CRTH2 Receptors Research Articles

Vasoactive Intestinal Peptide Receptor, CRTH2, Antagonist Treatment Improves Eosinophil and Mast Cell-Mediated Esophageal Remodeling and Motility Dysfunction in Eosinophilic Esophagitis.

Ultrasonography has shown that eosinophils accumulate in each segment of the esophageal mucosa in human EoE, ultimately promoting esophageal motility dysfunction; however, no mechanistic evidence explains how or why this accumulation occurs. Quantitative PCR, ELISA, flow cytometry, immunostaining, and immunofluorescence analyses were performed using antibodies specific to the related antigens and receptors. In deep esophageal biopsies of EoE patients, eosinophils and mast cells accumulate adjacent to nerve cell-derived VIP in each esophageal segment. qRT-PCR analysis revealed five- to sixfold increases in expression levels of VIP, CRTH2, and VAPC2 receptors and proteins in human blood- and tissue-accumulated eosinophils and mast cells. We also observed a significant correlation between mRNA CRTH2 levels and eosinophil- and nerve cell-derived VIPs in human EoE (p < 0.05). We provide evidence that eosinophil and mast cell deficiency following CRTH2 antagonist treatment improves motility dysfunction in a chronic DOX-inducible CC10-IL-13 murine model of experimental EoE. CRTH2 antagonist treatment is a novel therapeutic strategy for inflammatory cell-induced esophageal motility dysfunction in IL-13-induced chronic experimental EoE.

MiR-665 overexpression inhibits the apoptosis of luteal cells in small ruminants suppressing HPGDS

Evidence has shown that microRNA-665 (miR-665) is highly expressed in the mid-luteal phase compared with the early and end-luteal phase of the corpus luteum (CL) life cycle. However, whether miR-665 is a positive regulator of the life span of the CL is still unknown. The objective of this study is to explore the effect of miR-665 on the structural luteolysis in the ovarian CL. In this study, the targeting relationship between miR-665 and hematopoietic prostaglandin synthase (HPGDS) was firstly verified by dual luciferase reporter assay. Then, quantitative real-time PCR (qRT-PCR) was used to detect the expression of miR-665 and HPGDS in luteal cells. Following miR-665 overexpression, the apoptosis rate of the luteal cells was determined using flow cytometry; B-cell lymphoma-2 (BCL-2) and caspase-3 mRNA and protein were measured using qRT-PCR and Western blot (WB) analysis. Finally, the DP1 and CRTH2 receptors of PGD2, a synthetic product of HPGDS, were localized using immunofluorescence. Results confirmed that HPGDS was a direct target gene of miR-665, and miR-665 expression was negatively correlated with HPGDS mRNA expression in luteal cells. Meanwhile, after miR-665 was overexpressed, the apoptotic rate of the luteal cells showed a significant decrease (P < 0.05) and this was accompanied by elevated expression levels of anti-apoptotic factor BCL-2 mRNA and protein and decreased expression levels of apoptotic factor caspase-3 mRNA and protein (P < 0.01). Moreover, the immune fluorescence staining results showed that the DP1 receptor was also significantly decreased (P < 0.05), but the CRTH2 receptor was significantly increased (P < 0.05) in luteal cells. Overall, these results indicate that miR-665 reduces the apoptosis of luteal cells via inhibiting caspase-3 expression and promoting BCL-2 expression, and the biological function of miR-665 may be attributed to its target gene HPGDS which regulates the balance of DP1 and CRTH2 receptors expression in luteal cells. As a consequence, this study suggests that miR-665 might be a positive regulator of the life span of the CL rather than destroy the integrity of CL in small ruminants.

