RESUMEN
OBJECTIVE: Due to its potent neurotrophic activity, insulin-like growth factor I (IGF-I) has been proposed many times for therapeutic application in disorders of the central nervous system (CNS). However, insufficient brain delivery to yield beneficial central without peripheral side effects have prevented clinical development in most instances. DESIGN: We recently reported the generation of a polyethylene-glycol modified IGF-I variant (PEG-IGF-I) with prolonged half-life and less acute side effects, but with fully maintained slow anabolic activity. Here we investigated if these beneficial properties result in improved brain availability of the drug, thereby reaching therapeutically relevant steady-state concentrations to elicit beneficial effects on neuronal function. RESULTS: After a single subcutaneous injection, PEG-IGF-I reached much higher steady-state levels in brain tissue and cerebrospinal fluid compared with IGF-I. Two weeks treatment with PEG-IGF-I was sufficient to modulate brain plasticity processes, as judged by changes in synaptic proteins and related animal behavior. Furthermore, chronic treatment of a mouse model of brain amyloidosis with PEG-IGF-I reverted deficits in insulin/IGF-I signaling, synaptic proteins and cognitive performance. CONCLUSIONS: Our data generate the therapeutic potential for PEG-IGF-I to treat CNS disorders by systemic drug application, and in addition scientifically support its application in disorders of synaptic function and neuronal development.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/análogos & derivados , Fármacos Neuroprotectores/farmacocinética , Polietilenglicoles/farmacocinética , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Química Encefálica , Enfermedades del Sistema Nervioso Central/tratamiento farmacológico , Factor I del Crecimiento Similar a la Insulina/administración & dosificación , Factor I del Crecimiento Similar a la Insulina/análisis , Factor I del Crecimiento Similar a la Insulina/líquido cefalorraquídeo , Factor I del Crecimiento Similar a la Insulina/química , Factor I del Crecimiento Similar a la Insulina/farmacocinética , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratones , Ratones Endogámicos C57BL , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/análisis , Fármacos Neuroprotectores/farmacología , Polietilenglicoles/administración & dosificación , Polietilenglicoles/análisis , Polietilenglicoles/química , Polietilenglicoles/farmacología , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
RATIONALE: The neurokinin 3 (NK(3)) receptor is a novel target under investigation for improvement of the symptoms of schizophrenia due to its ability to modulate dopaminergic signaling. However, research on effects of NK(3) antagonism with animal models has been hindered because of species differences in the receptor between humans, rats, and mice. OBJECTIVES: The aim of the present study is to further knowledge on the role of NK(3) in cognitive functioning by testing the effect of knockout of the NK(3) receptor on tests of working memory, spatial memory, and operant responding. MATERIALS AND METHODS: NK(3) knockout mice generated on a C57Bl/6 background were tested in delayed matching to position (DMTP), spontaneous alternation, Morris water maze, and active avoidance tasks. RESULTS: NK(3) knockout mice showed better performance in the DMTP task, though not delay dependently, which points to an effect on operant performance but not on working memory. No differences were seen between the groups in spontaneous alternation, another indication that working memory is not affected in NK(3) knockouts. There was no impairment in knockout mice in Morris water maze training, and the mice also showed faster response latency in the active avoidance task during training. CONCLUSIONS: Collectively, these results support a role for the NK(3) receptor in performance of operant tasks and in spatial learning but not in working memory.
Asunto(s)
Cognición/fisiología , Receptores de Neuroquinina-3/genética , Receptores de Neuroquinina-3/fisiología , Animales , Clonación Molecular , Condicionamiento Operante/efectos de los fármacos , Femenino , Genotipo , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Memoria a Corto Plazo/efectos de los fármacos , Ratones , Ratones Noqueados , Refuerzo en Psicología , Percepción Espacial/fisiologíaRESUMEN
alpha-Synuclein (alpha-SYN) is deposited in intraneuronal cytoplasmic inclusions (Lewy bodies, LBs) characteristic for Parkinson's disease (PD) and LB dementias. alpha-SYN forms LB-like fibrils in vitro, in contrast to its homologue beta-SYN. Here we have investigated the solubility of SYNs in human LB diseases and in transgenic mice expressing human wild-type and PD-associated mutant [A30P]alpha-SYN driven by the brain neuron-specific promoter, Thy1. Distinct alpha-SYN species were detected in the detergent-insoluble fractions from brains of patients with PD, dementia with LBs, and neurodegeneration with brain iron accumulation type 1 (formerly known as Hallervorden-Spatz disease). Using the same extraction method, detergent-insolubility of human alpha-SYN was observed in brains of transgenic mice. In contrast, neither endogenous mouse alpha-SYN nor beta-SYN were detected in detergent-insoluble fractions from transgenic mouse brains. The nonamyloidogenic beta-SYN was incapable of forming insoluble fibrils because amino acids 73 to 83 in the central region of alpha-SYN are absent in beta-SYN. In conclusion, the specific accumulation of detergent-insoluble alpha-SYN in transgenic mice recapitulates a pivotal feature of human LB diseases.
