The effect of adrenergic agonists on BMDC potential to skew Tcells.

<p>Panel A. FACS plots of intracellular IFNγFand IL-4 in -OT-II T cells co-cultured with BMDC pre-treated with vehicle (Veh), epinephrine (Epine) or salbutamol (Sal) (all at 1 µM). Intracellular IFNγ FNr salarerer affected, while IL-4 production is increased after AR-βaffected, while IL-4 productB, histograms of % IFNγ positive T cells by FACS and by ELISA of day 4 of co-culture supernatant. Panel C, histograms of % IL-4 positive T cells by FACS and by ELISA. Data are expressed as % positive of CD4 gated T cells or as pg/ml and represent the mean ± SEM of three independent experiments representative of 4 experiments. ***p<0.001 (ANOVA).</p

Incubation of vehicle (veh), epinephrine (epine), and salbutamol (sal) of immature Ovalbumin loaded BMDC in a T cell proliferation assay.

<p>Cells were incubated during LPS maturation o/n. The cells were washed and freshly isolated naïve CD4 OT-II cells were stained with CFSE. Cells were co-cultured for 3 days and CFSE dilution was determined by flow cytometry. Overlays shown are representative of 3 independent experiments.</p

Adrenergic agonists exposure stimulates skewing of a Treg population.

<p>Panel A shows flow cytometry analyses of intracellular staining of Foxp3 positive T cells skewed by BMDC treated with indicated AR agonist. Panel B, histograms of Foxp3 positive T cells (left) and IL-10 (right) concentrations in supernatant representative of three independent experiments. Panel C, FACS analyses and, panel D histograms of T cells skewed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085086#pone-0085086-g007" target="_blank">fig. 7A</a> with added TGF-β, to stimulate Foxp3 differentiation. Panel E shows the concentrations of TGF-β analyses of intracellular staining of Foxp3 posVeh), epinephrine (Epine) and salbutamol (Sal). Data are expressed in % positive of CD4 gated T cells (left) or as pg/ml (right) and represent the mean ± SEM of three independent experiments representative of 4 experiments. *p<0.05 **p<0.01 (ANOVA).</p

Effect of adrenergic and cholinergic activation on cytokine production in maturing BMDC after 24

<p>Panel A, IL-10, IL-12p70 and IL-23 concentrations in ACh and Nicotine (Nic; 1 µM) pre-treated BMDC. Panel B, IL-10, IL-12p70 and IL-23 concentrations in epinephrine (epine), salbutamol (sal) and/or propranolol (prop) (all at 1 µM) pre-treated BMDC. The values are expressed in pg/ml and represent the mean ± SEM of three independent experiments representative of 5 experiments. * p<0.05, ** p<0.01, ***p<0.001 (ANOVA).</p

The relative mRNA expression levels of the AR-βe r<i>adrb1</i>) and AR-β an<i>adrb2</i>) in immature BMDC, and matured BMDC matured in the presence of epinephrine or salbutamol (Panel A).

<p>AR-β3 receptors are not expressed in BMDC. Panel B, the expression of the enzyme for catecholamine production, Tyrosine hydroxylase (TH) expression is not affected by epinephrine in matured BMDC. Panel C, blocking of endogenous TH activity by AMPT does not affect the potential of epinephrine or salbutamol to modulate IL-10 and IL-12p70 production. The data represent the mean ± SEM of three independent experiments representative of 5 experiments. * p<0.05, ** p<0.01, ***p<0.001 (ANOVA).</p

The effects of ACh, epinephrine, salbutamol and nicotine on BMDC endocytosis of FITC-Dextran.

<p>Panel A shows a time course of endocytosis in treated BMDCs. CD11c gated immature BMDC endocytosis was assessed using flow cytometry. Results are expressed as mean fluorescence intensity and are the mean ± SEM of three experiments. Statistical significance was calculated using one way ANOVA. * p<0.05, ***p<0.001 versus control (37°C). Panel B: overlay histograms of BMDC stained for MHCII and co-stimulatory molecules CD40, CD80 and CD86 after 24 hours of LPS stimulation were determined by flow cytometry. Cell populations in the grey area indicate the specific isotype control. Both MHCII and co-stimulatory molecules are up-regulated by immature BMDC compared to LPS stimulated matured BMDC. Incubation with the various neurotransmitters before or during maturation has no effect on the levels of maturation markers. Numbers on each histogram indicate the geometric mean fluorescence intensity of cell population for each molecule from representative data of three experimental groups.</p

Modulation of Compound 48/80-induced RBL-2H3 degranulation.

<p>Compound 48/80 treatment of RBL- 2H3 cells induced a concentration dependent release of β-hexosaminidase. 30 minutes pre-incubation with 10 and 100 µM fexofenadine reduced β-hexosaminidase release in control cells and cells treated with 500 and 1000 µg Compound 48/80/ml (figure A). Carebastine pretreatment did not show an effect on Compound 48/80 stimulated RBL-2H3 cells (figure B). Results are a representative example of 3 independent experiments and expressed as mean ± SEM (4 wells per condition, *<i>P</i><0.05).</p

In vivo post stress fexofenadine treatment.

<p>The visceromotor response to distension was measured pre-WA and 24 and 48 hours post-WA in NH and MS rats. Fexofenadine or vehicle was administered 3 times between 24- and 48 hours measurements (cumulative dosages 18 and 1.8 mg/kg). Responses to distension are depicted as AUC (left side histograms) and per volume (right side line-diagrams, corresponding statistics in lower right side tables). NH rats did not become hypersensitive to distension and fexofenadine treatment did not change sensitivity levels (figures A and B). In MS rats WA induced enhanced sensitivity to distension in all 3 treatment groups (figures C, D and E). Treatment with 18 and 1.8 mg fexofenadine/kg (figure D and E respectively) but not vehicle alone (C) was able to reverse stress induced visceral hypersensitivity. All data are presented as mean ± SEM, all groups n = 8 or 9 rats, *<i>P</i><0.05 and **<i>P</i><0.01.</p

Relative colonic expression values for the <i>histamine H1 receptor</i> gene.

<p>H1R mRNA expression was evaluated relative to the housekeeping gene <i>Ppib</i> in colonic samples of NH and MS rats. Tissue was collected 7 days post vehicle treatment and distensions. There were no significant differences.</p

Experimental overview.

<p>Experimental overview.</p