The effects of N-acetylcysteine and glutathione on smoke-induced changes in lung phagocytes and epithelial cells.

We studied the mechanism of the delay in neutrophil traffic in pulmonary microvasculature previously observed during cigarette smoking, the effect of cigarette smoke on lung phagocytes and epithelial cell function, and augmentation of the glutathione (GSH) antioxidant system using the thiol drug N-acetylcysteine. Using a micropore membrane system to mimic the dimensions of the average pulmonary capillary, we showed that cigarette smoke reduces cell deformability, increasing the difficulty experienced by the larger neutrophils in negotiating the smaller capillary segments, so delaying their passage during smoking. This effect is both diminished and recoverable by the addition of plasma, and by GSH in concentrations found in plasma. Cigarette smoke induces oxidative changes in both the cell membrane and the cell cytoskeleton, and diminishes the ability of neutrophils to release reactive oxygen intermediates. The injurious effect of oxidants can be measured in vitro by the detachment of 51Cr-radiolabeled alveolar epithelial cells grown in monolayers, an effect also diminished by the addition of GSH. Such epithelial cell detachment in vitro may be reflected as the epithelial permeability that occurs at an early stage in asymptomatic smokers. N-Acetylcysteine given orally (600 mg/day) increases both plasma and bronchoalveolar lavage GSH in normal subjects, but a sustained increase in plasma GSH requires higher dosage regimens in patients with chronic obstructive pulmonary disease (600 mg three times daily). Thus, the potential exists to enhance the antioxidant status of both plasma and the airspaces of the lungs against oxidant-induced injury.

Effect of cigarette smoke and its condensates on alveolar epithelial cell injury in vitro.

The oxidant-antioxidant balance in the airspaces of the lungs may be critical in protecting the lungs from the effects of cigarette smoke. We studied the effect of cigarette smoke and its condensates on the detachment, attachment, and proliferation of the A549 human alveolar epithelial cell line, in an in vitro model of cell injury and regeneration and the protective effects of antioxidants. Whole and vapor phase cigarette smoke decreased 51Cr-labeled A549 cell attachment, increased cell detachment, and decreased cell proliferation, as assessed by [3H]thymidine uptake. Freshly isolated rat type II alveolar epithelial cells showed an enhanced susceptibility to smoke-induced cell lysis when compared with the A549 cell line. Reduced glutathione (GSH) (400 microM) protected against the effects of cigarette smoke exposure on cell attachment, proliferation, and detachment. Depletion of intracellular GSH with buthionine sulfoxamine enhanced the epithelial cell detachment injury produced by smoke condensates. We conclude that cigarette smoke and its condensates cause an oxidant-induced injury to A549 human type II alveolar epithelial cells. Both intra- and extracellular GSH have important roles in protecting epithelial cells from the injurious effects of cigarette smoke.

Epithelial permeability, inflammation, and oxidant stress in the air spaces of smokers.

The mechanism responsible for the increased air-space permeability in cigarette smokers is unknown. The aim of this study was to assess the acute and chronic effects of cigarette smoking on epithelial permeability, inflammation, and oxidant stress in the air spaces of smokers. Fourteen cigarette smokers underwent 99mTc-diethylenetriamine pentaacetic acid (99mTc-DTPA) lung scans after abstaining from smoking for 12 h (chronic smoking) and 1 h after smoking two cigarettes (acute smoking). Each smoker also underwent bronchoscopy and bronchoalveolar lavage (BAL) after either chronic (n = 8) or acute smoking (n = 7). Seven nonsmokers also underwent bronchoscopy and BAL. The time to 50% clearance of 99mTc-DTPA (t50) after chronic smoking was 16.7 +/- 1. 3 min (mean +/- SE), and was further reduced after acute smoking to 14.8 +/- 1.0 min (p < 0.01). Neutrophil numbers were increased in bronchoalveolar lavage fluid (BALF) in the acute smoking group as compared with the nonsmokers (p < 0.05). Superoxide release from mixed BAL leukocytes was increased after chronic (p < 0.01) and acute (p < 0.001) smoking, as were thiobarbituric acid-reactive species (TBARS), providing evidence of lipid peroxidation in plasma (chronic, p < 0.05; acute, p < 0.05). Trolox equivalent antioxidant capacity (TEAC) was reduced in plasma (p < 0.001) and increased in BALF (p < 0.05) in both smoking groups. The study therefore showed an acute increase in epithelial permeability and an increase in the number of neutrophils in the air spaces of cigarette smokers concomitant with evidence of increased oxidant stress.

Changes in neutrophil morphology and morphometry following exposure to cigarette smoke.

