RESUMEN
TNF is a major mediator of inflammation, immunity, and apoptosis. Pre-exposure to TNF reduces sensitivity to restimulation, a phenomenon known as tolerance, considered as protective in sepsis, but also as a paradigm for immunoparalysis. Earlier experiments in TNF-tolerant cells display inhibition of NF-kappaB-dependent IL-8 gene expression at the transcriptional level with potential involvement of C/EBPbeta. In this study, we have shown that a kappaB motive was sufficient to mediate transcriptional inhibition under TNF tolerance conditions in monocytic cells. Furthermore, in tolerant cells, TNF-induced NF-kappaB p65 phosphorylation was markedly decreased, which was accompanied by the formation of C/EBPbeta-p65 complexes. Remarkably, in C/EBPbeta(-/-) cells incubated under the conditions of TNF tolerance, neither impairment of transcription nor inhibition of p65 phosphorylation was observed. Finally, we showed that C/EBPbeta overexpression reduced p65-mediated transactivation and that association of C/EBPbeta with p65 specifically prevented p65 phosphorylation. Our data demonstrate that C/EBPbeta is an essential signaling component for inhibition of NF-kappaB-mediated transcription in TNF-tolerant cells and suggest that this is caused by blockade of p65 phosphorylation. These results define a new molecular mechanism responsible for TNF tolerance in monocytic cells that may contribute to the unresponsiveness seen in patients with sepsis.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/fisiología , Tolerancia Inmunológica , FN-kappa B/antagonistas & inhibidores , Factor de Transcripción ReIA/antagonistas & inhibidores , Transcripción Genética/inmunología , Factor de Necrosis Tumoral alfa/fisiología , Proteína beta Potenciadora de Unión a CCAAT/deficiencia , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Línea Celular Tumoral , Silenciador del Gen , Células HeLa , Humanos , Tolerancia Inmunológica/genética , Interleucina-8/antagonistas & inhibidores , Interleucina-8/genética , Complejos Multiproteicos/metabolismo , FN-kappa B/fisiología , Fosforilación , Regiones Promotoras Genéticas , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Transactivadores/antagonistas & inhibidores , Transactivadores/metabolismo , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
There is some evidence that the potent cytokine tumor necrosis factor (TNF) is able to induce tolerance after repeated stimulation of cells. To investigate the molecular mechanisms mediating this phenomenon, the expression of interleukin-8 (IL-8), which is regulated by transcription factors NF-kappaB and C/EBPbeta, was monitored under TNF tolerance conditions. Pretreatment of monocytic cells for 72 h with low TNF doses inhibited TNF-induced (restimulation with a high dose) IL-8 promoter-dependent transcription as well as IL-8 production. Under these conditions neither activation of NF-kappaB nor IkappaB proteolysis was affected after TNF re-stimulation, albeit a slightly reduced IkappaB-alpha level was found in the TNF pretreated but not re-stimulated sample. Remarkably, in tolerant cells an increased binding of C/EBPbeta to its IL-8 promoter-specific DNA motif as well as an elevated association of C/EBPbeta protein with p65-containing NF-kappaB complexes was observed. Finally, overexpression of C/EBPbeta, but not p65 or Oct-1, markedly prevented TNF-induced IL-8 promoter-dependent transcription. Taken together, these data indicate that the expression of IL-8 is inhibited at the transcriptional level in TNF-tolerant cells and C/EBPbeta is involved under these conditions in mediating the negative-regulatory effects, a mechanism that may play a role in inflammatory processes such as sepsis.
