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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 754-759, (2002)
© 2002 American Thoracic Society

Original Article

Diminished Immunoreactivity of {gamma}-Glutamylcysteine Synthetase in the Airways of Smokers' Lung

Terttu Harju, Riitta Kaarteenaho-Wiik, Ylermi Soini, Raija Sormunen and Vuokko L. Kinnula

Departments of Internal Medicine and Pathology, University of Oulu, Oulu University Hospital; and Biocenter of Oulu, Oulu, Finland

Correspondence and requests for reprints should be addressed to Professor Vuokko L. Kinnula, Department of Internal Medicine, University of Oulu, P.O. Box 5000, 90014, Oulu, Finland. E-mail: vuokko.kinnula{at}oulu.fi


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glutathione (GSH) plays a major role in protecting the airways against oxidative stress. The rate-limiting enzyme in de novo GSH synthesis is {gamma}-glutamylcysteine synthetase ({gamma}-GCS), which is induced by acute exposure to GSH-depleting cytokines and oxidants, but downregulated by transforming growth factor ß and prolonged oxidant exposure, at least in vitro. Cell-specific expression or regulation of {gamma}-GCS may play an important role both in the defense against oxidants and in the pathogenesis of oxidant-associated airway diseases. In this study, the localizations of {gamma}-GCS heavy ({gamma}-GCS-HS) and light ({gamma}-GCS-LS) subunits were investigated by immunohistochemistry in 22 patients with chronic obstructive pulmonary disease (COPD), 20 smokers without COPD, and 13 lifelong nonsmokers. The ultrastructural distributions of both {gamma}-GCS subunits were assessed by immuno–electron microscopy. Both subunits were expressed most prominently in the large airways, and their ultrastructural localization was both cytoplasmic and along the plasma membrane. The expression of {gamma}-GCS-HS was stronger in the central bronchial epithelium than in the peripheral bronchioli (p = 0.020), or in alveolar macrophages (p = 0.008). The expression of {gamma}-GCS-HS in the central bronchial epithelium showed a tendency to be higher in nonsmokers compared with all smokers (p = 0.052). Alveolar macrophages of nonsmokers had higher levels of {gamma}-GCS-HS (p = 0.001) and {gamma}-GCS-LS (p = 0.001) than did smokers. The expression of {gamma}-GCS-HS in the central bronchial epithelium was more marked in nonsmokers than in patients with COPD (p = 0.015), the difference between smokers and patients with COPD was not significant. In conclusion, the heavy and light subunits of {gamma}-GCS are mainly expressed in the large airways. Their tendency to decrease in cigarette smokers may further predispose lung cells to ongoing oxidant stress, which contributes to the progression of lung injury.

Key Words: {gamma}-glutamylcysteine synthetase • smoking • chronic obstructive pulmonary disease • oxidant • glutathione


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The lung is exposed to a higher oxygen tension than most other tissues. Inflammatory mediators also lead to increased oxidative stress by recruitment of neutrophils and activation of inflammatory cells. Cigarette smoke is a very potent oxidant, as one puff contains 1014–1016 free radicals (1). Oxidants and inflammatory mediators, in turn, activate stress kinases and redox-sensitive transcription factors, such as NF-{kappa}B and AP-1, which regulate the genes of several antioxidant enzymes and related proteins, such as manganese superoxide dismutase (MnSOD), {gamma}-glutamylcysteine synthetase ({gamma}-GCS), also called as glutamate-cysteine ligase, and heme oxygenase-1 in the lung (2–4). Free radicals and antioxidants are also involved in fibrogenesis (5), which is why the effects of antioxidant enzymes on the pathogenesis of lung diseases with airway and parenchymal involvement are probably of great importance.

