Differential Effects of the Toll-like Receptor 2 Agonists, PGN and PAM3CSK4, on Substance P-Induced Human Mast Cell Activation

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.

Differential Effects of the Toll-Like Receptor 2 Agonists, PGN and Pam3CSK4 on Anti-IgE Induced Human Mast Cell Activation

“…Furthermore, 24 h pre-incubation of PGN and Pam3CSK4 inhibited anti-IgE induced calcium mobilization in LAD2 cells, which was a necessary upstream signaling mechanism required for antigen induced mast cells degranulation [15] . In contrast to our previous study which observed that only Pam3CSK4 but not PGN could suppress calcium increase induced by the neuropeptide, substance P [24] , the inhibitory action of PGN against anti-IgE induced calcium increase suggested that Pam3CSK4 and PGN modulate different sources of calcium. Since substance P induces mainly release of calcium from intracellular stores while FcεRI activation induces calcium influx through opening of cell membrane calcium channels, the activation of TLR2/6 heterodimer by PGN may thus suppress the functions of calcium channels.…”

“…Previously, we have demonstrated that PGN and Pam3CSK4 induced the release of IL-8 by employing different signaling pathways in LAD2 cells. Furthermore, activation of different signaling pathways induced by PGN and Pam3CSK4 also resulted in opposite modulatory effect on LAD2 cells activation in response to substance P [24] . In the current study, Pam3CSK4 but not PGN acted in synergy with anti-IgE for IL-8 release from LAD2 cells which is the reverse of the modulatory actions of the two TLR2 agonists on substance P activated LAD2 cells.…”

Go is required for the release of IL-8 and TNF-α, but not degranulation in human mast cells

“…Together with our previous study (Yu et al, 2013), these studies demonstrate the differential function of G o and G i protein in human mast cells.…”

“…Our previous study has demonstrated that substance P-induced degranulation and release of IL-8 from the human mast cell line LAD2 cells were both blocked by PTX, whereas BAC differentially inhibited degranulation but potentiated IL-8 release (Yu et al, 2013).These results strongly indicate that G i and G o could differentially regulate the activities of human mast cells.…”

“…In the previous study, we also demonstrated that synthesis of IL-8 induced by substance P was blocked by ciclosporin, a specific inhibitor of calcineurin, indicating the involvement of NFAT pathway in substance P-induced IL-8 release (Yu et al 2013). However, translocation of NFAT was not inhibited by knockdown of Gα o protein in LAD2 cells (Fig.…”