Induced sputum, but not BALF stimulated PMN ROS production is enhanced by ionomycin during COPD

Abstract

Reactive oxygen species (ROS) are important in COPD pathogenesis. Previously, we found that induced sputum (IS) and bronchoalveolar lavage fluid (BALF) stimulate ROS production in polymorphonuclear neutrophils (PMN). Ca2+ acts as a signal molecule during ROS synthesis. Still, effect of airway mucous according to intracellular Ca2+ is unknown. The aim – to evaluate a role of IS and BALF on ROS production in Ca2+ activated PMN. Methods. PMN were isolated by a density gradient from peripheral blood of 18 patients with moderate COPD and 10 healthy non-smoking individuals (HI). ROS production was induced by PMA (0.1-30 nM). Ionomycin (750 ng/ml) was added to increase intracellular Ca2+ concentration. IS or BALF pooled from 20 COPD patients was added to PMN suspensions (2x106/ml) alone (50%/RPMI); with ionomycin; with PMA (0.1-30 nM) or PMA/ionomycin. The relative ROS generation was measured by FACS using DHR-123 by fluorescence intensity in PMN population. Results. PMA and PMA/ionomycin induced higher ROS production in COPD group than in HI (p<0.05). IS and BALF itself stimulated ROS in COPD (38.7±3.9%, 30.1±4.3% respectively) and HI (23.7±4.8%, 15.5±2.5% respectively). Adding of ionomycin to IS enhanced ROS (COPD: 12.1±3.3%, HI: 6.1±2.1%, compared to stimulation without ionomycin); while to BALF – had no effect. IS/PMA/ionomycin stimulated ROS production and the max. increase was obtained at 3 nM PMA (COPD: 49.5±8.7%, HI: 28±4.4%, compared to stimulation without ionomycin). While, BALF/PMA/ionomycin had no effect compared to BALF/PMA in neither group. Conclusion. Ionomycin stimulates ROS production in PMN activated by IS, but not by BALF. This effect is stronger in PMN of COPD patients than HI.