RESUMEN

Growth arrest and DNA damage inducible protein 34 (GADD34) is induced by various cellular stresses, such as DNA damage, endoplasmic reticulum stress, and amino-acid deprivation. Although the major roles of GADD34 are regulating ER stress responses and apoptosis, a recent study suggested that GADD34 is linked to innate immune responses. In this report, we investigated the roles of GADD34 in inflammatory responses against bacterial infection. To explore the effects of GADD34 on systemic inflammation in vivo, we employed a lipopolysaccharide (LPS)-induced murine sepsis model and assessed the lethality, serum cytokine levels, and tissue injury in the presence or absence of GADD34. We found that GADD34 deficiency increased the lethality and serum cytokine levels in LPS-induced sepsis. Moreover, GADD34 deficiency enhanced tissue destruction, cell death, and pro-inflammatory cytokine expression in LPS-induced acute liver injury. Pro-inflammatory cytokine production after LPS stimulation is regulated by the Toll-like receptor 4 (TLR4)-mediated NF-κB signaling pathway. In vitro experiments revealed that GADD34 suppressed pro-inflammatory cytokine production by macrophages through dephosphorylation of IKKß. In conclusion, GADD34 attenuates LPS-induced sepsis and acute tissue injury through suppressing macrophage activation. Targeting this anti-inflammatory role of GADD34 may be a promising area for the development of therapeutic agents to regulate inflammatory disorders.

Asunto(s)

Lesión Pulmonar Aguda/inmunología , Macrófagos Peritoneales/inmunología , FN-kappa B/inmunología , Proteína Fosfatasa 1/inmunología , Sepsis/inmunología , Receptor Toll-Like 4/inmunología , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/patología , Animales , Línea Celular , Citocinas/genética , Citocinas/inmunología , Regulación de la Expresión Génica , Quinasa I-kappa B/genética , Quinasa I-kappa B/inmunología , Lipopolisacáridos , Activación de Macrófagos , Macrófagos Peritoneales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/genética , Cultivo Primario de Células , Proteína Fosfatasa 1/genética , Sepsis/inducido químicamente , Sepsis/genética , Sepsis/patología , Transducción de Señal , Receptor Toll-Like 4/genética

RESUMEN

While Gr1(+)CD11b(+) cells are known to regulate immune responses and accumulate in most cancer tissues, the function of Gr1(+)CD11b(+) cells in inflammation is poorly understood. We investigated the role of Gr1(+)CD11b(+) cells in a dextran sulphate sodium (DSS)-treated mouse model of ulcerative colitis (UC). C57BL/6 mice were treated with 2% DSS in drinking water for 5 days. Disease progression and recovery were assessed by body weight, disease activity index score (DAI) score and colon length. Splenic Gr1(+)CD11b(+) cell number was greatly increased during the recovery phase of DSS-induced colitis. DSS-derived splenic Gr1(+)CD11b(+) cells were administered intravenously to recipient (C57BL/6) mice during the early phase of DSS treatment. The transplanted splenic DSS-induced Gr1(+)CD11b(+) cells improved DSS-induced colitis and promoted efficient colonic mucosal healing. We found that the CD11b(+) single positive cells increased in the course of DSS-induced colitis in lamina propria. The transplantation of splenic Gr1(+)CD11b(+) cells induced feedback suppression of myeloid-lineage cell development. Namely, the transplantation of splenic Gr1(+)CD11b(+) cells greatly suppressed the migration of CD11b(+) single positive cells to the lamina propria. Further, transplantation of Gr-1(+)CD11b(+) cells greatly suppressed the increase of the same population, especially during the late phase of DSS colitis both in spleen and bone marrow.

