RESUMEN
We previously reported that macrophage activators such as LPS, IL-2, and IL-4 down-modulate the M-CSFR via a mechanism involving protein kinase C and phospholipase C. In this study, we showed that M-CSFR is shed from macrophage surface and identified the protease responsible for M-CSFR cleavage and down-modulation. The shedding of M-CSFR elicited by phorbol esters (tetradecanoylphorbol myristate acetate (TPA)) or LPS in murine BAC.1-2F5 macrophages was prevented by cation chelators, as well as hydroxamate-based competitive inhibitors of metalloproteases. We found that the protease cleaving M-CSFR is a transmembrane enzyme and that its expression is controlled by furin-like serine endoproteases, which selectively process transmembrane metalloproteases. M-CSFR down-modulation was inhibited by treating cells in vivo, before TPA stimulation, with an Ab raised against the extracellular, catalytic domain of proTNF-converting enzyme (TACE). TACE expression was confirmed in BAC.1-2F5 cells and found inhibited after blocking furin-dependent processing. Using TACE-negative murine Dexter-ras-myc cell monocytes, we found that in these cells TPA is unable to down-modulate M-CSFR expression. These data indicated that TACE is required for the TPA-induced M-CSFR cleavage. The possibility that the cleavage is indirectly driven by TACE via the release of TNF was excluded by treating cells in vivo with anti-TNF Ab. Thus, we concluded that TACE is the protease responsible for M-CSFR shedding and down-modulation in mononuclear phagocytes undergoing activation. The possible physiological relevance of this mechanism is discussed.
Asunto(s)
Activación de Macrófagos/inmunología , Macrófagos/enzimología , Macrófagos/inmunología , Metaloendopeptidasas/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas ADAM , Proteína ADAM17 , Animales , Anticuerpos Monoclonales/farmacología , Dominio Catalítico/inmunología , Línea Celular , Línea Celular Transformada , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/inmunología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/inmunología , Humanos , Hidrólisis , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Metaloendopeptidasas/inmunología , Metaloendopeptidasas/aislamiento & purificación , Ratones , Ratones Endogámicos A , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Monocitos/enzimología , Monocitos/inmunología , Receptor de Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
We evaluated the synthesis of nitric oxide (NO) and of the neurotoxic kynurenine metabolites 3OH-kynurenine and quinolinic acid (QUIN) in interferon-gamma (IFN-gamma)-activated macrophages of the murine BACl.2F5 cell line with the aim of investigating the roles of mononuclear phagocytes in inflammatory neurological disorders. IFN-gamma induced indoleamine 2,3-dioxygenase (IDO) and NO synthase (NOS) and increased the synthesis of 3OH-kynurenine, QUIN, and NO that accumulated in the incubation medium where they reached neurotoxic levels. Macrophage exposure to norharmane, an IDO inhibitor, resulted in a decreased formation of not only the kynurenine metabolites but also NO. The inhibition of NO synthesis could not be ascribed to reduced NADPH availability or decreased NOS induction. Norharmane inhibited NOS activity also in coronary vascular endothelial cells and in isolated aortic rings. Our findings suggest that activated macrophages release large amounts of neurotoxic molecules and that norharmane may represent a prototype compound to study macrophage involvement in inflammatory brain damage.
Asunto(s)
Interferón gamma/farmacología , Quinurenina/biosíntesis , Activación de Macrófagos , Macrófagos/fisiología , Óxido Nítrico Sintasa/fisiología , Ácido Quinolínico/metabolismo , Triptófano Oxigenasa/fisiología , Animales , Línea Celular , Indolamina-Pirrol 2,3,-Dioxigenasa , Activación de Macrófagos/efectos de los fármacos , Ratones , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II , Triptófano Oxigenasa/antagonistas & inhibidoresRESUMEN
The interference of low-molecular-weight phosphotyrosine protein phosphatase with the macrophage response to macrophage colony-stimulating factor was investigated. This paper shows that this phosphatase, already known to be involved in platelet-derived growth factor receptor signaling, is physiologically expressed in murine macrophages and dephosphorylates in vitro macrophage colony-stimulating factor receptor molecules immunoprecipitated from macrophage colony-stimulating factor-stimulated macrophages. We obtained the first demonstration that a phosphotyrosine-specific protein phosphatase dephosphorylates the macrophage colony-stimulating factor receptor in vivo and reduces the mitogenic response to macrophage colony-stimulating factor. The data indicate that low-molecular-weight phosphotyrosine protein phosphatase is a negative regulator of macrophage colony-stimulating factor receptor signaling.
Asunto(s)
Factor Estimulante de Colonias de Macrófagos/fisiología , Mitógenos/fisiología , Proteínas Tirosina Fosfatasas/biosíntesis , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/fisiología , Tirosina/metabolismo , Células 3T3 , Animales , Recuento de Células , Línea Celular , ADN/metabolismo , Humanos , Macrófagos/citología , Macrófagos/enzimología , Macrófagos/metabolismo , Ratones , Mitógenos/antagonistas & inhibidores , Peso Molecular , Fosforilación/efectos de los fármacos , Proteínas Tirosina Fosfatasas/fisiología , Receptor de Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Timidina/metabolismo , Transfección , Tritio/metabolismo , Tirosina/antagonistas & inhibidoresRESUMEN
The activation of macrophages interferes with their response to macrophage colony-stimulating factor (M-CSF), the main growth and differentiation factor for mononuclear phagocytes. We tested the rapid effects of interleukin-4 (IL-4), the alternative macrophage activator produced by Th2 helper lymphocytes, on the receptor for M-CSF (M-CSFR) expressed on the cell surface of murine macrophages. IL4 rapidly down-modulated M-CSFR in a dose-dependent fashion. This effect was unique to IL-4 among a number of Th2-produced cytokines, none of which, with the exception of IL4 itself, is able to activate macrophages. The down-modulation of M-CSFR by IL4 was partially prevented by the inhibition of the activity of phospholipase C or protein kinase C. The data are consistent with the hypothesis that the down-modulation of M-CSFR is a property common to, and exclusive of, macrophage activators, and is driven by different activators via a common mechanism.