Enhanced type III collagen gene expression during bleomycin induced lung fibrosis

Intratracheal instillation of bleomycin into mice leads to deposition of collagen in the lung and fibrosis, but the mechanism for this is poorly understood. Enhanced collagen gene expression, increased collagen synthesis, decreased collagen degradation, and proliferation of fibroblasts have all been proposed as possible contributors. To obtain information on the activity of collagen producing cells at an early stage in the development of pulmonary fibrosis in situ hybridisation was used to detect and localise products of the type III procollagen gene. In addition, assay of type III procollagen gene expression was performed using dot-blot analysis of lung RNA extracts.

Lung fibrosis was induced in mice by intratracheal instillation of bleomycin sulphate (6 mg/kg body weight) and tissues were examined after three, 10, 21 and 35 days. RNA-RNA hybridisation was accomplished with riboprobes labelled with sulphur-35 which were generated from a 1.7 kb mouse procollagen a1(III) cDNA. In situ hybridisation was performed on sections fixed in paraformaldehyde and embedded in paraffin wax and steady state values of type III procollagen mRNA were assayed by dot-blot analysis of total lung RNA extracted by guanidium isothiocyanate.

Data obtained using both techniques suggest that type III procollagen gene expression was enhanced in bleomycin induced fibrosis and that expression was maximal between 10 and 35 days after a single dose of bleomycin. The most active cells were located in interstitial areas around the conducting airways, although these cells were usually seen in areas with no histological evidence of fibrosis. Regions with the most advanced fibrosis, as assessed by histological methods, rarely contained cells with activity above the threshold detectable by this technique.

These results suggest that activation of interstitial fibroblasts, with enhanced type III collagen gene expression, forms at least part of the mechanism leading to increased collagen deposition in bleomycin induced fibrosis and that this occurs before fibrosis is detected by conventional histological staining.