Asunto(s)
Interleucina-8/biosíntesis , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Potenciadoras de Unión a CCAAT/fisiología , Línea Celular , Proteínas de Unión al ADN/fisiología , Tolerancia a Medicamentos/genética , Factor C1 de la Célula Huésped , Humanos , Interleucina-8/genética , Monocitos/efectos de los fármacos , Monocitos/metabolismo , FN-kappa B/fisiología , Factor 1 de Transcripción de Unión a Octámeros , Regiones Promotoras Genéticas/efectos de los fármacos , Factores de Transcripción/fisiologíaRESUMEN
BACKGROUND: The balance between proteinases and antiproteinases plays an important role in tissue destruction and remodelling. In chronic obstructive pulmonary disease (COPD) and emphysema, an imbalance between matrix metalloproteinases (MMPs) and inhibitors of tissue metalloproteinase (TIMPs) has been reported. Alveolar macrophages are considered to be the main source of MMPs. We therefore have analyzed the effects of free and liposomal all trans-retinoic acid (ATRA) on the expression of MMP-9 and TIMP-1 in bronchoalveolar lavage (BAL) cells from patients with COPD and patients with other lung diseases. MATERIAL AND METHODS: BAL cells were incubated 1-3 day with either liposomal or free ATRA. Supernatants were tested for MMP-9 and TIMP-1 protein in specific ELISA systems; mRNA analysis was performed by semi-quantitative RT-PCR and by quantitative LightCycler PCR. RESULTS: We demonstrate that either liposomal or free ATRA selectively down-regulates MMP-9 and up-regulates TIMP-1. At the protein level, MMP-9 is decreased 3-fold and TIMP-1 is increased 3.5-fold compared to the base line with empty liposomes or untreated cells. The ratio of MMP-9 and its inhibitor TIMP-1, which may be crucial to the overall proteolytic potential decreased by factor 8. That this countercurrent effect of ATRA is not due to an altered protein stability but to transcriptional regulation could be demonstrated by RT-PCR. Quantitative LightCycler analysis revealed a 2.5-fold decrease of MMP-9 mRNA and a 4.5 fold increase of TIMP- 1 mRNA. CONCLUSIONS: These data suggest that ATRA treatment via its impact on the proteinase/antiproteinase ratio may become a new therapeutic strategy for patients with inflammatory destructive lung diseases.
Asunto(s)
Regulación hacia Abajo , Metaloproteinasa 9 de la Matriz/biosíntesis , Inhibidor Tisular de Metaloproteinasa-1/biosíntesis , Tretinoina/farmacología , Regulación hacia Arriba , Antineoplásicos/farmacología , Líquido del Lavado Bronquioalveolar/citología , Relación Dosis-Respuesta a Droga , Humanos , Liposomas/metabolismo , Fosfatidilserinas/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
Strenuous, anaerobic exercise leads to an increase of leukocytes that are mobilized from the marginal pool. We have analyzed in human peripheral blood the effect of exercise on the number of CD14(+)CD16(+) monocytes as determined by two-color immunofluorescence and flow cytometry. We show herein that this type of monocyte responds with a dramatic up to 4.8-fold increase. Mobilization does not occur after 1 min at 100 or 200 W but 1 min at 400 W leads to a twofold increase of the CD14(+)CD16(+) monocytes immediately after exercise. The numbers remain high at 5 min and gradually decrease to reach the initial level at 20 min postexercise. After 20 min of rest, the CD14(+)CD16(+) monocytes can be mobilized again by a second exercise. The CD14(+)CD16(+) monocytes appear to be mobilized from the marginal pool where they preferentially home because of a higher expression of adhesion molecules like CD11d and very late antigen-4. Exercise goes along with an increase of catecholamines, and mobilization of the CD14(+)CD16(+) monocytes can be substantially reduced by treatment of donors with the beta-adrenergic receptor blocker propranolol. Mobilization of CD14(+)CD16(+) monocytes by a catecholamine-dependent mechanism may contribute to the increase of these cells in various clinical conditions.