Targeting the PGD2/CRTH2/DP1 Signaling Pathway in Asthma and Allergic Disease: Current Status and Future Perspectives

Prostaglandin D2 (PGD2) released by degranulating mast cells is believed to play a key role in orchestrating mechanisms of inflammation in allergies and asthma. The biological effects of PGD2 are mediated by D-prostanoid (DP1), CRTH2 (DP2), and thromboxane prostanoid (TP) receptors. The CRTH2 receptor is involved in induction of migration and activation of T helper type 2 (Th2) lymphocytes, eosinophils, and basophils; up-regulation of adhesion molecules; and promotion of pro-inflammatory Th2-type cytokines (interleukin [IL]-4, 5, 13), whereas the DP receptor is associated with relaxation of smooth muscles, vasodilation, inhibition of cell migration, and apoptosis of eosinophils. A number of CRTH2/PGD2 receptor antagonists have been investigated in asthma and allergic diseases. The CRTH2 antagonist (OC000459) or dual CRTH2 and TP receptor antagonist (ramatroban) were effective in reducing eosinophilia, nasal mucosal swelling, and clinical symptoms of allergic rhinitis, with the latter drug registered for clinical use in this indication. OC000459 and setipiprant reduced the late but not early phase of response in an allergen challenge in atopic asthmatics. In persistent asthma, some molecules induced limited improvement in lung function, quality of life, and asthma symptoms (OC000459, BI671800), but in other trials with AMG 853 and AZ1981 these findings were not confirmed. The clear discrepancy between animal studies and clinical efficacy of CRTH2 antagonism in allergic rhinitis, and lack of efficacy in a general cohort of asthmatics, highlight the issue of patient phenotyping. There is no doubt that the PGD2/CATH2/DP1 pathway plays a key role in allergic inflammation and further studies with selective or combined antagonisms in well defined cohorts of patients are needed.

Effect of prostaglandin D2 on VEGF release by nasal polyp fibroblasts

BackgroundVascular endothelial growth factor (VEGF) is known to be associated with the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). VEGF is produced by a variety of cells including fibroblasts. It was recently reported that prostaglandin (PG) E2 induces VEGF release by nasal polyp fibroblasts. However, little is known regarding possible regulation of VEGF by other PGs. We have reported that molecules that regulate PGD2 metabolism play roles in the pathogenesis of CRS including in local eosinophilia and type 2 cytokine production. In the present study, we sought to determine whether PGD2 regulates VEGF release by nasal polyp fibroblasts. MethodsNasal polyp fibroblasts were established from nasal polyps. These fibroblasts were stimulated with serial dilutions of PGD2 or PGD2 receptor (DP/CRTH2)-selective agonists in the presence or absence of receptor-selective antagonists. The concentration of VEGF in the culture supernatants was determined using ELISA. Results5 μM of PGD2 significantly induced VEGF release by nasal polyp fibroblasts. VEGF release was also obtained by stimulation with a DP receptor-selective, but not with a CRTH2 receptor-selective agonist. In addition, PGD2-induced VEGF release was significantly inhibited by pre-treatment with DP receptor-selective antagonists. In contrast, pre-treatment with a CRTH2 receptor-selective antagonist significantly enhanced PGD2-induced VEGF release. ConclusionsPGD2 stimulates VEGF production via DP but not CRTH2 receptors in nasal polyp fibroblasts.

S19 Increased CRTH2 expression in asthmatic bronchial epithelium

Introduction and ObjectivesProstaglandin-D2 (PGD2) mediates chemotaxis of Th2 cells, basophils and eosinophils through the chemoattractant receptor homologous-molecule expressed on T-helper-type-2 cells (CRTh2). Pulmonary PGD2 levels are increased in patients with...

Indomethacin inhibits eosinophil migration to prostaglandin D2: therapeutic potential of CRTH2 desensitization for eosinophilic pustular folliculitis

Indomethacin is a cyclo-oxygenase inhibitor, and shows therapeutic potential for various eosinophilic skin diseases, particularly eosinophilic pustular folliculitis. One of the unique characteristics of indomethacin is that, unlike other non-steroidal anti-inflammatory drugs, it is a potent agonist of chemoattractant receptor-homologous molecule expressed on T helper type 2 cells (CRTH2), a receptor for prostaglandin D2 (PGD2 ). This study investigated the pharmacological actions of indomethacin on eosinophil migration to clarify the actual mechanisms underlying the therapeutic effects of indomethacin on eosinophilic pustular folliculitis. Eosinophils exhibited chemokinetic and chemotactic responses to both PGD2 and indomethacin through CRTH2 receptors. Pre-treatment of eosinophils with indomethacin greatly inhibited eosinophil migration to PGD2 and, to a much lesser extent, to eotaxin (CCL11); these effects could be mediated by homologous and heterologous desensitization of eosinophil CRTH2 and CCR3, respectively, by agonistic effects of indomethacin on CRTH2. Indomethacin also cancelled a priming effect of Δ(12) -PGJ2 , a plasma metabolite of PGD2 , on eosinophil chemotaxis to eotaxin. Indomethacin down-modulated cell surface expression of both CRTH2 and CCR3. Hair follicle epithelium and epidermal keratinocytes around eosinophilic pustules together with the eccrine apparatus of palmoplantar lesions of eosinophilic pustular folliculitis were immunohistochemically positive for lipocalin-type PGD synthase. Indomethacin may exert therapeutic effects against eosinophilic skin diseases in which PGD2 -CRTH2 signals play major roles by reducing eosinophil responses to PGD2 .

