RESUMEN
Epstein-Barr virus (EBV) infection, history of infectious mononucleosis (IM) and HLA-A and DRB1 have all been proposed as risk factors for multiple sclerosis (MS). Our aim was to analyse possible interactions between antibodies against Epstein-Barr virus nuclear antigen 1 (EBNA1) or EBNA1 fragments, presence of DRB1*15 and absence of A*02. The study population includes newly diagnosed cases and matched controls. Interaction on the additive scale was calculated using attributable proportion due to interaction (AP), which is the proportion of the incidence among individuals exposed to two interacting factors that is attributable to the interaction per se. IM showed association with MS, odds ratio (OR)=1.89 (1.45-2.48% confidence interval (CI)), as did raised EBNA1 IgG OR=1.74 (1.38-2.18 95%CI). All EBNA1 fragment IgGs were associated with MS risk. However, EBNA1 fragment 385-420 IgG levels were more strongly associated to MS than total EBNA1 IgG, OR=3.60 (2.75-4.72 95%CI), and also interacted with both DRB1*15 and absence of A*02, AP 0.60 (0.45-0.76 95%CI) and AP 0.39 (0.18-0.61 95%CI), respectively. The observed interaction between HLA class I and II genotype and reactivity to EBV-related epitopes suggest that the mechanism through which HLA genes influence the risk of MS may, at least in part, involve the immune control of EBV infection.
Asunto(s)
Antígenos HLA/genética , Herpesvirus Humano 4/inmunología , Esclerosis Múltiple/genética , Esclerosis Múltiple/inmunología , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Antígenos HLA/inmunología , Antígenos HLA-A/genética , Antígenos HLA-A/inmunología , Cadenas HLA-DRB1/genética , Cadenas HLA-DRB1/inmunología , Humanos , Inmunoglobulina G/inmunología , Mononucleosis Infecciosa/genética , Mononucleosis Infecciosa/inmunología , Mononucleosis Infecciosa/virología , Persona de Mediana Edad , Esclerosis Múltiple/virología , Factores de Riesgo , Fumar/efectos adversos , Adulto JovenRESUMEN
BACKGROUND: Compared with quantitative observations, the search for qualitative changes that may characterize the immune response to Epstein-Barr virus (EBV) in multiple sclerosis (MS) has been less intense. OBJECTIVE: To examine the B-cell epitopes of antibodies against the Epstein-Barr nuclear antigen-1 (EBNA-1) and their relevance for MS, through a study in disease-discordant identical twins. METHODS: We evaluated the antibodies to all unique, maximally overlapping octapeptides of EBNA-1 in 12 pairs of monozygotic (MZ) twins (9 MS-discordant, 3 healthy), 3 non-twin patients and 2 healthy subjects. All except one of the patients were untreated. The EBV serology of these individuals had been assessed in advance using commercially available and in-house enzyme-linked immunosorbent assay (ELISA) kits, including assays for antibodies against select peptides of EBNA-1: EBNA-72 (GAGGGAGAGG) and EBNA-206 (EADYFEYHQEGGPDGE). RESULTS: The glycine-alanine rich domain of EBNA-1 was immunodominant in all subjects. Compared with healthy individuals, and similarly to what has been described in infectious mononucleosis (IM) patients, affected co-twins and non-twin patients had a significantly increased response to another EBNA-1 epitope (aa. 401-411). CONCLUSION: In a study that controls for confounders, our data focus an EBNA-1 specificity that may be associated with MS pathogenesis.
