The small lipid mediator prostaglandin D₂ (PGD₂) is the major cyclooxygenase metabolite of arachidonic acid and is released by activated mast cells in response to allergen exposure 1. PGD₂ has long been considered a potentially important mediator in several diseases such as asthma, allergic rhinitis, atopic dermatitis, and allergic conjunctivitis 2345. PGD₂ elicits biological responses by interaction with three specific seven-transmembrane receptors, referred to as DP/DP1, DP2/CRTH2, and TP (DP, D prostanoid receptor; CRTH2, chemoattractant receptor homologous molecule expressed on T helper type 2 cells; TP, thromboxane A₂ receptor) 678. Via interaction with one (or a combination) of its three specific receptors PGD₂ may contribute to bronchoconstriction, eosinophilia and mucus production in allergic asthma. However, assessment of actual roles of PGD₂ in allergic diseases has been hampered by its very short biological half-life and the lack of specific receptor antagonists suitable to uncover how signaling of individual PGD₂ receptors contribute to disease processes in vivo. It is also possible that the contribution of PGD2 to allergic airway inflammation is easily missed if the load of allergen in challenge studies is too large 9.

CRTH2 is expressed on eosinophils, TH₂ cells and basophils, which are all considered to contribute to the pathogenesis of allergic diseases 3101112131415. Several lines of evidence suggest that activation of CRTH2 in response to PGD₂ mediates recruitment of inflammatory cells in vitro and in vivo. In vitro, activation of CRTH2 induces chemotaxis of TH₂ cells, eosinophils, and basophils 716. In vivo, CRTH2 mediates mobilization of eosinophils from guinea-pig bone marrow 17, promotes eosinophilia and exacerbates pathology in mouse models of allergic asthma and atopic dermatitis 18, and induces eosinophil infiltration into the airways upon intratracheal administration of PGD₂ or a selective CRTH2 agonist 192021. Based on evidence supporting a pro-inflammatory role of CRTH2, this receptor has attracted great interest as a drug target for therapeutic intervention in allergic diseases. Confusingly, however, allergic mice that lack a functional CRTH2 receptor and hence are incapable to signal through CRTH2 have been reported to exhibit both increased 22 and reduced 2324 allergic inflammation in models of asthma 222324 and atopic dermatitis 23. These diverging reports involving gene-deficient animals further underscore the need to use specific CRTH2 antagonists to explore the in vivo function of CRTH2.

It was recently reported that ramatroban, which was initially developed as a TP antagonist and is now used for treatment of allergic rhinitis in Japan, also displays potent CRTH2 antagonistic activity 25. Consistent with this finding, ramatroban has been shown to abrogate blood eosinophilia induced by a CRTH2-specific agonist in rats 19 and to inhibit PGD₂-stimulated human eosinophil migration in vitro 25. While these data are congruent with the notion that ramatroban acts through inhibition of CRTH2 receptors, it is not clear whether its clinical efficacy in allergic rhinitis is due to inhibition of TP, CRTH2 or both receptors. We have recently reported a ramatroban analog (given internal code number TM30089) with high antagonistic potency on and selectivity for human CRTH2 and devoid of affinity to the TP receptor 2627.

Here we report that TM30089 is a highly potent antagonist on mouse CRTH2 and lacks affinity to mouse TP in agreement with its pharmacological properties on the human receptor orthologs. In addition, this study demonstrates that TM30089 does not display appreciable affinity to a range of other receptors and enzymes that have potentially important roles in allergic asthma. Furthermore, using a mouse model of allergic asthma involving two allergen challenges, we have discovered that TM30089 similar to ramatroban inhibits allergen challenge-induced airway tissue eosinophilia and mucus cell hyperplasia. These in vivo data support the promise of CRTH2 as a target for treatment of allergic airways diseases.

Emerging evidence suggests that PGD₂-activation of its high affinity receptor CRTH2 may be particularly critical in the pathogenesis of eosinophilic airway inflammation, since activation by PGD₂ of CRTH2 potently stimulates chemotaxis of eosinophils in vitro and in vivo 719202125414243. Herein, we demonstrate that the small molecule TM30089 is a highly potent and selective inhibitor of mouse CRTH2 function in vitro, and by using this antagonist demonstrate for the first time that inhibition of CRTH2 signaling in vivo suppresses the development of certain key features characteristic for allergic asthma.

