Abstract
Mast cells (MCs) are tissue resident immune cells that play important roles in the pathogenesis of allergic disorders. These responses are mediated via the cross-linking of cell surface high affinity IgE receptor (FcϵRI) by antigen resulting in calcium (Ca2+) mobilization, followed by degranulation and release of proinflammatory mediators. In addition to FcϵRI, cutaneous MCs express Mas-related G protein-coupled receptor X2 (MRGPRX2; mouse ortholog MrgprB2). Activation of MRGPRX2/B2 by the neuropeptide substance P (SP) is implicated in neurogenic inflammation, chronic urticaria, mastocytosis and atopic dermatitis. Although Ca2+ entry is required for MRGPRX2/B2-mediated MC responses, the possibility that calcium release-activated calcium (CRAC/Orai) channels participate in these responses has not been tested. Lentiviral shRNA-mediated silencing of Orai1, Orai2 or Orai3 in a human MC line (LAD2 cells) resulted in partial inhibition of SP-induced Ca2+ mobilization, degranulation and cytokine/chemokine generation (TNF-α, IL-8, and CCL-3). Synta66, which blocks homo and hetero-dimerization of Orai channels, caused a more robust inhibition of SP-induced responses than knockdown of individual Orai channels. Synta66 also blocked SP-induced extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt phosphorylation and abrogated cytokine/chemokine production. It also inhibited SP-induced Ca2+ mobilization and degranulation in primary human skin MCs and mouse peritoneal MCs. Furthermore, Synta66 attenuated both SP-induced cutaneous vascular permeability and leukocyte recruitment in mouse peritoneum. These findings demonstrate that Orai channels contribute to MRGPRX2/B2-mediated MC activation and suggest that their inhibition could provide a novel approach for the modulation of SP-induced MC/MRGPRX2-mediated disorders.
Highlights
Mast cells (MCs) are tissue resident immune cells best known for their roles in anaphylaxis and atopic disorders, which result from FcεRI/IgE-mediated histamine release and the generation of lipid mediators and cytokines [1]
Rabbit anti-Orai1, Orai2 and Orai3 antibodies from Alomone lab (Rockville, MD, USA), anti-extracellular signal-regulated kinase 1/2 (ERK1/2), anti-phospho-ERK1/2 (Thr-202/Tyr-204), anti-phospho-Akt (Ser-473), anti-Akt, b-Actin and goat antirabbit IgG-horseradish peroxidase (HRP) were obtained from Cell Signaling Technology (Danvers, MA, USA)
We found that Orai2 silencing resulted in ~50% reduction in protein expression (Figures 1A, B), this was associated with significant inhibition of Ca2+ influx (Figure 1E) with small reduction in degranulation (Figure 1F)
Summary
Mast cells (MCs) are tissue resident immune cells best known for their roles in anaphylaxis and atopic disorders, which result from FcεRI/IgE-mediated histamine release and the generation of lipid mediators and cytokines [1]. While all MCs are characterized by the expression of cell surface FcεRI, cutaneous MCs highly expresses a newly identified G protein-coupled receptor (GPCR) known as Mas-related GPCR-X2 (MRGPRX2, mouse counterpart MrgprB2) [9] Activation of this receptor by an increasing list of cationic ligands contributes to host defense, pseudoallergy and a number of chronic inflammatory diseases [9,10,11]. Activation of murine MCs by SP via MrgprB2 contributes to experimental neurogenic inflammation, pain, and atopic dermatitis [14, 15] Both anti-IgE and SP cause intracellular increase in Ca2+ concentration, the kinetics of Ca2+ mobilization varies for different stimuli. The mechanism via which SP activates MRGPRX2 to cause Ca2+ mobilization and mediator release is unknown
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
