RESUMEN
BACKGROUND: Increased vascular permeability is a fundamental characteristic of inflammation. Substances that are released during inflammation, such as prostaglandin (PG) E(2), can counteract vascular leakage, thereby hampering tissue damage. OBJECTIVE: In this study we investigated the role of PGE(2) and its receptors in the barrier function of human pulmonary microvascular endothelial cells and in neutrophil trafficking. METHODS: Endothelial barrier function was determined based on electrical impedance measurements. Neutrophil recruitment was assessed based on adhesion and transendothelial migration. Morphologic alterations are shown by using immunofluorescence microscopy. RESULTS: We observed that activation of E-type prostanoid (EP) 4 receptor by PGE(2) or an EP4-selective agonist (ONO AE1-329) enhanced the barrier function of human microvascular lung endothelial cells. EP4 receptor activation prompted similar responses in pulmonary artery and coronary artery endothelial cells. These effects were reversed by an EP4 antagonist (ONO AE3-208), as well as by blocking actin polymerization with cytochalasin B. The EP4 receptor-induced increase in barrier function was independent of the classical cyclic AMP/protein kinase A signaling machinery, endothelial nitric oxide synthase, and Rac1. Most importantly, EP4 receptor stimulation showed potent anti-inflammatory activities by (1) facilitating wound healing of pulmonary microvascular endothelial monolayers, (2) preventing junctional and cytoskeletal reorganization of activated endothelial cells, and (3) impairing neutrophil adhesion to endothelial cells and transendothelial migration. The latter effects could be partially attributed to reduced E-selectin expression after EP4 receptor stimulation. CONCLUSION: These data indicate that EP4 agonists as anti-inflammatory agents represent a potential therapy for diseases with increased vascular permeability and neutrophil extravasation.
Asunto(s)
Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Neutrófilos/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Actinas/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Permeabilidad Capilar/fisiología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Cultivadas , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citocalasina B/farmacología , Dinoprostona/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/inmunología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/inmunología , Humanos , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Éteres Metílicos/farmacología , Microvasos/efectos de los fármacos , Microvasos/inmunología , Microvasos/metabolismo , Naftalenos/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fenilbutiratos/farmacología , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/fisiología , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The use of the lipid lowering agent niacin is hampered by a frequent flush response which is largely mediated by prostaglandin (PG) D(2). Therefore, concomitant administration of the D-type prostanoid (DP) receptor antagonist laropiprant has been proposed to be a useful approach in preventing niacin-induced flush. However, antagonizing PGD(2), which is a potent inhibitor of platelet aggregation, might pose the risk of atherothrombotic events in cardiovascular disease. In fact, we found that in vitro treatment of platelets with laropiprant prevented the inhibitory effects of PGD(2) on platelet function, i.e. platelet aggregation, Ca(2+) flux, P-selectin expression, activation of glycoprotein IIb/IIIa and thrombus formation. In contrast, laropiprant did not prevent the inhibitory effects of acetylsalicylic acid or niacin on thrombus formation. At higher concentrations, laropiprant by itself attenuated platelet activation induced by thromboxane (TP) and E-type prostanoid (EP)-3 receptor stimulation, as demonstrated in assays of platelet aggregation, Ca(2+) flux, P-selectin expression, and activation of glycoprotein IIb/IIIa. Inhibition of platelet function exerted by EP4 or I-type prostanoid (IP) receptors was not affected by laropiprant. These in vitro data suggest that niacin/laropiprant for the treatment of dyslipidemias might have a beneficial profile with respect to platelet function and thrombotic events in vascular disease.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Plaquetas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Indoles/farmacología , Agregación Plaquetaria/efectos de los fármacos , Subtipo EP3 de Receptores de Prostaglandina E/biosíntesis , Plaquetas/citología , Calcio/metabolismo , Dislipidemias/tratamiento farmacológico , Dislipidemias/metabolismo , Femenino , Humanos , Hipolipemiantes/uso terapéutico , Indoles/uso terapéutico , Masculino , Niacina/uso terapéutico , Selectina-P/biosíntesis , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/biosíntesis , Prostaglandina D2/metabolismo , Trombosis/metabolismo , Trombosis/prevención & controlRESUMEN
