Gelatinase B is required for alveolar bronchiolization after intratracheal bleomycin

Abstract

Increased expression of matrix metalloproteinases, particularly gelatinase B (MMP-9), has been described in the lungs in pulmonary fibrosis. Intratracheal bleomycin is often used experimentally to produce lesions resembling human fibrosing alveolitis. To assess the role of gelatinase B in bleomycin-induced fibrosing alveolitis, we instilled bleomycin intratracheally into gelatinase B-deficient mice and gelatinase B+/+ littermates. Twenty-one days after bleomycin the two groups of mice were indistinguishable in terms of pulmonary histology and total lung collagen and elastin. However, the lungs of gelatinase B-deficient mice showed minimal alveolar bronchiolization, whereas bronchiolization was prominent in the lungs of gelatinase B+/+ mice. Gelatinase B was identified immunohistochemically in terminal bronchiolar cells and bronchiolized cells 7 and 14 days after bleomycin in gelatinase B+/+ mice, and whole lung gelatinase B mRNA was increased at the same times. Many bronchiolized cells displayed Clara cell features by electron microscopy. Some bronchiolized cells stained with antibody to helix transcription factor 4, a factor associated with the ciliated cell phenotype. Thus, fibrosing alveolitis develops after intratracheal bleomycin irrespective of gelatinase B. However, gelatinase B is required for alveolar bronchiolization, perhaps by facilitating migration of Clara cells and other bronchiolar cells into the regions of alveolar injury.

Figure 1.

Inflammatory cells in BAL fluid 21 days after bleomycin. Bleomycin-treated animals showed significant increases in macrophages, neutrophils, and lymphocytes in BAL fluid at 21 days after bleomycin instillation as compared with nontreated or saline-treated controls. No significant difference was observed in the number of inflammatory cells in BAL fluid between gelatinase B+/+ and −/− mice. Each bar represents the mean of four or five animals ± SEM. *, Indicates P < 0.05 versus gelatinase B+/+ controls; **, P < 0.05 versus gelatinase B−/− controls.

Figure 2.

Hydroxyproline and desmosine content of lung 21 days after bleomycin. Lung collagen and elastin, as measured by the amount of hydroxyproline and desmosine, respectively, were increased comparably in gelatinase B+/+ and gelatinase B−/− mice 21 days after bleomycin treatment compared to lungs in saline-treated mice. Each bar represents the mean of four or five animals ± SEM.

Figure 3.

RT-PCR analysis of lung gelatinase B mRNA after bleomycin treatment in gelatinase B+/+ mice. a: Autoradiograph of RT-PCR of whole lung RNA isolated from nontreated or bleomycin-treated mice at 7, 14, and 21 days after bleomycin. Each lane represents RT-PCR products from a single mouse. b: The mean values (± SEM) of five mice expressed as the ratio of the signal intensity of gelatinase B mRNA to that of GAPDH at each time point. Gelatinase B mRNA in the lungs was significantly up-regulated by bleomycin treatment at 7 and 14 days, and returned to normal levels at 21 days. *, Indicates P < 0.05 versus nontreated mice.

Figure 4.

RT-PCR analysis of TIMP-1 mRNA after bleomycin treatment in gelatinase B+/+ and gelatinase B−/− mice expressed as the ratio of TIMP-1 mRNA to GAPDH mRNA. Gelatinase B+/+ (black bar), gelatinase B−/− (hatched bar). Each group consisted of four or five animals ± SEM. *, Indicates P < 0.05 versus gelatinase B+/+ nontreated mice; **, P < 0.05 versus gelatinase B−/− nontreated mice.

Figure 5.

Immunohistochemistry for gelatinase B in gelatinase B+/+ mice after bleomycin. Lung tissue was harvested from control mice (a) and at 7 (b) and 14 days (c) after bleomycin treatment. Gelatinase B was found in an occasional cell in normal lungs. Gelatinase B was present in many nonciliated airway epithelial cells at 14 days after bleomycin. Most of the bronchiolized cells were positive for gelatinase B at 7 and 14 days after bleomycin instillation (arrows) (original magnification, × 400).

Figure 6.

Bronchiolization 21 days after bleomycin treatment. a: In fibrotic regions of gelatinase B+/+ lung, clusters of cuboidal bronchiolar-appearing epithelium were prominent adjacent to bronchioles or at their distal termination (“bronchiolization”). b: Alveolar bronchiolization was rarely observed in the gelatinase B−/− lung. c: Bronchiolization was present in the matrilysin−/− lung. (elastica-Masson Goldner stain; original magnification, ×400)

Figure 7.

Electron micrographs of terminal bronchioles after intratracheal bleomycin. Uranyl acetate and lead citrate. 1500×. a: Three days after bleomycin in gelatinase B+/+ lung there is swelling of Clara cells and a ciliated cell (arrow). *, Indicates tip of septum of alveolar duct at the entrance to the alveolus. b: Three days after bleomycin in gelatinase B−/− lung, there are swollen Clara cells and ciliated cells (arrows). *, Indicates entrance to the alveolus. c: Twenty-one days after bleomycin in gelatinase B+/+ there is bronchiolization consisting of many Clara cells and some ciliated cells (arrows). The capillary (Ca) of the original alveolar wall is covered with bronchiolized epithelial cells. d: Twenty-one days after bleomycin in gelatinase B−/− Clara cells and some ciliated cells (arrows) are located in the original terminal bronchiole. An alveolar type I cell and an alveolar type II cell (II) are observed in the region of alveoli. * indicates entrance to the alveolus.

Figure 8.

Expression of PCNA and HFH-4 21 days after bleomycin treatment. a: PCNA-positive cells are widespread in distal lesions (arrows) of grade IV bronchiolization, but not in the proximal lesions in gelatinase B+/+ mice. b: PCNA-stained cells are limited in low-grade bronchiolization (arrow) immediately adjacent to conducting airway in gelatinase B−/− mice. c: HFH-4 expression is present in ciliated airway epithelial and proximal sites (*) of high-grade alveolar bronchiolization in gelatinase B+/+. d: HFH-4 expression in gelatinase B−/− lung is limited to ciliated airway epithelium. Original magnification, ×400.