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Differential effects of the Toll-like receptor 2 agonists, PGN and Pam3CSK4 on anti-IgE induced human mast cell activation.

Yu Y, Yip KH, Tam IY, Sam SW, Ng CW, Zhang W, Lau HY - PLoS ONE (2014)

Bottom Line: Pretreatment of PGN and Pam3CSK4 inhibited anti-IgE induced calcium mobilization and degranulation without down-regulation of FcεRI expression.Pam3CSK4 but not PGN acted in synergy with anti-IgE for IL-8 release when the TLR2 agonist was added simultaneously with anti-IgE.Studies with inhibitors of key enzymes implicated in mast cell signaling revealed that the synergistic release of IL-8 induced by Pam3CSK4 and anti-IgE involved ERK and calcineurin signaling cascades.

View Article: PubMed Central - PubMed

Affiliation: Shenzhen Key Laboratory for Translational Medicine of Dermatology, Biomedical Research Institute, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen, Guangdong Province, China; School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, SAR, China.

ABSTRACT
Mast cells are pivotal in the pathogenesis of allergy and inflammation. In addition to the classical IgE-dependent mechanism involving crosslinking of the high-affinity receptor for IgE (FcεRI), mast cells are also activated by Toll-like receptors (TLRs) which are at the center of innate immunity. In this study, we demonstrated that the response of LAD2 cells (a human mast cell line) to anti-IgE was altered in the presence of the TLR2 agonists peptidoglycan (PGN) and tripalmitoyl-S-glycero-Cys-(Lys)4 (Pam3CSK4). Pretreatment of PGN and Pam3CSK4 inhibited anti-IgE induced calcium mobilization and degranulation without down-regulation of FcεRI expression. Pam3CSK4 but not PGN acted in synergy with anti-IgE for IL-8 release when the TLR2 agonist was added simultaneously with anti-IgE. Studies with inhibitors of key enzymes implicated in mast cell signaling revealed that the synergistic release of IL-8 induced by Pam3CSK4 and anti-IgE involved ERK and calcineurin signaling cascades. The differential modulations of anti-IgE induced mast cell activation by PGN and Pam3CSK4 suggest that dimerization of TLR2 with TLR1 or TLR6 produced different modulating actions on FcεRI mediated human mast cell activation.

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Effects of TLR2 ligands on anti-IgE induced calcium mobilization in LAD2 cells.(A, B) LAD2 cells were incubated with anti-IgE (○), PGN/Pam3CSK4 (◊), anti-IgE with PGN/Pam3CSK4 (▪), and calcium mobilization was measured at the same time. (C, D) Cells were incubated with PGN or Pam3CSK4 for 24 h prior to being challenged with anti-IgE (▪). Changes in [Ca2+]i were compared in the presence or absent of PGN or Pam3CSK4. Error bars were omitted for the clarity of the graph. The peak levels of calcium influx were compared with area under the curves analysis (E, F, G, H). Significant differences following student t-test were indicated by asterisks: *p<0.05, **p<0.01 (n = 4–6).
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pone-0112989-g003: Effects of TLR2 ligands on anti-IgE induced calcium mobilization in LAD2 cells.(A, B) LAD2 cells were incubated with anti-IgE (○), PGN/Pam3CSK4 (◊), anti-IgE with PGN/Pam3CSK4 (▪), and calcium mobilization was measured at the same time. (C, D) Cells were incubated with PGN or Pam3CSK4 for 24 h prior to being challenged with anti-IgE (▪). Changes in [Ca2+]i were compared in the presence or absent of PGN or Pam3CSK4. Error bars were omitted for the clarity of the graph. The peak levels of calcium influx were compared with area under the curves analysis (E, F, G, H). Significant differences following student t-test were indicated by asterisks: *p<0.05, **p<0.01 (n = 4–6).

Mentions: Increase in cytosolic calcium is essential both for degranulation and for release of de novo synthesized mediators in mast cells [15]. Anti-IgE induced an increase of [Ca2+]i with peak elevation at around 4 minutes after activation and gradually returned to normal level over the next 4 min. Pam3CSK4 induced a much lower but sustained level of [Ca2+]i that was significant at around 3 min after activation while PGN did not cause intracellular calcium increase (Fig. 3A, B). Increase in calcium influx by Pam3CSK4 was not due to cytotoxicity as Pam3CSK4 did not influence cell viability of LAD2 cells as determined by WST-1 cell proliferation assay and trypan blue exclusion test following incubation of LAD2 cells with Pam3CSK4 for 30 min and 24 h (data not shown). When added simultaneously with anti-IgE, PGN did not influence anti-IgE induced calcium mobilization (Fig. 3A, E) while Pam3CSK4 altered the pattern of calcium increase induced by anti-IgE to that induced by Pam3CSK4 alone with reduced peak but sustained calcium influx (Fig. 3B). The AUC analysis thus did not show significant difference between the influx of calcium induced by anti-IgE alone and that induced by anti-IgE with Pam3CSK4 (Fig. 3F). Following 24 h pre-incubation, PGN and Pam3CSK4 both significantly inhibited the peak anti-IgE induced calcium increase (Fig. 3C, D) as indicated by the AUC analysis (Fig. 3G, H).