Eosinophil extravasation across the endothelium is a key feature of allergic inflammation. Here, we investigated the role of PGE(2) and its receptor, E-type prostanoid receptor (EP)-4, in the regulation of eosinophil interaction with human pulmonary microvascular endothelial cells. PGE(2) and the EP4 receptor agonist ONO AE1-329 significantly reduced eotaxin-induced eosinophil adhesion to fibronectin, and formation of filamentous actin and gelsolin-rich adhesive structures. These inhibitory effects were reversed by a selective EP4 receptor antagonist, ONO AE3-208. PGE(2) and the EP4 agonist prevented the activation and cell-surface clustering of ß2 integrins, and L-selectin shedding of eosinophils. Under physiological flow conditions, eosinophils that were treated with the EP4 agonist showed reduced adhesion to endothelial monolayers upon stimulation with eotaxin, as well as after TNF-α-induced activation of the endothelial cells. Selective activation of EP1, EP2, and EP3 receptors did not alter eosinophil adhesion to endothelial cells, whereas the EP4 antagonist prevented PGE(2) from decreasing eosinophil adhesion. Finally, eosinophil transmigration across thrombin- and TNF-α-activated endothelial cells was effectively reduced by the EP4 agonist. These data suggest that PGE(2) -EP4 signaling might be protective against allergic responses by inhibiting the interaction of eosinophils with the endothelium and might hence be a useful therapeutic option for controlling inappropriate eosinophil infiltration.
Asunto(s)
Comunicación Celular , Células Endoteliales/metabolismo , Eosinófilos/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Antígenos CD18/metabolismo , Adhesión Celular/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CCL11/farmacología , Ciclopentanos/farmacología , Dinoprostona/farmacología , Células Endoteliales/citología , Eosinófilos/citología , Fibronectinas/metabolismo , Citometría de Flujo , Gelsolina/metabolismo , Humanos , Selectina L/metabolismo , Éteres Metílicos , Microscopía Confocal , Naftalenos/farmacología , Fenilbutiratos/farmacología , Subtipo EP1 de Receptores de Prostaglandina E/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP3 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/agonistas , Subtipo EP4 de Receptores de Prostaglandina E/antagonistas & inhibidores , Tioglicolatos/farmacología , Trombina/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Accumulation of eosinophils in tissue is a hallmark of allergic inflammation. Here we observed that a selective agonist of the PGE(2) 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 Ca(2+) mobilization. The involvement of the EP4 receptor was substantiated by a selective EP4 antagonist, which reversed the inhibitory effects of PGE(2) 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.
Asunto(s)
Regulación hacia Abajo , Eosinófilos/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Antígeno CD11b/metabolismo , Calcio/metabolismo , Canales de Calcio/química , Canales de Calcio/metabolismo , Movimiento Celular , Ciclopentanos/farmacología , Dinoprostona/farmacología , Eosinófilos/efectos de los fármacos , Eosinófilos/inmunología , Humanos , Inflamación/metabolismo , Inflamación/patología , Isoindoles/farmacología , Éteres Metílicos , Mucosa Nasal/metabolismo , Mucosa Nasal/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/agonistas , Subtipo EP4 de Receptores de Prostaglandina E/antagonistas & inhibidores , Sulfonamidas/farmacología , Tioglicolatos/farmacologíaRESUMEN
OBJECTIVE: Low concentrations of prostaglandin (PG) E(2) enhance platelet aggregation, whereas high concentrations inhibit it. The effects of PGE(2) are mediated through 4 G protein-coupled receptors, termed E-type prostaglindin (EP) receptor EP1, EP2, EP3, and EP4. The platelet-stimulating effect of PGE(2) has been suggested to involve EP3 receptors. Here we analyzed the receptor usage relating to the inhibitory effect of PGE(2). METHODS AND RESULTS: Using flow cytometry, we found that human platelets expressed EP4 receptor protein. A selective EP4 agonist (ONO AE1-329) potently inhibited the platelet aggregation as induced by ADP or collagen. This effect could be completely reversed by an EP4 antagonist, but not by PGI(2), PGD(2), and thromboxane receptor antagonists or cyclooxygenase inhibition. Moreover, an EP4 antagonist enhanced the PGE(2)-induced stimulation of platelet aggregation, indicating a physiological antiaggregatory activity of EP4 receptors. The inhibitory effect of the EP4 agonist was accompanied by attenuated Ca(2+) flux, inhibition of glycoprotein IIb/IIIa, and downregulation of P-selectin. Most importantly, adhesion of platelets to fibrinogen under flow and in vitro thrombus formation were effectively prevented by the EP4 agonist. In this respect, the EP4 agonist synergized with acetylsalicylic acid. CONCLUSIONS: These results are suggestive of EP4 receptor activation as a novel antithrombotic strategy.
