Signal transducer and activator of transcription factor 6 (Stat6)-deficient mice are protected from antigen-induced airway hyperresponsiveness and mucus production

Abstract

The pleiotropic cytokine interleukin 4 (IL-4) has been shown to regulate many processes thought to be important in the allergic diathesis. To determine the mechanism(s) by which IL-4 mediates allergic airway responses to inhaled allergens, we compared the effects of antigen sensitization and challenge on the development of allergic airway responses in mice in which the gene for the signal transducer and activator of transcription factor 6 (Stat6) was disrupted to those of their wild-type littermates. Strikingly, Stat6-deficient mice failed to develop airway hyperresponsiveness (AHR), which was observed in their wild-type littermates after allergen provocation. Moreover, antigen-induced increases in mucus-containing cells were found to be completely Stat6 dependent. Consistent with the lack of Th2 cytokine responses in Stat6-deficient mice, no ovalbumin-specific immunoglobulin (Ig)E was detected in their serum. In contrast, Stat6 signaling only partially mediated antigen-induced eosinophilia with no role in vascular adhesion molecule 1 expression. These results indicate that Stat6 signal transduction is critical in the development of allergen-induced AHR and that agents that specifically inhibit this pathway may provide a novel strategy for the treatment of allergic disorders.

Figure 1

Effects of Stat6 gene disruption on airway reactivity to intravenous acetylcholine (50 μg/kg) in mice 3 d after a single aspiration challenge with antigen (1.5% OVA) or PBS. Values shown are mean and SE (n = 9). * P value <0.05 versus all other groups; ⁺ P value <0.05 versus Ag-treated Stat6-deficient group. APTI, airway-pressure-time index, cm H₂O-sec.

Figure 2

Effects of Stat6 gene disruption on the numbers of BAL inflammatory cells recovered from mice 3 d after a single antigen or PBS aspiration challenge. Mice were treated as described in Fig. 1. Values shown are mean and SE (n = 9). * P value <0.05 versus PBS-treated groups.

Figure 3

Effect of Stat6 deficiency on IL-4 (A), IL-5 (B), and IFN-γ (C) protein levels in BAL fluids of mice 3 d after a single antigen or PBS aspiration challenge. Mice were treated as described in Fig. 1. Protein levels were analyzed by ELISA. OD readings were converted to picograms per milliliter by comparison with standard curves. Values shown are mean and SE (n = 9). * P value <0.05 versus PBS-treated groups; ⁺ P value <0.05 versus Ag-treated Stat6-deficient group.

Figure 4

Effect of Stat6 gene disruption on serum total IgE levels of mice 3 d after a single antigen or PBS aspiration challenge. Mice were treated as described in Fig. 1. Serum was diluted 1:50 for total IgE and analyzed by ELISA. OD readings were converted to nanograms per milliliter by comparison with a standard curve. Values are reported as mean and SE (n = 9). * P value <0.05 versus PBS-treated groups; ⁺ P value <0.05 versus Ag-treated Stat6-deficient animals.

Figure 5

Effect of Stat6 deficiency on serum OVA-specific IgG1 (A) and OVA-specific IgG2a (B) antibody levels of mice 3 d after a single antigen or PBS aspiration challenge. Mice were treated as described in Fig. 1. Serum was diluted 1:100, 1: 250, and 1:500 with FBS for analysis of OVA-specific IgG1 and IgG2a antibody levels by ELISA. Since recombinant OVA-specific antibodies were not available to generate a standard curve, relative antibody levels are reported using OD readings obtained after background absorbance was subtracted. Reported OD values are from samples diluted 1:250, all of which were below the saturation point of the assay as demonstrated by comparison to OD values obtained from samples diluted 1:100. Values are reported as mean and SE (n = 9). * P value <0.05 versus PBS-treated groups; ⁺ P value <0.05 versus OVA-treated Stat6-deficient animals.

Figure 6

Effects of Stat6 gene disruption on antigen-induced increases in the number of mucus-containing cells in the airway epithelium. Lung sections (n = 4/experimental group, four sections per animal) from OVA-treated wild-type (A) and Stat6-deficient (B) mice were stained with periodic acid Schiff. (A) Lung section of antigen-treated wild-type mouse demonstrating interstitial inflammatory cells and a large degree of goblet cell hyperplasia. (B) Lung section of antigen-treated Stat6-deficient mouse demonstrating interstitial inflammatory cells and an absence of mucus-containing cells. Bars, 100 μm.

Figure 7

Immunohistochemical detection of VCAM-1 expression in lung sections from antigen-treated wild-type and Stat6-deficient mice treated as described in Fig. 1 legend. Lung sections (n = 2, four sections per animal) from either antigen-treated wild-type (A and B) or Stat6-deficient animals (C and D) were incubated with either isotype-matched control IgG mAb (A and C) or anti–VCAM-1 mAb (B and D). Notice comparable VCAM-1–positive staining in sections from antigen-treated wild-type and Stat6-deficient mice (B and D). Notice lack of positive staining in sections incubated with the isotype-matched control antibody (A and C). Bars, 50 μm.