RATIONALE: We previously showed that PBMC from IgE+ (>100 IU/ml) adults with asthma produced significantly greater nitric oxide (NO) concentrations than IgE- (<100 IU/ml) after 5 days in culture. NO production further increased in the presence of cytokines (IL-15, IL-18, IFNγ) and vitamin D3 (CYT+D3). We now examined intracellular iNOS expression in CD33+ monocytes from IgE+ and IgE- adults after 18 hrs + CYT+D3.
METHODS: Blood from IgE+ (n=14) and IgE- (n=7) adults was collected at 9-10 AM. Exhaled breath NO and serum IgE levels were determined (Niox Vero; fluoroenzyme immunoassay). PBMC were incubated for 18 hrs ± hIL-15 (1 µg/ml), IL-18 (1 µg/ml), IFNγ (10 ng/ml) and vitamin D3 (20 pmol/ml). Cells were collected by vigorous pipetting and analyzed by flow cytometry (Fortessa) for surface CD33 expression (anti-CD33 PE, BD Biosciences) and intracellular iNOS expression (rabbit monoclonal anti-human iNOS) (abcam), followed by goat anti-rabbit IgG (Alexa Fluor 488)(abcam). Spearman and Pearson coefficients, t-test, and Mann-Whitney U-test were used in analysis.
RESULTS: Monocytes in peripheral blood were CD45+CD14+ (92.6+3.4%) and CD33+ (96.5+3.7%), and iNOS- (<2%) (n=5). Incubation for 18 hrs with CYT+D3 significantly increased CD33+iNOS+ monocytes in PBMC from IgE+ adults (27.5 vs 15.65%; p<0.016), but not iNOS expression in PBMC from IgE- subjects (6.5 vs 8.7%; p>0.05) (Mann-Whitney U-test)
CONCLUSIONS: Measurement of intracellular iNOS by flow cytometry in CD33+ monocytes detects increased expression in the presence of cytokines and vitamin D3 in IgE+ adults. Monocyte iNOS expression is a potential biomarker of systemic inflammation in IgE+ adults with asthma.