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.