Modulation of glutaredoxin in the lung and sputum of cigarette smokers and chronic obstructive pulmonary disease

The pathogenesis of chronic obstructive pulmonary disease (COPD) is probably strongly associated with reactive oxygen metabolites. Cigarette smoke not only contains high levels of oxidants, but it also leads to the accumulation of neutrophils and macrophages in the lung and to their activation [1‐3]. A number of studies have investigated antioxidant defense mechanisms in cigarette smoke exposed cells and in chronic cigarette smokers. These studies have found that glutathione (GSH), a thiol containing tripeptide present in the epithelial lining fluid (ELF) at high concentrations, plays an essential role in protecting human airways against exogenous and endogenous oxidants and cigarette smoke [1, 4, 5]. However, only some of the enzyme systems participating in GSH regulation, and thereby probably also participating in COPD pathogenesis, have been investigated in human lung.

Glutathione is present in increased concentrations in the ELF of chronic smokers [6] and both acute and chronic exposure of experimental animals to cigarette smoke causes depletion in the intracellular GSH concentration [7]. GSH is transported from cells by multiple mechanisms, while the plasma membrane is impermeable to GSH preventing its transportation back into cells. The replenishment of intracellular GSH is accomplished by the reduction of oxidized glutathione, i.e. glutathione disulphide (GSSG), release of GSH from the proteins and de novo GSH synthesis [8]. Enzyme mechanisms that are known to regulate GSH metabolism include the rate-limiting enzyme in GSH synthesis, glutamate cysteine ligase (GCL, also known as γ-glutamylcysteine synthetase, γ-GCS), glutathione peroxidases (GPx), glutathione reductase (GR), γ-glutamyltranspeptidase (γ-GT) and glutathione-S-transferases (GST). There appears to be increased mRNA expression of GCL, GPxs and some GSTs in the bronchial epithelium of chronic smokers, but decreased immunoreactivities or activities of several of these enzymes in cigarette smokers or during COPD progression [9]. Glutaredoxins (Grx) represent a redox modulatory protein family with potential effects on GSH regulation and homeostasis, but until now they have not been assessed in smoking related lung diseases.

Classical glutaredoxins are small thiol disulfide oxidoreductases with a conserved active site sequence -CXXC- and a GSH binding site. They belong to the thioredoxin fold superfamily [10, 11], thioredoxin being a known redox modulatory enzyme in human lung [12, 13]. There are two Grxs in humans, cytosolic Grx1 and mitochondrial Grx2 [14, 15]. They catalyze disulfide reductions, preferring GSH-mixed disulfides as substrates, by utilizing the reducing power of GSH in the presence of NADPH and glutathione reductase [16]. Grxs can be hypothesized to participate in the reduction of the GSH-mixed disulfides of thiol-containing proteins back to their active forms during and after oxidative stress in cigarette smokers and in COPD.

In this study the expression of Grxs was investigated in lung specimens of non-smokers and cigarette smokers and in different stages [17] of COPD or emphysema/COPD associated with α-1-antitrypsin deficiency (GOLD stages I, II and IV). The major focus was on macrophages, as Grx1 is mainly expressed in these cells [18] and since one typical feature in COPD is the accumulation of macrophages in the lung. Given that Grx1 may be present also in the extracellular space [19, 20], levels of Grx1 were also examined from induced sputum specimens, both cells and supernatants obtained from non-smokers, smokers and COPD patients.

The present study suggests that Grx1 is a potential redox modulatory protein regulating the homeostasis of glutathionylated proteins and GSH not only in healthy lung but also in cigarette smokers and in COPD patients. In addition, the presence of Grx1 in the extracellular space may play a role in the replenishment of glutathione in the airways, especially during acute exacerbations where the oxidant stress is increased.