Asunto(s)
Ejercicio Físico/fisiología , Receptores de Lipopolisacáridos/metabolismo , Monocitos/fisiología , Receptores de IgG/metabolismo , Administración Tópica , Antiinflamatorios/farmacología , Ciclismo , Moléculas de Adhesión Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Epinefrina/fisiología , Glucocorticoides , Humanos , Masculino , Metilprednisolona/farmacología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Norepinefrina/fisiología , Carrera , Factores de TiempoRESUMEN
IL-10 is a unique cytokine because it is anti-inflammatory and immunosuppressive. IL-10 is regulated at the level of transcription, but the critical motifs and the relevant transcription factors controlling this gene have remained elusive to date. We now report that a sequence at -120 bp in the human IL-10 promoter binds Stat3 but no other Stat proteins. Mutation of this motif abrogates LPS-induced trans-activation. Overexpression of dominant negative Stat3 suppresses promoter activity, while wild-type Stat3 leads to an enhancement of this activity. Our results show that Stat3, by binding to a single motif in the IL-10 promoter, is controlling expression of the human IL-10 gene.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica/inmunología , Interleucina-10/biosíntesis , Interleucina-10/genética , Lipopolisacáridos/farmacología , Transactivadores/fisiología , Línea Celular , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Vectores Genéticos/síntesis química , Humanos , Interleucina-10/química , Interleucina-10/fisiología , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas/inmunología , Factor de Transcripción STAT3 , Eliminación de Secuencia , Transducción de Señal/genética , Transducción de Señal/inmunología , Relación Estructura-Actividad , Transactivadores/biosíntesis , Transactivadores/genética , Transcripción Genética/inmunología , Activación Transcripcional/inmunología , Transfección/inmunologíaRESUMEN
At the Definition of Human Blood Monocytes conference held in Munich, Germany in October 1999, experts met to discuss data on the characterization of monocytes in health and disease. Emphasis was on how to best define these cells, on which subpopulations can be identified, and on how the monocytes can best be distinguished from other cells such as natural killer cells, granulocytes, and dendritic cell precursors.
Asunto(s)
Leucocitos/fisiología , Macrófagos/fisiología , Monocitos/fisiología , Antígenos CD/análisis , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Humanos , Leucocitos/clasificación , Leucocitos/citología , Macrófagos/clasificación , Monocitos/clasificaciónRESUMEN
The subset of human blood monocytes expressing low levels of CD14 and high levels of CD16 (CD14+CD16+) exhibits features resembling mature tissue macrophages and can be expanded in inflammatory conditions. We analyzed expression of CC chemokine receptors (CCR) in CD14+CD16+ versus CD14++ monocytes, which may be crucial for specific trafficking. Multicolor flow cytometric analysis of whole peripheral blood revealed that, as opposed to CD14++ monocytes, the CD14+CD16+ subset lacked surface expression of monocyte chemotactic protein-1 (MCP-1) receptor CCR2, however, it showed significantly higher surface expression of the macrophage inflammatory protein 1alpha (MIP-1alpha)/RANTES receptor CCR5. This was paralleled by differences in mRNA expression in the subsets, as shown by reverse transcriptase-polymerase chain reaction using sorted cells. In comparison to CD14++ monocytes, CD14+CD16+ cells expressed lower CCR2 but higher CCR5 transcript levels, whereas CCR1 levels were equivalent. Flow cytometric analysis of isolated human monocytes recovered after transendothelial chemotaxis assays revealed that the percentage of CD14+CD16+ cells was dramatically reduced in the fraction migrating toward MCP-1 compared with the fraction that did not migrate or the input, showing that polarized CCR2 expression was accompanied by a differential chemotactic responsiveness. Moreover, CD11b surface expression was preferentially up-regulated by MCP-1 in CD14++ cells but by MIP-1alpha in CD14+CD16+ monocytes, confirming the functional relevance of distinct CCR expression. The characteristics of CD14+CD16+ cells may reflect preactivation by cytokines and determine their predilective localization during specific inflammatory conditions or susceptibility to infection.