Asunto(s)
Anticuerpos Antivirales/inmunología , Linfocitos B/inmunología , Enfermedades en Gemelos , Infecciones por Virus de Epstein-Barr/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Epítopos Inmunodominantes , Esclerosis Múltiple/inmunología , Gemelos Monocigóticos , Adulto , Linfocitos B/virología , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/virología , Femenino , Humanos , Inmunidad Humoral , Italia , Masculino , Esclerosis Múltiple/genética , Esclerosis Múltiple/virología , Gemelos Monocigóticos/genéticaRESUMEN
To examine if uremia influences muscle interleukin-6 (IL-6) metabolism we studied the exchange of IL-6 across the forearm in 16 patients with chronic kidney disease (CKD) (stages 3 and 4), in 15 hemodialysis (HD)-treated end-stage renal disease (ESRD) patients (n=15), and in six healthy controls. In addition, we performed an analysis of both IL-6 protein and IL-6 mRNA expression in muscle of CKD (stage 4) patients showing evidence of inflammation and in controls. A release of IL-6 from the forearm was observed in patients with elevated IL-6 plasma levels. Arterial IL-6 was directly related to released IL-6 (r=0.69; P<0.004) in HD patients. Both IL-6 protein and IL-6 mRNA expression were increased in muscle of inflamed CKD patients vs controls (P<0.05). Although muscle net protein balance was similar in all patients, it was significantly more negative in HD patients with high than in those with low IL-6 plasma levels (P<0.05). In addition, net protein balance was related to the forearm release of IL-6 in HD patients only (r=0.47; P<0.038). These data demonstrate that IL-6 expression is upregulated in muscle, and that muscle tissue, by releasing this cytokine, may contribute to the inflammatory response in HD patients. The release of IL-6 from peripheral tissues is associated with an increase in muscle protein loss in HD patients, suggesting that muscle release of IL-6 is linked to protein catabolism in these patients. The release of IL-6 from peripheral tissues may act as a signal for the inflammatory response and contribute to functional dysregulation in uremia.
Asunto(s)
Interleucina-6/genética , Interleucina-6/metabolismo , Insuficiencia Renal Crónica/inmunología , Insuficiencia Renal Crónica/metabolismo , Anciano , Arterias , Biopsia , Enfermedades Cardiovasculares/inmunología , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/fisiopatología , Femenino , Antebrazo/irrigación sanguínea , Expresión Génica/inmunología , Humanos , Inmunohistoquímica , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/fisiopatología , Interleucina-1/sangre , Interleucina-10/sangre , Masculino , Persona de Mediana Edad , Músculo Esquelético/inmunología , Músculo Esquelético/patología , Fenilalanina/metabolismo , ARN Mensajero/metabolismo , Insuficiencia Renal Crónica/fisiopatología , Factor de Necrosis Tumoral alfa/metabolismo , Uremia/inmunología , Uremia/metabolismo , VenasRESUMEN
Extracellular nucleotides act as potent signaling molecules in the neuron-glia and glia-glia communication, via the activation of specific ligand-gated P2X and G-protein-coupled metabotropic P2Y receptors. Most of the data available about the effects of P2 receptor activation in the CNS concern astrocytes, microglia, and neurons. To gain insights into the role of purinergic receptors in oligodendrocyte development, we characterized the expression and functional activity of P2 receptors in rat oligodendrocyte progenitors (OPs) and investigated the effects of ATP and its breakdown products on their functions. We describe here that rat OPs express different types of P2 receptors and that nucleotide-induced Ca(2+) raises in these progenitor cells are mainly due to the activation of P2X(7) ionotropic and ADP-sensitive P2Y(1) metabotropic receptors. We also show that ATP and ADP stimulate OP migration, inhibit the mitogenic response of OPs to PDGF and promote oligodendrocyte differentiation. The pharmacological profile of the nucleotide-induced effects demonstrates the important regulatory role of P2Y(1) receptor signaling in OP functions. These findings suggest that ATP, which is released in high amounts under inflammatory conditions and following cell death, might regulate remyelination processes in inflammatory demyelinating diseases of the CNS, like multiple sclerosis.
Asunto(s)
Adenosina Trifosfato/farmacología , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Oligodendroglía/efectos de los fármacos , Receptores Purinérgicos P2/fisiología , Células Madre/efectos de los fármacos , Adenosina Trifosfato/antagonistas & inhibidores , Animales , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Interacciones Farmacológicas , Humanos , Modelos Biológicos , Oligodendroglía/citologíaRESUMEN
To gain insights into the role of purinergic receptors in oligodendrocyte development, we characterized the expression and functional activity of P2 receptors in cultured rat oligodendrocyte progenitors and investigated the effects of ATP and its breakdown products on the migration and proliferation of this immature glial cell population. Using Western blot analysis, we show that oligodendrocyte progenitors express several P2X (P2X(1,2,3,4,7)) and P2Y (P2Y(1,2,4)) receptors. Intracellular Ca(2+) recording by Fura-2 video imaging allowed to determine the rank potency order of the P2 agonists tested: ADPbetaS = ADP = Benzoyl ATP > ATP > ATPgammaS > UTP, alpha,beta-meATP ineffective. Based on the above findings, on pharmacological inhibition by the antagonists oxATP and MRS2179, and on the absence of alpha,betameATP-induced inward current in whole-cell recording, P2X(7) and P2Y(1) were identified as the main ionotropic and metabotropic P2 receptors active in OPs. As a functional correlate of these findings, we show that ATP and, among metabotropic agonists, ADP and the P2Y(1)-specific agonist ADPbetaS, but not UTP, induce oligodendrocyte progenitor migration. Moreover, ATP and ADP inhibited the proliferation of oligodendrocyte progenitors induced by platelet-derived growth factor, both in purified cultures and in cerebellar tissue slices. The effects of ATP and ADP on cell migration and proliferation were prevented by the P2Y(1) antagonist MRS2179. By confocal laser scanning microscopy, P2Y(1) receptors were localized in NG2-labeled oligodendrocyte progenitors in the developing rat brain. These data indicate that ATP and ADP may regulate oligodendrocyte progenitor functions by a mechanism that involves mainly activation of P2Y(1) receptors.