Asunto(s)

Trasplante de Células , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/terapia , Colon/patología , Bazo/metabolismo , Animales , Antígeno CD11b/biosíntesis , Recuento de Células , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/fisiopatología , Sulfato de Dextran/administración & dosificación , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Receptores de Superficie Celular/biosíntesis , Bazo/inmunología , Bazo/patología

RESUMEN

Manganese superoxide dismutase Mn-SOD plays a major role in protecting mitochondria from oxidative damage. Overexpression of Mn-SOD maintains cell survival under conditions that lead to apoptotic death. In addition to the antioxidative enzyme, platelet-derived growth factor (PDGF) is a principal survival factor that inhibits apoptosis and promotes proliferation by activating survival signaling pathways in various cells. Here we show that PDGF induced the expression of the Mn-SOD gene in NIH3T3 cells, and its induction was associated with early growth response-1 (Egr-1), a transcription factor. An electrophoretic mobility shift assay demonstrated that Egr-1 bound to the proximal promoter of the Mn-SOD gene in response to PDGF. The proximal promoter region of Mn-SOD was shown to be transcriptionally responsive to both basal and PDGF stimulation by transfection studies. Forced expression of Egr-1 in the cells activated Mn-SOD transcription in a dose-dependent manner. The pathway by which PDGF induced Egr-1 involved the mitogen-activated protein kinase kinase-1 (MEK1) and extracellular signal-regulated kinases 1 and 2 (ERK1/2), because the effect of PDGF on the induction of Egr-1 was blocked by U0126, a specific MEK1 inhibitor. These findings indicate that the induction of Mn-SOD is part of the anti-apoptotic properties mediated by PDGF.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Regulación Enzimológica de la Expresión Génica , Proteínas Inmediatas-Precoces , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Superóxido Dismutasa/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Células 3T3 , Animales , ADN , Proteína 1 de la Respuesta de Crecimiento Precoz , MAP Quinasa Quinasa 1 , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Transducción de Señal , Factor de Transcripción Sp1/metabolismo , Dedos de Zinc

RESUMEN

C methylation at genomic CpG dinucleotides has been implicated in the regulation of a number of genetic activities during vertebrate cell differentiation and embryo development. The methylated CpG could induce chromatin condensation through the recruitment of histone deacetylase (HDAC)-containing complexes by methyl-CpG-binding proteins. These proteins consist of the methylated-DNA binding domain (MBD). Unexpectedly, however, several studies have identified MBD-containing proteins encoded by genes of Drosophila melanogaster, an invertebrate species supposed to be void of detectable m(5)CpG. We now report the genomic structure of a Drosophila gene, dMBD2/3, that codes for two MBD-containing, alternatively spliced, and developmentally regulated isoforms of proteins, dMBD2/3 and dMBD2/3Delta. Interestingly, in vitro binding experiments showed that as was the case for vertebrate MBD proteins, dMBD2/3Delta could preferentially recognize m(5)CpG-containing DNA through its MBD. Furthermore, dMBD2/3Delta as well as one of its orthologs in mouse, MBD2b, could function in human cells as a transcriptional corepressor or repressor. The activities of HDACs appeared to be dispensable for transcriptional repression by dMBD2/3Delta. Finally, dMBD2/3Delta also could repress transcription effectively in transfected Drosophila cells. The surprisingly similar structures and characteristics of the MBD proteins as well as DNA cytosine (C-5) methyltransferase-related proteins in Drosophila and vertebrates suggest interesting scenarios for their roles in eukaryotic cellular functions.

Asunto(s)

Islas de CpG , Metilación de ADN , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica , Genes de Insecto , Proteínas de Insectos/fisiología , Proteínas Represoras/fisiología , Transcripción Genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromatina/metabolismo , Cromatina/ultraestructura , ADN/genética , ADN/metabolismo , ADN Complementario/genética , Proteínas de Unión al ADN/genética , Evolución Molecular , Histona Desacetilasas/fisiología , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/fisiología , Especificidad de la Especie

RESUMEN

Age-dependent changes in the axonal branching patterns of single locus coeruleus neurons, which innervate both the frontal cortex and hippocampus dentate gyrus, have been studied in male F344 rats. We used an electrophysiological approach involving antidromic activation to differentiate single from multi-threshold locus coeruleus neurons in each terminal field with age (7-27 mo of age). Most of these neurons have a single threshold in the young rats, whereas in the older brains, the neurons have multi-threshold responses. This implies an increased amount of axonal branching in the older brains. The time course of the increase differs in the two terminal fields, suggesting that the degree of plasticity or age-dependent increase in branching can differ across terminal fields.