Epithelial lining fluid (ELF) contains an over 140-fold level of glutathione (GSH) (L-{gamma}-glutamyl-L-cysteinyl-glycine) compared with plasma (6, 7), and GSH appears to be a critical antioxidant in protecting the airway epithelium from oxidant injury (8). The rate-limiting enzyme in GSH synthesis is {gamma}-GCS (9), which is a heterodimer consisting of heavy ({gamma}-GCS-HS, 73 kD) and light ({gamma}-GCS-LS, 27.7 kD) subunits (10). The heavy (catalytic) subunit contains all of the catalytic activity, and it has been investigated in more detail than the light (regulatory) subunit (11). Both subunits are induced by acute oxidant stress and inflammatory mediators (12–15). There are, however, studies suggesting that transforming growth factor ß (TGF-ß) and chronic exposure to oxidants can also downregulate {gamma}-GCS, at least in vitro (14, 16). There are two previous studies where the distribution of {gamma}-GCS has been investigated in human lung (17, 18). Soini and coworkers found that both subunits of {gamma}-GCS are mainly expressed in bronchial epithelium and also to variable degrees in alveolar macrophages (17). Rahman and coworkers reported a tendency toward elevated expression of the mRNA of the {gamma}-GCS-HS in the peripheral airways of patients with chronic obstructive pulmonary disease (COPD) compared with smokers and ex-smokers without COPD (18). There are, however, no systematic studies on the expression of the {gamma}-GCS protein in the airways of nonsmokers, smokers, or patients with COPD. It can be hypothesized that {gamma}-GCS may also be decreased in the human airways after chronic exposure to cigarette smoke, which, in turn, may have an important influence on the development of oxidant injury in smokers' lung.

This study was undertaken (1) to investigate the distribution and expression of {gamma}-GCS-HS and {gamma}-GCS-LS in normal human lung and to assess the possible differences in the expression of this enzyme in the large and small airways and the alveolar space; (2) to assess the ultrastructural localization of {gamma}-GCS in human lung cells; and (3) to compare the {gamma}-GCS expression patterns of nonsmokers, smokers without obstruction, and smokers with COPD. Immunohistochemistry allows the identification of {gamma}-GCS-HS and -LS directly from tissue sections and the precise localization of the enzyme in the cells. Because no previous studies on the ultrastructural compartmentalization of {gamma}-GCS are available, immuno–electron microscopy (IEM) provides important information about the localization of this enzyme at the ultrastructural level of human lung.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Lung tissue samples from 55 patients (22 smokers with COPD, 20 smokers with normal lung function, and 13 life-long nonsmokers) undergoing resection for lung tumor were drawn for immunohistochemical studies from the archives of the Department of Pathology, Oulu University Hospital. Given the fact that the resection of malignant tumors may theoretically have an influence on the adjacent structures, samples of nonmalignant lung obtained during surgery for hamartomas were additionally included (one in the nonsmoker group and two in the COPD group). Tissue specimens from tumor-free central bronchi and peripheral lung tissue were selected. The smokers were divided into smokers with or without COPD. COPD was defined on the basis of preoperative lung function: FEV1 less than 80% of reference, FEV1/FVC less than 70%, and no reversibility (bronchodilatation effect less than 12%). The patients were not under corticosteroid therapy (neither inhaled nor systemic) and did not suffer from asbestos-related disease. The clinical characteristics were obtained from the patient records (Table 1) . Lung tissue from a current smoker without COPD was used for IEM studies to localize both subunits of {gamma}-GCS at the ultrastructural level.


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  		TABLE 1. Patient characteristics

 
Immunohistochemistry
Formalin-fixed paraffin-embedded lung tissue specimens were identified by computerized records. Two tissue blocks from each patient were selected, one from the resection line with central cartilage-containing bronchus and the other from peripheral lung. Four-micrometer sections were cut for immunohistochemical analysis. The sections were deparaffinized in xylene and rehydrated in a descending ethanol series. Endogenous peroxidase was blocked by incubating the sections in 0.1% hydrogen peroxide in absolute methanol for 10 minutes. To enhance immunoreactivity, the sections were boiled in 10 mM citrate buffer (pH 6.0) for 2 minutes at 850 W and after that for 8 minutes at 150 W. To diminish background staining, the sections were then incubated with 2% milk powder followed by incubation with the primary antibodies for {gamma}-GCS-HS and {gamma}-GCS-LS with dilutions of 1:1000 (generous gifts from Dr. T Kavanagh, University of Washington, Seattle, WA). The antibodies against selected peptides of {gamma}-GCS subunits were raised in rabbits, and their specificity has been earlier tested in cultured cells (17, 19).

The immunostaining was done using the Histostain-Plus Kit (Zymed Laboratories Inc., San Francisco, CA), and the chromogen was aminoethyl carbazole (Zymed). In negative controls, the primary antibodies were substituted with phosphate-buffered saline (PBS) or rabbit primary antibody isotype control from Zymed.