Asunto(s)
Oligodendroglía/metabolismo , Agonistas del Receptor Purinérgico P2 , Células Madre/metabolismo , Adenosina Difosfato/fisiología , Adenosina Trifosfato/fisiología , Animales , Animales Recién Nacidos , Western Blotting , Señalización del Calcio/fisiología , Diferenciación Celular/fisiología , Movimiento Celular , Proliferación Celular , Supervivencia Celular/fisiología , Células Cultivadas , Cerebelo/citología , Cerebelo/crecimiento & desarrollo , Quimiotaxis de Leucocito/efectos de los fármacos , Electroforesis en Gel de Poliacrilamida , Electrofisiología , Inmunohistoquímica , Microscopía Confocal , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar , Receptores Purinérgicos P2/fisiología , Receptores Purinérgicos P2Y1RESUMEN
BACKGROUND: A 10-year experience of abdominal wall hernia repair performed with anterior tension-free mesh or plug technique under local anesthesia in end-stage renal failure patients submitted to continuous ambulatory peritoneal dialysis (CAPD) is described in order to assess the safety and effectiveness of this approach. METHODS: Between January 1993 and December 2002, 18 hernia repairs were performed under local anesthesia in 16 patients (14 males and two females) with a mean age of 70 years (48-78). One umbilical and three unilateral inguinal hernias were observed and repaired before starting peritoneal dialysis (PD), while two umbilical, eight unilateral, and two bilateral groin hernias developed and were then treated during PD. Repairs were performed electively in all but one case, which was an emergency operation for strangulation. An ipsilateral scrotal swelling was also present in two indirect unilateral inguinal hernias. In these cases, the hernia sac was ligated before entering, while in the others it was simply dissected and inverted. RESULTS: Patients were discharged the same day or the day after surgery. No local or general immediate or late complications occurred. CAPD in subjects operated on during PD treatment was resumed the same day of surgery. In no instance was hernia recurrence or leak of dialysis solution observed at follow-up examinations. CONCLUSIONS: The absence of surgical and general complications and the nearly immediate resumption of PD indicate the anterior tension-free repair under local anesthesia as a safe and effective technique for CAPD patients even in an ambulatory or day-surgery setting.
Asunto(s)
Anestesia Local , Hernia Abdominal/cirugía , Diálisis Peritoneal Ambulatoria Continua , Procedimientos Quirúrgicos Operativos/métodos , Anciano , Femenino , Hernia Abdominal/complicaciones , Humanos , Fallo Renal Crónico/complicaciones , Fallo Renal Crónico/terapia , Masculino , Persona de Mediana Edad , Mallas Quirúrgicas , Técnicas de Sutura , Resultado del TratamientoRESUMEN
Recent studies have provided new insights on the identity and mechanisms of action of growth factors implicated in myelination processes. Members of several different families of growth factors have been shown to affect oligodendrocyte survival, proliferation and differentiation and their manipulation in the experimental animal provides a means to clarify the molecular mechanisms underlying repair processes in the injured central nervous system. A future challenge is to use this knowledge to boost remyelination and ameliorate clinical symptoms in patients with demyelinating diseases, like multiple sclerosis.
