RESUMEN

Expression of major histocompatibility antigens class-2 (MHC-II) under non-inflammatory conditions is not usually associated with the nervous system. Comparative analysis of immunogenicity of human embryonic/fetal brain-derived neural stem cells (hNSCs) and human mesenchymal stem cells with neurogenic potential from umbilical cord (UC-MSCs) and paediatric adipose tissue (ADSCs), while highlighting differences in their immunogenicity, led us to discover subsets of neural cells co-expressing the neural marker SOX2 and MHC-II antigen in vivo during human CNS development. MHC-II proteins in hNSCs are functional, and differently regulated upon differentiation along different lineages. Mimicking an inflammatory response using the inflammatory cytokine IFNγ induced MHC-II up-regulation in both astrocytes and hNSCs, but not in UC-MSCs and ADSCs, either undifferentiated or differentiated, though IFNγ receptor expression was comparable. Together, hypoimmunogenicity of both UC-MSCs and ADSCs supports their suitability for allogeneic therapy, while significant immunogenicity of hNSCs and their progeny may at least in part underlie negative effects reported in some patients following embryonic neural cell grafts. Crucially, we show for the first time that MHC-II expression in developing human brains is not restricted to microglia as previously suggested, but is present in discrete subsets of neural progenitors and appears to be regulated independently of inflammatory stimuli.

Asunto(s)

Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica , Antígenos de Histocompatibilidad Clase II/genética , Interferón gamma/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Tejido Adiposo/citología , Astrocitos/citología , Astrocitos/metabolismo , Biomarcadores , Sangre Fetal/citología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Interferón gamma/farmacología , Neuronas/citología , Neuronas/metabolismo , Receptores de Interferón/metabolismo

RESUMEN

Macrophages come across active prostaglandin (PG) metabolism during inflammation, shunting early production of pro-inflammatory towards anti-inflammatory mediators terminating the process. This work for the first time provides evidence that a phytochemical may modulate the arachidonate (AA) metabolism in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, promoting the ultimate formation of anti-inflammatory cyclopentenone 15deoxy-PGJ2. Added 1 h before LPS, indicaxanthin from Opuntia Ficus Indica prevented activation of nuclear factor-κB (NF-κB) and over-expression of PGE2 synthase-1 (mPGES-1), but up-regulated cyclo-oxygenase-2 (COX-2) and PGD2 synthase (H-PGDS), with final production of the anti-inflammatory cyclopentenone. The effects were positively related with concentration between 50 and 100 µM. Indicaxanthin did not have any effect in the absence of LPS. A kinetic study investigating the redox status of LPS-stimulated macrophages between 0.5 and 12 h, either in the absence or in the presence of 50-100 µM indicaxanthin, revealed a differential control of ROS production, with early (0.5-3 h) modest inhibition, followed by a progressive (3-12 h) concentration-dependent enhancement over the level induced by LPS alone. In addition, indicaxanthin caused early (0.5-3 h) concentration-dependent elevation of conjugated diene lipid hydroperoxides, and production of hydroxynonenal-protein adducts, over the amount induced by LPS. In LPS-stimulated macrophages indicaxanthin did not affect PG metabolism when co-incubated with either an inhibitor of NADPH oxidase or vitamin E. It is concluded that LPS-induced pro-oxidant activity of indicaxanthin at the membrane level allows formation of signaling intermediates whose accumulation modulates PG biosynthetic pathway in inflamed macrophages.

Asunto(s)

Ácido Araquidónico/metabolismo , Betaxantinas/farmacología , Peróxidos Lipídicos/biosíntesis , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Oxidantes/farmacología , Prostaglandinas/biosíntesis , Piridinas/farmacología , Animales , Betaxantinas/química , Betaxantinas/aislamiento & purificación , Línea Celular , Relación Dosis-Respuesta a Droga , Frutas/química , Macrófagos/metabolismo , Ratones , Opuntia/química , Oxidantes/química , Oxidantes/aislamiento & purificación , Piridinas/química , Piridinas/aislamiento & purificación , Relación Estructura-Actividad

