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 [21
] 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.
GSTO1-1 was also detectable in extracellular fluids such as induced sputum supernatants, BALF, plasma and cell culture mediums. Previous studies have already indicated that GSTO1-1 is highly expressed in the liver, suggesting that plasma GSTO1-1 positivity may also be associated with hepatic secretion. All sputum and BALF supernatants were positive for GSTO1-1, but negative for several other markers of intracellular proteins including β-actin. Over 80% of the cells were viable which also argues against any leakage of GSTO1-1 through damaged cell membranes. It is therefore possible that GSTO1-1 is similar to Grx1, in being able to be excreted into the airways where it can participate in the maintenance of GSH homeostasis. GSH levels are increased in the BAL fluid representing epithelial lining fluid (ELF) of cigarette smokers [28
], but decreased in the ELF during COPD exacerbations [29
]. At present the regulation of GSH in the airway secretions/ELF is far from clear, but GSH synthesis in the ELF is very unlikely to occur. Significant amounts of GSH may be present, however, as protein-GSH mixed disulfides that are known to accumulate during oxidative stress both intracellularly and to the extracellular space [30
]. GSTO1-1 is one potential enzyme capable of participating in these reactions: it contains cysteine in its active site where Cys32 can form a disulphide bond with GSH and thereby function as a potential reservoir of GSH during oxidative stress. Previous structural studies have indicated that the active site of GSTO1-1 is relatively open and could potentially accommodate glutathionylated protein structures [31
] though the capacity of these reactions is probably diminished in COPD. Overall the regulation of GSH maintenance, binding of GSH to proteins, its oxidation and release is complicated. This study significantly extends earlier observations on these pathways and importance of extracellular fluids in these reactions; summary of these reactions has been gathered to Figure .
Figure 6 Suggested role of GSTO (glutathione transferase omega) in cigarette smoke induced oxidative stress. Enzymes maintaining GSH homeostasis are important in protecting lung against cigarette smoke induced oxidative stress. GSTO belongs to the glutathione-S-transferase (more ...)
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.