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Sistema Nervioso Central/efectos de los fármacos , Sustancias de Crecimiento , Regeneración Nerviosa/efectos de los fármacos , Animales , Sistema Nervioso Central/citología , Humanos , Esclerosis Múltiple/tratamiento farmacológico , Neuronas/efectos de los fármacos , Oligodendroglía/citología , Oligodendroglía/efectos de los fármacos , Células Madre/efectos de los fármacosRESUMEN
Major progress has been made over the last years in our understanding of the mechanisms underlying immune privilege and immune surveillance of the central nervous system (CNS). Once considered a passive process relying only on physical barriers, immune privilege is now viewed as a more complex phenomenon, which involves active regulation of immune reactivity by the CNS microenvironment. Evidence has also emerged that the immune system continuously and effectively patrols the CNS and that dysregulated immune responses against CNS-associated (exogenous or self) antigens are involved in the pathogenesis of various neurological diseases. In this article we shall briefly review current knowledge of how the immune response is regulated locally in the CNS and which cell types and molecular mechanisms are involved in shaping intracerebral immune responses.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Sistema Nervioso Central/inmunología , Leucocitos/inmunología , Sistema Linfático/inmunología , HumanosRESUMEN
During the past decade, mechanisms involved in the immune surveillance of the central nervous system (CNS) have moved to the forefront of neuropathological research mainly because of the recognition that most neurological disorders involve activation and, possibly, dysregulation of microglia, the intrinsic macrophages of the CNS. Increasing evidence indicates that, in addition to their well-established phagocytic function, microglia may also participate in the regulation of non specific inflammation as well as adaptive immune responses. This article focuses on the signals regulating microglia innate immune functions, the role of microglia in antigen presentation, and their possible involvement in the development of CNS immunopathology.
Asunto(s)
Comunicación Celular/inmunología , Sistema Nervioso Central/inmunología , Vigilancia Inmunológica/fisiología , Microglía/inmunología , Animales , Antígenos de Superficie/inmunología , Sistema Nervioso Central/citología , Quimiocinas/inmunología , Citocinas/inmunología , Humanos , Microglía/citología , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
Dendritic cells (DCs) are thought to be key elements in the initiation and maintenance of autoimmune diseases. In this study, we sought evidence that DCs recruited to the central nervous system (CNS), a site that is primarily devoid of resident DCs, play a role in the effector phase and propagation of the immune response in experimental autoimmune encephalomyelitis (EAE). After immunization of SJL mice with proteolipid protein 139-151 peptide, process-bearing cells expressing the DC markers DEC-205 and CD11c appeared early in the spinal cord. During acute, chronic, and relapsing EAE, DEC-205(+) DCs expressing a lymphostimulatory phenotype (including the mature DC marker MIDC-8, major histocompatibility complex class II, CD40, and CD86 molecules) accumulated within the CNS inflammatory cell infiltrates. More prominent infiltration of the spinal cord parenchyma by mature DCs was observed in mice with relapsing disease. Macrophage inflammatory protein 3alpha, a chemokine active on DCs and lymphocytes, and its receptor CCR6 were up-regulated in the CNS during EAE. These findings suggest that intracerebral recruitment and maturation of DCs may be crucial in the local stimulation and maintenance of autoreactive immune responses, and that therapeutic strategies aimed at manipulating DC migration could be useful in the treatment of CNS autoimmune disorders.
Asunto(s)
Sistema Nervioso Central/fisiopatología , Quimiocinas CC , Células Dendríticas/fisiología , Encefalomielitis Autoinmune Experimental/inmunología , Enfermedad Aguda , Animales , Senescencia Celular , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Quimiocina CCL20 , Plexo Coroideo/patología , Enfermedad Crónica , Células Dendríticas/inmunología , Progresión de la Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/fisiopatología , Femenino , Proteínas Inflamatorias de Macrófagos/metabolismo , Meninges/patología , Ratones , Ratones Endogámicos , Receptores CCR6 , Receptores de Quimiocina/metabolismo , Recurrencia , Médula Espinal/patología , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Interactions of CD4(+) T helper (Th) cells with microglia and astrocytes are likely to play an important role in regulating immune responses as well as tissue damage and repair during infectious and autoimmune central nervous system (CNS) diseases. T cells secreting Th1-type cytokines provide inducing signals for microglia to mature into functional antigen presenting cells (APC). The ability of microglia to act as efficient APC for the restimulation of Th1 cells suggests a role for these cells in the local amplification of pro-inflammatory immune responses. Conversely, the Th2-inducing capacity of microglia and astrocytes together with their ability to produce anti-inflammatory mediators could play a role in providing counter-regulatory signals limiting CNS inflammation. In this article, we review recent studies addressing the functional significance of T cell-CNS glia interactions and present new data on the expression of cyclooxygenase-2, the inducible enzyme involved in prostanoid biosynthesis, in microglia and astrocytes during the course of experimental allergic encephalomyelitis.
