Evaluation of IL-4Rα expression in IL-4Rα^−/−/SMP8–IL-4Rα^+/− mice. (A) Expression of IL-4Rα by IL-4Rα^+/+, IL-4Rα^+/−, IL-4Rα^−/−, and IL-4Rα^−/−/SMP8–IL-4Rα^+/− mice in spleen (top), tracheal smooth muscle cells (middle), or tracheal epithelial cells (bottom). Representative results of three to four replicate experiments with one mouse/group are shown. (B) IL-4Rα expression by CD45⁺CD3⁺ and CD45⁺CD3⁻ spleen cells from mice of the indicated genotype. Single experiment with four mice/group. *, P < 0.05 by one-tailed Student’s t test compared with IL-4Rα^−/− spleen cells. (C) IL-4Rα expression by macrophages in lung digests from the indicated genotype. Macrophages were identified as CD45⁺CD11b⁺ cells that displayed light scatter characteristics typical for macrophages. Single experiment with four mice/group. *, P < 0.05 by one-tailed Student’s t test compared with lung macrophages from IL-4Rα^−/− mice. (D) IL-4Rα expression by tracheal smooth muscle cells from mice of the indicated genotype. Two experiments with two mice/group each. *, P < 0.05 by one-tailed Student’s t test compared with tracheal smooth muscle cells from IL-4Rα^−/− mice. (B–D) Error bars indicate SEs. (E) YFP (green) expression in smooth muscle cells in bronchioles (top) or alveoli (middle) in Rosa/SMP8-Cre⁺ lung. Arrows point to YFP⁺ cells. Lack of YFP (green) expression in bronchioles from control (Rosa/SMP8-Cre⁻) lung (bottom) is also shown. Bars, 25 µm. (F) Comparison of IL-4Rα expression by tracheal smooth muscle cells from wild-type, SMP8-Cre/IL-4Rα^flox/−, IL-4Rα^−/−, and IL-4Rα^−/−/SMP8–IL-4Rα^+/− mice. Histograms of IL-4Rα expression of cells from IL-4Rα^−/− mice are shown as purple-filled black lines in all three panels; histograms of IL-4Rα expression by cells from two individual IL-4Rα^flox/−/SMP8-Cre^+/− mice (top), wild-type mice (middle), and IL-4Rα^−/−/SMP8–IL-4Rα^+/− mice (bottom) are as red and green lines. Data are from one of the two experiments used to prepare the graph in D.

i.t. inoculation of IL-4Rα^−/−/SMP8–IL-4Rα^+/− mice with IL-4C induces AHR but not goblet cell hyperplasia or BAL fluid eosinophilia. (A–C) IL-4Rα^+/−, IL-4Rα^−/−, and IL-4Rα^−/−/SMP8–IL-4Rα^+/− mice were inoculated daily i.t. with saline or IL-4C (2 µg IL-4 plus 10 µg BVD4-1D11) every other day for 7 d and tested for responsiveness to methacholine by barometric plethysmography (A; two pooled experiments, n = 8–10/group), flexiVent (B; one experiment, n = 4–8/group), and APTI (C; one experiment, n = 4/group). (D) In a separate experiment, IL-4Rα^+/−, IL-4Rα^−/−, and IL-4Rα^−/−/SMP8–IL-4Rα^+/− mice were inoculated daily with saline or IL-4C and evaluated by barometric plethysmography 1 d after the first and third inoculations for responsiveness to methacholine (two pooled experiments, n = 12/group). (E) Numbers of BAL neutrophils, lymphocytes, macrophages, and eosinophils (two pooled experiments, n = 8–9/group). (F) Lung sections were PAS stained and evaluated for percentage of PAS⁺ bronchial epithelial cells (two pooled experiments, n = 8–9/group). Data shown in B and C are from independent experiments. Error bars indicate SEs. *, P < 0.05 for IL-4C versus saline treatment.

