ENHANCED TYPE-III COLLAGEN GENE-EXPRESSION DURING BLEOMYCIN-INDUCED LUNG FIBROSIS

Background-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, increas ed collagen synthesis, decreased collagen degradation, and proliferati on of fibroblasts have all been proposed as possible contributors. To obtain information on the activity of collagen producing cells at an e arly stage in the development of pulmonary fibrosis in situ hybridisat ion was used to detect and localise products of the type III procollag en gene. In addition, assay of type III procollagen gene expression wa s performed using dot-blot analysis of lung RNA extracts. Methods-Lung fibrosis was induced in mice by intratracheal instillation of bleomyc in sulphate (6 mg/kg body weight) and tissues were examined after thre e, 10, 21 and 35 days. RNA-RNA hybridisation was accomplished with rib oprobes labelled with sulphur-35 which were generated from a 1.7 kb mo use procollagen alpha1(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. Results-Data obtained using both techniques suggest that type III pro collagen gene expression was enhanced in bleomycin induced fibrosis an d that expression was maximal between 10 and 35 days after a single do se of bleomycin. The most active cells were located in interstitial ar eas around the conducting airways, although these cells were usually s een in areas with no histological evidence of fibrosis. Regions with t he most advanced fibrosis, as assessed by histological methods, rarely contained cells with activity above the threshold detectable by this technique. Conclusions-These results suggest that activation of inters titial fibroblasts, with enhanced type III collagen gene expression, f orms at least part of the mechanism leading to increased collagen depo sition in bleomycin induced fibrosis and that this occurs before fibro sis is detected by conventional histological staining.