RESUMEN

An imbalanced immune system has long been known to influence a variety of mood disorders including anxiety, obsessive-compulsive disorders and depression. In this study, we sought to model the impact of an immunocompromised state on these emotional behaviors using RAG-1⁻/⁻ mice, which lack T and B cells. We also investigated the relative contribution of CD4⁺ or CD8⁺ T cells to these manifestations using RAG-1⁻/⁻/OT-II and RAG-1⁻/⁻/OT-I transgenic mice, respectively. Our results show that RAG-1⁻/⁻ mice present a significant increase in digging and marble-burying activities compared with wild-type mice. Surprisingly, these anxiety-like behaviors were significantly reverted in RAG-1⁻/⁻/OT-II but not RAG-1⁻/⁻/OT-I transgenic mice. Immunodepletion experiments with anti-CD4 or anti-CD8 in C57/BL6 mice or repopulation studies in RAG-1⁻/⁻ mice did not reproduce these findings. Microarray analysis of the brain of RAG-1⁻/⁻ and RAG-1⁻/⁻/OT-II mice revealed a significantly different gene fingerprint, with the latter being more similar to wild-type mice than the former. Further analysis revealed nine main signaling pathways as being significantly modulated in RAG-1⁻/⁻ compared with wild-type mice. Taken together, these results suggest that life-long rather than transient immunodeficient conditions influence the emotional behaviors in mice. Most interestingly, these effects seem to correlate with a specific absence of CD4⁺ rather than CD8⁺ T cells. Validation of these findings in man might provide new clues on the mechanism by which early life immune modulation might impact mood response in adults and provide a further link between immune and emotional well-being.

Asunto(s)

Conducta Animal/fisiología , Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/fisiología , Emociones/fisiología , Proteínas de Homeodominio/fisiología , Animales , Corticosterona/sangre , Citocinas/sangre , Citometría de Flujo , Huésped Inmunocomprometido/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

This study demonstrates that a long-lasting co-culture of neutrophil surrogates (HL-60 cells), minimally primed by platelet activating factor (PAF), and resting endothelial cells (EC) results in the elaboration of an hyper-adhesive endothelial surface, as measured by the increase in the expression of endothelial adhesion molecules E-Selectin, VCAM-1, and ICAM-1. This endothelial dysfunction is mediated by the activation of the redox-sensitive transcription factor NF-κB through an exclusive adhesion-driven mechanism active in the endothelial cell: reactive oxygen and nitrogen species, extracellularly released by minimally primed HL-60 cells, are not involved in the induction of the endothelial dysfunction. By exploring for the first time the potential for minimally primed neutrophil surrogates to induce endothelial dysfunction, this study suggests a novel mechanism which may be operative in pathologies, mediated by minimally primed neutrophils, such as hyperdyslipidemia and cardiovascular complications.

Asunto(s)

Células Endoteliales/fisiología , Oxidantes/metabolismo , Receptor Cross-Talk , Adhesión Celular , Línea Celular , Técnicas de Cocultivo , Selectina E/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células HL-60 , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo

RESUMEN

Lipopolysaccharide (LPS) or endotoxin, the major constituent of the outer membrane of Gram negative bacteria, has been implicated as the bacterial product responsible for the clinical syndrome of sepsis. LPS binding to the host receptor Toll-like receptor 4 (TLR4) triggers an inflammatory reaction characterised by the release of large number of inflammatory mediators that allow the host to respond to the invading pathogen. When this production becomes un-controlled and excessive, it leads to the development of septic shock. Despite decades of efforts in supporting therapies, sepsis remains the leading cause of death amongst critically ill patients. Unfortunately, the major factor contributing to the high morbidity and mortality of sepsis is the lack of the effective targeted treatment. Indeed, over 30 drugs for the treatment of sepsis have been developed: many of these target specific inflammatory mediators and have thus been, in general, unsuccessful since sepsis relies on the cross talk of several cytokines and the block of a single factor has been proven to be ineffective. More successful strategies include those modulating the early phase of LPS signalling such as the ones that prevent the binding of LPS to host cells and the subsequent cascade of detrimental events. In this light, effective LPS antagonists would represent invaluable tools to efficaciously manage sepsis. This review discusses the evolution of naturally occurring and synthetic LPS antagonists with emphasis on the development of several natural new molecules.

