Distinct roles of Ca2+mobilization and G protein usage on regulation of Toll‐like receptor function in human and murine mast cells

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Abstract
Summary: Toll‐like receptors (TLRs) expressed in mast cells play important roles in orchestrating host defence against bacterial pathogens. Previous studies demonstrated that TLR2 agonist tripalmitoyl‐S‐glycero‐Cys‐(Lys)4(Pam3Cys) stimulates both degranulation and cytokine production in human mast cells but only induces cytokine production in murine mast cells. To determine the molecular basis for this difference, we utilized a human mast cell line LAD 2, murine lung and bone marrow‐derived mast cells (MLMC and BMMC). We found that Pam3Cys caused a sustained Ca2+mobilization and degranulation in LAD 2 mast cells but not in MLMC or BMMC. Despite these differences, Pam3Cys stimulated equivalent chemokine CCL2 generation in all mast cell types tested. Cyclosporin A (CsA), an inhibitor of Ca2+/calcineurin‐mediated nuclear factor of activated T cells (NFAT) activation, blocked chemokine production in LAD 2 but not in MLMC or BMMC. In contrast, inhibitors of nuclear factor kappa B (NF‐κB) completely blocked CCL2 production in MLMC and BMMC but not in LAD 2 mast cells. Pertussis toxin and U0126, which, respectively, inhibit Gαi,extracellular signal‐regulated kinase (ERK) phosphorylation substantially inhibited Pam3Cys‐induced CCL2 generation in LAD 2 mast cells but had little or no effect on chemokine generation in MLMC and BMMC. These findings suggest that TLR2 activation in human LAD 2 mast cells and MLMC/BMMC promotes the release of different classes of mediators via distinct signalling pathways that depend on Ca2+mobilization and G protein usage.