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1.
J Immunol ; 184(9): 4852-62, 2010 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-20368273

RESUMEN

NK cells are renowned for their ability to kill virally infected or transformed host cells by release of cytotoxic granules containing granzymes and perforin. NK cells also have important regulatory capabilities chiefly mediated by secretion of cytokines, such as IFN-gamma and TNF. The secretory pathway for the release of cytokines in NK cells is unknown. In this study, we show localization and trafficking of IFN-gamma and TNF in human NK cells in compartments and vesicles that do not overlap with perforin or other late endosome granule markers. Cytokines in post-Golgi compartments colocalized with markers of the recycling endosome (RE). REs are functionally required for cytokine release because inactivation of REs or mutation of RE-associated proteins Rab11 and vesicle-associated membrane protein-3 blocked cytokine surface delivery and release. In contrast, REs are not needed for release of perforin from preformed granules but may be involved at earlier stages of granule maturation. These findings suggest a new role for REs in orchestrating secretion in NK cells. We show that the cytokines IFN-gamma and TNF are trafficked and secreted via a different pathway than perforin. Although perforin granules are released in a polarized fashion at lytic synapses, distinct carriers transport both IFN-gamma and TNF to points all over the cell surface, including within the synapse, for nonpolarized release.


Asunto(s)
Gránulos Citoplasmáticos/inmunología , Gránulos Citoplasmáticos/metabolismo , Pruebas Inmunológicas de Citotoxicidad , Interferón gamma/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Perforina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Adulto , Compartimento Celular/inmunología , Membrana Celular/inmunología , Membrana Celular/metabolismo , Polaridad Celular/inmunología , Células Cultivadas , Pruebas Inmunológicas de Citotoxicidad/métodos , Endosomas/inmunología , Endosomas/metabolismo , Humanos , Sinapsis Inmunológicas/metabolismo , Interferón gamma/biosíntesis , Células K562 , Activación de Linfocitos/inmunología , Perforina/biosíntesis , Transporte de Proteínas/inmunología , Vesículas Secretoras/inmunología , Vesículas Secretoras/metabolismo , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis
2.
Cell Rep ; 13(9): 1881-94, 2015 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-26655903

RESUMEN

In contrast to the class I phosphoinositide 3-kinases (PI3Ks), the organismal roles of the kinase activity of the class II PI3Ks are less clear. Here, we report that class II PI3K-C2ß kinase-dead mice are viable and healthy but display an unanticipated enhanced insulin sensitivity and glucose tolerance, as well as protection against high-fat-diet-induced liver steatosis. Despite having a broad tissue distribution, systemic PI3K-C2ß inhibition selectively enhances insulin signaling only in metabolic tissues. In a primary hepatocyte model, basal PI3P lipid levels are reduced by 60% upon PI3K-C2ß inhibition. This results in an expansion of the very early APPL1-positive endosomal compartment and altered insulin receptor trafficking, correlating with an amplification of insulin-induced, class I PI3K-dependent Akt signaling, without impacting MAPK activity. These data reveal PI3K-C2ß as a critical regulator of endosomal trafficking, specifically in insulin signaling, and identify PI3K-C2ß as a potential drug target for insulin sensitization.


Asunto(s)
Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Insulina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Autofagia , Glucemia/análisis , Células Cultivadas , Fosfatidilinositol 3-Quinasas Clase II/genética , Dieta Alta en Grasa , Endosomas/metabolismo , Hígado Graso/metabolismo , Hígado Graso/patología , Técnicas de Sustitución del Gen , Hepatocitos/citología , Hepatocitos/metabolismo , Insulina/sangre , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal
3.
Nat Commun ; 5: 3450, 2014 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-24625684

RESUMEN

Stroke is a major cause of death worldwide and the leading cause of permanent disability. Although reperfusion is currently used as treatment, the restoration of blood flow following ischaemia elicits a profound inflammatory response mediated by proinflammatory cytokines such as tumour necrosis factor (TNF), exacerbating tissue damage and worsening the outcomes for stroke patients. Phosphoinositide 3-kinase delta (PI3Kδ) controls intracellular TNF trafficking in macrophages and therefore represents a prospective target to limit neuroinflammation. Here we show that PI3Kδ inhibition confers protection in ischaemia/reperfusion models of stroke. In vitro, restoration of glucose supply following an episode of glucose deprivation potentiates TNF secretion from primary microglia-an effect that is sensitive to PI3Kδ inhibition. In vivo, transient middle cerebral artery occlusion and reperfusion in kinase-dead PI3Kδ (p110δ(D910A/D910A)) or wild-type mice pre- or post-treated with the PI3Kδ inhibitor CAL-101, leads to reduced TNF levels, decreased leukocyte infiltration, reduced infarct size and improved functional outcome. These data identify PI3Kδ as a potential therapeutic target in ischaemic stroke.


Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Accidente Cerebrovascular/enzimología , Accidente Cerebrovascular/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Fosfatidilinositol 3-Quinasa Clase I , Modelos Animales de Enfermedad , Inflamación/metabolismo , Masculino , Ratones
4.
Nat Commun ; 2: 491, 2011 Oct 04.
Artículo en Inglés | MEDLINE | ID: mdl-21971506

RESUMEN

Neurosecretory vesicles undergo docking and priming before Ca(2+)-dependent fusion with the plasma membrane. Although de novo synthesis of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P(2)) is required for exocytosis, its precise contribution is still unclear. Here we show that inhibition of the p110δ isoform of PI3-kinase by IC87114 promotes a transient increase in PtdIns(4,5)P(2), leading to a potentiation of exocytosis in chromaffin cells. We then exploit this pathway to examine the effect of a transient PtdIns(4,5)P(2) increase on neurosecretory vesicles behaviour, outside the context of a secretagogue stimulation. Our results demonstrate that a rise in PtdIns(4,5)P(2) is sufficient to promote the mobilization and recruitment of secretory vesicles to the plasma membrane via Cdc42-mediated actin reorganization. PtdIns(4,5)P(2), therefore, orchestrates the actin-based conveyance of secretory vesicles to the plasma membrane.


Asunto(s)
Actinas/metabolismo , Células Cromafines/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animales , Bovinos , Membrana Celular/metabolismo , Exocitosis , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Células PC12 , Transporte de Proteínas , Ratas
5.
J Cell Biol ; 190(6): 1053-65, 2010 Sep 20.
Artículo en Inglés | MEDLINE | ID: mdl-20837769

RESUMEN

Phosphoinositide 3-kinase (PI3K) p110 isoforms are membrane lipid kinases classically involved in signal transduction. Lipopolysaccharide (LPS)-activated macrophages constitutively and abundantly secrete proinflammatory cytokines including tumor necrosis factor-α (TNF). Loss of function of the p110δ isoform of PI3K using inhibitors, RNA-mediated knockdown, or genetic inactivation in mice abolishes TNF trafficking and secretion, trapping TNF in tubular carriers at the trans-Golgi network (TGN). Kinase-active p110δ localizes to the Golgi complex in LPS-activated macrophages, and TNF is loaded into p230-labeled tubules, which cannot undergo fission when p110δ is inactivated. Similar blocks in fission of these tubules and in TNF secretion result from inhibition of the guanosine triphosphatase dynamin 2. These findings demonstrate a new function for p110δ as part of the membrane fission machinery required at the TGN for the selective trafficking and secretion of cytokines in macrophages.


Asunto(s)
Aparato de Golgi/enzimología , Aparato de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase I , Dinaminas/metabolismo , Activación Enzimática/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Silenciador del Gen/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/enzimología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Macrófagos/metabolismo , Ratones , Inhibidores de las Quinasa Fosfoinosítidos-3 , Transporte de Proteínas/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Vías Secretoras/efectos de los fármacos , Red trans-Golgi/efectos de los fármacos , Red trans-Golgi/enzimología
6.
Immunobiology ; 214(7): 601-12, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19268389

RESUMEN

Cytokines and other immune mediators are secreted by cells of the immune system during immune responses and as a means of communication. While the functions of these cytokines, chemokines and mediators are well known, the intracellular pathways that lead to their secretion by different cells are only now being fully documented. Cytokines in some cells are released from secretory granules while in other cells they are released via constitutive secretory pathways that instead have more dynamic vesicular carriers. Recent studies have revealed that newly synthesized cytokines can be routed via compartments such as recycling endosomes prior to their secretion. Here we describe and show examples of some of the pathways used for cytokine trafficking and release in macrophages, including some of the cellular machinery required for this transport. Increasingly, these trafficking pathways are revealed as having important regulatory roles in the execution of immune responses.


Asunto(s)
Citocinas/metabolismo , Macrófagos/inmunología , Transporte de Proteínas , Animales , Comunicación Celular/inmunología , Endosomas , Humanos , Inmunidad Celular , Vías Secretoras/inmunología , Vesículas Secretoras
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