RESUMEN
Glucan phosphate has been shown to enhance antimicrobial immunity in a variety of experimental models. However, the mechanisms by which glucans enhance resistance to infection remain largely unknown. Interferon-gamma (IFN-gamma) is a key regulator of both innate and acquired immunity. Suppression of IFN-gamma production is a prominent feature of the altered immune response that follows major trauma or sepsis. The present studies were designed to determine the effect of glucan phosphate on IFN-gamma expression in normal mice and endotoxin [lipopolysaccharide (LPS)]-tolerant mice. The model of LPS tolerance was used because it results in patterns of cytokine expression similar to those commonly observed following severe trauma or sepsis. Glucan treatment potentiated LPS-induced IFN-gamma expression in control mice. The induction of LPS tolerance resulted in marked suppression of LPS-induced IFN-gamma production. However, co-administration of glucan with LPS, during the tolerance induction phase, attenuated the LPS-tolerant response. Interleukin-12 (IL-12) and IL-18 are important mediators of LPS-induced IFN-gamma production. LPS-induced IL-12 p40 mRNA expression was increased in the spleens of glucan-treated mice compared with controls. Induction of LPS tolerance caused marked suppression of IL-12 production, a response that was attenuated by glucan treatment. IL-18 was constitutively expressed in both control and LPS-tolerant mice, and LPS-induced serum levels of IL-18 were increased in mice treated with glucan. T cells isolated from glucan-treated mice exhibited increased IFN-gamma expression in response to IL-12 and IL-18, as well as increased expression of the IL-12 and IL-18 receptors. The ability of glucan to potentiate IFN-gamma expression in control mice provides a potential mechanism by which glucan enhances antimicrobial immunity. The ability of glucan to attenuate suppressed IFN-gamma expression in LPS-tolerant mice denotes its potential benefit for the treatment of trauma and sepsis-induced immunosuppression.
Asunto(s)
Antiinfecciosos/inmunología , Endotoxinas/inmunología , Glucanos/inmunología , Inmunocompetencia , Inductores de Interferón/inmunología , Interferón gamma/biosíntesis , beta-Glucanos , Animales , Sinergismo Farmacológico , Femenino , Regulación de la Expresión Génica/inmunología , Tolerancia Inmunológica , Interleucina-12/biosíntesis , Interleucina-18/biosíntesis , Interleucina-18/sangre , Células Asesinas Naturales/inmunología , Lipopolisacáridos/inmunología , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/genética , Bazo/inmunología , Linfocitos T/inmunologíaRESUMEN
Endotoxin (lipopolysaccharide [LPS]) tolerance is a state of altered immunity characterized, in part, by suppression of LPS-induced gamma interferon (IFN-gamma) expression. However, the cellular mediators regulating LPS-induced production of IFN-gamma in normal mice and the effect of LPS tolerance on these mediators has not been well characterized. Our studies show that macrophage dysfunction is the primary factor causing suppressed IFN-gamma expression in LPS-tolerant mice. Specifically, LPS-tolerant macrophages have a markedly impaired ability to induce IFN-gamma secretion by T cells and NK cells obtained from either control or LPS-tolerant mice. However, T cells and NK cells isolated from LPS-tolerant mice produce normal levels of IFN-gamma when cocultured with control macrophages or exogenous IFN-gamma-inducing factors. Assessment of important IFN-gamma-regulating factors showed that interleukin-12 (IL-12) and costimulatory signals provided by IL-15, IL-18, and CD86 are largely responsible for LPS-induced IFN-gamma expression in control mice. IL-10 is an inhibitor of IFN-gamma production in both the control and LPS-tolerant groups. Expression of IL-12 and the IL-12 receptor beta1 (IL-12Rbeta1) and IL-12Rbeta2 subunits are suppressed in the spleens of LPS-tolerant mice. LPS-tolerant splenocytes also exhibit decreased production of IL-15 and IL-15Ralpha. However, expression of IL-18 and the B7 proteins CD80 and CD86 are unchanged or increased compared to controls after induction of LPS tolerance. CD28, a major receptor for B7 proteins, is also increased in the spleens of LPS-tolerant mice. Expression of the inhibitory cytokine IL-10 and the IL-10R are sustained after induction of LPS tolerance. These data show that suppression of IFN-gamma production in LPS-tolerant mice is largely due to macrophage dysfunction and provide insight into the cellular alterations that occur in LPS tolerance. This study also better defines the factors that mediate LPS-induced IFN-gamma production in normal mice.
