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.

Figure 1.

Bacterial recovery from the lungs of mice exposed to E. coli. Mice were killed immediately after bacterial exposure to determine the initial bacterial inoculation at 0 h (n = 4). Mice were killed at 6 h (n = 6) or at 24 h (n = 10) to determine remaining bacterial burden as the lungs cleared the infection. Results are representative of two separate experiments.

Figure 2.

Bacterial inhalation induces airspace inflammation. Hematoxylin and eosin staining was performed on lung sections from mice exposed to inhaled E. coli. Mice killed at 0 h (A) showed only occasional airway macrophages. At 24 h post-treatment (B), increased numbers of white blood cells were found in the airspaces. The majority of these cells were neutrophils (C), although macrophages were also present. Bar, 50 μm.

Figure 3.

BALF PMNs and macrophages are increased after bacterial inoculation. BALF was recovered from mouse lungs as described in Materials and Methods, and total numbers of PMNs and macrophages (A) were determined by differential counting. MPO activity (B) and MMP-9 levels (C) were determined. *P < 0.01 compared with 0 h, ANOVA and Tukey's post-test. **P < 0.05 compared with 0 and 6 h, ANOVA and Tukey's post-test. ***P < 0.05 compared with 0 h, Student's t test.

Figure 4.

EC-SOD accumulates in the BALF 24 h after inoculation with E. coli. Western blots were performed on equivalent protein amounts of BALF. (A) Total ECSOD is increased in the BALF 24 h after inoculation. (B) The majority of accumulating EC-SOD is proteolyzed (lacks heparin-binding domain). (C) There was no significant difference in total EC-SOD at 6 h. Results are representative of two separate experiments. *P < 0.05, Student's t test.

Figure 5.

EC-SOD is not depleted from lung homogenates 24 h after bacterial inoculation. Western blots were performed on equivalent protein amounts of lung homogenate. Total EC-SOD in the lung is unchanged 24 h after inoculation (P = 0.15, Student's t test).

Figure 6.

EC-SOD levels in the lung parenchyma are unchanged after E. coli inoculation. Paraffin-embedded lung sections (5 μm thick) from mice killed at 0 h (A and C) or at 24 h (B and D) were stained with antibody against EC-SOD (A and B) or with normal rabbit serum (C and D). (E) Macrophages (arrow) and PMN (arrowheads) stained strongly for EC-SOD. (F) EC-SOD labeling intensity is unchanged 24 h after inoculation (P = 0.54, Student's t test). Bar, 50 μm.

Figure 7.

Human EC-SOD produced under the SPC promoter in transgenic mice is not found in BALF of asbestos-treated transgenic mice. Western blots for EC-SOD were performed on lung homogenates (A) and BALF (B) from SPC EC-SOD transgenic (Trg) and wild-type (WT) mice. (A) Trg mice produce the human and mouse forms of EC-SOD (distinguishable on reducing SDS-PAGE) in the lung, whereas inflammatory cells express only mouse EC-SOD. Fourteen days after exposure to asbestos (B) or 3 d after exposure to bleomycin (C), there is BALF accumulation of only mouse EC-SOD in the Trg mice, suggesting an extrapulmonary source of EC-SOD (i.e., infiltrating inflammatory cells).

Figure 8.

EC-SOD knockout mice have increased neutrophils in response to inflammatory lung injury. C57BL/6 wild-type (WT) mice and EC-SOD knockout (KO) mice were intratracheally instilled with 0.04 units bleomycin (A) or with 1 × 10⁵ bacteria (B). Bacteria consisted of heat-killed K. pneumoniae. Percentages of neutrophils in the BALF were determined through differential counting. *P < 0.05, one-way ANOVA and Tukey's post test, bleomycin-treated EC-SOD KO mice compared with bleomycin-treated wild-type mice.