An involvement of neurokinin-1 receptor in FcεRΙ-mediated RBL-2H3 mast cell activation

Abstract

Objective and design: To determine whether the neurokinin-1 receptor (NK1R) plays a role in the activation of RBL-2H3 mast cells after FcεRΙ aggregation. Materials and methods: NK1R expression in RBL-2H3 cells was inhibited by small hairpin RNA (shRNA) against NK1R, and determined by western blotting. For activation, both NK1R knockdown and control RBL-2H3 cells were sensitized by dinitrophenol (DNP)-specific IgE and stimulated with the antigen DNP-bovine serum albumin (BSA). Following the activation of RBL-2H3 cells, monocyte chemoattractant protein (MCP-1) production and intracellular calcium flux were monitored by ELISA and confocal microscopy assay, respectively. For investigation of the signaling mechanism, phosphorylation of mitogen-activated protein kinases (MAPKs) after RBL-2H3 cell activation was assessed by western blotting. Results: shRNA-NK1R mediated an effective inhibition of NK1R expression in RBL-2H3 cells. Protein production of MCP-1 was reduced by more than 55 % in NK1R knockdown RBL-2H3 cells compared with control RBL-2H3 cells. In addition, both calcium mobilization and phosphorylation levels of MAPKs (Erk1/2, JNK, and p38) after DNP-BSA stimulation (via FcεRΙ) were decreased due to the inhibition of NK1R expression. Conclusion: NK1R is required for the activation of RBL-2H3 cells following FcεRΙ engagement and involved in the regulation of MAPK signaling pathways. © 2012 The Author(s).