Abstract
BackgroundAsthma 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-1β, TNF-α, and IFN-γ) 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.MethodsConfluent 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.ResultsIn 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.ConclusionWe 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.
Highlights
Asthma is a chronic airway inflammatory disease characterized by an imbalance in both Th1 and Th2 cytokines
Activation of eosinophils, T lymphocytes, neutrophils and macrophages are all involved in airway inflammation, which can trigger the release of mediators and cytokines that contribute to the clinical syndrome of asthma
Small airway epithelial cells differentiation Immunofluorescent staining for MUC5AC, β – Tubulin IV and zonula occludens-1 (ZO-1) was detected after 7 days of air-liquid interface (ALI)
Summary
Asthma is a chronic airway inflammatory disease characterized by an imbalance in both Th1 and Th2 cytokines. We hypothesized that IL-13 or cytomix (IL-1β, TNF-α, and IFN-γ) 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. Asthma is a chronic inflammatory disease characterized by airway hyperresponsiveness and variable airflow obstruction. Activation of eosinophils, T lymphocytes, neutrophils and macrophages are all involved in airway inflammation, which can trigger the release of mediators and cytokines that contribute to the clinical syndrome of asthma. Cytokines secreted by type 1 lymphocytes (Th1) and macrophages, including IL-1β, TNF-α, and IFN-γ, may be increased in asthma subjects and contribute to the inflammatory process [2]. Because of the relative inaccessibility of small airways, examination of the inflammatory process is mainly limited to post-mortem or bronchial biopsy analysis while dynamic in vivo assessment remains limited to estimates of small airway and alveolar concentration based on exhaled NO levels and mathematical models [4]
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