Thoracic radiotherapy (RT) is often limited by lung toxicity (pulmonary fibrosis). Pulmonary fibrosis ensues after an unexplained latent period which follows the acute reaction and is characterized by reduction in pulmonary vital capacity and exertional dyspnea. Elevation of fibrogenic cytokines, most notably, TGF-B1 and B2 (TGF-B1-B2), and fibroblast proliferation that extends from irradiated to adjacent lung, are features at the molecular and cellular levels. The principal investigator recently demonstrated that intratracheal (IT) injection of manganese superoxide dismutase-plasmid/liposomes (MnSOD-PL) protects the murine lung from irradiation-induced organizing alveolitis/fibrosis induced by single dose or fractionated irradiation. The proposed research will use validated, genetically modified animal models along with quantitative molecular methods to elucidate the cellular mechanism of irradiation lung fibrosis and the level(s) at which epitope-hemagglutinin (HA)-tagged MnSOD transgene therapy protects. The first specific aim tests the hypothesis that organizing alveolitis/fibrosis is initiated by accumulation of macrophage attractant, VCAM-1 in endothelial cells at 80-100 days after irradiation. The second specific aim tests the hypothesis that TGF-B1-B2 production by bone marrow-derived bronchoalveolar macrophages (BAMs) mediates fibroblast recruitment and proliferation. The third specific aim tests the hypothesis that circulating fibroblast progenitor cells, also of bone marrow origin, home to, and proliferate in irradiated lung to produce organizing alveolitis/fibrosis. Methods include BrdU in situ labelling, histopathology, green fluorescent protein-positive (GFP+) male hematopoietic stem cell (macrophage progenitor) engraftment to GFP- female mice and transplantation of GFP+ purified bone marrow stromal cells (BMSCs), continuous anti-TGF-B antibody or soluble TGF-B receptor (TGF-B-R) delivery, injection of HA-MnSOD-PL, fractionated irradiation, and in situ assays of DNA damage. These experiments will provide substantial, new insight into the basic pathogenesis of the pulmonary irradiation response. A comprehensive understanding of the underlying mechanisms is critical for identifying novel targets for intervention. This project may facilitate development of specific strategies to minimize pulmonary irradiation toxicity, thereby making RT safer and more effective.