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)D 2 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. PGD 2 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. PGD 2 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 asthReceived: April 13, 2010 Accepted: April 13, 2010 Published online: June 16, 2010 Univ. Prof. Dr. Akos Heinemann Institute of Experimental and Clinical Pharmacology, Medical University of Graz Universitätsplatz 4 AT–8010 Graz (Austria) Tel. +43 316 380 4508, Fax +43 316 380 9645, E-Mail akos.heinemann @ medunigraz.at © 2010 S. Karger AG, Basel 0031–7012/10/0856–0372$26.00/0 Accessible online at: www.karger.com/pha D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /4 /2 01 7 6: 06 :4 8 P M PGD 2 Receptors as Therapeutic Targets Pharmacology 2010;85:372–382 373 frequently ineffective or, like corticosteroids, might have serious side effects [1, 2] . Therefore, demand persists for additional treatments of allergic diseases, particularly for those patients who need to avoid corticosteroids (e.g. children), and those who are insufficiently treated by standard therapeutic regimens. In the case of bronchial asthma, these patients rely mainly on bronchodilator therapy and are at risk of suffering both from side effects of these drugs and uncontrolled pulmonary inflammation leading to airway remodeling [3] . The discovery of the orphan G protein-coupled receptor GPR44, its expression pattern (basophils, eosinophils and type 2 T helper, Th2, cells) [4] and its ensuing deorphanization as a receptor for the mast cell-derived mediator prostaglandin (PG)D 2 [5] has fueled additional research in the field. This novel receptor, meanwhile termed ‘chemoattractant receptor-homologous molecule expressed on Th2 cells’ (CRTH2), has thus provided an innovative and promising target for antiinflammatory drug development. Role of PGD 2 in Allergic Inflammation Like other prostanoids, PGD 2 is synthesized by phospholipase A 2 , cyclooxygenase and specific terminal synthases upon activation of cells by different stimuli such as allergens, oxidative stress or cytokines. PGD 2 is the major mast cell product that is released during anaphylaxis [6, 7] and substantial evidence has accumulated in the last few years that PGD 2 might be involved in the initiation and perpetuation of allergic inflammation. However, PGD 2 is produced in significant amounts also by dendritic cells, macrophages, eosinophils, Th2 cells and endothelial cells [8–14] . A prominent contribution of PGD 2 to the late-phase allergic reaction is, on the one hand, suggested by enhanced eosinophilic lung inflammation and cytokine release in transgenic mice overexpressing PGD synthase [15] . Moreover, PGD 2 enhances leukotriene (LT) C 4 synthesis by eosinophils during allergic inflammation [16] . Conversely, an inhibitor of hematopoietic PGD synthase attenuates the antigen-induced production of PGD 2 and ameliorates airway inflammation in vivo in mice [17] . Interestingly, the biosynthesis of PGD 2 increases after the intake of cyclooxygenase inhibitors in aspirin-intolerant patients [18–20] . The resulting imbalance between PGD 2 and PGE 2 has been suggested to favor the development of asthma and nasal polyposis [21] . On the other hand, PGD 2 has been suggested to play a beneficial role in the resolution phase of inflammation by controlling Th1-mediated mechanisms [22, 23] . Receptors for PGD 2 The biological effects of PGD 2 are, in principle, mediated by two distinct G protein-coupled receptors, D-type prostanoid receptor (DP) and CRTH2. Moreover, at higher concentrations, PGD 2 is a ligand for the thromboxane (TX) receptor, which mediates the bronchoconstrictor effect of PGD 2 [24, 25] . Chemoattractant Receptor-Homologous Molecule Expressed on Th2 Cells CRTH2, also known as DP 2 , GPR44 or CD294, has initially been found on Th2 cells, eosinophils and basophils [4] , and mediates their chemotaxis towards PGD 2 [5, 26] . However, the CRTH2 receptor is activated not only by PGD 2 but also by several PGD 2 metabolites – including 13,14-dihydro-15-keto-PGD 2 , PGJ 2 , 12 PGJ 2 , 12 PGD 2 , 9 ,11 -PGF 2 and 15-deoxy-PGJ 2 [27–30] –, by PGF 2 [30] , a thromboxane metabolite, 11-dehydroTXB 2 [31] , and also by the precursor of all 2-series PG and TX, PGH 2 [32] . In animal models, exogenously administered CRTH2 agonists can induce eosinophil infiltration into the lungs and skin, and aggravate the pathology of allergic responses [33–35] . Moreover, several experimental studies in mice have shown that CRTH2 antagonists can ameliorate allergen-induced cutaneous, pulmonary and upper respiratory inflammation [36–41] . In humans, sequence variants of the gene encoding CRTH2 are associated with asthma and allergic phenotypes [42] . In addition, recent data suggest that CRTH2 is also expressed on monocytes [43] and macrophages, and mediates their migration induced by PGD 2 and endotoxin [8, 44] . Strikingly, PGD 2 levels in bronchoalveolar lavage fluid of patients with chronic obstructive pulmonary disease correlate