Barrier function of airway epithelium: effects of radiation and protection by keratinocyte growth factor

In patients undergoing radiation therapy in the thoracic region, ionizing radiation causes immediate damage to pulmonary endothelial and epithelial cells. We have recently shown that keratinocyte growth factor (KGF) protects against increases in permeability induced by hydrogen peroxide in human airway epithelial cells. Since radiation injury involves the production of oxygen free radicals, we tested the hypothesis that KGF would protect against radiation-induced increases in permeability. Two lines of human airway epithelial cells (Calu-3 and 16HBE14o-) were grown on collagen-coated polyester membranes (Transwell, Costar) and the permeability of the monolayers was determined by measuring the flux of tracers from the top chamber to the bottom chamber as a function of time. Changes in permeability were apparent 4 h after exposure. Increasing doses of radiation (2-10 Gy) stimulated significant increases in permeability compared with control monolayers (P < 0. 05, n=5-10) in Calu-3 and 16HBE14o- cells. KGF (50 ng/ml) alone reduced permeability significantly compared with controls, protected against increases in permeability with low doses of radiation and provided partial protection at higher doses. KGF also provided a significant effect in cells irradiated with 10 Gy (n=5, P < 0. 05) when given for the 4 h immediately after irradiation. The effects of KGF were sustained. After a full 24-h pretreatment with KGF, cells were incubated in medium without KGF for 8 or 12 h prior to 10 Gy irradiation. Both of these treatments significantly reduced permeability to albumin in sham-irradiated and irradiated cells (n=3, P < 0.05). To investigate the signal transduction pathways through which KGF mediates protection, permeability was measured in the presence of the protein kinase C (PKC) inhibitor, calphostin C, or the tyrosine kinase inhibitor, genistein. Inhibition of PKC blocked the decrease in basal tracer flux caused by KGF treatment in both cell types and removed the KGF-mediated protection against radiation. Incubation with genistein completely blocked the KGF-mediated decrease in the baseline tracer flux, as well as the ameliorating effect observed after irradiation. Rhodamine-phalloidin staining of the F-actin cytoskeleton showed disruption of the cytoskeleton with radiation exposure, increased density of F-actin filaments with KGF treatment, and resistance to disruption when cells were pretreated with KGF and exposed to radiation. Our results suggest that KGF regulates permeability in airway epithelium through a pathway mediated by PKC and tyrosine kinase that stabilizes the F-actin cytoskeleton.