Nicotine effects on neutrophil F-actin formation and calcium release: implications for tobacco use and pulmonary diseases.

Abstract

Alterations in neutrophil functions by tobacco components may play a pivotal role in pulmonary emphysema. This study examined the role of nicotine in altering F-actin formation and calcium (Ca2+) release (two early events in neutrophil motility). The effects of these alterations on the motile function of phagocytosis were also examined. Human peripheral neutrophils from medically healthy nonsmoking subjects were incubated with nicotine at concentrations normally encountered during acute exposure to cigarette smoke (10(-2) to 10(-5) M) and/or the chemotactic peptide FLPEP (10(-7) M). Relative F-actin stain was determined by NBD phallacidin staining followed by flow cytometry. Intracellular Ca2+ was determined by INDO-1 AM loading followed by emission ratio quantitation by fluorometry. Phagocytosis was determined by the % phagocytic cells with carboxylated microspheres. Incubation of neutrophils with varying concentrations of nicotine resulted in a significant elevation of the relative F-actin stain at 30 s at 10(-2) and 10(-3) M (p < .05, ANOVA) and at 30 min at 10(-2) to 10(-4) M (p < 0.05). In time course studies with 10(-7) M FLPEP stimulation, there was a approximately 325% rise in relative F-actin stain at 30-60 s, followed by a gradual decrease to near baseline levels. There was an immediate rise in Ca2+ to approximately 150% over baseline values, followed by a gradual decrease to baseline. By contrast, stimulation with nicotine demonstrated a approximately 105% increase in relative F-actin staining at 10(-2) M (p < .001, ANOVA) and a smaller increase at 10(-3) M, which remained elevated up to 600 s. Intracellular Ca2+ levels also rose in a dose-dependent manner with an increased of 700% over baseline with 10(-2) M nicotine, and remained elevated up to 600 s. Coincubation with both FLPEP and nicotine demonstrated additive effects in relative F-actin staining at both maximal and submaximal concentrations. Preincubation with 10(-2) or 10(-3) M nicotine suppressed the % phagocytic cells by 32% and 16%, respectively (p < .001, ANOVA) with only a 1-4% reduction in cell viability (trypan blue exclusion). The results demonstrate that the concentration of nicotine during acute cigarette exposure can directly stimulate neutrophil F-actin formation and intracellular Ca2+ release by a mechanism different from peptide stimulation. The alteration of these two pivotal neutrophil signaling events by nicotine may in turn alter other neutrophil functions in tobacco-related pulmonary emphysema.