Purpose: Activation of TGF-β1 pathway after radiation is implicated in lung fibrosis. We previously found that radiation induces lung TNF-α action and inhibition of TNFR1 is effective in reducing early apoptosis and improving lung function. Here, we investigated the roles of both cytokines and their potential interaction in lung collagen production after radiation.
 Experiment Design: C57BL/J6 mice lung were irradiated with various doses or regimen, assessed for TNF-α and TGF-β1, and measured for collagen production by hydroxyproline assay or western blotting. Inhibition of TNF-α action by etanercept (for TNF-α) or genetic knock-out (for TNFR1) was used to establish its role in collagen production and potential effect on increasing TGF-β1. Finally, production of cytokines by radiation and their effects on collagen gene expression through TβR-mediated Smad3 phosphorylation were studied in various lung cell cultures including macrophages (MH-S), epithelial cells (NL-20), microvascular endothelial cells (HPMVEC), and fibroblasts (IMR-90).
 Results: A single dose radiation with 15 Gy transiently increased lung TNF-α and TGF-β1 by ~ 25% (3 h) and ~160% (24 h), respectively. These inductions were greater (~75% and ~210%, respectively) at day 7 when fractionated radiation (3 x 5 Gy) was used, correlating with the sustained elevation of lung collagen content. Induction of collagen III protein by fractionated radiation was dramatically reduced or diminished in TNFR1 or Smad3 knock-out mice (TNFR1-/- or Smad3-/-). Pre-treatment with etanercept in C57BL/J6 mice, which effectively decreased apoptosis in the irradiated lung, reduced lung TGF-β1 production, similar to the reduction observed in irradiated TNFR1-/- mice. The reduction of TGF-β1 by etanercept correlated with decreases in collagen III expression and Smad3 phosphorylation in irradiated lung. In cell cultures, radiation with 4 Gy increased production of both cytokines in MH-S cells by ~50%, the induction that was mostly diminished in cells pre-treated with etanercept or cells deficient in TNFR1. Collagen production was most abundant and inducible by TNF-α or TGF-β1 (2 ng/ml) in fibroblasts (IMR-90), correlating with the induction of collagen III mRNA. Finally, TNF-α action had no effect on Smad3 activation in all cells while TGF-β1 activated Smad3 phosphorylation, drastically or moderately, in IMR-90 or HPMVEC cells.
 Conclusion: Both TNF-α and TGF-β1 were increased by the lung radiation, correlating with an increase in collagen production. Inhibition of TNF-α pathway decreased TGF-β1 production in irradiated lung or irradiated lung macrophages. TGF-β1 or TNF-α induced collagen production in lung fibroblasts with or without involving Smad3 phosphorylation.

99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA