RESUMEN
For B cells to make antibodies against most antigens, they require help from T cells. T cell help is delivered as two signals to the B cell, one of which is via CD40 and the other can be through receptors for any of a variety of soluble cytokines. We have constructed recombinant vaccinia viruses that express the ligand for CD40 and have shown that the growth of these viruses is dramatically controlled in vivo, even in mice that lack T or B cells. In this paper, we also describe our attempts to analyse the CD40 ligand-mediated antiviral activity by studying the clearance of these viruses in mice that are deficient in important antiviral mechanisms. Thus, the antiviral activity of CD40L may represent a surprising and potent effector mechanism of T cells activated during a virus infection.
Asunto(s)
Antivirales/inmunología , Antígenos CD40/inmunología , Animales , Linfocitos B/inmunología , Antígenos CD40/farmacología , Células Cultivadas , Humanos , Interferón gamma/inmunología , Ligandos , Ratones , Ratones Desnudos , Proteínas Recombinantes/inmunología , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Virus Vaccinia/inmunologíaRESUMEN
The antiviral nature of tumor necrosis factor (TNF) is generally well accepted. TNF appears to induce multiple antiviral mechanisms, and to synergize with interferon (IFN)-gamma in promoting antiviral activities. We infected TNF receptor (TNFR)-deficient mice with the virulent murine pathogen, ectromelia virus (EV), and observed that otherwise resistant mice were susceptible to lethal infection. To study the molecular basis of the antiviral action of TNF, mice were infected with a recombinant vaccinia virus encoding murine TNF (VV-HA-TNF). In normal mice, the replication of VV-HA-TNF was highly attenuated. In contrast, mice in which the TNFR type 1 (p55) or the TNFR type 2 (p75) were genetically disrupted showed a moderate defect in their capacity to clear the TNF-encoding virus. The contribution of both TNF receptors to the control of VV-HA-TNF was confirmed by the enhanced replication of VV-HA-TNF in mice deficient for both p55 and p75. These observations were corroborated by infecting TNFR-deficient mice with EV. For both infections, the p55 and p75 TNFRs were necessary to maintain normal levels of resistance. Thus, the antiviral activity of TNF is mediated via both TNFRs in vivo. Furthermore, these studies establish that TNF is an important component of the host response to a natural virus infection.
Asunto(s)
Antivirales/farmacología , Receptores del Factor de Necrosis Tumoral/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Ectromelia Infecciosa/genética , Ectromelia Infecciosa/inmunología , Ectromelia Infecciosa/virología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Recombinación Genética , Factor de Necrosis Tumoral alfa/genética , Vaccinia/genética , Vaccinia/inmunología , Vaccinia/virología , Virus Vaccinia/efectos de los fármacos , Virus Vaccinia/genética , Virus Vaccinia/patogenicidad , Replicación Viral/efectos de los fármacosRESUMEN
SWR/J transgenic (tg) mice were generated expressing the TCR beta chain derived from an anticollagen type II (CII) arthritogenic T cell clone. The SWR/J strain was selected because it is resistant to collagen-induced arthritis (CIA) and lacks the V beta gene segment used by the T cell clone. Expression of the tg beta chain on all thymocytes and peripheral lymph node T cells led to a more efficient anti-CII immune response, but did not confer CIA susceptibility to SWR/J mice. Nevertheless, this tg beta chain enhanced predisposition to CIA as (DBA/1 x SWR) F1 beta tg mice were more susceptible than normal F1 littermates. Our results demonstrate that the expression of the tg beta chain contributes to CIA susceptibility, but by itself it is not sufficient to overcome CIA resistance in the SWR/J strain.
Asunto(s)
Artritis/inmunología , Colágeno/fisiología , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Linfocitos T/inmunología , Animales , Células Clonales , Expresión Génica , Humanos , Hibridomas , Inmunidad Innata , Ratones , Ratones Endogámicos DBA , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismoRESUMEN
In mice double transgenic for functionally rearranged T cell receptor (TCR) V beta 2 and V beta 8.2 genes we found that most T lymphocytes express both TCR beta chains simultaneously. These T cells show no abnormality in thymic selection in vivo and their TCRs are capable of transducing activation signals in vitro. These results indicate that multispecific T cells may appear in the periphery if allelic exclusion of TCR beta genes is not established at the level of gene rearrangement.
Asunto(s)
Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Linfocitos T/inmunología , Alelos , Animales , Antígenos CD4/inmunología , Antígenos CD8/inmunología , Cruzamientos Genéticos , Citometría de Flujo , Regulación de la Expresión Génica , Genotipo , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Transducción de Señal , Timo/inmunologíaRESUMEN
In normal mice neonatal injection of staphylococcal enterotoxin B (SEB) induces tolerance in T cells that express reactive T cell receptor (TCR) V beta regions. To determine if a T cell neonatal defect was present in MRL-lpr/lpr mice, 20 micrograms of SEB was injected intraperitoneally every other day into V beta 8.2 TCR transgenic and nontransgenic MRL(-)+/+ and MRL-lpr/lpr mice from birth to 2 wk of age. At 2 wk of age, V beta 8+ T cells were depleted, and SEB reactivity was lost, in spleen, lymph node, and thymus. These effects were equivalent in +/+ and lpr/lpr SEB-tolerized mice. However, MRL-lpr/lpr mice failed to maintain neonatal tolerance. By 4 wk of age, there was a dramatic increase in T cells expressing V beta 8.2 in the peripheral lymph nodes of MRL-lpr/lpr mice but not MRL(-)+/+ mice. In vitro stimulation with SEB or TCR crosslinking revealed a total loss of neonatal tolerance 2 wk after cessation of SEB treatment in lpr/lpr mice, but not +/+ mice. The time-course of recovery of V beta 8+ T cells and reactivity to SEB and TCR crosslinking in the thymus of MRL-lpr/lpr mice was similar to that in the lymph node. Thymectomy at 2 wk of age eliminated tolerance loss in lymph nodes of MRL-lpr/lpr mice at 4 wk of age, indicating that loss of peripheral tolerance was due to the emigration of untolerized T cells from the thymus. Challenge of neonatally tolerized MRL-lpr/lpr mice with SEB (100 micrograms, i.p.) at 8 wk of age resulted in a dramatic onset of T cell-mediated autoimmune disease characterized by 30% weight loss and 60% morality. This indicated that loss of tolerance to SEB also occurred in vivo. In contrast, neonatally tolerized MRL(-)+/+ mice remained totally unresponsive to SEB challenge and did not undergo any detectable weight loss. These results suggest that there is normal induction of neonatal tolerance to SEB in lpr/lpr mice, but that tolerance is not maintained after the tolerizing antigen is removed. This loss of neonatal tolerance can lead to severe weight loss and death on exposure to the tolerizing antigen later in life.
Asunto(s)
Enterotoxinas/inmunología , Tolerancia Inmunológica/genética , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Animales Recién Nacidos , Antígenos CD4/análisis , Antígenos CD8/análisis , Cruzamientos Genéticos , Replicación del ADN , Citometría de Flujo , Interferón gamma/biosíntesis , Interleucina-2/biosíntesis , Ganglios Linfáticos/inmunología , Activación de Linfocitos , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Staphylococcus aureus/inmunologíaRESUMEN
Interleukin 12 (IL-12) produced by macrophages immediately after infection is considered essential for activation of a protective immune response against intracellular pathogens. In the murine Mycobacterium bovis Bacillus Calmette-Guérin (BCG) model we assessed whether early IL-12 production by macrophages depends on other cytokines. In vitro, murine bone marrow-derived macrophages produced IL-12 after infection with viable M. bovis BCG or stimulation with LPS, however, priming with recombinant interferon gamma (rIFN-gamma) was necessary. In addition, IL-12 production by these macrophages was blocked by specific anti-tumor necrosis factor alpha (TNF-alpha) antiserum. Macrophages from gene deletion mutant mice lacking either the IFN-gamma receptor or the TNF receptor 1 (p55) failed to produce IL-12 in vitro after stimulation with rIFN-gamma and mycobacterial infection. In vivo, IL-12 production was induced in spleens of immunocompetent mice early during M. bovis BCG infection but not in those of mutant mice lacking the receptors for IFN-gamma or TNF. Our results show that IL-12 production by macrophages in response to mycobacterial infection depends on IFN-gamma and TNF. Hence, IL-12 is not the first cytokine produced in mycobacterial infections.
