BARRIER FUNCTION OF AIRWAY EPITHELIUM - EFFECTS OF RADIATION AND PROTECTION BY KERATINOCYTE GROWTH-FACTOR

In patients undergoing radiation therapy in the thoracic region, ioniz ing radiation causes immediate damage to pulmonary endothelial and epi thelial cells. We have recently shown that keratinocyte growth factor (KGF) protects against increases in permeability induced by hydrogen p eroxide in human airway epithelial cells. Since radiation injury invol ves the production of oxygen free radicals, we tested the hypothesis t hat KGF would protect against radiation-induced increases in permeabil ity. Two lines of human airway epithelial cells (Calu-3 and 16HBE14o(- )) were grown on collagen-coated polyester membranes (Transwell, Costa r) and the permeability of the monolayers was determined by measuring the flux of tracers from the top chamber to the bottom chamber as a fu nction of time. Changes in permeability were apparent 4 h after exposu re. Increasing doses of radiation (2-10 Gy) stimulated significant inc reases in permeability compared with control monolayers (P < 0.05, n = 5-10) in Calu-3 and 16HBE14o(-) cells. KGF (50 ng/ml) alone reduced p ermeability significantly compared with controls, protected against in creases in permeability with low doses of radiation and provided parti al 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. Af ter 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 tr eatments significantly reduced permeability to albumin in sham-irradia ted and irradiated cells (n = 3, P < 0.05). To investigate the signal transduction pathways through which KGF mediates protection, permeabil ity was measured in the presence of the protein kinase C (PKC) inhibit or, calphostin C, or the tyrosine kinase inhibitor, genistein. Inhibit ion of PKC blocked the decrease in basal tracer flux caused by KGF tre atment in both cell types and removed the KGF-mediated protection agai nst radiation. Incubation with genistein completely blocked the KGF-me diated decrease in the baseline tracer flux, as well as the ameliorati ng effect observed after irradiation. Rhodamine-phalloidin staining of the F-actin cytoskeleton showed disruption of the cytoskeleton with r adiation exposure, increased density of F-actin filaments with KGF tre atment, and resistance to disruption when cells were pretreated with K GF and exposed to radiation. Our results suggest that KGF regulates pe rmeability in airway epithelium through a pathway mediated by PKC and tyrosine kinase that stabilizes the F-actin cytoskeleton. (C) 1998 by Radiation Research Society.