Glutathione S-transferase omega in the lung and sputum supernatants of COPD patients

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Figure 1

Immunohistochemical staining for GSTO1-1 (1:200) in the peripheral lung of non-smoker (A), COPD stage I-II (B) and COPD stage IV (C). Negative control, COPD stage IV (D). GSTO1-1 was mainly expressed in alveolar macrophages.

Figure 2

A. The numbers of alveolar macrophages were increased in smokers and in stage I-II COPD compared to non-smokers and stage IV COPD (p = 0.009). The post-hoc comparison was significant at 0.05 level between non-smokers and stage I-II COPD (*). B. The mean percentage of GSTO1-1 positive macrophages was higher in severe stage IV COPD compared to smokers or COPD stage I-II (p = 0.004). The difference between non-smokers and other groups was not statistically significant. The post-hoc comparison was significant at 0.05 level between stage IV COPD and smokers and between stage IV and stage I-II COPD (*).

Figure 3

The GSTO1-1 immunoreactivity was most prominent in alveolar macrophages varying from negative to moderate/intense and GSTO1-1 expression was either absent or weak in other cell types. Alveolar epithelium was always positive in stage IV COPD. Bars represent means, error bars standard error of mean.

Figure 4

Macrophages in induced sputum exhibited positive GSTO1-1 reactivity. Representative sputum cytospins from a smoker (A) and stage II COPD (B) are shown.

Figure 5

A. Western blotting for GSTO1-1 in induced sputum of non-smokers, smokers and COPD-patients showed an decreased immunoreacitivity in patients with COPD compared to non-smokers (p = 0.023). B. GSTO1-1 could be detected in the plasma samples and bronchoalveolar lavage fluid. Representative Western blots from three non-smokers are shown. C. GSTO1-1 was also expressed in the culture medium of U937 monocytes and BEAS-2B cells. D. Lung tissue homogenates showed higher level of GSTO1-1 in the specimens obtained from non-smokers or smokers with normal lung function when compared to COPD, p = 0.003.

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 family (GST) that detoxifies toxic substrates present in tobacco smoke by a GSH-dependent mechanism. GSTO contains an N-terminal glutathione-binding domain and is able to bind and release GSH. GSH is one of the major antioxidants in airway secretions, and it can be hypothesized that GSTO participates in the maintenance of GSH homeostasis not only intracellularly but also in the extracellular space. ROS, reactive oxygen species; RNS, reactive nitrogen species; GSH, reduced glutathione; GSSG, oxidized glutathione; GR glutathione reductase; GCL, glutamate cysteine ligase; Grx, glutaredoxin; Prxe, peroxiredoxin, GPXe, glutathione peroxidase (extracellular); GSX, glutathione; ECSOD, extracellular superoxide dismutase; GSNO, nitrosoglutathione; GS, glutathione syntethase.