IL-3 induces granzyme B expression in human basophils. (A) Dose-dependent induction of granzyme B in basophils stimulated with IL-3. (i) Basophils were cultured with different concentrations of unglycosylated E coli–derived or glycosylated CHO-derived recombinant human IL-3 for 24 hours. The GzmB content was then determined by a specific ELISA in cell lysates. A representative experiment (mean of triplicates) out of 3 is shown. (ii) Basophils were cultured without or with different concentrations of IL-3 as indicated for 24 hours. GzmB expression was analyzed in cellular protein extracts by Western blotting. Actin is shown as a loading control. The variability of GzmB induction at a maximally effective concentration of IL-3 in basophils isolated from different donors is shown in Figure S1. (B) Expression of GzmB by NK cells and human basophils, but not neutrophils. Basophils were cultured in the absence or presence of IL-3 (10 ng/mL) for 20 hours. Protein extracts prepared from cultured basophils or freshly isolated neutrophils (N) and NK cells (NK) were analyzed by Western blotting using anti-GzmB mAb (i), and anti–α-defensin-1/3 mAb (ii). The number of cells corresponding to the protein amount loaded on a gel is indicated above the lines. Actin is shown as a loading control. Results are representative of 3 independent experiments with cells from different donors. (C) Heterogeneous expression of GzmB in basophils. Intracellular expression of GzmB (i) and cell surface expression of ST2 (ii) were analyzed by flow cytometry in basophils cultured for 20 hours without and with IL-3 (10 ng/mL). (D) GzmB staining shows a cytoplasmic granular pattern. Cytospin preparations of basophils cultured for 20 hours with IL-3 (10 ng/mL) were stained with anti-GzmB mAb (green, top) or isotype-matched control (mouse IgG1) (green, bottom). DNA was stained with DAPI (blue). Shown are merged images created in Adobe Photoshop 8.0.1 (Adobe Systems, San Jose, CA) without any alteration of the original digital images.