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. 2012;7(11):e50734.
doi: 10.1371/journal.pone.0050734. Epub 2012 Nov 30.

Chemoattractant receptor homologous to the T helper 2 cell (CRTH2) is not expressed in human amniocytes and myocytes

Lynne Sykes  1 , Yun Lee, Shirin Khanjani, David A Macintyre, Xiao J Yap, Sathana Ponnampalam, Tiong Ghee Teoh, Phillip R Bennett
Affiliations

Chemoattractant receptor homologous to the T helper 2 cell (CRTH2) is not expressed in human amniocytes and myocytes

Lynne Sykes et al. PLoS One. 2012.

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. 15dPGJ2 reduced NF-κB p65 activity in amniocytes and myocytes.
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, (A), myocytes (B). Immunoblots were re-probed for β-actin as an internal loading control.
Figure 2
Figure 2. Pyl A has no effect on NF-κB p65 activity in amniocytes and myocytes.
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, (A) and myocytes (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 (C, E) or myocytes (D, F). 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; *P<0.05.
Figure 3
Figure 3. CRTH2 mRNA is expressed in amniocytes and myocytes.
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. (A,B): Non-template control (lane 1), amniocytes (lane 2), choriodecidua (lane 3), myocytes (lane 4), placenta (lane 5); (C): Reverse transcriptase controls (lanes 1,3,5,7,9), PBMCs (lane 2), amniocytes (lanes 4,6), myocytes (lanes 8,10).
Figure 4
Figure 4. Western blot of protein lysate from PBMCs, amniocytes, myocytes.
50 µg of whole cell lysate from PBMCs, amniocytes, myocytes were subject to western analysis. Membranes were probed with the primary antibody -SC-23092 to detect a Mr∼34 000 product (A; Lanes 1–3). The secondary antibody control blot is also shown (B; Lanes 1–3). β-actin was used as a loading control. Lane 1 = PBMCs, lane 2 = amniocytes, lane 3 = myocytes. Multiple bands are seen on the blot. A band appears at Mr∼34 000 in amniocytes, faintly in the myocyte lane, but is absent in the positive control PBMCs lane. However, the strongest bands appear at Mr∼15 000 and at just above Mr∼43 000 in all lanes. Myocytes from passage zero, one, two, three and four (lanes 1–5 respectively) were also examined for CRTH2 expression showing a very faint band at Mr∼34 000, with no effect of passage number (C).
Figure 5
Figure 5. Detection of radiolabelled CRTH2 in pSG5 expression vector by x-ray and immunoblot.
The in vitro transcription translation kit was used with 35S 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 (A). 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: 35S Methionine labelled CRTH2 (Lane 3), Cold Methionine CRTH2 protein product (lane 4). Membranes were probed with 3 commercial antibodies; SC-23092 (B), SC-21798 (C), ProSci 4027 (D). None of the antibodies detected CRTH2.
Figure 6
Figure 6. Flow cytometry for the detection of endogenous CRTH2.
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+/CD4+ lymphocytes (A). Histograms showing no staining, isotype control and CRTH2+ labelled lymphocytes (B), amniocytes (C) and myocytes (D), (n = 5).
Figure 7
Figure 7. Flow cytometry for the detection of transfected CRTH2.
Amniocytes (A) and myocytes (B) were transfected using gene juice and electroporation respectively with CRTH2 PSG5 expression vector and labeled with anti-CRTH2-PE. A small shift to the right is seen in the histogram showing expression of CRTH2 in transfected cells with an increase in the mean fluorescence intensity from 87.75 in the isotype control to 143.49 in amniocytes, and from 68.84 to 96.97 in myocytes. An example of a GFP control is given for the myocyte transfection, with cells imaged with phase contrast and with the fluorescent microscope to detect GFP transfected cells (C). A direct comparison is also shown with non-GFP transfected cells (D).
Figure 8
Figure 8. The effect of 15dPGJ2 on basal NF-κB p65 phosphorylation in peripheral blood mononuclear cells.
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 (A). 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). J2 = 15dPGJ2, GSKX = GSKCRTH2X. Effect of treatment was examined for statistical significance using ANOVA of repeated measures with Bonferroni’s multiple comparison test; **** P<0.0001.
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This study was funded by a Wellbeing of Women research training fellowship, grant 148 (to LS). PRB is funded by the Imperial College NIHR Biomedical Research Centre. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.