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

Abstract

Background: Exhaled nitric oxide is increased in asthma, but the mechanisms controlling its production, including the effects of T-helper type 2 (Th2) cytokines, are poorly understood. In mouse and submerged human epithelial cells, Th2 cytokines inhibit expression of inducible nitric oxide synthase (iNOS). Arginases have been proposed to contribute to asthma pathogenesis by limiting the arginine substrate available to NOS enzymes, but expression of any of these enzymes has not been extensively studied in primary human cells.

Objectives: We hypothesized that primary human airway epithelial cells in air-liquid interface (ALI) culture would increase iNOS expression and activity in response to IL-13, while decreasing arginase expression.

Methods: iNOS and arginase mRNA (real-time PCR) and protein expression (Western blot and immunofluorescence) as well as iNOS activity (nitrite levels) were measured in ALI epithelial cells cultured from bronchial brushings of normal and asthmatic subjects following IL-13 stimulation.

Results: IL-13 up-regulated iNOS mRNA primarily at a transcriptional level in epithelial cells. iNOS protein and activity also increased, arginase1 protein expression decreased while arginase 2 expression did not change. The changes in iNOS protein correlated strongly with changes in nitrites, and inclusion of arginase (1 or 2) did not substantially change the relationship. Interestingly, iNOS mRNA and protein were not correlated.

Conclusions: These results contrast with many previous results to confirm that Th2 stimuli enhance iNOS expression and activity. While arginase 1 protein decreases in response to IL-13, neither arginase appears to substantially impact nitrite levels in this system.

Fig. 1.

(a) IL-13 (10 ng/mL for 10 days) significantly increased inducible nitric oxide synthase (iNOS) mRNA (n = 33, P<0.0001) compared with cells stimulated with media alone. (b) IL-13 (10 ng/mL for 10 days) significantly increased iNOS protein levels (n = 32, P < 0.0001). Representative Western blots are shown above the graph. *Positive control = submerged primary human epithelial cells stimulated with IL-1β, TNF-α and INF-γ (50 ng/mL each) for 24 h.

Fig. 2.

Representative immunofluorescence of bronchial epithelial inducible nitric oxide synthase (iNOS) protein from five independent subjects. Original magnification is × 200. (a) Secondary antibody alone with DAPI stain on unstimulated cells at day 10 of ALI. (b) Basal expression of iNOS in unstimulated cells at day 10. (c) Secondary antibody alone for IL-13-stimulated cells at day 10. (d) IL-13 induced expression of iNOS after 10 days. The green and blue colors represent iNOS antibody and nuclear DAPI staining, respectively.

Fig. 3.

IL-13 (10 ng/mL for 10 days) increased nitrite levels in the apical supernatants. IL-13 increased nitrite levels in normal control (P = 0.0009) and asthmatic subjects (P<0.01). The increase and the absolute nitrite levels in the normal controls were significantly greater than those observed in asthmatic subjects (P<0.02). Changes in nitrite values were significantly greater in normal controls compared with asthmatics (P<0.05).

Fig. 4.

(a) Dose response: IL-13 (0.5–20 ng/mL) added to the cultures. IL-13 increased inducible nitric oxide synthase (iNOS) mRNA and peaks at 10 ng/mL (n = 4, P<0.01 over all). (b) Time course study: IL-13 (10 ng/mL) added from 1 to 10 days before harvest. IL-13 increased iNOS mRNA which peaked at 7 days (n = 4, P<0.01 over all). In small inset, increases in mRNA were consistently seen as early as 5 h after stimulation (baseline 0.02 ± 0.01, 5 h is 1.3 ± 0.5 [iNOS mRNA relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), P = 0.0002, (n = 9)]. (c) Representative Western blots for iNOS protein (denatured gels) (upper blots). IL-13 increased iNOS protein in dose and time dependent manner. Non-denatured gels (lower blots) demonstrating IL-13 induced dose and time dependent increases in the higher molecular weight iNOS homodimer (~250 kDa), with concomitant decreases in the monomeric form (130 kDa). (d) Quantitative densitometric analysis of the dose dependent IL-13 induced increase in iNOS protein. (P = 0.02). (e) Quantitative densitometric analysis of the time dependent IL-13 induced increase in iNOS protein (P = 0.03).

Fig. 5.

(a) Stability of IL-13-induced iNOS mRNA expression appears to be similar to that induced by IFN-γ. Act D was added after 12 h of stimulation, the RNA harvested at 0, 1, 6 and 12 h. There is no significant difference between IL-13 and IFN-γ at any time points (n = 3 for IL-13 group and n = 7 for IFN-γ group). (b) Nuclear run on assay on cells stimulated with IL-13 (10 ng/mL for 10 days) and treated with NTPs (or not) for 30 min. Data are presented means ± SE. All data are expressed as percentage of baseline (no NTPs added group, n = 5).

Fig. 6.

Arginase 2 mRNA and protein are constitutively expressed in primary human bronchial cells. (a) IL-13 (10 ng/mL for 10 days) did not change arginase 2 mRNA expression (P = 0.37, n = 26). (b) Arginase 2 Western blots (representative from 3 subjects). Densitometry analysis of arginase 2 protein normalized by β-actin (n = 25). IL-13 did not impact arginase 2 protein expression (P = 0.09). IL-13 marginally increased arginase 2 protein expression. *Positive control for arginase 2 = rat kidney lysate [24].

Fig. 7.

(a) Representative Western blot of arginase 1. IL-13 (10 ng/mL for 10 days) decreased arginase 1 expression. (b) IL-13 decreased arginase 1 protein expression in 19 subjects with detectable baseline levels of arginase 1 (P = 0.03). *Positive control for arginase 1 = rat liver lysate [24].