Involvement of EP2 and EP4 Receptors in Eosinophilic Esophagitis: A Pilot Study

Ten patients with clinically, endoscopically, and histologically confirmed EoE (46.7 ± 13.7 [mean age ± SD]; 7 males/3 females), and eight healthy control subjects (33 ± 13; 3 males/5 females) were included in the study. Diagnosis of EoE was established by clinical, endoscopic, and histological criteria. Conditions and criteria of diagnosis followed the recommendations according to the current published guidelines [2]. From EoE patients, blood and esophageal mucosal biopsies (from the proximal and distal part of the esophagus) were collected (see Table 1 for patient characteristics). From healthy control subjects, only blood was collected. EoE patients were recruited from the Department of Internal Medicine, and healthy volunteers were recruited from the Division of Pharmacology at the Medical University of Graz. Eosinophils isolated from healthy volunteers served as controls for flow cytometry experiments and for migration and adhesion assays. Subjects with significant comorbidities, intercurrent illness (such as infections), and pregnant women were excluded from the study. Blood was collected in Vacuette^® EDTA blood tubes (Greiner-Bio-One, Austria) and immediately processed for flow cytometric experiments. Collected biopsies were immediately frozen (two/patient) or fixed (two/patient) in 10% phosphate-buffered formalin for histochemical analysis. Two subjects of the EoE cohort were on proton pump inhibitors at the time of blood collection, while subjects who had biopsies taken were on no therapy at the time of collection (see EoE patient characteristics in Table 1).

Human primary esophageal epithelial cells were purchased from Cell Biologics (Chicago, IL, USA; Cat.# H-6046), cultivated in complete human epithelial cell medium provided by the vendor and used until passage seven.

Whole blood from EoE patients or healthy individuals was lysed with 1x BD FACS lysing solution to remove erythrocytes. Samples were washed once in PBS, resuspended in fixative solution, and kept on ice for 10 min. After washing with PBS, cells were blocked with Ultra-V Block (Lonza; Allendale, NJ, USA) for 30 min at 4 °C. For flow cytometric visualization of the DP1 receptor, cells were incubated with mouse polyclonal antibody against DP1 (20 μg/ml; Sigma-Aldrich, St. Louis, MO, USA), or normal mouse IgG (20 μg/ml; Sigma-Aldrich) for 30 min at 4 °C. For flow cytometric visualization of the DP2 receptor, cells were stained with Alexa Fluor 647-conjugated rat anti-human DP2 antibody or Alexa Fluor 647-conjugated rat IgG2a isotype control (10 μg/ml; BD Biosciences; San Jose, CA, USA) for 30 min at 4 °C. Cells were washed in PBS, resuspended in antibody Diluent (Dako, Glostrup, Denmark) and incubated for 30 min at 4 °C with the secondary antibody (1:2000; Alexa Fluor 488-conjugated rabbit anti-mouse secondary antibody; Life Technologies, Carlsbad, CA, USA). For flow cytometric visualization of the EP receptors, cells were incubated with rabbit polyclonal antibodies against EP1, EP2, and EP3 (20 μg/ml; Alomone Labs, Jerusalem, Israel) and mouse polyclonal antibody against EP4 (20 μg/ml; Alomone Labs, Jerusalem, Israel), or normal rabbit or mouse IgG (20 μg/ml; Santa Cruz Biotechnology, Dallas, TX, USA) for 30 min at 4 °C. Cells were washed in PBS, resuspended in antibody Diluent (Dako, Glostrup, Denmark), and incubated for 30 min at 4 °C with the secondary antibody (1:2000; PE-conjugated goat anti-rabbit or rabbit anti-mouse secondary antibody; Life Technologies, Carlsbad, CA, USA). Finally, all samples were washed with PBS, fixative solution was added, and samples were kept on ice until analyzed on a FACSCanto II flow cytometer. Eosinophils were identified on the basis of forward versus side scatter parameters and autofluorescence. Receptor expression was recorded as mean fluorescence intensity and expressed as fold increase over isotype control signals.

Migration assays were performed with peripheral blood eosinophils in 96-well Transwell plates with 5-µm membrane inserts (Corning Inc., Lowell, MA, USA). Eosinophils from healthy donors were incubated with the following compounds: EP4 agonist ONO-AE1-329 (100 nM) or EP4 antagonist ONO-AE3-208 (100 nM; ONO compounds were a generous gift from ONO Pharmaceutical, Osaka, Japan), EP2 agonist butaprost (100 and 300 nM; Cayman Chemicals, Ann Arbor, MI, USA), DP1 agonist BW245C (100 nM), and DP1 antagonist MK0524 (1 µM; Cayman Chemicals). Thereafter, a suspension of 5 × 10⁴ eosinophils was placed in the upper well. Supernatant from the esophageal epithelial cell culture was added into the bottom well as a chemoattractant. Eosinophils were allowed to migrate for 1 h at 37 °C and 5% CO2 in a humidified incubator and evaluated by flow cytometry. Each migration experiment was performed in triplicates. The average number of migrated cells was determined from at least five independent experiments.

Electric cell-substrate impedance sensing (ECIS^®, Applied Biophysics, Troy, NY, USA) with gold electrode arrays of the type 8W10E + was used to monitor the integrity of esophageal epithelial cell monolayers at real time in vitro as previously described [23]. ECIS^® array wells were activated with l-cysteine (10 mM) and precoated with 1% gelatine for 30 min at 37 °C. Then, wells were allowed to equilibrate with 200 µL cell medium for 15 min at 37 °C before esophageal epithelial cells were added in additional 200 µL medium to each well (1.5 × 10⁵ cells/well). Monolayer formation was followed by impedance measurements. Cells were treated with either 100 or 300 nM of EP4 agonist ONO-AE1-329 or EP2 agonist butaprost. Data are expressed as normalized resistance over time.