Asunto(s)

Lipopolisacáridos/antagonistas & inhibidores , Sepsis/tratamiento farmacológico , Animales , Humanos , Lípido A/análogos & derivados , Lípido A/antagonistas & inhibidores , Lípido A/farmacología , Lipopolisacáridos/química , Lipopolisacáridos/farmacología , Estructura Molecular , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología

RESUMEN

Although thiazolidinediones were designed as specific peroxisome proliferator-activated receptor (PPAR)-gamma-ligands, there is evidence for some off-target effects mediated by a non-PPARgamma mechanism. Previously we have shown that rosiglitazone has antiinflammatory actions not explicable by activation of PPARgamma,but possibly by the glucocorticoid receptor (GR). Rosiglitazone induces nuclear translocation both of GR-green fluorescent protein, and endogenous GR in HeLa and U20S cells but with slower kinetics than dexamethasone. Rosiglitazone also induces GR phosphorylation (Ser211), a GR ligand-binding-specific effect. Rosiglitazone drives luciferase expression from a simple glucocorticoid-response element containing reporter gene in a GR-dependent manner (EC50 4 microm), with a similar amplitude response to the partial GR agonist RU486. Rosiglitazone also inhibits dexamethasone-driven reporter gene activity (IC50 2.9 microm) in a similar fashion to RU486, suggesting partial agonist activity. Importantly we demonstrate a similar effect in PPARgamma-null cells, suggesting both GR dependence and PPARgamma independence. Rosiglitazone also activates a GAL4-GR chimera, driving a upstream activating sequence promoter, demonstrating DNA template sequence independence and furthermore enhanced steroid receptor coactivator-1-GR interaction, measured by a mammalian two-hybrid assay. Both ciglitazone and pioglitazone, structurally related to rosiglitazone, show similar effects on the GR. The antiproliferative effect of rosiglitazone is increased in U20S cells that overexpress GR, suggesting a biologically important GR-dependent component of rosiglitazone action. Rosiglitazone is a partial GR agonist, affecting GR activation and trafficking to influence engagement of target genes and affect cell function. This novel mode of action may explain some off-target effects observed in vivo. Additionally, antagonism of glucocorticoid action may contribute to the antidiabetic actions of rosiglitazone.

Asunto(s)

PPAR gamma/fisiología , Receptores de Glucocorticoides/agonistas , Tiazolidinedionas/farmacología , División Celular/efectos de los fármacos , Línea Celular Tumoral , Dexametasona/antagonistas & inhibidores , Dexametasona/farmacología , Activación Enzimática , Genes Reporteros , Células HeLa , Antagonistas de Hormonas/farmacología , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Neoplasias Pulmonares , Mifepristona/farmacología , Receptores de Glucocorticoides/metabolismo , Rosiglitazona , Transfección

RESUMEN

The glucocorticoids are the most potent anti-inflammatory drugs that we possess and are effective in a wide variety of diseases. Although their action is known to involve receptor mediated changes in gene transcription, the exact mechanisms whereby these bring about their pleiotropic action in inflammation are yet to be totally understood. Whilst many different genes are regulated by the glucocorticoids, we have identified one particular protein-annexin A1 (Anx-A1)-whose synthesis and release is strongly regulated by the glucocorticoids in many cell types. The biology of this protein, as revealed by studies using transgenic animals, peptide mimetics and neutralizing antibodies, speaks to its role as a key modulator of both of the innate and adaptive immune systems. The mechanism whereby this protein exerts its effects is likely to be through the FPR receptor family-a hitherto rather enigmatic family of G protein coupled receptors, which are increasingly implicated in the regulation of many inflammatory processes. Here we review some of the key findings that have led up to the elucidation of this key pathway in inflammatory resolution.

Asunto(s)

Anexina A1/fisiología , Antiinflamatorios/farmacología , Glucocorticoides/farmacología , Inmunidad Innata/efectos de los fármacos , Inflamación/inmunología , Animales , Anexina A1/metabolismo , Humanos , Sistema Inmunológico/efectos de los fármacos , Inmunidad Innata/inmunología

RESUMEN

OBJECTIVE: Annexin-1 (Anx-A1) has been recently shown to play a key role in T-cell activation and to be highly expressed in T cells from RA patients. Here, we investigated the effects of glucocorticoids (GCs) on Anx-A1 expression in T cells in vitro and in vivo. METHODS: To evaluate the effects of dexamethasone (Dex) on Anx-A1 expression, human peripheral blood T cells were incubated with Dex and then analysed by real-time PCR and western blotting. Similar experiments were carried out in vivo by measuring Anx-A1 levels in T cells from patients with RA before and after administration of steroids. RESULTS: Incubation of T cells with Dex decreased Anx-A1 levels in a time-dependent fashion and almost abolished its expression after 12 h. Stimulation of T cells pre-incubated with Dex for 12 h with anti-CD3/CD28 led to significant reduction of IL-2 production. Addition of human recombinant Anx-A1 to Dex-treated cells reversed the inhibitory effects of the steroids on anti-CD3/CD28-induced IL-2 production. Treatment of RA patients with steroid decreased Anx-A1 expression in T cells. CONCLUSIONS: GCs suppress Anx-A1 expression in T cells in vitro and in vivo. These results provide evidence for a novel pathway by which steroids regulate the adaptive immune response and suggest that Anx-A1 may represent a target for the treatment of autoimmune diseases.