Immunoreactivity was assessed semiquantitatively by grading the staining intensity of epithelium or macrophages as negative (0), light (1), moderate (2), or intense (3). All analyses were conducted blindly without information about the clinical characteristics of the subjects by two experienced lung pathologists independently, and interobserver repeatability, measured using Cohen's kappa statistics (20), was moderate ({kappa} = 0.44).

CD8-Positive Lymphocytes
Frozen sections of peripheral lung tissue from five patients were cut into 4-µm slices, and mouse monoclonal antibodies were used to identify CD8-positive (CD8+) lymphocytes (anti-CD8, M707; Dako A/S, Glostrup, Denmark). Monoclonal antibody binding was detected with the alkaline phosphatase anti-alkaline phosphatase method (APAAP kit system; Dako) and fast-red substrate.

IEM
The lung tissue was fixed in 4% paraformaldehyde in 0.1 M phosphate buffer with 2.5% sucrose (pH 7.4) for 1 hour, immersed in 2.3 M sucrose, and frozen in liquid nitrogen. Thin cryosections were cut with a Leica Ultracut UCT ultramicrotome. For immunolabeling, the sections were first incubated in 0.05 M glycine in PBS and then in 5% bovine serum albumin (BSA) in PBS with 0.1% cold water fish skin gelatin (Aurion, Wageningen, The Netherlands). Antibodies and gold conjugate were diluted in 0.1% BSA-C (Aurion) in PBS. All washings were performed in 0.1% BSA-C in PBS. The sections were then incubated with antibodies to {gamma}-GCS-HS and {gamma}-GCS-LS for 60 minutes followed by protein A-gold complex (size 10 nm) for 30 minutes, prepared as described by Slot and Geuze (21). The controls were prepared by carrying out the labeling procedure without the primary antibody. The sections were embedded in methylcellulose and examined under a Philips CM100 transmission electron microscope.

Statistical Methods
The statistical analyses were performed with the SPSS for Windows software (SPSS, Chicago, IL). Continuous data were compared using analysis of variance. When analysis of variance results indicated that groups differed, post hoc comparisons were performed using two-tailed t tests. Categorical data were compared using Fisher exact test designed for small sample groups. p Values less than 0.05 were considered statistically significant.

Ethical Considerations
The study protocol was accepted by the ethical committee of the University of Oulu and Oulu University Hospital.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Distribution of {gamma}-GCS in the Large and Small Airways
When analyzed from the whole material (n = 55), the expression of {gamma}-GCS-HS was stronger in the central bronchial epithelium than in the peripheral bronchiolar epithelium (p = 0.020), or in the alveolar macrophages (p = 0.008) (Figure 1) . Also, {gamma}-GCS-LS was expressed most prominently in the central bronchial epithelium, the expression being significantly weaker (p = 0.0004) in the peripheral bronchiolar epithelium and in the alveolar macrophages (p = 0.03) (Fisher's exact test).



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  		Figure 1. Expression of {gamma}-GCS-HS and -LS in the epithelium of central bronchus and peripheral bronchiolus, as well as in alveolar macrophages. Immunoreactivity was assessed from the lung tissue samples of 55 subjects, and the results are presented as mean ± SEM of all cases investigated. The differences between the heavy subunit (open bars) expression in central bronchial epithelium and peripheral bronchiolar epithelium (p = 0.020), as well as in central bronchial epithelium and alveolar macrophages (p = 0.008) were statistically significant. Corresponding differences for the light subunit (closed bars) expression were also statistically significant, p = 0.0004 and p = 0.03, respectively (Fisher's exact test).

 
Distribution of {gamma}-GCS in Healthy Lung from Nonsmokers
Both subunits of {gamma}-GCS were expressed in the bronchial epithelium in the large, cartilage-containing bronchi and the small peripheral bronchioli of nonsmokers' lungs. The intensity in the large airways varied from weak (1/13) to moderate or strong (12/13) for the heavy subunit, the corresponding figures for the light subunit being 6/13 and 7/13, respectively. The bronchiolar epithelium of the small airways showed weak reactivity of the heavy chain in 9/13 cases and moderate to strong reactivity in 4/13 cases, the corresponding figures for the light chain being 10/13 and 3/13, respectively. Alveolar epithelium showed no reactivity of either subunit. Alveolar macrophages were weakly positive for the heavy chain in 6/13 cases and for the light chain in 6/13 cases, and the staining was moderate or strong in all the other cases. Vascular endothelial cells and fibroblasts were mainly negative. These results are summarized in Figure 2 (left-sided panels).




