The absence of reactive oxygen species production protects mice against bleomycin-induced pulmonary fibrosis

Abstract

Background: Reactive oxygen species and tissue remodeling regulators, such as metalloproteinases (MMPs) and their inhibitors (TIMPs), are thought to be involved in the development of pulmonary fibrosis. We investigated these factors in the fibrotic response to bleomycin of p47phox -/- (KO) mice, deficient for ROS production through the NADPH-oxidase pathway.

Methods: Mice are administered by intranasal instillation of 0.1 mg bleomycin. Either 24 h or 14 days after, mice were anesthetized and underwent either bronchoalveolar lavage (BAL) or lung removal.

Results: BAL cells from bleomycin treated WT mice showed enhanced ROS production after PMA stimulation, whereas no change was observed with BAL cells from p47phox -/- mice. At day 1, the bleomycin-induced acute inflammatory response (increased neutrophil count and MMP-9 activity in the BAL fluid) was strikingly greater in KO than wild-type (WT) mice, while IL-6 levels increased significantly more in the latter. Hydroxyproline assays in the lung tissue 14 days after bleomycin administration revealed the absence of collagen deposition in the lungs of the KO mice, which had significantly lower hydroxyproline levels than the WT mice. The MMP-9/TIMP-1 ratio did not change at day 1 after bleomycin administration in WT mice, but increased significantly in the KO mice. By day 14, the ratio fell significantly from baseline in both strains, but more in the WT than KO strains.

Conclusions: These results suggest that NADPH-oxidase-derived ROS are essential to the development of pulmonary fibrosis. The absence of collagen deposition in KO mice seems to be associated with an elevated MMP-9/TIMP-1 ratio in the lungs. This finding highlights the importance of metalloproteinases and protease/anti-protease imbalances in pulmonary fibrosis.

Figure 1

Production of reactive oxygen species (ROS) by bronchoalveolar lavage (BAL) cells, 1 day (A) and 14 days (B) after intranasal administration of bleomycin (0.1 mg, BLM) or saline (Control) to mice with the p47^phox subunit of NADPH-oxidase deleted (p47^phox -/- knockout mice (KO): solid bars), compared with "Wild Type" p47^phox +/+ mice (WT: blank bars). Cells were stimulated in vitro with PMA (0.8 μM) and ROS production was evaluated by chemiluminescence. Data were collected at the time of maximum light emission. Results are expressed as relative light units (mean ± SEM). ***: p < 0.001 comparison with control mice exposed to saline solution alone. ###: p < 0.001 for KO mice compared with WT mice. n = 5–9.

Figure 2

Level of IL-6 (pg/mL) in BAL fluids, 1 day after intranasal administration of bleomycin (BLM, 0.1 mg/mouse) or saline vehicle (Control), to p47^phox +/+ WT mice (blank bars) and p47^phox -/- KO mice (solid bars). Results are presented as the mean ± SEM. ** : p < 0.01 compared with control mice exposed to saline solution alone. #: p < 0.05 for p47^phox -/- KO mice compared with p47^phox +/+ WT mice. n = 4–9.

Figure 3

Representative gelatin zymogram of BAL supernatant fluids, 1 and 14 days after intranasal administration of bleomycin (BLM, 0.1 mg/mouse) or saline vehicle (Control) to p47^phox +/+ WT mice (A) and p47^phox -/- KO mice (B). The following gelatinolytic bands were identified as MMP activity: pro-MMP-9 (105 kDa), MMP-9 (86 kDa), pro-MMP-2 (70 kDa), and MMP-2 (64 kDa). M: molecular weight marker.

Figure 4

Quantification by densitometry of 105-kDa pro-MMP-9 (A), and 64-kDa MMP-2 (B) gelatinase activity on zymograms of BAL fluid, performed 1 day or 14 days after intranasal administration of bleomycin (BLM, 0.1 mg/mouse) or saline vehicle (Control), to p47^phox +/+ WT mice (blank bars) and p47^phox -/- KO mice (solid bars). Results are represented as the mean of relative intensity ± SEM. * : p < 0.05, ** : p < 0.01, *** : p < 0.001 compared with control mice exposed to saline solution alone. ###: p < 0.001 for p47^phox -/- KO mice compared with p47^phox +/+ WT mice. n = 8–10.

Figure 5

Quantification by densitometry of Pro-MMP-9 (105 kDa, A) and MMP-2 (64 kDa, B) gelatinase activity on zymograms of lung homogenates, performed 14 days after intranasal administration of bleomycin (BLM, 0.1 mg/mouse) or saline vehicle (Control) to p47^phox -/- KO mice (solid bars) and p47^phox +/+ WT mice (blank bars). Results are represented as the mean of relative intensity ± SEM. * : p < 0.05, ** : p < 0.01, compared with control mice exposed to saline solution alone. ##: p < 0.01 for p47^phox -/- [KO] mice compared with p47^phox +/+ WT mice. n = 3–5.