Asunto(s)
Monocitos/clasificación , Monocitos/inmunología , Receptores de Quimiocina/sangre , Receptores de Quimiocina/genética , Antígenos CD/sangre , Quimiotaxis de Leucocito , Regulación de la Expresión Génica/inmunología , Humanos , Receptores de Lipopolisacáridos/sangre , Antígeno de Macrófago-1/sangre , ARN Mensajero/genética , Receptores CCR2 , Receptores CCR5/sangre , Receptores CCR5/genética , Receptores de IgG/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción GenéticaRESUMEN
Cells of the weakly CD14 positive human B cell line RPMI 8226, clone 1, will mobilize NF-kappaB (p50/p65 and p50/p50) proteins and produce TNF mRNA when stimulated with lipopolysaccharide (LPS). When such cells are precultured with a low amount of LPS (50-250 ng/ml) for 3 - 4 days followed by a secondary stimulation with a high dose of LPS (1 microg/ml) then the cytokine expression is strongly reduced, i. e. the cells have become tolerant. Western blot analysis of proteins of the NF-kappaB/rel family demonstrates cytoplasmic p50 and p65 for naive B cells plus a low level of p52. While with tolerance induction the pattern of p50 and p65 proteins remains essentially unchanged, the LPS tolerant 8226 cells show a dramatic increase of both p52 protein and its p100 precursor in the cytosol. This p52 is found strongly upregulated in Western blots of extracts from purified nuclei of tolerant cells. Also, gelshift analysis with the -605 kappaB motif of the human TNF 5'-region shows an additional high mobility complex in LPS tolerant cells -a complex that is supershifted with an anti-p52 antibody. Functional analysis with the -1064 TNF 5'-region in front of the luciferase reporter gene demonstrates that transactivation of the TNF promoter is strongly reduced in tolerant cells. Also, overexpression of p52 will suppress activity of TNF promoter reporter gene constructs. Taken together these data show that tolerance to LPS in the human RPMI 8226 B cell line involves upregulation of the p52 (NF-kappaB2) gene, which appears to be instrumental in the blockade of TNF gene expression.
Asunto(s)
Lipopolisacáridos/farmacología , FN-kappa B/genética , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/genética , Linfocitos B , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Receptores de Lipopolisacáridos/análisis , Subunidad p50 de NF-kappa B , ARN Mensajero/genética , Transcripción Genética/efectos de los fármacosRESUMEN
The CD14(+)/CD16(+) subset of human blood monocytes, which expresses low levels of the lipopolysaccharide receptor CD14 and high levels of the Fc receptor CD16 and exhibits features of mature tissue macrophages, is expanded in certain inflammatory conditions and may be relevant in atherosclerosis. Scavenger receptors (ScR) are important for lipid accumulation into macrophage-derived foam cells in atherogenesis and for the clearance of pathogens. Hence, we compared the function and expression of ScR in CD33(low) CD16(+) and CD33(high) CD14(++) monocyte subsets. Double immunofluorescence analysis of isolated monocytes revealed that the CD33(low) subset showed lower specific, ScR-mediated binding of DiI-labeled modified low-density lipoproteins (LDL) than CD33(high) cells. Differences in modified LDL binding between subsets were accompanied by changes in mRNA expression. RT-PCR in sorted cells indicated lower ScR class A type I/II (ScR-AI/II) mRNA levels in CD14(+)/CD16(+) than in CD14(++) cells, whereas CD36 transcripts were unaltered. This was paralleled by findings in mostly CD16(+) monocyte-derived macrophages showing a marked reduction in ScR-mediated binding of acetylated LDL, but not in the binding of oxidized LDL, and lower expression of ScR-AI/II mRNA, but not CD36 transcripts, after exposure to tumor necrosis factor-alpha for 48 h in vitro. Thus the subset of CD14(+)/CD16(+) monocytes shows distinct ScR function and expression, possibly reflecting a preactivation by cytokines with a predilection for specific inflammatory or vascular conditions, e.g., atherogenesis.
