Comparison of the effects of mepyramine, JNJ 7777120, and mepyramine+JNJ 7777120 on parameters of murine OVA-induced experimental asthma.

<p>The effects of mepyramine, JNJ 7777120, or mepyramine plus JNJ 7777120 applied during either the provocation phase or the sensitization phases on selective parameters observed in the experimental asthma model in this study were compared to the treatment with the solvent DMSO in the respective phase, and evaluated as not altered [<b>→</b>], slightly reduced [(<b>↓</b>)], reduced [<b>↓</b>], and strongly reduced [<b>↓↓</b>]. (mepy.: mepyramine, JNJ7777: JNJ 7777120).</p

Effect of mepyramine and JNJ 7777120 on BAL-fluid cellularity.

<p>Mice were sensitized to, and provoked with, OVA and treated with DMSO, mepyramine, JNJ 7777120, or mepyramine plus JNJ 7777120 either 30 min before each provocation cycle (<i>A</i>) or 30 min before and 2 h after each injection for sensitization (<i>B</i>). As control, mice were sensitized with PBS (sham), provoked with OVA, and treated with DMSO. Cells in BAL-fluids, collected 24 h after the final challenge, were differentially counted. Data shown are means ± SD (n_(sham) = 3, n_(DMSO) = 7, n_{(mepyramine, JNJ 7777120, or mepyramine+JNJ 7777120)} = 8; *, p≤0.05; **, p≤0.01; ***, p≤0.005).</p

Effect of mepyramine and JNJ 7777120 on Th2 cytokine and anti-OVA IgE concentrations in sera.

<p>Mice were sensitized to, and provoked with, OVA and treated with DMSO, mepyramine, JNJ 7777120, or mepyramine plus JNJ 7777120 either 30 min before each provocation cycle (<i>A, B</i>) or 30 min before and 2 h after each injection for sensitization (<i>C, D</i>). In sera, collected 24 h after the final provocation, IL-13 concentrations were measured by FlowCytomix (<i>A, C</i>) and anti-OVA IgE titers were determined by ELISA (<i>B, D</i>). Data shown are means ± SD (n_(sham) = 3, n_(DMSO) = 7, n_{(mepyramine, JNJ 7777120, or mepyramine+JNJ 7777120)} = 8; *, p≤0.05; **, p≤0.01; ***, p≤0.005).</p

Effect of mepyramine and JNJ 7777120 on the T cell cytokine production of lymph node cells after in vitro re-stimulation.

<p>Mice were sensitized to OVA and treated with DMSO, mepyramine, JNJ 7777120, or mepyramine plus JNJ 7777120 30 min before and 2 h after each injection for sensitization. 48 h after the last injection, single cells suspension out of mesenteric lymph nodes were re-stimulated <i>in vitro</i> with OVA for 48 hours. Thereafter, accumulated cytokines in the supernatants were quantified by a multiplex assay. Data shown are means ± SD (n_{(each group)} = 3*, p≤0.05; **, p≤0.01; ***, p≤0.005).</p

Histamine regulates EGR-1 mRNA expression.

<p>HEK293 mH₁R cells (A) and HEK293 mH₄R cells (B) were incubated without any inhibitor (Ø) or with 50 µM PD 980598 (PD) for 1 h, with 2 µM SB 203580 (SB) for 30 min, or 25 µM KG-501 (KG) for 20 min and then incubated with or without histamine for 4 h. Total RNA was extracted from the cells and reverse transcribed into cDNA. Expression of EGR-1 was determined by real time PCR using TagMan probes. Data shown are means ± SD of the relative EGR-1 expression in relation to unstimulated cells (basal) (n = 3, each consisting of 2 replicates; **: p≤0.01; ***: p≤0.005; ns: not significant).</p

Pertussis toxin selectively blocks signaling of the mH₄R.

<p>HEK293 mH₁R cells (A+C) and HEK293 mH₄R cells (B+D) were incubated in the presence or absence of 50 ng/ml pertussistoxin (PTX) for 18 h. Changes in [Ca²⁺]_i were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107481#pone-0107481-g002" target="_blank">Figure 2</a> after stimulation with 100 µM histamine (HA) or 10 µM ATP as indicated on the x-axis (A+B). Intracellular cAMP concentrations were analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107481#pone-0107481-g003" target="_blank">Figure 3</a> after stimulation for 10 min with 100 µM forskolin (Forsk) and histamine (HA) (C+D). Forskolin-induced cAMP concentrations were 2385±160 pmol/mg protein in HEK293 H₁R and 2164±436 pmol/mg protein in HEK 293 H₄R cells, respectively. Data shown are means ± SD (n = 2, each consisting of 2–3 replicates; *: p≤0.05; **: p≤0.01; ***: p≤0.005; ns: not significant).</p

Mobilization of [Ca²⁺]_i by histamine is mediated by H₁R and H₄R.

