Intracellular glutathione and bronchoalveolar cells in fibrosing alveolitis: effects of N-acetylcysteine.

Extracellular glutathione deficiency and exaggerated oxidative stress may contribute to the pathogenesis of fibrosing alveolitis (FA). High-dose N-acetylcysteine (NAC) supplementation partially reverses extracellular glutathione depletion and oxidative damage, but effects on intracellular glutathione are unknown. Intracellular total glutathione (GSHt) and activation of bronchoalveolar lavage cells (BAC) obtained from 18 FA patients (9 males, aged 52+/-2 yrs), before and after 12 weeks of oral NAC (600 mg t.i.d.), were assessed. Eight healthy nonsmokers (2 males, aged 36+/-6 yrs) served as a control group. Intracellular GSHt was decreased in FA (1.57+/-0.20 nmol 1x10(6) BAC(-1) versus 2.78+/-0.43 nmol x 10(6) BAC(-1)). After NAC treatment, the intracellular GSHt content increased (1.57+/-0.20 versus 1.87+/-0.19 nmol x 1 x 10(6) BAC(-1)). The spontaneous oxidative activity of BAC decreased after NAC treatment (2.7+/-0.8 versus 1.0+/-0.2 nmol x 1 x 10(6) BAC(-1) x h(-1)). Interleukin-8 concentration (82.1+/-31.5 versus 80.0+/-22.6 pg x mL bronchoalveolar fluid (BALF), nonsignificant (NS)) and myeloperoxidase activity (1.93+/-0.64 versus 1.55+/-0.47 mU x mL(-1) BALF, NS) did not change significantly, but were found to be inversely correlated to intracellular GSHt. In conclusion, high-dose N-acetylcysteine supplementation increases intracellular glutathione levels slightly. This increase is associated with a mild reduction of oxidative activity but not with a reduction of bronchoalveolar cell activation in these patients.

Pulmonary glutathione levels in acute episodes of Farmer's lung.

Acute episodes of farmer's lung (FL) are associated with activation and migration of neutrophils into the lungs, causing oxidative stress. We conducted a study to evaluate the effect of episodes of FL on antioxidant defense of the lung by glutathione (GSH). A total of 15 patients with symptomatic FL (one female and 14 males, age 42 +/- 1 yr [mean +/- SEM]) underwent a standardized hay exposure test for 1 h and were then monitored through lung function measurements for 6 h, after which bronchoalveolar lavage (BAL) was performed. As a control, 10 asymptomatic farmers (AF) (two males and eight females, age 43 +/- 1 yr) underwent the same diagnostic procedures. At 3 to 6 h after antigen exposure, the lung function of FL patients was significantly impaired (VC: -31 +/- 4%; single-breath diffusing capacity of carbon monoxide [DL(CO)]: -17 +/- 3%; and Pa(O(2)): -14 +/- 2%, all versus baseline, whereas in AF, only minor changes occurred VC: -4 +/- 5%; DL(CO): -9 +/- 3%, and Pa(O(2)): -5 +/- 2%, all versus baseline). The number of neutrophils in bronchoalveolar lavage fluid was increased in FL patients as compared with AF (29 +/- 7 x 10(4)/ml versus 10 +/- 7 x 10(4)/ml, p < 0.05). The concentrations of total and reduced glutathione (GSH(T) and GSH, respectively) in epithelial lining fluid were decreased in FL patients and increased in AF (GSH(T): 292.5 +/- 27.5 microM versus 1, 185.0 +/- 189.9 microM, respectively, p < 0.001; GSH: 256.8 +/- 22.1 microM versus 1,054.5 +/- 172.9 microM, respectively, p < 0.001). These findings suggest that the individual ability to upregulate GSH in the alveolar space in response to an inflammatory stimulus may have implications for the development of symptomatic FL. We conclude that intrapulmonary GSH levels are distinctly different in patients with FL and AF, and that the regulation of GSH may play an important role in the pathogenesis of FL.

Antioxidative and clinical effects of high-dose N-acetylcysteine in fibrosing alveolitis. Adjunctive therapy to maintenance immunosuppression.

