Glutathione S-transferase omega in the lung and sputum supernatants of COPD patients
© Harju et al. 2007
Received: 29 January 2007
Accepted: 06 July 2007
Published: 06 July 2007
The major contribution to oxidant related lung damage in COPD is from the oxidant/antioxidant imbalance and possibly impaired antioxidant defence. Glutathione (GSH) is one of the most important antioxidants in human lung and lung secretions, but the mechanisms participating in its homeostasis are partly unclear. Glutathione-S-transferase omega (GSTO) is a recently characterized cysteine containing enzyme with the capability to bind and release GSH in vitro. GSTO has not been investigated in human lung or lung diseases.
GSTO1-1 was investigated by immunohistochemistry and Western blot analysis in 72 lung tissue specimens and 40 sputum specimens from non-smokers, smokers and COPD, in bronchoalveolar lavage fluid and in plasma from healthy non-smokers and smokers. It was also examined in human monocytes and bronchial epithelial cells and their culture mediums in vitro.
GSTO1-1 was mainly expressed in alveolar macrophages, but it was also found in airway and alveolar epithelium and in extracellular fluids including sputum supernatants, bronchoalveolar lavage fluid, plasma and cell culture mediums. The levels of GSTO1-1 were significantly lower in the sputum supernatants (p = 0.023) and lung homogenates (p = 0.003) of COPD patients than in non-smokers.
GSTO1-1 is abundant in the alveolar macrophages, but it is also present in extracellular fluids and in airway secretions, the levels being decreased in COPD. The clinical significance of GSTO1-1 and its role in regulating GSH homeostasis in airway secretions, however, needs further investigations.
Several studies suggest the importance of oxidative stress in the pathogenesis of chronic obstructive pulmonary disease (COPD). Cigarette smoke not only contains high levels of oxidants, but it also activates oxidant producing pathways in the lungs [1, 2]. The oxidant/antioxidant imbalance present in the lungs of these patients also results from the impaired capacity of the antioxidant/detoxification enzymes to detoxify the harmful reactive oxygen metabolites [3–8]. Very little is known about specific changes in the major antioxidant defence mechanisms in mild or severe COPD.
One of the major antioxidants in human airways is glutathione (GSH) (L-γ-glutamyl-L-cysteinyl-glycine); however the regulatory mechanisms controlling the intra- and extra-cellular concentrations of GSH are not completely understood [9–11]. The rate limiting enzyme in GSH biosynthesis, glutamate cysteine ligase (GCL) is induced by cigarette smoke , but controversially shown to either increase or decrease in COPD [5, 13, 14]. GCL levels alone do not explain the changes observed in the free GSH levels of airways in smokers or COPD . Other enzymes that can participate in GSH homeostasis in the lung and airway secretions include glutathione peroxidases (Gpx); for example Gpx2 is induced in experimental mice model by smoke exposure  and Gpx3 is increased in the bronchial epithelium and epithelial lining fluid of smokers . Another additional group of enzymes that is associated with GSH homeostasis in human airways is glutaredoxin (Grx) family of enzymes. The classical member of this family, Grx1, is regulated in bronchial epithelial cells by oxidants and cigarette smoke in vitro , but has also been shown to be present in the extracellular fluids including sputum supernatants [17–19]. One important function of glutaredoxins is their thioltransferase activity and the subsequent effects on the glutathionylation state of proteins in the lung. It has become apparent that there is another thioltransferase i.e. glutathione-S-transferase omega (GSTO) in mammalian cells which may have potential role in regulating GSH homeostasis. This enzyme belongs to the glutathione-S-transferase family (GST) that detoxify toxic substrates present in tobacco smoke by a GSH-dependent mechanism [20, 21]. GSTO contains an N-terminal glutathione-binding domain suggesting its role in the metabolism and maintenance of GSH levels in intact cells [20, 22]. Since GSH is one of the major antioxidants of the airways, it can be hypothesized that GSTO may participate in the maintenance of GSH not only intracellularly, but also in the extracellular space and this may be modulated by oxidative stress.
The present study was undertaken 1) to investigate the cell specific distribution and expression of GSTO1-1 in healthy human lung, 2) to compare the GSTO1-1 expression patterns in the lung of non-smokers, smokers without obstruction and smokers with variable severities of COPD, 3) to assess whether GSTO1-1 is associated with COPD severity and 4) to analyze whether GSTO1-1 can be detected in airway secretions/induced sputum supernatants, bronchoalveolar lavage fluid (BALF) or plasma.