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  		Figure 2. Expression of {gamma}-GCS-HS (A) and -LS (B) in the epithelium of central bronchus (open bars) and peripheral bronchiolus (epithelial expression) (closed bars), as well as in alveolar macrophages (shaded bars) in nonsmokers, smokers with normal lung function and smokers with COPD. Immunoreactivity was assessed from the lung tissue samples of 13 nonsmokers, 20 smokers, and 22 smokers with COPD. The results are presented as mean ± SEM. The statistically significant p values in the {gamma}-GCS-HS expression were: 0.015 nonsmokers versus smokers with COPD in central bronchial epithelium; 0.000 between nonsmokers and smokers without COPD for alveolar macrophages. The statistically significant p-values in the {gamma}-GCS-LS expression were the following: 0.021 for nonsmokers versus smokers without COPD in central bronchial epithelium; 0.006 for nonsmokers versus smokers with COPD; 0.000 between nonsmokers and smokers without COPD for alveolar macrophages (Fisher's exact test).

 
Distribution of {gamma}-GCS in Smokers with Normal Lung Function
Both heavy and light subunits were expressed in the bronchial epithelium in the large, cartilage-containing bronchi as well as in the small peripheral bronchioli. The intensity in the large airways varied from weak (6/20) to moderate or strong (14/20) for the heavy subunit the corresponding figures for the light subunit being 18/20 and 2/20, respectively. Bronchiolar epithelium of the small airways showed weak reactivity for the heavy subunit in 12/20 cases and moderate to strong reactivity in 8/20 cases, the figures for the light subunit being 20/20 and 0/20, respectively. Alveolar epithelium showed no reactivity for either subunit. Alveolar macrophages were weakly positive for the heavy chain in 19/20 cases and for the light chain in 20/20 cases. Vascular endothelial cells and fibroblasts were mainly negative. The results are presented in Figure 2 (panels in the middle).

Distribution of {gamma}-GCS in Smokers with COPD
In the central bronchi, the intensity of {gamma}-GCS-HS varied from weak (11/22) to moderate or strong (11/22), the corresponding values for {gamma}-GCS-LS being 20/22 and 2/22, respectively. The bronchiolar epithelium showed weak reactivity for the heavy subunit in 12/22 cases and moderate to strong reactivity in 10/22 cases, the corresponding figures for the light subunit being 22/22 and 0/22, respectively. Occasionally, metaplastic alveolar epithelium could be detected, and these areas were positively stained. Normal alveolar epithelium showed no reactivity for either subunit in any case. Alveolar macrophages were weakly positive for the heavy subunit in 18/22 cases and for the light subunit in 16/22 cases. Vascular endothelial cells were faintly positive, and fibroblasts were mainly negative. CD8+ cells were found in the alveolar walls and, occasionally, in the bronchial epithelium, but the localization was different from the {gamma}-GCS localization. The results are summarized in Figure 2 (right-sided panels), and the representative immunohistochemical findings are illustrated in Figures 3 and 4 .



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  		Figure 3. (A) Strong immunoreactivity for {gamma}-GCS-HS in the epithelium of a central bronchus of a nonsmoker. Scale bar = 40 µm. (B) The same case as in (A). Immunoperoxidase stain, serum isotype control with hematoxylin counterstain. Scale bar = 40 µm. (C) Positive staining for {gamma}-GCS-LS in the epithelium of a central bronchus in a nonsmoker. Scale bar = 40 µm. (D) The same case as in (C). Immunoperoxidase stain, serum isotype control with hematoxylin counterstain. Scale bar = 40 µm. (E) Faint immunoreactivity for {gamma}-GCS-HS in the epithelium of a peripheral bronchiolus of a nonsmoker. Scale bar = 80 µm. (F) Immunoreactivity for {gamma}-GCS-LS in the epithelium of a peripheral bronchiolus of a nonsmoker. Scale bar = 40 µm.

