Figure 5.

Local and paracrine effects of pneumocyte irradiation (IR). A) C57Bl/6NCr mice were exposed to 0 Gy, 5 Gy, or 17.5 Gy thoracic IR (n = 3 per group). At 4 weeks, samples of lung tissue were collected and dihydroethidium (DHE) oxidation was assessed. Scale bars = 16 μm. B) Primary pneumocytes were plated and exposed to graded doses of irradiation. DHE oxidation was assessed at 3 days. C and D) Primary pneumocytes were exposed to 0 Gy (control), 5 Gy, or 17.5 Gy of irradiation and assessed at 3 days. Pneumocytes were treated with diphenyleneiodonium (DPI; 100nM) immediately before and after IR. DHE oxidation was assessed and cells were costained for β-galactosidase (β-gal) activity. The percentage of pneumocytes staining with DHE and β-gal activity by treatment at 3 days are shown in (C). Scale bars = 16 μm. The rate of costaining for β-gal and DHE is shown in (D). The low rate of β-gal activity in DPI-treated pneumocytes precluded assessment of colocalization. E and F) Green fluorescent protein (GFP)-expressing unirradiated primary pneumocytes were cocultured with irradiated primary pneumocytes (17.5 Gy). Cells were fixed after 3 days and costained for β-gal and pro-surfactant C (pro-SPC). Arrows in (E) point to senescent unirradiated GFP-expressing pneumocytes. Scale bars = 16 μm. The percentage of GFP + type II airway epithelial cells (AECII) staining positive for β-gal activity at 3 days are shown in (F). G and H) Primary pneumocytes were plated and exposed to 0 Gy, 5 Gy, or 17.5 Gy. Conditioned media was collected after 3 days. The effects of treatment with irradiated pneumocyte conditioned media on fibroblast proliferation and collagen production was assessed after 72 hours. Bars represent the mean. Error bars represent the standard deviation. Brackets indicate P < .05 by analysis of variance. All statistical tests were two-sided. Scale bar = 40 μm.