Asunto(s)
Tolerancia Inmunológica , Interferón gamma/metabolismo , Células Asesinas Naturales/inmunología , Lipopolisacáridos/inmunología , Macrófagos Peritoneales/inmunología , Linfocitos T/inmunología , Animales , Antígeno B7-1/biosíntesis , Antígeno B7-1/metabolismo , Antígenos CD28/metabolismo , Citocinas/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Interferón gamma/biosíntesis , Interferón gamma/genética , Interleucinas/biosíntesis , Células Asesinas Naturales/efectos de los fármacos , Lipopolisacáridos/administración & dosificación , Macrófagos Peritoneales/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunología , Linfocitos T/efectos de los fármacosRESUMEN
Anovel nuclear factor kappaB (NF-kappaB) binding site has been identified within the promoter region of the mouse gene encoding choline acetyltransferase (ChAT), the enzyme that synthesizes acetylcholine and has been implicated in the cognitive deficits associated with aging and Alzheimer's disease. This binding site, which is located within the nerve growth factor (NGF)-responsive enhancer element, was recognized by the NF-kappaB protein p49 but not p65 or p50. p49 from both basal forebrain and PC12 nuclear extracts interacted with this specific sequence in electrophoretic mobility shift assays. Mutation of the NF-kappaB site caused an increase in NGF-induced promoter activation, whereas overexpression of p49 in NGF-differentiated PC12 cells caused a decrease in endogenous ChAT enzyme activity and a decrease in promoter activity that was specifically mediated through this NF-kappaB binding site. Treatment of PC12 cells with NGF resulted in a drastic reduction in nuclear p49 binding to the ChAT NF-kappaB site after 24 h, but nuclear p49 levels were not altered, suggesting that late NGF-mediated events prevent binding of p49 to the ChAT promoter by an unknown mechanism other than nuclear translocation. Decreased ChAT expression and increased NF-kappaB activity in the brain are associated with aging and Alzheimer's disease. These data indicate that p49 is a negative regulator of ChAT expression and suggest a possible mechanism for aging-associated declines in cholinergic function.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Colina O-Acetiltransferasa/genética , FN-kappa B/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Regiones Promotoras Genéticas , Envejecimiento/metabolismo , Grupos de Población Animal , Animales , Sitios de Unión/genética , Química Encefálica , Línea Celular , Colina O-Acetiltransferasa/biosíntesis , Huella de ADN , Elementos de Facilitación Genéticos/efectos de los fármacos , Masculino , Mutagénesis Sitio-Dirigida , Factor de Crecimiento Nervioso/farmacología , Proteínas Nucleares/metabolismo , Células PC12 , Prosencéfalo/química , Prosencéfalo/metabolismo , Isoformas de Proteínas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
There are age-associated cognitive and cholinergic deficits in the neurotrophin-dependent cholinergic basal forebrain neurons (CBFNs). There are also increases in the activity of the transcription factor NF-kappaB in the aged rodent brain that may reflect chronic enhancement of stress response signaling. We used partial immunolesions (PIL) to CBFN to examine the role of endogenous NGF on choline acetyltransferase (ChAT) activity and NGF-mediated NF-kappaB alteration after cholinergic deafferentation. We injected 192 IgG-saporin, an immunotoxin selectively taken up by neurotrophin receptor p75(NTR)-bearing neurons, into lateral ventricles, followed by infusions of anti-NGF to assess NF-kappaB, ChAT and NGF responses to PIL after anti-NGF infusion. Treatment with anti-NGF decreased ChAT activity by 17-34% in the cortex, hippocampus, and olfactory bulb and PIL decreased ChAT activity by 47-73%. Changes in AChE activity levels paralleled those observed for ChAT after PIL. NGF protein levels in the olfactory bulb, but not the cortex or hippocampus, increased significantly after PIL treatment. Infusion of anti-NGF abolished the PIL-induced eight-fold NGF increase in CNS. NF-kappaB binding activity to the IgG-kappaB and ChAT specific NF-kappaB consensus sequences, increased in the cortex but not hippocampus after PIL followed by anti-NGF infusion. It is likely that immunolesion-induced changes in ambient NGF levels may perturb NF-kappaB activity.