Asunto(s)
Coagulación Sanguínea , Plaquetas/metabolismo , Dinoprostona/metabolismo , Agregación Plaquetaria , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Trombosis/prevención & control , Aspirina/farmacología , Coagulación Sanguínea/efectos de los fármacos , Plaquetas/efectos de los fármacos , Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Fibrinógeno/metabolismo , Fibrinolíticos/farmacología , Citometría de Flujo , Humanos , Éteres Metílicos/farmacología , Naftalenos/farmacología , Selectina-P/metabolismo , Fenilbutiratos/farmacología , Adhesividad Plaquetaria , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/efectos de los fármacos , Trombosis/sangreRESUMEN
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 asthma. Here, we revisit the roles of PGD(2) receptors in the regulation of eosinophil and Th2 cell function and the efforts towards developing candidate compounds for clinical evaluation.
Asunto(s)
Antiinflamatorios/uso terapéutico , Hipersensibilidad Inmediata/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Prostaglandina D2/metabolismo , Receptores Inmunológicos/antagonistas & inhibidores , Receptores de Prostaglandina/antagonistas & inhibidores , Animales , Antiinflamatorios/farmacología , Eosinófilos/inmunología , Eosinófilos/metabolismo , Humanos , Hipersensibilidad Inmediata/inmunología , Hipersensibilidad Inmediata/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Ratones , Receptores Inmunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
BACKGROUND: Enhanced eosinophil migration from the blood into the tissue is a hallmark of allergic diseases. Prostaglandin (PG) I(2) is the major prostanoid released by endothelial cells. Mice deficient in PGI(2) receptors (IPs) show exaggerated eosinophilic inflammation in response to allergen. OBJECTIVE: We set out to determine the role of PGI(2) in eosinophil trafficking. METHODS: Human lung microvascular endothelial cells and purified human eosinophils were used to study adhesion and transendothelial migration. Morphologic studies were performed with fluorescence microscopy. RESULTS: PGI(2) markedly attenuated the migration of eosinophils through cell-free filters but had no effect on neutrophil migration. The inhibitory effect of PGI(2) on eosinophils was prevented by the IP antagonist Cay10441 and the adenylyl cyclase inhibitor SQ22536. Similarly, PGI(2) prevented the adhesion of eosinophils to fibronectin and the rapid upregulation and activation of the adhesion molecule CD11b. IP expression on eosinophils was confirmed by means of flow cytometry and Western blotting. Furthermore, when endothelial cells were treated with the COX inhibitor diclofenac to abolish PGI(2) production, adhesion of eosinophils to endothelial monolayers and subsequent transendothelial migration were markedly enhanced. Similarly, the IP antagonist enhanced eosinophil adhesion to endothelial cells. Inhibition of PGI(2) biosynthesis decreased the electrical resistance of endothelial monolayers and compromised the texture of adherent junctions, as visualized by means of VE-cadherin and F-actin staining. CONCLUSION: We propose that endothelium-derived PGI(2) might be fundamental for the maintenance of the endothelial barrier function against infiltrating cells. These results suggest that selective IP agonists might have beneficial effects in allergic inflammation.
Asunto(s)
Quimiotaxis de Leucocito/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Eosinófilos/fisiología , Epoprostenol/farmacología , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales/citología , Endotelio Vascular/metabolismo , Eosinófilos/efectos de los fármacos , Epoprostenol/metabolismo , Humanos , Pulmón/irrigación sanguínea , Pulmón/citología , Pulmón/inmunologíaRESUMEN
Rat gastric mucosal damage was induced by ischemia-reperfusion. The 5-lipoxygenase inhibitors MK886 and A63162, the 12-lipoxygenase inhibitor baicalein, the 15-lipoxygenase inhibitor PD146176 and the lipoxin (LX) A(4)/annexin 1 antagonist Boc1 increased mucosal damage in a dose-dependent manner. Low doses of these compounds, which have no effects on mucosal integrity, cause severe damage when combined with low doses of indomethacin, celecoxib or dexamethasone. 16,16-Dimethylprostaglandin (PG) E(2) and LXA(4) can replace each other in preventing mucosal injury induced by either cyclooxygenase or lipoxygenase inhibitors. The results suggest that not only cyclooxygenases, but also lipoxygenases have a role in limiting gastric mucosal damage during ischemia-reperfusion.