Asunto(s)
Enfermedad por Cuerpos de Lewy/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Aminoácidos/genética , Animales , Sitios de Unión/genética , Western Blotting , Encéfalo/metabolismo , Encéfalo/patología , Detergentes , Modelos Animales de Enfermedad , Humanos , Enfermedad por Cuerpos de Lewy/genética , Enfermedad por Cuerpos de Lewy/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Solubilidad , Fracciones Subcelulares , Sinaptosomas/metabolismo , Sinucleínas , alfa-SinucleínaRESUMEN
Environmental and genetic factors that contribute to the pathogenesis of Parkinson's disease are discussed. Mutations in the alpha-synuclein (alphaSYN ) gene are associated with rare cases of autosomal-dominant Parkinson's disease. We have analysed the dopaminergic system in transgenic mouse lines that expressed mutant [A30P]alphaSYN under the control of a neurone-specific Thy-1 or a tyrosine hydroxylase (TH) promoter. The latter mice showed somal and neuritic accumulation of transgenic [A30P]alphaSYN in TH-positive neurones in the substantia nigra. However, there was no difference in the number of TH-positive neurones in the substantia nigra and the concentrations of catecholamines in the striatum between these transgenic mice and non-transgenic littermates. To investigate whether forced expression of [A30P]alphaSYN increased the sensitivity to putative environmental factors we subjected transgenic mice to a chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) regimen. The MPTP-induced decrease in the number of TH-positive neurones in the substantia nigra and the concentrations of catecholamines in the striatum did not differ in any of the [A30P]alphaSYN transgenic mouse lines compared with wild-type controls. These results suggest that mutations and forced expression of alphaSYN are not likely to increase the susceptibility to environmental toxins in vivo.
Asunto(s)
1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Proteínas del Tejido Nervioso/genética , Enfermedad de Parkinson/genética , Ácido 3,4-Dihidroxifenilacético/metabolismo , Sustitución de Aminoácidos , Animales , Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Ácido Homovanílico/metabolismo , Humanos , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/fisiología , Neuritas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Trastornos Parkinsonianos/inducido químicamente , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/patología , Regiones Promotoras Genéticas , Sustancia Negra/metabolismo , Sinucleínas , Antígenos Thy-1/genética , Tirosina 3-Monooxigenasa/genética , alfa-SinucleínaRESUMEN
Mutations in the alpha-synuclein (alphaSYN) gene are associated with rare cases of familial Parkinson's disease, and alphaSYN is a major component of Lewy bodies and Lewy neurites. Here we have investigated the localization of wild-type and mutant [A30P]alphaSYN as well as betaSYN at the cellular and subcellular level. Our direct comparative study demonstrates extensive synaptic colocalization of alphaSYN and betaSYN in human and mouse brain. In a sucrose gradient equilibrium centrifugation assay, a portion of betaSYN floated into lower density fractions, which also contained the synaptic vesicle marker synaptophysin. Likewise, wild-type and [A30P]alphaSYN were found in floating fractions. Subcellular fractionation of mouse brain revealed that both alphaSYN and betaSYN were present in synaptosomes. In contrast to synaptophysin, betaSYN and alphaSYN were recovered from the soluble fraction upon lysis of the synaptosomes. Synaptic colocalization of alphaSYN and betaSYN was directly visualized by confocal microscopy of double-stained human brain sections. The Parkinson's disease-associated human mutant [A30P]alphaSYN was found to colocalize with betaSYN and synaptophysin in synapses of transgenic mouse brain. However, in addition to their normal presynaptic localization, transgenic wild-type and [A30P]alphaSYN abnormally accumulated in neuronal cell bodies and neurites throughout the brain. Thus, mutant [A30P]alphaSYN does not fail to be transported to synapses, but its transgenic overexpression apparently leads to abnormal cellular accumulations.