Asunto(s)

Anexina A1/análisis , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/inmunología , Linfocitos T CD4-Positivos/química , Dexametasona/uso terapéutico , Glucocorticoides/uso terapéutico , Anciano , Anciano de 80 o más Años , Anexina A1/metabolismo , Anticuerpos Monoclonales/farmacología , Western Blotting , Complejo CD3/inmunología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Depresión Química , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Interleucina-2/inmunología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

B-lymphocytes play a key role in the pathogenesis of many immune-mediated diseases, such as autoimmune and atopic diseases. Therefore, targeting B-lymphocytes provides a rationale for refining strategies to treat such diseases for long-term clinical benefits and minimal side effects. In this study we describe a protocol for repopulating irradiated mice with B-lymphocytes engineered for restricted expression of transgenes using haematopoietic stem cells. A self-inactivating lentiviral vector, which encodes enhanced green fluorescence protein (EGFP) from the spleen focus-forming virus (SFFV) promoter, was used to generate new vectors that permit restricted EGFP expression in B-lymphocytes. To achieve this, the SFFV promoter was replaced with the B-lymphocyte-restricted CD19 promoter. Further, an immunoglobulin heavy chain enhancer (Emu) flanked by the associated matrix attachment regions (MARs) was inserted upstream of the CD19 promoter. Incorporation of the Emu-MAR elements upstream of the CD19 promoter resulted in enhanced, stable and selective transgene expression in human and murine B-cell lines. In addition, this modification permitted enhanced selective EGFP expression in B-lymphocytes in vivo in irradiated mice repopulated with transduced bone marrow haematopoietic stem cells (BMHSCs). The study provides evidence for the feasibility of targeting B-lymphocytes for therapeutic restoration of normal B-lymphocyte functions in patients with B-cell-related diseases.

Asunto(s)

Antígenos CD19/genética , Linfocitos B/inmunología , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Células Madre Hematopoyéticas/metabolismo , Regiones Promotoras Genéticas , Animales , Antígenos CD19/inmunología , Linfocitos B/metabolismo , Línea Celular , Citometría de Flujo , Expresión Génica , Ingeniería Genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Trasplante de Células Madre Hematopoyéticas , Humanos , Lentivirus/genética , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Transducción Genética/métodos , Transgenes

RESUMEN

Double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) has been generally thought to be solely regulated by dsRNA, an intermediate in the replication of many viruses. However, the notion that PKR acts solely as a sensor for viral infection has been challenged by recent findings that alteration of PKR activity has effects on cellular growth and by the discovery of a virus-independent activator of PKR, a cellular protein called PACT (PKR-activating protein). The activation of the transcription factor nuclear factor kappa B (NF-kappaB) by PKR has been shown to account for the host antiviral response. We summarize the most recent findings on the molecular mechanisms leading to the activation of NF-kappaB by PKR and discuss three major unanswered questions. First, is PACT an alternative to dsRNA as a direct activator of the PKR-NF-kappaB pathway? Second, how is PACT itself activated and targeted to PKR? And third, what are the biological functions of PKR in the absence of viral infection?

Asunto(s)

Proteínas Portadoras/fisiología , FN-kappa B/fisiología , Proteínas de Unión al ARN , Ribonucleoproteínas/fisiología , Virus/enzimología , eIF-2 Quinasa/fisiología , Animales , Apoptosis/inmunología , Apoptosis/fisiología , Activación Enzimática/fisiología , Humanos , FN-kappa B/metabolismo , eIF-2 Quinasa/metabolismo

RESUMEN

In the present study we investigated in the reverse passive Arthus reaction elicited in the rat skin the anti-inflammatory effect of double-stranded oligodeoxynucleotides (ODN) with consensus nuclear factor-kappaB (NF-kappaB) sequence as transcription factor decoys (TFD) to inhibit NF-kappaB binding to native DNA sites. Local administration of wild-type-, but not mutant-decoy ODN, dose-dependently reduced both plasma leakage and neutrophil infiltration in rat skin. Molecular analysis performed on soft tissue obtained from rat skin demonstrated: (1) an inhibition of NF-kappaB/DNA binding activity; (2) a decreased nuclear level of p50 and p65 NF-kappaB subunits; (3) an inhibition of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) protein expression, two inflammatory enzymes transcriptionally controlled by NF-kappaB. Furthermore, SN-50, a cell-permeable peptide capable of inhibiting the nuclear translocation of NF-kappaB complexes, as well as ammonium pyrrolidine dithiocarbamate, an inhibitor of NF-kappaB activation, exhibited a similar profile of activity of decoy ODN. Our results indicate that decoy ODN, acting as an in vivo competitor for the transcription factor's ability to bind to cognate recognition sequence, may represent a novel strategy to modulate immune reactions.