Asunto(s)
Anexinas/metabolismo , Mucosa Gástrica/efectos de los fármacos , Lipooxigenasa/fisiología , Receptores de Formil Péptido/fisiología , Receptores de Lipoxina/fisiología , Daño por Reperfusión/tratamiento farmacológico , 16,16-Dimetilprostaglandina E2/farmacología , Acetamidas/farmacología , Animales , Antiulcerosos/farmacología , Celecoxib , Inhibidores de la Ciclooxigenasa/farmacología , Dexametasona/farmacología , Sinergismo Farmacológico , Flavanonas/farmacología , Fluorenos/farmacología , Mucosa Gástrica/irrigación sanguínea , Mucosa Gástrica/patología , Glucocorticoides/farmacología , Indoles/farmacología , Indometacina/farmacología , Inhibidores de la Lipooxigenasa/farmacología , Masculino , Oligopéptidos/farmacología , Éteres Fenílicos , Antagonistas de Prostaglandina/farmacología , Pirazoles/farmacología , Ratas , Ratas Wistar , Receptores de Lipoxina/antagonistas & inhibidores , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Sulfonamidas/farmacologíaRESUMEN
The role of cyclooxygenases and prostaglandins in experimental models of gastroprotection is well established. We investigated the effects of the 5-lipoxygenase inhibitor A63162, the 12-lipoxygenase inhibitor baicalein and the 15-lipoxygenase inhibitor PD146176 as well as the nonspecific lipoxin A(4)/annexin-1 antagonist Boc1 on adaptive protection induced by 20% ethanol against 70% ethanol, and on protection induced by sodium salicylate against the mucosal-damage-aggravating effects of celecoxib and dexamethasone during local ischemia-reperfusion in rats. It was found that both types of gastroprotection were antagonized by the lipoxygenase inhibitors and the lipoxin A(4)/annexin-1 antagonist in doses that have no direct damaging effect on gastric mucosa. The results suggest that not only cyclooxygenases, but also active lipoxygenases and, possibly, annexin-1 are required for these types of gastroprotection to occur.
Asunto(s)
Anexinas/metabolismo , Inhibidores de la Ciclooxigenasa/farmacología , Etanol/farmacología , Mucosa Gástrica/efectos de los fármacos , Lipooxigenasa/fisiología , Receptores de Formil Péptido/fisiología , Receptores de Lipoxina/antagonistas & inhibidores , Salicilato de Sodio/farmacología , Acetamidas/farmacología , Animales , Celecoxib , Inhibidores de la Ciclooxigenasa 2/farmacología , Dexametasona/farmacología , Interacciones Farmacológicas , Flavanonas/farmacología , Fluorenos/farmacología , Mucosa Gástrica/irrigación sanguínea , Mucosa Gástrica/patología , Glucocorticoides/farmacología , Inhibidores de la Lipooxigenasa/farmacología , Masculino , Oligopéptidos/farmacología , Éteres Fenílicos , Antagonistas de Prostaglandina/farmacología , Pirazoles/farmacología , Ratas , Ratas Wistar , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Daño por Reperfusión/prevención & control , Sulfonamidas/farmacologíaRESUMEN
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.