Asunto(s)
Encéfalo/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Enfermedad de Parkinson/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Humanos , Ratones , Ratones Transgénicos , Microscopía Confocal , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Neuritas/metabolismo , Enfermedad de Parkinson/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Mutación Puntual , Fracciones Subcelulares/metabolismo , Sinapsis/metabolismo , Sinaptosomas/metabolismo , Sinucleínas , alfa-SinucleínaRESUMEN
Interleukin-18 (IL-18) is a recently identified proinflammatory cytokine. Its ability to induce interferon-g suggests a potential virustatic effect. On the other hand, it stimulates NFkB - an activator of HIV replication. Recently, stimulation of HIV-1 in monocytic cells has been demonstrated. In the present study, the influence of IL-18 on HIV-1 replication in lymphatic cells was investigated. Hut78 cells were infected with HIV-1 in the presence of recombinant human IL-18 expressed either in E. coli or eucaryotically by baculovirus in Sf9 cells. HIV-1 replication was monitored by p24 ELISA and endpoint titration of culture supernatants on C8166 cells. The addition of IL-18 led to a 3- to 15-fold enhancement of HIV replication in Hut78 cells. By addition of neutralising monoclonal anti-IL-18 antibodies, this effect of IL-18 was reduced by 75%. Exposure of Hut78 to IL-18 prior to HIV infection could exclude the possibility that IL-18 promotes infection of cells. Taken together, these data provide direct evidence for an IL-18-mediated enhancement of HIV-1 replication in lymphatic cells.
Asunto(s)
VIH-1/fisiología , Interleucina-18/farmacología , Replicación Viral/efectos de los fármacos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Cartilla de ADN , Escherichia coli , Proteína p24 del Núcleo del VIH/biosíntesis , VIH-1/efectos de los fármacos , Humanos , Cinética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/farmacología , Spodoptera , Linfocitos T , TransfecciónRESUMEN
The 15-20 kDa synuclein (SYN) phosphoproteins are abundantly expressed in nervous tissue. Members of the family include alpha- and beta-SYN, and the more distantly related gamma-SYN and synoretin. SYN genes have been identified in Torpedo, canary, and several mammalian species, indicating an evolutionary conserved role. Expression of alpha-SYN was found to be modulated in situations of neuronal remodeling, namely, songbird learning and after target ablation of dopaminergic striatonigral neurons in the rat. The presynaptic localization of alpha-SYN is further supportive of a direct physiological role in neuronal plasticity. The extensive synaptic co-localization of alpha- and beta-SYN might indicate functional redundancy of these highly homologous synucleins. However, alpha-SYN was the only family member identified in Lewy bodies and cytoplasmic inclusions characteristic for multiple system atrophy. Moreover, alpha-SYN was genetically linked to familial Parkinson's disease. The two Parkinson's disease-associated mutations accelerated the intrinsic aggregation property of alpha-SYN in vitro. Post-translational modifications, such as phosphorylation and proteolysis, and/or interaction with other proteins, might regulate alpha-SYN fibril formation in vivo. Cytoskeletal elements and signal transduction intermediates have been recently identified as binding partners for alpha-SYN. Preliminary data available from transgenic mice suggest that (over)expressed human alpha-SYN proteins are less efficiently cleared from the neuronal cytosol. Thus, Parkinson's disease-associated mutations might perturb axonal transport, leading to somal accumulation of alpha-SYN and eventually Lewy body formation.
Asunto(s)
Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Enfermedad de Parkinson/fisiopatología , Secuencia de Aminoácidos , Animales , Células Cultivadas , Humanos , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Neuronas/patología , Enfermedad de Parkinson/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Sinapsis/fisiología , Sinucleínas , alfa-SinucleínaRESUMEN
The role of interleukin-12 (IL-12) was investigated in different shock models using anti-IL-12 reagents. IL-12 is composed of two disulfide-bonded subunits, p35 and p40. The IL-12 p40 homodimer (p40)2 has been shown to be a potent IL-12 antagonist in vitro. We investigated its in vivo inhibitory capacity in different shock models of mice. We could demonstrate that (p40)2 is able to protect mice from septic shock in primarily IL-12-dependent models such as the Shwartzman reaction and lipopolysaccharide (LPS)-induced shock, whereas (p40)2 has no effect in the tumor necrosis factor alpha-dependent LPS/D-GalN shock model. In IL-12-dependent shock models, (p40)2 inhibits IL-12-induced gamma interferon production and thereby interferes with the cascade of cytokine release, finally leading to death.