inversely with lung function [45] . Therefore, using CRTH2 antagonists is currently being considered as a highly promising approach to the treatment of allergic diseases and asthma. Interestingly, a CRTH2 antagonist is currently being evaluated in phase II trials in patients with chronic obstructive pulmonary disease (clinical trial registration No. NCT00690482, NCT00766415). CRTH2 signals through G i proteins, leading to inhibition of cAMP formation and increases in intracellular Ca 2+ [5] . In eosinophils, however, we observed that CRTH2 stimulation leads to pertussis-toxin-insensitive activation of phosphatidylinositol 3-kinase (PI3K), phospholipase C, p38 mitogen-activated protein kinase (MAPK) and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase [46] . These pathways mediate eosinophil shape change, actin polymerization D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /4 /2 01 7 6: 06 :4 8 P M Schuligoi et al. Pharmacology 2010;85:372–382 374 and CD11b upregulation [26, 46] . The migration and survival of Th2 cells in response to PGD 2 -induced stimulation of CRTH2 depend on PI3K, while PGD 2 -evoked cytokine release involves PI3K, calcineurin and nuclear factor of activated T cells [47] . On the other hand, preactivation of p38 MAPK and glycogen synthase kinase-3 restrain the CRTH2-mediated migration of eosinophils and the cytokine section of Th2 cells, respectively [47, 48] . A recent study revealed that, unexpectedly, CRTH2 is not phosphorylated upon agonist stimulation, a mechanism by which the activity of most G protein-coupled receptors is typically regulated. Instead, the C terminus of CRTH2 inhibits G i signaling, which may compensate for the absence of the classical phosphorylation-dependent signal attenuation [49] . D-Type Prostanoid Receptor DP, also referred to as DP 1 , is expressed more widely than CRTH2, including platelets, several types of leukocytes, the vasculature, the central nervous system, retina, nasal mucosa, lungs and intestine [5, 50–54] . Activation of the DP receptor results in cutaneous and pulmonary venous vasodilatation, arterial hypotension, mucin secretion, inhibition of platelet aggregation and lowering of intraocular pressure [54–60] . DP has been described to play both antiinflammatory and proinflammatory roles: on the one hand, DP activation inhibits the function of neutrophils, basophils, dendritic cells, Langerhans cells, Th1 cells and natural killer cells [43, 61–67] . In vivo, inhalation of a selective DP agonist suppresses asthma in a murine model by downmodulation of lung dendritic cell function and induction of regulatory T cells [68] . In addition, PGD 2 but not CRTH2 agonists inhibit the scratching response of mice with atopic dermatitis [69] . Stimulation of DP enhances the barrier function of endothelial cells [70] and confers protection to neurons [71] . On the other hand, PGD 2 and DP receptors contribute to astrogliosis and demyelination in the twitcher mouse model [72] . PGD 2 synergizes with tumor necrosis factor via DP receptors to promote the production of monocyte chemotactic protein-1 and interleukin (IL)-8 in monocytic cells [73] . In vivo, a DP antagonist blocks antigen-induced rhinitis, conjunctivitis and eosinophil infiltration into the lung of the guinea pig [74, 75] and experimental asthma in sheep [76] . DP-deficient mice exhibit reduced pulmonary inflammation in response to allergens [77] . The DP receptor is expressed on bronchiole epithelial cells in antigen-challenged mice [77] , as well as nasal epithelial goblet cells in normal human volunteers [52] , suggesting that DP might stimulate mucus secretion in allergy. Moreover, an association of polymorphisms of the DP promoter and gene with asthma has been detected in humans [78] . These findings hence point to a proinflammatory role of DP, but are difficult to reconcile with the known functional responses to DP receptor activation. Despite the fact that it has comparable avidity for PGD 2 , the DP receptor shows little similarity with the CRTH2 receptor. In addition to PGD 2 , 12 PGD 2 , PGJ 2 and PGH 2 are also potent DP agonists, while other metabolites of PGD 2 such as 13,14-dihydro-15-keto-PGD 2 and 15-deoxy-PGJ 2 are selective for CRTH2 [5, 27, 29, 32, 79] . DP receptor activation leads to G s -mediated increases in intracellular cAMP although an agonistevoked Ca 2+ flux has also been observed in heterologous expression systems [50, 80] . The effects exerted by PGD 2 on dendritic cells are associated with the phosphorylation of cAMP response element-binding protein, but do not parallel with a deactivation of nuclear factor B and MAPK pathways [43] . So far, further downstream signaling pathways subsequent to DP activation have received very little attention. Interestingly, PGD 2 stimulation of fibroblast repair in an in vitro collagen contraction assay is mediated by DP and Ca 2+ -independent protein kinase C, but it is independent of cAMP and protein kinase A [81] . Regulation of Eosinophil Responses by PGD 2 Eosinophils play