Acute cigarette smoking delays neutrophils within the pulmonary circulation in some smokers. Evidence from an in-vitro Micropore filter model of the pulmonary capillaries indicates that this may be due to a smoke induced decrease in cell deformability. In order to determine whether changes in cell shape are associated with the observed decrease in neutrophil deformability following smoke exposure, cell morphology, using scanning electron microscopy, and morphometric measurements, made using transmission electron microscopy, were performed on aliquots of neutrophils harvested from whole blood in non-smoking subjects before and after exposure in vitro to cigarette smoke. Smoke exposure increased the maximum diameter and circumference of neutrophils, without changing their area. There was also a change in the maximum to minimum cell diameter ratio, which indicated that the cells had become less spherical. Scanning electron microscopy showed that smoke exposed cells had developed blebbing of their surface membranes, suggestive of an oxidative injury to the cell membrane rather than the shape changes associated with cell activation. These changes in the morphology and morphometry of smoke exposed neutrophils may contribute to the reduction in cell deformability induced by cigarette smoke.

Neutrophil retention in the lungs of patients with chronic obstructive pulmonary disease.

In order to study neutrophil traffic in the lungs of humans, we harvested autologous neutrophils and radiolabeled them with indium-111 prior to reinjection. The passage of these [111In]neutrophils through the pulmonary vasculature was compared with that of [99mTc]erythrocytes in normal elderly subjects and in patients with chronic obstructive pulmonary disease (COPD). Neutrophil sequestration within the lungs of seven normal subjects, 10 min after reinjection, correlated with local erythrocyte transit times in the lungs (tau = 0.72, p less than 0.001). This relationship was lost in patients with COPD. In seven patients studied during an acute exacerbation of COPD, neutrophil retention was higher during the first passage through the lungs (mean, 22.0 SD 14.1%) compared with 14 patients studied when their condition was stable (16.3 SD 3.4%, p less than 0.001), or to the normal elderly subjects (13.7 SD 7.0%, p less than 0.001). In addition, the subsequent rate of neutrophil washout from the lungs was slower in patients with acute COPD (1.93 SD 0.66 x 10(-3)s1) than in those with stable disease (3.08 SD 1.8 x 10(-3)s-1, p less than 0.02). Neutrophil retention in the lungs correlated inversely with the extent of emphysema, assessed quantitatively by CT scanning (tau = 0.68, p less than 0.05). Thus, patients presenting with acute exacerbations of COPD have an increased neutrophil burden in the pulmonary vasculature with the potential for increased lung proteolysis.

Changes in neutrophil deformability following in vitro smoke exposure: mechanism and protection.

We have previously demonstrated a reduction in the deformability of neutrophils, exposed to whole particulate cigarette smoke in vitro, by measuring their ability to filter through a micropore membrane with pore dimensions similar to those of the average pulmonary capillary segment. In this study, we exposed neutrophils to the vapor phase of cigarette smoke and investigated the mechanism of the reduction in neutrophil filterability. Although both stimulated neutrophils and smoke-exposed neutrophils demonstrated an increase in filtration pressures, and thus a reduction in cell deformability, compared with control untreated cells, the spontaneous release of the reactive oxygen intermediates hydrogen peroxide and the superoxide anion was depressed following in vitro smoke exposure and there was no shape change to suggest that smoke-exposed cells were activated. The presence of erythrocytes, plasma, or the antioxidants albumin and glutathione prevented the reduction in cell filterability following smoke exposure, suggesting that in vitro smoke exposure, in our system, was mediated by oxidants. Indeed, the increase in filtration pressures, produced by smoke, could be mimicked by the addition of the oxidant hypochlorous acid. The cytoskeletal inhibitors cytochalasin B and D improved the filterability of smoke-exposed cells, suggesting that smoke may change neutrophil deformability through an effect on the actin component of the cytoskeleton. By contrast, colchicine, a specific inhibitor of the microtubules, had no effect. Preincubation with a monoclonal antibody to the CD18 antigen, to block this major neutrophil adhesive glycoprotein, did not alter the filtration pressure developed by stimulated or smoke-exposed neutrophils, suggesting that increased adhesivity was not the mechanism of the increase in filtration pressures observed following smoke exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of effect of N-acetylcysteine on the release of oxygen radicals from neutrophils and alveolar macrophages.

N-acetylcysteine (NAC) is rapidly de-acetylated in vivo to cysteine (CYSH), a precursor of glutathione (GSH) which is an antioxidant in cells and body fluids. We investigated the effect of oral administration of N-acetyl cysteine for 5 days on the spontaneous and stimulated generation of hydrogen peroxide (H2O2) and superoxide anion (O2-) from human and rat phagocytic leucocytes. Alveolar macrophages (AM) were obtained by bronchoalveolar lavage (BAL) in control rats and rats given NAC in their drinking water. Neutrophils (PMNL) were harvested from whole blood in normal nonsmoking volunteers before and after NAC was given by mouth. The stimulated release of H2O2 and O2 from both rat AM and human PMN was not changed by administration of NAC. However, a small but significant increase was observed in both the spontaneous generation of O2- from rat AM and the spontaneous generation of H2O2 from human PMNL. Administration of NAC significantly increased cysteine levels in human plasma and rat BAL, but the levels in human PMNL and rat AM after NAC did not differ from control levels. GSH levels were not altered significantly by NAC.