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Linfocitos T CD4-Positivos/citología , Comunicación Celular/inmunología , Enfermedades Autoinmunes Desmielinizantes SNC/inmunología , Microglía/citología , Neuroinmunomodulación/inmunología , Astrocitos/citología , HumanosRESUMEN
To understand the mechanisms underlying spontaneous remission of proteolipid protein (PLP) 139-151 peptide-induced experimental allergic encephalomyelitis (EAE), an acute autoimmune disease of SJL mice resembling human multiple sclerosis, we examined both the effector response site in the central nervous system (CNS) and the immunization site at different phases of the disease. In the CNS, the frequency of PLP 139-151 peptide-specific IFN-gamma-producing T cells as well as the amount of infiltrating CD4(+) and CD11b(+) cells decreased with recovery. However, IL-4-producing cells were always rare and cyclooxygenase-2(+) cells were numerous only at disease peak in the CNS, suggesting that T(h)2 cytokines and prostaglandins did not determine remission of EAE. By looking at the s.c. site of PLP 139-151 peptide plus adjuvant injection, we found that, although the inflammatory infiltrate was abundant, CD11b(+) cells started to decrease already during disease acute phase and DEC-205(+) cells were numerous only at early time points. We propose that immunization site inflammation is short-lived in PLP 139-151 peptide-induced EAE, and this leads to a temporary autoreactive T cell stimulation and to a self-limited disease.
Asunto(s)
Antígenos CD , Encefalomielitis Autoinmune Experimental/inmunología , Lectinas Tipo C , Enfermedad Aguda , Animales , Antígenos CD11/análisis , Antígenos CD4/análisis , Sistema Nervioso Central/inmunología , Ciclooxigenasa 2 , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inducido químicamente , Femenino , Inmunización , Técnicas para Inmunoenzimas , Inmunohistoquímica , Interferón gamma/análisis , Interleucina-4/análisis , Isoenzimas/análisis , Glicoproteínas de Membrana/análisis , Ratones , Antígenos de Histocompatibilidad Menor , Proteína Proteolipídica de la Mielina , Fragmentos de Péptidos , Prostaglandina-Endoperóxido Sintasas/análisis , Receptores de Superficie Celular/análisis , Piel/inmunología , Médula Espinal/inmunología , Factores de TiempoRESUMEN
Human immunodeficiency virus (HIV) infection of the central nervous system (CNS) affects primarily microglial cells and astrocytes. Infection of these latter cells occurs independently of CD4 and is characterised by preferential accumulation of 2 Kb mRNA, encoding mostly Nef, and by low levels of 4.5 and 9 Kb RNAs. We have investigated the potential role of chronic HIV infection of human astrocytic cells on the expression of pro-inflammatory cytokines, chemokines and their receptors by comparing the infected TH4-7-5 with its parental uninfected 85HG66 cell lines. Upregulated levels of tumour necrosis factor-alpha (TNF-alpha) and of certain chemokines, namely interleukin-8 (IL-8) and regulated upon activation normal T cell expressed and secreted (RANTES), were observed in the infected versus uninfected cells, whereas monocyte chemotactic protein-1 (MCP-1) was comparably expressed in both cell lines. This pattern of expression was confirmed in primary foetal astrocytes transiently transfected with HIV. In addition, CXCR1, CXCR2 and CCR2b, receptors for IL-8 and MCP-1, respectively, were also found to be upregulated in TH4-7-5 versus 85HG66. CXCR4, the receptor of stromal cell derived factor-1 (SDF-1) and co-receptor for syncytium inducing HIVs, was comparably expressed in infected and uninfected astrocytic cells, whereas CCR5 was not detected in either cell line. Furthermore, treatment of TH4-7-5 cells with TNF-alpha or IL-1beta stimulated RNA and protein secretion of IL-8, MCP-1, and RANTES as well as HIV expression. Thus, our findings suggest that HIV infection of astrocytic cells can contribute to the establishment of a chronic inflammatory state in the CNS, eventually resulting in HIV encephalitis, by increasing the secretion of pro-inflammatory cytokines, such as TNF-alpha and several chemokines. Overexpression of chemokine receptors including CCR2b, CXCR1 and CXCR2 in infected astrocytic cells may contribute to HIV-induced damage of the CNS via autocrine/paracrine activation of astrocytes.
