Tochino, Y; Trudeau, JB; Yamamoto, M; Zhao, J; Wenzel, SE
Introduction: Elevations in exhaled nitric oxide (FeNO) generally reflect allergic inflammation in asthmatics, yet it does not reflect disease severity. We previously reported IL-13 induced iNOS expression in well-differentiated primary human airway epithelial cells in vitro. In addition iNOS protein expression in freshly brushed epithelial cells increases with disease severity. However, in severe asthmatics, where studies suggest a high level of oxidative stress, the correlation between FeNO and iNOS protein is poor. In the presence of oxidative stress, Nitric oxide (NO) is likely metabolized to peroxynitrite, leading to nitrosylated tyrosine residues, which can be measured in tissues and cells.
Hypothesis: Increasing asthma severity is associated with increased iNOS expression and activation which better correlates with nitrotyrosine levels as compared to FeNO.
Methods: Fresh bronchial epithelial cells were obtained from 20 Normal Controls (NC), 5 Mild Asthmatics (MiA), not on inhaled corticosteroids (ICS), 14 Mild to Moderate Asthmatics (MMA) on ICS, and 25 Severe Asthmatic (SA) subjects. The relative expression of iNOS mRNA was measured by real time PCR and protein by western blot. FeNO was measured the bronchoscopy day as ppb at 50ml/s. Nitrotyrosine was measured as the sum of the band densities of the Western blot. While iNOS protein level was compared to FeNO for all subjects, limitations of sample only allowed nitrotyrosine comparisons with 13 of these subjects (3 NC and 10 asthmatics).
Results: When compared to each group, iNOS protein and mRNA values in SA were the highest and significantly increased compared to NC. While iNOS protein and mRNA were also significantly higher in SA than MMA (protein; p=0.04, mRNA; p=0.01), FeNO was not significantly different between SA and MMA (p=0.21). FeNO in MiA was significantly higher than in NC (p=0.03), but there were no other intergroup difference. While FeNO significantly correlated to iNOS protein among all groups (n=34; r=0.56, p=0.01) and in asthmatics alone (n=22; r=0.49, p=0.02), FeNO did not correlate to iNOS protein in SA (n=11; r=0.51, p=0.11). Nitrotryosine levels correlated with iNOS protein among all groups (n=13; r=0.64, p=0.02) and tended to correlate in asthmatics alone (n=10; r=0.57, p=0.08).
Conclusion: FeNO does not distinguish asthma severity, while iNOS protein expression does differentiate severity. This disconnect may be due to the diversion of NO to peroxynitrite/nitrotyrosine in subjects with severe asthma.
