Ionomycin stimulates a synergistic effect of IL-8 and induced sputum on PMN chemotaxis during COPD

Abstract

Migration of polymorphonuclear neutrophils (PMN) from blood to the airway is increased during COPD. This process is regulated by chemotactic factors and intracellular Ca2+ concentration, as well. The role of chemokines and airway mucous in PMN activated by increased intracellular Ca2+ is unknown. The aim was to evaluate a role of both IL-8 in combination with induced sputum (IS) and increased Ca2+ on PMN chemotaxis. Methods. PMN were isolated by high density gradient from peripheral blood of 21 patients with moderate COPD and 10 healthy non-smoking individuals (HI). PMN chemotaxis was evaluated in 10-well chemotaxis chamber. The lower and upper wells were separated with polycarbonate membrane. Ionomycin (750 ng/ml) was added to increase intracellular Ca2+ concentration. IL-8 (10, 30, 100 ng/ml) and a pool of IS from 20 COPD (50%/RPMI) were used to stimulate PMN chemotaxis. The lower wells were filled with: (1) IL-8 or IL-8/ionomycin, (2) IS or IS/ionomycin (3) IL-8/IS or IL-8/IS/ionomycin. The upper wells were filled with 2.5x106/ml PMN and incubated for 2h. PMN chemotaxis was evaluated by FACS using reference cell beads. Results. Ionomycin enhanced IL-8 (max. 100 ng/ml: COPD: 79±5% vs 63±4%, HI: 42±5% vs 36±3% p<0.05) and IS (COPD: 60±7% vs 45±5%, HI: 32±6% vs 17±4% p<0.05) stimulated PMN chemotaxis. IL-8 (30-100 ng/ml)/IS induced a higher PMN migration than IL-8 or IS alone (p<0.05). The highest PMN chemotaxis was induced by combination of IL-8 (30 ng/ml)/IS/ionomycin both in COPD group (91±3%) and HI (78±3%). Conclusions. IL-8 and IS acted synergistically on PMN chemotaxis. Ionomycin enhanced a synergistic effect of IL-8 and IS and it was higher in COPD group.