Asunto(s)
Receptores de Lipopolisacáridos/análisis , Proteínas de la Membrana , Monocitos/inmunología , Monocitos/metabolismo , Receptores de IgG/análisis , Receptores Inmunológicos/metabolismo , Receptores de Lipoproteína , Animales , Antígenos CD36 , Línea Celular , Humanos , Lipoproteínas LDL/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Monocitos/citología , Monocitos/efectos de los fármacos , ARN Mensajero/metabolismo , Receptores Inmunológicos/genética , Receptores Depuradores , Receptores Depuradores de Clase A , Receptores Depuradores de Clase B , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Extensively oxidized low density lipoprotein (ox-LDL), a modulator of atherogenesis, down-regulates the lipopolysaccharide (LPS)-induced activation of transcription factor NF-kappaB. We investigated whether 4-hydroxynonenal (HNE), a prominent aldehyde component of ox-LDL, represents one of the inhibitory substances. NF-kappaB activation by stimuli such as LPS, interleukin (IL)-1beta, and phorbol ester, but not tumor necrosis factor (TNF), was reversibly inhibited by HNE in a dose-dependent manner in human monocytic cells, whereas AP-1 binding was unaffected. Using similar HNE concentrations, LPS-induced kappaB- and TNF or IL-8 promoter-dependent transcription was prevented. Furthermore, pretreatment with HNE suppressed TNF production but not lactate dehydrogenase levels. Under these conditions the binding of LPS to monocytic cells was not significantly affected. However, induced proteolysis of the inhibitory proteins IkappaB-alpha, IkappaB-beta, and, at a later time point, IkappaB-epsilon was prevented. This is not due to inhibition of the proteasome, the major proteolytic activities of which remain unaffected, but rather to a specific prevention of the activation-dependent phosphorylation of IkappaB-alpha. This is the first report which demonstrates that HNE specifically inhibits the NF-kappaB/Rel system. Down-modulation of NF-kappaB-regulated gene expression may contribute at certain stages of atherosclerosis to low levels of chronic inflammation and may also be involved in other inflammatory/degenerative diseases.
Asunto(s)
Aldehídos/farmacología , Proteínas de Unión al ADN/antagonistas & inhibidores , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Línea Celular , Cisteína Endopeptidasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Humanos , Hidrólisis , Quinasa I-kappa B , Proteínas I-kappa B , Elastasa de Leucocito/metabolismo , Complejos Multienzimáticos/metabolismo , Ácido Ocadaico/farmacología , Fosforilación , Complejo de la Endopetidasa Proteasomal , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Monocytes respond to lipopolysaccharide (LPS) stimulation with a rapid expression of the tumor necrosis factor (TNF) gene. Upon repeated LPS stimulation there is, however, little production of TNF mRNA and protein; i.e., the cells are tolerant to LPS. Analysis of NF-kappaB proteins in gel shift assays demonstrated that the DNA binding activity that is induced by LPS stimulation in tolerant cells consists mainly of p50-p50 homodimers. Since p50 can bind to DNA but lacks a transactivation domain, this may explain the blockade of TNF gene expression. We now show that in the monocytic cell line Mono Mac 6, this inability to respond can be largely ascribed to NF-kappaB, since a reporter construct directed by a trimeric NF-kappaB motif is strongly transactivated by LPS stimulation of naive cells whereas LPS-tolerant cells exhibit only low activity. Also, Western blot analyses of proteins extracted from purified nuclei showed mobilization of threefold-higher levels of p50 protein in tolerant compared to naive cells, while mobilization of p65 was unaltered. Overexpression of p50 in HEK 293 cells resulted in a strong reduction of p65-driven TNF promoter activity at the levels of both luciferase mRNA and protein. These data support the concept that an upregulation of p50 is instrumental in LPS tolerance in human monocytes.
Asunto(s)
Tolerancia Inmunológica , Lipopolisacáridos/inmunología , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/genética , Línea Celular , Línea Celular Transformada , Núcleo Celular/metabolismo , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , FN-kappa B/genética , Subunidad p50 de NF-kappa B , Regiones Promotoras Genéticas , Factor de Transcripción ReIA , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/inmunología , Regulación hacia ArribaRESUMEN
Glucocorticoids (GC) are potent anti-inflammatory and immunosuppressive agents that act on many cells of the body, including monocytes. Here we show that a 5-day course of high dose GC therapy differentially affected the CD14++ and the CD14+ CD16+ monocyte subpopulations in 10 patients treated for multiple sclerosis. While the classical (CD14++) monocytes exhibited a substantial increase from 495 +/- 132 to 755 +/- 337 cells/microl, the CD14+ CD16+ monocytes responded with a pronounced decrease from 36 +/- 15 to 2 +/- 3 cells/microl (P < 0.001). In 4/10 patients the CD14+ CD16+ monocytes fell below detection limits (<0.2 cells/microl). This observation was confirmed when the CD14+ CD16+ monocytes were identified by virtue of their low CD33 expression as these cells decreased as well. After discontinuation of GC therapy the CD14+ CD16+ monocytes reappeared and reached normal levels after 1 week. The profound depletion of CD14+ CD16+ monocytes by GC as described here is a novel effect of GC action in vivo and may contribute to GC-mediated immunosuppression. Determination of the number of this monocyte subset may also serve to monitor the effectiveness of GC therapy in patients requiring immunosuppressive treatment.