Asunto(s)

Antioxidantes/farmacología , Reacción de Arthus/metabolismo , FN-kappa B/metabolismo , Piel/efectos de los fármacos , Tiocarbamatos/farmacología , Factores de Transcripción/farmacología , Análisis de Varianza , Animales , Ciclooxigenasa 2 , ADN/metabolismo , Interacciones Farmacológicas , Isoenzimas/antagonistas & inhibidores , Masculino , Prostaglandina-Endoperóxido Sintasas , Pirrolidinas/farmacología , Ratas , Ratas Wistar , Piel/metabolismo

RESUMEN

In this study we have investigated the relationship between neointima formation and NF-kappaB activation in a model of endothelial denudation of rat carotid artery (balloon angioplasty) using the antioxidant pyrrolidine dithiocarbamate as inhibitor of NF-kappaB activation. Furthermore, we have correlated NF-kappaB activation to the expression of inducible isoforms of both nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) in injured carotids. In control group a significant proliferation of neointima was observed 14 days after balloon angioplasty, which was correlated to an increase of NF-kappaB/DNA binding activity as well as p50/p65 nuclear levels compared to those observed in the carotids from naive or sham-operated rats. Furthermore, NF-kappaB activation was correlated to increased iNOS and COX-2, but not beta-actin, protein expression. Treatment of rats for 14 days with the antioxidant agent pyrrolidine dithiocarbamate (50, 100, 200 mg/kg per os and day) caused a significant inhibition of all the parameters assayed, except beta-actin protein expression. These results indicate that prevention of NF-kappaB activation may lead to the inhibition of neointima formation and suggest that antioxidant agents may have therapeutic relevance for the prevention of human restenosis.

Asunto(s)

Vasos Sanguíneos/lesiones , FN-kappa B/fisiología , Angioplastia de Balón , Animales , Antioxidantes/farmacología , Vasos Sanguíneos/efectos de los fármacos , Western Blotting , Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/fisiología , Ciclooxigenasa 2 , Densitometría , Ensayo de Cambio de Movilidad Electroforética , Endotelio Vascular/efectos de los fármacos , Isoenzimas/biosíntesis , Masculino , FN-kappa B/antagonistas & inhibidores , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo II , Pruebas de Precipitina , Antígeno Nuclear de Célula en Proliferación/metabolismo , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Pirrolidinas/farmacología , Ratas , Ratas Wistar , Tiocarbamatos/farmacología

RESUMEN

Stimulation of J774 macrophages with lipopolysaccharide (LPS) leads to the release of large amounts of prostaglandins (PGs) generated by the inducible isoform of cyclooxygenase (COX-2). Nitric oxide (NO), a pleiotropic free radical, has been demonstrated to modulate the release of a broad range of inflammatory mediators, amongst these PGs. In the present study we investigated the molecular mechanism by which NO affects cyclooxygenase pathway. Incubation of J774 cells with LPS caused an increase of prostaglandin E2 production and COX-2 protein expression which was prevented in a concentration-dependent fashion by pre-incubating cells with sodium nitroprusside (SNP) and S-nitroso-glutathione (GSNO), two NO-generating agents. Electrophoretic mobility shift assay indicated that both NO-generating agents blocked LPS-induced activation of nuclear factor-kappaB (NF-kappaB) by increasing IkappaB-alpha protein expression and blocking nuclear translocation of NF-kappaB subunits p50 and p65. SNP and GSNO also inhibited nuclear factor-interleukin-6 (NF-IL6) activation. These results show for the first time that SNP and GSNO down-regulate LPS-induced COX-2 expression by inhibiting NF-kappaB and NF-IL6 activation and suggest a negative feed-back mechanism that may be important for limiting excessive or prolonged PGs production in pathological events.

