RESUMO
Infiltration of monocytes and macrophages into the site of inflammation is critical in the progression of inflammatory diseases such as atherosclerosis. Cell migration is dependent on the continuous organization of the actin cytoskeleton, which is regulated by members of the small Rho GTPase family (RhoA, Cdc42, Rac) that are also important for the regulation of signal transduction pathways. We have recently reported on reduced plaque formation in an atherosclerotic mouse model transplanted with bone marrow from adipose triglyceride lipase-deficient (Atgl-/-) mice. Here we provide evidence that defective lipolysis in macrophages lacking ATGL, the major enzyme responsible for triacylglycerol hydrolysis, favors an anti-inflammatory M2-like macrophage phenotype. Our data implicate an as yet unrecognized principle that insufficient lipolysis influences macrophage polarization and actin polymerization, resulting in impaired macrophage migration. Sustained phosphorylation of focal adhesion kinase [due to inactivation of its phosphatase by elevated levels of reactive oxygen species (ROS)] results in defective Cdc42, Rac1 and RhoA activation and in increased and sustained activation of Rac2. Inhibition of ROS production restores the migratory capacity of Atgl-/- macrophages. Since monocyte and macrophage migration are a prerequisite for infiltrating the arterial wall, our results provide a molecular link between lipolysis and the development of atherosclerosis.
Assuntos
Lipólise , Macrófagos/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Movimento Celular , Polaridade Celular , Feminino , Lipase/deficiência , Lipase/metabolismo , Macrófagos/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismoRESUMO
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.
Assuntos
Regulação para Baixo , Eosinófilos/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Antígeno CD11b/metabolismo , Cálcio/metabolismo , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Movimento Celular , Ciclopentanos/farmacologia , Dinoprostona/farmacologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/imunologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Isoindóis/farmacologia , Éteres Metílicos , Mucosa Nasal/metabolismo , Mucosa Nasal/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Prostaglandina E Subtipo EP4/agonistas , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidores , Sulfonamidas/farmacologia , Tioglicolatos/farmacologiaRESUMO
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.
Assuntos
Quimiotaxia de Leucócito/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Eosinófilos/fisiologia , Epoprostenol/farmacologia , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/citologia , Endotélio Vascular/metabolismo , Eosinófilos/efeitos dos fármacos , Epoprostenol/metabolismo , Humanos , Pulmão/irrigação sanguínea , Pulmão/citologia , Pulmão/imunologiaRESUMO
Microglia, the immunocompetent cells of the CNS, are rapidly activated in response to injury and microglia migration towards and homing at damaged tissue plays a key role in CNS regeneration. Lysophosphatidic acid (LPA) is involved in signaling events evoking microglia responses through cognate G protein-coupled receptors. Here we show that human immortalized C13NJ microglia express LPA receptor subtypes LPA(1), LPA(2), and LPA(3) on mRNA and protein level. LPA activation of C13NJ cells induced Rho and extracellular signal-regulated kinase activation and enhanced cellular ATP production. In addition, LPA induced process retraction, cell spreading, led to pronounced changes of the actin cytoskeleton and reduced cell motility, which could be reversed by inhibition of Rho activity. To get an indication about LPA-induced global alterations in protein expression patterns a 2-D DIGE/LC-ESI-MS proteomic approach was applied. On the proteome level the most prominent changes in response to LPA were observed for glycolytic enzymes and proteins regulating cell motility and/or cytoskeletal dynamics. The present findings suggest that naturally occurring LPA is a potent regulator of microglia biology. This might be of particular relevance in the pathophysiological context of neurodegenerative disorders where LPA concentrations can be significantly elevated in the CNS.
Assuntos
Movimento Celular , Citoesqueleto/metabolismo , Microglia/citologia , Microglia/metabolismo , Proteoma/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Linhagem Celular , Regulação da Expressão Gênica , Glicólise , Humanos , Lisofosfolipídeos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptores de Ácidos Lisofosfatídicos/genética , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
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.
Assuntos
Quimiotaxia de Leucócito/imunologia , Dinoprostona/imunologia , Eosinófilos/imunologia , Receptores de Prostaglandina E/imunologia , Alprostadil/análogos & derivados , Alprostadil/farmacologia , Animais , Células Cultivadas , Quimiotaxia de Leucócito/efeitos dos fármacos , Dinoprostona/metabolismo , Inibidores Enzimáticos/farmacologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Cobaias , Humanos , Imuno-Histoquímica , Camundongos , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C/imunologia , Proteína Quinase C/metabolismo , Receptores de Prostaglandina E/metabolismo , Receptores de Prostaglandina E Subtipo EP2RESUMO
In order to engineer the choline oxidase from Arthrobacter nicotianae (An_CodA) for the potential application as biological bleach in detergents, the specific activity of the enzyme toward the synthetic substrate tris-(2-hydroxyethyl)-methylammonium methylsulfate (MTEA) was improved by methods of directed evolution and rational design. The best mutants (up to 520% wt-activity with MTEA) revealed mutations in the FAD- (A21V, G62D, I69V) and substrate-binding site (S348L, V349L, F351Y). In a separate screening of a library comprising of randomly mutagenised An_CodA, with the natural substrate choline, four mutations were identified, which were further combined in one clone. The constructed clone showed improved activity towards both substrates, MTEA and choline. Mapping these mutation sites onto the structural model of An_CodA revealed that Phe351 is positioned right in the active site of An_CodA and very likely interacts with the bound substrate. Ala21 is part of an alpha-helix which interacts with the diphosphate moiety of the flavin cofactor and might influence the activity and specificity of the enzyme.
Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Arthrobacter/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Corantes/metabolismo , Engenharia de Proteínas , Substituição de Aminoácidos , Sítios de Ligação , Evolução Molecular Direcionada , Metilaminas/metabolismo , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Estrutura Terciária de Proteína , Ésteres do Ácido Sulfúrico/metabolismoRESUMO
At the sites of inflammation, leukocytes are confronted with mediators which induce different cellular responses like chemotaxis, degranulation and respiratory burst. Morphologically, these responses are accompanied by changes in the cells' shape. In this study, we investigated the involvement of the actin cytoskeleton and Ca2+ in the shape change responses of human eosinophils and neutrophils to chemoattractants and correlated the obtained findings to degranulation and respiratory burst using flow cytometry. Shape change was recorded as an increase in forward scatter. Degranulation was measured as the cell surface upregulation of the granule-associated marker CD63. Respiratory burst was determined fluorimetrically as the oxidation of dihydrorhodamine 123. The involvement of actin filaments and phospholipase C (PLC) was investigated with the actin inhibitor cytochalasin B and the selective PLC inhibitor U-73122, respectively. The data that we obtained demonstrated that granulocytes exhibit 2 distinct types of shape change responses when stimulated with chemoattractants: (i) one type is induced by chemokines like eotaxin and interleukin 8, which are poor degranulators, and also by classical chemoattractants, C5a and formyl-methionyl-leucyl-phenylalanine; this shape change depends on the activation of PLC and functional actin filaments, but does not require Ca2+ influx from outside; (ii) the second type of shape change is not stimulated by chemokines, but can be seen with classical chemoattractants which are also potent inducers of degranulation and respiratory burst. This type of shape change does not require any functional actin filaments, but appears to be a consequence of degranulation and depends essentially on the activation of PLC and Ca2+ influx from the extracellular space.
Assuntos
Citoesqueleto de Actina/fisiologia , Cálcio/fisiologia , Eosinófilos/citologia , Eosinófilos/fisiologia , Neutrófilos/citologia , Neutrófilos/fisiologia , Citoesqueleto de Actina/efeitos dos fármacos , Antígenos CD , Cálcio/metabolismo , Cálcio/farmacologia , Moléculas de Adesão Celular/metabolismo , Degranulação Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Quimiocinas/farmacologia , Fatores Quimiotáticos/farmacologia , Citocalasina B/farmacologia , Eosinófilos/efeitos dos fármacos , Estrenos/farmacologia , Humanos , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Leucócitos/fisiologia , Neutrófilos/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Glicoproteínas da Membrana de Plaquetas , Pirrolidinonas/farmacologia , Explosão Respiratória , Tetraspanina 30 , Fosfolipases Tipo C/efeitos dos fármacos , Fosfolipases Tipo C/metabolismoRESUMO
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.
Assuntos
Quimiotaxia , Eosinófilos/citologia , Eosinófilos/metabolismo , Prostaglandinas D/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Animais , Medula Óssea , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Eosinófilos/efeitos dos fármacos , Fêmur/citologia , Cobaias , Humanos , Ligantes , Receptores Imunológicos/genética , Receptores de Prostaglandina/genéticaRESUMO
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.
Assuntos
Fatores Quimiotáticos de Eosinófilos/imunologia , Quimiotaxia de Leucócito/imunologia , Eosinófilos/imunologia , Prostaglandina D2/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Ácidos Araquidônicos/imunologia , Ácidos Araquidônicos/metabolismo , Carbazóis/farmacologia , Quimiocina CCL11 , Quimiocinas CC/imunologia , Quimiocinas CC/metabolismo , Fatores Quimiotáticos de Eosinófilos/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Humanos , Inibidores da Agregação Plaquetária/farmacologia , Prostaglandina D2/metabolismo , Sulfonamidas/farmacologiaRESUMO
We investigated the actions of a panel of nonsteroidal anti-inflammatory drugs on eosinophils, basophils, neutrophils, and monocytes. Indomethacin alone was a potent and selective inducer of eosinophil and basophil shape change. In eosinophils, indomethacin induced chemotaxis, CD11b up-regulation, respiratory burst, and L-selectin shedding but did not cause up-regulation of CD63 expression. Pretreatment of eosinophils with indomethacin also enhanced subsequent eosinophil shape change induced by eotaxin, although treatment with higher concentrations of indomethacin resulted in a decrease in the expression of the major eosinophil chemokine receptor, CCR3. Indomethacin activities and cell selectivity closely resembled those of prostaglandin D(2) (PGD(2)). Eosinophil shape change in response to eotaxin was inhibited by pertussis toxin, but indomethacin- and PGD(2)-induced shape change responses were not. Treatment of eosinophils with specific inhibitors of phospholipase C (U-73122), phosphatidylinositol 3-kinase (LY-294002), and p38 mitogen-activated protein kinase (SB-202190) revealed roles for these pathways in indomethacin signaling. Indomethacin and its analogues may therefore provide a structural basis from which selective PGD(2) receptor small molecule antagonists may be designed and which may have utility in the treatment of allergic inflammatory disease.