Abstract 354: Prostaglandin D 2 -F Type Prostanoid Receptor Signaling Is a Novel Cardiomyocyte Survival Pathway

Background: Lipocalin-type prostaglandin D synthase (L-PGDS) is abundantly expressed on cardiomyocytes. We recently demonstrated that dexamethasone stimulates PGD 2 -dominated activation of prostanoid biosynthesis, thereby protecting hearts against ischemia/reperfusion injury. Here, we examined the downstream signaling responsible for cardioprotection mediated through PGD 2 -dominated activation. Methods and Results: (1) In cultured neonatal rat cardiomyocytes, PGD 2 strongly activates ERK in a dose-dependent manner, although canonical PGD 2 receptors, including DP (PGD 2 receptor) and CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) receptors, are hardly expressed on cardiomyocytes. (2) Interestingly, PGD 2 bounds to FP receptor (the canonical PGF 2 α receptor) with an affinity comparable to that for the DP receptor, and the FP receptor is abundantly expressed on cardiomyocytes. (3) PGD 2 -induced ERK activation is completely blocked by FP antagonist or siRNA-mediated knockdown of the FP, but not by DP and CRTH2 antagonist and siRNA-mediated knockdown of DP and CRTH2. (4) PGD 2 activates ERK in Langendorff perfused DP-knock out (KO) and CRTH2-KO mice hearts to comparable levels as those observed for wild-type hearts, while cannot activate it in FP-KO hearts. (5) Consistently, the cardioprotective effect of PGD 2 -dominated activation by dexamethasone was blunted in FP KO hearts. (6) Furtermore, genomewide gene expression profiles by microarray analysis and quantitative real-time RT-PCR analysis identified that Nrf-2 was the downstream target of L-PGDS-mediated PGD 2 biosynthesis. (7) In cultured cardiomyocytes, FP agonist stimulated Nrf2 nuclear translocation and consequently induced Nrf2-target genes expression in an ERK-dependent manner. (8) Finally, The cardioprotective effect by dexamethasone was completely abolished in Nrf-2 KO hearts. Conclusion: FP serves as a functionally relevant PGD 2 receptor in the hearts and PGD 2 -FP signaling plays a substantial role in the improvement of functional recovery after ischemia-reperfusion injury in the heart. Nrf-2 is a major effector molecule responsible for the cardioprotecton elicited by L-PGDS-derived PGD 2 .

Abstract 9955: Cooperative Operation of PGD 2 /FP and 15d-PGJ 2 /nrf2 Pathway in the L-PGDS-Mediated Cardioprotection

