The Y500F mutation selectively impairs activation of the IRS-2/AKT signaling cascade. (A–D) IL-4–induced signaling events in WT and Y500F T cells. (A) Jak1 and IL-4Rα tyrosine phosphorylation. (Top) Lysates of T cell lymphoblasts were directly blotted with an anti–phospho-Jak1 antibody (pJak1) then reprobed with an anti-Jak1 protein antibody. (Bottom) IL-4Rα immunoprecipitates were probed with an antiphosphotyrosine antibody (pY-IL-4Rα) then reprobed with an anti–IL-4Rα antibody. The IL-4Rα Y500 lysates were overloaded to reveal low level phosphorylation of mutant IL-4Rα in response to IL-4 treatment. (B) IRS-2 and Dok-R tyrosine phosphorylation. IRS-2 and Dok-R immunoprecipitates were probed with an antiphosphotyrosine antibody followed by anti–IRS-2 and anti–Dok-R antibodies, respectively. (C) Akt phosphorylation. Cell lysates were directly blotted with an anti–phospho-Akt serine 473 antibody (pAkt) and then reprobed with an anti-Akt protein antibody. (D) Time course of Stat6 phosphorylation. Stat6 immunoprecipitates were probed with an anti–phospho-Stat6 antibody (pY-Stat6) and then reprobed with an anti-Stat6 antibody. (E–H) IL-4–induced signaling events in WT and Y500F splenic B cells. (E) Tyrosine phosphorylation of IRS-2 (left) and Dok1 (right). (F) p46Shc phosphorylation. Lysates were probed with anti–phospho-Shc antibody and then reprobed with an anti-Shc antibody. (G) Akt phosphorylation on serine 473. (H) Time course of Stat6 phosphorylation.

Impact of the Y500F mutation on Th cytokine production. (A) In vitro Th cell polarization. Naive WT and Y500F splenic T cells were differentiated into Th1 or Th2 cells and then stimulated with anti-TCRβ mAB for 48 h. Culture supernatants were then harvested and examined for IL-4, IL-5, IL-13, and IFN-γ production by ELISA. (B) Cytokine production in response to antigenic stimulation. Splenocytes and mesenteric LN cells of OVA-immunized WT and Y500F mice were cultured for 4 d in the presence of 50 μg/ml of OVA. Culture supernatants were assayed for IL-4, IL-5, IL-13, and IFN-γ by ELISA. In both panels, results are means ± SE (n = 4–5 mice/group) and are representative of three experiments. *P < 0.001; **P = 0.013; ***P = 0.002.

The Y500F mutation augments isotype switching. (A) Production of IgG1 and IgE by cultured splenocytes treated with an anti-CD40 mAb + IL-4. Levels of IgG1 and IgE antibodies present in IgG1 and IgE in supernatants of unstimulated cells were <8 ng/ml, respectively. Results are means ± SE antibody (4 mice/group). Similar results were found in three separate experiments. (B) Molecular events in isotype switching. RT-PCR analysis of μ, γ1, and ɛ germline (GLT) and γ1 and ɛ postswitch transcripts (Iμ→γ1 and Iμ→ɛ, respectively) in splenic B cells treated for 48 h with IL-4, anti-CD40 mAB, or both. Results are representative of four pairs of WT and mutant mice from two independent experiments. (C) Sμ-Sγ1 and Sμ-Sɛ deletional switch recombination measured by DC-PCR at day 6 after treatment with anti-CD40 mAb + IL-4. PCR was performed using 5 ng (lanes 1 and 3) or 20 ng (lanes 2 and 4) of template DNA.

