Eva M. Sturm

CRTH2 and D-type prostanoid receptor antagonists as novel therapeutic agents for inflammatory diseases.

Accumulation of type 2 T helper (Th2) lymphocytes and eosinophils is a hallmark of bronchial asthma and other allergic diseases, and it is believed that these cells play a crucial pathogenic role in allergic inflammation. Thus, Th2 cells and eosinophils are currently considered a major therapeutic target in allergic diseases and asthma. However, drugs that selectively target the accumulation and activation of Th2 cells and eosinophils in tissues are unavailable so far. Prostaglandin (PG)D2 is a key mediator in various inflammatory diseases including allergy and asthma. It is generated by activated mast cells after allergen exposure and subsequently orchestrates the recruitment of inflammatory cells to the tissue. PGD2 induces the chemotaxis of Th2 cells, basophils and eosinophils, stimulates cytokine release from these cells and prolongs their survival, and might hence indirectly promote IgE production. PGD2 mediates its biologic functions via 2 distinct G protein-coupled receptors, D-type prostanoid receptor (DP), and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). DP and CRTH2 receptors are currently being considered as highly promising therapeutic targets for combating allergic diseases and asthma. Here, we revisit the roles of PGD2 receptors in the regulation of eosinophil and Th2 cell function and the efforts towards developing candidate compounds for clinical evaluation.

Prostaglandin E2 Inhibits Eosinophil Trafficking through E-Prostanoid 2 Receptors

The accumulation of eosinophils in lung tissue is a hallmark of asthma, and it is believed that eosinophils play a crucial pathogenic role in allergic inflammation. Prostaglandin (PG) E2 exerts anti-inflammatory and bronchoprotective mechanisms in asthma, but the underlying mechanisms have remained unclear. In this study we show that PGE2 potently inhibits the chemotaxis of purified human eosinophils toward eotaxin, PGD2, and C5a. Activated monocytes similarly attenuated eosinophil migration, and this was reversed after pretreatment of the monocytes with a cyclooxygenase inhibitor. The selective E-prostanoid (EP) 2 receptor agonist butaprost mimicked the inhibitory effect of PGE2 on eosinophil migration, whereas an EP2 antagonist completely prevented this effect. Butaprost, and also PGE2, inhibited the C5a-induced degranulation of eosinophils. Moreover, selective kinase inhibitors revealed that the inhibitory effect of PGE2 on eosinophil migration depended upon activation of PI3K and protein kinase C, but not cAMP. In animal models, the EP2 agonist butaprost inhibited the rapid mobilization of eosinophils from bone marrow of the in situ perfused guinea pig hind limb and prevented the allergen-induced bronchial accumulation of eosinophils in OVA-sensitized mice. Immunostaining showed that human eosinophils express EP2 receptors and that EP2 receptor expression in the murine lungs is prominent in airway epithelium and, after allergen challenge, in peribronchial infiltrating leukocytes. In summary, these data show that EP2 receptor agonists potently inhibit eosinophil trafficking and activation and might hence be a useful therapeutic option in eosinophilic diseases.

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.

The basophil activation test in the diagnosis of allergy: technical issues and critical factors.

Background:  The basophil activation test (BAT) is a widely validated and reliable tool especially for the diagnosis of hymenoptera venom allergy. Nevertheless, several pitfalls have to be considered and outcomes may differ because of diverse in‐house protocols and commercially available kits. We aimed to identify the factors that may influence results of the CD63‐based BAT.

CD203c‐based basophil activation test in allergy diagnosis: Characteristics and differences to CD63 upregulation

The basophil activation test (BAT) based on CD203c upregulation has been validated as a reliable tool for the diagnosis of IgE‐mediated allergies. Nevertheless, CD203c‐based BAT is hardly comparable with that of CD63‐based tests, as the mechanisms of CD203c versus CD63 induction differ considerably. The aim of the present study was to identify potent influencing factors of the CD203c‐based BAT and to emphasize differences between CD63 and CD203c detection.

Inconsistent Results of Diagnostic Tools Hamper the Differentiation between Bee and Vespid Venom Allergy

