Original Contribution
Smoking and COPD increase sputum levels of extracellular superoxide dismutase

https://doi.org/10.1016/j.freeradbiomed.2011.05.008Get rights and content

Abstract

Extracellular superoxide dismutase (ECSOD) is the major superoxide-scavenging enzyme in the lung. Certain ECSOD polymorphisms are protective against COPD. We postulated that smokers and COPD subjects would have altered levels of ECSOD in the lung, airway secretions, and/or plasma. Lung tissue ECSOD was evaluated from nonsmokers, smokers, and subjects with mild to very severe COPD by Western blot, immunohistochemistry, and ELISA. ECSOD levels in plasma, bronchoalveolar lavage fluid (BALF), and induced-sputum supernatants were analyzed by ELISA and correlated with smoking history and disease status. Immunohistochemistry identified ECSOD in extracellular matrix around bronchioles, arteries, and alveolar walls, with decreases seen in the interstitium and vessels of severe COPD subjects using digital image analysis. Plasma ECSOD did not differ between COPD subjects and controls nor based on smoking status. ECSOD levels in induced sputum supernatants were elevated in current smokers and especially in COPD subjects compared to nonsmokers, whereas corresponding changes could not be seen in the BALF. ECSOD expression was reduced around vessels and bronchioles in COPD lungs. Substantial increases in sputum ECSOD in smokers and COPD is interpreted as an adaptive response to increased oxidative stress and may be a useful biomarker of disease activity in COPD.

Introduction

The pathogenesis of chronic obstructive pulmonary disease (COPD) has been strongly linked with oxidative stress [1], [2], [3], [4], [5], [6]. One of the major antioxidant enzymes of the human lung parenchyma that scavenges superoxide radicals is extracellular superoxide dismutase (ECSOD or SOD3) [7], [8]. Genetic studies have suggested that a functional polymorphism of ECSOD, the Arg213Gly mutation (associated with elevated circulating ECSOD levels) and other ECSOD polymorphisms are protective against COPD in smokers [9], [10], [11], [12]. However, based on current knowledge, the Arg213Gly polymorphism can explain only a small proportion of protection against COPD development given that only 25% of smokers develop COPD [13]. It is unclear whether lung ECSOD tissue levels or airway-secreted ECSOD levels are directly associated with COPD and its severity.

Three SOD enzymes (CuZnSOD, MnSOD, and ECSOD) have been characterized in mammalian cells, and they have a cell-specific distribution in human lung. The regulation and roles of CuZnSOD and MnSOD in nonmalignant and malignant lung diseases have been previously studied and reviewed in detail [14], [15]. However, the significance of ECSOD especially in human lung diseases remains unclear. Experimental studies have shown that mice lacking ECSOD survive but are vulnerable to oxidative stress [16], [17], [18], [19]; transgenic mice overexpressing ECSOD, in contrast, are protected against oxidative stress [17]. ECSOD plays a major role in protecting lung against hyperoxia [17] and asbestos fibers [18], [19]. In experimental studies on asbestos exposure, ECSOD is redistributed and secreted to the bronchoalveolar lavage fluid (BALF) [19]. To our knowledge only two studies have investigated ECSOD in experimental cigarette smoke exposure by using ECSOD (SOD3) transgenic and knockout mice [20.21]. These studies concluded that ECSOD protected against smoke- and elastase-induced emphysema [20] and that macrophages from ECSOD-overexpressing mice had less oxidative stress and lower levels of inflammatory cytokines in response to cigarette smoke [21].

ECSOD is a homotetrameric glycoprotein in which the monomer is secreted in one of two forms: with a polybasic binding tail present or with it proteolytically cleaved [22]. Tetramers consisting solely of monomers lacking the binding tail are postulated to not bind to tissue extracellular proteins, and the lack of tissue binding has in turn been suggested to reduce the local protective effect of the enzyme [23], [24]. ECSOD is expressed in lung tissue, with high levels being found in the vasculature and epithelium [7], [25]. It has an affinity for binding to the negatively charged extracellular macromolecules of collagen, hyaluronan, and heparan sulfate [26]. The Arg213Gly mutation that is associated with protection against COPD in smokers modifies the binding tail, affecting binding to collagen and increasing plasma levels of ECSOD to levels that are 6- to 10-fold higher than normal [27], [28]. It remains unclear how ECSOD modifies the risk of COPD in smokers with and without this mutation and whether there are differences in ECSOD expression in lung tissue or airways.

Only a few studies have been conducted on ECSOD in human lung diseases. Lung ECSOD is low/undetectable in fibroblastic foci lesions of idiopathic pulmonary fibrosis (IPF) [29], and its expression is very low in human lung cancers [30]. A previous study of ECSOD found no changes in lung ECSOD immunoreactivity in mild–moderate COPD [31].

We hypothesized that ECSOD would be secreted into the lung extracellular fluids and that it may differ in extracellular fluids such as sputum and BALF and circulating blood in response to oxidative stress from smoking and/or COPD. Levels of ECSOD in these secretions may provide useful biomarkers to detect early lung injury related to smoking. Plasma and sputum levels of ECSOD were compared in smokers with and without COPD to those in nonsmokers. Sputum and BALF levels of ECSOD were compared to assess their relative utility as biomarkers of disease activity.

Section snippets

Material and methods

These studies were carried out collaboratively in both Finland and the United States from complementary populations. ELISA measurements were performed in Denver, Colorado, on human samples (plasma, sputum, and BALF) from both Finland and Denver. Lung tissue was studied in two cohorts: the U.S. source was the Lung Tissue Research Consortium (LTRC) and the other was patients from Helsinki. COPD was evaluated by the GOLD criteria [32]. In this classification the various severities include GOLD

Immunohistochemical analysis reveals cell-specific declines in ECSOD in the COPD lung

Immunohistochemistry confirmed previous results in ECSOD distribution in human lung [25], [45], i.e., ECSOD was strongly expressed in the extracellular matrix of the perivascular areas and around the bronchioles, consistent with its known binding to negatively charged extracellular proteins such as collagen and proteoglycans (Fig. 1). It also was seen in the alveolar walls and occasionally as high expression in alveolar type II cells. Within the bronchiolar lumen there was staining along the

Discussion

ECSOD can be detected in lung tissue, in specific cell types within the lung parenchyma, and in airway lining fluid, sputum, and plasma. Under normal conditions levels of ECSOD in each of these tissues or fluids represent a combination of constitutive secretion and response to local regulatory factors. Our results suggest that both cigarette smoking and the disease processes associated with COPD affect ECSOD expression and secretion into specific lung compartments. They further suggest that

Acknowledgments

This study utilized biological specimens and data provided by the Lung Tissue Research Consortium supported by the National Heart, Lung, and Blood Institute. Adjunct Professor Marjukka Myllärniemi is acknowledged for helping with the image analysis, and Mrs. Tiina Marjomaa for her skillful technical assistance. Funding was provided by the Monfort Foundation (E.A.R., E.M., J.D.C., R.P.B.), the Flight Attendant Medical Research Institute (R.P.B.), a government subsidy for health science research

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