Asunto(s)
Quimiotaxis , Eosinófilos/fisiología , Prostaglandina H2/fisiología , Receptores Inmunológicos/fisiología , Receptores de Prostaglandina/fisiología , Basófilos/efectos de los fármacos , Basófilos/fisiología , Proteínas Sanguíneas/farmacología , Proteínas Sanguíneas/fisiología , Calcio/fisiología , Carbazoles/farmacología , Cationes Bivalentes , Degranulación de la Célula , Línea Celular , Colágeno/metabolismo , Eosinófilos/efectos de los fármacos , Humanos , Hidantoínas/farmacología , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Oxidorreductasas Intramoleculares/metabolismo , Lipocalinas/antagonistas & inhibidores , Lipocalinas/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Piperidinas/farmacología , Prostaglandina H2/farmacología , Receptores Inmunológicos/agonistas , Receptores Inmunológicos/antagonistas & inhibidores , Receptores de Prostaglandina/agonistas , Receptores de Prostaglandina/antagonistas & inhibidores , Sulfonamidas/farmacologíaRESUMEN
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) E(2) exerts anti-inflammatory and bronchoprotective mechanisms in asthma, but the underlying mechanisms have remained unclear. In this study we show that PGE(2) potently inhibits the chemotaxis of purified human eosinophils toward eotaxin, PGD(2), 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 PGE(2) on eosinophil migration, whereas an EP2 antagonist completely prevented this effect. Butaprost, and also PGE(2), inhibited the C5a-induced degranulation of eosinophils. Moreover, selective kinase inhibitors revealed that the inhibitory effect of PGE(2) 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.
Asunto(s)
Quimiotaxis de Leucocito/inmunología , Dinoprostona/inmunología , Eosinófilos/inmunología , Receptores de Prostaglandina E/inmunología , Alprostadil/análogos & derivados , Alprostadil/farmacología , Animales , Células Cultivadas , Quimiotaxis de Leucocito/efectos de los fármacos , Dinoprostona/metabolismo , Inhibidores Enzimáticos/farmacología , Eosinófilos/efectos de los fármacos , Eosinófilos/metabolismo , Cobayas , Humanos , Inmunohistoquímica , Ratones , Fosfatidilinositol 3-Quinasas/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/inmunología , Proteína Quinasa C/metabolismo , Receptores de Prostaglandina E/metabolismo , Subtipo EP2 de Receptores de Prostaglandina ERESUMEN
Perfluorooctanoic acid (PFOA) and agonists of peroxisome proliferator-activated receptors (PPAR) alpha and gamma were investigated for potential anti-inflammatory effects in cerulein-induced acute pancreatitis in rats. PFOA significantly reduced both leukocyte accumulation and prostanoid synthesis. The PPAR-alpha agonist clofibrate had no effect on leukocyte activation but significantly inhibited prostanoid synthesis whereas the PPAR-gamma agonist rosiglitazone significantly reduced leukocyte activation but did not affect synthesis of prostaglandins in the pancreas. Neither PFOA, nor clofibrate or rosiglitazone had an effect on the formation of the inflammatory edema or elevated levels of lipase activity in the blood serum. In summary, PFOA attenuates the accumulation of activated leukocytes and reduces the synthesis of prostanoids in the pancreas during cerulein-induced acute pancreatitis. An activation of PPAR-alpha causes inhibition of prostanoid synthesis while activation of PPAR-gamma inhibits leukocyte activation.
Asunto(s)
Antiinflamatorios/farmacología , Caprilatos/farmacología , Fluorocarburos/farmacología , PPAR alfa/fisiología , PPAR gamma/fisiología , Pancreatitis/tratamiento farmacológico , Enfermedad Aguda , Animales , Femenino , Rayos gamma , Leucocitos/fisiología , Lipasa/metabolismo , Páncreas/metabolismo , Páncreas/patología , Pancreatitis/etiología , Pancreatitis/metabolismo , Prostaglandinas/biosíntesis , Ratas , Ratas Sprague-DawleyRESUMEN
Systemic inflammation leads to increased expression of spinal cyclooxygenase (COX)-2 and to a subsequent increase of prostaglandin (PG) biosynthesis, which contribute to the development of hyperalgesia and allodynia. In this study, endotoxin caused a sequential induction of membrane bound prostaglandin E synthase-1 and lipocalin-type PGD synthase (L-PGDS) in the mouse spinal cord. L-PGDS expression was detected in the leptomeninges, oligodendrocytes, and interestingly, in discrete perivascular cells. Endotoxin-caused increase was most prominent in oligodendrocytes. Endotoxin-induced COX-2 and membrane bound prostaglandin E synthase-1 were restricted to the leptomeninges and perivascular cells. COX-1 was not influenced by endotoxin. We found COX-1 expressed in microglia, some of them in close proximity to L-PGDS-positive oligodendrocytes and co-localization of COX-1 with L-PGDS in perivascular and leptomeningeal cells under control conditions. It can be assumed, that PGD2 biosynthesis under control conditions is mediated via COX-1 and that during inflammation, increased PGD2 is dependent on COX-2. We found the PGD2 receptors DP1 and chemoattractant receptor homologous molecule expressed on T helper type 2 cells (CRTH2) localized in neurons of the dorsal, and motoneurons in the ventral horn. The localization of the PGD2 receptors DP1 and CRTH in spinal cord neurons, particularly in neurons of lamina I and II involved in the processing of nociceptive stimuli, supports a role of PGD2 under inflammatory conditions.
