(A) WT or Itch^–/– mice (4 mice for each group) were injected with high-dose soluble OVA or PBS as control on day 0 and day 3. On day 9, the mice were sensitized with OVA in alum, followed by aerosol OVA exposure for 4 consecutive days from day 19. A portion of lung tissues before or after OVA aerosol challenge was processed for H&E staining. (B) The lung sections stained with H&E were scored for severity of inflammation on a scale of 0–5; at least 500 fields were scored per mouse. The data represent the means of 4 mice per group. (C) Lung sections were stained with PAS, and PAS⁺ cells were counted; 1,000 goblet cells were counted. The results represent the mean percentage of positive cells from 4 mice. (D) Sera collected from the mice were analyzed for OVA-specific IgE by ELISA. (E) BAL fluid was analyzed for granulocyte and lymphocyte infiltration by flow cytometry. FSC, forward scatter; SSC, side scatter. (F) Total cell numbers and the percentage of lymphocytes and eosinophils were determined. (G) The cytokine concentrations for IL-4, IL-5, and IL-13 in BAL fluid were measured by ELISA. (H) T cells from bronchial lymph nodes of WT and Itch^–/– mice were stimulated in vitro with OVA plus APCs, and the cell proliferation was measured by thymidine incorporation. A–D are representatives of 3 repeated experiments with 4 mice per group.