RNA isolation, reverse transcription (RT-PCR) and quantitative real-time PCR (qPCR) were performed as previously described [24] using primers for EP1, EP2, EP3, EP4, DP1, DP2, and β-actin (all from Bio-Rad; listed in Table 2). Relative gene expression was assessed according to the ΔΔCq method.

Paraffin-embedded sections of biopsies from EoE patients (n = 3) were cut (5 µm) and deparaffinized. For immunohistochemistry, sections were microwaved for 2 × 5 min cycles in 10 mM citrate buffer, and processed by ABC method according to the manufacturer’s protocol (Vectastain ABC kit; Vector Laboratories, Burlingname, CA, USA). Sections were incubated with rabbit polyclonal antibodies against EP2 (1:500) (Alomone Labs, Jerusalem, Israel), EP4 (1:500), and DP1 (1:100) (Cayman Chemicals), visualized with 3-3′-diaminobenzidine (DAB) or Vector^® ImmPact™ NovaRED HRP substrate kit according to the manufacturer’s protocol (Vector Laboratories), and counterstained with hematoxylin. As a negative control, primary antibodies were omitted during the staining procedure. Stainings were evaluated by two investigators. Images were taken with a high-resolution digital camera (Olympus UC 90) and analyzed by CellSense^® standard 1.17 imaging software (Olympus, Vienna, Austria). Only contrast, brightness and color balance of images were adjusted. Sirius Red (Direct Red 80^®, Sigma) was used to stain eosinophils in deparaffinized sections.

Data are described as mean ± SEM (or otherwise stated) of at least n = 5 observations or otherwise stated. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test, or by Student’s t test run on GraphPad Prism^® software. p values of < 0.05 were considered significant.

We first evaluated EP and DP receptor expression in peripheral blood eosinophils of EoE patients and healthy control subjects. We observed significant differences in EP2 (p = 0.0055) and EP4 (p = 0.0206) expression between EoE patients and control subjects but no changes in the expression levels of EP1 and EP3 (Fig. 1). Also, no differences were observed for DP1 and DP2 expression between EoE patients and control subjects (Fig. 1).

Next, we tested whether EP2 and EP4 receptors influence the migration and adhesion capacity of eosinophils to human primary esophageal epithelial cells and their supernatant. To this end, eosinophils were incubated with 100 or 300 nM of the EP4 agonist ONO-AE1-329 or the EP4 antagonist ONO-AE3-208, with 100 and 300 nM of the EP2 agonist butaprost, and, since flow cytometry data suggested a slight increase in DP1 expression in the eosinophils (Fig. 1), with 100 nM of the DP1 agonist BW245C or 1 µM of DP1 antagonist MK0524. Using supernatant from human primary esophageal epithelial cells as a chemoattractant, only the EP4 agonist (Fig. 2a), but not the EP2 and DP1 agonists (Fig. 2b, c), had an effect on the chemotactic behavior of eosinophils. In adhesion assays, however, both the EP4 agonist ONO-AE1-329 (Fig. 2d) and EP2 agonist butaprost (Fig. 2e) dose-dependently decreased the adhesion of eosinophils to primary esophageal epithelial cells, whereas the DP1 agonist BW245 clearly enhanced eosinophil adhesion (Fig. 2f).

Expression levels of EP and DP receptors were investigated by qPCR in esophageal mucosal biopsies and by immunohistochemistry in histological sections of esophageal mucosa from EoE patients. Among EP receptors, highest gene expression was observed for EP4, while expression levels of EP2 and EP3 were very low (Fig. 3a). qPCR in esophageal squamous epithelial cell cultures showed that gene expression of EP4 was the highest among EP receptors, while levels of DP1 and DP2 were extremely low (Fig. 3b), indicating that infiltrated rather than esophageal epithelial cells are responsible for the DP1 expression measured in mucosal biopsies from EoE patients (Fig. 3a).

In accordance with these findings, we observed DP1 immunostaining in eosinophils (arrows) but hardly any in epithelial cells (Fig. 4a). EP4 immunostaining was present in eosinophils (Fig. 4b, arrow) but also in epithelial cells (Fig. 4b, arrowhead), confirming our qPCR data. Although EP2 transcripts are present in esophageal epithelial cells (Fig. 3b), they were barely detectable in the qPCR of EoE biopsies (Fig. 3a). However, EP2 immunostaining could be observed in eosinophils (Fig. 4c, arrows) and epithelial cells of EoE biopsies (Fig. 4c, arrowhead).

Since EP receptors were also observed in esophageal epithelial cells (Figs. 3b, 4b and c), we investigated their functional implication by assessing their role in barrier integrity. We conducted ECIS^® impedance assays in esophageal epithelial cells incubated with EP4 agonist ONO-AE1-329 (Fig. 5a; 10 and 300 nM) and EP2 agonist butaprost (Fig. 5b; 10 and 300 nM). A decrease in resistance correlates with a decrease in the monolayer’s integrity. As a result of the treatments, the EP4 agonist dose-dependently decreased resistance in comparison with vehicle (control) (Fig. 5a), while a decrease in resistance after incubation with the EP2 agonist was marginal though significant (Fig. 5b).