Asunto(s)

Envejecimiento/fisiología , Axones/fisiología , Giro Dentado/citología , Lóbulo Frontal/citología , Locus Coeruleus/citología , Animales , Giro Dentado/fisiología , Electrofisiología , Lóbulo Frontal/fisiología , Locus Coeruleus/fisiología , Masculino , Vías Nerviosas , Plasticidad Neuronal/fisiología , Ratas , Ratas Endogámicas F344

RESUMEN

Expression of the manganese superoxide dismutase (Mn-SOD) is induced by tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and lipopolysaccharide (LPS). Recently, a TNF-responsive element (TNFRE) was identified within the second intron of the murine Mn-SOD gene. The 5' CCAAT/enhancer binding protein (C/EBP)-related region within the TNFRE was responsive to TNF, whereas the 3' NF-kappaB-related region alone was not. This report describes the minimal promoter region of the Mn-SOD gene and investigates the cis-acting elements and trans-acting factors responsible for TNF-alpha-induced Mn-SOD gene expression. Reporter plasmid transfection studies demonstrated that inducible transcription factors enhanced the transcriptional activity of the Mn-SOD gene through the intronic enhancer region. Electrophoretic mobility shift assays demonstrated that after TNF-alpha stimulation, p50 and p65 NF-kappaB subunits bound specifically to the newly identified NF-kappaB transcription factor-binding site, distinct from the previously described NF-kappaB site, within the intronic enhancer region. In addition, site-directed mutagenesis and cotransfection studies demonstrated that the NF-kappaB p65 subunit enhanced the transcriptional activity of the Mn-SOD gene through the newly identified NF-kappaB site. These results show that a NF-kappaB p65 subunit is mainly involved in the molecular mechanisms controlling TNF-alpha-mediated Mn-SOD gene transcription.

Asunto(s)

FN-kappa B/fisiología , Superóxido Dismutasa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Células 3T3 , Animales , Secuencia de Bases , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática , Intrones , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis , FN-kappa B/química , FN-kappa B/metabolismo , Subunidad p50 de NF-kappa B , Proteínas Nucleares/metabolismo , Plásmidos , Regiones Promotoras Genéticas , Unión Proteica , Factor de Transcripción Sp1/metabolismo , Superóxido Dismutasa/genética , Factores de Tiempo , Factor de Transcripción ReIA , Transcripción Genética , Transfección

RESUMEN

Crescentic glomerulonephritis leads to a rapid loss of renal function. Although glomerular crescents are rich in extracellular matrix (ECM), the composition and genesis of the ECM are incompletely understood. Heparan sulfate (HS) is a major ECM molecule and has polymeric structure of great variability. Recent findings that alterations in HS epitopes are associated with renal pathology prompted us to hypothesize that specific HS epitopes might be expressed in the evolution of crescents. We reviewed clinical records of 724 patients who underwent renal biopsy and found 21 patients with rapidly progressive glomerulonephritis. Immunohistochemistry was performed using monoclonal antibodies (mAbs) against well-defined HS epitopes. One mAb was directed against unsaturated uronic acid residues generated during the selective removal of HS by heparitinase (a), and a further two different mAbs against N-sulfate-enriched and O-sulfate-poor portions of HS (b). Results showed that mAb (a) reacted to ECM of normal, sclerosed and crescentic glomeruli and that mAbs (b) reacted strongly to ECM of fibrocellular crescents but not to fibrous crescents, the periglomerular areas and noncrescentic intraglomerular areas. We concluded there are regional differences in HS epitope expression, although HS are ubiquitous components of glomerular ECM. N-sulfate-enriched and O-sulfate-poor portions of HS might play a role in crescent formation.