Asunto(s)
Colina O-Acetiltransferasa/metabolismo , FN-kappa B/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Neuronas/enzimología , Acetilcolinesterasa/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Biomarcadores , Colinérgicos/farmacología , Proteínas de Unión al ADN/metabolismo , Desnervación , Electroforesis , Inmunotoxinas/farmacología , Microinyecciones , N-Glicosil Hidrolasas , Factor de Crecimiento Nervioso/inmunología , Prosencéfalo/citología , Unión Proteica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Proteínas Inactivadoras de Ribosomas Tipo 1 , SaporinasRESUMEN
Apoptosis in the nervous system is a necessary event during the development of the nervous system and is also present after genotoxic events, be they chronic as in aging or more acute after trauma and ischemia. Apoptotic events reflect an interplay between intrinsic signaling events that rely on cytokines, neurotransmitters, and growth factors and responses to extrinsic events that increase levels of radical oxygen species. Both intrinsically and extrinsically driven signal-transduction pathways act via transcription factors that regulate the coordinated timely expression of stress-response genes as part of a decision-making process that can commit cells to apoptosis or survival. Here we discuss the role of two transcription factors that participate in apoptosis in the nervous system: the activator protein AP-1 and nuclear factor kappaB.
Asunto(s)
Apoptosis/fisiología , Degeneración Nerviosa/fisiopatología , Neuronas/citología , Transducción de Señal/fisiología , Animales , HumanosRESUMEN
The cholinergic neurons of the basal forebrain (CNBF) are the major source of cholinergic innervation of the cortex and hippocampus. In Alzheimer's disease and aged brain, there are severe losses of cholinergic neurons in the nucleus basalis of Meynert, leading to a reduction of cortical cholinergic activity which correlates with the severity of cognitive deficits. While there is evidence that aged central nervous system (CNS) displays impaired stress response signaling, pharmacologic treatments with neurotrophic factors appear to ameliorate these age-associated cholinergic deficits. To mimic these cholinergic deficits in experimental animals and study the acute effects of nerve growth factor (NGF), we induced a partial lesion of CBFNs by the intracerebroventricular (i.c.v.) injection of the cholinergic immunotoxin 192IgG-saporin, in groups of 3- and 30-month-old rats. The lesion was followed 14 days later by i.c.v. administration of NGF, known to restore partial immunolesion-induced cholinergic deficits in rat CNS, and all rats were killed 2 days after the NGF treatment. Here we report the effects of partial immunolesions on the levels of choline acetyltransferase (ChAT) activity and NGF receptor mRNA levels in the basal forebrain of 3- and 30-month-old rats. Because of their presence in the promoters of the NGF, NGF receptors, and ChAT genes, we also measured DNA-binding activity of the transcription factors NFB and AP-1 in the cortex and hippocampus. We discuss these findings in the context of endogenous NGF-mediated signal transduction mechanisms and conclude that we have evidence for age-associated decreases in endogenous NGF responses to partial deafferentation of the basal forebrain cholinergic projections.
Asunto(s)
Envejecimiento/fisiología , Anticuerpos Monoclonales/toxicidad , Colinérgicos/toxicidad , Inmunotoxinas/toxicidad , Factores de Crecimiento Nervioso/farmacología , Prosencéfalo/efectos de los fármacos , Animales , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/enzimología , Corteza Cerebral/inmunología , Corteza Cerebral/metabolismo , Colina O-Acetiltransferasa/metabolismo , ADN/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/enzimología , Hipocampo/inmunología , Hipocampo/metabolismo , Inyecciones Intraventriculares , Masculino , N-Glicosil Hidrolasas , FN-kappa B/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Bulbo Olfatorio/efectos de los fármacos , Bulbo Olfatorio/enzimología , Bulbo Olfatorio/inmunología , Bulbo Olfatorio/metabolismo , Prosencéfalo/enzimología , Prosencéfalo/inmunología , Prosencéfalo/metabolismo , Unión Proteica , ARN Mensajero/análisis , Ratas , Ratas Endogámicas F344 , Receptor de Factor de Crecimiento Nervioso , Receptor trkA/genética , Receptor trkA/metabolismo , Receptores de Factor de Crecimiento Nervioso/genética , Receptores de Factor de Crecimiento Nervioso/metabolismo , Proteínas Inactivadoras de Ribosomas Tipo 1 , Saporinas , Factor de Transcripción AP-1/metabolismoRESUMEN
Age-related cognitive deficits are often associated with loss of cholinergic activity within the neurotrophin-dependent cholinergic neurons that project from the basal forebrain to the hippocampus. The cause of reduced cholinergic function is unknown, but alterations in transcription factor-signaling pathways causing altered gene expression may cause decreased specific tissue function, resulting in loss of cholinergic activity. We measured transcription factor Nuclear Factor kappa B by electrophoretic mobility shift assay and Western analysis in young and aged rat brain tissues and report that basal levels of Nuclear Factor kappa B DNA-binding activity increase in the hippocampus and basal forebrain with age to significantly higher levels at 30 months of age. This age-associated increase in binding activity is associated with increased translocation of p65 to the nucleus. These data show an age-associated alteration in Nuclear Factor kappa B signal transduction pathways that may contribute to age-associated decreases in specific tissue function.