Δ12-Prostaglandin D2 is a potent and selective CRTH2 receptor agonist and causes activation of human eosinophils and Th2 lymphocytes

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Abstract

Prostaglandin D2 (PGD2) is a lipid mediator produced by mast cells, macrophages and Th2 lymphocytes and has been detected in high concentrations in the airways of asthmatic patients. There are two receptors for PGD2, namely the D prostanoid (DP) receptor and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). The proinflammatory effects of PGD2 leading to recruitment of eosinophils and Th2 lymphocytes into inflamed tissues is thought to be predominantly due to action on CRTH2. Several PGD2 metabolites have been described as potent and selective agonists for CRTH2. In this study we have characterized the activity of Δ12-PGD2, a product of PGD2 isomerization by albumin. Δ12-PGD2 induced calcium mobilization in CHO cells expressing human CRTH2 receptor, with efficacy and potency similar to those of PGD2. These effects were blocked by the TP/CRTH2 antagonist ramatroban. Δ12-PGD2 bound to CRTH2 receptor with a pKi of 7.63, and a 55-fold selectivity for CRTH2 compared to DP. In Th2 lymphocytes, Δ12-PGD2 induced calcium mobilization with high potency and an efficacy similar to that of PGD2. Δ12-PGD2 also caused activation of eosinophils as measured by shape change. Taken together, these results show that Δ12-PGD2 is a potent and selective agonist for CRTH2 receptor and can cause activation of eosinophils and Th2 lymphocytes. These data also confirm the selective effect of other PGD2 metabolites on CRTH2 and illustrate how the metabolism of PGD2 may influence the pattern of leukocyte infiltration at sites of allergic inflammation.

Introduction

Prostaglandin D2 (PGD2) is an eicosanoid, a class of lipid mediator synthesised by cells in response to local tissue damage, or hormonal stimuli. Eicosanoids bind to specific cell surface receptors on a wide variety of tissues throughout the body and mediate diverse effects in these tissues. PGD2 is known to be produced by mast cells, macrophages and Th2 lymphocytes and has been detected in high concentrations in the airways of allergic asthmatic patients challenged with antigen [1]. Instillation of PGD2 into airways can provoke many features of the asthmatic response, including bronchoconstriction [2], [3] and eosinophil accumulation [4]. The potential of exogenously applied PGD2 to cause inflammatory responses has been confirmed by the use of transgenic mice overexpressing human PGD synthase, where endogenous production of PGD2 is linked to exaggerated eosinophilic lung inflammation and Th2 cytokine production in response to antigen [5]. In addition, PGD2 was shown to enhance production of IL-4 and IL-5 and accumulation of eosinophils, lymphocytes, and macrophages in the lung of ovalbumin-sensitized wild-type mice [6]. The first receptor specific for PGD2 to be discovered was the D prostanoid (DP) receptor, which is linked to the elevation of intracellular cAMP [7], [8]. However, PGD2 is thought to mediate much of its proinflammatory activity through interaction with another G protein-coupled receptor termed CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) which is expressed by Th2 lymphocytes, eosinophils and basophils [9], [10]. Both DP and CRTH2 receptors have been postulated to contribute to the development of allergic inflammation [8], [11], [12]. The exact role played by each receptor in the process of inflammation remains unclear but the general view suggests that signals through CRTH2 receptor are pro-inflammatory, whereas signals through DP receptor may either be pro-inflammatory or anti-inflammatory, depending on the setting and the target cells [11], [12], [13]. However, with respect to activation of Th2 lymphocytes and eosinophils, the effects of PGD2 are mediated by CRTH2 [11].

PGD2, like other prostanoids is rapidly metabolized in vivo and has an estimated half-life of 1.5 min in the blood [14]. It is therefore conceivable that some of the biological effects of PGD2 might be due to the activity of some of its metabolites rather than to PGD2 itself. Indeed, several PGD2 metabolites have been recently characterized as potent eosinophil activators, including 13,14 dihydro-15-keto-PGD2 (dk-PGD2), 15-deoxy-Δ12,14-PGJ2, and 15-deoxy-Δ12,14-PGD2 [9], [10], [15]. In addition, dk-PGD2 was reported to be selective for CRTH2 over DP receptor [9], [16]. Furthermore, Δ12-PGJ2, a plasma metabolite of PGD2 [17], [18] that can be detected in relevant amounts in human urine [19], can induce mobilization of eosinophils from the bone marrow and prime eosinophils for chemotaxis [20]. These data suggest that (i) the metabolism of PGD2 may be important in defining the pattern of leukocyte infiltration in allergic disease, and (ii) CRTH2 is important in mediating both the local and systemic effect of PGD2 and its metabolites.

In the present study, we report the activity of several PGD2 metabolites, and show that Δ12-PGD2, a product of PGD2 isomerization by albumin, is a potent CRTH2 agonist that can cause activation of both human eosinophils and Th2 lymphocytes. Our study also demonstrates that Δ12-PGD2 binds selectively to CRTH2 compared to DP receptor.

Section snippets

Materials

Calcium-3 dye was purchased from Molecular Devices (Wokingham, UK). Mono-poly resolving medium was obtained from Dainippon Pharmaceuticals (Osaka, Japan). MACS anti-CD 16 microbeads were from Miltenyi Biotec (Bisley, Surrey). Poly-d-lysine coated 96-well plates were obtained from Greiner (Gloucestershire, UK). [3H]-PGD2 was from Amersham Biosciences (Buckinghamshire, UK). PGD2 and its metabolites were obtained from Cayman Chemical (Ann Arbor, MI) or from StylaCats Limited (Cheshire, UK).

Calcium mobilization in CHO/CRTH2

We first investigated the effect of PGD2 and Δ12-PGD2 on intracellular calcium mobilization, using CHO/CRTH2 cells. Both compounds induced an increase in intracellular calcium mobilization, in a concentration-dependent manner, with pEC50 values of 8.52 ± 0.06 and 8.45 ± 0.13 (n = 4), for PGD2 and Δ12-PGD2, respectively (Fig. 1a, Table 1). Δ12-PGD2 behaved as a full agonist (compared to PGD2, Fig. 1a). The effect of Δ12-PGD2 and PGD2 was completely abolished by Bordella Pertussis toxin (PTX)

Discussion

In the present study, we have analysed the pharmacological properties of Δ12-PGD2, a metabolite of PGD2 that can be generated by isomerization of PGD2 by albumin [23]. Our data constitute compelling evidence that Δ12-PGD2 is a potent and selective agonist for CRTH2 receptor, in both primary leukocytes and transfected mammalian cells. In CHO cells transfected with CRTH2 receptor, Δ12-PGD2 mimicked the effect of PGD2, stimulating calcium mobilization with potency comparable to that of PGD2. The

Acknowledgements

We are grateful to Evotec-OAI for the generation of stable cell lines and the synthesis of ramatroban. We thank Caroline Sell for her assistance with tissue culture.

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