Asunto(s)

Glomerulonefritis/metabolismo , Heparitina Sulfato/análisis , Animales , Anticuerpos Monoclonales/inmunología , Epítopos , Heparitina Sulfato/inmunología , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Ratas , Ratas Wistar

RESUMEN

We have established a novel injury model in the central nervous system by a stereotaxic injection of ethanol into rat striatum to induce necrosis. With this model, we clarify a function of inducible nitric oxide synthase (iNOS) in a healing mechanism around a necrotic lesion. A semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that the iNOS mRNA arose at 6 h, peaked at 24 h, and declined to a lower level 48 h after an intrastriatal 5-microL ethanol injection. From in situ hybridization, this iNOS mRNA was expressed in the area surrounding the injury. By immunohistochemistry, mononuclear cells at this boundary area of necrosis were stained with anti-iNOS antibody on the first day after the injury. These cells turned out to be reactive microglia from the positive staining of GSA-I-B4, ED-1 and OX-42. Haematoxylin-eosin (HE) staining showed that neurons in this boundary area gradually disappear up to 5 days after the injury with an increment of microglial cells, and this area became cavernous. Nuclei of neurons in this area were stained positive by the terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end-labelling (TUNEL) assay on the first day after the injury. These TUNEL-positive neurons gradually disappeared toward the third day, while microglial cells increased. L-Ng-nitro-arginine methylester (L-NAME), a competitive NOS inhibitor, administration diminished the elimination of neurons by microglia in this boundary area surrounding necrosis. Microglial NO may act as a neurotoxic agent to eliminate damaged neurons near the necrosis in the form of delayed neuronal death, and may reintegrate the neuronal circuits with functionally intact neurons.

Asunto(s)

Cuerpo Estriado/efectos de los fármacos , Etanol/toxicidad , Microglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Óxido Nítrico/fisiología , Animales , Muerte Celular/efectos de los fármacos , Cuerpo Estriado/lesiones , Cuerpo Estriado/patología , Fragmentación del ADN , Inhibidores Enzimáticos/farmacología , NG-Nitroarginina Metil Éster/farmacología , Necrosis , Neuronas/patología , Óxido Nítrico Sintasa/análisis , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Reacción en Cadena de la Polimerasa/métodos , Ratas , Técnicas Estereotáxicas , Transcripción Genética

RESUMEN

In osteoblast-like MC3T3-E1 cells, we recently reported that PGE1 and PGF2alpha induce interleukin (IL)-6 synthesis via activation of protein kinase A and protein kinase C, respectively. Moreover, in the case of IL-1-induced IL-6 synthesis in these cells, we showed that protein kinase C activation by IL-1 limits the IL-6 synthesis. In the present study, we investigated the effect of T3 on IL-6 synthesis induced by these agonists in MC3T3-E1 cells. T3, which by itself had little effect on IL-6 synthesis, significantly reduced the IL-6 synthesis induced by PGE1 in a dose-dependent manner in the range between 10 pM and 10 nM. T3 also reduced PGE1-induced activation of protein kinase A. T3 inhibited the IL-6 synthesis induced by cholera toxin, an activator of Gs, or forskolin, which directly activates adenylate cyclase. However, T3 did not affect (Bu)2cAMP-induced IL-6 synthesis. In addition, T3 reduced PGF2alpha-induced IL-6 synthesis dose dependently in the range between 10 pM and 10 nM. T3 also inhibited IL-6 synthesis induced by 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C. On the other hand, T3 markedly enhanced IL-1-induced IL-6 synthesis. This enhancement by T3 was potentiated in protein kinase C down-regulated cells. T3 hardly affected the protein kinase C activation induced by PGF2alpha or IL-1. These results strongly suggest that T3 modulates IL-6 synthesis at two points in osteoblasts as follows; one is exerted at the point between adenylate cyclase and protein kinase A, and the other is at a point downstream from protein kinase C activation.

