The Adaptive Response Of Smokers To Oxidative Stress

Editorials

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The Adaptive Response of Smokers to Oxidative Stress Moving from Culture to Tissue An efficient antioxidant defense is essential to protect the lung against the continuous threat posed by exogenous and endogenous oxidants. Major airborne sources of oxidants that may cause injury to the lung are cigarette smoke and air pollutants, whereas inflammatory cells may constitute an endogenous source of oxidants. Oxidative stress is considered to be an important element in the pathogenesis of a variety of inflammatory lung diseases, including idiopathic pulmonary fibrosis, acute respiratory distress syndrome, and chronic obstructive pulmonary disease (COPD) (1, 2). The relative contribution of inhaled (exogenous) and inflammatory cellderived (endogenous) oxidants differs among these conditions. The ability of oxidants to cause direct injury to the epithelium of the lung and to inactivate proteinase inhibitors, and their involvement in mucus hypersecretion, inflammatory gene expression, and neutrophil recruitment has provided a rationale for the development of antioxidants as a treatment for inflammatory lung disease. Current therapeutic strategies to protect the lung against oxidative stress, however, are far from optimal. Lung tissue has the capacity to mount an adaptive response quickly to oxidative stress by recruitment of antioxidant defenses. Various antioxidant mechanisms are operative

in the lung, and include the action of glutathione (GSH), the predominant nonprotein antioxidant in the lung. This tripeptide has been shown to play a key role in the lung’s defense against oxidative stress (3), as demonstrated by studies showing its critical involvement in the regulation of oxidant-induced apoptosis in lung epithelial cells (4). There is a substantial turnover of GSH in the lung, which requires an efficient system to maintain GSH levels. De novo synthesis is an important mechanism to maintain and increase GSH levels in epithelial lining fluid and involves the action of various components, among which ␥-glutamylcysteine synthetase (␥-GCS) appears to play a key role as a rate-limiting enzyme (3). In vitro studies have revealed that a variety of mediators, including oxidants and proinflammatory cytokines, is able to increase expression of ␥-GCS, usually secondary to depletion of intracellular GSH stores. In contrast, transforming growth factor-␤ was found to decrease ␥-GCS expression (3). This is important because the expression of transforming growth factor-␤ is increased in various inflammatory lung disorders, including COPD and idiopathic pulmonary fibrosis. ␥-GCS is a heterodimer in which the heavy subunit contains the catalytic activity, whereas the light subunit serves as a regulatory chain. Interestingly, expression of

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these subunits differs in various tissues, and the genes encoding the subunits are localized on different chromosomes. Although the molecular mechanisms involved in GSH production in cell cultures are beginning to be unraveled, much less is known about the mechanisms that regulate epithelial lining fluid GSH levels in humans. Therefore, the study by Harju and coworkers in this issue of AJRCCM (pp. 754–759) is important because it characterizes the protein expression of ␥-GCS in resected lung tissue obtained from smokers and nonsmokers (5). The authors explain that ␥-GCS protein is predominantly expressed in bronchial and bronchiolar epithelium, whereas its expression in alveolar epithelium is restricted to metaplastic and/or dysplastic areas. In addition, they demonstrate that ␥-GCS expression in bronchial epithelium and alveolar macrophages is higher in nonsmokers than in smokers. How do we interpret the findings of Harju and coworkers (5)? First, their observation that ␥-GCS is predominantly present in bronchial/bronchiolar epithelium but absent in normal alveolar epithelium is unexpected. Various studies, using cultured alveolar epithelial cells and demonstrating ␥-GCS heavy-subunit mRNA in alveolar epithelium by in situ hybridization indicate that the large quantities of GSH in epithelial lining fluid are derived in large part from type II alveolar epithelial cells (6–8). It cannot be excluded that the absence of detectable ␥-GCS protein in alveolar epithelium is the result of insufficient sensitivity of the methods used by Harju and coworkers to demonstrate this protein in the periphery of the lung (5). Nevertheless, their finding of differential expression in different parts of the lung warrants further investigation. The second important observation made by the authors is that ␥-GCS protein (both heavy and light subunit) expression is lower in smokers with or without airflow obstruction, as compared with nonsmokers. This would indicate that the antioxidant defenses in smokers are impaired, which would put them at increased risk for the deleterious effects of oxidative stress. In contrast to Rahman and coworkers (8), Harju and coworkers did not find differences in ␥-GCS expression between smokers with and without airflow obstruction. In their study, Rahman and coworkers showed that the expression of ␥-GCS heavy subunit mRNA is higher in alveolar epithelial cells in (ex) smokers with COPD as compared with that observed in smokers without COPD (8). Increased ␥-GCS expression in patients with COPD is in line with the observation that GSH levels in bronchoalveolar lavage fluid from COPD patients are inversely related to FEV1 (9), suggesting an adaptive (but insufficient?) response to oxidative stress in patients with COPD. Indications for an adaptive response in smokers come from studies demonstrating that acute smoking depletes the lung of GSH, whereas chronic smoking increases GSH levels in epithelial lining fluid (10). Based on these considerations and current insights into the key role of ␥-GCS in GSH production and its regulation by oxidants, the observed lower expression of ␥-GCS protein in smokers and its virtual absence in alveolar epithelium are unexpected. How do we reconcile this apparent contradictory finding on ␥-GCS and GSH levels? A plausible explanation is the one suggested by the authors: ␥-GCS expression does not explain the higher levels of GSH in epithelial lining fluid in chronic smokers (9, 10). This means that other mechanisms involved in maintaining pulmonary GSH levels may be more

