Jiang Jingjing, Malavia Nikita, Suresh Vinod, George Steven C
Department of Biomedical Engineering, University of California Irvine, Irvine, CA, USA.
Respir Res. 2009 Jan 19;10(1):3. doi: 10.1186/1465-9921-10-3.
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.
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.
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.
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.
哮喘是一种慢性气道炎症性疾病，其特征为Th1和Th2细胞因子失衡。哮喘患者呼出的一氧化氮（NO）水平升高，是气道炎症潜在的有用非侵入性标志物。然而，呼出NO个体间变异性的起源和潜在机制尚未完全明确。我们之前曾描述过正常人支气管上皮细胞（NHBEs，气管来源）释放气相NO的情况。然而，小气道是哮喘发病的主要部位。我们推测，在培养中用白细胞介素-13（IL-13）或细胞混合物（IL-1β、肿瘤坏死因子-α和干扰素-γ）刺激分化的小气道上皮细胞（SAECs，第10 - 12代）和A549细胞（II型肺泡细胞的模型细胞系）会增强气相NO的释放。
将SAECs和A549细胞的汇合单层培养于Transwell板中，使SAECs在气液界面分化为纤毛细胞和黏液分泌细胞。然后用IL-13（10 ng/mL）或细胞混合物（每种细胞因子10 ng/mL）刺激细胞。使用化学发光分析仪在48小时内测量细胞上方顶空气相中的NO释放量。
与我们之前在NHBE中的结果相反，SAECs和A549细胞的基线NO释放量可忽略不计。然而，细胞混合物可显著增加NO释放（分别为0.51±0.18和0.29±0.20皮升·秒-1·平方厘米-2），在约10小时达到峰值。诱导型一氧化氮合酶（iNOS）蛋白表达在时间和幅度上均呈一致模式增加。相比之下，IL-13仅适度增加SAECs中的NO释放，达到峰值（0.06±0.03皮升·秒-1·平方厘米-2）的速度较慢（30至48小时），且不改变A549细胞中的NO释放。
我们得出结论，气道上皮可能是呼出气体中NO的来源，个体间变异性可能部分归因于炎症类型（Th1与Th2）和部位（大气道与小气道）的变异性。
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