptosis with IL-33 and IL-5 were synergistic. In contrast, percentages of cells exhibiting reduced mitochondrial membrane potential were greater with IL-33 than IL-5 and effects of these cytokines were also synergistic. Antimycin, an inhibitor of mitochondrial electron transport, almost completely inhibited 2C4-induced apoptosis with either IL-33 or IL-5. Surprisingly, 2C4-induced eosinophil ROS production was significantly enhanced with IL-5 but not IL-33. Siglec-8-mediated apoptosis in the presence of IL-33 was more sensitive in magnitude than IL-5 to inhibition by the pan-caspase inhibitor Z-VAD-FMK, yet both cytokine conditions were associated with PARP cleavage. These data demonstrate that IL-33 is as effective but less potent than IL-5 in enhancing Siglec-8-mediated eosinophil apoptosis, and can synergize with IL-5. Eosinophils primed by IL-33 and/or IL-5 in vivo would be expected to display enhanced susceptibility to undergoing Siglec-8-induced apoptosis.
IL-33 通过 ST2 受体直接激活嗜酸性粒细胞。与 IL-5 一样，IL-33 在小鼠模型中诱导嗜酸性粒细胞增多和嗜酸性气道炎症，并激活人嗜酸性粒细胞的反应。此前，我们报道 IL-5 可增强 Siglec-8 介导的线粒体和活性氧（ROS）依赖性嗜酸性粒细胞凋亡，并消除该细胞死亡过程对半胱天冬酶的依赖。但是否像 IL-5 一样，IL-33 增强涉及 Siglec-8 等受体的促凋亡途径以及以类似的方式尚未得到探索。用人嗜酸性粒细胞预先孵育一系列浓度的 IL-33 和/或 IL-5，在存在或不存在 Siglec-8 单克隆抗体（mAb）2C4 和半胱天冬酶抑制剂的情况下，通过 Annexin-V 标记检测细胞凋亡。使用四甲基罗丹明作为线粒体膜电位丧失和损伤的标志物。通过测量超氧化物歧化酶抑制的细胞色素 c 还原来确定 ROS 的产生。使用 Western blot 评估多聚（ADP-核糖）聚合酶（PARP）的切割。单独培养或用 mAb 2C4 培养的嗜酸性粒细胞在 24 小时时发生低水平的凋亡。用 IL-33 或 IL-5 培养 24 小时后，2C4 诱导的嗜酸性粒细胞凋亡明显且同等增强，尽管 IL-5 更强效。IL-33 和 IL-5 对凋亡的影响具有协同作用。相比之下，IL-33 引起的减少的线粒体膜电位百分比大于 IL-5，并且这些细胞因子的作用也是协同的。抗霉素，一种线粒体电子传递抑制剂，几乎完全抑制了 IL-33 或 IL-5 诱导的 2C4 凋亡。令人惊讶的是，IL-5 但不是 IL-33 可显著增强 2C4 诱导的嗜酸性粒细胞 ROS 产生。在 IL-33 存在下，Siglec-8 介导的凋亡对泛半胱天冬酶抑制剂 Z-VAD-FMK 的敏感性大于 IL-5，但两种细胞因子条件均与 PARP 切割有关。这些数据表明，IL-33 与 IL-5 一样有效，但作用较弱，可增强 Siglec-8 介导的嗜酸性粒细胞凋亡，并与 IL-5 协同作用。体内由 IL-33 和/或 IL-5 诱导的嗜酸性粒细胞预计会表现出对 Siglec-8 诱导的凋亡的增强易感性。
