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 [7, 19–21, 25, 41–43]. 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 [25, 26] (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 [44–50], 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 . 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 . Thus, the marked protection against allergen-induced asthma observed in DP-/- mice was completely lost when more than three allergen challenges were used . 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 . 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 . 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 [18–21, 51], 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 [11–13], 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 , but also been effective in inhibiting eosinophil infiltration into the nasal mucosa in patients suffering from allergic rhinitis . 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 [7, 52]. 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 [3, 53]. 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 , 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 . Unfortunately, data on airway tissue eosinophils were not presented . On the other hand and in apparent contrast, CRTH2-deficient mice from another laboratory were protected from bronchial hyperresponsiveness, and mucus production . Furthermore, from a third laboratory IgE-mediated skin eosinophilic inflammation was significantly reduced in CRTH2-deficent mice compared to wild type animals . These latter authors  also reported that they could not confirm the previously reported increase in lung eosinophilia  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 PGF2α, 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.