RESUMO
Identifying new target molecules through which eosinophils secrete their stored proteins may reveal new therapeutic approaches for the control of eosinophilic disorders such as host immune responses to parasites. We have recently reported the expression of the purinergic P2Y12 receptor (P2Y12R) in human eosinophils; however, its functional role in this cell type and its involvement in eosinophilic inflammation remain unknown. Here, we investigated functional roles of P2Y12R in isolated human eosinophils and in a murine model of eosinophilic inflammation induced by Schistosoma mansoni (S. mansoni) infection. We found that adenosine 5'-diphosphate (ADP) induced human eosinophils to secrete eosinophil peroxidase (EPO) in a P2Y12R dependent manner. However, ADP did not interfere with human eosinophil apoptosis or chemotaxis in vitro. In vivo, C57Bl/6 mice were infected with cercariae of the Belo Horizonte strain of S. mansoni. Analyses performed 55 days post infection revealed that P2Y12R blockade reduced the granulomatous hepatic area and the eosinophilic infiltrate, collagen deposition and IL-13/IL-4 production in the liver without affecting the parasite oviposition. As found for humans, murine eosinophils also express the P2Y12R. P2Y12R inhibition increased blood eosinophilia, whereas it decreased the bone marrow eosinophil count. Our results suggest that P2Y12R has an important role in eosinophil EPO secretion and in establishing the inflammatory response in the course of a S. mansoni infection.
Assuntos
Eosinófilos/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Schistosoma mansoni/patogenicidade , Difosfato de Adenosina/farmacologia , Animais , Células da Medula Óssea/citologia , Sobrevivência Celular/efeitos dos fármacos , Colágeno/metabolismo , Modelos Animais de Doenças , Peroxidase de Eosinófilo/metabolismo , Eosinófilos/efeitos dos fármacos , Eosinófilos/imunologia , Humanos , Inflamação , Interleucina-13/análise , Interleucina-13/sangue , Interleucina-4/análise , Interleucina-4/sangue , Fígado/metabolismo , Fígado/parasitologia , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores Purinérgicos P2Y12/química , Receptores Purinérgicos P2Y12/genética , Esquistossomose mansoni/imunologia , Esquistossomose mansoni/parasitologia , Esquistossomose mansoni/patologia , Células Th2/imunologiaRESUMO
Cysteinyl leukotrienes (cysLTs) are cell membrane-impermeant lipid mediators that play major roles in the pathogenesis of eosinophilic inflammation and are recognized to act via at least 2 receptors, namely, cysLT1 receptor (cysLT1R) and cysLT2 receptor (cysLT2R). Eosinophils, which are granulocytes classically associated with host defense against parasitic helminthes and allergic conditions, are distinguished from leukocytes by their dominant population of cytoplasmic crystalloid (also termed secretory, specific, or secondary) granules that contain robust stores of diverse preformed proteins. Human eosinophils are the main source of cysLTs and are recognized to express both cysLTs receptors (cysLTRs) on their surface, at the plasma membrane. More recently, we identified the expression of cysLTRs in eosinophil granule membranes and demonstrated that cysLTs, acting via their granule membrane-expressed receptors, elicit secretion from cell-free human eosinophil granules. Herein, we review the multifaceted roles of cysLTs in eliciting eosinophil granule protein secretion. We discuss the intracrine and autocrine/paracrine secretory responses evoked by cysLTs in eosinophils and in cell-free extracellular eosinophil crystalloid granules. We also discuss the importance of this finding in eosinophil immunobiology and speculate on its potential role(s) in eosinophilic diseases.
Assuntos
Eosinófilos/imunologia , Regulação da Expressão Gênica/imunologia , Leucotrienos/imunologia , Receptores de Leucotrienos/imunologia , Vesículas Secretórias/imunologia , Animais , Comunicação Autócrina/imunologia , Membrana Celular/imunologia , Humanos , Comunicação Parácrina/imunologiaRESUMO
Cell-free granules, upon extrusion from human eosinophils, remain fully competent to secrete granule-derived proteins in receptor-mediated processes in response to different stimuli. However, in order to avoid the shrinkage and damage of granules, as well as preserve their structure, properties, and functionality, the use of an optimized process of subcellular fractionation using an isoosmotic density gradient is needed. Here, we describe a detailed protocol of subcellular fractionation of nitrogen-cavitated eosinophils on an isoosmotic iodinated density gradient (iodixanol) and the isolation of well-preserved and functional membrane-bound specific granules.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Eosinófilos/citologia , Eosinófilos/metabolismo , Animais , HumanosRESUMO
Eosinophils store a wide range of preformed proteins, including cationic proteins and cytokines, within their morphologically unique granules. Recently, we have demonstrated that cell-free eosinophil granules are functional, independent, secretory organelles and that clusters of cell-free granules are commonly found at tissue sites associated with various pathologic conditions. Cytolytic release of intact eosinophil granules produces extracellular organelles that are fully capable of ligand-elicited, active, secretory responses and are hence able to act as 'cluster bombs' that amplify the differential secretory properties of eosinophils. Herein, we review recent progress in elucidating the molecular mechanisms involved in the cytolytical release of intact cell-free functional eosinophil granules in a process associated with the liberation of eosinophil DNA traps (nets), a known aspect of the innate response recognized in various immune cells and pathological conditions. We also discuss the importance of clusters of cell-free eosinophil granules trapped in eosinophil DNA nets in disease and speculate on their potential role(s) in immunity as well as compare available data on DNA-releasing neutrophils.