Transforming growth factor alpha is a critical mediator of radiation lung injury

Abstract

Radiation fibrosis of the lung is a late toxicity of thoracic irradiation. Epidermal growth factor (EGF) signaling has previously been implicated in radiation lung injury. We hypothesized that TGF-α, an EGF receptor ligand, plays a key role in radiation-induced fibrosis in lung. Mice deficient in transforming growth factor (TGF-α(-/-)) and control C57Bl/6J (C57-WT) mice were exposed to thoracic irradiation in 5 daily fractions of 6 Gy. Cohorts of mice were followed for survival (n ≥ 5 per group) and tissue collection (n = 3 per strain and time point). Collagen accumulation in irradiated lungs was assessed by Masson's trichrome staining and analysis of hydroxyproline content. Cytokine levels in lung tissue were assessed with ELISA. The effects of TGF-α on pneumocyte and fibroblast proliferation and collagen production were analyzed in vitro. Lysyl oxidase (LOX) expression and activity were measured in vitro and in vivo. Irradiated C57-WT mice had a median survival of 24.4 weeks compared to 48.2 weeks for irradiated TGF-α(-/-) mice (P = 0.001). At 20 weeks after irradiation, hydroxyproline content was markedly increased in C57-WT mice exposed to radiation compared to TGF-α(-/-) mice exposed to radiation or unirradiated C57-WT mice (63.0, 30.5 and 37.6 μg/lung, respectively, P = 0.01). C57-WT mice exposed to radiation had dense foci of subpleural fibrosis at 20 weeks after exposure, whereas the lungs of irradiated TGF-α (-/-) mice were largely devoid of fibrotic foci. Lung tissue concentrations of IL-1β, IL-4, TNF-α, TGF-β and EGF at multiple time points after irradiation were similar in C57-WT and TGF-α(-/-) mice. TGF-α in lung tissue of C57-WT mice rose rapidly after irradiation and remained elevated through 20 weeks. TGF-α(-/-) mice had lower basal LOX expression than C57-WT mice. Both LOX expression and LOX activity were increased after irradiation in all mice but to a lesser degree in TGF-α(-/-) mice. Treatment of NIH-3T3 fibroblasts with TGF-α resulted in increases in proliferation, collagen production and LOX activity. These studies identify TGF-α as a critical mediator of radiation-induced lung injury and a novel therapeutic target in this setting. Further, these data implicate TGF-α as a mediator of collagen maturation through a TGF-β independent activation of lysyl oxidase.

FIG. 1

Effects of TGF-α deficiency on radiation-induced lung injury. C57-WT and TGF-α^−/− mice were exposed to 5 × 6 Gy of thoracic irradiation. Panel A. Lung tissue was collected at the indicated time points (n = 3 mice per strain per time point) after irradiation and subjected to ELISA to determine the concentration of TGF-α. *P < 0.05 for the comparison to other strain at the same time point; points: mean, error bars: SD. Panel B: Kaplan-Meier plot of survival of irradiated mice. Panel C: Lung tissue collected at 20 weeks after irradiation from C57-WT and TGF-α^−/− mice was analyzed for hydroxyproline content (n = 3 mice per strain per time point). Columns: mean, error bars: SD. Panel D: Masson’s trichrome staining of lung tissue collected at 20 weeks after irradiation. The lungs of C57-WT mice harbor extensive foci of subpleural fibrosis while those of TGF-α^−/− mice exhibit minimal fibrosis. Collagen: blue, nuclei: purple, cytoplasm/epithelia: pink. Scale bar: 50 µm.

FIG. 2

Effects of TGF-α deficiency on radiation-induced pulmonary inflammation. C57-WT and TGF-α^−/− mice were exposed to 5 × 6 Gy of thoracic irradiation. Lung tissue was collected at multiple time points after irradiation (n = 3 mice per strain per time point) and processed for immunohistochemical evaluation of macrophage (panel A), neutrophil (panel B) and T-cell (panel C) infiltration. DAB (brown) was used as the chromogen in each example (right panels) with hematoxylin as the counterstain. Columns: mean, error bars: SD, brackets: P < 0.05, scale bars: 12 µm, high power field (HPF) (63×); NE: neutrophil elastase.

FIG. 3

Effects of TGF-α deficiency on radiation-induced inflammatory cytokine expression. C57-WT and TGF-α^−/− mice were exposed to 5 × 6 Gy of thoracic irradiation. Lung tissue was collected at multiple time points weeks after irradiation (n = 3 mice per strain per time point) and processed for ELISA. One hundred milligrams of lung was homogenized in 1 mL of lysis buffer and analyzed for the concentration of IL-1β (panel A), IL-4 (panel B), TNF-α (panel C), IL-6 (panel D), TGF-β (panel E) and EGF (panel F). No significant differences between strains were seen for any analyte.