Asunto(s)

Proteína beta Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Inhibidores de la Ciclooxigenasa/farmacología , FN-kappa B/antagonistas & inhibidores , Óxido Nítrico/fisiología , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Animales , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Línea Celular , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Dinoprostona/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Inducción Enzimática/efectos de los fármacos , Inducción Enzimática/fisiología , Isoenzimas/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Macrófagos/metabolismo , Ratones , FN-kappa B/metabolismo , Donantes de Óxido Nítrico/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo

RESUMEN

We investigated the role of constitutive transcription factor nuclear factor kappaB (NF-kappaB) in nitric oxide (NO)-mediated apoptosis in J774 macrophages. Our results show that NF-kappaB is present in untreated J774 cells in a form constitutively active. Incubation of cells with sodium nitroprusside (SNP) and S-nitroso-glutathione (GSNO), two NO-generating compounds, caused: (a) inhibition of constitutive NF-kappaB/DNA binding activity; (b) decrease of cell viability; (c) DNA fragmentation; (d) ApopTag positivity. Pyrrolidine dithiocarbamate (PDTC) and N-alpha-para-tosyl-L-lysine chloromethyl ketone (TLCK), two inhibitors of NF-kappaB activation, showed the same effects of both NO-generating compounds. Furthermore, SNP and GSNO as well as PDTC and TLCK significantly increased the cytoplasmic level of IkappaBalpha. All together these results demonstrate that constitutive NF-kappaB protects J774 macrophages from NO-induced apoptosis. Moreover, these findings show, for the first time, that NO-generating compounds may induce apoptosis in J774 macrophages by down-regulating constitutive NF-kappaB/DNA binding activity and suggest a novel mechanism by which NO induces apoptosis.

Asunto(s)

Apoptosis/fisiología , Supervivencia Celular/fisiología , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas I-kappa B , Macrófagos/fisiología , FN-kappa B/fisiología , Óxido Nítrico/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Animales , Línea Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Fragmentación del ADN/fisiología , Proteínas de Unión al ADN/efectos de los fármacos , Ratones , Inhibidor NF-kappaB alfa , Nitroprusiato/farmacología

RESUMEN

Transcription of hypoxia-inducible genes is regulated by hypoxia response elements (HREs) located in either the promoter or enhancer regions. Analysis of these elements reveals the presence of one or more binding sites for hypoxia-inducible factor 1 (HIF-1). Hypoxia-inducible genes include vascular endothelial growth factor (VEGF), erythropoietin, and glycolytic enzyme genes. Site-directed mutational analysis of the VEGF gene promoter revealed that an HIF-1 binding site (HBS) and its downstream HIF-1 ancillary sequence (HAS) within the HRE are required as cis-elements for the transcriptional activation of VEGF by either hypoxia or nitric oxide (NO). The core sequences of the HBS and the HAS were determined as TACGTG and CAGGT, respectively. These elements form an imperfect inverted repeat, and the spacing between these motifs is crucial for activity of the promoter. Gel shift assays demonstrate that as yet unknown protein complexes constitutively bind to the HAS regardless of the presence of these stimuli in several cell lines, in contrast with hypoxia- or NO-induced activation of HIF-1 binding to the HBS. A common structure of the HRE, which consists of the HBS and the HAS, is seen among several hypoxia-inducible genes, suggesting the presence of a novel mechanism mediated by the HAS for the regulation of these genes.

Asunto(s)

Hipoxia de la Célula , Proteínas de Unión al ADN/fisiología , Factores de Crecimiento Endotelial/genética , Regulación de la Expresión Génica/fisiología , Linfocinas/genética , Óxido Nítrico/fisiología , Proteínas Nucleares/fisiología , Factores de Transcripción , Secuencia de Bases , Sitios de Unión , Análisis Mutacional de ADN , Cartilla de ADN , Proteínas de Unión al ADN/química , Eritropoyetina/genética , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Proteínas Nucleares/química , Activación Transcripcional , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

The transcription factor nuclear factor-kappaB (NF-kappaB) plays a key role in the expression of several genes involved in the inflammatory process. In the present study we investigated in an acute model of inflammation, the carrageenin-induced hind paw edema, the anti-inflammatory effect of double stranded oligodeoxynucleotides (ODN) with consensus nuclear factor-kappaB (NF-kappaB) sequence as transcription factor decoys (TFD) to inhibit NF-kappaB binding to native DNA sites. Local administration of wild-type, but not mutant-ODN decoy, dose-dependently inhibited edema formation induced by carrageenin in rat paw. Molecular analysis performed on soft tissue obtained from inflamed paw demonstrated: (1) an inhibition of NF-kappaB DNA binding activity; (2) a decreased nuclear level of p50 and p65 NF-kappaB subunits; (3) an inhibition of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) protein expression, two inflammatory enzymes transcriptionally controlled by NF-kappaB. Furthermore, SN-50, a cell-permeable peptide capable of inhibiting the nuclear translocation of NF-kappaB complexes, exhibited a similar profile of activity of ODN decoy. Our results indicate for the first time that ODN decoy, acting as an in vivo competitor for the transcription factor's ability to bind to cognate recognition sequence, may represent a novel strategy to modulate acute inflammation.