Background Double sensitization (DS) to bee and vespid venom is frequently observed in the diagnosis of hymenoptera venom allergy, but clinically relevant DS is rare. Therefore it is sophisticated to choose the relevant venom for specific immunotherapy and overtreatment with both venoms may occur. We aimed to compare currently available routine diagnostic tests as well as experimental tests to identify the most accurate diagnostic tool. Methods 117 patients with a history of a bee or vespid allergy were included in the study. Initially, IgE determination by the ImmunoCAP, by the Immulite, and by the ADVIA Centaur, as well as the intradermal test (IDT) and the basophil activation test (BAT) were performed. In 72 CAP double positive patients, individual IgE patterns were determined by western blot inhibition and component resolved diagnosis (CRD) with rApi m 1, nVes v 1, and nVes v 5. Results Among 117 patients, DS was observed in 63.7% by the Immulite, in 61.5% by the CAP, in 47.9% by the IDT, in 20.5% by the ADVIA, and in 17.1% by the BAT. In CAP double positive patients, western blot inhibition revealed CCD-based DS in 50.8%, and the CRD showed 41.7% of patients with true DS. Generally, agreement between the tests was only fair and inconsistent results were common. Conclusion BAT, CRD, and ADVIA showed a low rate of DS. However, the rate of DS is higher than expected by personal history, indicating that the matter of clinical relevance is still not solved even by novel tests. Furthermore, the lack of agreement between these tests makes it difficult to distinguish between bee and vespid venom allergy. At present, no routinely employed test can be regarded as gold standard to find the clinically relevant sensitization.

Influence of total IgE levels on the severity of sting reactions in Hymenoptera venom allergy.

Background:  Detection of specific IgE for Hymenoptera venoms and skin tests are well established diagnostic tools for the diagnosis of insect venom hypersensitivity. The aim of our study was to analyze the effect of total IgE levels on the outcome of generalized anaphylactic reactions after a Hymenoptera sting.

Hierarchy of eosinophil chemoattractants: role of p38 mitogen‐activated protein kinase

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) D2. 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 PGD2 enhancement of eosinophil migrations. In contrast, eotaxin or 5‐oxo‐ETE inhibited the migration of eosinophils towards PGD2. 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 mitogen‐activated protein kinase, and inhibition of p38 mitogen‐activated protein kinase by SB‐202190 converted the effect of 5‐oxo‐ETE on the chemotaxis to PGD2 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 PGD2 were unaltered. In conclusion, PGD2 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.

EP4 receptor stimulation down-regulates human eosinophil function

Accumulation of eosinophils in tissue is a hallmark of allergic inflammation. Here we observed that a selective agonist of the PGE2 receptor EP4, ONO AE1-329, potently attenuated the chemotaxis of human peripheral blood eosinophils, upregulation of the adhesion molecule CD11b and the production of reactive oxygen species. These effects were accompanied by the inhibition of cytoskeletal rearrangement and Ca2+ mobilization. The involvement of the EP4 receptor was substantiated by a selective EP4 antagonist, which reversed the inhibitory effects of PGE2 and the EP4 agonist. Selective kinase inhibitors revealed that the inhibitory effect of EP4 stimulation on eosinophil migration depended upon activation of PI 3-kinase and PKC, but not cAMP. Finally, we found that EP4 receptors are expressed by human eosinophils, and are also present on infiltrating leukocytes in inflamed human nasal mucosa. These data indicate that EP4 agonists might be a novel therapeutic option in eosinophilic diseases.

Prostaglandin H2 induces the migration of human eosinophils through the chemoattractant receptor homologous molecule of Th2 cells, CRTH2

The major mast cell product PGD2 is released during the allergic response and stimulates the chemotaxis of eosinophils, basophils, and Th2‐type T lymphocytes. The chemoattractant receptor homologous molecule of Th2 cells (CRTH2) has been shown to mediate the chemotactic effect of PGD2. PGH2 is the common precursor of all PGs and is produced by several cells that express cyclooxygenases. 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 (+)‐3‐[[(4‐fluorophenyl)sulfonyl] methyl amino]‐1,2,3,4‐tetrahydro‐9H‐carbazole‐9‐acetic acid (Cay10471) but not by the hematopoietic PGD synthase inhibitor 4‐benzhydryloxy‐1‐[3‐(1H‐tetrazol‐5‐yl)‐propyl]piperidine (HQL79). In chemotaxis assays, eosinophils showed a pronounced migratory response toward PGH2, but 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 D‐type prostanoid (DP) receptor antagonist 3‐[(2‐cyclohexyl‐2‐hydroxyethyl)amino]‐2,5‐dioxo‐1‐(phenylmethyl)‐4‐imidazolidine‐heptanoic acid (BWA868c). Each of these effects of PGH2 was enhanced in the presence of plasma and/or albumin. In eosinophils, PGH2‐induced calcium ion (Ca2+) flux was subject to homologous desensitization with PGD2. Human embryo kidney (HEK)293 cells transfected with human CRTH2 or DP likewise responded with Ca2+ flux, and untransfected HEK293 cells showed no response. These data indicate that PGH2 causes activation of the PGD2 receptors CRTH2 and DP via a dual mechanism: by interacting directly with the receptors and/or by giving rise to PGD2 after catalytic conversion by plasma proteins.