TNF Production in Activated RBL-2H3 Cells Requires Munc13-4
Tolulope E. Ayo1, Pratikshya Adhikari1, Shuzo Sugita2,3 and Hao Xu1
1Department of Cell and Molecular Biology, School of Biological, Environmental, and Earth Sciences, University of Southern Mississippi, Hattiesburg, Mississippi
2Division of Fundamental Neurobiology, University Health Network, Toronto, ON, Canada
3Department of Physiology, Faculty of Medicine, University of Toronto, ON, Canada
The stimulation of mast cells leads to the release of vast amount of mediators that various studies have shown to be of great medical importance. One of these mediators is Tumor Necrosis Factor (TNFα). The purpose of this study is to show the connection that exists between TNF production and secretion in stimulated mast cells using a tumor homolog of mucosal mast cells, RBL-2H3(Rat basophilic leukemia cells). Munc13-4(an important regulatory protein in mast cell exocytosis) knock-out RBL-2H3 cells were created and IgE/antigen dependent stimulation produced high amount of TNF in wild type but not in munc13-4 knock-out cells. The production of TNF was rescued upon re-introduction of Munc13-4 indicating our knock-out procedure was target specific. Also, pre-incubation of cells with R-7050, an inhibitor of TNF receptor signaling pathway before stimulation blocked TNF production without affecting TNF release. Therefore, we proposed that there is a feedback loop in which TNF released upon mast cell stimulation binds to TNF receptor to boost TNF production.