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

Abstract

Background 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. Methods 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. Results In CF and control cultures the level of accumulated gNO under baseline conditions was low (<20 ppb). Treatment with interferon gamma (IFNγ) 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 IFNγ treatment of control cells (576 ppb) was threefold greater than that from CF cells (192 ppb). Conclusions 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 IFNγ, supporting the hypothesis that the regulation of NO production is altered in CF. The results also demonstrate that IFNγ 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.

Temporal structure/function variation in cultured differentiated human nasal epithelium associated with acute single exposure to tobacco smoke or E-cigarette vapor

“…Cellular NO production was determined by positioning three ALI cultures in a vessel (Cytometric Sciences LLC, Chapel Hill, NC) designed for cellular NO measurement (Ostrowski et al, 2012). Transwells seeded with cells and transwells without cells to serve as controls were incubated in vessels for 6h at 35 °C after which NO concentration was determined using a Sievers 270B NO analyzer at a sampling flow rate of 500 ml/min.…”

“…Moreover, the advent of e-cigarettes and other nicotine “vaping” devices has led to the commercial promotion of these products as having reduced risk for adverse health effects associated with tobacco products. In addition to published studies pointing to dysmorphology and dysfunction of mucociliary clearance mechanisms associated with tobacco smoke exposure, there is a growing awareness of the role of the regulation of ciliary function modulation by nitric oxide (NO) (Jain et al, 1993; Jiao et al, 2011; Maniscalco et al, 2016; Ostrowski et al, 2012; Sisson et al, 2009; Vleeming et al, 2002; Wyatt, 2015; Zhou et al, 2009, 2011). While a variety of agents such as alcohol, tobacco smoke and microbiological products have been shown to affect this mechanism, there is little data to inform how NO modulation of ciliary function might be associated with e-cigarette use (Hariri et al, 2016; Sisson et al, 2009; Zhou et al, 2009).…”

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

“…We describe the culturing and re-differentiating of NECs, and not bronchial epithelial cells, which have been used in many previously published studies [24][25][26][27] . We see various reasons why the use of NECs can be advantageous as compared to the use of bronchial ECs.…”

Characterizing well-differentiated culture of primary human nasal epithelial cells for use in wound healing assays

“…Growing epithelial cells at the air-liquid interface (ALI) allows for differentiation into a pseudostratified epithelium with basal cells, cilia projecting from the apical surface, and goblet cells producing mucus [7,8]. Many studies confirm ALI as a physiologically relevant platform for studying epithelial function and mechanisms associated with respiratory diseases and represents an excellent and more realistic model for airway epithelial function experiments [9][10][11][12][13][14][15]. Differentiation of nasal epithelial cells at ALI takes at least 21 days after "lift" as recommended by the media manufactures [16] but much conjecture about this differentiation period exists in the literature.…”