TNF Production in Activated RBL-2H3 Cells Requires Munc13-4

Mast cell activation triggers intricate signaling pathways that promote the expression and/or release of a wide range of mediators including tumor necrosis factor (TNF; also known as TNFα). In this study, we investigated the connection between TNF secretion and TNF production, exploiting RBL-2H3 cells (a tumor analog of mucosal mast cells) that are depleted of Munc13-4, a crucial component of the mast cell exocytic machinery. We showed that antigen/IgE elicited robust TNF production in RBL-2H3 cells, but not in Munc13-4 knockout cells. The production defect was corrected when Munc13-4 was reintroduced into the knockout cell line, suggesting that the phenotype was not caused by any secondary effect derived from the knockout approach. Furthermore, pre-incubation of RBL-2H3 cells with R-7050, an antagonist of TNF receptor-dependent signaling, was shown to block TNF production without inhibiting TNF release. These observations provide fresh evidence for a robust feed-back loop to boost TNF production in activated mast cells.