Elevated Exhaled Nitric Oxide in Allergen-Provoked Asthma Is Associated with Airway Epithelial iNOS

Background
Fractional exhaled nitric oxide is elevated in allergen-provoked asthma. The cellular and molecular source of the elevated fractional exhaled nitric oxide is, however, uncertain.
Objective
To investigate whether fractional exhaled nitric oxide is associated with increased airway epithelial inducible nitric oxide synthase (iNOS) in allergen-provoked asthma.
Methods
Fractional exhaled nitric oxide was measured in healthy controls (n = 14) and allergic asthmatics (n = 12), before and after bronchial provocation to birch pollen out of season. Bronchoscopy was performed before and 24 hours after allergen provocation. Bronchial biopsies and brush biopsies were processed for nitric oxide synthase activity staining with nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), iNOS immunostaining, or gene expression analysis of iNOS by real-time PCR. NADPH-d and iNOS staining were quantified using automated morphometric analysis.
Results
Fractional exhaled nitric oxide and expression of iNOS mRNA were significantly higher in un-provoked asthmatics, compared to healthy controls. Allergic asthmatics exhibited a significant elevation of fractional exhaled nitric oxide after allergen provocation, as well as an accumulation of airway eosinophils. Moreover, nitric oxide synthase activity and expression of iNOS was significantly increased in the bronchial epithelium of asthmatics following allergen provocation. Fractional exhaled nitric oxide correlated with eosinophils and iNOS expression.
Conclusion
Higher fractional exhaled nitric oxide concentration among asthmatics is associated with elevated iNOS mRNA in the bronchial epithelium. Furthermore, our data demonstrates for the first time increased expression and activity of iNOS in the bronchial epithelium after allergen provocation, and thus provide a mechanistic explanation for elevated fractional exhaled nitric oxide in allergen-provoked asthma.

背景
变应原诱发哮喘患者的呼出气一氧化氮分数（fractional exhaled nitric oxide）水平升高，但该水平升高的细胞与分子来源尚未明确。

研究目的
本研究旨在探讨变应原诱发哮喘患者的呼出气一氧化氮分数是否与气道上皮诱导型一氧化氮合酶（inducible nitric oxide synthase, iNOS）表达上调相关。

研究方法
本研究于非花粉季对14名健康对照者与12名过敏性哮喘患者分别开展桦树花粉支气管激发试验前后，对其呼出气一氧化氮分数进行检测。分别于变应原激发前及激发后24小时实施支气管镜检查，采集支气管活检组织与刷检标本，分别采用烟酰胺腺嘌呤二核苷酸磷酸黄递酶（nicotinamide adenine dinucleotide phosphate diaphorase, NADPH-d）染色检测一氧化氮合酶活性、iNOS免疫染色，以及通过实时聚合酶链反应（real-time PCR）开展iNOS基因表达分析。采用自动化形态计量分析对NADPH-d染色与iNOS染色结果进行定量。

研究结果
与健康对照者相比，未接受变应原激发的哮喘患者呼出气一氧化氮分数与iNOS mRNA表达水平均显著升高。过敏性哮喘患者在变应原激发后，其呼出气一氧化氮分数显著升高，同时气道嗜酸性粒细胞出现蓄积。此外，变应原激发后哮喘患者的支气管上皮内一氧化氮合酶活性与iNOS表达均显著上调。呼出气一氧化氮分数与嗜酸性粒细胞计数及iNOS表达水平呈显著相关。

结论
哮喘患者体内更高的呼出气一氧化氮分数水平与支气管上皮内iNOS mRNA表达上调显著相关。此外，本研究数据首次证实变应原激发后哮喘患者支气管上皮内iNOS表达与活性均出现上调，从而为变应原诱发哮喘患者呼出气一氧化氮分数升高提供了机制层面的解释。