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

Abstract

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.

Diaphragm Muscle Contraction Decrease in a Mouse Model of Ovalbumin-Induced Allergic Airway Inflammation

“…There were previously reported that OVA increased the breathing resistance of the mice [11], resistive breathing induced IL-4 expression in diaphragm muscle [12], Th2 cytokines such as IL-4 and IL-13 induce iNOS expression through the STAT-6 pathway [13][14][15][16][17], iNOS reduced diaphragm muscle contraction, inhibition of iNOS induction and inhibition of NOS activity prevented diaphragm muscle contractile dysfunction [18]. Study results of ours are in agreement with study that previously reported.…”

Receptor-Interacting Protein 2 Gene Silencing Attenuates Allergic Airway Inflammation

“…IL-13, IL-1b, and TNF-a have been shown to induce iNOS expression in human bronchial epithelial cells, leading to elevated NO production (45,46). In addition, iNOS gene expression is regulated by the NF-kB pathway (40).…”

IL‐13 induced increases in nitrite levels are primarily driven by increases in inducible nitric oxide synthase as compared with effects on arginases in human primary bronchial epithelial cells

“…In contrast, in these same systems, Th2 cytokines particularly IL-4 and IL-13, inhibit iNOS expression and nitrite production, or modestly increase it in combination with IFN-g [7,[9][10][11]. Recently, IL-13 was reported to variably induce iNOS mRNA and protein expression in 2 of 3 normal human bronchial epithelial cell lines in air-liquid interface (ALI) culture, but no mechanisms for the increase were reported [12].…”