Background Lipocalin-type prostaglandin D synthase (L-PGDS) is abundantly expressed in adult cardiomyocytes. We recently demonstrated that dexamethasone stimulates PGD2-dominated activation of prostanoid biosynthesis via induction of phospholipase A2 and cyclooxygenase-2, thereby protecting hearts against ischemia/reperfusion injury. Here, we examined the downstream signaling responsible for cardioprotection mediated through PGD2-dominated activation of prostanoid biosynthesis. Methods and Results (1) In cultured neonatal rat cardiomyocytes, PGD2 strongly activated ERK in a dose-dependent manner, although canonical PGD2 receptors, including DP (PGD2 receptor) and CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) receptors, were hardly expressed on cardiomyocytes. (2) Interestingly, PGD2 bound to FP receptor (the canonical PGF2α receptor) with an affinity comparable to that for the DP receptor, and the FP receptor was abundantly expressed in cardiomyocytes. (3) PGD2-induced ERK activation was completely blocked by FP antagonist or siRNA-mediated knockdown of the FP, but not by DP and CRTH2 antagonist and siRNA-mediated knockdown of DP and CRTH2. (4) PGD2 activated ERK in Langendorff perfused DP-knock-out (KO) and CRTH2-KO mice hearts to comparable levels as those observed for wild-type hearts, while it did not activate ERK in FP-KO hearts. (5) Dexamethasone protected hearts against ischemia-reperfusion injury in L-PGDS-dependent manner. The dexamethasone-mediated cardioprotection was partially cancelled in FP-KO mice. (6) Gene-chip and Q-PCR analysis revealed that dexamethasone induced Nrf2-dependent antioxidant gene expression in wild-type mice, but not in L-PGDS KO mice. Dexamethasone-mediated cardioprotection against ischemia-reperfusion injury was partially attenuated in Nrf-2 KO mice. (7) In cultured cardiomyocytes, PGD2 had little effect on Nrf2 activation, while 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), which is formed from PGD2, strongly activated Nrf2. Conclusion PGD2 and 15d-PGJ2 are cooperatively involved in the L-PGDS-mediated cardioprotection against ischemia-reperfusion injury via FP receptor and Nrf-2 activation, respectively.

Distinct roles of prostaglandin D2 receptors in chronic skin inflammation

Prostaglandin D2 (PGD2) is a prostanoid implicated in allergic inflammation. However, the roles of PGD2 in immune and allergic responses remain controversial. PGD2 exerts its effect through the CRTH2 and DP receptors. To elucidate functional differences of PGD2 and its receptors in chronic skin inflammation, chronic contact hypersensitivity (chronic CHS) and IgE-mediated chronic allergic skin inflammation (IgE-CAI) were induced in mice deficient in the CRTH2 and/or DP genes. DP (−/−) mice and CRTH2 (−/−)/DP (−/−) mice showed exacerbated chronic CHS, and conversely, CRTH2 (−/−) mice exhibited diminished skin responses. Skin responses correlated with local levels of IL-13, CCL11, and CCL22. These phenotypic changes in chronic CHS of mutant mice were similar to those in acute CHS despite the differences in the cytokine milieus; chronic CHS and acute CHS were mediated by Th2 and Th1/Th17 immunity, respectively. However, in IgE-CAI, DP (−/−) mice showed comparable skin responses to wild-type mice. Alleviation of IgE-CAI was observed in CRTH2 (−/−) mice, and as a consequence, CRTH2 (−/−)/DP (−/−) mice exhibited diminished IgE-CAI compared with wild-type mice. IgE-CAI in mutant mice correlated with local IL-4 and CCL22 production. Consistent with these results, a CRTH2-specific antagonist exerted inhibitory effects in both chronic CHS and IgE-CAI. The present study demonstrates that functional roles of PGD2 and its receptors appear to depend on the nature of the inflammation. Nevertheless, tools targeted against PGD2-CRTH2 signals could offer therapeutic potential for both types of chronic skin inflammation.

D-type prostanoid receptor enhances the signaling of chemoattractant receptor–homologous molecule expressed on TH2 cells

Prostaglandin (PG) D(2) is substantially involved in allergic responses and signals through the 7 transmembrane-spanning/G protein-coupled receptors, chemoattractant receptor-homologous molecule expressed on T(H)2 cells (CRTH2), and D-type prostanoid (DP) receptor. Although the proinflammatory function of CRTH2 is well recognized and CRTH2 is hence considered an important emerging pharmacotherapeutic target, the role of the DP receptor in mediating the biological effects of PGD(2) in patients with allergic inflammation has remained unclear. The cross-talk of CRTH2 and DP receptors was investigated by using both a recombinant HEK293 cell model and human eosinophils in Ca(2+) mobilization assays, coimmunoprecipitation, Western blotting, radioligand binding, and immunofluorescence. We show that CRTH2 and DP receptors modulate one another's signaling properties and form CRTH2/DP heteromers without altering their ligand-binding capacities. We find that the DP receptor amplifies the CRTH2-induced Ca(2+) release from intracellular stores and coincidentally forfeits its own signaling potency. Moreover, desensitization or pharmacologic blockade of the DP receptor hinders CRTH2-mediated signal transduction. However, CRTH2 internalization occurs independently of the DP receptor. In cells that express both receptors, pharmacologic blockade of Gα(q/11) proteins abolishes the Ca(2+) response to both CRTH2 and DP agonists, whereas inhibition of Gα(i) proteins selectively attenuates the CRTH2-mediated response but not the DP signal. Our data demonstrate the capacity of DP receptors to amplify the biological response to CRTH2 activation. Therefore the CRTH2/DP heteromer might not only represent a functional signaling unit for PGD(2) but also a potential target for the development of heteromer-directed therapies to treat allergic diseases.

