Induction of GzmB by cytokines and products of activated mast cells. (A) Effect of different cytokines on the induction of GzmB and on the response of basophils to C5a. Basophils were cultured overnight in medium alone or with IL-3 (10 ng/mL), GM-CSF (50 ng/mL), IL-5 (50 ng/mL), NGF (50 ng/mL), IL-2 (100 U/mL), IFN-α (1000 U/mL), or IFN-γ (1000 U/mL). Intracellular GzmB was analyzed by flow cytometry. The Δ mean fluorescence (mean fluorescence of the specific mAb minus fluorescence of control antibody for each condition) is shown. (Lower panels) Following overnight culture with indicated cytokines, basophils were stimulated with C5a (10 nM) for 30 minutes to induce degranulation. Released (▪) and cell-associated (□) GzmB, LTC₄, and histamine release is shown. No histamine or LTC₄ release was detected when the stimulation with C5a was omitted (not shown). Mean values of triplicates of a representative experiment are shown. Experiments performed with basophils from 3 different donors showed an identical pattern of results and the same order of efficacy of the cytokines. Also note the congruent results for GzmB induction determined by flow cytometry and ELISA that were performed in independent experiments. (B) Relationship between induction of GzmB and “late priming” with different concentrations of IL-3. Basophils were incubated in medium or different concentrations of IL-3. After 24 hours, cells were stimulated with 10 nM C5a for 30 minutes. Released and cell-associated GzmB and histamine and leukotriene formation is shown (mean values of triplicate determinations of a representative experiment out of 3). (C) The products of IgE-activated mast cells induce GzmB in basophils. Western blots of cell extracts of basophils cultured overnight with supernatants of human mast cells (MC supernatants) that have been activated by IgE-receptor cross-linking (a-IgER) or not (buffer) are shown (“Materials and methods”). For comparison, extracts of the same basophil preparation of freshly isolated basophils, or basophils cultured in medium (buffer) or with IL-3 (10 ng/mL), are included in the blot. Where indicated by +, a neutralizing polyclonal goat anti–IL-3 antibody (a-IL-3; 10 μg/mL) was added. An irrelevant control goat IgG had no effect on GzmB expression (data not shown). Tyr694 phosphorylation of Stat5 is shown as another independent indicator for the stimulation of basophils by mast cell–derived IL-3. Total Stat5 levels were identical under all the conditions (data not shown).