Asunto(s)
Interferón gamma/fisiología , Interleucina-12/biosíntesis , Macrófagos/metabolismo , Infecciones por Mycobacterium/inmunología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Secuencia de Bases , Células Cultivadas , Femenino , Interferón gamma/farmacología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mycobacterium bovis , Proteínas RecombinantesRESUMEN
The Nur77/Nurr1 family of DNA binding proteins has been reported to be required for the signal transduction of CD3/T cell receptor (TCR)-mediated apoptosis in T cell hybridomas. To determine the role of this family of DNA-binding proteins in thymic clonal deletion, transgenic (Tg) mice bearing a dominant negative mutation were produced. The transgene consisted of a truncated Nur77 (deltaNur77) gene encoding the DNA-binding domain of Nur77 ligated to a TCR-beta enhancer resulting in early expression in thymocytes. Apoptosis of CD4+CD8+ thymocytes mediated by CD3/TCR signaling was greatly inhibited in the deltaNur77 Tg mice, compared with non-Tg littermates, after treatment with anti-CD3 or anti-TCR antibody in vivo and in vitro. Clonal deletion of self-reactive T cells was investigated in deltaNur77-Db/HY TCR-alpha/beta double Tg mice. There was a five-fold increase in the total number of thymocytes expressing self-reactive Db/HY TCR-alpha/beta in the deltaNur77-TCR-alpha/beta double Tg male mice. Deficient clonal deletion of self-reactive thymocytes was demonstrated by a 10-fold increase in the CD4+CD8+ thymocytes that expressed Tg TCR-alpha/beta. There was an eightfold increase in the CD8+, Db/HY TCR-alpha/beta T cells in the lymph nodes (LN) of delta Nur77-Db/HY TCR-alpha/beta double Tg compared with Db/HY TCR-alpha/beta Tg male mice. In spite of defective clonal deletion, the T cells expressing the Tg TCR were functionally anergic. In vivo analysis revealed increased activation and apoptosis of T cells associated with increased expression of Fas and Fas ligand in LN of deltaNur77-Db/HY TCR-alpha/beta double male mice. These results indicate that inhibition of Nur77/Nurr1 DNA binding in T cells leads to inefficient thymic clonal deletion, but T cell tolerance is maintained by Fas-dependent clonal deletion in LN and spleen.
Asunto(s)
Apoptosis , Supresión Clonal , Proteínas de Unión al ADN/metabolismo , Autotolerancia , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Complejo CD3 , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Citotoxicidad Inmunológica , Proteínas de Unión al ADN/genética , Femenino , Activación de Linfocitos , Depleción Linfocítica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Datos de Secuencia Molecular , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Receptores de Antígenos de Linfocitos T , Receptores Citoplasmáticos y Nucleares , Receptores de Esteroides , Eliminación de Secuencia , Subgrupos de Linfocitos T , Factores de Transcripción/genéticaRESUMEN
Using mice double deficient for tumor necrosis factor (TNF) and lymphotoxin alpha (LT alpha), we demonstrated that TNF and/or LT alpha are necessary for development of a normal splenic microarchitecture and for isotype switch after immunization with sheep red blood cells (SRBC). In the present study, we extended these observations by determining which TNF receptor (TNFR) is involved in morphological and functional differentiation of the spleen. Spleen morphology and antibody response were investigated in wild-type, TNFR1-/-, TNFR2-/- and TNF/LT alpha-/- mice immunized with SRBC. TNF/LT alpha-/- mice, which have a complete disruption of the TNF/LT alpha signaling system including the LT beta-receptor pathway, displayed an abnormal microarchitecture, and isotype switch did not take place. TNFR1-/- and TNFR2-/- mice displayed a normal spleen microarchitecture and mounted an IgM and IgG antibody response to SRBC. However, the IgG production in TNFR1-/- mice was minimal, with citers leveling off 6 d after immunization. In this strain, immunofluorescence revealed a lack of follicular dendritic cells (FDC) network, detected with FDC-M1 as well as anti-CR1, and a lack of germinal centers, detected with peanut agglutinin. In conclusion, whereas normal splenic microarchitecture and isotype switch might require the LT beta receptor, differentiation of FDC network, development of germinal centers, and full IgG response depend on signaling via TNFR1.
