13 research outputs found

    Changes in the Th1 : Th2 Cytokine Bias in Pregnancy and the Effects of the Anti-Inflammatory Cyclopentenone Prostaglandin 15-Deoxy-Δ12,14-Prostaglandin J2

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    Pregnancy is a complex immunological state in which a bias towards T helper 2 (Th2) protects the fetus. Evidence suggests that proinflammatory cytokines increase the risk of poor neonatal outcome, independently of the direct effect of preterm labour. The anti-inflammatory prostaglandin 15-deoxy-Δ12,14-Prostaglandin J2 (15dPGJ2) inhibits nuclear factor Kappa B (NF-κB) in amniocytes and myocytes in vitro and is a ligand for the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) receptor. Here we examine the Th1:Th2 cytokine bias in pregnancy and whether 15dPGJ2 could be used to inhibit the production of the proinflammatory cytokines through inhibition of NF-κB while simultaneously promoting Th2 interleukin 4 (IL-4) synthesis via CRTH2 in T helper cells. Peripheral blood mononuclear cells (PBMCs) from women at 28 weeks, term pre-labour, term labour as well as non-pregnant female controls were cultured with 15dPGJ2 or vehicle control and stimulated with phorbol myristyl acetate (PMA)/ionomycin. The percentage of CD4+ cells producing interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) in response to PMA/ionomycin was significantly reduced in pregnancy. 15dPGJ2 reduced IFN-γ and TNF-α production in stimulated T helper cells, but did not alter IL-4 production in CRTH2+ve cells. 15dPGJ2 also reduced phospho-p65 in stimulated PBMCs. In summary, 15dPGJ2 suppresses the Th1 response of PBMCs during pregnancy and active labour whilst maintaining the Th2 response suggesting a therapeutic benefit in reducing neonatal morbidity in inflammation-induced PTL

    Chemoattractant Receptor Homologous to the T Helper 2 Cell (CRTH2) Is Not Expressed in Human Amniocytes and Myocytes

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    BACKGROUND: 15-deoxy-Δ 12,14- Prostaglandin J2 (15dPGJ2) inhibits Nuclear factor kappa B (NF-κB) in human myocytes and amniocytes and delays inflammation induced preterm labour in the mouse. 15dPGJ2 is a ligand for the Chemoattractant Receptor Homologous to the T helper 2 cell (CRTH2), a G protein-coupled receptor, present on a subset of T helper 2 (Th2) cells, eosinophils and basophils. It is the second receptor for Prostaglandin D2, whose activation leads to chemotaxis and the production of Th2-type interleukins. The cellular distribution of CRTH2 in non-immune cells has not been extensively researched, and its identification at the protein level has been limited by the lack of specific antibodies. In this study we explored the possibility that CRTH2 plays a role in 15dPGJ2-mediated inhibition of NF-κB and would therefore represent a novel small molecule therapeutic target for the prevention of inflammation induced preterm labour. METHODS: The effect of a small molecule CRTH2 agonist on NF-κB activity in human cultured amniocytes and myocytes was assessed by detection of p65 and phospho-p65 by immunoblot. Endogenous CRTH2 expression in amniocytes, myocytes and peripheral blood mononuclear cells (PBMCs) was examined by PCR, western analysis and flow cytometry, with amniocytes and myocytes transfected with CRTH2 acting as a positive control in flow cytometry studies. RESULTS: The CRTH2 agonist had no effect on NF-κB activity in amniocytes and myocytes. Although CRTH2 mRNA was detected in amniocytes and myocytes, CRTH2 was not detectable at the protein level, as demonstrated by western analysis and flow cytometry. 15dPGJ2 inhibited phospho-65 in PBMC'S, however the CRTH2 antagonist was not able to attenuate this effect. In conclusion, CRTH2 is not expressed on human amniocytes or myocytes and plays no role in the mechanism of 15dPGJ2-mediated inhibition of NF-κB

    CRTH2 mRNA is expressed in amniocytes and myocytes.