In fibrosing alveolitis (FA), activated phagocytes cause excessive oxidative stress in the lower respiratory tract. Additionally, levels of glutathione, a major antioxidant of the human lung, are markedly reduced. Since N-acetylcysteine (NAC) is a known precursor for glutathione synthesis, we investigated the effect of NAC on redox balance and lung function in FA. Eighteen patients with an established diagnosis of FA were treated with 600 mg NAC three times daily for 12 wk in addition to their latest immunosuppressive therapy. Before and after NAC therapy, pulmonary function tests (PFTs) and bronchoalveolar lavage (BAL) were performed. BAL fluid was analyzed with regard to cell differential, glutathione status, and methionine sulfoxide content of BAL proteins (Met(O)), as an indicator of oxidative stress at the alveolar surface. There was an increase of total glutathione (GSHt = GSH +/- 2 x GSSG: 3.43 +/- 0.30 microM versus 4.20 +/- 0.66 microM, p < 0.05) and of reduced glutathione (GSH: 2.58 +/- 0.24 microM versus 3.42 +/- 0.54 microM, p < 0.005) in native BAL fluid and in the epithelial lining fluid (GSHt: 267.3 +/- 26.0 microM versus 367.1 +/- 36.0 microM, p < 0.005; GSH: 204.5 +/- 20.7 microM versus 302.9 +/- 32.2 microM, p < 0.005). The increase of GSH was accompanied by a decrease of Met(O) (6.83 +/- 0.71% versus 4.60 +/- 0.40%, p < 0.005). PFTs significantly improved during NAC treatment. We conclude that high-dose NAC significantly improved the antioxidant screen of the lungs by elevating GSH levels. Moreover, the decrease of Met(O) levels indicated an antioxidant effect at the alveolar surface. These biochemical changes were accompanied by an improvement of PFTs in patients under maintenance immunosuppression. NAC supplementation should, therefore, be considered as an adjunct therapy for FA.

Increased oxidation of extracellular glutathione by bronchoalveolar inflammatory cells in diffuse fibrosing alveolitis.

An unbalanced oxidative stress is thought to be an important element in the pathogenesis of diffuse fibrosing alveolitis (DFA). The purpose of our study was to investigate the role of reactive oxygen metabolites (ROMs) released from cultured bronchoalveolar inflammatory cells (BA-cells) on glutathione oxidation. We studied bronchoalveolar lavage samples from 10 healthy controls and from 20 patients with diffuse fibrosing alveolitis (all were nonsmokers). BA-cells obtained by bronchoalveolar lavage (BAL) were incubated with 50 microM of reduced glutathione (GSH). Oxidation of GSH to glutathione disulphide (GSSG) by BA-cell derived oxidants was detected as a decline of GSH in the supernatants. Total glutathione (GSHtot = GSH + 2 GSSG) and GSSG in the epithelial lining fluid (ELF), and methionine sulphoxide (Met(O)) content of BAL proteins were determined. In diffuse fibrosing alveolitis the oxidative activity of BA-cells was enhanced, GSHtot and GSH were decreased, whereas the GSSG:GSH ratio was increased. The oxidative activity of BA-cells correlated positively with the GSSG:GSH ratio, but not with the methionine sulphoxide content. The methionine sulphoxide content was elevated in diffuse fibrosing alveolitis and inversely correlated with GSHtot. The methionine sulphoxide content also correlated positively with the percentage of BAL neutrophils. We conclude that BA-cell-derived reactive oxygen species are capable of oxidizing extracellular GSH in vitro. The positive correlation between the BA-cell oxidative activity in vitro and GSSG:GSH ratio in ELF suggests that a similar oxidative effect on extracellular GSH may also occur in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

[Pulmonary manifestations of systemic scleroderma: pathophysiologic and clinical significance of the activation of lung fibroblasts]. / Die pulmonale Manifestation der systemischen Sklerodermie: Pathophysiologische und klinische Bedeutung der Aktivierung von Lungenfibroblasten.

Fibrosing alveolitis (FA) is a common and often fatal complication of systemic sclerosis (SSC). The purpose of this study was to characterize the fibrotic process within the lungs using bronchoalveolar lavage fluid (BALF). We investigated 25 healthy controls (CON) and 85 SSC patients. In 61 patients (72%) lung function tests, clinical, and radiological findings indicated manifest FA, whereas 24 patients (28%) where free of significant lung disease. Of the latter, 12 had pathologic BAL differential cell counts (= subclinical alveolitis; SUB), 12 had normal BAL cytology (NOR). BAL samples were analysed for chemoattractant activity (CAA) for fibroblasts using Boyden chambers. Procollagen-III-Peptide (P-III-P) and Laminin fragment P1 (Lam-P1) were measured radioimmunologically. CAA (expressed as % of the effect of conditioned medium) was increased in FA and SUB (CON: 17.3 +/- 3.2; FA: 40.8 +/- 5.8, p < 0.01 vs. CON; SUB: 58.6 +/- 11.8, p < 0.01 vs. CON; NOR: 23.7 +/- 6.3; n.s.). Lam-P1 [U/ml ELF] was also elevated in FA and SUB patients (CON: 0.90 +/- 0.17; FA: 2.07 +/- 0.48, p < 0.05 vs. CON; SUB: 2.61 +/- 1.14, p < 0.05 vs. CON; NOR: 1.05 +/- 0.35, n.s. vs. CON). P-III-P [U/ml ELF] was elevated in FA patients (CON: 8.3 +/- 1.1; FA: 26.9 +/- 5.5, p < 0.001 vs. CON) but not in SUB or NOR (SUB: 10.2 +/- 0.7, NOR: 7.9 +/- 2.9; n.s.). There was no significant relationship between P-III-P and LAM-P1 values in ELF and serum, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)