Asunto(s)
Formación de Anticuerpos , Células Dendríticas/citología , Células Dendríticas/inmunología , Linfotoxina-alfa/genética , Receptores del Factor de Necrosis Tumoral/fisiología , Animales , Diferenciación Celular , Cruzamientos Genéticos , Técnica del Anticuerpo Fluorescente , Cambio de Clase de Inmunoglobulina , Inmunoglobulina G/sangre , Inmunoglobulina G/clasificación , Inmunoglobulina M/sangre , Linfotoxina-alfa/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Mutantes , Transducción de Señal , Especificidad de la Especie , Bazo/citología , Bazo/inmunologíaRESUMEN
Aplastic anemia may be associated with persistent viral infections that result from failure of the immune system to control virus. To evaluate the effects on hematopoiesis exerted by sustained viral replication in the presence of activated T cells, blood values and bone marrow (BM) function were analyzed in chronic infection with lymphocytic choriomeningitis virus (LCMV) in perforin-deficient (P0/0) mice. These mice exhibit a vigorous T cell response, but are unable to eliminate the virus. Within 14 d after infection, a progressive pancytopenia developed that eventually was lethal due to agranulocytosis and thrombocytopenia correlating with an increasing loss of morphologically differentiated, pluripotent, and committed progenitors in the BM. This hematopoietic disease caused by a noncytopathic chronic virus infection was prevented by depletion of CD8+, but not of CD4+, T cells and accelerated by increasing the frequency of LCMV-specific CD8+ T cells in T cell receptor (TCR) transgenic (tg) mice. LCMV and CD8+ T cells were found only transiently in the BM of infected wild-type mice. In contrast, increased numbers of CD8+ T cells and LCMV persisted at high levels in antigen-presenting cells of infected P0/0 and P0/0 x TCR tg mice. No cognate interaction between the TCR and hematopoietic progenitors presenting either LCMV-derived or self-antigens on the major histocompatibility complex was found, but damage to hematopoiesis was due to excessive secretion and action of tumor necrosis factor (TNF)/lymphotoxin (LT)-alpha and interferon (IFN)-gamma produced by CD8+ T cells. This was studied in double-knockout mice that were genetically deficient in perforin and TNF receptor type 1. Compared with P0/0 mice, these mice had identical T cell compartments and T cell responses to LCMV, yet they survived LCMV infection and became life-long virus carriers. The numbers of hematopoietic precursors in the BM were increased compared with P0/0 mice after LCMV infection, although transient blood disease was still noticed. This residual disease activity was found to depend on IFN-gamma-producing LCMV-specific T cells and the time point of hematopoietic recovery paralleled disappearance of these virus-specific, IFN-gamma-producing CD8+ T cells. Thus, in the absence of IFN-gamma and/or TNF/LT-alpha, exhaustion of virus-specific T cells was not hampered.
Asunto(s)
Anemia Aplásica/inmunología , Linfocitos T CD8-positivos/inmunología , Coriomeningitis Linfocítica/inmunología , Anemia Aplásica/complicaciones , Anemia Aplásica/patología , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Médula Ósea/inmunología , Médula Ósea/virología , Linfocitos T CD4-Positivos/inmunología , Línea Celular , Citocinas/inmunología , Modelos Animales de Enfermedad , Recuento de Eritrocitos , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Coriomeningitis Linfocítica/complicaciones , Coriomeningitis Linfocítica/virología , Linfotoxina-alfa/inmunología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Pancitopenia/etiología , Pancitopenia/inmunología , Perforina , Recuento de Plaquetas , Proteínas Citotóxicas Formadoras de Poros , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/inmunología , Receptores Tipo I de Factores de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/inmunología , Latencia del Virus , Replicación ViralRESUMEN
Interferon-gamma (IFN-gamma) exerts pleiotropic effects, including antiviral activity, stimulation of macrophages and natural killer cells, and increased expression of major histocompatibility complex antigens. Mice without the IFN-gamma receptor had no overt anomalies, and their immune system appeared to develop normally. However, mutant mice had a defective natural resistance, they had increased susceptibility to infection by Listeria monocytogenes and vaccinia virus despite normal cytotoxic and T helper cell responses. Immunoglobulin isotype analysis revealed that IFN-gamma is necessary for a normal antigen-specific immunoglobulin G2a response. These mutant mice offer the possibility for the further elucidation of IFN-gamma-mediated functions by transgenic cell- or tissue-specific reconstitution of a functional receptor.
Asunto(s)
Inmunidad , Interferón gamma/fisiología , Receptores de Interferón/fisiología , Animales , Anticuerpos Antivirales/sangre , Inmunoglobulina G/sangre , Listeriosis/inmunología , Subgrupos Linfocitarios , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Receptores de Interferón/genética , Linfocitos T Citotóxicos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Vaccinia/inmunología , Virosis/inmunología , Replicación ViralRESUMEN
Benzodiazepine tranquilizers are used in the treatment of anxiety disorders. To identify the molecular and neuronal target mediating the anxiolytic action of benzodiazepines, we generated and analyzed two mouse lines in which the alpha2 or alpha3 GABAA (gamma-aminobutyric acid type A) receptors, respectively, were rendered insensitive to diazepam by a knock-in point mutation. The anxiolytic action of diazepam was absent in mice with the alpha2(H101R) point mutation but present in mice with the alpha3(H126R) point mutation. These findings indicate that the anxiolytic effect of benzodiazepine drugs is mediated by alpha2 GABAA receptors, which are largely expressed in the limbic system, but not by alpha3 GABAA receptors, which predominate in the reticular activating system.
