Chloride Channel 3 Channels in the Activation and Migration of Human Blood Eosinophils in Allergic Asthma

Abstract

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.

ClC-3 promotes angiotensin II-induced reactive oxygen species production in endothelial cells by facilitating Nox2 NADPH oxidase complex formation

“…Quantification data are presented as the mean ± SEM in relative units of this work, however, is that these changes induced by Ang II are significantly inhibited by ClC-3 knockdown and further potentiated by ClC-3 overexpression, suggesting the requirement of ClC-3 for NADPH oxidase-derived ROS generation in endothelial cells. Over the past decade, the correlation between ClC-3 and intracellular ROS generation has been suggested in several studies, in which ClC-3 has been shown to be involved in Noxdependent oxidative stress in vascular smooth muscle cells, neutrophils, eosinophils and endothelial progenitor cells [10][11][12][13]. The present study, to our knowledge, is the first to provide evidence that Ang II-induced Nox activity and signaling require ClC-3 in endothelial cells.…”

“…Deficiency in ClC-3 inhibits Nox1-derived ROS production induced by cytokines in the endosomes of vascular smooth muscle cells [11]. The dependence of NADPH oxidase activity on ClC-3 was also recently described in human blood eosinophils [12]. Consistent with these findings, our recent study showed that a lack of ClC-3 inhibited angiotensin II (Ang II)induced endothelial progenitor cell apoptosis by suppressing ROS generation from NADPH oxidase [13].…”

“…An important question that remains to be answered is how Nox is regulated by ClC-3. Although the previous studies mentioned the above reported modulation of Nox-derived ROS generation by ClC-3 [10][11][12][13], the underlying mechanisms remained to be determined. It has been speculated that ClC-3 might work to compensate for the charge imbalance generated by O 2 − production from Nox1 and prevent the accumulation of negative charges in the endosomes of vascular smooth muscle cells [11].…”

The R213G polymorphism in SOD3 protects against allergic airway inflammation

“…Allergen exposure drives oxidative stress by causing airway epithelium and smooth muscle cells to produce ROS, particularly O 2 -, through activation of NADPH oxidase (4,5). Other sources of ROS include inflammatory cells like eosinophils (6), neutrophils (7), and monocytes/macrophages (8). Asthmatics have increased hydrogen peroxide (9) and nitric oxide (10) as exhaled gases supporting the role of oxidative stress in asthma.…”

Interleukin‐5 drives glycolysis and reactive oxygen species‐dependent citric acid cycling by eosinophils

“…Furthermore, the initiation of apoptosis has been reported as an alternative role to respiration for eosinophil mitochondria . As eosinophils produce large amounts of nicotinamide adenine dinucleotide phosphate oxidase (NOX)2‐dependent extracellular reactive oxygen species (ROS) upon activation, it is commonly thought that oxygen consumption by eosinophils supports ROS production rather than oxidative phosphorylation (OXPHOS). Contrary to this, human eosinophils are sensitive to oligomycin (mitochondrial ATP synthase inhibitor), suggesting that in addition to glycolysis, mitochondria can indeed contribute, at least in part, to ATP production .…”