Asunto(s)

Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Interleucina-6/biosíntesis , Osteoblastos/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Triyodotironina/farmacología , Alprostadil/farmacología , Animales , Bucladesina/farmacología , Colforsina/farmacología , Dinoprost/farmacología , Interleucina-1/farmacología , Ratones , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

Murine interferon-gamma (IFN-gamma) stimulates the murine macrophage tumour cell line RAW264-7 to produce nitric oxide (NO). IFN-gamma induces expression of inducible NO synthase (iNOS), manganese superoxide dismutase (Mn-SOD) and copper zinc SOD (CuZn-SOD) in these cells. To investigate the mechanism of induction of SOD expression, we added S-nitroso-N-acetyl penicillamine (SNAP) to RAW264-7 cells. SNAP enhanced the expression of Mn-SOD and CuZn-SOD. These results suggest that when producing NO, RAW264-7 cells express SOD that might protect them from NO toxicity.

Asunto(s)

Macrófagos/enzimología , Óxido Nítrico/metabolismo , Penicilamina/análogos & derivados , Superóxido Dismutasa/metabolismo , Vasodilatadores/farmacología , Animales , Cobre/metabolismo , Expresión Génica , Interferón gamma/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Compuestos de Manganeso/metabolismo , Ratones , Penicilamina/farmacología , ARN Mensajero/genética , S-Nitroso-N-Acetilpenicilamina , Superóxido Dismutasa/genética , Células Tumorales Cultivadas , Compuestos de Zinc/metabolismo

RESUMEN

In CD2/protein kinase C alpha (PKC alpha)-overexpressing human CD2/rabbit PKC alpha transgenic mice, an aging-dependent increase in PKC alpha expression and a decrease in proliferative responsiveness of splenic T cells were promoted. We found that an aging-associated accumulation of CD44(high) CD45RB(low) memory CD4+ T cells in exchange for CD44(low) CD45RB(high) naive CD4+ T cells was promoted in transgenic mice. A disequilibrium between Ag-dependent generation and subsequent elimination of memory T cells in these mice was shown to underlie this phenomenon. When stimulated with Ag, the PKC alpha transgenic mice responded poorly regarding Ab production and produced cytokines biased for high IFN-gamma/IL-12 and low IL-4/IL-10 levels. These results prove, for the first time, a causal role for chronic signal transduction through PKC alpha in aging-associated immunodysfunction and provide the first animal model for genetically promoted immunosenescence.

Asunto(s)

Envejecimiento/inmunología , Linfocitos T CD4-Positivos/citología , Regulación del Desarrollo de la Expresión Génica , Síndromes de Inmunodeficiencia/etiología , Isoenzimas/fisiología , Proteína Quinasa C/fisiología , Transducción de Señal/fisiología , Envejecimiento/genética , Animales , Linfocitos T CD4-Positivos/enzimología , Linfocitos T CD4-Positivos/inmunología , División Celular , Citocinas/biosíntesis , Citocinas/genética , Inducción Enzimática , Humanos , Receptores de Hialuranos/análisis , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Isoenzimas/genética , Antígenos Comunes de Leucocito/análisis , Ratones , Ratones Transgénicos , Proteína Quinasa C/genética , Proteína Quinasa C-alfa , Conejos , Proteínas Recombinantes de Fusión/fisiología , Organismos Libres de Patógenos Específicos , Bazo/citología

RESUMEN

Sodium butyrate, a histone deacetylase inhibitor, has been shown to induce differentiation of many cancer cells and senescence-like state of human fibroblasts. Here we show that sodium butyrate also induces senescence-like state of NIH3T3 cells. The treated cells were blocked at G1 phase and featured morphologically like senescent cells with enlarged cytoplasm and multiple nuclei. The expression of p21(WAF/CIP1) (p21) increased after sodium butyrate treatment at transcriptional level. To analyze the induction of promoter activity, we isolated 4.6 kb of murine p21 promoter and inserted it upstream of a luciferase reporter gene. When this construct was transiently transfected into NIH3T3 cells, sodium butyrate enhanced the luciferase activity. p53 independency of sodium butyrate-inducible p21 promoter activity was confirmed by using the deletion mutants lacking p53 binding sites and p53 deficient cells in transfection experiments.