Asunto(s)
Glucocorticoides/administración & dosificación , Terapia de Inmunosupresión , Inmunosupresores/administración & dosificación , Receptores de Lipopolisacáridos/inmunología , Monocitos/inmunología , Esclerosis Múltiple/sangre , Esclerosis Múltiple/tratamiento farmacológico , Receptores de IgG/inmunología , Adulto , Femenino , Citometría de Flujo , Humanos , Inyecciones Intravenosas , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/inmunologíaRESUMEN
OBJECTIVES: Major operative trauma like aorta-coronary bypass operation may lead to postoperative immunodisturbance, putting the patient at an increased risk for infection and sepsis. The monocyte/macrophage system and the endotoxin receptor CD14 are important in the early recognition and elimination of invading bacteria. The aim of this study was to analyze changes in membrane-associated CD14 and soluble CD14 during and after cardiac involving cardiopulmonary bypass. METHODS: We studied numbers of leukocytes, monocytes, and monocyte subpopulations, expression of monocyte membrane-associated CD14 and plasma levels of soluble CD14 in 10 patients (63 +/- 8 years of age), who underwent elective cardiopulmonary bypass. RESULTS: Cardiopulmonary bypass induced marked postoperative monocytosis, which was maximal 20 hours after the operation (485 +/- 242 cells/microl before, 1080 +/- 264 cells/microl 20 hours after surgery). Expression of membrane-associated CD14 on classical CD14++ monocytes decreased significantly by 40%, reaching a nadir 20 hours after surgery (p < 0.05). At the time of maximal membrane-associated CD14 suppression, the levels of soluble CD14 measured by enzyme-linked immunosorbent assay were clearly increased (3.2 +/- 1.0 microg/ml before versus 5.6 +/- 1.0 microg/ml 20 hours after, p < 0.001). No significant change of the percentage of small (alpha) and large (beta) forms of soluble CD14 was found. CONCLUSIONS: Cardiopulmonary bypass leads to reduced membrane-associated CD14 expression on peripheral blood monocytes and increased levels of soluble CD14 through shedding or secretion of membrane-associated CD14 from the cell surface. These findings indicate that bypass is associated with significant monocyte activation.
Asunto(s)
Puente Cardiopulmonar , Puente de Arteria Coronaria/métodos , Enfermedad Coronaria/cirugía , Regulación hacia Abajo/fisiología , Receptores de Lipopolisacáridos/metabolismo , Monocitos/metabolismo , Western Blotting , Enfermedad Coronaria/sangre , Enfermedad Coronaria/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Estudios de Seguimiento , Humanos , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Monocitos/citología , Monocitos/inmunología , Receptores de IgG/metabolismoRESUMEN
We investigated the effect of proteasome inhibitors on the lipopolysaccharide (LPS)-induced expression of several monocytic cytokines, which may be dependent on the transcription factor, nuclear factor-kappaB (NF-kappaB). Exposure of human monocytic THP-1 cells to ALLN and Mu873 prevented the LPS-induced degradation of IkappaB-alpha and -beta, as did the more potent proteasome inhibitor, PSI, whereas several calpain inhibitors were ineffective. This was accompanied by the inhibition of nuclear NF-kappaB binding activity and NF-kappaB transcriptional activation. At the mRNA level, the inhibitors blocked the expression of tumor necrosis factor (TNF) and interleukin-1beta (IL-1beta), whereas IL-8 remained unaffected by ALLN and was only partially reduced by the highest dose of PSI. The latter effect appears to be due to an increase in IL-8 mRNA stability in the presence of proteasome inhibitors. Furthermore, the production of TNF was efficiently suppressed by ALLN and PSI, less by Mu873, and not at all by calpain inhibitors. In primary human blood monocytes ALLN also prevented the LPS-induced degradation of IkappaB-alpha and -beta, efficiently blocked the production of TNF and, to a lesser extent, IL-1beta, whereas that of IL-8 was not inhibited. The expression of NF-kappaB-dependent monocytic cytokines may be selectively controlled by the proteasome, offering a potential therapeutic target in inflammatory disease.