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Astrocitos/metabolismo , Quimiocina CCL5/metabolismo , VIH-1/metabolismo , Interleucina-8/metabolismo , Receptores de Quimiocina/metabolismo , Astrocitos/citología , Astrocitos/virología , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL5/genética , Quimiocina CXCL12 , Quimiocinas/metabolismo , Quimiocinas CXC/metabolismo , Humanos , Inflamación/metabolismo , Interleucina-1/metabolismo , Interleucina-1/farmacología , Interleucina-8/genética , ARN Mensajero/biosíntesis , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Analysis of the mechanisms underlying CNS immune surveillance and immunopathology have provided new insights into the intracerebral regulation of immune responses. Here, Francesca Aloisi, Francesco Ria and Luciano Adorini review the role of CNS antigen presenting cells and focus on the control of Th1 and Th2 responses by microglia and astrocytes.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Astrocitos/inmunología , Microglía/inmunología , Linfocitos T/inmunología , Animales , Sistema Nervioso Central/inmunología , Humanos , Modelos Biológicos , NeuroinmunomodulaciónRESUMEN
In astrocytes the activity of the Na+,K(+)-ATPase pump maintains an inwardly directed electrochemical sodium gradient used by the Na+-dependent transporters and regulates the extracellular K+ concentration essential for neuronal excitability. We show here that incubation of cultured rat astrocytes with angiotensin II (Ang II) modulates Na+,K(+)-ATPase activity, in a dose- and time-dependent manner. Na+,K(+)-ATPase activation was mediated by binding of Ang II to AT1 receptors as it was completely blocked by DuP 753, a specific AT1 receptor subtype antagonist. Stimulation of Na+,K(+)-ATPase activity by Ang II was dependent on protein kinase C (PKC) activation because PKC antagonists abolished the inducing effect of Ang II and the PKC activator phorbol 12-myristate 13-acetate enhanced transporter activity. Ang II stimulated translocation of PKC-delta but not that of other PKC isoforms from the cytosol to the plasma membrane. These results indicate that the activity of Na+,K(+)-ATPase in astrocytes is increased by physiological concentrations of Ang II and that the AT1 receptor subtype mediates the Na+,K(+)-ATPase response to Ang II via PKC-delta activation.
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Angiotensina II/farmacología , Astrocitos/enzimología , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Receptores de Angiotensina/fisiología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Astrocitos/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Activación Enzimática/fisiología , Proteína Quinasa C-delta , Ratas , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2RESUMEN
A precise knowledge of the early events inducing maturation of resting microglia into a competent APC may help to understand the involvement of this cell type in the development of CNS immunopathology. To elucidate whether signals from preactivated T cells are sufficient to induce APC features in resting microglia, microglia from the adult BALB/c mouse CNS were cocultured with Th1 and Th2 lines from DO11.10 TCR transgenic mice to examine modulation of APC-related molecules and Ag-presenting capacity. Upon Ag-specific interaction with Th1, but not Th2, cells, microglia strongly up-regulated the surface expression of MHC class II, CD40, and CD54 molecules. Induction of CD86 on mouse microglia did not require T cell-derived signals. Acutely isolated adult microglia stimulated Th1 cells to secrete IFN-gamma and, to a lesser extent, IL-2, but were inefficient stimulators of IL-4 secretion by Th2 cells. Microglia exposed in vitro to IFN-gamma showed enhanced expression of MHC class II, CD40, and CD54 molecules and became able to restimulate Th2 cells. In addition to IFN-gamma, GM-CSF increased the ability of microglia to activate Th1, but not Th2, cells without up-regulating MHC class II, CD40, or CD54 molecules. These results suggest that interaction with Th1 cells and/or Th1-secreted soluble factors induces the functional maturation of adult mouse microglia into an APC able to sustain CD4+ T cell activation. Moreover, GM-CSF, a cytokine secreted by T cells as well as reactive astrocytes, could prime microglia for Th1-stimulating capacity, possibly by enhancing their responsiveness to Th1-derived signals.
