Ostrowski, LE; Stewart, D; Hazucha, MJ; Knowles, MR
INTRODUCTION: Nitric oxide (NO) is produced by multiple cell types in the airways and has been proposed to perform many functions, including acting as a vasodilator, modulating inflammatory responses, and possessing anti-microbial effects. The level of NO, measured in exhaled breath or from the nasal cavity, has been shown to be altered in different disease states. In asthmatics, the level of exhaled NO is higher than in control groups, and this is likely due to increased inflammation. In primary ciliary dyskinesia (PCD) and cystic fibrosis (CF), the levels of NO are lower than in control groups. The regulation of NO production and the potential role of altered NO production in disease pathogenesis is not completely understood.
RATIONALE: To understand the mechanism responsible for the low levels of gas-phase NO observed in CF and PCD patients, we have begun to study the production of gas-phase NO directly from fully-differentiated, air/liquid interface cultures of human bronchial epithelial (HBE) cells under several different conditions.
METHODS: Passage 1 HBE cells were cultured at an air/liquid interface (ALI) under conditions that promote differentiation into a mostly ciliated, pseudo-stratified epithelium similar to that of the in vivo airway. Cultures were incubated in gas-tight Teflon chambers and the concentration of gas-phase NO was measured using a Sievers 270B nitric oxide analyzer. Gas-phase NO levels were measured from differentiated and undifferentiated cultures, with and without treatment with interferon-gamma (INFg), under both ALI and submerged conditions.
RESULTS: In control cultures (not CF, PCD, or asthma), the level of accumulated gas-phase NO under baseline conditions was very low (<20 ppb). Treatment with IFNg induced iNOS expression, but not eNOS or nNOS, and stimulated NO levels significantly in differentiated cultures, while having no significant effect on undifferentiated cultures. Elimination of the air/liquid interface by submersion with apical fluid drastically reduced the level of gas-phase NO production. In preliminary studies, CF cells showed a trend toward lower levels of NO.
CONCLUSIONS: The results show that well-differentiated primary cultures of HBE cells produce low levels of gas-phase NO that can be induced to high levels (>1000 ppb) following treatment with IFNg. The results also show that differentiated cells respond to IFNg more robustly and release higher levels of NO than undifferentiated cells, and that a layer of fluid on the apical surface of differentiated cells drastically reduces the amount of gas-phase NO, possibly by limiting the availability of oxygen.