Asunto(s)
Ansiolíticos/farmacología , Diazepam/farmacología , Receptores de GABA-A/metabolismo , Animales , Ansiolíticos/metabolismo , Conducta Animal/efectos de los fármacos , Sitios de Unión , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Células Cultivadas , Diazepam/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Marcación de Gen , Hipocampo/citología , Potenciales de la Membrana/efectos de los fármacos , Ratones , Técnicas de Placa-Clamp , Fenobarbital/farmacología , Mutación Puntual , Células Piramidales/efectos de los fármacos , Células Piramidales/fisiología , Receptores de GABA-A/química , Receptores de GABA-A/genética , Transmisión Sináptica , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Tetracycline-regulated gene expression in eukaryotic cell lines, plants, and transgenic mice has become a powerful tool for the analysis of eukaryotic gene expression and function. The system consists of two plasmids, one encoding the transactivator protein under control of a viral cytomegalovirus promoter, and the second being the tet-operator minimal promoter driving the gene of interest. Here we show that these control elements, when integrated in cis on a single plasmid, allow efficient and tight control of reporter gene expression in vitro and in vivo. Dependent on the route of administration of tetracycline, gene expression can be partially or fully repressed in transgenic mice, whereas removal of the antibiotic induces the reporter gene in various tissues to levels up to 800-fold more than the two-plasmid system. In addition, crossing and analysis of animals transgenic for the individual components of the system are unnecessary, and genetic segregation of the control elements during breeding is prevented.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Represoras/genética , Tetraciclina/farmacología , Animales , Secuencia de Bases , Biotecnología , Células CHO , Cricetinae , Cartilla de ADN/genética , Genes Reporteros , Luciferasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Plásmidos/genética , Reacción en Cadena de la Polimerasa , TransfecciónRESUMEN
Lymphocytic infiltrates of BALB/c C4 hyperplastic alveolar nodule (HAN) have elevated natural killer (NK) activity, which correlates positively with the progression of C4 HAN to tumor: C4 HAN produces an infectious mouse mammary tumor virus, MMTV(C4), which encodes a superantigen that activates and deletes T-cells with the V beta 2 segment in the T-cell receptor. In this report, NK activation by both MMTV(C4) and MMTV(C4) superantigen was tested. NK activity was measured in naive BALB/c mice, BALB/c mice depleted of V beta 2+ T-cell, or V beta 2-transgenic mice after they received injections of either purified MMTV(C4) or MMTV(C4)-infected splenocytes. Elevated NK activity was observed in BALB/c mice receiving MMTV(C4) or MMTV(C4)-infected splenocytes. Depletion of V beta 2+, but not V beta 8+, T-cells by specific anti-V beta hybridoma before injection of MMTV(C4)-infected cells reduced but did not eliminate NK activation. NK activation in V beta 2-transgenic mice occurred before massive CD4 T-cell deletion took place and was more pronounced than that in the nontransgenic littermates. These results indicate that MMTV activates NK cells through superantigen-dependent and -independent pathways and supports the role of MMTV(C4) in the augmented NK activity observed in C4 HAN infiltrates. The progression of C4 HAN to tumor represents a model system for the analysis of how tumorigenesis may be affected by lesion-associated viruses.
