Figure 3

Btk deficiency impacts on Ag-induced cytokine secretion in a dose-dependent manner. (a,b) WT (red), Btk−/− (blue), Ship1−/− (green), and DKO (yellow) BMMCs or (c,d) WT and Ship1−/− BMMCs, pre-treated for 30 min with 0.3 µM Ibrutinib or vehicle (DMSO), were left untreated (con) or stimulated with the indicated concentrations of Ag (DNP-HSA). (e,f) WT and Ship1−/− BMMCs were pre-loaded with IgE overnight (for the following DNP-HSA stimulation) or left without IgE (for the following IgE stimulation). Subsequently, the cells were treated for 30 min with 0.3 µM Ibrutinib or vehicle (DMSO). Cells were then left unstimulated (con) or stimulated with Ag (DNP-HSA [DNP]; 2 ng/ml) or with 2 µg/ml IgE (SPE-7). Supernatants were subjected to IL-6 (a,c,e) and TNF-α (b,d,f) ELISAs. Each bar is the mean of triplicates ± SD; comparable results were obtained in at least three experiments with different BMMC cultures. Statistical data were analysed for (a) n = 5, (b) n = 4, (c) n = 6 or n = 4 (2000 ng/ml DNP-HSA), (d) n = 5 or n = 3 (2000 ng/ml DNP-HSA), (e) and (f) n = 3 experiments with n.s. (non significant), *p < 0.05, **p < 0.005, ***p < 0.0005.