 


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  		Figure 4. (A) Immunoreactivity for {gamma}-GCS-HS in the epithelium of a central bronchus of a patient with COPD. Scale bar = 40 µm. (B) Positive staining for {gamma}-GCS-LS in the epithelium of a central bronchus of a patient with COPD. Scale bar = 40 µm. (C) Immunoreactivity for {gamma}-GCS-HS in the epithelium of a peripheral bronchiolus of a patient with COPD. Scale bar = 40 µm. (D) Immunoreactivity for {gamma}-GCS-LS in the epithelium of a peripheral bronchiolus of a patient with COPD. Scale bar = 40 µm. (E) Positive staining for {gamma}-GCS-HS in alveolar macrophages in a patient with COPD. Scale bar = 40 µm. (F) Positive staining for {gamma}-GCS-LS in alveolar macrophages in a patient with COPD. Scale bar = 40 µm.

 
Comparison of {gamma}-GCS between Nonsmokers, Smokers, and Patients with COPD
The expression of {gamma}-GCS-HS in the central bronchial epithelium appeared to be higher in nonsmokers compared with all smokers, but the difference did not reach statistical significance (p = 0.052). The expression of {gamma}-GCS-HS in the peripheral bronchiolar epithelium (p = 0.036) and in the alveolar macrophages (p = 0.001) was significantly higher in nonsmokers. Also, the expression of {gamma}-GCS-LS in the central bronchial epithelium (p = 0.002), in the peripheral bronchiolar epithelium (p = 0.011), and in alveolar macrophages (p = 0.001) of nonsmokers was higher than the corresponding values in all smokers.

The expression of {gamma}-GCS-HS in the central bronchial epithelium was significantly higher in nonsmokers than in patients with COPD (p = 0.015) but not in smokers without COPD. The expression in macrophages was higher in nonsmokers than in smokers with normal lung function (p = 0.000) and marginally higher in nonsmokers than in patients with COPD (p = 0.053).

The intensity of {gamma}-GCS-LS in the central bronchial epithelium in nonsmokers was stronger than in smokers with or without COPD (p = 0.006 and 0.021, respectively). Also, the expression in macrophages was stronger in nonsmokers than in smokers with normal lung function (p = 0.000). No significant difference was seen in the immunoreactivities of the peripheral airways.

Ultrastructural Localization of {gamma}-GCS in Lung Cells
IEM studies on bronchial epithelium confirmed the localization of both subunits in the basal and apical bronchial epithelial cells and goblet cells (Figure 5) . Labeling was seen diffusely in the cytoplasm, vesicular structures in goblet cells, and along the plasma membrane. Also, macrophages and plasma cells showed cytoplasmic labeling of both subunits. No nuclear or mitochondrial labeling could be detected.



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  		Figure 5. Ultrastructural localization of {gamma}-GCS-HS in the central bronchial epithelium is mainly restricted to the vesicular structures in goblet cells (A) and to the small vesicles and plasma membranes in epithelial cells (B). Scale bar = 200 nm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although the regulation of {gamma}-GCS in cultured cells has been investigated in numerous studies, very few if any systematic studies have been conducted to assess its distribution and expression in healthy human lung or oxidant-related lung diseases. Nor have there been any studies in which the expression of this or some other antioxidant enzymes have been compared at various levels of human airways. This is the first study where the expression of both subunits of {gamma}-GCS has been compared in healthy human lung from nonsmokers, smokers, and patients with COPD. Given the fact that most lung tissue studies on patients with COPD have used material obtained from lung cancer surgery, we also included lung tissue specimens obtained from normal lung of patients with nonmalignant diseases. Our study shows that both subunits of this enzyme are expressed most prominently in the cytoplasmic compartments of basal and apical bronchial epithelial cells and goblet cells of the large airways. The high level of expression of both subunits, especially in the large airways, suggests the importance of this enzyme in the first-line defense of human lung against oxidants. Peripheral lung had lower levels of both subunits than the large airways. In addition, normal alveolar epithelium was negative, with the exception of intensive positivity in metaplastic and/or dysplastic epithelium. The latter finding may have multiple implications concerning the malignant transformation of lung epithelial cells.