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Citocinas/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Proteínas I-kappa B , Monocitos/fisiología , Complejos Multienzimáticos/metabolismo , FN-kappa B/biosíntesis , Línea Celular , Núcleo Celular/fisiología , Dactinomicina/farmacología , Humanos , Interleucina-1/biosíntesis , Interleucina-8/biosíntesis , Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Inhibidor NF-kappaB alfa , Complejo de la Endopetidasa Proteasomal , ARN Mensajero/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Transcripción Genética/efectos de los fármacos , Transcripción Genética/fisiología , Transfección , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
BACKGROUND: Functional heterogeneity in the tumor necrosis factor alpha (TNF-alpha) gene may be responsible for the TNF-alpha response in infectious and autoimmune diseases. Recently, the TNF-238 promoter polymorphism was observed as being associated with a more destructive disease in rheumatoid arthritis (RA). To determine the relation between TNF-238 and disease progression, the extent of joint destruction in a cohort of 101 RA patients followed for 12 years was analyzed. Furthermore, we have attempted to link this polymorphism to TNF-alpha gene transcription in monocytes and lymphocytes in vitro. PATIENTS, MATERIALS, AND METHODS: The extent of joint destruction determined on X-rays of hands and feet assessed after 0, 3, 6, and 12 years was compared with TNF-238 genotypes. Functional consequences of TNF-alpha gene polymorphisms using reporter gene constructs were analyzed in cells of the monocyte and lymphocyte lineage by means of transient transfection systems. RESULTS: The rate of joint damage in -238GA patients was lower than that in the -238GG patients, independent of HLA-DR4. Damage after 12 years was 76 +/- 30 for the -238GA versus 126 +/- 13 for the -238GG patients as determined by the van der Heijde's modification of Sharp's method. Furthermore, TNF-238A was found to be in linkage disequilibrium with an additional polymorphism at position -376. Functional assays revealed no significant differences in the level of inducible reporter gene expression between the TNF-238/-376 promoter constructs in the cell types tested. CONCLUSION: In a prospective study, we show that the TNF-238GG genotype contributes to progression of joint destruction in RA, independent of the presence of HLA-DR4. However, in vitro transfection assays indicate that TNF-238A by itself or in combination with TNF-376A is not likely to be of direct functional relevance for transcriptional activation. Therefore, these polymorphisms may serve as markers for additional polymorphisms in the TNF/LT locus or neighboring genes that may influence disease severity.
Asunto(s)
Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Polimorfismo Genético , Regiones Promotoras Genéticas , Factor de Necrosis Tumoral alfa/genética , Adulto , Alelos , Artritis Reumatoide/diagnóstico por imagen , Estudios de Casos y Controles , Línea Celular , Estudios de Cohortes , Frecuencia de los Genes , Genotipo , Humanos , Desequilibrio de Ligamiento , Linfocitos/inmunología , Persona de Mediana Edad , Monocitos/inmunología , Estudios Prospectivos , Radiografía , Transcripción GenéticaRESUMEN
We describe a patient with self-induced disease who presented with repeated urinary tract infection and sepsis due to intravesical and intravenous injection of feces. Sepsis occurred repeatedly such that the patient exhibited 10 bouts of fever > 40 degrees C in a single month. This bacterial challenge led to massive activation of the monocyte system with high levels of TNF-alpha, IL-6, and monocyte colony-stimulating factor (M-CSF). This cytokine response was followed by strong expansion of the novel CD14+CD16+ monocyte subset. These results suggest that cytokines induce the development of CD14+CD16+ cells in human septicemia and that CD14+CD16+ cells may serve as indicator for previous bouts of excessive inflammation.