Asunto(s)

Butiratos/farmacología , Senescencia Celular/efectos de los fármacos , Ciclinas/genética , Regiones Promotoras Genéticas , Células 3T3 , Animales , Ácido Butírico , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Regulación de la Expresión Génica/efectos de los fármacos , Genes p53 , Ratones , ARN Mensajero/genética , Eliminación de Secuencia

RESUMEN

Radiation pneumonitis is a major complication of radiation therapy. To elucidate the mechanisms of radiation-induced pneumonitis, we studied nitric oxide (NO) produced from lung tissues using a model of unilaterally irradiated rats. Our results demonstrated that alveolar macrophages (AM) produced NO after irradiation, and the expression of inducible NO synthase (NOS) in both AM and alveolar epithelial cells was increased. Furthermore, the progression of radiation pneumonitis was reduced with the in vivo treatment of the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). The effect of L-NAME was further confirmed by the inhibition of mRNA expression for procollagen-alpha1 type III of the lung. With these results, NO produced from AM and alveolar epithelial cells after irradiation may be an important mediator in the progression of radiation pneumonitis.

Asunto(s)

Mediadores de Inflamación/fisiología , Óxido Nítrico/fisiología , Neumonitis por Radiación/fisiopatología , Animales , Inducción Enzimática , Inhibidores Enzimáticos/farmacología , Pulmón/enzimología , Pulmón/efectos de la radiación , Macrófagos Alveolares/metabolismo , Masculino , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitritos/metabolismo , Procolágeno/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar

RESUMEN

We have used the yeast one-hybrid system to clone transcription factors that bind to specific sequences in the proximal promoters of the type I collagen genes. We utilized as bait the sequence between -180 and -136 in the pro-alpha2(I) collagen promoter because it acts as a functional promoter element and binds several DNA-binding proteins. Three cDNA clones were isolated that encoded portions of the mouse SPR2 transcription factor, whereas a fourth cDNA contained a potential open reading frame for a polypeptide of 775 amino acids and was designated BFCOL1. Recombinant BFCOL1 was shown to bind to the -180 to -152 segment of the mouse pro-alpha2(I) collagen proximal promoter and to two discrete sites in the proximal promoter of the mouse pro-alpha1(I) gene. The N-terminal portion of BFCOL1 contains its DNA-binding domain. DNA transfection experiments using fusion polypeptides with the yeast GAL4 DNA-binding segment indicated that the C-terminal part of BFCOL1 contained a potential transcriptional activation domain. We speculate that BFCOL1 participates in the transcriptional control of the two type I collagen genes.

Asunto(s)

Colágeno/genética , Proteínas de Unión al ADN/genética , Regiones Promotoras Genéticas/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Clonación Molecular , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Ratones , Datos de Secuencia Molecular

RESUMEN

Alveolar macrophages (AM) from normal rats had immunosuppressive activity to mitogen-induced proliferative responses of splenic lymphocytes. We studied the mechanism and the implication of the nitric oxide synthetase pathway in AM-mediated suppression of concanavalin A (Con A)-induced lymphocyte proliferation. The culture supernatant from AM cultures alone did not have immunosuppressive activity to Con A-induced proliferative responses of non-adherent spleen cells (n-ad SC), but the culture supernatant from co-culture of AM and autologous n-ad SC had this activity. Con A-pulsed AM also liberated the immunosuppressive factor. When AM and autologous n-ad SC were cultured separately under the condition that medium could freely communicate, the culture supernatant did not suppress the Con A-induced proliferative response of n-ad SC. This indicated that the immunosuppressive factor was liberated when AM was activated by cell-to-cell contact with n-ad SC. Further, we examined the immunosuppressive activity of the culture supernatant of co-culture of AM and autologous n-ad SC to Con A-induced responses of allogeneic n-ad SC and xenogeneic murine n-ad SC, and allogeneic mixed leucocyte reaction, and found that this culture supernatant could suppress all these proliferative responses. Nitrate (NO2-) synthesis was markedly augmented in the culture supernatants of Con A-pulsed AM and co-culture of AM and n-ad SC. NG-monomethyl-L-arginine (MMA), a specific competitive inhibitor of the nitric oxide synthetase pathway (NOSP), extinguished both NO2- synthesis by AM and AM-mediated immunosuppressive activity. These data suggest that NOSP was important in AM-mediated suppression of Con A-induced lymphocyte proliferation.