Effect of i.t. allergen administration on IL-4Rα^−/−/SMP8–IL-4Rα^+/− mice reconstituted with IL-4Rα^+/+ allergen–immune lymphoid cells. Wild-type BALB/c mice were immunized i.p. with dust mite allergen adsorbed to alum once a week for 3 wk and then inoculated i.t. with dust mite allergen every other day for 18 d. (A and B) 5 × 10⁶ bronchial lymph node cells prepared from these mice were injected i.v. into IL-4Rα^+/−, IL-4Rα^−/−, and IL-4Rα^−/−/SMP8–IL-4Rα^+/− mice, which were then inoculated three times per week i.t. for 3 wk with saline or dust mite allergen and tested for responsiveness to methacholine by barometric plethysmography (A; two pooled experiments, n = 8–12/group), flexiVent analysis (A; one experiment, n = 3–6/group), or APTI (B; one separate experiment, n = 3/group for saline and 5–6/group for house dust mite). (C) BAL was performed, and numbers of BAL neutrophils, lymphocytes, macrophages, and eosinophils were determined (two pooled experiments, n = 6–11/group). (D) Lung sections were PAS stained and evaluated for percentage of PAS⁺ bronchial epithelial cells (two pooled experiments, n = 9–13/group). (E) Serum IgE levels were determined by ELISA (two pooled experiments, n = 5–13/group). Error bars indicate SEs. *, P < 0.05 for dust mite allergen versus saline treatment.

IL-13 induces allergic airway disease in mice that selectively lack IL-4Rα in smooth muscle. (A, top) Mice of the indicated genotype were inoculated i.t. with saline or 3 µg IL-13 thrice weekly for 3 wk and then analyzed for AHR by barometric plethysmography (two pooled experiments, n = 6–14/group). (bottom) Mice were then inoculated with an additional dose of IL-13 and analyzed for AHR with a flexiVent apparatus (two pooled experiments, n = 6–14/group). (B) BAL cell numbers and differential counts (one experiment, n = 7–8/group). (C) Percentage of PAS⁺ bronchial epithelial cells (one experiment, n = 7–8/group). Error bars indicate SEs. Asterisks indicate a significant increase (*, P < 0.05) for IL-13 versus saline treatment.

i.t. inoculation of IL-4Rα^−/−/SMP8–IL-4Rα^+/− mice with IL-13 induces AHR but not goblet cell hyperplasia or BAL fluid eosinophilia. (A–C) IL-4Rα^+/−, IL-4Rα^−/−, and IL-4Rα^−/−/SMP8–IL-4Rα^+/− mice were inoculated daily i.t. with saline or 2 or 3 µg IL-13 for a consecutive 7 d and tested for responsiveness to methacholine by barometric plethysmography (A; three experiments, pooled data, n = 11–13/group), flexiVent (B; one experiment, n = 4–7/group), and APTI (C; one experiment, n = 3–5/group). (D) Numbers of BAL neutrophils, lymphocytes, macrophages, and eosinophils (three experiments, pooled data, n = 11–12/group). (E) Lung sections were PAS stained and evaluated for percentage of PAS⁺ bronchial epithelial cells (one of three experiments with similar results shown, n = 4–5/group). Error bars indicate SEs. *, P < 0.05 for IL-13 versus saline treatment.

Effect of i.t. allergen administration on IL-4Rα^−/−/SMP8–IL-4Rα^+/− mice. (A–C) IL-4Rα^+/−, IL-4Rα^−/−, and IL-4Rα^−/−/SMP8–IL-4Rα^+/− mice were inoculated three times per week i.t. for 3 wk with saline or dust mite allergen and then tested for responsiveness to methacholine by barometric plethysmography (A; two pooled experiments, n = 9–12/group), flexiVent (B; two pooled experiments, n = 9–12/group), and APTI (C; one separate experiment, n = 4–6/group). (D) Serum IgE levels were determined by ELISA (two pooled experiments, n = 8–11/group). (E) Lung sections were PAS stained and evaluated for percentage of PAS⁺ bronchial epithelial cells (two pooled experiments, n = 9–11/group). (F) Numbers of BAL neutrophils, lymphocytes, macrophages, and eosinophils (two pooled experiments, n = 9–12/group). (G) Production of IL-4 and serum levels of IL-13–sIL-13Rα2 complexes (two pooled experiments, n = 11–14/group). Error bars indicate SEs. *, P < 0.05 for dust mite allergen versus saline treatment.