Asunto(s)
Células Asesinas Naturales/inmunología , Células Asesinas Naturales/microbiología , Activación de Linfocitos/inmunología , Virus del Tumor Mamario del Ratón , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/fisiología , Animales , Secuencia de Bases , Femenino , Hiperplasia/microbiología , Células Asesinas Naturales/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/fisiología , Depleción Linfocítica , Masculino , Neoplasias Mamarias Experimentales/microbiología , Virus del Tumor Mamario del Ratón/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Datos de Secuencia Molecular , Alveolos Pulmonares/microbiología , Alveolos Pulmonares/patología , Infecciones por Retroviridae/inmunología , Infecciones por Retroviridae/microbiología , Bazo/citología , Bazo/metabolismo , Superantígenos/inmunología , Superantígenos/farmacología , Linfocitos T/citología , Linfocitos T/inmunología , Infecciones Tumorales por Virus/inmunología , Infecciones Tumorales por Virus/microbiologíaRESUMEN
A s.c. injection of a mouse colon adenocarcinoma cell line, colon 26 clone 20, induced cachexia, as evidenced by progressive weight loss and severe hypoglycemia. Several lines of evidence indicate that a pro-inflammatory cytokine, interleukin 6 (IL-6), plays a major role, albeit partially, in the establishment of cachexia in this model. Because IL-10 can potentially inhibit the production of pro-inflammatory cytokines including IL-6, we evaluated the effects of IL-10 gene transfer on the establishment of cachexia. IL-6 transcript was detected at tumor sites of mice inoculated with parental or control vector transfectant cells, and serum IL-6 levels were markedly increased in these mice. The injection of parental cells into IL-6-deficient mice induced cachexia with elevated serum IL-6 levels comparable to wild-type mice, indicating that tumor cells are a major source of IL-6. The inoculation of IL-10-transfectant cells kept IL-10 mRNA expression at tumor sites and induced the elevation in serum IL-10 levels without affecting the growth rates of colon 26 cells both in vitro and in vivo. However, the implantation with IL-10-transfectant cells reduced the expression of IL-6 mRNA at the tumor sites and the elevation in serum IL-6 levels. Concomitantly, mice inoculated with IL-10-transfectant cells did not exhibit progressive weight loss, a reduction in food intake, or severe hypoglycemia, which was observed in mice inoculated with parental or control vector-transfectant cells. Collectively, these results suggest that IL-10 gene transfer prevented the occurrence of cachexia with a concomitant inhibition of IL-6 production at the tumor sites.
Asunto(s)
Caquexia/prevención & control , Neoplasias del Colon/complicaciones , Interleucina-10/genética , Transfección , Animales , Caquexia/genética , Caquexia/metabolismo , Neoplasias del Colon/metabolismo , Femenino , Interferón gamma/metabolismo , Interleucina-1/metabolismo , Interleucina-10/metabolismo , Interleucina-10/fisiología , Interleucina-6/deficiencia , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales/complicaciones , Neoplasias Experimentales/metabolismo , ARN Mensajero/metabolismo , Organismos Libres de Patógenos Específicos , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Recent studies in mice harboring a targeted disruption of genes encoding TNF receptor 1 (TNFR-1) or Interleukin 6 (IL-6) suggested a critical role for TNF and IL-6 in initiation of liver regeneration after 2/3 partial hepatectomy. However, hepatocyte proliferation can also occur following treatment with agents that do not induce tissue loss (primary mitogens). To determine whether the above cytokines could also be involved in mitogen-induced liver cell proliferation, we studied the hepatocyte proliferative response after treatment with primary mitogens in mice knock-out for TNFR-1 or IL-6. Our results showed no difference in the proliferative response of the liver between the wild type and the knock-out mice following treatment with the mitogens 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), or the peroxisome proliferator, ciprofibrate, suggesting that TNF or IL-6 may not play a major role in this type of proliferation. Gel shift assay indicated that TCPOBOP-induced hepatocyte proliferation is not associated with activation of STAT3 transcription factor, a major target of IL-6 and other growth factors/cytokines. Our results thus indicate that hepatocyte proliferation can be induced by at least two different pathways; compensatory regeneration being TNF and IL-6-dependent, and mitogen-induced direct hyperplasia which does not require TNF or IL-6.
Asunto(s)
Interleucina-6/fisiología , Hígado/citología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Antígenos CD/genética , División Celular/efectos de los fármacos , Ácido Clofíbrico/análogos & derivados , Ácido Clofíbrico/farmacología , Proteínas de Unión al ADN/metabolismo , Ácidos Fíbricos , Hepatectomía , Interleucina-6/genética , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nafenopina/farmacología , Piridinas/farmacología , Receptores del Factor de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral , Factor de Transcripción STAT3 , Transactivadores/metabolismo , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Tenascin-R (TN-R), an extracellular matrix glycoprotein of the CNS, localizes to nodes of Ranvier and perineuronal nets and interacts in vitro with other extracellular matrix components and recognition molecules of the immunoglobulin superfamily. To characterize the functional roles of TN-R in vivo, we have generated mice deficient for TN-R by homologous recombination using embryonic stem cells. TN-R-deficient mice are viable and fertile. The anatomy of all major brain areas and the formation and structure of myelin appear normal. However, immunostaining for the chondroitin sulfate proteoglycan phosphacan, a high-affinity ligand for TN-R, is weak and diffuse in the mutant when compared with wild-type mice. Compound action potential recordings from optic nerves of mutant mice show a significant decrease in conduction velocity as compared with controls. However, at nodes of Ranvier there is no apparent change in expression and distribution of Na+ channels, which are thought to bind to TN-R via their beta2 subunit. The distribution of carbohydrate epitopes of perineuronal nets recognized by the lectin Wisteria floribunda or antibodies to the HNK-1 carbohydrate on somata and dendrites of cortical and hippocampal interneurons is abnormal. These observations indicate an essential role for TN-R in the formation of perineuronal nets and in normal conduction velocity of optic nerve.