A randomized, double‐blind, placebo‐controlled study of the CRTH2 antagonist OC000459 in moderate persistent asthma

CRTH2 is a G-protein-coupled receptor that mediates the activation of Th2 lymphocytes, eosinophils and basophils in response to prostaglandin D(2) and may be involved in the pathogenesis of airway inflammation and dysfunction in asthma. To evaluate the effects of a potent and selective CRTH2 antagonist, OC000459, on the lung function, symptoms and eosinophilic airway inflammation in a double-blind, parallel group trial in steroid-free subjects with moderate persistent asthma. Adult subjects were randomized to oral OC000459 200 mg twice daily (N=65) or a placebo (N=67) for 28 days. The primary end-point was the change from baseline in pre-bronchodilator forced expiratory volume in 1 s (FEV(1) ); eosinophilic airway inflammation was assessed by induced sputum differential eosinophil count. The trial was registered on the clinicaltrials.gov database (Identifier NCT01057927). Data were analysed for both the Full Analysis (FA) population and the Per Protocol (PP) population (55 treated with OC000459 and 52 with placebo), which excluded non-compliant subjects. In the FA population, the mean change in FEV(1) was 7.1% on OC000459 compared with 4.3% on placebo (not significant); in the PP population, the mean changes were 9.2% and 1.8%, respectively (P=0.037). Improvement in quality of life was apparent in both FA and PP populations [difference from the placebo in AQLQ(S) total score of 0.29, P=0.0113 and 0.37, P=0.0022, respectively]. OC000459 also improved the night-time symptom scores (mean reduction of 0.36 vs. 0.11, P=0.008, FA population; 0.37 vs. 0.12, P=0.022, PP population). The geometric mean sputum eosinophil count reduced from 2.1% to 0.7% (P=0.03) after OC000459, but this effect was not significant when compared with the change on placebo (P=0.37). Adverse events on OC000459 were comparable to those on placebo; respiratory infections were notably less common during OC000459 than the placebo treatment. This study provides the first clinical evidence that CRTH2 receptors contribute to airflow limitation, symptoms and eosinophilic airway inflammation in asthma. OC000459 shows promise as a novel oral treatment for asthma and related disorders.

Cross-talk of PGD2 receptors: the DP receptor modulates signaling and trafficking of CRTH2

Background Prostaglandin (PG) D2 is substantially involved in allergic inflammation and signals via the seven transmembrane (7TM) spanning/G protein-coupled receptors (GPCRs) chemoattractant receptor homologous molecule expressed on Th2 cells (CRTH2) and D-type prostanoid (DP) receptor. Both receptors are co-expressed in eosinophils among other immune cells and have emerged as therapeutic targets in allergic diseases. While a proinflammatory function of the CRTH2 receptor is well recognized, the role of the DP receptor in allergic inflammation, however, remains unclear. As it has been shown for many other 7TM/GPCRs, we believe that intermolecular cross-talk occurs between CRTH2 and DP receptors which might be essential for receptor function and regulation.

