Time course of IL-3–induced expression of granzyme B, and its exocytosis in response to different basophil agonists. (A) Release of granzyme B by degranulation of basophils upon IgE-dependent or IgE-independent stimulation. Purified basophils were cultured in medium alone or exposed to a maximally effective concentration of IL-3 (10 ng/mL) for the indicated time periods and subsequently stimulated with buffer (control), anti-FcϵRIα mAb (100 ng/mL, aIgER), or C5a (10 nM) for 30 minutes. GzmB was measured in the cell supernatants and in pellets by ELISA. The whole bar gives an estimation of total GzmB for every condition. Histamine was measured in the supernatants and in the pellets as an indicator of basophil degranulation. A representative experiment (mean of triplicates) is shown. In experiments performed with cells from different donors, GzmB released by basophils cultured with IL-3 for 24 hours was on average 1870 pg GzmB/10⁶ basophils (range, 1150-4600 pg; 9 donors) upon stimulation with C5a, and 2150 pg GzmB/10⁶ basophils (range, 1310-4920 pg; 5 donors) upon IgE-receptor cross-linking. (B) Exocytosis of GzmB is induced by diverse agonists. Basophils were cultured with or without IL-3 (10 ng/mL) for 20 hours before stimulation with anti-FcϵRIα mAb (100 ng/mL; aIgER), C5a (10 nM), fMLP (2.5 μM), MCP-1 (100 nM), or eotaxin (100 nM) for 30 minutes. Released GzmB, histamine, and LTC₄ were measured in the cell supernatants. Mean values of triplicates of a representative experiment are shown. An identical pattern of results with the same order of efficacy of these agonists was observed with cells from 3 different donors.