In vitro, TM30089, which is structurally closely related to ramatroban, was found to bind mouse CRTH2 with nanomolar affinity and potently inhibit its signaling in cells overexpressing the receptor. In addition, TM30089 completely lacks affinity to the mouse TP receptor, unlike ramatroban which represents a dual TP/CRTH2 antagonist on both human and mouse receptor orthologs 2526 (this study). Furthermore TM30089 in concentrations up to 10 μM did not show significant binding to or inhibition of selected chemokine receptors, anaphylatoxin receptors, the other high affinity PGD₂ receptor DP as well as the cyclooxygenases 1 and 2. Owing to its high selectivity over all other tested receptors and enzymes, and the fact that its chemical structure is closely related to ramatroban which has proven efficacious in various animal and human studies of allergic rhinitis and asthma 44454647484950, TM30089 emerges a suitable research tool to explore the contribution of CRTH2-signaling in allergic airway inflammation in vivo.

We found that administration of TM30089 displayed anti-inflammatory efficacy in a mouse model of allergic asthma that mimics some of the major histopathological features characteristic of human asthma such as eosinophilia and mucus cell hyperplasia 38. In the present study we used allergen challenges during only two consecutive days which proved to be sufficient for causing goblet cell hyperplasia as well as a significant accumulation of eosinophils in airway-pulmonary tissues. More challenges for more days would have increased the eosinophilia further. However, the present model was preferred because it had been reported that efficacy of interference with PGD₂ is dependent on the allergen load 9. Thus, the marked protection against allergen-induced asthma observed in DP-/- mice was completely lost when more than three allergen challenges were used 9. It is of note that although the present number of provocations was low the load of allergen would still be far greater than the level of allergens human asthmatics are exposed to.

The present observation of increased BALF eosinophils agrees with previous data indicating that already during the build-up of tissue eosinophilia by allergen exposure these cells begin to be lost into the airway lumen 32. Yet, studies resorting exclusively to the determination of BALF eosinophilia run the risk of drawing incorrect conclusions about changes in eosinophil numbers in the most important locale, the airway-pulmonary tissues. For example, we and others have previously observed in allergic mice that drug interventions may inhibit lumen eosinophilia whilst the tissue eosinophilia remains unchanged or is, indeed, increased as reviewed in 34. It was, therefore, important to note in this study that both drug treatments tended to reduce BALF eosinophils demonstrating that increased elimination of these cells into the airway lumen could not explain the present inhibitory effects of these drugs on airway tissue eosinophilia. Using TM30089, we thus unravel that CRTH2-signaling appears integral to the recruitment of eosinophils to the airways in vivo. So far, it has only been demonstrated that externally administered PGD₂ is able to induce local eosinophilia in different models of inflammation 1819202151, and that CRTH2 is the cellular mediator for this effect. However, whether CRTH2 signaling is relevant in a disease paradigm has not been established to date. The present finding that a small molecule inhibitor of CRTH2 is effectively attenuating eosinophil trafficking to the airway tissues hence suggests that CRTH2 is an important effector in this OVA-induced asthma model and regulates allergic inflammation in vivo. Since recruitment of eosinophils to inflammatory sites is considered a critical parameter in asthma and other allergic diseases 111213, our findings also highlight the potential importance of CRTH2 as a novel therapeutic target.

Interestingly, the selective CRTH2 antagonist TM30089 equals the dual TP/CRTH2 antagonist ramatroban regarding inhibition of eosinophil recruitment in vivo. Ramatroban has previously not only been shown to attenuate airway inflammation in guinea pig and mouse asthma models 45, but also been effective in inhibiting eosinophil infiltration into the nasal mucosa in patients suffering from allergic rhinitis 48. It remained elusive, however, whether inhibition of CRTH2 or TP or both receptors accounted for its anti-inflammatory efficacy in these studies. Potentially, inhibition by ramatroban of eosinophil trafficking may be explained by two different mechanisms: (i) blockade of TXA₂-mediated expression of adhesion molecules on endothelial cells and/or (ii) direct inhibition of CRTH2-dependent eosinophil migration 752. The present finding that both, a dual TP/CRTH2 antagonist as well as a selective CRTH2 antagonist are comparable in their ability to prevent peribronchial eosinophil infiltration in OVA-sensitized mice therefore is indicative of the notion that exclusive inhibition of CRTH2 may also be sufficient to prevent eosinophil infiltration into the airway tissues in allergic humans. Thus, it is tempting to speculate that efficacy of ramatroban is likely related to inhibition of CRTH2 rather than inhibition of TP both in rodent asthma models and in humans.