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    <p>mRNA was isolated from cultured amniocytes, myocytes, PBMCs, choriodecidual and placental extracts and converted to cDNA (n = 6). Qualitative PCR was used with three primer sets to amplify CRTH2 showing product sizes of 309 bp, 265 bp, and 114 bp. Non-template and reverse transcriptase negative controls were used and mRNA from placenta, choriodecidua and peripheral blood mononuclear cells were used as a positive controls. (<b>A</b>,<b>B</b>): Non-template control (lane 1), amniocytes (lane 2), choriodecidua (lane 3), myocytes (lane 4), placenta (lane 5); (<b>C</b>): Reverse transcriptase controls (lanes 1,3,5,7,9), PBMCs (lane 2), amniocytes (lanes 4,6), myocytes (lanes 8,10).</p

    Flow cytometry for the detection of endogenous CRTH2.

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    <p>Lymphocytes were gated according to forward scatter and side scatter and T helper cells were identified using CD4 as a cell surface marker (n = 6). A representative cytogram is presented with the right upper quadrant showing CRTH2<sup>+</sup>/CD4<sup>+</sup> lymphocytes (<b>A</b>). Histograms showing no staining, isotype control and CRTH2<sup>+</sup> labelled lymphocytes (<b>B</b>), amniocytes (<b>C</b>) and myocytes (<b>D</b>), (n = 5).</p

    15dPGJ2 reduced NF-κB p65 activity in amniocytes and myocytes.

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    <p>Protein was extracted from IL-1β stimulated and 15dPGJ2 treated cells and levels of nuclear p65 were examined using immunoblotting. A dose response of 0.1–32 µM of 15dPGJ2 was used (n = 3). Representative immunoblots are shown for amniocytes, (<b>A</b>), myocytes (<b>B</b>). Immunoblots were re-probed for β-actin as an internal loading control.</p

    Pyl A has no effect on NF-κB p65 activity in amniocytes and myocytes.

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    <p>Protein was extracted from IL-1β stimulated and Pyl treated cells and levels of nuclear p65 and phosphorylated p65 (p-p65) were examined using immunoblotting. A dose response of 0.1–32 µM of Pyl A was used. Representative immunoblots are shown for amniocytes, (<b>A</b>) and myocytes (<b>B</b>). Immunoblots were re-probed for β-actin as an internal loading control. Densitometric analysis of the immunoblots was conducted revealing no effect of Pyl A on p65 or p-p65 in amniocytes (<b>C</b>, <b>E</b>) or myocytes (<b>D</b>, <b>F</b>). NS = non-stimulated (non-IL-1β treated cells). Effect of treatment was examined for statistical significance using ANOVA of repeated measures with Bonferroni’s multiple comparison test; *<i>P</i><0.05.</p

    Detection of radiolabelled CRTH2 in pSG5 expression vector by x-ray and immunoblot.

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    <p>The in vitro transcription translation kit was used with 35<sup>S</sup> Methionine to demonstrate the expression of CRTH2 and to provide a positive control for detection of CRTH2. A Mr∼34 000 product was detected by x-ray (Lane 2). The progesterone expression vector was used as a control for the TNT kit (<b>A</b>). The protein lysate from the TNT kit was subjected to western analysis; Negative Control: TNT kit with no plasmid DNA (lane 1), Negative control: Progesterone PSG5 Expression vector (lane 2), Positive control: 35<sup>S</sup> Methionine labelled CRTH2 (Lane 3), Cold Methionine CRTH2 protein product (lane 4). Membranes were probed with 3 commercial antibodies; SC-23092 (<b>B</b>), SC-21798 (<b>C</b>), ProSci 4027 (<b>D</b>). None of the antibodies detected CRTH2.</p

    The effect of 15dPGJ2 on basal NF-κB p65 phosphorylation in peripheral blood mononuclear cells.

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    <p>PBMCs were treated with 32 µM of 15dPGJ2 for 2 hours with or without preincubation with 2 µM of GSKCRTH2X for 45 mins. Whole cell protein lysate was examined for levels of phosphorylated p65 (p-p65) using immunoblotting. A representative immunoblot from n = 6 samples are shown (<b>A</b>). Immunoblots were reprobed for β-actin as an internal loading control. Densitometric analysis of the immunoblots was conducted revealing a complete inhibition of p-p65 levels with 15dPGJ2. Pre-incubation with the CRTH2 antagonist GSKCRTH2X had no effect on inhibition (<b>B</b>). J2 = 15dPGJ2, GSKX = GSKCRTH2X. Effect of treatment was examined for statistical significance using ANOVA of repeated measures with Bonferroni’s multiple comparison test; **** <i>P</i><0.0001.</p
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