Matrix metalloproteinases promote inflammation and fibrosis in asbestos-induced lung injury in mice

Abstract

Inhalation of asbestos fibers causes pulmonary inflammation and eventual pulmonary fibrosis (asbestosis). Although the underlying molecular events are poorly understood, protease/antiprotease and oxidant/antioxidant imbalances are believed to contribute to the disease. Implicated in other forms of pulmonary fibrosis, the matrix metalloproteinases (MMPs) have not been examined in asbestosis. We therefore hypothesized that MMPs play a pathogenic role in asbestosis development. Wild-type C57BL/6 mice were intratracheally instilled with 0.1 mg crocidolite asbestos, causing an inflammatory response at 1 d and a developing fibrotic response at 7, 14, and 28 d. Gelatin zymography demonstrated an increase in MMP-9 (gelatinase B) during the inflammatory phase, while MMP-2 (gelatinase A) was profoundly increased in the fibrotic phase. Immunohistochemistry revealed MMP-9 in and around bronchiolar and airspace neutrophils that were often associated with visible asbestos fibers. MMP-2 was found in fibrotic regions at 7, 14, and 28 d. No increases in RNA levels of MMP-2, MMP-9, or MMP-8 were found, but levels of MMP-7, MMP-12, and MMP-13 RNA did increase at 14 d. The MMP inhibitors, TIMP-1 and TIMP-2, were also increased at 7-28 d after asbestos exposure. To confirm the importance of MMP activity in disease progression, mice exposed to asbestos were given daily injections of the MMP inhibitor, GM6001. MMP inhibition reduced inflammation and fibrosis in asbestos-treated mice. Collectively, these data suggest that MMPs contribute to the pathogenesis of asbestosis through effects on inflammation and fibrosis development.

Figure 1.

MMP-2 and MMP-9 are increased in lung homogenates after asbestos exposure. Wild-type C57BL/6 mice were exposed to asbestos and killed at 1, 7, 14, and 28 d. Gelatin zymography of lung homogenates was performed as described in Materials and Methods. A representative gel is shown, but densitometry was performed on zymograms comparing controls and asbestos samples at each time point (n = 3–6). Densitometry was normalized as the average intensity of the asbestos bands divided by the average of the control (titanium dioxide, TiO2) bands at the same time point. If the asbestos levels of MMPs were equal to control levels, the value would therefore equal 1.0. The identification of bands as MMP-2 and MMP-9 was made through comparison with purified MMP-2 and MMP-9 (+). Latent and active MMP-2 were increased at all time points, while MMP-9 was significantly increased at 24 h only. *P < 0.05 Student's t test, comparing asbestos with control.

Figure 2.

MMP-2 and MMP-9 are increased in BAL fluid after asbestos exposure. Gelatin zymography and densitometry was performed as in Figure 1. Only the latent forms of MMP-2 and MMP-9 were found in BAL fluid. There are significant increases in MMP-2 and MMP-9 at all time points after asbestos exposure. *P < 0.05, Student's t test, comparing asbestos to the control (titanium dioxide, TiO2).

Figure 3.

Immunohistochemistry for MMP-9 in asbestos-exposed mouse lungs. Sections were incubated with anti–MMP-9 antibody (A–D) or with normal IgG as a negative control (E, F). (A, B) One day after asbestos exposure, bronchiolar neutrophils surrounding visible asbestos fibers (fibers at arrow) stained strongly for MMP-9 (red color). (C) Bronchiolar neutrophils retained MMP-9–positive staining at 14 d. However, no interstitial MMP-9 was observed, either in normal alveolar septa or in areas of fibrosis (asterisk). Identical staining patterns were observed at 7 and 28 d. (D) Titanium dioxide–treated mice did not display any MMP-9 staining. Macrophages that have engulfed these particles are clearly visible (arrowhead). Normal goat IgG revealed no staining at 24 h (E), 14 d (F), or 7 d (data not illustrated). Bar equals 50 μm.

Figure 4.

Immunohistochemistry for MMP-2 in asbestos-exposed mouse lungs. (A) Titanium dioxide–treated mice exhibited punctuate localization of MMP-2 in type II epithelial cells (arrowheads). (B) At 1 d, asbestos-treated mice exhibited the same type II cell staining, but in addition had a more diffuse staining over alveolar septa (arrows). (C) Also at 1 d, asbestos-treated mice had MMP-2 around bronchiolar neutrophils. (D) After 7 d, there is a significant increase in interstitial MMP-2 in areas of fibrosis. (E) As late as 28 d, fibrotic regions retain MMP-2 expression, here in fibrosis with visible asbestos fibers (arrows). (F) Normal goat IgG revealed no staining. Bar equals 50 μm.

Figure 5.

Quantitative RT-PCR for MMP-2, MMP-7, MMP-9, MMP-8, MMP-12, and MMP-13 in asbestos-exposed mouse lungs. (A) Crossing thresholds for MMP-9 were divided by crossing thresholds (ct) for GAPDH and did not reveal any significant differences between groups. Results were averaged for each group (n = 1–3 for each group). Lower crossing thresholds denote greater RNA expression of the target gene. (B) MMP-2/GAPDH ct was significantly greater at 1 d (24 h) asbestos compared with 24 h titanium dioxide (TiO2), denoting less MMP-2 RNA at that time point. (C) Ct of MMP-7 RNA was lower 14 d after asbestos, revealing a significant increase in MMP-7 levels in the 14 d asbestos group compared with both 1 d titanium dioxide and 1 d asbestos. (D) MMP-8/GAPDH ct did not reveal any significant differences. (E) MMP-12/GAPDH ct and (F) MMP-13/GAPDH ct levels showed significantly increased RNA levels of these MMPs at 14 d. *P < 0.05, as determined by one-way ANOVA and Tukey's post test.

Figure 6.

TIMP-1 and TIMP-2 are increased after asbestos exposure. Western blotting was performed on lung homogenates to determine levels of TIMP-1 and TIMP-2. Positive controls are on the right side of the blot. Both TIMPs showed increases that were most pronounced at 7–28 d after asbestos exposure. Densitometry was normalized to β-actin as a loading control. *P < 0.05, one-way ANOVA followed by Tukey's post test, asbestos compared with control (titanium dioxide).

Figure 7.

MMP inhibition reduces inflammation and fibrosis after asbestos exposure. Wild-type mice were injected daily (intraperitoneally) with the broad-spectrum MMP inhibitor GM6001 beginning on Day 0. Mice were exposed to asbestos on Day 1 and killed on Day 13. (A) Total BAL fluid cells were not significantly increased in the asbestos + GM6001 group compared with TiO2 + vehicle controls. (B) BAL fluid neutrophils were significantly decreased in the asbestos + GM6001 group compared with asbestos + vehicle group. (C) Hydroxyproline levels were also significantly decreased after MMP inhibition, indicating decreased fibrosis. (D) The decrease in fibrosis was also observed using blinded histologic scoring. *P < 0.05 one-way ANOVA and Tukey's post test.