Asunto(s)
Axones/fisiología , Sistema Nervioso Central/fisiopatología , Proteínas de la Matriz Extracelular/deficiencia , Proteínas del Tejido Nervioso/deficiencia , Conducción Nerviosa/fisiología , Tenascina/deficiencia , Animales , Células Cultivadas , Ratones , Ratones Noqueados , Microscopía Electrónica , Glicoproteína Asociada a Mielina/fisiología , Red Nerviosa/fisiología , Proteínas del Tejido Nervioso/análisis , Nervio Óptico/ultraestructura , Nódulos de Ranvier/fisiología , Células Madre/fisiologíaRESUMEN
We have used site-directed mutagenesis in conjunction with homologous recombination to generate two mouse lines carrying point mutations in the glycine binding site of the NMDAR1 subunit (Grin1). Glycine concentration-response curves from acutely dissociated hippocampal neurons revealed a 5- and 86-fold reduction in receptor glycine affinity in mice carrying Grin1(D481N) and Grin1(K483Q) mutations, respectively, whereas receptor glutamate affinity remained unaffected. Homozygous mutant Grin1(D481N) animals are viable and fertile and appear to develop normally. However, homozygous mutant Grin1(K483Q) animals are significantly lighter at birth, do not feed, and die within a few days. No gross abnormalities in CNS anatomy were detected in either Grin1(D481N) or Grin1(K483Q) mice. Interestingly, in situ hybridization and Western blot analysis revealed changes in the expression levels of NMDA receptor subunits in Grin1(D481N) mice relative to wild type that may represent a compensatory response to the reduction in receptor glycine affinity. Grin1(D481N) mice exhibited deficits in hippocampal theta burst-induced long-term potentiation (LTP) and spatial learning and also a reduction in sensitivity to NMDA-induced seizures relative to wild-type controls, consistent with a reduced activation of NMDA receptors. Mutant mice exhibited normal prepulse inhibition but showed increased startle reactivity. Preliminary analysis indicated that the mice exhibit a decreased natural aversion to an exposed environment. The lethal phenotype of Grin1(K483Q) animals confirms the critical role of NMDA receptor activation in neonatal survival. A milder reduction in receptor glycine affinity results in an impairment of LTP and spatial learning and alterations in anxiety-related behavior, providing further evidence for the role of NMDA receptor activation in these processes.
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Glicina/fisiología , Mutación Puntual/fisiología , Receptores de Glicina/genética , Receptores de Glicina/fisiología , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/fisiología , Animales , Autorradiografía , Conducta Animal/fisiología , Southern Blotting , Western Blotting , Calcio/fisiología , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Marcación de Gen , Hipocampo/citología , Hipocampo/metabolismo , Homocigoto , Interpretación de Imagen Asistida por Computador , Hibridación in Situ , Potenciación a Largo Plazo/fisiología , Ratones , Técnicas de Placa-Clamp , Mutación Puntual/genética , Reflejo de Sobresalto/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Convulsiones/inducido químicamente , Convulsiones/genética , Convulsiones/fisiopatologíaRESUMEN
The major histocompatibility complex (MHC) glycoproteins, MHC1 and MHC2, play a key role in the presentation of antigen and the development of the immune response. In the current study we examined the regulation of the MHC2 in the mouse brain after facial axotomy. The normal facial motor nucleus showed very few slender and elongated MHC2+ cells. Transection of the facial nerve led to a gradual but strong upregulation in the number of MHC2+ cells, beginning at day 2 and reaching a maximum 14 days after axotomy, correlated with the induction of mRNA for tumor necrosis factor (TNF) alpha, interleukin (IL) 1beta and interferon-gamma (IFNgamma) and a peak in neuronal cell death. In almost all cases, MHC2 immunoreactivity was restricted to perivascular macrophages that colocalized with vascular basement membrane laminin and macrophage IBA1-immunoreactivity, with no immunoreactivity on phagocytic microglia, astrocytes or invading T-cells. Heterologous transplantation and systemic injection of endotoxin or IFNgamma did not affect this perivascular MHC2 immunoreactivity, and transgenic deletion of the IL1 receptor type I, or TNF receptor type 1, also had no effect. However, the deletion of IFNgamma receptor subunit 1 caused a significant increase, and that of TNF receptor type 2 a strong reduction in the number of MHC2+ macrophages, pointing to a counter-regulatory role of IFNgamma and TNFalpha in the immune surveillance of the injured nervous system.