Other characteristic features of allergic asthma are mucus hypersecretion and airway remodeling 353. Mice challenged by inhalation of aerosolized ovalbumin showed marked goblet cell hyperplasia, which was significantly attenuated in animals treated with the dual TP/CRTH2 antagonist ramatroban or the selective CRTH2 antagonist TM30089. Interestingly, exclusive inhibition of CRTH2 by TM30089 was equally effective as compared with dual TP/CRTH2 antagonsim in ameliorating goblet cell hyperplasia. This finding is intriguing since corticosteroids, which now are mainstay asthma treatment, have variable effects on suppression of goblet cell hyperplasia 54, and offers the exciting perspective that selective inhibition of CRTH2 may be beneficial to achieve clinical improvement of allergic asthma. Further studies are warranted to investigate the molecular basis for the beneficial effects of CRTH2 antagonists in experimental allergic asthma in more detail.

Recent reports dealing with CRTH2-deficient mice have generated a highly inconsistent picture of receptor function. On one hand, OVA-sensitized mice lacking CRTH2 displayed enhanced occurrence of eosinophils in BALF 22. Unfortunately, data on airway tissue eosinophils were not presented 22. On the other hand and in apparent contrast, CRTH2-deficient mice from another laboratory were protected from bronchial hyperresponsiveness, and mucus production 24. Furthermore, from a third laboratory IgE-mediated skin eosinophilic inflammation was significantly reduced in CRTH2-deficent mice compared to wild type animals 23. These latter authors 23 also reported that they could not confirm the previously reported increase in lung eosinophilia 22 and they stated that only further studies including CRTH2 antagonists will contribute to final conclusions in this field. Although the specific contribution of CRTH2 in allergic inflammation thus is inconsistent in studies using mice with targeted gene disruption, the findings of the present study are congruent with the notion that CRTH2 represents an eosinophilotactic receptor and provide strong support for the concept that CRTH2-signaling in vivo is an important molecular step in the pathogenesis of allergic asthma.

It is intriguing to note that CRTH2 is not only activated by PGD₂ and several of its metabolites the generation of which is dependent on the enzyme PGD synthase, but also by products of the arachidonic acid cascade that are generated independent of PGD₂ production. Among the latter are the TXA₂ metabolite 11-dehydro-TXB₂, and PGF_2α, that have recently been demonstrated to activate eosinophils and basophils [43, 55]. The possibility that CRTH2 can be activated in cellular contexts where PGD synthase is not present, i.e. in the absence of PGD₂ production, further corroborates its importance as a regulator of allergic inflammation and underscores the potential usefulness of CRTH2 antagonists as anti-asthmatic agents.

Our study is the first to demonstrate efficacy of a small molecule selective CRTH2 antagonist in an experimental model of eosinophilic airway inflammation, and indicates that CRTH2-signaling is integral for orchestrating some of the pathological features characteristic for allergic asthma such as recruitment of eosinophils and mucus cell hyperplasia. Although we cannot rule out at present that inhibition of both TP and CRTH2 may be superior to obtain clinical efficacy in allergic diseases, our data certainly suggest that blockade of CRTH2 alone is sufficient to yield anti-inflammatory efficacy in experimental asthma, even to the extent that this mechanism may explain the efficacy of ramatroban. Although the OVA-induced asthma model does not reproduce all the features of the human disease, we propose that selective CRTH2 antagonists represent a novel and promising therapeutic approach to treat allergic asthma and related inflammatory diseases.

The author(s) declare that they have no competing interests.

LU, JMM, LA, MK, TU, TH, GA, CGAP, EK participated in the design and performance of the study and LU, JMM, TU, CGAP and EK drafted and wrote the manuscript. All authors read and approved the final manuscript.