CRTH2 mediates the activation of human Th2 cells in response to PGD2 released from IgE/anti‐IgE treated nasal polyp tissue

Mast cells release mediators upon stimulation that contribute to the pathogenesis of chronic airway disease, including the recruitment and activation of Th2 lymphocytes. The objective was to determine the involvement of prostaglandin D(2) (PGD(2)) and its receptors in the chemotaxis of Th2 cells, using nasal polyp tissue. Tissue explants from ten patients with nasal polyposis were incubated with RPMI alone or RPMI containing IgE/anti-IgE for 30 min. Some samples were treated with diclofenac to inhibit the production of PGD(2). Supernatants were assayed for PGD(2) content and for their ability to promote human Th2 cell chemotaxis in the presence and absence of a CRTH2 antagonist. Transcript levels of D protanoid receptor type 1 (DP(1)), chemoattractant receptor-homologous receptor expressed on Th2 cells (CRTH2) and PGD(2) synthase were analysed by real time PCR. Increased release of PGD(2) by nasal polyp tissue treated with IgE/anti-IgE was significantly inhibited by preincubation of the tissue with diclofenac. Transcript levels of PGD(2) synthase, DP(1) and CRTH2 receptors increased after stimulation with IgE/anti-IgE. Supernatants from IgE/anti-IgE-stimulated nasal polyp tissue caused significantly increased chemotaxis of Th2 cells. The levels of PGD(2) produced and the degree of Th2 cell chemotaxis were highly correlated. Diclofenac inhibited the production of Th2 cell chemotactic activity, and the chemotactic effect of the supernatant on Th2 cells was inhibited by the CRTH2 antagonist ramatroban. These data suggest that in immunologically activated nasal polyp tissue, PGD(2) produced by mast cells promotes the migration of Th2 cells through a CRTH2 dependent mechanism.

CRTH2 antagonist ameliorates airway inflammation in rats with asthma

To investigate the effect of prostaglandin D2 receptor antagonists on the airway inflammation in rats with asthma. Forty male SD rats were randomly divided into four groups: Group A (normal control), Group B (asthma group), Group C (CRTH2 antagonist BAYu3405 treatment group), Group D (DP1 antagonist BWA868C treatment group). Asthma was induced by ovalbumin (OVA) challenge. The rats in each group were sacrificed 24 h after the last challenge of OVA.DP1/CRTH2 receptors on eosinophils (EOS) were measured by radiological binding assay (RBA). The left lungs were used for histological examinations and bronchoalveolar lavage fluid (BALF) was collected from the right lungs. The total cell numbers, EOS absolute count and differential cell counts in BALF were performed. Serum concentrations of IL-4, 5 and IFN-gamma were measured by ELISA. Rats in BAYu3405 treatment group showed profoundly decreased infiltrates of EOS and lymphocytes in the wall of bronchus when compared with those of asthma group and BWA868C treatment group. Serum concentrations of IFN-gamma in rats of BAYu3405 treatment group increased, but IL-4 and IL-5 decreased significantly when compared with those in rats of asthma group and BWA868C treatment group (P<0.01), and BALF EOS count was decreased significantly (P<0.01). Peripheral blood EOS count was higher than that in rats of normal control group, but was not significantly different from that in rats of asthma group and BWA868C treatment group. The combining capacity of CRTH2 and DP total combining capacity on EOS in asthma group, BAYu3405 treatment group and BWA868C treatment group were significantly higher than those in Group A (P<0.01). There was no significant difference in DP1 among all the groups (P>0.05). CRTH2, but not DP1 antagonist can effectively ameliorate airway inflammation in rats with asthma.

Effects of Anti-Asthma Agents on Cytokine and Prostaglandin Production in Ovalbumin-Sensitized Splenocytes

The cytokines which is produced by allergen-specific T helper (Th) cells play a pivotal role in the pathogenesis of asthma. Asthma is caused by exaggerated T-helper 2 (Th2)-based immune responses. It is suggested that controlling such Th2-based response is necessary for asthma therapy. The current therapies for asthma focus primarily on control of symptoms and suppression of inflammation, without affecting the underlying cause. So, we examined that anti-asthmatic drugs might have play a certain role in Th2/Th1 balance. Splenocytes isolated from ovalbumin (OVA)-sensitized mice cultured with anti-asthmatic drugs. It is well known that Th2 and Th1 immune responses can balance one another, as Th2 mediators suppress Th1 responses and Th1 mediators similarly inhibit Th2 responses. But salmeterol inhibits both of Th1 and Th2 mediators, which salmeterol is a suppressor of immune responses not only a suppressor of Th2-based immune responses. Aminophylline is a weak suppressor of immune responses. But ipratropium and cromoglycate don't have any suppressor effect to Th2-driven responses. They only have suppressor effect to Th1 immune responses. Salmeterol, ipratropium, aminophylline, and cromoglycate augmented mRNA levels of CRTH2, EP2, and IP2 receptors in OVA-sensitized splenocytes. It is well known that the up-regulation of CRTH2 - PGD2 receptor - results in restraint of eosinophil recruitment and that the increment of IP and EP2 - PGI2 and PGE2 receptor, respectively - may induce the accumulation of cAMP that decrease the effector function of T cells. Moreover salmeterol and cromoglycate increase the mRNA expression of PGD2 synthase. These findings indicate that anti-asthma agents may alleviate the immunological responses that cause the asthmatic diseases.

