No abstract

Mast cells play important roles in innate immunity through their activation via toll-like receptors (TLRs) but also contribute to neuroimmunological responses and inflammation through their activation by the neuropeptide substance P (SP) via Ga ilo proteins. This study aims to compare the effects of the TLR2 agonists peptidoglycan (PGN) and tripalmitoyl-S-glycero-Cys-(Lys)4 (Pam3CSK4) on SP-induced human mast cell activation. The human mast cell line LAD2 was employed and mast cell activation was determined by assays of p-hexosaminidase, IL-8 and intracellular calcium. TLR2 agonists did not cause degranulation, but induced the release oflL-8. Pretreatment ofPGN and Pam3CSK4 inhibited SP induced degranulation but only Pam3CSK4 blocked SP induced calcium mobilization. SP-induced IL-8 release was synergistically enhanced by PGN but abolished by Pam3CSK4. Studies with inhibitors of key enzymes implicated in mast cell signaling revealed that synergistic release oflL-8 induced by PGN and SP involved calcineurin, ERK, NF-KB and PI3K signaling cascades whereas Pam3CSK4 inhibited SP induced mast cell activation by interfering with the interaction between SP and Ga ilo proteins. These findings suggest that activation of human mast cells can be differentially modified by TLR2 agonists via distinct signaling pathways through facilitating formation of different TLR2 heterodimers with other TLRs.

Abstract. We performed this study to demonstrate the applicability of the microelectrode array (MEA) to study electrophysiological changes of rat peritoneal mast cells in the presence of compound 48 / 80 under normal, Ca 2+-free, Ca 2+-free with EDTA, and Cl − -free conditions. The use of high extracellular K + (KCl, 150 mM), charybdotoxin (ChTX, 100 nM), and Cl − -free containing ChTX buffers verified that the hyperpolarizing signal was due to the activation of mainly K + and, to a lesser extent, Cl − channels. Compound 48 / 80 concentration-dependently shortened the latent periods (the onset of response) and increased both the spatial (the K + and Cl − hyperpolarizing field potentials, HFP) and temporal measurements (the duration of response). Ca 2+ -free buffer had no effect on the latent period of compound 48 / 80 but increased the HFP at high concentrations. The latent period increased while the HFP diminished when cells were equilibrated in Ca 2+-free buffer containing EDTA. Durations of the HFP were generally longer when cells were in either Ca 2+-free or Ca 2+-free containing EDTA buffers than when cells were in normal buffer. The EC 50 values confirmed that effects were only affected in Ca 2+-free buffer containing EDTA but not in Ca 2+-free or Cl − -free buffers, further reinforcing the hypothesis that the presence of Ca 2+ is not essential to the action of compound 48 / 80. The present study is the first application of MEA to study rat peritoneal mast cells, and our results indicate that it could be of value in future pharmacological research on other non-excitable cells.