Asunto(s)
Interleucina-12/fisiología , Choque Séptico/prevención & control , Factor de Necrosis Tumoral alfa/fisiología , Animales , Citocinas/fisiología , Femenino , Interferón gamma/fisiología , Interleucina-12/antagonistas & inhibidores , Interleucina-18 , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos C57BL , Ratas , Choque Séptico/etiología , Fenómeno de Shwartzman/mortalidadRESUMEN
The mRNA and protein expression of the alpha- and beta-chains of IFN-gammaR were evaluated on a panel of human Th1 and Th2 clones. When cultured in IL-2-conditioned medium, both types of clones expressed mRNA for the alpha- and beta-chains, and both chains were present in the cytoplasm. Membrane expression of the alpha-chain was higher on Th2 than on Th1, whereas the beta-chain was poorly expressed on both types but increased following IL-2 withdrawal or PHA stimulation. In addition, both types of clones overexpressed MHC class I glycoproteins following IFN-gammaR triggering by exogenous IFN-gamma, although the kinetics was slower in Th1, and this exposure induced mRNA for IRF-1. When their TCR was triggered in the absence of APC, Th1 only underwent apoptosis. This activation-induced apoptosis was prevented by blocking of the alpha-chain or by IFN-gamma neutralization. Addition of IFN-gamma triggered the apoptosis of Th2 clones. Apoptosis of both types of clones was mediated by autocrine or exogenous IFN-gamma through the up-regulation of Fas-L expression, since anti-IFN-gammaR alpha mAb inhibited its expression on Th1 and exogenous IFN-gamma increased its expression on Th2. These results indicate that activated human Th1 and Th2 lymphocytes express IFN-gammaR alpha- and beta-chains and are both sensitive to signals provided by IFN-gamma. Data also suggest that IFN-gamma is critical for switching off their responses.
Asunto(s)
Apoptosis , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Interferón/metabolismo , Células TH1/patología , Células Th2/patología , Células Clonales , Humanos , ARN Mensajero/análisis , Transducción de Señal , Células TH1/metabolismo , Células Th2/metabolismo , Receptor de Interferón gammaRESUMEN
Studies of hamster-human and mouse-human somatic fibroblast hybrids and transfected mouse fibroblasts have demonstrated that signaling through the human interferon-gamma receptor (hu-IFN-gammaR) requires the formation of a complex consisting of ligand (IFN-gamma), a ligand binding receptor chain (IFN-gammaR1), and a signal transducing receptor chain (IFN-gammaR2). To date, the ability of this receptor complex to transduce the full repertoire of biological signals has been difficult to assess due to the limited number of activities that IFN-gamma can exert on fibroblasts. The current report assesses the ability of hu-IFN-gammaR chains to transduce signals in the absence of background human gene products by expressing hu-IFN-gammaR2 in a transformed macrophage cell line (F10/96) derived from a hu-IFN-gammaR1 transgenic mouse. Our results indicate that F10/96 clones expressing both human receptor proteins bind hu-IFN-gamma with an affinity comparable to that of human cells. Binding of either human or mouse IFN-gamma to its respective receptor elicits classic IFN-gamma responses such as up-regulation of major histocompatibility complex antigens, enhanced expression of IRF-1, and increased production of NO2- radicals, interleukin-6, tumor necrosis factor-alpha, and granulocyte macrophage-colony stimulating factor. However, hu-IFN-gamma could not fully protect the clones from cytopathic effects of encephalomyocarditis virus and vesicular stomatitis virus while mo-IFN-gamma could. These results demonstrate that while co-expression of hu-IFN-gammaR1 and hu-IFN-gammaR2 is necessary and sufficient for most IFN-gamma-induced responses, it is not sufficient to confer a generalized antiviral state. These findings further suggest that additional species-specific accessory factor(s) are necessary for full signaling potential through the IFN-gamma receptor complex. The nature and potential role of such factors in IFN-gammaR signaling is discussed.