<p>HEK293 mH₁R cells (A+D), HEK293 mH₄R cells (B+E), and empty vector-transfected HEK293 cells (C) were labelled with Fura 2-AM and stimulated with different concentrations of histamine (HA) (A–C) or with a constant HA concentration in combination with DMSO or varying concentrations of mepyramine (MEP) or JNJ7777120 (JNJ) (D+E). Changes in [Ca²⁺]_i were determined by fluorescence measurements at an emission wavelength of 508 nm and excitation wavelengths of 340 nm and 380 nm. Data shown are means ± SD (n = 2–3, each consisting of 2 replicates).</p

Histamine induces phosphorylation of MAP-kinases <i>via</i> H₁R and H₄R.

<p>HEK293 empty vector cells, HEK293 mH₁R cells, and HEK293 mH₄R cells were incubated with or without 10 µM HA for 5 min and lysed afterwards. Phosphorylation of different serine/threonine kinases in these lysates were detected by MAPK array. Phosphorylation intensity was quantified by analysis of the pixel density of every single spot (A). Shown are the differences of the densities of corresponding spots obtained using lysates of cells with and without HA stimulation. Data shown are means ± SD (n = 2, each measured in 2 replicates; *: p≤0.05; **: p≤0.01; ***: p≤0.005). In B, exemplarily the spots of ERK1 and ERK2 after histamine induction in the cells as indicated on the left, and quantification control spots (ctr.) are demonstrated.</p

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Objective<p>Rheumatoid arthritis (RA) is a chronic and progressive joint disease. It appears that anti-inflammatory feedback mechanisms that could restrain joint inflammation and restore homeostasis are insufficient to perform this control. In this study, we investigated the contribution of the MER tyrosine kinase-mediated anti-inflammatory response on arthritis and whether targeting MER could be a valid approach to treat RA.</p>Methods<p>KRN serum transfer arthritis (KRN STA) was induced in either Mertk-deficient mice or in mice that adenovirally overexpressed Pros1. Human synovial micromasses were treated with MER-specific antibodies or PROS1. Collagen-induced arthritis (CIA) mice were treated with MER-specific agonistic antibodies or by viral overexpression of Pros1.</p>Results<p>Mertk^−/− mice showed exacerbated arthritis pathology, whereas Pros1 overexpression diminished joint pathology in KRN STA. Human synovial micromasses challenged with MER-specific antibodies enhanced the secretion of inflammatory cytokines, whereas stimulating MER with PROS1 reduced the secretion of these cytokines, confirming the protective role of MER. Next, we treated CIA mice with MER-specific agonistic antibodies, and this unexpectedly resulted in exacerbated arthritis pathology. This was associated with increased numbers of apoptotic cells in their knee joints and higher serum levels of interleukin (IL)-16C, a cytokine released by secondary necrotic neutrophils. Apoptotic cell numbers and IL-16C levels were enhanced during arthritis in Mertk^−/− mice and reduced in Pros1-overexpressing mice.</p>Conclusion<p>MER plays a protective role during joint inflammation and activating MER by its ligand PROS1 ameliorates disease. Treatment of mice with MER receptor agonistic antibodies is deleterious due to its counterproductive effect of blocking efferocytosis in the arthritic joint.</p

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Objective<p>Rheumatoid arthritis (RA) is a chronic and progressive joint disease. It appears that anti-inflammatory feedback mechanisms that could restrain joint inflammation and restore homeostasis are insufficient to perform this control. In this study, we investigated the contribution of the MER tyrosine kinase-mediated anti-inflammatory response on arthritis and whether targeting MER could be a valid approach to treat RA.</p>Methods<p>KRN serum transfer arthritis (KRN STA) was induced in either Mertk-deficient mice or in mice that adenovirally overexpressed Pros1. Human synovial micromasses were treated with MER-specific antibodies or PROS1. Collagen-induced arthritis (CIA) mice were treated with MER-specific agonistic antibodies or by viral overexpression of Pros1.</p>Results<p>Mertk^−/− mice showed exacerbated arthritis pathology, whereas Pros1 overexpression diminished joint pathology in KRN STA. Human synovial micromasses challenged with MER-specific antibodies enhanced the secretion of inflammatory cytokines, whereas stimulating MER with PROS1 reduced the secretion of these cytokines, confirming the protective role of MER. Next, we treated CIA mice with MER-specific agonistic antibodies, and this unexpectedly resulted in exacerbated arthritis pathology. This was associated with increased numbers of apoptotic cells in their knee joints and higher serum levels of interleukin (IL)-16C, a cytokine released by secondary necrotic neutrophils. Apoptotic cell numbers and IL-16C levels were enhanced during arthritis in Mertk^−/− mice and reduced in Pros1-overexpressing mice.</p>Conclusion<p>MER plays a protective role during joint inflammation and activating MER by its ligand PROS1 ameliorates disease. Treatment of mice with MER receptor agonistic antibodies is deleterious due to its counterproductive effect of blocking efferocytosis in the arthritic joint.</p