IL-13–induced pulmonary smooth muscle gene expression. (A) Mice of the indicated genotype were inoculated i.t. with normal saline or 3 µg IL-13 for a consecutive 7 d. Lung RNA was prepared and analyzed in an initial experiment by gene scan to identify genes that were increased at least twofold in IL-13– versus saline-treated IL-4Rα^−/−/SMP8–IL-4Rα^+/− mice and at least twofold in IL-13–treated IL-4Rα^−/−/SMP8–IL-4Rα^+/− versus IL-4Rα^−/− mice and for genes that were expressed >10-fold more in IL-13–treated IL-4Rα^+/− versus IL-4Rα^−/−/SMP8–IL-4Rα^+/− mice (gene scan performed with two separate groups of four mice each for each genotype and treatment). Results of the gene scan were verified by real-time PCR with the same RNA samples and with RNA prepared from lungs from two additional experiments. Total of 18–22 mice for each genotype/treatment group. To pool data from different experiments, the mean value for each gene in the IL-4Rα^+/−/saline group in each experiment was given an arbitrary value of 1, and all individual values were adjusted accordingly. Geometric means and SEs were then determined. Log values were converted back to linear values for graphing. A one-tailed Student’s t test was used to test the hypotheses that IL-13 induced greater gene expression than saline in IL-4Rα^−/−/SMP8–IL-4Rα^+/− mice and that IL-13 induced greater gene expression in IL-4Rα^−/−/SMP8–IL-4Rα^+/− than in IL-4Rα^−/− mice. *, P < 0.05 for IL-13– versus saline-treated IL-4Rα^−/−/SMP8–IL-4Rα^+/− mice; †, P < 0.05 for IL-13–treated IL-4Rα^−/−/SMP8–IL-4Rα^+/− versus IL-4Rα^−/− mice. (B) Age- and sex-matched IL-4Rα^+/−, IL-4Rα^−/−, and SMP8-Cre^+/−/IL-4Rα^flox/− mice were inoculated with saline or IL-13 as in A. RNA was prepared from their lungs and assayed for expression levels of the genes shown by real-time PCR. Data were pooled from two experiments with similar results (n = 13–14 mice/group). A one-tailed Student’s t test was used to compare gene expression in saline- versus IL-13–treated IL-4Rα^−/−/SMP8–IL-4Rα^+/− mice and in IL-13–treated IL-4Rα^+/− versus IL-4Rα^−/− and SMP8-Cre^+/−/IL-4Rα^flox/− mice. *, P < 0.05 for IL-13– versus saline-treated IL-4Rα^+/− or SMP8-Cre^+/−/IL-4Rα^flox/− mice; †, P < 0.05 for IL-13–treated SMP8-Cre^+/−/IL-4Rα^flox/− versus IL-4Rα^−/− mice.

House dust mite allergen induces allergic airway disease in mice that selectively lack IL-4Rα in smooth muscle. (A, top) Mice of the indicated genotype were inoculated i.t. with saline or 50 µg house dust mite allergen thrice weekly for 3 wk and then analyzed for AHR by barometric plethysmography (one experiment, n = 5–8/group). (bottom) Mice were then inoculated with an additional dose of house dust mite allergen and analyzed for AHR with a flexiVent apparatus (one experiment, n = 5–8/group). (B and C) Percentage of PAS⁺ bronchial epithelial cells (B; one experiment, n = 5–8/group) and BAL cell numbers and differential counts (C; one experiment, n = 5–8/group). (D) Serum IgE levels evaluated by ELISA (one experiment, n = 5–8/group). Error bars indicate SEs. Asterisks indicate a significant increase (*, P < 0.05) for IL-13 versus saline treatment. All data were obtained from the same set of mice. Similar results were observed in a second experiment.