Susceptibility of cyclooxygenase-2-deficient mice to pulmonary fibrogenesis

Abstract

The cyclooxygenase (COX)-2 enzyme has been implicated as an important mediator of pulmonary fibrosis. In this study, the lung fibrotic responses were investigated in COX-1 or COX-2-deficient (-/-) mice following vanadium pentoxide (V(2)O(5)) exposure. Lung histology was normal in saline-instilled wild-type and COX-deficient mice. COX-2(-/-), but not COX-1(-/-) or wild-type mice, exhibited severe inflammatory responses by 3 days following V(2)O(5) exposure and developed pulmonary fibrosis 2 weeks post-V(2)O(5) exposure. Western blot analysis and immunohistochemistry showed that COX-1 protein was present in type 2 epithelial cells, bronchial epithelial cells, and airway smooth muscle cells of saline or V(2)O(5)-exposed wild-type and COX-2(-/-) mice. COX-2 protein was present in Clara cells of wild-type and COX-1(-/-) terminal bronchioles and was strongly induced 24 hours after V(2)O(5) exposure. Prostaglandin (PG) E(2) levels in the bronchoalveolar lavage (BAL) fluid from wild-type and COX-1(-/-) mice were significantly up-regulated by V(2)O(5) exposure within 24 hours, whereas PGE(2) was not up-regulated in COX-2(-/-) BAL fluid. Tumor necrosis factor-alpha was elevated in the BAL fluid from all genotypes after V(2)O(5) exposure, but was significantly and chronically elevated in the BAL fluid from COX-2(-/-) mice above wild-type or COX-1(-/-) mice. These findings indicate that the COX-2 enzyme is protective against pulmonary fibrogenesis, and we suggest that COX-2 generation of PGE(2) is an important factor in resolving inflammation.

Figure 1.

Lung histopathology showing increased injury and inflammation in COX-2^−/− mice relative to wild-type and COX-1^−/− mice 3 days following intratracheal instillation of V₂O₅. A: Saline-instilled wild-type. B: V₂O₅-instilled wild-type. Inset shows mononuclear cell infiltration (arrow). C: Saline-instilled COX-1^−/−. D: V₂O₅-instilled COX-1^−/−. Arrow in inset indicates mononuclear cell. E: Saline-instilled COX-2^−/−. F: V₂O₅-instilled COX-2^−/−. Arrow in inset indicates multinucleated giant cell amid numerous mononuclear cells. All sections were stained with hematoxylin and eosin. Magnification, ×80, except for insets which are ×400.

Figure 2.

Trichrome staining of lung sections showing enhanced fibrosis in COX-2^−/− mice relative to wild-type and COX-1^−/− mice 15 days following intratracheal instillation of V₂O₅. The magnification of each panel is ×80. A: Saline-instilled wild-type. B: V₂O₅-instilled wild-type. C: Saline-instilled COX-1^−/−. D: V₂O₅-instilled COX-1^−/−. E: Saline-instilled COX-2^−/−. F: V₂O₅-instilled COX-2^−/−.

Figure 3.

Hydroxyproline content in lungs of wild-type, COX-1^−/−, and COX-2^−/− mice instilled with saline or V₂O₅. Lungs were harvested 15 days after instillation and digested for hydroxyproline content using the colorimetric assay described in Materials and Methods. Numbers in parentheses above each bar indicate number of animals in that group. *, P < 0.05 compared to saline.

Figure 4.

COX-1 and COX-2 protein expression 24 hours following saline and V₂O₅ instillation in wild-type, COX-1^−/−, and COX-2^−/− mice. The abundance of COX-1 and COX-2 protein in the lungs was determined by Western blotting using immunospecific antibodies as described in Materials and Methods. A: Representative Western blot. B: Scanning densitometry of COX-1 and COX-2 expression. Data are the mean ± range of two separate experiments.

Figure 5.

Immunohistochemistry showing COX-1 localization in type II epithelial cells, airway smooth muscle cells, and airway epithelium. A: Saline-instilled wild-type showing numerous COX-1 positive cells residing within the alveolar walls of the lung parenchyma (arrows); the inset panel shows the results of SP-A immunostaining that confirmed these cells as type II pneumocytes. Airway epithelial cells (barbed arrow) and airway smooth muscle cells (arrowhead) were also positive for COX-1. B: V₂O₅-instilled wild-type showing COX-1 localized in type II epithelial cells (arrows), airway epithelium (barbed arrows), and airway smooth muscle (arrowhead). C: Saline-instilled COX-1 null. D: V₂O₅-instilled COX-1 null. E: Saline-instilled COX-2 null showing COX-1 localized in type II cells (arrows) but not in Clara cells of terminal bronchiole. F: V₂O₅-instilled COX-2 null showing COX-1 localized in airway epithelium (barbed arrow), airway smooth muscle (arrowhead), and type II cells (arrows). Note the absence of COX-1 immunostaining in the COX-1 null mice (C and D). Magnification of all panels ×160.

Figure 6.

Immunohistochemistry showing COX-2 localization in Clara cells of the distal airways and terminal bronchioles. A: Saline-instilled wild-type showing weak COX-2 immunostaining in Clara cells. B: V₂O₅-instilled wild-type showing increased COX-2 immunostaining in epithelial cells of terminal bronchioles that were confirmed as Clara cells by immunostaining with a CC10 antibody (inset). Arrows indicate the distal end of the terminal bronchiole in serial sections stained for COX-2 or CC10. C: Saline-instilled COX-1 null. D: V₂O₅-instilled COX-1 null showing increased COX-2 immunostaining in Clarac cells of terminal bronchioles (inset shows CC10 immunostaining on a serial section and arrows indicate the same bronchiole that is positive for both COX-2 and CC10). E: Saline-instilled COX-2 null. F: V₂O₅-instilled COX-2 null. Note the absence of COX-2 immunostaining in the COX-2 null mice. All panels ×160.

Figure 7.

PGE₂ levels in BAL fluid in wild-type, COX-1^−/−, and COX-2^−/− mice following instillation with saline or V₂O₅. BAL fluid was collected at the indicated time points post-instillation and PGE₂ measured by an enzyme immunoassay. Each V₂O₅ group represents 5 to 6 animals and each saline group represents 3 to 4 animals. *, P < 0.05 vs. all saline-instilled groups. **, P < 0.01 vs. all saline-instilled groups. ^†, P < 0.05 vs. COX-1^−/− or COX-2^−/− V₂O₅ group.

Figure 8.

TNF-α levels in BAL fluid in wild-type, COX-1^−/− and COX-2^−/− mice following instillation with saline or V₂O₅. BAL fluid was collected at the indicated time points post-instillation and TNF-α was measured by ELISA. Each V₂O₅ group represents 5 to 6 animals and each saline group represents 3 to 4 animals. *P < 0.05 vs. all saline-instilled groups. **, P < 0.01 vs. all saline-instilled groups. ^§, P < 0.05 vs. wild-type V₂O₅ group. ^†, P < 0.01 vs. wild-type V₂O₅ group.