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Células Presentadoras de Antígenos/inmunología , Comunicación Celular/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Microglía/inmunología , Células TH1/inmunología , Adyuvantes Inmunológicos/fisiología , Animales , Células Presentadoras de Antígenos/citología , Antígenos CD/biosíntesis , Moléculas de Adhesión Celular/biosíntesis , Diferenciación Celular/inmunología , Separación Celular , Sistema Nervioso Central/citología , Sistema Nervioso Central/inmunología , Técnicas de Cocultivo , Femenino , Antígenos de Histocompatibilidad Clase II/biosíntesis , Sueros Inmunes/farmacología , Inmunofenotipificación , Interferón gamma/inmunología , Interferón gamma/fisiología , Interfase/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Microglía/citología , Células TH1/metabolismo , Células Th2/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
We have compared the efficiency of central nervous system and peripheral antigen-presenting cells (APC) in T cell priming and restimulation. OVA peptide 323 - 339-dependent activation of DO11.10 TCR-transgenic naive CD4+ and polarized Th1 or Th2 cells was assessed in the presence of microglia and astrocytes from the neonatal mouse brain as well as dendritic cells (DC) and B cells purified from adult mouse lymph nodes. DC were the most efficient in inducing naive T cell proliferation, IL-2 secretion and differentiation into Th1 cells, followed by IFN-gamma-preactivated microglia, large and small B cells. Astrocytes failed to activate naive T cells. IFN-gamma-pretreated microglia were as efficient as DC in the restimulation of Th1 cells, whereas IFN-gamma-pretreated astrocytes, large and small B cells were much less efficient. Conversely, Th2 cells were efficiently restimulated by all the APC types examined. During T cell priming, DC secreted more IL-12 than microglia but similar amounts of IL-12 were secreted by the two cell types upon interaction with Th1 cells. The hierarchy of APC established in this study indicates that DC and microglia are the most efficient in the stimulation of naive CD4(+) T cells and in the restimulation of Th1 cells, suggesting that activated microglia may effectively contribute to Th1 responses leading to central nervous system inflammation and tissue damage. These potentially pathogenic responses could be counteracted by the high efficiency of astrocytes as well as microglia in restimulating Th2 cells.
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Astrocitos/inmunología , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Activación de Linfocitos/inmunología , Microglía/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Astrocitos/metabolismo , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Microglía/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
In the human immunodeficiency virus type 1 (HIV-1)-infected brain, the virus does not replicate in astrocytes, but a synthesis of viral regulatory proteins occurs in these cells, leading to accumulation of Nef. As an approach to understand the effects of Nef on astrocyte functional activity, we analyzed whether intracellular Nef interferes with the expression and activation of the enzyme protein kinase C (PKC), which is an important regulator of astroglial functions and HIV-1 replication. Astrocytoma clones (U251 MG) not expressing Nef (Neo), or expressing wild-type Nef (Bru) or nonmyristoylated Nef (TH) were used to monitor the expression and activation of 10 PKC isoforms. The same clones were used to evaluate the effect of Nef on the viral long terminal repeat (LTR) promoter after activation of PKC with the phorbol ester 12-myristate 13-acetate (PMA). PKC intracellular distribution and activation were evaluated by Western blot analysis of cytosolic and membrane fractions of control and Nef-expressing clones. PMA-induced LTR activation was analyzed in clones transfected with a plasmid encoding for the CAT reporter gene controlled by the LTR promoter, by using an enzyme-linked immunosorbent assay to measure CAT expression. Nef selectively downregulated the expression and activation of betaII and epsilon PKC isoforms in astrocytoma cells. Such downregulation correlated with an inhibition of LTR activation after PMA stimulation. The myristoylation of Nef and its membrane localization were essential for these effects. These results suggest that Nef may alter astrocytic functions by interfering with PKC expression and activation and contribute to the restriction of HIV-1 replication in astrocytes.
