Alterations of neutrophil oxidative burst by in vitro smoke exposure: implications for oral and systemic diseases.

Alterations of neutrophil functions by tobacco products may play a central role in the pathogenesis of periodontal diseases and several smoking-related systemic diseases. In the present study, we examined the in vitro effects of cigarette smoke on neutrophils at times and concentrations that may be encountered during smoke exposure. We measured the level of smoke exposure in the in vitro system by measuring the levels of nicotine and comparing these to levels in the oral cavity in smokers before and after smoking. We examined both the unstimulated and stimulated release of 2 oxidative burst products: superoxide (O-2) and hydrogen peroxide (H2O2). Salivary washings were collected from 7 smokers (> 1 pack/day) before smoking a cigarette. Immediately after they smoked a cigarette, a second set of washings was collected. In vitro exposure to smoke involved incubating aliquots of neutrophils in phosphate-buffered saline for 1 to 5 minutes. Nicotine and cotinine levels were quantitated using gas chromatography, with detection by electron impact mass spectrometry. Peripheral neutrophils were isolated from medically healthy non-smoking volunteers via a double-density gradient technique and incubated in vitro with whole cigarette smoke for 0 to 5 minutes. Phorbol myristate acetate (PMA; 10(-7) M) was used to stimulate half of the cell aliquots. Superoxide generation was assessed through the superoxide dismutase (SOD) inhibitable reduction of ferricytochrome c. H2O2 production was assessed through the H2O2-dependent breakdown of dichlorofluorescin diacetate to its fluorophore and measured by flow cytometry. There was a marked elevation in salivary nicotine concentration from before smoking (mean: 80.8 ng) to after smoking (mean 1,685 ng/mL). In the in vitro smoke box system, there was a time-related elevation in nicotine from 1 to 5 minutes (50-->136 ng/mL). In PMA-stimulated cells exposed to smoke, there was a time-related inhibition of both superoxide and H2O2 production. However, in unstimulated cells exposed to smoke, there was a time-related increase in the release of superoxide and H2O2. A novel finding in unstimulated cells exposed to smoke was that there appeared to be 2 distinct populations of cells--one of "high" H2O2 producers and one of "low" H2O2 producers. The proportion of high H2O2 producers increased relative to smoke exposure. The relative production of H2O2 in the unstimulated high producers was comparable to PMA-stimulated cells at 5 minutes. This release of superoxide and H2O2 in unstimulated cells exposed to smoke may alter the pathogenic processes both in periodontal diseases and other systemic diseases.