Kano Gen, Bochner Bruce S, Zimmermann Nives
Division of Allergy and Immunology, Cincinnati Children's Hospital Medical Center, United States; Department of Pediatrics, Kyoto Prefecture University of Medicine, Japan.
Division of Allergy-Immunology, Northwestern University Feinberg School of Medicine, United States.
Immunobiology. 2017 Feb;222(2):343-349. doi: 10.1016/j.imbio.2016.09.006. Epub 2016 Sep 20.
Siglec-8 is a surface receptor predominantly expressed on human eosinophils where its ligation induces reactive oxygen species (ROS) formation and cell death. Since Siglec-8 has intracellular tyrosine-based motifs, we hypothesized that Src family kinases (SFKs) are involved in ROS formation and cell death induced by Siglec-8 engagement.
Human peripheral blood eosinophils were purified and incubated with anti-Siglec-8 monoclonal antibodies (mAb, agonist), IL-5, and SFK pharmacological inhibitors. We focused on Siglec-8-induced cell death in short-term IL-5-activated cells leading to a regulated necrosis-type cell death. ROS production was determined by dihydrorhodamine (DHR) 123 labeling and flow cytometry, or by chemiluminescence using Amplex red. Activation of SFK was determined using phospholuminex and Western blotting.
In order to determine cellular localization of ROS production, we measured intra and extracellular ROS. While an ETosis stimulus (calcium ionophore A23187) led to extracellular ROS (ecROS) production, Siglec-8-engagement in short-term IL-5 activated cells led to intracellular ROS (icROS) accumulation. Consistently, inhibition of extracellular ROS by catalase inhibited ETosis, but not IL-5-primed Siglec-8-induced cell death. In order to determine signaling events for Siglec-8, we performed Western blotting and found SFK phosphorylation in lysates from eosinophils stimulated with anti-Siglec-8 mAb±IL-5. In order to identify which SFKs were involved, we used the phospholuminex assay and found increased levels of phosphorylated Fgr in the cytoplasmic fraction of cells co-stimulated with anti-Siglec-8 and IL-5 for 3 hours compared with cells stimulated with IL-5 alone. To test the involvement of SFKs in ROS production and cell death, we used SFK inhibitors PP2 and dasatinib, both of which completely inhibited eosinophil ROS production and cell death induced by anti-Siglec-8 and IL-5 co-stimulation.
Siglec-8 engagement in short-term IL-5-activated eosinophils causes icROS production and SKF phosphorylation, and both are essential in mediating Siglec-8-induced cell death.
唾液酸结合免疫球蛋白样凝集素8（Siglec-8）是一种主要在人类嗜酸性粒细胞上表达的表面受体，其结合可诱导活性氧（ROS）形成和细胞死亡。由于Siglec-8具有基于酪氨酸的细胞内基序，我们推测Src家族激酶（SFK）参与了Siglec-8结合诱导的ROS形成和细胞死亡。
纯化人类外周血嗜酸性粒细胞，并用抗Siglec-8单克隆抗体（mAb，激动剂）、白细胞介素-5（IL-5）和SFK药理抑制剂进行孵育。我们重点研究了Siglec-8在短期IL-5激活的细胞中诱导的细胞死亡，这种死亡导致一种调节性坏死型细胞死亡。通过二氢罗丹明（DHR）123标记和流式细胞术，或使用Amplex red通过化学发光法测定ROS产生。使用磷酸荧光素酶检测和蛋白质免疫印迹法测定SFK的激活情况。
为了确定ROS产生的细胞定位，我们测量了细胞内和细胞外的ROS。虽然一种嗜酸性粒细胞脱颗粒刺激物（钙离子载体A23187）导致细胞外ROS（ecROS）产生，但Siglec-8在短期IL-5激活的细胞中的结合导致细胞内ROS（icROS）积累。一致地，过氧化氢酶抑制细胞外ROS可抑制嗜酸性粒细胞脱颗粒，但不能抑制IL-5预处理的Siglec-8诱导的细胞死亡。为了确定Siglec-8的信号转导事件，我们进行了蛋白质免疫印迹，发现在用抗Siglec-8 mAb±IL-5刺激的嗜酸性粒细胞裂解物中有SFK磷酸化。为了确定涉及哪些SFK，我们使用了磷酸荧光素酶检测法，发现与单独用IL-5刺激的细胞相比，在用抗Siglec-8和IL-5共同刺激3小时的细胞的细胞质部分中，磷酸化Fgr水平升高。为了测试SFK在ROS产生和细胞死亡中的作用，我们使用了SFK抑制剂PP2和达沙替尼，两者均完全抑制了抗Siglec-8和IL-5共同刺激诱导的嗜酸性粒细胞ROS产生和细胞死亡。
Siglec-8在短期IL-5激活的嗜酸性粒细胞中的结合导致icROS产生和SKF磷酸化，两者在介导Siglec-8诱导的细胞死亡中均至关重要。