Asunto(s)
Astrocitoma/patología , Neoplasias Encefálicas/patología , Regulación Neoplásica de la Expresión Génica , Productos del Gen nef/fisiología , VIH-1/fisiología , Isoenzimas/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Proteína Quinasa C/biosíntesis , Secuencias Repetidas Terminales , Complejo SIDA Demencia/enzimología , Complejo SIDA Demencia/virología , Acilación , Astrocitoma/enzimología , Neoplasias Encefálicas/enzimología , Cloranfenicol O-Acetiltransferasa/biosíntesis , Inducción Enzimática/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Productos del Gen nef/química , Genes Reporteros , Humanos , Isoenzimas/genética , Ácido Mirístico/metabolismo , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Regiones Promotoras Genéticas , Proteína Quinasa C/genética , Proteína Quinasa C beta , Proteína Quinasa C-epsilon , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/biosíntesis , Acetato de Tetradecanoilforbol/farmacología , Transfección , Células Tumorales Cultivadas/efectos de los fármacos , Replicación Viral , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
Following brain injury, microglial cells produce pro- and anti-inflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin-10 (IL-10). IL-10 provides an efficient autocrine mechanism for controlling microglia activation. To elucidate the mechanisms that regulate the cytokine profile of microglia, we examined the effects of several immunomodulators on IL-10 and TNF production by cultured mouse microglia. Lipopolysaccharide (LPS) was the only inducer of IL-10 and TNF gene expression and secretion. The T helper 1-type cytokine interferon-gamma (IFN-gamma) induced TNF transcripts, but not TNF secretion, and suppressed LPS-induced IL-10 mRNA and secretion by microglia. Opposite to IFN-gamma, the lipid mediator prostaglandin E2 (PGE2) enhanced the LPS-induced production of IL-10 and inhibited that of TNF. The effects of PGE2 on cytokine gene expression and secretion were antagonized by IFN-gamma. Agents that increase cAMP levels mimicked the action of PGE2 on cytokine secretion, indicating the involvement of cAMP-coupled prostaglandin receptors. In conclusion, IFN-gamma and PGE2, two mediators released at inflammatory sites, differentially regulate the production of a proinflammatory and an anti-inflammatory cytokine in microglia. We suggest that the activity and role of microglia in the damaged CNS could be finely tuned by the local concentration ratio of these mediators.
Asunto(s)
Dinoprostona/farmacología , Regulación de la Expresión Génica/inmunología , Interferón gamma/farmacología , Interleucina-10/genética , Microglía/inmunología , Factor de Necrosis Tumoral alfa/genética , Animales , Animales Recién Nacidos , Células Cultivadas , Citocinas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Inflamación/prevención & control , Interferón gamma/antagonistas & inhibidores , Isoproterenol/farmacología , Cinética , Lipopolisacáridos/farmacología , Ratones , Microglía/citología , Microglía/efectos de los fármacos , Prosencéfalo/citología , Prosencéfalo/inmunología , ARN Mensajero/genética , Proteínas Recombinantes , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/efectos de los fármacos , Transcripción Genética/inmunologíaRESUMEN
To identify a good system to introduce foreign genes into normal and tumoral astrocytes, we studied the efficiency of two chemical methods, calcium phosphate precipitation and lipofection, and of a viral-mediated transfer by a vector derived from the highly attenuated modified vaccinia virus Ankara (MVA). Using the beta-galactosidase (beta-gal) gene (lacZ) as reporter, we searched for optimal experimental conditions to obtain an efficient gene transfer into human embryonic and neonatal rat astrocytes and into a human astrocytoma cell line (U373 MG). The beta-gal protein production was evaluated by cytochemical staining and enzymatic activity assay. Among chemical methods, lipofection was the most efficient system to transfect astrocytes in providing up to 60% of beta-gal-positive cells in all the cell types analyzed. MVA infection also proved to be an efficient system to introduce heterologous genes into human embryonic astrocytes that appeared 80-100% positive 48-96 hr after an infection at a multiplicity of 1-10. In contrast, only a limited infection was observed with rat astrocytes, human astrocytoma cells, and human leptomeningeal cells. A recombinant MVA vector expressing the human immunodeficiency virus-1 (HIV-1) regulatory protein Nef was used to transfect human embryonic astrocytes, and the resulting Nef expression was compared with that detected after lipofection in the same cells. By Western blot analysis, Nef expression was observed in human astrocytes 24-96 hr after infection and was similar to that present in stably HIV-1-infected astrocytoma cells. Lipofection resulted in lower Nef expression. In spite of these promising results, the negative effects of MVA infection on cell viability and the possibility that a productive infection occurs in human embryonic astrocytes limit the use of this vector for gene delivery in developmentally immature human glial cells.