Tissue, induced sputum, bronchoalveolar lavage and plasma specimens
The characteristics of the patients in the immunohistochemistry studies
Non-smoker N = 16
Smoker N = 22
COPD N = 34
The characteristics of the patients in Western blotting for whole lung homogenates
Non-smoker N = 9
Smoker N = 5
COPD N = 17
The characteristics of the patients in the sputum study
Non-smoker N = 6
Smoker N = 5
COPD N = 15
Bronchoalveolar lavage (BAL) had been obtained from 3 non-smokers and 3 smokers who had been investigated for minor respiratory symptoms of unknown etiology. Fiberoptic bronchoscopy for sampling BAL fluid was performed under local anaesthesia with lignocaine and the fluid was collected after installation of 10 aliquots of 20 ml from the right middle lobe. The cytocentrifuge preparation indicated a normal cell differential count with over 90% of the cells being macrophages. After centrifugation (400 × g for 15 minutes), the cells and supernatant were collected, frozen and stored at -80 C.
Plasma samples were collected from 4 non-smokers, 4 healthy smokers and 4 patients with stage I-II COPD.
Human histiocytic lymphoma (U937) cells were obtained from the American Type Culture Collection . The cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Monocyte-macrophage differentiation was induced by phorbol 12-myristate 13-acetate (PMA) at concentrations of 100 ng/ml. Human non-malignant bronchial epithelial (BEAS-2B) cells (American Type Culture Collection, Rockville, MD, USA) were cultured in Bronchial Epithelial Growth Medium (BEGM) (Clonetics Corporation, Walkersville, MD, USA) and subcultured before reaching confluence.
Immunohistochemistry and cytochemistry of GSTO1-1 in the tissues and sputum specimens
One tissue block from each patient was selected from peripheral lung. Four-μm 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 3% hydrogen peroxide in absolute methanol for 15 minutes. The sections were incubated with the primary antibody for GSTO1-1 using a dilution of 1:200. The immunostaining was done using the Histostain-Plus Kit (Zymed Laboratories Inc., San Francisco, CA), and the chromogen was aminoethyl carbazole (AEC) (Zymed Laboratories Inc.). In negative controls, the primary antibody was substituted with phosphate-buffered saline (PBS) or rabbit primary antibody isotype control from Zymed Laboratories Inc.
The number of macrophages was calculated using the Zeiss AxioHOME Morphometry program (Zeiss, Jena, Germany). GSTO-positive macrophages were counted by two techniques and by three investigators, first by calculating the number in 10 high power fields of the specimen (YS) and secondly by using the Zeiss AxioHOME Morphometry program (Zeiss) (PR, KS). Immunoreactivity was also assessed semiquantitatively by grading the staining intensity of the macrophages, bronchial, bronchiolar or alveolar epithelium or vascular endothelium as negative (0), weak (1) or moderate/intense (2) (YS). GSTO1-1 positive and negative cells in the sputum specimens were counted (400 cells/cytospin).
The cytospin samples were treated with Ortho Permeafix (Ortho Diagnostic Systems Inc., UK) and for immunostaining, Zymed ABC Histostain-Plus Kit was used according to the manufacturer's protocol. The samples were incubated with an antibody against GSTO1-1 and negative control samples with Zymed Rabbit Isotype Control and PBS, and stained with AEC (Zymed Laboratories Inc.) and thereafter with Mayer's haematoxylin.
Western blot analysis
Western blot analysis from tissue homogenates and sputum supernatants was conducted as described earlier  with 1:2000–1:5000 dilution of GSTO1-1 antibody. In previous studies from our laboratory and others [17, 26, 27] β-actin has shown high individual variability, especially in tissue samples from the diseased lung. Instead of using β-actin as a loading control, the protein concentration was measured carefully as triplicates and equal loading was ensured by staining the blotted membranes with Ponceau S (Sigma Aldrich, St. Louis, MO, USA).
The statistical analyses were performed with the SPSS for Windows software (SPSS, Chicago, IL, USA). Continuous data were compared using analysis of variance (ANOVA). When ANOVA results indicated that groups differed, post hoc comparisons were performed using two-tailed t-tests. Categorical data were compared using Fisher's exact test designed for small sample groups. P-values less than 0.05 were considered statistically significant. Correlations to lung functions were analyzed with the Pearson correlation test.
