Modulation of NF-κB and hypoxia-inducible factor--1 by S-nitrosoglutathione does not alter allergic airway inflammation in mice

Abstract

Induction of nitric oxide synthase (NOS)-2 and production of nitric oxide (NO) are common features of allergic airway disease. Conditions of severe asthma are associated with deficiency of airway S-nitrosothiols, a biological product of NO that can suppress inflammation by S-nitrosylation of the proinflammatory transcription factor, NF-κB. Therefore, restoration of airway S-nitrosothiols might have therapeutic benefit, and this was tested in a mouse model of ovalbumin (OVA)-induced allergic inflammation. Naive or OVA-sensitized animals were administered S-nitrosoglutathione (GSNO; 50 μl, 10 mM) intratracheally before OVA challenge and analyzed 48 hours later. GSNO administration enhanced lung tissue S-nitrosothiol levels and reduced NF-κB activity in OVA-challenged animals compared with control animals, but did not lead to significant changes in total bronchoalveolar lavage cell counts, differentials, or mucus metaplasia markers. Administration of GSNO also altered the activation of hypoxia-inducible factor (HIF)-1, leading to HIF-1 activation in naive mice, but suppressed HIF-1 activation in OVA-challenged mice. We assessed the contribution of endogenous NOS2 in regulating NF-κB and/or HIF-1 activation and allergic airway inflammation using NOS2(-/-) mice. Although OVA-induced NF-κB activation was slightly increased in NOS2(-/-) mice, associated with small increases in bronchoalveolar lavage neutrophils, other markers of allergic inflammation and HIF-1 activation were similar in NOS2(-/-) and wild-type mice. Collectively, our studies indicate that instillation of GSNO can suppress NF-κB activation during allergic airway inflammation, but does not significantly affect overall markers of inflammation or mucus metaplasia, thus potentially limiting its therapeutic potential due to effects on additional signaling pathways, such as HIF-1.

Figures

Figure 1.

Instillation of S-nitrosoglutathione (GSNO) enhances protein S-nitrosylation in the mouse airway. Naive C57BL/6J mice were administered oropharingeal GSNO (50 mM; 50 μl), and lung tissues were collected after various time periods (15 min, 1 h, and 4 h) for analysis of protein S-nitrosylation. Whole lung tissue was analyzed by the biotin switch assay for changes in global protein S-nitrosylation (A) and S-nitrosylation of NF-κB p65 (SNO-p65) (B). Ctl, control.

Figure 2.

Inhibition of NF-κB activity in ovalbumin (OVA)-sensitized and -challenged mice by administration of GSNO. Naive or sensitized C57BL/6J mice were challenged with OVA on 3 consecutive days, and, where indicated, mice were also administered oropharingeal GSNO (10 mM, 50 μl; 30 minutes before OVA challenge). Lung tissues were collected 48 hours after the last OVA challenge, and nuclear extracts were prepared for analysis of NF-κB p65 DNA binding assay (A) and Western blot (B). *P < 0.01 compared with aluminum hydroxide (Alum)/OVA PBS group; #P < 0.05 compared with the OVA/OVA PBS group using ANOVA (n = 8). (C) S-Nitrosylated NF-κB p65 (SNO-p65) was assessed by biotin switch assay in whole lung tissue 48 hours after the latest OVA challenge. (D) Densitometric analysis of S-nitrosylated (SNO)-p65 to total p65 ratio. *P < 0.05 compared with the corresponding Alum/OVA control group using ANOVA (n = 6).

Figure 3.

Effects of GSNO administration on airway inflammation, bronchoalveolar lavage (BAL) protein content, and Ig levels. Naive or sensitized C57BL/6J mice were challenged with OVA and/or GSNO on 3 consecutive days, and bronchoalveolar lavage (BAL) fluid was collected 48 hours after the last aerosolized OVA challenge for the assessment of total (A) and differential (B) cell counts. (C) Sections generated from paraffin-embedded lungs were stained with hematoxylin and eosin (H&E) and evaluated by light microscopy to visualize inflammation by assessing the extent of infiltrates around airways or vasculature. (D) Protein concentrations were measured in the BAL and assessed as a marker of vascular permeability. OVA-specific IgE (E) and IgG1 (F) were analyzed from serum by ELISA. *P < 0.05 compared with the corresponding Alum/OVA control group using ANOVA (n = 8).

Figure 4.

Evaluation of mucus metaplasia by periodic acid-Schiff (PAS) staining. (A) Paraffin-embedded lungs were stained using PAS reagent to visualize mucus-producing cells (stained in pink) in the airways, and were scored (B) for the extent of reactivity, as described in Materials and Methods. *P < 0.05 compared with corresponding Alum/OVA control group using ANOVA (n = 6).

Figure 5.

The transcription factor, hypoxia-inducible factor (HIF)–1, is activated in the OVA model of allergic airway disease, and is modified by administration of GSNO. (A) Mice were treated as described in Materials and Methods, and HIF-1 DNA binding was evaluated in nuclear extracts. (B) HIF-1α protein levels were analyzed by Western analysis in whole lung protein lysates. (C) Expression levels of the HIF-1α transcript were determined by quantitative real-time PCR from cDNA generated from whole mouse lung RNA. *P < 0.05 compared with Alum/OVA PBS group; #P < 0.05 compared with OVA/OVA PBS group using ANOVA (n = 8).

Figure 6.

Effect of nitric oxide synthase (NOS)–2 deficiency on NF-κB and HIF-1 activity in response to OVA challenge. C57BL/6J and NOS2−/− mice were sensitized and challenged with OVA, as described in the Materials and Methods. Whole-lung nuclear extracts were analyzed by NF-κB p65 DNA binding assay (A) and Western blot (B). *P < 0.05 compared with the corresponding Alum/OVA control group using ANOVA (n = 5). (C) SNO-p65 levels were analyzed in whole lung tissue 48 hours after the latest OVA challenge by the biotin switch technique, and the SNO-p65 to total p65 ratio was determined by densitometric analysis (D). *P < 0.05 compared with the corresponding Alum/OVA control group using ANOVA (n = 5). (E) HIF-1 DNA binding activity was evaluated in nuclear extracts from whole lung and (F) HIF-1α protein levels in whole lung lysates were analyzed by Western analysis. *P < 0.05 compared with corresponding Alum/OVA control group using ANOVA (n = 5).

Figure 7.

Effect of NOS2-deficiency on OVA-induced allergic airway inflammation and markers of mucus metaplasia. C57BL/6J and NOS2−/− mice were sensitized and challenged with OVA, as described in Materials and Methods. Total (A), differential (B), and neutrophil (C) cell counts were assessed from the BAL. *P < 0.05, compared with corresponding Alum/OVA control group using ANOVA (n = 5); #P < 0.05 compared with the wild-type (C57BL/6J) OVA/OVA group using ANOVA (n = 5). Reactivity of PAS was evaluated (D) and scored (E) in paraffin-embedded lung sections to visualize mucus-producing cells in the airways of OVA-sensitized (or control) and -challenged, wild-type and NOS2−/− mice. *P < 0.05 compared with corresponding Alum/OVA control group using ANOVA (n = 5).