Production of IL-33 by B cells upon activation of Notch signaling. (A) IL-33 expression by splenic CD19⁺RFP⁺ B cells from NICD1 mice and controls, as examined by intracellular flow cytometric analysis. (B) Representative images of splenocytes of NICD1 mice immunofluorescently stained for IL-33 and RFP. Cells were stained for intracellular IL-33 and then cytospin slides prepared. Slides were then counterstained with DAPI. White arrowheads indicate RFP⁺ cells. (C) CD80, CD86, and CD95 expression profiles of splenic CD19⁺RFP⁺ B cells from NICD1 mice were compared between cells within the highest quartile and those within lower 3 quartiles of intracellular IL-33 expression. (D) Comparison of mean forward scatter of splenic CD19⁺RFP⁺ B cells from NICD1 mice between cells within the highest quartile and those within lower 3 quartiles of intracellular IL-33 expression. In panels C-D, the mean percentage of cells from 3 individual mice per group is shown. In panels A-D, data in the bar graphs are expressed as the mean and SEM from 3 individual mice. **P < .01 and ***P < .001 (Student t test). (E) IL-33 production by splenic CD19⁺ B cells from WT B6 mice stimulated with LPS in vitro and cultured in the presence of L cells expressing Dll1, Dll4, and Jagged1 was compared with that cultured in the presence of mock-transduced L cells. (F) Inhibition of Dll1-mediated IL-33 production by WT B cells upon addition of a Notch-signaling inhibitor, DAPT. Splenic CD19⁺ B cells from WT B6 mice were stimulated with LPS in vitro and cultured in the presence of L cells with or without Dll1 expression. (G) Dll1-mediated induction of NRARP, Notch3, and IL33 gene expression in WT B cells, as evaluated by quantitative PCR. Splenic CD19⁺ B cells from WT B6 mice were stimulated with LPS in vitro and cultured in the presence of L cells with or without Dll1 expression. Dll1-Notch signaling was inhibited by the addition of an anti–Dll1-blocking antibody. Expression levels are demonstrated relative to those detected in B cells cocultured with mock-transduced L cells and without a Dll1-blocking antibody. In panels E-G, data in the bar graphs are expressed as the mean and SEM from 3 independent experiments. (E) *P < .05 (Dunnett test). (F-G) *P < .05, **P < .01, and ***P < .01 (Tukey-Kramer test). DMSO, dimethyl sulfoxide; MFI, mean fluorescence intensity.