The study protocol was approved by the ethical committees of Oulu University Hospital and Helsinki University Hospital and it is in accordance with the ethical standards of the Helsinki declaration of 1975.
Immunohistochemistry from the tissue specimens
There was a negative correlation between FEV1 and % of GSTO1-1 positive macrophages in all COPD cases (r = -0.533, p = 0.002) and in severe COPD (r = -0.794, p = 0.011). There was no correlation between the pack-years or the dosage of inhaled corticosteroid and percentage of GSTO1-1 positive macrophages.
Induced sputum extracellular fluids and tissue homogenates
To confirm the presence of GSTO1-1 in extracellular fluids, GSTO1-1 was analyzed also from plasma and BALF samples of non-smokers and smokers and from the mediums of the cell cultures (U937 and BEAS2B). GSTO1-1 could be detected in all of samples (representative samples are shown in Figure 5B,5C). Tissue homogenates that contain both lung cells and extracellular matrix, exhibited a lower level of GSTO1-1 in the specimens obtained from the COPD cases compared to the non-smokers and smokers with normal lung function (p = 0.003) (Fig 5D). In Western analysis, there was no correlation between the lung function parameters and relative intensity in the lung homogenates
Glutathione related mechanisms that function both intra- and extracellularly are known to be crucial in the pulmonary defense against oxidants and probably also against cigarette smoke. Here we show that GSTO1-1 has a highly specific localization in the lung, being expressed mainly in alveolar macrophages, but also weakly in other cell types such as airway/alveolar epithelial cells. Importantly GSTO1-1 could also be detected in extracellular fluids including sputum supernatants, BALF, plasma and the cell culture mediums of cultured monocytes and bronchial epithelial cells. This finding strongly supports the idea that the regulation of the GSH homeostasis is not only regulated by intracellular antioxidant enzymes, but is associated with extracellular thiol-modulating proteins that participate in GSH binding and release.
Previously the distribution of GSTO1-1 has been investigated in one human study  which showed it to be abundant in a wide range of normal tissues, particularly in the liver but also in the lung (three specimens); in the lung GSTO1-1 could be found only in macrophages. The results of the present study are in line with these findings, but also found GSTO1-1 immunoreactivity in the bronchial and alveolar epithelium. The immunoreactivity was also confirmed in BEAS-2B bronchial epithelial cells in culture.
Tissue studies showed elevated percentage of GSTO1-1 positive macrophages in peripheral lung in severe COPD compared to smokers or stage I-II COPD with negative correlation to lung function parameters. Furthermore, alveolar epithelium was always found to be GSTO1-1 positive in severe COPD. These results may refer to continuing efforts to protect inflamed, remodelled alveolar epithelium and possibly also macrophages against oxidative stress. Overall the synthesis/level of GSTO1-1 may be enhanced in smoker's lung/COPD but together with other antioxidant enzymes is not sufficient to maintain adequate levels of free GSH against oxidative stress.
Tissue homogenates that contain both the cells and matrix showed decreased levels of GSTO1-1 in COPD. These changes in the GSTO1-1 levels in COPD may be partly associated both with decreased levels of macrophages in severe COPD, but also with the presence of all tissue components including the matrix in the lung homogenates and lowered GSTO1-1 levels in the extracellular space.
In summary, this study significantly extends earlier understanding about the antioxidant defence in human lung and extracellular fluids. The important new finding of the presence of GSTO1-1 in extracellular fluids will require further investigation to elucidate the role of GSTO1-1 there since the regulation of GSH levels in these fluids is still poorly understood. The regulation of GSTO is also unknown and there is no literature concerning the effect of oxidative stress on GSTO expression. This is also a critical area requiring research in future investigations.
This work was supported by grants from the Finnish Anti-Tuberculosis Association Foundation, Finnish Association of Respiratory Medicine, Sigrid Juselius Foundation, Ahokas Foundation, the Australian National Health and Medical Council, the Academy of Finland, the Magnus Ehrnrooth Foundation, the Finnish Cultural Foundation and the Funding of Helsinki University Hospital (HUCH EVO). We are grateful to Ms Kirsi Kvist-Mäkelä, Ms Tiina Marjomaa, Ms Heta Merikallio and Mr Manu Tuovinen for their excellent technical assistance.
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