Gaurav Rohit, Bewtra Againdra K, Agrawal Devendra K
Department of Biomedical Sciences, Internal Medicine and Center of Clinical and Translational Sciences, Creighton University School of Medicine, Omaha, Nebraska.
Am J Respir Cell Mol Biol. 2015 Aug;53(2):235-45. doi: 10.1165/rcmb.2014-0300OC.
Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is responsible for respiratory burst in immune cells. Chloride channel 3 (CLC3) has been linked to the respiratory burst in eosinophils and neutrophils. The effect of cytokines and the involvement of CLC3 in the regulation of NADPH-dependent oxidative stress and on cytokine-mediated migration of eosinophils are not known. Human peripheral blood eosinophils were isolated from healthy individuals and from individuals with asthma by negative selection. Real-time PCR was used to detect the expression of NADPH oxidases in eosinophils. Intracellular reactive oxygen species (ROS) measurement was done with flow cytometry. Superoxide generation was measured with transforming growth factor (TGF)-β, eotaxin, and CLC3 blockers. CLC3 dependence of eosinophils in TGF-β- and eotaxin-induced migration was also examined. The messenger RNA (mRNA) transcripts of NADPH oxidase (NOX) 2, dual oxidase (DUOX) 1, and DUOX2 were detected in blood eosinophils, with very low expression of NOX1, NOX3, and NOX5 and no NOX4 mRNA. The level of NOX2 mRNA transcripts increased with disease severity in the eosinophils of subjects with asthma compared with healthy nonatopic volunteers. Change in granularity and size in eosinophils, but no change in intracellular ROS, was observed with phorbol myristate acetate (PMA). PMA, TGF-β, and eotaxin used the CLC3-dependent pathway to increase superoxide radicals. TGF-β and eotaxin induced CLC3-dependent chemotaxis of eosinophils. These findings support the requirement of CLC3 in the activation and migration of human blood eosinophils and may provide a potential novel therapeutic target to regulate eosinophil hyperactivity in allergic airway inflammation in asthma.
烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶负责免疫细胞中的呼吸爆发。氯离子通道3（CLC3）与嗜酸性粒细胞和中性粒细胞的呼吸爆发有关。细胞因子的作用以及CLC3在NADPH依赖性氧化应激调节和细胞因子介导的嗜酸性粒细胞迁移中的参与情况尚不清楚。通过阴性选择从健康个体和哮喘患者中分离出人外周血嗜酸性粒细胞。采用实时聚合酶链反应（PCR）检测嗜酸性粒细胞中NADPH氧化酶的表达。用流式细胞术测量细胞内活性氧（ROS）。用转化生长因子（TGF）-β、嗜酸性粒细胞趋化因子和CLC3阻滞剂测量超氧化物的产生。还研究了嗜酸性粒细胞在TGF-β和嗜酸性粒细胞趋化因子诱导的迁移中对CLC3的依赖性。在血液嗜酸性粒细胞中检测到了NADPH氧化酶（NOX）2、双氧化酶（DUOX）1和DUOX2的信使核糖核酸（mRNA）转录本，NOX1、NOX3和NOX5的表达非常低，且未检测到NOX4 mRNA。与健康的非特应性志愿者相比，哮喘患者嗜酸性粒细胞中NOX2 mRNA转录本的水平随疾病严重程度增加。用佛波酯肉豆蔻酸酯（PMA）处理后，嗜酸性粒细胞的粒度和大小发生变化，但细胞内ROS无变化。PMA、TGF-β和嗜酸性粒细胞趋化因子利用依赖CLC3的途径增加超氧自由基。TGF-β和嗜酸性粒细胞趋化因子诱导嗜酸性粒细胞的CLC3依赖性趋化作用。这些发现支持了CLC3在人血嗜酸性粒细胞激活和迁移中的必要性，并可能为调节哮喘过敏性气道炎症中嗜酸性粒细胞的过度活性提供一个潜在的新治疗靶点。