Asunto(s)
Antígenos CD/fisiología , Efecto Citopatogénico Viral , Interferón gamma/fisiología , Macrófagos/fisiología , Receptores de Interferón/fisiología , Animales , Células de la Médula Ósea , Proteínas de Unión al ADN/fisiología , Expresión Génica , Factor Estimulante de Colonias de Granulocitos/metabolismo , Humanos , Factor 1 Regulador del Interferón , Interleucina-6/metabolismo , Ligandos , Sustancias Macromoleculares , Activación de Macrófagos , Ratones , Ratones Transgénicos , Óxido Nítrico/metabolismo , Fosfoproteínas/fisiología , ARN Mensajero/genética , Transducción de Señal , Transfección , Factor de Necrosis Tumoral alfa/metabolismo , Receptor de Interferón gammaRESUMEN
To find out how physiologically secreted IFN-gamma controls either the proliferation or the apoptosis of human T lymphocytes, the kinetics of expression of the alpha- and beta-chains of its receptor (IFN-gamma R) were sequentially followed on T lymphocytes first activated with PHA and then cultured in the presence of IL-2, and related to the kinetics of expression of Fas, Bcl-2, and IL-2R p55 chain. Both IFN-gamma R chains were poorly expressed on the membrane of resting T lymphocytes. Following their stimulation with PHA, IFN-gamma R alpha but not IFN gamma R beta-chain up-modulated before T lymphocyte entry into the S phase, and then IFN-gamma R alpha down-modulated when they passed through the S and G2/M. The ensuing proliferative response was inhibited by an anti-IFN-gamma R alpha mAb that impeded the binding of IFN-gamma. When PHA-activated T lymphoblasts were cultured for 16 days with IL-2, IFN-gamma R alpha expression increased, whereas that of the beta-chain remained barely detectable. Fas and Bcl-2 were both highly expressed. When these T lymphoblasts were restimulated by PHA, OKT3, or Staphylococcus enterotoxin beta-pokeweed mitogen, both chains up-modulated and most cells underwent apoptosis in a way apparently independent of Bcl-2, but not of Fas. This apoptosis, too, was prevented by the anti-IFN-gamma R alpha mAb. Physiologically secreted IFN-gamma is thus involved in the activation of resting T lymphocytes and in the apoptosis of reactivated lymphoblasts. However, high expression of IFN-gamma R beta took place when IFN-gamma induced apoptosis, but not when it induced proliferation. In conclusion, a correlation exists between differential expression of the IFN-gamma R beta-chain and the delivery by IFN-gamma of proliferative or apoptotic signals.
Asunto(s)
Antígenos CD/biosíntesis , Apoptosis/efectos de los fármacos , Interferón gamma/metabolismo , Interferón gamma/farmacología , Activación de Linfocitos/efectos de los fármacos , Receptores de Interferón/biosíntesis , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Anticuerpos Monoclonales/farmacología , Antígenos CD/química , Enterotoxinas/farmacología , Humanos , Interleucina-2/farmacología , Cinética , Fitohemaglutininas/farmacología , Mitógenos de Phytolacca americana/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Receptores de Interferón/química , Receptores de Interleucina-2/biosíntesis , Staphylococcus aureus/inmunología , Superantígenos/inmunología , Receptor fas/biosíntesis , Receptor de Interferón gammaRESUMEN
The injection of 20 ng of mouse interleukin-12 (IL-12) protects mice from a lethal infection with encephalomyocarditis virus. In vitro, an anti-gamma interferon (anti-IFN-gamma) monoclonal antibody but not an anti-IL-12 monoclonal antibody neutralizes the antiviral activity present in the supernatants of splenocytes stimulated with IL-12. Finally, IL-12 fails to protect 129 Sv/Ev IFN-gamma R0/0 mice against encephalomyocarditis virus infection. These results demonstrate that IL-12 exerts its antiviral activity through the induction of endogenous IFN-gamma.