Asunto(s)
Lipopolisacáridos/administración & dosificación , Prostaglandina D2/metabolismo , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/metabolismo , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/metabolismo , Receptores de Prostaglandina/biosíntesis , Receptores de Prostaglandina/metabolismo , Médula Espinal/enzimología , Animales , Citocinas/biosíntesis , Citocinas/genética , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Mediadores de Inflamación/metabolismo , Inyecciones Intraperitoneales , Masculino , Ratones , Ratones Endogámicos BALB C , Prostaglandina-Endoperóxido Sintasas/genética , Receptores Inmunológicos/genética , Receptores de Prostaglandina/genética , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Factores de TiempoRESUMEN
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.
Asunto(s)
Quimiotaxis , Eosinófilos/citología , Eosinófilos/metabolismo , Prostaglandinas D/metabolismo , Receptores Inmunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Animales , Médula Ósea , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Eosinófilos/efectos de los fármacos , Fémur/citología , Cobayas , Humanos , Ligandos , Receptores Inmunológicos/genética , Receptores de Prostaglandina/genéticaRESUMEN
Prostaglandin (PG)D(2), an important mediator in allergic diseases, is rapidly transformed in plasma to active metabolites that bind and activate two distinct receptors, DP1 and CRTH2. Since the rate of PGD(2) degradation and the bioactivity of the resulting metabolites are still unclear, the aim of our study was to analyze the kinetics and biological effects of PGD(2) metabolites formed in plasma. Eosinophil shape change was taken as a parameter of chemotactic activation mediated by CRTH2 whereas inhibition of platelet aggregation served as a measure of DP1 activity. PGD(2) was degraded in plasma with an apparent half-life of approximately 30 min, accompanied by a loss of potency in inhibiting platelet aggregation as well as inducing eosinophil stimulation. Incubation of PGD(2) in plasma for 120 min caused an increase in the IC(50) for platelet aggregation by a factor of 6.5 and an increase of the EC(50) for eosinophil shape change by a factor of 7.2. However, tandem mass spectrometry analysis showed that incubation of PGD(2) in plasma for 120 min resulted in clearance of PGD(2) of more than 92%, which was mirrored by a continuous formation of Delta(12)-PGD(2) and Delta(12)-PGJ(2), whereas only small amounts of 15d-PGD(2) and 15d-PGJ(2) were detected. Interestingly, a rapid degradation of PGD(2) was also observed in serum, which was not prevented by pepsin digestion of serum preceding the addition of PGD(2). Therefore, despite extensive non-enzymatic metabolization of PGD(2) in plasma, its biological activity with respect to DP1 and CRTH2 is maintained through the formation of bioactive metabolites.
Asunto(s)
Prostaglandina D2/sangre , Receptores Inmunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Forma de la Célula/efectos de los fármacos , Forma de la Célula/fisiología , Factores Quimiotácticos Eosinófilos/metabolismo , Quimiotaxis/efectos de los fármacos , Quimiotaxis/fisiología , Colágeno/farmacología , Eosinófilos/efectos de los fármacos , Eosinófilos/metabolismo , Humanos , Cinética , Leucocitos , Agregación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/fisiología , Prostaglandina D2/análisis , Prostaglandina D2/farmacología , Receptores Inmunológicos/efectos de los fármacos , Receptores de Prostaglandina/efectos de los fármacos , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Factores de TiempoRESUMEN
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.