Targeted knock-in mutagenesis of murine IL-4Rα gene. (A) Targeting strategy. WT exon 12 was replaced with one carrying an A to T substitution in the second position of codon 500 in exon 12 of IL4RA, changing its specificity from a tyrosine (Y) to a phenylalanine (F). The mutant exon is marked with an asterisk. Successful targeting introduced within intron 11 a floxed Neo cassette flanked by EcoRI sites. Subsequent treatment with Cre recombinase removed the neo cassette, leaving one loxP sequence and the EcoRI site in place. Restriction enzyme abbreviations: B, BamHI; R, EcoRI; and X, XhoI. □ and ▪ denote no-coding and coding exonic sequences, respectively. (B) Southern blot analysis of EcoRI-digested mouse tail genomic DNA of WT, heterozygous (HET), and homozygous (HOMO) mutant mice. The introduction of a novel EcoRI site in intron 11 of targeted IL4A reduces the size of an EcoRI genomic fragment detected with an exon 4 probe from 12.5 kb in the WT allele to 8.5 kb in the mutant allele. (C) PCR analysis of mouse tail DNA using Y500 and F500 allele-specific primers (top and middle, respectively) or primers spanning the residual 34-bp loxP site left in intron 11 after Cre-mediated excision of the floxed Neo cassette. (D, top) RT-PCR analysis of IL-4Rα mRNA expression in splenocytes of WT and Y500F homozygous mutant mice. Amplified GAPDH transcripts were used as controls. (Bottom) Sequence analysis of RT-PCR products of IL-4Rα transcripts of WT and Y500F mutant mice. (E) Flow cytometric analysis of IL-4Rα expression in IL-4RαY500F lymphocytes. Splenic B cells of WT, IL-4Rα knock-out, and IL-4Rα Y500F mice were stained with PE-conjugated rat antimurine IL-4Rα mAb (mIL4R-M1). Control staining was performed on WT splenic B cells using a PE-conjugated, isotype-matched rat IgG2a antibody.

Effect of the Y500F mutation on IL-4–induced lymphocyte proliferation and gene expression. (A) T cell proliferation. (Left) CD4⁺ splenic T cells of WT and homozygous Y500F littermate mice were cultured with plate-bound anti-TCRβ mAb (100 ng/ml) and IL-4 as indicated. Proliferation was assessed by ³H-thymidine incorporation. Results are mean responses of triplicate cultures ± SE and are representative of three experiments. (Right) Purified CD4⁺ T cells were cultured with PMA (25 ng/ml) and ionomycin (1 μM) or with plate-bound anti-TCRβ and IL-2 (100 U/ml). (B) B cell proliferation. (Left) Splenic B cells were cultured with anti-CD40 mAb (1 μg/ml) and IL-4 as indicated. (Right) Splenic B cells were cultured with IL-4 (10 ng/ml), anti-IgM antibodies (10 μg/ml), or IL-4 and anti-IgM antibodies as indicated. (C) IL-4–induced up-regulation of CD23 and MHC class II antigens. Splenic B cells were cultured for 48 h in the absence (thin line) or in the presence (thick line) of IL-4 at 50 ng/ml and then examined for CD23 and I-A^d expression by flow cytometry. Results are representative of five experiments.

The Y500F mutation up-regulates total IgE production and antigen-specific IgG1, IgG2a, and IgE antibody responses. Mice were immunized with OVA (100 μg) mixed with alum or sham immunized with saline/alum on day 0 and 14. Total (A) and OVA-specific Ig isotypes (B) were determined on day 28 postimmunizations by ELISA. Results are means ± SE (n = 6/group).

The Y500F mutation enhances AHR, tissue eosinophilia, and goblet cell metaplasia in an antigen-induced model of allergic airway inflammation. (A) AHR to aerosolized methacholine in WT and Y500F mutant mice (n = 5–6/group) immunized with OVA/alum or sham immunized with saline/alum mix and then subsequently challenged with aerosolized OVA. AHR was assessed by enhanced respiratory pause (Penh). Results are means ± SE; ^†P = 0.03 (versus IL-4Rα Y500F OVA). (B) Differential count of inflammatory cells found in BAL fluid of mice tested in A. *P = 0.04 (versus IL-4Rα Y500F OVA). (C) Lung histology of WT and IL-4Rα Y500F mutant mice sensitized and challenged with OVA. H&E, hematoxylin-eosin; PAS, periodic acid-schiff reagent. (D) The number of mucin-positive cells in the bronchi of OVA-treated WT and Y500F animals. PAS-stained lung sections were scored for the number of mucin-secreting cells per high power field (HPF, 400×) in central medium-sized bronchi. Three representative fields were counted for each of three mice in each group. **P < 0.0001 (versus IL-4Rα Y500F OVA). (E) AHR to aerosolized methacholine in WT and Y500F mutant mice (n = 14–15 mice/group) tested after intranasal instillation of IL-4, IL-13, or BSA at 5 μg each. (F) Differential count of inflammatory cells found in BAL fluid of mice tested in E. ^§P = 0.03; ^¶P = 0.016 versus IL-4Rα Y500F IL-13 at 100 and 333 mg methacholine/ml, respectively.