Inflammatory Cells as a Source of Airspace Extracellular Superoxide Dismutase after Pulmonary Injury

Abstract

Extracellular superoxide dismutase (EC-SOD) is an antioxidant abundant in the lung. Previous studies demonstrated depletion of lung parenchymal EC-SOD in mouse models of interstitial lung disease coinciding with an accumulation of EC-SOD in airspaces. EC-SOD sticks to the matrix by a proteolytically sensitive heparin-binding domain; therefore, we hypothesized that interstitial inflammation and matrix remodeling contribute to proteolytic redistribution of EC-SOD from lung parenchyma into the airspaces. To determine if inflammation limited to airspaces leads to EC-SOD redistribution, we examined a bacterial pneumonia model. This model led to increases in airspace polymorphonuclear leukocytes staining strongly for EC-SOD. EC-SOD accumulated in airspaces at 24 h without depletion of EC-SOD from lung parenchyma. This led us to hypothesize that airspace EC-SOD was released from inflammatory cells and was not a redistribution of matrix EC-SOD. To test this hypothesis, transgenic mice with lung-specific expression of human EC-SOD were treated with asbestos or bleomycin to initiate an interstitial lung injury. In these studies, EC-SOD accumulating in airspaces was entirely the mouse isoform, demonstrating an extrapulmonary source (inflammatory cells) for this EC-SOD. We also demonstrate that EC-SOD knockout mice possess greater lung inflammation in response to bleomycin and bacteria when compared with wild types. We conclude that the source of accumulating EC-SOD in airspaces in interstitial lung disease is inflammatory cells and not the lung and that interstitial processes such as those found in pulmonary fibrosis are required to remove EC-SOD from lung matrix.

Extracellular Superoxide Dismutase in Macrophages Augments Bacterial Killing by Promoting Phagocytosis

“…The expression of EC-SOD in the airspace and lung parenchyma was evaluated in wild-type mice via Western blot analyses of the BALF and lung homogenates. Consistent with previous work, 19 bacterial pneumonia leads to a significant increase in EC-SOD in the alveolar lining fluid (data not shown).…”

“…The entire left lung was removed and placed in 1 mL sterile water for homogenization as previously described. 19 Bronchoalveolar lavage fluid (BALF) was collected via instillation and recovery of 0.6 mL 0.9% saline solution from the right lung, and the right lung was then inflation-fixed with 10% formalin for histologic analyses.…”

“…12 BALF supernatants were stored at Ϫ70°C until analyses. The left lung was homogenized as previously described, 19 and the homogenates were separated into aliquots for quantitative cultures of bacteria, Western blot analysis, and myeloperoxidase assays. In brief, for Western blot analyses, buffer was added to an aliquot of lung homogenate to a final concentration of buffer containing 0.5% Triton X-100, 150 nmol/L NaCl, 15 mmol/L Tris, 1 mmol/L Ca Cl 2 , and 1 mmol/L MgCl 2 (pH 7.4), incubated on ice for 30 minutes, and centrifuged at 10,000 ϫ g for 20 minutes.…”

“…[7][8][9][10] EC-SOD acts as both an anti-inflammatory and antifibrotic agent in a number of pulmonary diseases including bleomycin-and asbestos-induced pulmonary fibrosis, [11][12][13][14] hyperoxia, [15][16][17] lipopolysaccharide-induced inflammation, 18 and pulmonary infection. 19,20 One mechanism by which EC-SOD inhibits inflammation is directly binding to and inhibiting oxidative fragmentation of several components in the extracellular matrix including collagen, heparan sulfate, and hyaluronan after interstitial lung injury. 11,[21][22][23][24] The importance of endogenous pulmonary EC-SOD was recently highlighted in a study that demonstrated that acute loss of EC-SOD resulted in 85% mortality secondary to the spontaneous development of acute respiratory distress syndrome.…”

“…In addition to its localization on matrices and in extracellular fluids, EC-SOD is also present intracellularly in neutrophils and macrophages. 13,19,26 However, the local-ization and role of EC-SOD in these inflammatory cells have not been investigated.…”