FIG. 4

Effects of TGF-α on proliferation and collagen production in vitro. NIH-3T3 cells and primary AEC were cultured in reduced serum media (1% FBS) overnight, treated with TGF-α (0, 1, 10 ng/mL) and incubated for 72 h. Panel A: Proliferation was evaluated by MTT assay and normalized to the proliferation of the vehicle treated control for each cell type. Panel B: Salt soluble collagen (no pepsin extraction) and mature collagen (cell pellet and scrapings of matrix adherent to culture plastic, with acid-pepsin extraction) production in NIH-3T3 cells were evaluated with a Sirius Red collagen assay. Values were normalized to the vehicle treated control. Panel C: Hydroxyproline content of NIH-3T3 media and cell/matrix (cell pellet and scrapings of matrix adherent to culture plastic) was assessed. Panel D: TGF-β concentration in culture supernatants was measured with ELISA at 1, 24 and 72 h after TGF-α treatment. Cell lysates were adjusted to a common protein concentration to account for differences in cell number. All in vitro experiments were performed in duplicate and validated in three separate experiments. Columns: mean, error bars: SD, brackets: P < 0.05 for comparisons between doses of TGF-α.

FIG. 5

Effects of TGF-α treatment on LOX activity in fibroblast cultures. NIH-3T3 cells were cultured in reduced serum media (1% FBS) overnight, treated with TGF-α (0–10 ng/mL) and incubated for 72 h. Cell pellets were collected for Western blotting analysis of LOX expression (panel A) or LOX activity (panel B). To exclude that TGF-β expression mediated the effects of TGF-α in regards to LOX activity, a TGF-β neutralizing antibody (1 µg/mL) was added to NIH-3T3 cells 1 h prior to TGF-α treatment (0–10 ng/mL), and LOX inhibitor BAPN (7.5 µM) was used for negative control. To confirm the importance of LOX in TGF-α mediated collagen accumulation, the LOX inhibitor BAPN (7.5 and 30 µM) was added to NIH-3T3 cells 1 h prior to TGF-α treatment (10 ng/mL). The LOX inhibitor BAPN was added to NIH-3T3 cells in varying concentrations 1 h prior to TGF-α or vehicle treatment (10 µM). Levels of LOX activity (panel C) and mature collagen (panel D) were assessed at 72 h. Hydroxyproline content was assessed after acid-pepsin extraction of the cells and matrix adherent to the tissue culture plastic and was normalized to the total protein content of each sample. Relative values of LOX activity were normalized to vehicle treated control. Cell lysate volumes were adjusted to a common protein concentration to account for differences in cell number. All in vitro experiments were performed in duplicate and validated in three separate experiments. Columns: mean, error bars: SD; *P < 0.05 for the comparison to vehicle at the same dose of TGF-α, brackets: P < 0.05 for the comparison.

FIG. 6

Effects of TGF-α deficiency on LOX expression and activity in murine lungs. C57-WT mice and TGF-α^−/− mice were exposed to 5 × 6 Gy of thoracic irradiation. Lung tissue (n = 3 mice per strain and treatment) was collected at 20 weeks after irradiation and LOX expression was assessed by (panel A) immunohistochemistry or (panel B) Western blotting. Panel C: Densitometric analysis of Western blotting (n = 5 mice per strain and treatment) for LOX expression. Panel D: Lung tissue from C57-WT mice and TGF-α^−/− mice was collected at multiple time points after irradiation (n = 3 per strain and time point), homogenized and assessed for LOX activity. Columns: mean, error bars: SD; brackets: P < 0.05; points: mean, *P < 0.05 for the comparison between strains at the time point.

FIG. 7

The effect of down-stream inhibitors on TGF-α mediated profibrotic signaling. NIH-3T3 cells were cultured in reduced serum media (1% FBS) overnight and treated with the indicated inhibitor in DMSO (1 µM) or DMSO as a vehicle control. One hour after the addition of the inhibitors, TGF-α (0 or 10 ng/mL) was added. Cell pellets and cell culture supernatants were collected 72 h after the addition of TGF-α. Cell lysate volumes were adjusted to a common protein concentration to account for differences in cell number. Media was analyzed without dilution. Panel A: LOX expression was evaluated by Western blotting. Panel B: LOX activity was assessed in NIH-3T3 cell culture supernatants and cell lysates. Panel C: Media soluble collagen (no pepsin extraction) and mature collagen (cell pellet and scrapings of matrix adherent to culture plastic, with acid-pepsin extraction) production in NIH-3T3 cells were evaluated with a Sirius Red collagen assay. Values were normalized to the vehicle treated control. Panel D: Hydroxyproline content of NIH-3T3 media and cell/matrix (cell pellet and scrapings of matrix adherent to culture plastic) was assessed. Values were normalized to total protein in each sample. All in vitro experiments were performed in duplicate and validated in three separate experiments. Columns: mean, error bars: SD, brackets: P < 0.05 for the comparison.