Asunto(s)
Antivirales/uso terapéutico , Infecciones por Cardiovirus/terapia , Virus de la Encefalomiocarditis , Interferón gamma/farmacología , Interleucina-12/uso terapéutico , Animales , Anticuerpos Monoclonales/farmacología , Antivirales/inmunología , Infecciones por Cardiovirus/inmunología , Relación Dosis-Respuesta a Droga , Humanos , Interferón Tipo I/inmunología , Interferón Tipo I/farmacología , Interferón gamma/inmunología , Interleucina-12/inmunología , Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Pruebas de Neutralización , Proteínas Recombinantes , Bazo/inmunología , Factores de TiempoRESUMEN
Several cytokines, in particular tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), have been shown to be responsible for pathological reactions which may lead to shock and death observed in infection with Gram-negative bacteria and in response to endotoxins (lipopolysaccharides, LPS). Priming of mice with the avirulent Bacille Calmette Guérin (BCG) vaccine strain of Mycobacterium bovis increases the sensitivity of mice to the lethal effect of LPS and results in an efficient priming for cytokine production. In response to low doses (1 microgram/mouse) of LPS, BCG-primed mice produce interleukin-12 (IL-12) which controls IFN-gamma production, as demonstrated by the ability of neutralizing anti-IL-12 antibodies to suppress IFN-gamma production. However, the concentration of the biologically active IL-12 p70 heterodimer is similar in the serum of both BCG-primed or unprimed mice, reaching levels of 1-3 ng/ml at 3-6 h after LPS injection, whereas IFN-gamma production was observed only in BCG-primed mice. The priming effect of BCG on IFN-gamma production appears to be mostly due to its ability to increase TNF-alpha production, which acts as cofactor with LPS-induced IL-12 in inducing IFN-gamma production, as shown by the ability of injection of TNF-alpha and LPS (1 microgram/mouse), but not LPS alone, to induce IFN-gamma production. However, in addition to TNF-alpha, other LPS-induced cofactor(s) are required in cooperation with IL-12 to induce optimal IFN-gamma production, because co-injection of TNF-alpha and IL-12, sufficient to induce serum concentrations of both cytokines higher and more persistent than those obtained by injection of LPS, was not sufficient to induce IFN-gamma production in vivo. Neutralizing anti-IL-12 antibodies, in addition to inhibiting the in vivo LPS-induced IFN-gamma production, also completely protect BCG-primed mice injected with up to 10 micrograms of LPS from shock-induced death. Thus, IL-12 is required for IFN-gamma production and lethality in an endotoxic shock model in mice.
Asunto(s)
Interferón gamma/biosíntesis , Interleucina-12/biosíntesis , Interleucina-12/inmunología , Choque Séptico/inmunología , Choque Séptico/mortalidad , Animales , Anticuerpos Monoclonales/inmunología , Lipopolisacáridos , Ratones , Ratones Endogámicos , Ratones SCIDRESUMEN
Female NZB/W F1 mice develop an autoimmune disease similar to human systemic lupus erythematosus (SLE), and ultimately die of glomerulonephritis. Starting at the age of 16 weeks NZB/W F1 mice were treated for a period of 19 weeks with soluble interferon-gamma receptor (sIFN-gamma R), anti-IFN-gamma monoclonal antibody (mAb) or IFN-gamma. All mice treated with sIFN-gamma R or anti-IFN-gamma mAb were alive 4 weeks after the treatment was discontinued, whereas 50% of mice died in the placebo groups and 78% of the mice died in the IFN-gamma-treated group. Histologically, there was severe membrano-proliferative glomerulonephritis in IFN-gamma- and placebo-treated mice, and minimal or no mesangioproliferative disease in mice receiving sIFN-gamma R or anti-IFN-gamma mAb. The renal mononuclear infiltrate (T lymphocytes and monocytes), expression of major histocompatibility complex class II antigen and glomerular immunoglobulin and complement deposition were reduced in those mice. These data suggest that an IFN-gamma inhibitor, such as the soluble IFN-gamma R, can be used for SLE therapy in the early stages of the disease.
