Figure 1.
IgE alone induces mast cell degranulation.
Mast cells were incubated with different concentrations of IgE (white bars) for 1 h. Anti-IgE-induced histamine release (black bar) is shown as a positive control (A). Mast cells were stimulated with IgE at 5 µg/mL for indicated times of incubation (B). Results are presented as the mean ± SEM of at least four independent experiments and each experiment was carried out in duplicate (n≥4). *p<0.05, **p<0.01.
Figure 2.
IgE alone stimulates mast cells to cysLT synthesis.
Mast cells were incubated with different concentrations of IgE (white bars) for 1 h. Anti-IgE-induced cysLT secretion (black bar) is presented as a positive control. Results are shown as the mean ± SEM of four independent experiments and each experiment was carried out in duplicate (n = 4). ***p<0.001.
Figure 3.
IgE alone induces de novo TNF synthesis in mast cells.
Mast cells were incubated with different concentrations of IgE (white bars) for 6 h. Anti-IgE-induced TNF secretion (black bar) is shown as a positive control (A). For TNF mRNA assessment mast cells were incubated with IgE (white bars) or anti-IgE (black bars) both at 5 µg/mL for the indicated times (B). TNF mRNA levels were determined by qRT-PCR and presented as fold-increase over the value of cytokine mRNA expression in non-stimulated cells after normalization with the transcript level of the housekeeping gene rat Actb. Results are presented as the mean ± SEM of at least three separate experiments performed in duplicate (A) or triplicate (B) (n≥3). *p<0.05, **p<0.01, ***p<0.001.
Figure 4.
IgE alone affects mast cell migration.
Mast cells were pretreated with IgE at 1 µg/mL or 5 µg/mL (IgE-coated mast cells) or medium (native mast cells) for 1 h before being placed into the upper wells, whereas medium (spontaneous migration), CCL5 or TNF had been added to lower wells of Boyden microchamber. Results are presented as the mean ± SEM of at least four independent experiments and each experiment was carried out in duplicate (n≥4). ***p<0.001.
Table 1.
Inhibition of IgE-induced mast cell histamine release, cysLT generation and TNF synthesis by various inhibitors.
Figure 5.
Cell signaling inhibitors influence IgE-affected both spontaneous and induced mast cell migration.
Mast cells were preincubated with U73122 (0.1 µM), Src I-1 (0.01 µM), LY294002 (50 µM) or medium alone for 1 h (none). Then, mast cells were treated with IgE at 5 µg/mL, washed and spontaneous (A), CCL5-induced (B) and TNF-induced (C) migration was examined in a Boyden microchamber. Bars for the positive controls demonstrate the same data set as in figure 4. Results are presented as the mean ± SEM of four independent experiments and each experiment was carried out in duplicate (n = 4). *p<0.05, **p<0.01, ***p<0.001.
Figure 6.
IgE alone stimulates phosphorylation of ERK, p38 and PI3K in mast cells.
Mast cells were treated with IgE alone at 5 µg/mL for the indicated times. Total cell lysates (50 µg) were subjected to Western blotting analysis using anti-phospho antibodies specific to ERK1/2 (pERK1/2), p38 (pp38) or PI3K (pPI3K). The same blots were reprobed with respective antibodies that recognize these signaling molecules, irrespective of the phosphorylation states. Results are representative of 3 independent experiments.
Figure 7.
IgE alone up-regulates FcεRI expression on rat mast cells.
Mast cells were incubated with IgE at 1 µg/mL (white bars) and 5 µg/mL (black bars) or without IgE (control FcεRI expression on native mast cells referred to as 100%) for 1, 6 and 24 h. Surface FcεRI expression was analyzed by flow cytometry using mouse anti-rat FcεRI primary antibodies (10 µg/mL) and FITC-labeled goat anti-mouse secondary antibodies (5 µg/mL) (A). Representative flow cytometry histogram showing FcεRI expression on native mast cells as well as on mast cells treated with 5 µg/mL for 1 h (B). Results are presented as the mean ± SEM of four independent experiments (n = 4). *p<0.05.