Our results suggest the expression of {gamma}-GCS to be more intense in the airway epithelium and alveolar macrophages of healthy nonsmokers than in smokers or patients with COPD. This result contrasts with the only available in situ study, which revealed a tendency toward elevated expression of the mRNA of {gamma}-GCS-HS in the peripheral human lung of smokers and ex-smokers with COPD compared with smokers or ex-smokers without the disease (18). The mRNA levels do not necessarily correlate with the enzyme protein. As observed here, the expression of this enzyme may also be different at various levels of the airways. In addition, the regulation of {gamma}-GCS-HS is complex, and the effects of acute and chronic exposure may differ significantly. It is known that {gamma}-GCS is transcriptionally, post-transcriptionally, and post-translationally regulated (22). The promoters of {gamma}-GCS-HS and {gamma}-GCS-LS also contain several different cis regulatory elements, whose activities are differentially regulated (22). Several studies show that acute (< 48 hours) exposure of cultured lung cells to oxidants, such as quinones, or to hyperoxia leads to a transient increase in {gamma}-GCS activity via a transcriptional mechanism (23–25). Furthermore, TGFß results in lowered mRNA and activity of {gamma}-GCS and decreased GSH content in human lung cells in culture (16). On the basis of these in vitro results, one can expect that long-lasting oxidant exposure with variable activation of the TGFß-associated pathways, as is the case with cigarette smokers and patients with COPD, may lead to downregulation of this and other antioxidant enzymes in human lung. In agreement with this hypothesis, our present study showed lower {gamma}-GCS immunoreactivities in smokers and patients with COPD than in healthy bronchial epithelium.

Several studies have shown that ELF of patients with COPD elevates the GSH content (6), which has been suggested to result from oxidant stress and induction of {gamma}-GCS (12). It is known that {gamma}-GCS is regulated by GSH homeostasis, and high GSH level should downregulate the enzyme activity. Apart from COPD, ELF GSH levels are also elevated in asthma and in chronic beryllium disease (26, 27), but in both of these cases, GSH level correlates with extracellular glutathione peroxidase In addition to {gamma}-GCS and extracellular glutathione peroxidase, other enzymes may also be associated with the GSH content of human lung, the most important of these enzymes being glutathione-S transferases and multidrug-resistance proteins. Further, {gamma}-glutamyl transpeptidase is a membrane-associated enzyme that also participates in the maintenance of the intracellular GSH level by decomposing extracellular GSH, which, in turn, facilitates GSH synthesis intracellularly (28). The regulatory mechanisms that maintain the ELF GSH content in vivo are still unresolved, but based on our study, {gamma}-GCS may not be responsible for the high ELF GSH content in COPD.

Typical features in the lungs of patients with COPD include accumulation of neutrophils, macrophages, and lymphocytes, especially CD8+ T-lymphocytes. The characteristics of this inflammatory process differ between the smokers who develop symptoms of chronic bronchitis and chronic airway limitation (COPD) and those who remain asymptomatic with normal lung function. Furthermore, severe COPD is associated with higher concentrations of goblet cells, CD8+ cells, and neutrophils than those in mild disease (29, 30). Inflammatory process is present both in the large and peripheral airways (31) as well as in lung parenchyma (32). The present investigation showed that neutrophils and CD8+ T-lymphocytes were negative for {gamma}-GCS reactivity, whereas both subunits could be detected in alveolar macrophages. In contrast to neutrophils, monocytes and/or macrophages also contain significant levels of other antioxidant enzymes, such as MnSOD, CuZnSOD, and glutathione peroxidase (33). The low antioxidant capacity of neutrophils further potentiates the oxidant burden locally, which may contribute to the progression of oxidant-enhanced lung damage in this and other diseases with neutrophil predominance.

In conclusion, {gamma}-GCS, the rate-limiting enzyme in GSH synthesis, is expressed most prominently in the basal and apical epithelial cells of the large airways. Both subunits are expressed most intensely in nonsmokers and appear to be downregulated in the airways of both smokers and patients with COPD. Because {gamma}-GCS expression patterns of smokers and patients with COPD were similar, this enzyme alone cannot predict the development of this disease. Downregulation of {gamma}-GCS in smokers' lung may, however, further enhance lung damage in cigarette smoke-related lung diseases, where multiple mechanisms in addition to the antioxidant defense are evidently involved.


   Acknowledgments
 
:

The authors are grateful to Ms. Päivi Koukkula for her technical assistance.

This work was supported by grants from the Finnish Anti-Tuberculosis Association Foundation and Sigrid Juselius Foundation.

Received in original form December 5, 2001; accepted in final form April 16, 2002


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 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
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