Lee Y A, Shin M H
Department of Environmental Medical Biology, Institute of Tropical Medicine, Yonsei University College of Medicine, Seoul, Korea.
J Investig Allergol Clin Immunol. 2009;19(3):188-94.
Eosinophils are important effector cells in the pathogenesis of allergic diseases such as bronchial asthma. Oxidative stress in the form of cellular reactive oxygen species (ROS) has been implicated in the pathogenesis of several allergic diseases. Recently, it has become evident that mitochondrial-derived ROS are important transducers of apoptosis and intracellular signaling. In this study, we investigated the role of mitochondrial ROS in the activation of extracellular signal-regulated kinases (ERK) 1 and 2-mitogen-activated protein kinase (MAPK) and caspase-3 in human eosinophils stimulated with H2O2.
Human eosinophils were purified using immunomagnetic negative selection and then stimulated with H2O. H2O2-induced eosinophil apoptosis was measured by staining cells with annexin V. Activation of ERK1/2 MAPK and caspases was assessed by Western blotting. Eosinophils were pretreated with rotenone, an inhibitor of the mitochondrial electron transport chain, before H2O2 was added.
Treatment with 1 mM H2O2 induced externalization of phosphatidylserine (PS) and activation of caspases in eosinophils. H2O2-triggered PS externalization and cleavage of caspase-3 were markedly inhibited by pretreatment with the mitochondrial ROS scavenger N-acetyl-L-cysteine. In addition, H2O2 strongly induced phosphorylation of ERK1/2, but not ERK5, in eosinophils. Hydrogen peroxide-triggered activation of caspase-3 and ERK1/2 was attenuated by pretreatment with rotenone.
These results suggest that mitochondrial respiration is essential for activation of ERK1/2 and caspase-3 in human eosinophils stimulated with H2O2.
嗜酸性粒细胞是支气管哮喘等过敏性疾病发病机制中的重要效应细胞。细胞活性氧（ROS）形式的氧化应激与多种过敏性疾病的发病机制有关。最近，线粒体衍生的ROS已成为细胞凋亡和细胞内信号传导的重要转导因子。在本研究中，我们调查了线粒体ROS在过氧化氢刺激的人嗜酸性粒细胞中细胞外信号调节激酶（ERK）1和2-丝裂原活化蛋白激酶（MAPK）以及半胱天冬酶-3激活中的作用。
使用免疫磁珠阴性选择法纯化人嗜酸性粒细胞，然后用H2O刺激。用膜联蛋白V染色细胞来检测H2O2诱导的嗜酸性粒细胞凋亡。通过蛋白质印迹法评估ERK1/2 MAPK和半胱天冬酶的激活情况。在添加H2O2之前，先用线粒体电子传递链抑制剂鱼藤酮预处理嗜酸性粒细胞。
用1 mM H2O2处理可诱导嗜酸性粒细胞中磷脂酰丝氨酸（PS）的外化和半胱天冬酶的激活。线粒体ROS清除剂N-乙酰-L-半胱氨酸预处理可显著抑制H2O@触发的PS外化和半胱天冬酶-3的切割。此外，H2O2强烈诱导嗜酸性粒细胞中ERK1/2的磷酸化，但不诱导ERK5的磷酸化。鱼藤酮预处理可减弱过氧化氢触发的半胱天冬酶-3和ERK1/2的激活。
这些结果表明，线粒体呼吸对于过氧化氢刺激的人嗜酸性粒细胞中ERK1/2和半胱天冬酶-3的激活至关重要。