Mast Cells and Arachidonic Acid Cascade in Inflammation

Prostaglandin D2 PGD2 is a major cyclooxygenase metabolite of arachidonic acid produced by mast cells and it is released following allergen challenge in diseases, such as allergic diseases. PGD2 may act as a neuromodulator and as an allergic and inflammatory mediator. In allergic diseases, activated mast cell synthesizes prostaglandin D2 (first cyclo-oxygenate mediator) which has bronchoconstrictive and vasodilating effects and attracts several leukocytes. It has been found that activated mast cells, challenged with physiological and non- physiological secretagogues, release elevated histamine and tryptase and chymase, leukotrienes B4, C4 and D4, 5-hydroxyeicosatetraenoic acid, PGD2, Platelet Activating Factor (PAF), heparin, and high-molecular-weight neutrophil chemotactic factor and cytokines/chemokines. PGD2 exerts its biological activity through the DP and CRTH2 receptors and their cDNA cloning which were characterized 15 years ago. In this report, we revisited the biological effects of arachidonic acid compounds released by activated mast cells in allergic and inflammatory states.

Evidence that 13–14 di-hydro, 15-keto prostaglandin D2-induced airway eosinophilia in guinea-pigs is independent of interleukin-5

Prostaglandin D(2) (PGD(2)) has been shown to cause eosinophil, basophil and Th2 cell chemotaxis in vitro and in vivo through an action on the prostaglandin CRTH2 receptor. In the present study, the dependence of PGD(2)-induced eosinophil accumulation in vivo on interleukin-5 (IL-5) blood eosinophilia was investigated. Guinea-pigs were exposed to aerosols of 13,14di-hydro 15-keto PGD(2) (DK-PGD(2)) or platelet activating factor (PAF) and the eosinophil content of the bronchoalveolar lavage fluid or blood determined. In some experiments, DK-PGD(2) was administered systemically and eosinophilia measured. Aerosols of DK-PGD(2) caused eosinophil accumulation in the lungs 24h after exposure. DK-PGD(2) (10 microg x ml(-1)) -induced airway eosinophilia was inhibited when animals were treated with the CRTH2 receptor antagonist ramatroban. 1-4h after exposure to DK-PGD(2) a significant decrease in blood eosinophil count was measured. The anti-IL-5 antibody TRFK-5 had no inhibitory effect of DK-PGD(2)-induced airway eosinophilia, but abolished airway eosinophilia induced by exposure of guinea-pigs to aerosols of PAF. Intracardiac injection of DK-PGD(2) induced a dose-related increase in blood eosinophil numbers. It is concluded that, in the guinea-pig, DK-PGD(2)-induced airway eosinophilia is mediated by an action on prostaglandin CRTH2 receptors and that this response appears to be independent of IL-5.

Effects of PGH2 and PGD2 on CRTH2 and DP receptors in primary cells and co-expressed in HEK293 cells

Prostaglandin (PG) D2 is a PGH2 metabolite deriving from the cyclooxygenase pathway and the major prostanoid released from activated human mast cells. The biological effects of PGD2 are mediated by the G protein-coupled receptors (GPCRs) D-type prostanoid receptor (DP) and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). Eosinophils, important effector cells in allergy, express both receptors. Activation of CRTH2 has been shown to result in pro-inflammatory responses, while the role of DP in allergic inflammation is still unclear. In this study we show that PGH2 selectively stimulates human peripheral blood eosinophils and basophils, but not neutrophils, and this effect is prevented by the CRTH2 receptor antagonist, Cay10471. In chemotaxis assays, eosinophils showed a pronounced migratory response towards PGH2, while eosinophil degranulation was inhibited by PGH2. Moreover, collagen-induced platelet aggregation was inhibited by PGH2 in platelet-rich plasma, which was abrogated in the presence of the DP antagonist, BWA868c. HEK293 cells transfected with either human CRTH2 or DP responded with Ca2+ flux, while untransfected HEK293 cells showed no response. These data indicate that PGH2 causes activation of the PGD2 receptors, CRTH2 and DP, even in the absence of functional PGD synthase. In further experiments, CRTH2 and DP receptors were stably co-expressed in HEK293 cells as a tool to explore receptor signalling and to investigate possible receptor heterodimerization. Data will be shown that demonstrate possible combinatorial effects of CRTH2 and DP to selective and non-selective agonists and antagonists in Ca2+ signalling assays.

