Nitric oxide gas phase release in human small airway epithelial cells.
BACKGROUND
Asthma is a chronic airway inflammatory disease characterized by an imbalance in both Th1 and Th2 cytokines. Exhaled nitric oxide (NO) is elevated in asthma, and is a potentially useful non-invasive marker of airway inflammation. However, the origin and underlying mechanisms of intersubject variability of exhaled NO are not yet fully understood. We have previously described NO gas phase release from normal human bronchial epithelial cells (NHBEs, tracheal origin). However, smaller airways are the major site of morbidity in asthma. We hypothesized that IL-13 or cytomix (IL-1beta, TNF-alpha, and IFN-gamma) stimulation of differentiated small airway epithelial cells (SAECs, generation 10-12) and A549 cells (model cell line of alveolar type II cells) in culture would enhance NO gas phase release.


METHODS
Confluent monolayers of SAECs and A549 cells were cultured in Transwell plates and SAECs were allowed to differentiate into ciliated and mucus producing cells at an air-liquid interface. The cells were then stimulated with IL-13 (10 ng/mL) or cytomix (10 ng/mL for each cytokine). Gas phase NO release in the headspace air over the cells was measured for 48 hours using a chemiluminescence analyzer.


RESULTS
In contrast to our previous result in NHBE, baseline NO release from SAECs and A549 is negligible. However, NO release is significantly increased by cytomix (0.51 +/- 0.18 and 0.29 +/- 0.20 pl.s-1.cm-2, respectively) reaching a peak at approximately 10 hours. iNOS protein expression increases in a consistent pattern both temporally and in magnitude. In contrast, IL-13 only modestly increases NO release in SAECs reaching a peak (0.06 +/- 0.03 pl.s-1.cm-2) more slowly (30 to 48 hours), and does not alter NO release in A549 cells.


CONCLUSIONS
We conclude that the airway epithelium is a probable source of NO in the exhaled breath, and intersubject variability may be due, in part, to variability in the type (Th1 vs Th2) and location (large vs small airway) of inflammation.
Jiang
Jingjing
J
Department of Biomedical Engineering, University of California Irvine, Irvine, CA, USA. jingjinj@uci.edu
Malavia
Nikita
N
Suresh
Vinod
V
George
Steven C
SC
D001249:Asthma / Q000175:diagnosis / Q000201:enzymology / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism; D001944:Breath Tests; D001980:Bronchi / Q000201:enzymology / Q000378:metabolism*; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000201:enzymology / Q000378:metabolism*; D005740:Gases; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D007371:Interferon-gamma / Q000378:metabolism; D018793:Interleukin-13 / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D015854:Up-Regulation
D015415:Biomarkers; D005740:Gases; D018836:Inflammation Mediators; D018793:Interleukin-13; D053583:Interleukin-1beta; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D007371:Interferon-gamma; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II