important roles in late-phase reactions by releasing bronchoconstrictor mediators such as LTC 4 and other chemoattractants which cause further influx of inflammatory cells into the tissue [82] . Like Th2 cells, eosinophils produce and release proinflammatory cytokines and growth factors including the immunoregulatory type 2 cytokines IL-4, IL-5, IL-10 and IL-13 [83] . Mucosal damage in chronic asthma is associated with cytotoxic and proinflammatory mediators that are released by activated eosinophils. Consequently, eosinophils also play a role in airway remodeling and angiogenesis in chronically inflamed tissue [84] . Importantly, it was shown that asthmatic patients who receive treatment based on eosinophil counts in sputum have significantly fewer severe asthma exacerbations than patients treated according to standard management therapy [85] . Moreover, animal studies also demonstrated that genetically modified mice lacking eosinophils are protected against allergen-induced lung injury and asthma [86, 87] . Therefore, eosinophils are currently considered a major therapeutic target in allergic diseases and asthma [88] , but they D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /4 /2 01 7 6: 06 :4 8 P M PGD 2 Receptors as Therapeutic Targets Pharmacology 2010;85:372–382 375 might also play pathogenic roles in several other diseases such as eosinophilic esophagitis [89] , colitis ulcerosa [90] , hypereosinophilic syndrome [91] or renal disease [92] . In many eosinophilic inflammatory diseases, eosinophilia has been attributed to reduced eosinophil apoptosis [93] . The Th2 cytokine IL-5 augments eosinophil recruitment and survival in experimental models of allergic inflammation. However, the limited clinical efficacy of antibodies against IL-5 in ameliorating asthma symptoms [94] suggests that additional mediators are also involved, such as granulocyte-macrophage colonystimulating factor or a plethora of chemoattractants that can stimulate the locomotion of eosinophils. In particular, these chemoattractants comprise the ligands of the chemokine receptor CCR3 [95] including eotaxin-1, -2 and -3, RANTES and the monocyte chemotactic peptides monocyte chemotactic protein-3 and -4. Moreover, activated complement factors such as C5a, and lipid mediators such as LTB 4 [96] , platelet activation factor [97] and 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxoETE) are also potent chemoattractants for eosinophils [98–100] . As mentioned above, PGD 2 and several of its metabolites are potent chemoattractants for eosinophils and this effect mainly relies on CRTH2 activation [5, 28, 32] . Moreover, we observed that PGD 2 and its metabolites are capable of stimulating the release of mature eosinophils from bone marrow [28, 101] . However, PGD 2 not only stimulates eosinophil trafficking but also activates eosinophils with respect to the respiratory burst and release of eosinophil cationic protein, and delays their apoptosis [28, 53] . While most PGD 2 effects in eosinophils are mediated by CRTH2, we could demonstrate that DP receptors are also involved in eosinophil chemotaxis, respiratory burst and mobilization from bone marrow [101, 102] . In contrast, the antiapoptotic effect of PGD 2 in eosinophils is solely mediated by DP receptors [53] . In addition to directly stimulating eosinophil migration, we also demonstrated that PGD 2 very efficiently primes eosinophils for other chemoattractants like eotaxin, 5-oxo-ETE or C5a [28, 48] . The priming effect of PGD 2 is exclusively mediated via CRTH2 receptors. In contrast, eosinophil migration towards PGD 2 is strikingly decreased by other chemoattractants such as eotaxin or 5-oxo-ETE, and PI3K as well as p38 MAPK are involved in these inhibitory effects. Conversely, the priming effect of PGD 2 is not reversed by inhibition of PI3K, p38 MAPK or peroxisome proliferator-activated receptor, which has been described to mediate the priming effect of the PGD 2 metabolite 15-deoxy12,14 -PGJ 2 [103] . Interestingly, available data also suggest that PGD 2 might inhibit eosinophil degranulation, as investigated by the C5a-induced upregulation of the granule-associated marker CD63 on eosinophils and the release of eosinophil peroxidase [32, 48, 101] . This effect is likewise mediated exclusively by CRTH2 receptors. From these studies we inferred that there is a hierarchy among eosinophil chemoattractants: PGD 2 might be an initial chemoattractant since it maintains its potency also in whole blood [48] and augments the responsiveness of eosinophils to other chemoattractants, but restrains eosinophil degranulation. In contrast, eotaxin seems to be an end point chemoattractant since it has a reduced efficacy in blood, is capable of downmodulating eosinophil responsiveness to other chemoattractants and stimulates eosinophil degranulation [48, 104] . CRTH2 expression levels are likely to be elevated in eosinophils from patients with allergic diseases, as suggested by observations in atopic dermatitis [105] . Role of PGD 2 Receptors in Regulation of Th2