Neutrophil superoxide production in the presence of cigarette smoke extract, nicotine and cotinine

Abstract

To determine the effect of cigarette smoke extract, nicotine and cotinine on lucigenin-detectable neutrophil superoxide production. Neutrophils from periodontally healthy individuals were treated with aqueous smoke extract, nicotine and cotinine, prior to stimulation or at the same time as stimulation with Fusobacterium nucleatum, IgG-opsonized Staphylococcus aureus and Escherichia coli Lipopolysaccharide (LPS). Superoxide generation was determined by lucigenin chemiluminescence. Smoke extract induced superoxide release from neutrophils (p <0.0001) in a dose-dependent manner. By contrast, superoxide generation by neutrophils in response to pathologically relevant stimuli was inhibited by pre-treatment with smoke extract (p <0.01). This inhibition did not require the continued presence of the extract. A similar reduction in stimulated superoxide production by smoke extract was detected when neutrophils were simultaneously exposed to the extract and stimuli. Nicotine and cotinine (0-10 μg/ml) had no effect on superoxide release from unstimulated or stimulated neutrophils. Stable water-soluble components of cigarette smoke directly induce superoxide generation by otherwise unstimulated neutrophils, but reduce superoxide responses of cells to pathologically relevant stimuli. These data suggest potential neutrophil-mediated mechanisms by which smoking may initiate and maintain oxidative stress at periodontally healthy sites and participate in disease progression, by reducing innate immune responses.