Asunto(s)

Tolerancia Inmunológica/inmunología , Macrófagos/inmunología , Óxido Nítrico/inmunología , Alveolos Pulmonares/inmunología , Animales , Comunicación Celular/inmunología , Células Cultivadas , Medios de Cultivo , Prueba de Cultivo Mixto de Linfocitos , Activación de Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratas , Ratas Endogámicas , Bazo/inmunología

RESUMEN

By adding IL-2 (supernatant of culture of concanavalin A-activated rat spleen cells) on Day 3 of mixed leucocyte cultures (MLC) we managed to fully activate multiple minor histocompatibility antigen (MIHA)-specific cytotoxic T-lymphocyte precursors (CTLp). In this newly developed system we studied genetic and stimulator cell requirements for the generation and activation of MIHA-specific memory CTLp. Memory CTLp were activated to generate effector CTL in MLC only when major histocompatibility complex (MHC)-compatible MIHA-allogeneic cells were used as stimulators. In contrast, memory CTLp were generated in mice that were primed by injection of either MHC-compatible or incompatible MIHA-allogeneic spleen cells. A surprisingly small number (10(4] of MHC-disparate cells cross-primed mice effectively. For priming, no special accessory cell types were required as stimulators, and 10(4) adherent cell-depleted spleen cells primed mice as well. These results contrasted to another finding that sonication-disrupted 10(6) stimulator cells did not prime mice effectively, and antigens shed from 10(7) live stimulator cells failed to sensitize host antigen-presenting cells for priming. It is suggested from these results that the mode of recognition of MIHA by virgin CTLp is unique or that an as yet unknown unusual stimulation pathway works for the priming.

Asunto(s)

Antígenos de Histocompatibilidad/inmunología , Memoria Inmunológica , Sitios Menores de Histocompatibilidad , Linfocitos T Citotóxicos/inmunología , Animales , Células Cultivadas , Interleucina-2/farmacología , Prueba de Cultivo Mixto de Linfocitos , Complejo Mayor de Histocompatibilidad , Ratones , Ratones Endogámicos

RESUMEN

The Thy-1 gene is expressed in a tissue- and stage-specific pattern and has a typical 1.6kb methylation-free island (MFI) covering about 600bp upstream and downstream of the two alternative first exons. By microinjection of a mouse Thy-1.1/human Thy-1 gene into fertilized eggs, we were able to show that the MFI is restored in the transgenic mice. The flanking sequence became methylated, but the MFI remains unmethylated in all tissues of transgenic mice at different developmental stages tested, irrespective of the site of expression of the gene. There is one exception, in extra-embryonal tissues of 14.5 day embryos a small percentage of the islands were methylated. We conclude that maintenance of the MFI is regulated by cis-acting sequences present within the gene, and indicates that the unmethylated state of the islands is consistent with a necessary but not sufficient condition for expression of the gene.

Asunto(s)

Antígenos de Superficie/genética , Genes , Hibridación Genética , Ratones Endogámicos/genética , Animales , ADN/aislamiento & purificación , Exones , Humanos , Metilación , Ratones , Antígenos Thy-1

