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

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 Ca ²⁺/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 Gα_i, extracellular signal-regulated kinase (ERK) phosphorylation substantially inhibited Pam ₃Cys-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.