ICAM-1 deficiency impairs ILC2 function in response to IL-33 challenge. (A and B) BM cells from WT mice were cultured in the presence of IL-7 (10 ng/ml) with PBS or IL-33 (10 ng/ml) treatment. 2 d later, the expression of ICAM-1 (A) or LFA-1 (B) on BM iILC2s was evaluated by flow cytometry. (C) Human PBMCs were cultured in the presence of recombinant human IL-2 (20 ng/ml) and IL-7 (20 ng/ml) with PBS or human IL-33 (20 ng/ml) treatment for 3 d. The MFI levels of ICAM-1 and LFA-1 on human ILC2s (CD45⁺Lin⁻CD127⁺CRTH2⁺) were evaluated by flow cytometry. (D) Equal number of purified lung ILC2s from WT and ICAM-1^−/− mice were cultured in the presence of IL-2 (10 ng/ml), IL-7 (20 ng/ml), and IL-33 (20 ng/ml) for 3 d, and the amount of IL-5 and IL-13 in the supernatants was evaluated by ELISA. (E–I) WT and ICAM-1^−/− mice were intranasally administered with IL-33 (500 ng/mouse/d) or PBS for three consecutive days (n = 6 for each group). Mice were sacrificed on day 4. (E) Representative flow cytometric analysis of ILC2, pregated on CD45⁺Lin⁻, and the number of ILC2 in lung from WT and ICAM-1^−/− mice after PBS or IL-33 treatment. (F) Frequencies of IL-5⁺IL-13⁺ in lung ILC2s after cell stimulation cocktail treatment for 4 h. IL-33-challenged groups were analyzed. (G) The amount of IL-5 and IL-13 in BAL was determined by ELISA. (H) The number of eosinophils (CD45⁺CD11c⁻Siglec-F⁺) in BAL was shown. (I) Representative H&E staining of lung sections (bars, 100 µm). Data are representative of two (E–I) to three (A–D) independent experiments. Error bars show mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001 by unpaired Student's t test. ns, not significant. Numbers within flow plots indicate the percentages of cells gated.