Asunto(s)

Antiinflamatorios no Esteroideos/uso terapéutico , Inflamación/prevención & control , FN-kappa B/genética , Oligonucleótidos/uso terapéutico , Enfermedad Aguda , Animales , Unión Competitiva , Carragenina , Ciclooxigenasa 2 , Inflamación/inducido químicamente , Inflamación/metabolismo , Isoenzimas/metabolismo , Masculino , FN-kappa B/metabolismo , Óxido Nítrico Sintasa/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Ratas , Ratas Wistar

RESUMEN

Activation of the transcription factor nuclear factor (NF)-kappaB by proinflammatory stimuli leads to increased expression of genes involved in inflammation. Activation of NF-kappaB requires the activity of an inhibitor of kappaB (IkappaB)-kinase (IKK) complex containing two kinases (IKKalpha and IKKbeta) and the regulatory protein NEMO (NF-kappaB essential modifier). An amino-terminal alpha-helical region of NEMO associated with a carboxyl-terminal segment of IKKalpha and IKKbeta that we term the NEMO-binding domain (NBD). A cell-permeable NBD peptide blocked association of NEMO with the IKK complex and inhibited cytokine-induced NF-kappaB activation and NF-kappaB-dependent gene expression. The peptide also ameliorated inflammatory responses in two experimental mouse models of acute inflammation. The NBD provides a target for the development of drugs that would block proinflammatory activation of the IKK complex without inhibiting basal NF-kappaB activity.

Asunto(s)

FN-kappa B/metabolismo , Péptidos/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Células COS , Células Cultivadas , Selectina E/biosíntesis , Selectina E/genética , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Quinasa I-kappa B , Inflamación/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutación , Péptidos/química , Mutación Puntual , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo

RESUMEN

We investigated the effect of cyclolinteinone, a sesterterpene from Caribbean sponge Cacospongia linteiformis, on inducible NO synthase (iNOS) and cyclo-oxygenase-2 (COX-2) protein expression in lipopolysaccharide (LPS)-stimulated J774 macrophages. Incubation of J774 cells with LPS (1 microgram/ml) caused an increase of both iNOS and COX-2 protein expression, which was prevented in a concentration-dependent fashion by cyclolinteinone (12.5, 25 and 50 microM). Electrophoretic mobility-shift assay indicated that cyclolinteinone blocked the activation of nuclear factor-kappaB (NF-kappaB), a transcription factor necessary for either iNOS or COX-2 induction. Cyclolinteinone also blocked disappearance of I(kappa)B-alpha from cytosolic fraction and nuclear translocation of NF-kappaB subunits p50 and p65. These results show that cyclolinteinone down-regulates iNOS and COX-2 protein expression by inhibiting NF-kappaB activation and suggest that it may represent a novel anti-inflammatory compound capable of controlling the excessive production of prostaglandins and nitric oxide occurring in several inflammatory diseases.

Asunto(s)

4-Butirolactona/análogos & derivados , Ciclohexanonas/farmacología , Inhibidores Enzimáticos/farmacología , Isoenzimas/efectos de los fármacos , Macrófagos/efectos de los fármacos , FN-kappa B/antagonistas & inhibidores , Óxido Nítrico Sintasa/antagonistas & inhibidores , Prostaglandina-Endoperóxido Sintasas/efectos de los fármacos , 4-Butirolactona/farmacología , Animales , Transporte Biológico , Línea Celular , Núcleo Celular/metabolismo , Ciclooxigenasa 2 , Hidrólisis , Isoenzimas/metabolismo , Macrófagos/enzimología , Macrófagos/metabolismo , Ratones , FN-kappa B/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Prostaglandina-Endoperóxido Sintasas/metabolismo

