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Measurement of IL-13–Induced iNOS-Derived Gas Phase Nitric Oxide in Human Bronchial Epithelial Cells

Department of Biomedical Engineering, and Department of Chemical Engineering and Materials Science, University of California Irvine, Irvine, California

Correspondence and requests for reprints to be addressed to Steven C. George, M.D., Ph.D., Department of Biomedical Engineering, 3120 Natural Sciences II, University of California Irvine, Irvine, CA 92697-2715. E-mail: scgeorge{at}uci.edu

Exhaled nitric oxide (NO) is altered in numerous diseases including asthma, and is thought broadly to be a noninvasive marker of inflammation. However, the precise source of exhaled NO has yet to be identified, and the interpretation is further hampered by significant inter-subject variation. Using fully differentiated normal human bronchial epithelial (NHBE) cells, we sought to determine (1) the rate of NO release (flux, pl·s^–1.cm^–2) into the gas; (2) the effect of IL-13, a prominent mediator of allergic inflammation, on NO release; and (3) inter-subject/donor variability in NO release. NHBE cells from three different donors were cultured at an air–liquid interface and stimulated with different concentrations of IL-13 (0, 1, and 10 ng/ml) for 48 h. Gas phase NO concentrations in the headspace over the cells were measured using a chemiluminescence analyzer. The basal NO flux from the three donors (0.05 ± 0.03) is similar in magnitude to that estimated from exhaled NO concentrations, and was significantly increased by IL-13 in a donor-specific fashion. The increase in NO release was strongly correlated with inducible nitric oxide synthase (iNOS) gene and protein expression. There was a trend toward enhanced production of nitrate relative to nitrite as an end product of NO metabolism in IL-13–stimulated cells. NO release from airway epithelial cells can be directly measured. The rate of release in response to IL-13 is strongly dependent on the individual donor, but is primarily due to the expression of iNOS.

Key Words: asthma • cytokines • inflammation • allergy

CLINICAL RELEVANCE The results from this study provide a direct in vitro link between inducible nitric oxide synthase expression in the human bronchial epithelium and nitric oxide gas phase release. The release is increased by IL-13, a prominent Th2 cytokine present in asthma