One recent microarray study found no change in Grx1 mRNA levels in the bronchial epithelium of healthy smokers compared to non-smokers [30]. In another study on cultured bronchial epithelial cells, Grx was found to be elevated by 10 fold during the first 10 hours of exposure to cigarette smoke [31]. However, to date no human studies are available on the protein levels or the regulation of Grxs in smokers' lung or in COPD patients. It is well known that cigarette smoke can increase GSH levels in the epithelial lining fluid and cause depletion of intracellular free GSH [6, 7]. It appears that after initial GSH depletion intracellular GSH levels increase for reasons that are unknown, one mechanism being increased GSH synthesis [4, 7, 9]. Oxidative stress also causes accumulation of protein-GSH mixed disulfides [32]. Since the intracellular concentration of cysteine-residues in proteins is higher than the concentration of free GSH, the formation of protein-GSH mixed disulfides has the potential to serve as a significant depository of GSH in living cells. Furthermore it has been shown that in an oxidizing environment, a major fraction of GSH is in fact present as mixed disulfides with proteins [33]. Formation of these mixed disulfides between GSH and proteins may serve both a regulatory and an antioxidant function by protecting the enzymes from irreversible oxidation that might lead to enzyme inactivation. Once oxidative stress has been alleviated the protein-GSH mixed disulfides are efficiently reduced by Grxs liberating free glutathione.

The present study suggests that Grx1 may be involved in the regulation of glutathionylated protein levels and GSH homeostasis in human lung (Fig 7). Immunohistochemical studies showed that Grx1 was mainly expressed in the alveolar macrophages of non-smokers and smokers and at different stages of COPD and that the level of Grx1 decreased according to the severity of the disease. These conclusions are supported by Western blot analysis. Not all macrophages in the sections were Grx1 positive, probably due to presence of various macrophage populations and the age of these cells. Overall, these changes are apparently related both to the total number macrophages present in lung as well as to the differences in the expression levels of Grx in macrophages.

The present study confirmed that Grx1 can be detected from the plasma, as shown previously [19, 20], and more importantly demonstrated that Grx1 could also be detected from sputum, both from the cells and supernatants. The latter observation suggests that Grx1 may be exported from alveolar macrophages to the extracellular space (i.e. sputum extract). The levels of Grx1 in the sputum supernatants were significantly higher in acute exacerbations of COPD than in the controls. This suggests that extracellular Grx1 attempts to reduce GSH-mixed disulfides during the oxidative stress in COPD to restore active proteins and to increase the concentration of free glutathione.

The assessment of functional Grx activity from the lung or sputum specimens is difficult since the assays available may not to be specific for Grx alone. In our recent paper [34] we described a new method for measuring the deglutathionylation activity in vitro, but also this assay is unsuitable for measuring the glutaredoxin activity in tissue or cell homogenates. In that particular study also other thioredoxin family members showed deglutathionylation activity, further suggesting that the current methods are not specific for the assessment of Grx activity in vivo. Supporting the findings in the present study, our recent observations showed that the activity of E. coli Grx1, which is similar to human Grx1, is markedly reduced under conditions mimicking oxidative stress [35].

Grx2 expression could not be detected reliably with the methods used here. However, this does not rule out the importance of Grx2 in the antioxidant defense of lung. Grx2 is a mitochondrial protein [14, 15] and therefore it might have a role in the regulation of the mitochondrial redox state and apoptotic events. Grx2 clearly needs further investigation in order to better understand the role of thiol oxidoreductases in the antioxidative defense mechanisms in human lung and COPD progression.

The regulation of Grx1 is poorly understood, but recent results of our laboratory and others have suggested minor or non-significant Grx1 induction by oxidants [35, 36] and significant Grx1 downregulation by transforming growth factor-beta (TGF-β) [18]. Grx1 is, however, only one of the enzymes that regulate GSH homeostasis in human lung. The rate limiting enzyme in GSH synthesis is GCL and this enzyme has been shown to be transiently induced by oxidative stress, cigarette smoke and COPD [1, 8, 30, 37] but decreased by TGF-β in vitro [38] and during COPD progression in vivo [9, 39]. The decrease of Grx1 in severe COPD/emphysema found here may in fact be associated with a simultaneous downregulation of other GSH associated enzymes in COPD thus further increasing the oxidant burden in the lung. These regulatory pathways clearly need to be studied in more detail.

In conclusion, Grx1 is a protein closely associated with GSH and reduction of GSH-mixed disulfides. Grx1 is located mainly in alveolar macrophages and the levels are decreased according to severity of COPD. Grx1 can also be detected from sputum supernatants, the levels being higher in acute exacerbations of COPD than in healthy controls. This increase in Grx1 levels may serve as an attempt to restore the functional activity of glutathionylated proteins and to increase the extracellular level of free glutathione. Overall these findings suggest that Grx1 is involved in the regulation of both intracellular and extracellular GSH homeostasis, and associated with the decrease of the GSH dependent antioxidant defense in severe COPD.