Repetitive intratracheal bleomycin models several features of idiopathic pulmonary fibrosis

Abstract

Single-dose intratracheal bleomycin has been instrumental for understanding fibrotic lung remodeling, but fails to recapitulate several features of idiopathic pulmonary fibrosis (IPF). Since IPF is thought to result from recurrent alveolar injury, we aimed to develop a repetitive bleomycin model that results in lung fibrosis with key characteristics of human disease, including alveolar epithelial cell (AEC) hyperplasia. Wild-type and cell fate reporter mice expressing β-galactosidase in cells of lung epithelial lineage were given intratracheal bleomycin after intubation, and lungs were harvested 2 wk after a single or eighth biweekly dose. Lungs were evaluated for fibrosis and collagen content. Bronchoalveolar lavage (BAL) was performed for cell counts. TUNEL staining and immunohistochemistry were performed for pro-surfactant protein C (pro-SP-C), Clara cell 10 (CC-10), β-galactosidase, S100A4, and α-smooth muscle actin. Lungs from repetitive bleomycin mice had marked fibrosis with prominent AEC hyperplasia, similar to usual interstitial pneumonia (UIP). Compared with single dosing, repetitive bleomycin mice had greater fibrosis by scoring, morphometry, and collagen content; increased TUNEL+ AECs; and reduced inflammatory cells in BAL. Sixty-four percent of pro-SP-C+ cells in areas of fibrosis expressed CC-10 in the repetitive model, suggesting expansion of a bronchoalveolar stem cell-like population. In reporter mice, 50% of S100A4+ lung fibroblasts were derived from epithelial mesenchymal transition compared with 33% in the single-dose model. With repetitive bleomycin, fibrotic remodeling persisted 10 wk after the eighth dose. Repetitive intratracheal bleomycin results in marked lung fibrosis with prominent AEC hyperplasia, features reminiscent of UIP.

Fig. 1.

Trichrome blue-stained lung sections post-bleomycin. A and C: patchy areas of fibrosis were noted on trichrome-stained lung sections from mice 2 wk after a single dose of 0.04 units of bleomycin. B and D: in contrast, fibrosis was more prominent in trichrome-stained lung sections from mice 2 wk after the 8th biweekly repetitive dose of 0.04 units of bleomycin. Furthermore, alveolar epithelial cell (AEC) hyperplasia was prominent with the repetitive model. Magnification in A and B, ×40; magnification in C and D, × 400.

Fig. 2.

Lung fibrosis was greater in the repetitive bleomycin model compared with the single-dose model. A: lung fibrosis was scored on trichrome blue-stained lung sections, revealing a greater score with the repetitive model. N = 5 for each column. B: morphometry measurements of lung sections revealed a greater percentage of lung tissue affected by fibrosis in the repetitive model. N = 5 for each column. C: by HPLC assay, hydroxyproline content was greater in lungs from the repetitive model. N = 3 for untreated and 6 for single and repetitive. *P < 0.05 compared with single dose.

Fig. 3.

TUNEL+ cells were greater in the repetitive bleomycin model compared with the single-dose model. Representative images from lung sections from 2 wk after a single dose of 0.04 units of bleomycin (A) and 2 wk after the 8th biweekly repetitive dose of 0.04 units of bleomycin (B). Magnification, ×800. Arrows point to TUNEL+ cells. C: when quantitated per high-power field, a greater percentage of cells were TUNEL+ in the repetitive bleomycin model. N = 5 per column. #P < 0.01 compared with single dose. *P < 0.01 compared with untreated.

Fig. 4.

For bronchoalveolar lavage (BAL) cell studies, total leukocyte (A), macrophage (B), neutrophil (C), and lymphocyte (D) counts in BAL were less in the repetitive model compared with the single-dose model. E: eosinophil counts were not different among the groups. F: neutrophil counts per high-power field were lower in the repetitive bleomycin group compared with the single-dose group. N = 5 per column. *P < 0.05 compared with other columns.

Fig. 5.

Epithelial-mesenchymal transition (EMT)-derived cells are detected in the lung following bleomycin treatment. Confocal microscopy was performed to detect fluorescent immunostaining for β-gal (green), S100A4 (red), and DAPI (blue) in R26Rosa.Stop.LacZ.SPC.Cre reporter mice. A–D: representative images from lung sections from mice 2 wk after the 8th biweekly repetitive dose of 0.04 units of bleomycin. Dual fluorescent (yellow) cells indicate S100A4+ fibroblasts that are derived from lung epithelial lineage. DIC, differential interference contrast. Magnification, ×600. Comparing the single-dose bleomycin model to the repetitive-dose bleomycin model, quantitation of the number of S100A4+ cells per high-power field (HPF) in areas of fibrosis was not statistically different between the 2 groups (E). However, the number of S100A4+/β-gal+ cells per HPF was higher in the repetitive group. F: the percentage of S100A4+ cells that were also β-gal+ in the repetitive-dose group approached 50% compared with ∼33% in the single-dose group. N = 3 per column. *P < 0.05 compared with single dose.

Fig. 6.

Hyperplastic AECs in the repetitive model express both pro-surfactant protein C (pro-SP-C) and Clara cell 10 (CC-10). A: immunohistochemistry (IHC) on lung sections from the repetitive model revealed that the hyperplastic AECs lining the areas of lung fibrosis were immunostained positive for pro-SP-C, a pattern similar to the hyperplastic epithelial cells noted in a UIP lung biopsy (B). C: by IHC, many of the hyperplastic AECs also immunostained positive for CC-10. D: in contrast, only rare hyperplastic AECs in UIP lung biopsies were positive for CC-10 by IHC. Magnification, ×600.

Fig. 7.

Characterization of bronchoalveolar stem cell (BASC)-like cells in areas of fibrosis. Confocal microscopy was performed to detect fluorescent immunostaining for pro-SP-C (green) and CC-10 (red). A–C: in lungs from mice 2 wk after a single dose of 0.04 units of bleomycin, occasional patches of dual fluorescent (yellow) cells that were pro-SP-C+/CC-10+ were noted. D–F: in contrast, many of the hyperplastic AECs lining areas of fibrosis in the repetitive-dose bleomycin model were dual pro-SP-C+/CC-10+ by immunofluorescence. Magnification in A–F, ×600. G: quantitation of the percentage of pro-SP-C+ cells that also expressed CC-10 revealed a greater percentage of dual-positive cells in lungs from the repetitive model. N = 5 per column. *P < 0.02 compared with single dose.