Altered expression of extracellular superoxide dismutase in mouse lung after bleomycin treatment

Abstract

The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is highly expressed in the extracellular matrix of lung tissue and is believed to protect the lung from oxidative damage that results in diseases such as pulmonary fibrosis. This study tests the hypothesis that proteolytic removal of the heparin-binding domain of EC-SOD results in clearance of the enzyme from the extracellular matrix of pulmonary tissues and leads to a loss of antioxidant protection. Using a polyclonal antibody to mouse EC-SOD, the immunodistribution of EC-SOD in normal and bleomycin-injured lungs was examined. EC-SOD labeling was strong in the matrix of vessels, airways, and alveolar surfaces and septa in control lungs. At 2 d post-treatment, a slight increase in EC-SOD staining was evident. In contrast, lungs examined 4 or 7 d post-treatment, showed an apparent loss of EC-SOD from the matrix and surface of alveolar septa. Notably, at 7 d post-treatment, the truncated form of EC-SOD was found in the bronchoalveolar lavage fluid of bleomycin-treated mice, suggesting that EC-SOD is being removed from the extracellular matrix through proteolysis. However, loss of EC-SOD through proteolysis did not correlate with a decrease in overall pulmonary EC-SOD activity. The negligible effect on EC-SOD activity may reflect the large influx of intensely staining inflammatory cells at day 7. These results indicate that injuries leading to pulmonary fibrosis have a significant effect on EC-SOD distribution due to proteolytic removal of the heparin-binding domain and may be important in enhancing pulmonary injuries by altering the oxidant/antioxidant balance in alveolar interstitial spaces.

Introduction

Reactive oxygen species such as superoxide and hydrogen peroxide are important pathogenic mediators of many diseases including pulmonary diseases [1], [2], [3]. Under normal conditions, the tissue damage caused by reactive oxygen species can be prevented by endogenous antioxidant mechanisms. However, when these mechanisms no longer compensate for large amounts of damaging oxidants, irreversible tissue damage can occur.

Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that catalyzes the dismutation of superoxide into hydrogen peroxide and oxygen [4]. The most recently characterized of the three superoxide dismutases, it shares this ability with two other SOD isozymes: the cytoplasmic and nuclear CuZn-SOD [5] and the mitochondrial Mn-SOD [6]. EC-SOD is a 135 kDa protein that exists primarily as a tetramer of four identical subunits [4]. This tetramer consists of two dimers, each containing a disulfide bond that links the heparin-binding domains in the C-terminus of two subunits together [7], [8], [9]. EC-SOD is found predominantly in the extracellular matrix of tissues and to a lesser extent in extracellular fluids [10], [11], [13]. The heparin-binding domain of EC-SOD interacts with heparin sulfate in the extracellular matrix of tissues and cell surfaces. This affinity to heparin sulfate is believed to be important in determining the specific location of EC-SOD in the extracellular matrix and allowing it to act as an efficient antioxidant. Heparin-binding affinity can be modulated through proteolysis of the heparin-binding domain and is believed to be important in regulating the distribution of EC-SOD in various tissues [7], [14]. Notably, the activity of many proteolytic enzymes is increased during inflammation. This increase in proteolytic activity may further exacerbate the oxidative stress associated with inflammation by removing EC-SOD from the affected area.

While EC-SOD is ubiquitously expressed in mammalian tissues, both species and tissue heterogeneity exist. Mice contain high levels of EC-SOD in their lungs compared to other mammals [10]. Increased levels of this antioxidant enzyme in the mouse lung suggests an important role for extracellular superoxide scavenging in this organ, possibly as a protection against the relatively high levels of oxygen to which the lung is continually exposed. The bleomycin-treated mouse is a well-described model system for oxidative stress–induced pulmonary fibrosis, and the abundance of EC-SOD in the mouse lung makes this a good system to study the role of EC-SOD in the development of pulmonary fibrosis. Treatment of mice with bleomycin results in a well characterized fibrotic response that occurs in two distinct phases. First, there is an acute phase characterized by an influx of inflammatory cells, in particular macrophages and polymorphonuclear leukocytes (PMN). This is followed by a chronic stage characterized by extracellular matrix remodeling and collagen deposition [15], [16]. EC-SOD is known to colocalize with type I collagen in the lung [13]. This relationship of EC-SOD and collagen may be particularly important since collagen is sensitive to degradation by superoxide. Collagen fragments can function as chemoattractants and activators of macrophages and neutrophils [17], [18], [19], [20]. Thus, proteolytic removal of EC-SOD from matrices rich in type I collagen may potentiate inflammation by increasing oxidative stresses leading to collagen fragmentation.

This study tests the hypothesis that insults leading to pulmonary inflammation and fibrosis may alter the distribution of EC-SOD in the extracellular matrix of the lung via enhanced proteolysis of the heparin-binding domain of EC-SOD and the subsequent clearance of this enzyme from the extracellular space. This will result in a change in the oxidant/antioxidant balance in the pulmonary extracellular matrix and would potentially contribute to a proinflammatory environment. The effect of bleomycin treatment on EC-SOD activity and immunodistribution in mouse lung is examined.