Asunto(s)
Lupus Eritematoso Sistémico/terapia , Nefritis Lúpica/prevención & control , Receptores de Interferón , Animales , Anticuerpos Antinucleares/sangre , Anticuerpos Monoclonales/uso terapéutico , Femenino , Inmunoglobulina G/sangre , Interferón gamma/antagonistas & inhibidores , Interferón gamma/fisiología , Nefritis Lúpica/inmunología , Nefritis Lúpica/patología , Ratones , Ratones Endogámicos NZB , Proteinuria/inmunología , Receptor de Interferón gammaRESUMEN
The Shwartzman reaction is elicited by two injections of lipopolysaccharide (LPS) in mice. The priming LPS injection is given in the footpad, whereas the lethal LPS challenge is given intravenously 24 h later. The injection of interferon gamma (IFN-gamma) or interleukin 12 (IL-12) instead of the LPS priming injection induced the lethal reaction in mice further challenged with LPS. Antibodies against IFN-gamma when given together with the priming agent, prevented the lethal reaction in mice primed with either LPS, IL-12, or IFN-gamma. Antibodies against IL-12, when given together with the priming agent, prevented the lethal reaction in mice primed with either LPS or IL-12 but not with IFN-gamma. These results strongly suggest that LPS induces the release of IL-12, that IL-12 induces the production of IFN-gamma, and that IFN-gamma is the cytokine that primes macrophages and other cell types. Upon LPS challenge, the lethal Shwartzman reaction is induced by a massive production of inflammatory cytokines that act on the target sites already sensitized by IFN-gamma. If mixtures of TNF and IL-1 or mixtures of TNF and IFN-gamma are used to challenge mice previously primed with IFN-gamma or IL-12, mortality is induced. In the same conditions, the individual cytokines or a mixture of IL-1 and IFN-gamma do not replace the LPS challenge. When the mice are primed with LPS, the combination of TNF, IL-1, and IFN-gamma induced only a partial mortality incidence suggesting that the involvement of other LPS-induced factors.
Asunto(s)
Interferón gamma/fisiología , Interleucinas/fisiología , Fenómeno de Shwartzman/etiología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Anticuerpos Monoclonales/inmunología , Femenino , Interferón gamma/toxicidad , Interleucina-1/biosíntesis , Interleucina-12 , Interleucina-6/biosíntesis , Interleucinas/toxicidad , Lipopolisacáridos/toxicidad , Ratones , Ratas , Factor de Necrosis Tumoral alfa/toxicidadRESUMEN
Acute phase proteins are good markers of inflammatory processes. To clarify whether Serum Amyloid Protein (SAP) can be a marker for the onset of SLE disease in mice, we measured constitutive and inducible SAP levels in normal mice of different strains, in C57Bl/6 lpr/lpr (B6lpr) and [NZB x NZW]F1 (NZB/W) SLE-prone mice, in mice that develop Lupus-like syndrome during chronic Graft versus Host (GvH) reaction and in mice suffering acute GvH reaction. In comparison to B6lpr, NZB/W mice showed higher blood levels of SAP but those levels did not correlate with autoimmune parameters. In B6lpr, the SAP levels steadily increased with age and correlated with some of the parameters used for monitoring the SLE disease. High levels of SAP were also found in mice suffering acute GvH reaction whereas the lupus-like chronic GvH disease was associated with limited increase of SAP levels.
Asunto(s)
Enfermedades Autoinmunes/metabolismo , Proteína Amiloide A Sérica/metabolismo , Edad de Inicio , Animales , Anticuerpos/química , Arteria Basilar/química , Biomarcadores/análisis , Enfermedad Injerto contra Huésped/metabolismo , Reacción Injerto-Huésped/inmunología , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Endogámicos NZB , Ratones Endogámicos , Conejos , Proteína Amiloide A Sérica/análisis , Proteína Amiloide A Sérica/inmunología , Proteína Amiloide A Sérica/aislamiento & purificaciónRESUMEN
Interferon-gamma (IFN-gamma) induces the expression of a set of early response genes by tyrosine phosphorylation of latent transcription factors such as p91. Although the tyrosine kinases, Jak1 and Jak2, have recently been shown to be critical for signal transduction by IFN-gamma, evidence is lacking for both tyrosine phosphorylation of the IFN-gamma receptor (IFN-gamma R) and the interaction between Jak1, Jak2, and the IFN-gamma R. In this report, we show that binding of IFN-gamma to HeLa cells initiated a series of events that resulted in the extremely rapid (15 s) tyrosine phosphorylation of not only Jak1, Jak2, and p91 but also the IFN-gamma R. Coimmunoprecipitation experiments revealed that Jak1 was associated with the IFN-gamma R prior to ligand binding, whereas Jak2 became part of the IFN-gamma R-Jak1 complex immediately after ligand binding. H2O2/vanadate treatment of cells for 15 min resulted in only the tyrosine phosphorylation of Jak1 and IFN-gamma R. Only after 60 min of this treatment did we observe tyrosine phosphorylation of Jak2 and p91 and assembly of the transcription factor complex FcRF gamma that binds to the promoter of the fcgr1 gene. These data suggest that JAK1 associates with the IFN-gamma R prior to ligand binding. IFN-gamma treatment of cells results in recruitment of JAK2 into the IFN-gamma R-Jak1 complex followed by assembly of the transcription factor FcRF gamma complex.