Prostaglandin D2 Receptors Control Osteoclastogenesis and the Activity of Human Osteoclasts

We recently showed that human osteoblasts synthesize prostaglandin D(2) (PGD(2)) and express both DP and CRTH2 receptors. Activation of the DP receptor decreased osteoprotegerin production, whereas activation of the CRTH2 receptor induced osteoblast chemotaxis and decreased RANKL expression. Our objectives in this study were to determine the presence, distribution, and action of these receptors in the functions of human osteoclasts and in osteoclastogenesis. Immunohistochemistry was used to detect the presence of DP and CRTH2 in in vitro-differentiated human osteoclasts in culture and in osteoclasts in situ. The effects of the activation of PGD(2) receptors on the cytoskeleton were determined by fluorescence microscopy. Specific agonists and antagonists allowed the study of the roles of these receptors on bone resorption and osteoclast differentiation. Our results show that in vitro-differentiated human osteoclasts and authentic fetal osteoclasts express both DP and CRTH2 receptors, as shown by immunocytochemistry. Similar results were obtained in osteoclasts from normal, osteoporotic, pagetic, and osteoarthritic adult bone tissues. Stimulation of osteoclasts with PGD(2) induced a robust reorganization of the cytoskeleton with a decrease in the number of cells presenting actin rings and an increase of lamellipodia, effects mediated by the DP and CRTH2 receptors, respectively. PGD(2) showed an inhibitory effect on bone resorption activity acting through the DP receptor. In vitro osteoclastogenesis from peripheral blood mononuclear cells cultured in the presence of RANKL and macrophage-colony stimulating factor was decreased by activation of either DP or CRTH2 receptors. These results suggest that PGD(2) receptors could be useful targets in certain bone diseases because their specific activation/inhibition leads to a decrease in osteoclastogenesis and to inhibition of bone resorption by osteoclasts.

The Role of the Prostaglandin D2 Receptor, DP, in Eosinophil Trafficking

Prostaglandin (PG) D2 is a major mast cell product that acts via two receptors, the D-type prostanoid (DP) and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) receptors. Whereas CRTH2 mediates the chemotaxis of eosinophils, basophils, and Th2 lymphocytes, the role of DP has remained unclear. We report in this study that, in addition to CRTH2, the DP receptor plays an important role in eosinophil trafficking. First, we investigated the release of eosinophils from bone marrow using the in situ perfused guinea pig hind limb preparation. PGD2 induced the rapid release of eosinophils from bone marrow and this effect was inhibited by either the DP receptor antagonist BWA868c or the CRTH2 receptor antagonist ramatroban. In contrast, BWA868c did not inhibit the release of bone marrow eosinophils when this was induced by the CRTH2-selective agonist 13,14-dihydro-15-keto-PGD2. In additional experiments, we isolated bone marrow eosinophils from the femoral cavity and found that these cells migrated toward PGD2. We also observed that BWA868c inhibited this response to a similar extent as ramatroban. Finally, using immunohistochemistry we could demonstrate that eosinophils in human bone marrow specimens expressed DP and CRTH2 receptors at similar levels. Eosinophils isolated from human peripheral blood likewise expressed DP receptor protein but at lower levels than CRTH2. In agreement with this, the chemotaxis of human peripheral blood eosinophils was inhibited both by BWA868c and ramatroban. These findings suggest that DP receptors comediate with CRTH2 the mobilization of eosinophils from bone marrow and their chemotaxis, which might provide the rationale for DP antagonists in the treatment of allergic disease.