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.

Several chemoattractants can regulate the recruitment of eosinophils to sites of inflammation, but the hierarchy among them is unknown. We observed here that eosinophil chemotaxis towards eotaxin or 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) was amplified up to sixfold in the presence of prostaglandin (PG) D 2 . This effect was only seen in eosinophils, and not in neutrophils or basophils. Pretreatment with the chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2) antagonist ramatroban prevented the PGD 2 enhancement of eosinophil migrations. In contrast, eotaxin or 5-oxo-ETE inhibited the migration of eosinophils towards PGD 2 . 5-oxo-ETE enhanced the chemotaxis to eotaxin, while eotaxin had no effect on 5-oxo-ETE-induced migration. 5-oxo-ETE induced the phosphorylation of p38 mitogenactivated protein kinase, and inhibition of p38 mitogen-activated protein kinase by SB-202190 converted the effect of 5-oxo-ETE on the chemotaxis to PGD 2 from inhibition to enhancement. The presence of blood or plasma markedly decreased the sensitivity of eosinophils to eotaxin or 5-oxo-ETE, while responses to PGD 2 were unaltered. In conclusion, PGD 2 might be an initial chemoattractant, since it maintains its potency in the circulation and augments the responsiveness of eosinophils to other chemoattractants. In contrast, eotaxin seems to be an end-point chemoattractant, since it has reduced efficacy in blood and is capable of down-modulating eosinophil responsiveness to other chemoattractants. IntroductionAccumulation of eosinophils at sites of allergic reactions, such as eczema or asthma, is associated with tissue injury and lung dysfunction [1]. It has been shown recently that asthmatic patients that received treatment based on eosinophil counts in sputum had significantly fewer severe asthma exacerbations than patients treated according to standard management therapy [2]. Moreover, genetically modified mice lacking eosinophils are protected against allergen-induced lung injury and asthma [3,4].Although several chemoattractants can mediate eosinophil recruitment, none of them could be identified so far as a predominating factor governing eosinophil infiltration, suggesting a co-operative action of multiple chemoattractants in disease. Among these are the CC-chemokines, such as eotaxin, RANTES, or MCP-4 acting through the chemokine receptor CCR3 [5,6]. Prostaglandin (PG) D 2 , a major mast cell mediator released during the allergic response [7] might be of particular importance, since PGD 2 is capable of inducing the chemotaxis of eosinophils, basophils and Th2-type * These authors contributed equally to this study. T cells via a novel receptor, chemoattractant receptorhomologous molecule expressed on TH2 cells (CRTH2) [8,9]. Besides chemotaxis, we have shown that activation of CRTH2 also causes eosinophil respiratory burst and the release of eosinophils from the bone marrow [10]. In vivo, PGD 2 can induce pulmonary eosinophilia [11], and a significant contribution of PGD 2 to the late phase allergi...

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These data further emphasize the importance of CRTH2 in eosinophil function and show that Cay10471 is a highly potent and selective antagonist of PGD(2)-induced eosinophil responses. Cay10471 might hence be a useful compound for the treatment of allergic diseases.

Chemoattractant receptor-homologous molecule expressed on T helper 2 cells (CRTH2) has attracted interest as a potential therapeutic target in inflammatory diseases. Ramatroban, a thromboxane A 2 receptor antagonist with clinical efficacy in allergic rhinitis, was recently found to also display potent CRTH2 antagonistic activity. Here, we present the pharmacological profile of three ramatroban analogs that differ chemically from ramatroban by either a single additional methyl group (TM30642), or an acetic acid instead of a propionic acid side chain (TM30643), or both modifications (TM30089). All three compounds bound to human CRTH2 stably expressed in human embryonic kidney 293 cells with nanomolar affinity. [ 3 H]Prostaglandin D 2 (PGD2) saturation analysis reveals that ramatroban and TM30642 decrease PGD2 affinity, whereas TM30643 and TM30089 exclusively depress ligand binding capacity (B max ). Each of the three compounds acted as potent CRTH2 antagonists, yet the nature of their antagonism differed markedly. In functional assays measuring inhibition of PGD2-mediated 1) guanosine 5Ј-O-(3-thio)triphosphate binding, 2) ␤-arrestin translocation, and 3) shape change of human eosinophils endogenously expressing CRTH2, ramatroban, and TM30642 produced surmountable antagonism and parallel rightward shifts of the PGD2 concentration-response curves. For TM30643 and TM30089, this shift was accompanied by a progressive reduction of maximal response. Binding analyses indicated that the functional insurmountability of TM30643 and TM30089 was probably related to long-lasting CRTH2 inhibition mediated via the orthosteric site of the receptor. A mechanistic understanding of insurmountability of CRTH2 antagonists could be fundamental for development of this novel class of anti-inflammatory drugs.Prostaglandin D 2 (PGD2) is the major prostanoid released by activated mast cells and is implicated as proinflammatory mediator in diseases such as allergic rhinitis, atopic dermatitis, and asthma (Hata and Breyer, 2004). The prime mode of PGD2 action is through two G protein-coupled receptors referred to as DP/DP1 and CRTH2/DP2, respectively (Boie et al., 1995;Hirai et al., 2001). Both PGD2 receptors transduce extracellular signals predominantly by coupling to heterotrimeric G proteins. DP is positively linked to adenylyl cyclases via G␣ s proteins.

AZ11665362 is a potent CRTH2 antagonist that is capable of blocking the migration of eosinophils and basophils, and the rapid mobilization of eosinophils from bone marrow. AZ11665362 might hence be useful for the treatment of allergic diseases.

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