Asunto(s)
Interferón gamma/farmacología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Receptores de Interferón/metabolismo , Tirosina/análogos & derivados , Electroforesis en Gel de Poliacrilamida , Células HeLa , Humanos , Immunoblotting , Janus Quinasa 1 , Janus Quinasa 2 , Fosforilación , Fosfotirosina , Proteínas Tirosina Quinasas/aislamiento & purificación , Receptores de Interferón/aislamiento & purificación , Proteínas Recombinantes , Tirosina/análisis , Tirosina/metabolismo , Receptor de Interferón gammaRESUMEN
Antibody neutralization studies have established interferon gamma (IFN-gamma) as a critical mediator of endotoxic shock. The advent of IFN-gamma receptor negative (IFN gamma R-/-) mutant mice has enabled a more direct assessment of the role of IFN-gamma in endotoxin (lipopolysaccharide [LPS]-induced shock. We report that IFN gamma R-/- mice have an increased resistance to LPS-induced toxicity, this resistance manifesting well before the synthesis and release of LPS-induced IFN-gamma. LPS-induced lymphopenia, thrombocytopenia, and weight loss seen in wild-type mice were attenuated in IFN gamma R-/- mice. IFN gamma R-/- mice tolerated 100-1,000 times more LPS than the minimum lethal dose for wild-type mice in a D-galactosamine (D-GalN)/LPS model. Serum tumor necrosis factor (TNF) levels were 10-fold reduced in mutant mice given LPS or LPS/D-GalN. Bone marrow and splenic macrophages from IFN gamma R-/- mice had a four- to sixfold decreased LPS-binding capacity which correlated with similar reduction in CD14. Serum from mutant mice reduced macrophage LPS binding by a further 50%, although LPS binding protein was only 10% reduced. The expression of TNF receptor I (p55) and II (p75) was identical between wild-type and mutant mice. Thus, depressed TNF synthesis, diminished expression of CD14, and low plasma LPS-binding capacity, in addition to blocked IFN-gamma signaling in the mutant mice, likely to combine to manifest in the resistant phenotype of IFN gamma R-/- mice to endotoxin.
Asunto(s)
Receptores de Interferón/deficiencia , Choque Séptico/inmunología , Animales , Peso Corporal , Endotoxinas , Inmunidad Innata , Lipopolisacáridos/toxicidad , Ratones , Datos de Secuencia Molecular , Receptores de Interferón/inmunología , Factor de Necrosis Tumoral alfa/análisis , Receptor de Interferón gammaRESUMEN
With the purpose to use a soluble receptor as an IFN gamma antagonist in vivo, we assessed the immunogenicity, the half-life, the bioavailability, and the activity of the soluble receptor after injection into mice. No significant immunogenicity was detected after multiple chronic injections of mouse (Mo)IFN gamma-R or injections of MoIFN gamma-R in emulsion with CFA. Pharmacokinetic experiments using radiolabeled MoIFN gamma-R revealed that the half-life of the soluble MoIFN gamma-R is 3 h in the blood and 6 h in the lymphoid organs. When the MoIFN gamma-R protein was traced by the capacity of the animal sera to displace the binding of radiolabeled MoIFN gamma-R from affinity purified rabbit anti-MoIFN gamma-R antibodies, a blood half-life of 1 h was determined. Finally, the capacity of the injected receptor to bind and neutralize the IFN gamma-mediated antiviral activity was assessed in vivo. Treatment with IFN-gamma or IFN-alpha A/D protected the mice from the lethal infection with encephalomyocarditis virus. Mice treated with MoIFN-gamma-R neutralized the protective effect of IFN-gamma.