Asunto(s)
Factores Quimiotácticos Eosinófilos/inmunología , Quimiotaxis de Leucocito/inmunología , Eosinófilos/inmunología , Prostaglandina D2/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Ácidos Araquidónicos/inmunología , Ácidos Araquidónicos/metabolismo , Carbazoles/farmacología , Quimiocina CCL11 , Quimiocinas CC/inmunología , Quimiocinas CC/metabolismo , Factores Quimiotácticos Eosinófilos/metabolismo , Quimiotaxis de Leucocito/efectos de los fármacos , Eosinófilos/efectos de los fármacos , Eosinófilos/metabolismo , Humanos , Inhibidores de Agregación Plaquetaria/farmacología , Prostaglandina D2/metabolismo , Sulfonamidas/farmacologíaRESUMEN
Although prostaglandin (PG)D2 is one of the main metabolites of the cyclooxygenase (COX) pathway of arachidonate metabolism in the brain, relatively little is known about the regulation of PGD2 biosynthesis in the spinal cord during systemic inflammation. Therefore, the present study was aimed at investigating the effect of endotoxin treatment on spinal PGD2 biosynthesis in BALB/c mice. Spinal inflammatory response to systemic endotoxin was verified by determination of spinal TNFalpha and IL-1beta mRNA. COX-1, COX-2, membrane-bound prostaglandin E synthase-1 (mPGES-1), and lipocalin-type prostaglandin D synthase (L-PGDS) mRNA and protein were determined by RT-PCR and western blot, respectively. The concentrations of immunoreactive PGD2 and PGE2 were measured in superfusion media of spinal cord samples in-vitro. Endotoxin treatment (1 mg/kg; 24 h before) enhanced the expression of COX-2, mPGES-1, and L-PGDS mRNA and protein in spinal cord, while there was no significant effect on COX-1 mRNA and protein. In superfusion media of spinal cord samples obtained from endotoxin treated mice, the concentrations of immunoreactive PGE2 and PGD2 were higher than in the control group suggesting enhanced spinal PG biosynthesis after endotoxin treatment. Addition of the selective COX-2 inhibitor lumiracoxib (100 nM) to the superfusion medium did not significantly affect PGE2 or PGD2 release in spinal cord obtained from non-treated mice. In spinal cord of endotoxin-treated mice, lumiracoxib (100 nM) attenuated PGE2 and PGD2 release to values similar to those observed in tissue obtained from non-endotoxin-treated mice. These results show enhanced expression of spinal L-PGDS and increased spinal PGD2 biosynthesis during systemic inflammation whereby enhanced biosynthesis seems to be dependent primarily on COX-2 activity.
Asunto(s)
Ciclooxigenasa 2/fisiología , Prostaglandina D2/biosíntesis , Médula Espinal/metabolismo , Animales , Western Blotting , Inhibidores de la Ciclooxigenasa 2/farmacología , Citocinas/biosíntesis , Diclofenaco/análogos & derivados , Técnicas para Inmunoenzimas , Interleucina-1/biosíntesis , Masculino , Ratones , Ratones Endogámicos BALB C , Compuestos Orgánicos/farmacología , ARN Mensajero/biosíntesis , Radioinmunoensayo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Neuropeptide-Y (NPY) is involved in the regulation of ingestive behaviour and energy homeostasis. Since deletion of the NPY Y2 and Y4 receptor gene increases and decreases food intake, respectively, we examined whether water intake during the light and dark phases is altered in Y2 and Y4 receptor knockout mice. The water consumption of mice staying in their home cages was measured by weighing the water bottles at the beginning and end of the light phase during 4 consecutive days. Control, Y2 and Y4 receptor knockout mice did not differ in their water intake during the light phase. However, during the dark phase Y2 and Y4 receptor knockout mice drank significantly more (46-63%, P<0.05) water than the control mice. The total daily water intake over 24 h was also enhanced. The enhanced water intake during the dark phase was not altered by the beta-adrenoceptor antagonist propranolol or the angiotensin AT1 receptor antagonist telmisartan (each injected intraperitoneally at 10 mg/kg). These data indicate that NPY acting via Y2 and Y4 receptors plays a distinctive role in the regulation of nocturnal water consumption. While beta-adrenoceptors and angiotensin AT1 receptors do not seem to be involved, water intake in Y2 and Y4 receptor knockout mice may be enhanced because presynaptic autoinhibition of NPY release and inhibition of orexin neurons in the central nervous system are prevented.