RESUMEN

The effect of four macrolide antibiotics (roxithromycin, clarithromycin, erythromycin, and azithromycin) on the generation of some mediators and cytokines involved in the inflammatory process has been studied both in vivo and in vitro. Rat carrageenin pleurisy was used as a model of acute inflammation, and the macrolides were administered (10, 20, and 40 mg/kg p.o.) 1 h before the carrageenin challenge. Exudate volume and leukocyte accumulation were both dose-dependently reduced by roxithromycin, clarithromycin and erythromycin in either normal or adrenalectomized animals. Furthermore, in normal rats, prostaglandin (PG)E(2), nitrate plus nitrite, and tumor necrosis factor-alpha levels in pleural exudate were significantly reduced by these macrolides. Roxithromycin appeared more effective than erythromycin and clarithromycin, whereas azithromycin only slightly affected the inflammatory reaction. None of the macrolides were able to modify leukotriene B(4) exudate levels. In vitro experiments have shown that the four macrolides (5-80 microM) reduced in a concentration-dependent manner the production of 6-keto-PGF(1alpha), NO(2)(-), tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 by lipopolysaccharide-stimulated J774 macrophages. In J774 cells, the inhibition of 6-keto-PGF(1alpha) and NO(2)(-) production by roxithromycin and erythromycin was not dependent on direct inhibition of cyclooxygenase-2 and inducible nitric oxide synthase activity because it appears to be related to the inhibition of cyclooxygenase-2 and inducible nitric oxide synthase protein expression. In conclusion, the present study shows that macrolide antibiotics have anti-inflammatory activity, which likely depends on their ability to prevent the production of proinflammatory mediators and cytokines, and suggest that these agents, particularly roxithromycin, can exert therapeutic effects independently of their antibacterial activity.

Asunto(s)

Antibacterianos/farmacología , Antiinflamatorios/farmacología , 6-Cetoprostaglandina F1 alfa/biosíntesis , Adrenalectomía , Anestesia , Animales , Azitromicina/farmacología , Carragenina , Línea Celular , Claritromicina/farmacología , Ciclooxigenasa 2 , Citocinas/biosíntesis , Dinoprostona/metabolismo , Relación Dosis-Respuesta a Droga , Eritromicina/farmacología , Mediadores de Inflamación/metabolismo , Isoenzimas/metabolismo , Leucotrieno B4/metabolismo , Macrófagos/metabolismo , Ratones , Nitratos/metabolismo , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Nitritos/metabolismo , Pleuresia/inducido químicamente , Pleuresia/prevención & control , Prostaglandina-Endoperóxido Sintasas/metabolismo , Ratas , Roxitromicina/farmacología , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Nitric oxide (NO) regulates production of vascular endothelial growth factor (VEGF) by normal and transformed cells. We demonstrate that NO donors may up-regulate the activity of the human VEGF promoter in normoxic human glioblastoma and hepatoma cells independent of a cyclic guanosine monophosphate-mediated pathway. Deletion and mutation analysis of the VEGF promoter indicates that the NO-responsive cis-elements are the hypoxia-inducible factor-1 (HIF-1) binding site and an adjacent ancillary sequence that is located immediately downstream within the hypoxia-response element (HRE). This work demonstrates that the HRE of this promoter is the primary target of NO. In addition, VEGF gene regulation by NO, as well as by hypoxia, is potentiated by the AP-1 element of the gene. Our study also reveals that NO and hypoxia induce an increase in HIF-1 binding activity and HIF-1alpha protein levels, both in the nucleus and the whole cell. These results suggest that there are common features of the NO and hypoxic pathways of VEGF induction, while in part, NO mediates gene transcription by a mechanism distinct from hypoxia. This is demonstrated by a difference in sensitivity to guanylate cyclase inhibitors and a different pattern of HIF-1 binding. These results show that there is a primary role for NO in the control of VEGF synthesis and in cell adaptations to hypoxia. (Blood. 2000;95:189-197)

Asunto(s)

Hipoxia de la Célula/fisiología , Proteínas de Unión al ADN/metabolismo , Factores de Crecimiento Endotelial/genética , Regulación de la Expresión Génica , Linfocinas/genética , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico/fisiología , Proteínas Nucleares/metabolismo , Penicilamina/análogos & derivados , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción Genética , Secuencia de Bases , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Cinética , Luciferasas/biosíntesis , Luciferasas/genética , Datos de Secuencia Molecular , Mutagénesis , Penicilamina/farmacología , Proteínas Recombinantes de Fusión/biosíntesis , S-Nitroso-N-Acetilpenicilamina , Eliminación de Secuencia , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular