IL-3–induced sST2 is sorted to basophils granules and released by triggers of exocytosis. (A) Immunofluorescence analysis of ST2 expression and release in human basophils. Cytospin preparations of basophils cultured for 20 hours in the presence of IL-3, with or without triggering degranulation by 10 nM C5a for 30 minutes, were stained with anti-ST2 mAb (green, top left panel) and anti-GzB mAb (green, bottom left panel). DNA was stained with Hoechst (blue, middle panels). Images were acquired using constant settings on a Nikon Eclipse E600 fluorescence microscope (Nikon, Tokyo, Japan) equipped with a 60×/1.4 NA oil-immersion objective lens using FITC and DAPI filter sets. A Nikon DXM 1200 digital camera was used to capture images. Merged images (right panels) were created in Adobe Photoshop 8.0.1 (Adobe Systems, San Jose, CA) without any alteration of the original digital images. (B,C) Degranulation of basophils triggers the release of sST2. (B) Protein extracts derived from basophils cultured overnight with or without IL-3 followed by stimulation with C5a or anti-FcϵRIα mAb (100 ng/mL; anti-IgER) for 30 minutes as indicated were analyzed for the presence of ST2 and Granzyme B (GzB) by Western blotting. (C) Purified basophils were cultured in medium, IL-3 or IL-33 (50 ng/mL) for 20 hours and subsequently stimulated with C5a or anti-FcϵRIα mAb as indicated. sST2 was measured in cell supernatants by specific ELISA. Mean values of triplicates are shown. The representative data shown in panels A, B, and C were from separate experiments with cells from different donors.