Interferon γ Stimulates Accumulation of Gas Phase Nitric Oxide in Differentiated Cultures of Normal and Cystic Fibrosis Airway Epithelial Cells

Exhaled nitric oxide (NO) levels have been reported to be lower in patients with cystic fibrosis (CF) than in controls; however the mechanism(s) responsible and the effect on pathogenesis are unclear. The objective of these studies was to determine if the low levels of gas phase NO (gNO) could be reproduced in well-differentiated air-liquid interface (ALI) cultures of normal and CF cells. Human bronchial epithelial (HBE) cells from CF and control tissues were cultured under ALI conditions that promote differentiation into a mostly ciliated, pseudostratified epithelium similar to that of the in vivo airway. Cultures were incubated in gas tight chambers and the concentration of gNO was measured using a Sievers nitric oxide analyzer. In CF and control cultures the level of accumulated gNO under baseline conditions was low (< 20 ppb). Treatment with interferon gamma (IFN gamma) induced iNOS expression and increased gNO significantly in differentiated cultures, while having no significant effect on undifferentiated cultures. Submersion of the apical surface with fluid drastically reduced the level of gNO. Importantly, the average level of gNO measured after IFN gamma treatment of control cells (576 ppb) was threefold greater than that from CF cells (192 ppb). The results demonstrate that the lower level of exhaled NO observed in CF patients is reproduced in well-differentiated primary cultures of HBE cells treated with IFN gamma, supporting the hypothesis that the regulation of NO production is altered in CF. The results also demonstrate that IFN gamma treatment of differentiated cells results in higher levels of gNO than treatment of undifferentiated cells, and that a layer of fluid on the apical surface drastically reduces the amount of gNO, possibly by limiting the availability of oxygen.