TGF-beta1-mediated alterations of rat lung fibroblast differentiation resulting in the radiation-induced fibrotic phenotype

Abstract

Purpose : To study the influence of TGF-beta1 and TGF-beta-neutralizing antibodies on the clonogenic activity and terminal differentiation of rat lung fibroblasts following radiation exposure. Material and methods : Early passage rat lung fibroblasts were used in this study. Colony formation assays were applied to determine the radiation sensitivity as well as radiation-induced alterations in differentiation pattern. Based on a TGF-beta1-specific ELISA system, the amount of TGF-beta1 in the culture medium of sham-irradiated and irradiated cultures was determined. Results : Applying immediate (ip) and delayed plating (dp) procedures for cells irradiated in the subconfluent state, distinct differences in the radiation dose–response curves could be observed (SF2 ip 0.20 +/- 0.025 versus SF2 dp 0.51 +/- 0.0077). Upon irradiation with a single dose of 4 Gy the level of TGF-beta1 found in the culture medium increased by about 60%. Radiation-induced terminal differentiation of progenitor fibroblasts (MF) to postmitotic fibrocytes (PMF) was expressed by the change of the ratio of PMF:MF (0.8 at 0 Gy versus 3.0 at 4 Gy). Neutralizing antibodies directed against TGF-beta inhibited both the radiation-induced reduction in clonogenic activity of rat lung fibroblasts as well as the radiation-induced terminal differentiation of MF progenitor fibroblasts to PMF postmitotic fibrocytes. Conclusion : The results indicate the important role of TGF-beta1 in triggering radiation-induced inhibition of clonogenic activity as well as terminal differentiation of rat lung fibroblasts. The data presented support the hypothesis that terminal differentiation is an essential cellular process in the development of radiation-induced fibrosis in the lung.