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Traumatismos del Nervio Facial/metabolismo , Genes MHC Clase II/fisiología , Macrófagos/metabolismo , Receptores de Interferón/fisiología , Receptores del Factor de Necrosis Tumoral/fisiología , Animales , Axotomía/métodos , Nervio Facial/metabolismo , Traumatismos del Nervio Facial/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Interferón/deficiencia , Receptores de Interferón/genética , Receptores del Factor de Necrosis Tumoral/deficiencia , Receptores del Factor de Necrosis Tumoral/genética , Factor 1 Asociado a Receptor de TNF/deficiencia , Factor 1 Asociado a Receptor de TNF/genética , Factor 1 Asociado a Receptor de TNF/fisiología , Factor 2 Asociado a Receptor de TNF/deficiencia , Factor 2 Asociado a Receptor de TNF/genética , Factor 2 Asociado a Receptor de TNF/fisiología , Receptor de Interferón gammaRESUMEN
Cytokines are known to be key players in host response to infection, immunological disorders, and tissue injury in the attempt of an organism to overcome the insult and restore homeostasis. Another important aspect of cytokines, however, is their normal physiological role during development in the unchallenged organism. The most elegant way to analyze both of these functions is to introduce targeted mutations in embryonic stem cells in order to create new mouse strains deficient for a given cytokine and identify the functions that are consequently impaired or lost. This review summarizes the mutant phenotypes of mice carrying a null mutation in the cytokine IL-6 gene or the tumor necrosis factor receptor 1 (Tnfr1) gene. Results for interferon-- and interferon-gamma receptor-deficient mice are included for comparison.
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Reacción de Fase Aguda/fisiopatología , Infecciones Bacterianas/fisiopatología , Interleucina-6/fisiología , Receptores del Factor de Necrosis Tumoral/fisiología , Choque Séptico/fisiopatología , Virosis/inmunología , Reacción de Fase Aguda/sangre , Animales , Anticuerpos Antivirales/metabolismo , Linfocitos B/fisiología , Infecciones Bacterianas/sangre , Infecciones Bacterianas/inmunología , Galactosamina , Inmunidad Celular , Inmunoglobulina G/metabolismo , Inmunoglobulina M/metabolismo , Interferón gamma/fisiología , Interleucina-1/administración & dosificación , Interleucina-6/deficiencia , Interleucina-6/genética , Interleucina-6/metabolismo , Dosificación Letal Mediana , Activación de Linfocitos , Virus de la Coriomeningitis Linfocítica/inmunología , Activación de Macrófagos , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Receptores del Factor de Necrosis Tumoral/deficiencia , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Choque Séptico/sangre , Factor de Necrosis Tumoral alfa/administración & dosificación , Virus Vaccinia/inmunologíaRESUMEN
Using the tumor necrosis factor receptor beta (TNFR beta) cDNA as a probe, overlapping clones from a genomic phage library were isolated which encompass the murine TNF receptor beta gene. Analysis of the gene led to the identification of 10 exons, most of which were concentrated in two clusters. The boundaries of the exons do not match protein domains or characteristic motifs of the extracellular region of the TNFR beta. The 5'-flanking region of the gene shows a high density of G and C nucleotides with a strong overrepresentation of CpG dinucleotides. Most of the analyzed CpG were found to be nonmethylated, suggesting that this region is an HTF island. We revealed at least three transcriptional start sites which is likely due to the absence of classical TATA and CAAT sequences from the putative promoter region. CAT assays confirmed promoter activity of the 5'-flanking sequences. Surprisingly, some successively shortened promoter constructs displayed higher relative promoter activity than a full length clone. Preliminary experiments indicate that the promoter region of the TNFR beta gene does not respond to a variety of cytokines. In summary, the structural and functional analysis suggest that the TNFR beta expression is directed by a non-inducible housekeeping-type promoter.