Differential effects of the Toll-like receptor 2 agonists, PGN and Pam3CSK4 on anti-IgE induced human mast cell activation.

Yu Y, Yip KH, Tam IY, Sam SW, Ng CW, Zhang W, Lau HY - PLoS ONE (2014)

Effects of TLR2 ligands on anti-IgE induced calcium mobilization in LAD2 cells.(A, B) LAD2 cells were incubated with anti-IgE (○), PGN/Pam3CSK4 (◊), anti-IgE with PGN/Pam3CSK4 (▪), and calcium mobilization was measured at the same time. (C, D) Cells were incubated with PGN or Pam3CSK4 for 24 h prior to being challenged with anti-IgE (▪). Changes in [Ca2+]i were compared in the presence or absent of PGN or Pam3CSK4. Error bars were omitted for the clarity of the graph. The peak levels of calcium influx were compared with area under the curves analysis (E, F, G, H). Significant differences following student t-test were indicated by asterisks: *p<0.05, **p<0.01 (n = 4–6).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232580&req=5

pone-0112989-g003: Effects of TLR2 ligands on anti-IgE induced calcium mobilization in LAD2 cells.(A, B) LAD2 cells were incubated with anti-IgE (○), PGN/Pam3CSK4 (◊), anti-IgE with PGN/Pam3CSK4 (▪), and calcium mobilization was measured at the same time. (C, D) Cells were incubated with PGN or Pam3CSK4 for 24 h prior to being challenged with anti-IgE (▪). Changes in [Ca2+]i were compared in the presence or absent of PGN or Pam3CSK4. Error bars were omitted for the clarity of the graph. The peak levels of calcium influx were compared with area under the curves analysis (E, F, G, H). Significant differences following student t-test were indicated by asterisks: *p<0.05, **p<0.01 (n = 4–6).
Mentions: Increase in cytosolic calcium is essential both for degranulation and for release of de novo synthesized mediators in mast cells [15]. Anti-IgE induced an increase of [Ca2+]i with peak elevation at around 4 minutes after activation and gradually returned to normal level over the next 4 min. Pam3CSK4 induced a much lower but sustained level of [Ca2+]i that was significant at around 3 min after activation while PGN did not cause intracellular calcium increase (Fig. 3A, B). Increase in calcium influx by Pam3CSK4 was not due to cytotoxicity as Pam3CSK4 did not influence cell viability of LAD2 cells as determined by WST-1 cell proliferation assay and trypan blue exclusion test following incubation of LAD2 cells with Pam3CSK4 for 30 min and 24 h (data not shown). When added simultaneously with anti-IgE, PGN did not influence anti-IgE induced calcium mobilization (Fig. 3A, E) while Pam3CSK4 altered the pattern of calcium increase induced by anti-IgE to that induced by Pam3CSK4 alone with reduced peak but sustained calcium influx (Fig. 3B). The AUC analysis thus did not show significant difference between the influx of calcium induced by anti-IgE alone and that induced by anti-IgE with Pam3CSK4 (Fig. 3F). Following 24 h pre-incubation, PGN and Pam3CSK4 both significantly inhibited the peak anti-IgE induced calcium increase (Fig. 3C, D) as indicated by the AUC analysis (Fig. 3G, H).

Bottom Line: Pretreatment of PGN and Pam3CSK4 inhibited anti-IgE induced calcium mobilization and degranulation without down-regulation of FcεRI expression.Pam3CSK4 but not PGN acted in synergy with anti-IgE for IL-8 release when the TLR2 agonist was added simultaneously with anti-IgE.Studies with inhibitors of key enzymes implicated in mast cell signaling revealed that the synergistic release of IL-8 induced by Pam3CSK4 and anti-IgE involved ERK and calcineurin signaling cascades.

View Article: PubMed Central - PubMed

Affiliation: Shenzhen Key Laboratory for Translational Medicine of Dermatology, Biomedical Research Institute, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen, Guangdong Province, China; School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, SAR, China.

ABSTRACT
Mast cells are pivotal in the pathogenesis of allergy and inflammation. In addition to the classical IgE-dependent mechanism involving crosslinking of the high-affinity receptor for IgE (FcεRI), mast cells are also activated by Toll-like receptors (TLRs) which are at the center of innate immunity. In this study, we demonstrated that the response of LAD2 cells (a human mast cell line) to anti-IgE was altered in the presence of the TLR2 agonists peptidoglycan (PGN) and tripalmitoyl-S-glycero-Cys-(Lys)4 (Pam3CSK4). Pretreatment of PGN and Pam3CSK4 inhibited anti-IgE induced calcium mobilization and degranulation without down-regulation of FcεRI expression. Pam3CSK4 but not PGN acted in synergy with anti-IgE for IL-8 release when the TLR2 agonist was added simultaneously with anti-IgE. Studies with inhibitors of key enzymes implicated in mast cell signaling revealed that the synergistic release of IL-8 induced by Pam3CSK4 and anti-IgE involved ERK and calcineurin signaling cascades. The differential modulations of anti-IgE induced mast cell activation by PGN and Pam3CSK4 suggest that dimerization of TLR2 with TLR1 or TLR6 produced different modulating actions on FcεRI mediated human mast cell activation.

Show MeSH
Related in: MedlinePlus