Elevated exhaled nitric oxide in allergen-provoked asthma is associated with airway epithelial iNOS

Abstract

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.

Figure 1. Higher concentration of fractional exhaled nictric oxide (FENO) among allergic asthmatics.

FENO was assessed in exhaled breath of healthy controls (n = 14) and asthmatics allergic to birch pollen (n = 12). Bars indicate mean value of all individuals, errorbars indicate SEM. Significant differences between asthmatic individuals and healthy controls were assessed using unpaired t-test. * indicates p<0.05. ppb, parts per billion.

Figure 2. Expression of inducible nitric oxide synthase mRNA (NOS2) is increased in the bronchial epithelium of allergic asthmatics.

RNA was extracted from cells isolated from bronchial brush biopsies collected during fiberoptic bronchoscopy of healthy controls (n = 4), and allergic asthmatics (n = 6). The integrity of RNA was assessed, and degraded samples were excluded from the analysis. Gene expression of NOS2 (coding for iNOS) relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was analyzed by semi-quantitative real-time PCR. Bars indicate means, errorbars indicate SEM. Significant differences were assessed using unpaired t-test. ** indicates p<0.01.

Figure 3. Elevated fractional exhaled nitric oxide (FENO) in allergic asthmatics following bronchial allergen provocation.

FENO was assessed in exhaled breath of asthmatics allergic to birch pollen (n = 13), before (light grey), 1 hour after (grey) and 24 hours after (dark grey) bronchial provocation with birch pollen. Bars indicate mean value of all individuals. Significant differences were assessed using paired t-test. ** indicates p<0.01. ppb, parts per billion.

Figure 4. Increased airway eosinophils correlates with elevated fractional exhaled nitric oxide (FENO).

Bronchoalveolar lavage (BAL) fluid was collected during fiberoptic bronchoscopy in allergic asthmatics (n = 13), before (light grey) and after allergen provocation (dark grey). BAL eosinophils were enumerated and correlated to FENO using Pearson correlation test of linear regression. ppb, parts per billion.

Figure 5. Activity of nitric oxide (NO) synthases is increased in the bronchial epithelium of allergic asthmatics following allergen provocation.

Bronchial biopsies were collected during fiberoptic bronchoscopy of allergic asthmatics (n = 9), before (light grey) and 24 hours after allergen provocation (dark grey). Representative image of the epithelial (Ep) activity of NO synthases was assessed by nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) staining (purple) in bronchial biopsies from allergic asthmatics A) before and B) after allergen provocation. C) Morphometric analysis was performed. Samples where intact bronchial epithelium could not be identified were omitted from the analysis. Epithelial cells detached from the basal membrane were excluded. Percentage of bronchial epithelium stained positive for NAPDH-d. Significant differences were assessed using paired t-test. Scale bars indicate 200 µm. * indicates p<0.05.

Figure 6. Expression of inducible nitric oxide synthase (iNOS) is increased in the bronchial epithelium of allergic asthmatics following allergen provocation.

Bronchial biopsies were collected during fiberoptic bronchoscopy of allergic asthmatics (n = 9), before (light grey) and 24 hours after allergen provocation (dark grey). Representative image of the epithelial (Ep) expression of iNOS (brown color) was assessed by immunohistochemistry in bronchial biopsies from allergic asthmatics A) before and B) after allergen provocation. Cell nuclei were counterstained with Mayer's haematoxylin (blue stain). Morphometric analysis was performed. Samples where intact bronchial epithelium could not be identified were omitted from the analysis. Epithelial cells detached from the basal membrane were excluded (indicated by arrow). C) Percentage of bronchial epithelium positive for iNOS immunoreactivity and D) correlation analysis of the percentage of positive epithelium and fractional exhaled nitric oxide (FENO) using Pearson correlation test of linear regression. Significant differences were detected using paired t-test. Scale bars indicate 200 µm. *** indicates p<0.001.