Figure 1.
Relative expression of TLRs (1–9) in CTLMC and MLMC.
RNA purified from CTLMC and MLMC was subjected to quantitative real-time PCR using SYBR Green for the indicated TLRs. The relative gene expression levels were determined in triplicates by difference in Ct numbers and results were described as in relation to Gmol β actin. Data are plotted as mean ± SEM and derived from 4–6 independent experiments.
Figure 2.
Pre-treatment with TLR agonists increase FcεRI-mediated degranulation in CTLMC and MLMC.
Cells were treated with different TLRs agonists for 24 h (A and C) or 96 h (B and D) and were sensitized with IgE followed by antigen. Degranulation was determined by measuring the release of β-hexosaminidase in duplicates. The release was calculated as a percentage of total β-hexosaminidase content released following activation. Results are represented as the mean ±SEM for 5 independent experiments. *p<0.05, **p<0.01 significantly different from the respective controls.
Figure 3.
Pre-treatment with TLR agonists increase leukotriene products in CTLMC and MLMC.
Cells were treated with different TLRs agonists for 24 h (A, C, E, G) and 96 h (B, D, F, H), CTMLC (A, B, E, F) and MLMC (C, D, G, H) were sensitized with IgE by antigen. CysLT and LTB4 were measured in the culture supernatant by ELISA in duplicates. Results are represented as the mean ±SEM for 5 independent experiments. *p<0.05, **p<0.01 significantly different from the control cells without IgE receptor cross-linking.
Figure 4.
IgE-receptor induced pro-inflammatory cytokine and chemokine production by mast cells are enhanced upon pre-treatment with TLRs agonists.
CTLMC (A,C,E) and MLMC (B,D,F) were treated for 96 h with different TLR agonists and then sensitized with IgE followed by antigen for 6 h. Culture supernatants were analysed for the presence of IL-6 (A,B), MIP-1α (C,D) and MCP-1 (E,F) by Luminex in duplicates. Data are mean ± SEM and representative of 5 independent experiments. *p<0.05, **p<0.01 significantly different from the respective controls.
Figure 5.
Pre-treatment with LPS did not increase FcεRI-mediated degranulation and leukotrienes release in MyD88−/− MLMC. MLMCs were treated with different LPS for 96 h and were sensitized with IgE followed by antigen.
Degranulation was determined by measuring the release of β-hexosaminidase in duplicates. The release was calculated as a percentage of total β-hexosaminidase content released following activation (A). CysLT and LTB4 also exhibited the similar pattern as measured in the culture supernatant by ELISA (B and C). Results are represented as the mean ±SEM for 5 independent experiments. *p<0.05, **p<0.01 significantly different from the respective controls.
Figure 6.
MAPK activation induced by LPS or IgE cross-linking.
(A) MLMC were treated with LPS (1 µg/ml) for 96 h, IgE cross-linking (IgE-XL) or combination with LPS and IgE-XL (10 min). Phosphorylation of ERK, p38 MAPK and JNK was detected by phosphospecific antibodies against the respective kinases by Western blot with reference to the total kinases present in whole cell lysate. Lane 1: Control resting cells; Lane 2: control cells with IgE-XL; Lane 3: LPS treated cells; Lane 4: LPS treated cells exposed to IgE-XL (B) To explore the role of MAPKs involved in LPS triggered induction of degranulation from activated mast cells following IgE-XL, MLMC were preincubated with JNK inhibitor SP 600125 (1 µM), ERK inhibitor PD 98059 (1 µM) and p38 MAPK inhibitor SB 203580 (10 µM) in presence and absence of LPS (1 µg/ml) for 96 h. The cells were then sensitized challenged with IgE for 90 min and further treated with antigen for 30 min. Supernatants were collected in duplicates to evaluate β-hexosaminidase activity. Release was calculated as a percentage of total β-hexosaminidase. Results are presented as the mean ± SEM for 5 independent experiments. **p<0.01.
Table 1.
Sequences of TLR and β-actin primer pairs.