RESUMEN

Thy-1 antigens are the only cell membrane antigens known to be able to induce primary antibody responses in vitro. We have shown that antigens from the thymocytes of mice and rats were highly immunogenic in cultures of murine spleen cells for the induction of Thy-1.1-specific plaque-forming cell responses, whereas antigens from other tissues, including brains and bone marrow, were poorly immunogenic, if at all. The thymocyte-specific Thy-1 immunogenicity was carried by disrupted cell membranes, and the specific activity for inducing responses was closely linked to Thy-1. We then tried to determine the mechanism of anti-Thy-1 antibody responses in vitro that were induced by the uniquely immunogenic thymocyte antigens. The thymocyte Thy-1 antigens behaved as T cell-independent class 2 (TI-2) antigens: they induced responses in athymic nude mice but not in CBA/N mice with a B cell defect. The apparent TI-2 responses to thymocyte Thy-1 did, however, require Thy-1+ cells in the responder, similar to anti-DNP-Ficoll responses. The full development of the anti-Thy-1 responses required the participation of splenic adherent cells (SAC). Nevertheless, the mechanism of the SAC dependency of anti-Thy-1 responses did not involve antigen presentation to lymphocytes by antigen-pulsed SAC, which contrasted with the finding that the presentation of antigen by live SAC to lymphocytes was indispensable for responses to DNP-Ficoll. The poor Thy-1 responsiveness of SAC-depleted spleen cells was fully restored by the addition of soluble factors (IL 1-like molecules) released from SAC into the culture, which did not replace the SAC-requirement of responses to DNP-Ficoll. It was concluded from these results that Thy-1 or Thy-1-linked structures on thymocyte membranes have an intrinsic activity to directly signal either TI-2 B cells or immature T cells, or both, for activation in the presence of soluble factors released from adherent accessory cells. This conclusion is discussed in relation to a hypothetical view that the thymocyte Thy-1 would physiologically mediate cell-to-cell interactions among special subsets of lymphocytes under thymic influence.

Asunto(s)

Antígenos de Superficie/inmunología , Suero Antilinfocítico/inmunología , Linfocitos T/inmunología , Animales , Células Productoras de Anticuerpos/inmunología , Antígenos T-Independientes/inmunología , Técnica de Placa Hemolítica , Inmunidad Celular , Activación de Linfocitos , Cooperación Linfocítica , Macrófagos/inmunología , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Ratones Desnudos , Ratas , Ratas Endogámicas , Antígenos Thy-1

RESUMEN

Murine allogeneic red blood cells (RBC) induce primary IgM antibody responses to H-2 alloantigens T-cell-independently (TI). In this study we showed that bacterial lipopolysaccharide (LPS), which should activate B lymphocytes polyclonally, could not trigger an anti-H-2d plaque-forming cell response. We then demonstrated that administration of LPS (on days 0-1) with allogeneic RBC suppressed the response of mice to H-2d, while giving LPS 4-6 days after RBC augmented the response. In contrast, LPS did not enable allogeneic spleen cells to induce an anti-H-2d response. Additional experiments showed that the allogeneic RBC behave as a TI class 2 antigen. It was concluded from these results that allogeneic RBC display a peculiar activity that exclusively triggers a TI type-2 B cell response that cannot be initiated by LPS and is modulated by LPS in an abnormal fashion. The possible significance of this finding in the mechanism of occurrence of natural H-2-specific IgM alloantibodies in aged mice is discussed.

Asunto(s)

Linfocitos B/inmunología , Eritrocitos/inmunología , Antígenos H-2/inmunología , Lipopolisacáridos/farmacología , Animales , Formación de Anticuerpos , Femenino , Inmunoglobulina M/inmunología , Masculino , Ratones , Ratones Endogámicos , Ratones Desnudos , Linfocitos T/inmunología , Ensayo de Placa Viral

RESUMEN

Antigenic requirements for the induction of T cell-independent primary splenic IgM antibody responses (plaque-forming cell responses) to H-2Dd alloantigens were studied. Results show that some functional activity or structural property of the donor cells is required for immunogenicity, because antigens are not active in subcellular forms. An unexpected finding was that allogeneic red blood cells were exceptionally highly immunogenic, and any lymphoid tissues including purified macrophages and tumor cell lines that were not contaminated with red blood cells were virtually nonimmunogenic. The definite role of red blood cells in donor tissues as immunogens was confirmed by water or ammonium chloride treatment that abolished immunogenicity, as well as by phenotyping of the immunogenic cells with antisera. Thus immunogenic cells were positive for erythrocyte-specific and H-2D antigens and negative for Thy-1, Ig, and NK-1. The possible roles of erythrocytes in induction and regulation of transplantation immunity and in B cell activation in general are discussed.

Asunto(s)

Eritrocitos/inmunología , Antígenos H-2/inmunología , Inmunoglobulina M/inmunología , Animales , Antígenos T-Independientes/inmunología , Linfocitos B/inmunología , Femenino , Masculino , Ratones , Ratones Endogámicos