Notch Signaling Enhances FcεRI-Mediated Cytokine Production by Mast Cells through Direct and Indirect Mechanisms

Th2-type cytokines and TNF-α secreted by activated mast cells upon cross-linking of FcεRI contribute to the development and maintenance of Th2 immunity to parasites and allergens. We have previously shown that cytokine secretion by mouse mast cells is enhanced by signaling through Notch receptors. In this study, we investigated the molecular mechanisms by which Notch signaling enhances mast cell cytokine production induced by FcεRI cross-linking. FcεRI-mediated production of cytokines, particularly IL-4, was significantly enhanced in mouse bone marrow-derived mast cells by priming with Notch ligands. Western blot analysis showed that Notch signaling augmented and prolonged FcεRI-mediated phosphorylation of MAPKs, mainly JNK and p38 MAPK, through suppression of the expression of SHIP-1, a master negative regulator of FcεRI signaling, resulting in the enhanced production of multiple cytokines. The enhancing effect of Notch ligand priming on multiple cytokine production was abolished by knockdown of Notch2, but not Notch1, and FcεRI-mediated production of multiple cytokines was enhanced by retroviral transduction with the intracellular domain of Notch2. However, only IL-4 production was enhanced by both Notch1 and Notch2. The enhancing effect of Notch signaling on IL-4 production was lost in bone marrow-derived mast cells from mice lacking conserved noncoding sequence 2, which is located at the distal 3' element of the Il4 gene locus and contains Notch effector RBP-J binding sites. These results indicate that Notch2 signaling indirectly enhances the FcεRI-mediated production of multiple cytokines, and both Notch1 and Notch2 signaling directly enhances IL-4 production through the noncoding sequence 2 enhancer of the Il4 gene.