Surface TLR2 and TLR4 Expression on Mature Rat Mast Cells Can Be Affected by Some Bacterial Components and Proinflammatory Cytokines

The aim of our study was to determine whether some bacterial components as well as some proinflammatory cytokines can affect surface mast cell levels. By the use of flow cytometry technique, we documented that freshly isolated mature rat peritoneal mast cells do express surface TLR2 and TLR4 protein, but not CD14 molecules, and respond to stimulation with TLR2 and TLR4 ligands by cysteinyl leukotriene generation. The level of TLR2 protein is modulated by PGN and CCL5 treatment, but not by LPS, LAM, TNF, or IL-6. Surface mast cell TLR4 expression is affected by LPS, LAM, IL-6, and CCL5. Considering that TLR-mediated activation conditions not only engaged these cells in antibacterial defense and development of inflammation but also might influence allergic processes, our observations that surface TLR2 and TLR4 expression can be regulated both bacterial components and proinflammatory cytokines seem to be very intriguing and importance

IgE by itself affects mature rat mast cell preformed and de novo-synthesized mediator release and amplifies mast cell migratory response.

BACKGROUND: Immunoglobulin E (IgE) binds to high affinity receptor FcεRI numerously expressed on mast cells. Recent findings have revealed that IgE by itself may regulate various aspects of mast cell biology, however, detailed data is still limited. METHODOLOGY/FINDINGS: Here, we have examined the influence of IgE alone, used at different concentrations, on mast cell activity and releasability. For the study we have employed in vivo differentiated mature tissue mast cells isolated from rat peritoneal cavity. Mast cells were exposed to IgE alone and then the release of preformed and de novo-synthesized mediators, surface FcεRI expression and mast cell migratory response were assessed. IgE by itself was found to up-regulate FcεRI expression and activate mast cells to degranulation, as well as de novo synthesis and release of cysteinyl leukotrienes and TNF. We have provided evidence that IgE alone also amplified spontaneous and CCL5- or TNF-induced migration of mast cells. Importantly, IgE was effective only at concentrations ≥ 3 µg/mL. A molecular basis investigation using an array of specific inhibitors showed that Src kinases, PLC/PLA2, MAP kinases (ERK and p38) and PI3K were entirely or partially involved in IgE-induced mast cell response. Furthermore, IgE alone stimulated the phosphorylation of MAP kinases and PI3K in rat mast cells. CONCLUSION: Our results clearly demonstrated that IgE by itself, at higher concentrations, influences mast cell activity and releasability. As there are different conditions when the IgE level is raised it might be supposed that in vivo IgE is one of the important factors modulating mast cell biology within tissues

Inhibition of IgE-induced mast cell histamine release, cysLT generation and TNF synthesis by various inhibitors.

<p>Inhibition of IgE-induced mast cell histamine release, cysLT generation and TNF synthesis by various inhibitors.</p

IgE alone stimulates mast cells to cysLT synthesis.

<p>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.</p

Distinct inhibitory efficiency of siRNAs and DNAzymes to β1 integrin subunit in blocking tumor growth

Receptors of the β1 integrin family are involved in many tumor-promoting activities. There are several approaches currently used to control integrin activity, and thus to potentially restrain tumor metastasis and angiogenesis. In this study, we compared inhibitory efficiencies of siRNA and DNAzymes against the β1 integrin subunit (DEβ1), in a mouse xenograft model. Both inhibitors were used under their most favorable conditions, in terms of concentrations, incubation time and lack of cytotoxic effects. Transfection of siRNAβ1 or DEβ1 remarkably inhibited the growth of both PC3 and HT29 colon cancer cells in vitro, and decreased their capability of initiating tumor formation in the mouse xenograft model. siRNAβ1 appeared to be slightly more efficient than DEβ1 when tested in vitro, however it was comparably less proficient in blocking the tumor growth in vivo. We conclude the DNAzyme, due to its greater resistance to degradation in extra- and intracellular compartments, to be a superior inhibitor of tumor growth in long lasting experiments in vivo when compared to siRNA, while the latter seems to be more efficient in blocking β1 expression during in vitro experiments using cell cultures

IgE alone affects mast cell migration.

<p>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.</p

Cell signaling inhibitors influence IgE-affected both spontaneous and induced mast cell migration.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079286#pone-0079286-g004" target="_blank">figure 4</a>. 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.</p

IgE alone induces mast cell degranulation.

<p>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.</p

IgE alone induces <i>de novo</i> TNF synthesis in mast cells.

<p>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.</p

IgE alone stimulates phosphorylation of ERK, p38 and PI3K in mast cells.

<p>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.</p