important. Clearly, it is important to study the relationship between expression of ␥-GCS and local GSH levels in the lungs, as in material collected by bronchoscopy. Such studies are mandatory to explore the nature of the relationship between GSH production and ␥-GCS expression in vivo, to confirm these findings, and to exclude that these results are affected by the disease (mostly lung carcinoma) that was the indication for lung resection surgery. Furthermore, it is essential to study both extracellular and intracellular GSH levels, especially because most in vitro studies on GSH regulation have focused on intracellular GSH levels. In conclusion, despite the recent advancement in understanding the molecular aspects of GSH production, the role of ␥-GCS in regulating pulmonary GSH levels in vivo remains incompletely understood. Studies such as those presented in this issue of AJRCCM will contribute to our further understanding of the regulation of antioxidant defense systems and provide evidence for a failure of antioxidant defense mechanisms in smokers. Pieter S. Hiemstra, Ph.D. Department of Pulmonology Leiden University Medical Center Leiden, The Netherlands

References 1. Repine JE, Bast A, Lankhorst I, and The Oxidative Stress Study Group. Oxidative stress in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1997;156:341–357. 2. Rahman I, MacNee W. Oxidative stress and regulation of glutathione in lung inflammation. Eur Respir J 2000;16:534–554. 3. Rahman I, MacNee W. Lung glutathione and oxidative stress: implications in cigarette smoke-induced airway disease. Am J Physiol 1999; 277:L1067–L1088. 4. Lavrentiadou SN, Chan C, Kawcak T, Ravid T, Tsaba A, van der Vleit A, Rasooly R, Goldkorn T. Ceramide-mediated apoptosis in lung epithelial cells is regulated by glutathione. Am J Respir Cell Mol Biol 2001;25:676–684. 5. Harju T, Kaarteenaho-Wiik R, Soini Y, Sormunen R, Kinnula VL. Diminished immunoreactivity of ␥-glutamylcysteine synthetase in the airways of smokers’ lung. Am J Respir Crit Care Med 2002;166:754– 759. 6. Cantin AM, North SL, Hubbard RC, Crystal RG. Normal alveolar epithelial lining fluid contains high levels of glutathione. J Appl Physiol 1987; 63:152–157. 7. Rahman I, Smith CAD, Lawson MF, Harrison DJ, MacNee W. Induction of gamma-glutamylcysteine synthetase by cigarette smoke is associated with AP-1 in human alveolar epithelial cells. FEBS Lett 1996;396: 21–25. 8. Rahman I, van Schadewijk AA, Hiemstra PS, Stolk J, van Krieken JH, MacNee W, De Boer WI. Localization of gamma-glutamylcysteine synthetase messenger RNA expression in lungs of smokers and patients with chronic obstructive pulmonary disease. Free Radic Biol Med 2000; 28:920–925. 9. Linden M, Rasmussen JB, Piitulanen E, Tunek A, Larson M, Tegner H, Venge P, Laitinen LA, Brattsand R. Airway inflammation in smokers with nonobstructive and obstructive chronic bronchitis. Am Rev Respir Dis 1993;148:1226–1232. 10. Morrison D, Rahman I, Lannan S, MacNee W. Epithelial permeability, inflammation, and oxidant stress in the air spaces of smokers. Am J Respir Crit Care Med 1999;159:473–479.

DOI: 10.1164/rccm.2205024