Leukocyte-derived extracellular superoxide dismutase does not contribute to airspace EC-SOD after interstitial pulmonary injury

Abstract

The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is abundant in the lung and is known to limit inflammation and fibrosis following numerous pulmonary insults. Previous studies have reported a loss of full-length EC-SOD from the pulmonary parenchyma with accumulation of proteolyzed EC-SOD in the airspace after an interstitial lung injury. However, following airspace only inflammation, EC-SOD accumulates in the airspace without a loss from the interstitium, suggesting this antioxidant may be released from an extrapulmonary source. Because leukocytes are known to express EC-SOD and are prevalent in the bronchoalveolar lavage fluid (BALF) after injury, it was hypothesized that these cells may transport and release EC-SOD into airspaces. To test this hypothesis, C57BL/6 wild-type and EC-SOD knockout mice were irradiated and transplanted with bone marrow from either wild-type mice or EC-SOD knockout mice. Bone marrow chimeric mice were then intratracheally treated with asbestos and killed 3 and 7 days later. At both 3 and 7 days following asbestos injury, mice without pulmonary EC-SOD expression but with EC-SOD in infiltrating and resident leukocytes did not have detectable levels of EC-SOD in the airspaces. In addition, leukocyte-derived EC-SOD did not significantly lessen inflammation or early stage fibrosis that resulted from asbestos injury in the lungs. Although it is not influential in the asbestos-induced interstitial lung injury model, EC-SOD is still known to be present in leukocytes and may play an influential role in attenuating pneumonias and other inflammatory diseases.

Fig. 1.

Generation and treatment of bone marrow chimeric (BMC) mice. BMC mice were generated by the irradiation of wild-type (WT) and extracellular superoxide dismutase (EC-SOD) knockout (KO) recipients and subsequent transplantation with WT and EC-SOD KO bone marrow from donor mice (A). After irradiation and bone marrow transplantation, BMC mice undergo several procedures and treatments: verification of chimerism to ensure efficient reconstitution of donor bone marrow in the recipient mice, intratracheal instillation of liposomal clodronate to deplete resident alveolar macrophages and replace them with donor-derived cells, and finally intratracheal treatment with asbestos to induce interstitial lung injury (B).

Fig. 2.

Immunostaining of EC-SOD in lung tissue from asbestos-treated BMC mice. EC-SOD in the lungs of the BMC mice was assessed 7 days after asbestos exposure to evaluate the localization of EC-SOD. In the WT/WT mice, EC-SOD expression is present in the alveolar parenchyma as well as resident alveolar macrophages (arrow) and infiltrating inflammatory cells. EC-SOD was present in the alveolar parenchyma but not in resident alveolar macrophages (arrow) or infiltrating inflammatory cells of WT/KO mice. Conversely, KO/WT mice have no pulmonary EC-SOD expression, but EC-SOD is expressed inside of inflammatory cells in the airspaces as well as resident alveolar macrophages (arrow). KO/KO mice did not have any EC-SOD present in the lungs. Positive staining was not present in the rabbit IgG control for each chimeric group.

Fig. 3.

Absence of airspace EC-SOD in KO/WT mice at both 3 and 7 days postasbestos exposure. Western blotting for EC-SOD was performed on equal protein amounts of bronchoalveolar lavage fluid (BALF, 8 μg) from BMC mice 3 and 7 days following intratracheal treatment with asbestos (A). The absence of EC-SOD in the KO/WT BALF indicates that leukocyte-derived EC-SOD does not contribute to the EC-SOD that accumulates in the airspace after asbestos-induced lung injury. The effect of clodronate on EC-SOD expression was also assessed by analyzing BALF from asbestos-treated WT/WT and WT/KO mice with and without preceding clodronate treatment (B). The levels of EC-SOD in the BALF of the WT/WT and WT/KO mice was also compared with nontransplanted WT mice 3 and 7 days after asbestos exposure (C). EC-SOD positive control (+) was 8 μg of WT lung homogenate.

Fig. 4.

Leukocyte-derived EC-SOD does not alter leukocyte accumulation in response to asbestos. Total white blood cells and cell differentials in the BALF of the BMC mice were measured 3 (n = 5–6 mice/group, A) and 7 (n = 3–5 mice/group, B) days after asbestos treatment. Cellular infiltrates in the BALF of nontransplanted WT mice were also analyzed 3 (n = 3/group, C) and 7 (n = 3/group, D) days following asbestos or inert particulate control treatment. Total cells were calculated by multiplying the cell concentration (cells/ml) by the total volume of BALF recovered. Data were analyzed using a one-way ANOVA with Tukey's posttest and are means ± SE. *P < 0.05.

Fig. 5.

Leukocyte-derived EC-SOD does not lessen the early fibrotic response to asbestos-induced interstitial lung injury. BMC mice were killed 7 days after intratracheal treatment with asbestos. Hematoxylin and eosin staining of lung sections revealed that leukocyte-derived EC-SOD did not attenuate asbestos-induced fibrosis, since the amount of fibrosis in the lungs was similar for all chimeric mice (A). Average histology score also showed no differences in fibrosis (A). The lungs of nontransplanted WT mice 7 days after asbestos or inert particulate control intratracheal treatment were also analyzed (B). Representative images are shown, and data are means ± SE; n = 3–5 mice/group.