Steroid-resistant neutrophilic inflammation in a mouse model of an acute exacerbation of asthma

Abstract

Neutrophilic inflammation in acute exacerbations of asthma tends to be resistant to treatment with glucocorticoids. This may be related to decreased activity and expression of histone deacetylase-2 (HDAC2), which down-regulates expression of proinflammatory genes via recruitment to the glucocorticoid receptor complex. We assessed airway inflammation and response to steroid treatment in a novel mouse model of an acute exacerbation of chronic asthma. Systemically sensitized mice received low-level challenge with aerosolized ovalbumin for 4 weeks, followed by a single moderate-level challenge to induce enhanced inflammation in distal airways. We assessed the effects of pre-treatment with dexamethasone on the accumulation of inflammatory cells in the airways, airway responsiveness to methacholine, expression and enzymatic activity of nuclear proteins including histone acetyl transferase (HAT) and HDAC2, and levels of transcripts for neutrophil chemoattractant and survival cytokines. Dexamethasone suppressed inflammation associated with eosinophil and T-lymphocyte recruitment, but did not prevent neutrophil accumulation or development of airway hyperresponsiveness. Increased activity of HAT was suppressed by steroid treatment, but the marked diminution of HDAC2 activity and increased activity of nuclear factor-kappaB were not reversed. Correspondingly, elevated expression of mRNA for TNF-alpha, granulocyte-macrophage colony-stimulating factor, IL-8, and p21(waf) were also not suppressed by dexamethasone. Levels of lipid peroxidation and protein nitration products were elevated in the acute exacerbation model. We conclude that impaired nuclear recruitment of HDAC2 could be an important mechanism of steroid resistance of the neutrophilic inflammation in exacerbations of asthma. Oxidative stress may contribute to decreased HDAC2 activity.

Figure 1.

Profile density of (A) eosinophils, (B) CD3⁺ T cells, and (C) neutrophils, in lung tissue of mice from the acute exacerbation model treated with vehicle or with dexamethasone, compared with naïve animals. Significant differences compared with naïve animals shown as ***P < 0.001 and compared with mice treated with vehicle shown as ^###P < 0.001.

Figure 2.

Immunostaining for Gr-1⁺ neutrophils in a frozen section of lung tissue of an animal from dexamethasone-treated group, demonstrating the location of the cells within the airway and alveolar walls. Immunoperoxidase, methyl green counterstain.

Figure 3.

(A) GR activity and (B) HAT activity in nuclear extracts of lung tissue of mice from the acute exacerbation model treated with vehicle or with dexamethasone, compared with naïve animals. Significant differences compared with naïve animals shown as **P < 0.01, ***P < 0.001 and compared with mice treated with vehicle shown as ^##P < 0.01, ^###P < 0.001.

Figure 4.

(A) Western blotting of nuclear extracts of lung tissue of mice for HDAC2 and the reference protein lamin A/C. Lanes 1 and 2 are samples from individual naïve animals; lanes 3 and 4 are from individual vehicle-treated animals; lanes 5 and 6 are from dexamethasone-treated animals. (B) Relative expression of HDAC2 protein in nuclear extracts of lung tissue of mice from the three groups. Significant differences compared with naïve animals shown as ***P < 0.001.

Figure 5.

NF-κB activity in nuclear extracts of lung tissue of mice from the acute exacerbation model treated with vehicle or with dexamethasone, compared with naïve animals. Significant differences compared with naïve animals shown as **P < 0.01.

Figure 6.

Relative expression of mRNA for (A) TNF-α; (B) GM-CSF; (C) KC, the murine functional homolog of IL-8; and (D) p21^waf, a marker of severe asthma, in nuclear extracts of lung tissue of mice from the acute exacerbation model treated with vehicle or with dexamethasone, compared with naïve animals. Significant differences compared with naïve animals shown as *P < 0.05, **P < 0.01.

Figure 6.

Relative expression of mRNA for (A) TNF-α; (B) GM-CSF; (C) KC, the murine functional homolog of IL-8; and (D) p21^waf, a marker of severe asthma, in nuclear extracts of lung tissue of mice from the acute exacerbation model treated with vehicle or with dexamethasone, compared with naïve animals. Significant differences compared with naïve animals shown as *P < 0.05, **P < 0.01.

Figure 7.

Relative levels of (A) lipid peroxidation products and (B) nitrotyrosine in cytoplasmic extracts of lung tissue of mice from the acute exacerbation model treated with vehicle or with dexamethasone, compared with naïve animals. Significant differences compared with naïve animals shown as **P < 0.01, ***P < 0.001.