Effects of PGN and Pam3CSK4 on the expression of FcεRI on LAD2 cells.

<p>LAD2 cells (without IgE sensitization) were incubated with PGN (50 µg/ml) (A) and Pam3CSK4 (20 µg/ml) (B) for 24 h and FcεRI surface expression was analyzed by flow cytometry after cells were incubated with FITC-conjugated anti-human FcεRI antibody, FITC-conjugated mouse IgG2b isotype control or FACS buffer for specific labelling of FcεRI, isotype and blank control respectively. The FcεRI expression of cells that were not treated (grey curve) or treated (blank curve) with the TLR2 ligands was not different as shown. Results were representative of four independent experiments.</p

Effects of inhibitors of MAPKs on TLR2 ligands and anti-IgE induced IL-8 release from LAD2 cells.

<p>(A) ERK inhibitor, PD98059 (10 µM), (B) JNK inhibitor, SP600125 (20 µM) or (C) p38 inhibitor, SB2023580 (10 µM) was incubated with LAD2 cells for 30 min before the addition of anti-IgE (2 µg/ml), PGN (50 µg/ml), Pam3CSK4 (20 µg/ml), anti-IgE with PGN or Pam3CSK4 for 24 h to induce the release of IL-8. The amounts of IL-8 release from activated LAD2 cells pre-incubated with an inhibitor and the corresponding control pre-incubated in culture medium were compared with student <i>t</i>-test. *p<0.05, **p<0.01, ***p<0.01 (n = 4–6).</p

Effects of TLR2 ligands on anti-IgE induced calcium mobilization in LAD2 cells.

<p>(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 <i>t</i>-test were indicated by asterisks: *p<0.05, **p<0.01 (n = 4–6).</p

Effects of inhibitors of calcineurin, NF-κB and PI3K on TLR2 ligands and anti-IgE induced IL-8 release from LAD2 cells.

<p>(A) ciclosporin (2 µg/ml), (B) Bay 11-7821 (10 µM) or (C) wortmannin (0.5 µM) was incubated with LAD2 cells for 30 min before the addition of anti-IgE (2 µg/ml), PGN (50 µg/ml), Pam3CSK4 (20 µg/ml), anti-IgE with PGN or Pam3CSK4 for 24 h to induce the release of IL-8. The amounts of IL-8 release from activated LAD2 cells pre-incubated with an inhibitor and the corresponding control pre-incubated in culture medium were compared with student <i>t</i>-test. *p<0.05, **p<0.01, ***p<0.001 (n = 4–5).</p

Effect of TLR2 ligands on anti-IgE induced degranulation and IL-8 release from LAD2 cells.

<p>(A, B) LAD2 cells were incubated with only PGN or Pam3CSK4 for 30 min (○). LAD2 cells were incubated with anti-IgE (2 µg/ml) at the same time (▴) or after 24 h pre-incubation (•) with PGN or Pam3CSK4. The levels of β-hex release induced by anti-IgE alone and in the present of TLR2 ligands were compared with one-way ANOVA and Dunnett’s multiple comparison tests. *p<0.05, **p<0.01, ***p<0.001 (n = 3–5). (C, D) LAD2 cells were incubated alone with anti-IgE (2 µg/ml, •), PGN/Pam3CSK4 (▪) or combination of anti-IgE with PGN/Pam3CSK4 (▴) for 24 h. Two-way ANOVA and Bonferroni posttests were applied to compare the actual amount of IL-8 released with the predicted value obtained by adding the individual amounts released by anti-IgE and PGN or Pam3CSK4 (○). **p<0.01 (n = 5).</p