Asunto(s)
Ritmo Circadiano/fisiología , Ingestión de Líquidos/genética , Receptores de Neuropéptido Y/deficiencia , Antagonistas Adrenérgicos beta/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Conducta Animal , Bencimidazoles/farmacología , Benzoatos/farmacología , Ritmo Circadiano/efectos de los fármacos , Ingestión de Líquidos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Propranolol/farmacología , Telmisartán , Factores de TiempoRESUMEN
BACKGROUND: 5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a chemoattractant for eosinophils and neutrophils, and the messenger RNA for its receptor, the oxo-eicosatetraenoic acid receptor (OXE), has been detected in several tissues. OBJECTIVES: This study aimed at clarifying the role of 5-oxo-ETE in the regulation of basophil function. METHODS: Basophil responses were determined in assays of flow-cytometric shape change, Ca(2+) flux, chemotaxis, and histamine release. Messenger RNA for OXE was detected by real-time PCR. RESULTS: We observed that human eosinophils were 3 to 10 times more sensitive to 5-oxo-ETE than neutrophils in flow-cytometric shape change and Ca(2+) flux assays, as estimated from the half-maximal responses of the cells. Basophils responded to 5-oxo-ETE in the shape change assay with a sensitivity similar to that of eosinophils. 5-Oxo-ETE was a weak inducer of Ca(2+) flux in basophils and did not cause histamine release but was a highly effective chemoattractant for basophils in the low nanomolar concentration range in a pertussis toxin-sensitive manner. In agreement with these functional studies, the messenger RNA for the 5-oxo-ETE receptor, OXE, was detectable in basophils as in monocytes, eosinophils, and neutrophils, but not in fibroblasts. Specimens from sinus mucosa, tonsils, and adenoids also contained detectable levels of messenger RNA for OXE. CONCLUSION: Our data suggest that 5-oxo-ETE is potentially involved in the regulation of basophil recruitment and might hence be a useful therapeutic target in atopic disease.
Asunto(s)
Ácidos Araquidónicos/farmacología , Basófilos/efectos de los fármacos , Basófilos/fisiología , Factores Quimiotácticos/farmacología , Quimiotaxis de Leucocito , Ácidos Araquidónicos/administración & dosificación , Basófilos/citología , Basófilos/metabolismo , Calcio/metabolismo , Forma de la Célula/efectos de los fármacos , Eosinófilos/efectos de los fármacos , Eosinófilos/metabolismo , Eosinófilos/fisiología , Citometría de Flujo , Humanos , Monocitos/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Neutrófilos/fisiología , Concentración Osmolar , Toxina del Pertussis/farmacología , ARN Mensajero/metabolismo , Receptores Eicosanoides/genéticaRESUMEN
BACKGROUND: Little is known about the mechanisms that regulate the selective recruitment of basophils to sites of allergic inflammation. OBJECTIVE: Here we examine the role of stem cell factor (SCF) in the regulation of basophil function. METHODS: Human basophils were isolated from peripheral blood, and their migration was investigated in chemotaxis assays. Apoptosis was detected by means of annexin V and propidium iodide staining. The expression of cell-surface molecules was measured by means of flow cytometry. RESULTS: SCF amplified the chemotactic responsiveness of human peripheral blood basophils to the chemoattractants eotaxin, monocyte chemotactic protein 2 and macrophage inflammatory protein 1alpha, and C5a, without being chemotactic or chemokinetic by itself. SCF synergized with chemoattractants in causing basophil upregulation of the integrin CD11b, and this effect was inhibited by a c-kit antibody, the tyrosine kinase inhibitor imatinib mesylate (STI-571), and a phosphatidylinositol 3 kinase inhibitor but not by inhibitors of p38 mitogen-activated protein kinase or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. Basophils bound fluorescence-labeled SCF and expressed its receptor, c-kit, which was markedly upregulated in culture for 24 to 48 hours in the presence of IL-3. Moreover, SCF prolonged basophil survival in concert with IL-3 by delaying apoptosis. These effects of SCF were selective for basophils because chemotaxis and CD11b upregulation of eosinophils or neutrophils were unchanged. CONCLUSION: SCF might be an important selective modulator of basophil function through a phosphatidylinositol 3 kinase-dependent pathway.