Asunto(s)
Citocinas/biosíntesis , Receptores de Lipopolisacáridos/sangre , Monocitos/inmunología , Receptores de IgG/sangre , Sepsis/inmunología , Adulto , Regulación de la Temperatura Corporal/fisiología , Femenino , Humanos , Interleucina-6/sangre , Monocitos/metabolismo , Fenotipo , Sepsis/sangre , Sepsis/etiología , Factores de TiempoRESUMEN
The vitamin D3 derived hormone 1.25 (OH)2 vitamin D3 (1,25 D3) is able to induce growth arrest and differentiation in myelomonocytic leukaemia cells. In order to allow for specific delivery to leukaemic cells the lipophilic compound was incorporated into the lipid membranes of liposomes. Liposomal 1.25 D3 reduced proliferation as measured by 3H-thymidine incorporation in HL60 leukaemia cells by up to 60%. When liposomes were prepared at different concentrations of 1,25 D3 65% inhibition was achieved at 48 nM. The MC 1288 stereoisomer of 1,25 D3 was more potent and had the same activity at 4.8 nM. The effect of the liposomal compounds was specific to myeloid cells as they reduced proliferation in myelomonocytic HL60, monoblastic U937 and monocytic Mono Mac 6 cells but not in the T-cell lines Jurkat and Molt 4. The antiproliferative effect of liposomal 1,25 D3 was associated with an induction of differentiation since treated HL60 cells showed a monocytic morphology, increased expression of CD14 and decreased expression of CD33. When peripheral blood leukaemic cells from M4 and M5 acute myeloid leukaemia (AML) patients were admixed with liposomal compounds an antiproliferative effect was seen in all five cases, including the two cases where free compounds led to enhanced growth. Liposomal delivery of 1,25 (OH)2 vitamin D3 may offer a novel approach to treatment of myelomonocytic leukaemia.
Asunto(s)
Leucemia Monocítica Aguda/patología , Leucemia Mielomonocítica Aguda/patología , Esteroide Hidroxilasas/farmacología , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Colestanotriol 26-Monooxigenasa , Relación Dosis-Respuesta a Droga , Células HL-60/patología , Humanos , Liposomas , Esteroide Hidroxilasas/administración & dosificaciónRESUMEN
Steroid hormones including sex hormones are known to influence cytokine production by cells in vitro. We investigated whether there are differences in cytokine production in vivo and ex vivo during the menstrual cycle in five ovulating women compared with five pregnant women and nine males. Interleukin 6 (IL-6) in plasma changed periodically during 12 of 13 cycles in five women. The IL-6 levels were lowest in the luteal phase when progesterone levels were elevated and highest preovulatory when progesterone levels were low (P < 0.009). This phenomenon was unrelated to changes in haematocrit or albumin and independent of cortisone, growth hormone, luteinizing or follicle stimulating hormone and testosterone. In contrast to IL-6, the soluble IL-6 receptor did not vary significantly during the menstrual cycle. In comparison, nine males and five pregnant women had low plasma IL-6 levels comparable with women during the luteal phase. In addition, levels of IL-6, IL-10 and TNF were determined after whole blood stimulation with lipopolysaccharide ex vivo during a menstrual cycle. Neither the number of CD-14++ or CD14/CD16+ cells nor the amounts of IL-6, IL-10 and TNF after stimulation showed cyclic changes. We suggest that sex hormones, especially oestrogen and progesterone, may influence immune responses by decreasing basal IL-6 levels in vivo.
Asunto(s)
Interleucina-6/sangre , Ciclo Menstrual/sangre , Femenino , Humanos , EmbarazoRESUMEN
Individuals with a consistently lower immune response may be more susceptible to infection but less prone to autoimmune disease or severe sepsis. The molecular mechanisms determining the low responder status are, however, unclear. We have screened 60 male donors for tumour necrosis factor (TNF) protein levels after stimulation of monocytes with lipopolysaccharide (LPS). Among these we identified three donors each that consistently had a level of less than 20% (low responders; LR) or of more than 80% (high responders; HR) of the maximum response seen in this population. Northern blot analysis of TNF mRNA after LPS stimulation revealed lower transcript levels in LR. Half life determination after actinomycin D blockade showed a similar decay rate for LR and HR and after blockade of degradation by cycloheximide treatment mRNA levels increased but LR remained lower compared to HR. These data indicate that the lower TNF mRNA levels in LR are not due to a more rapid mRNA degradation but rather a result of lower transcription. Transcripts for interleukin 6 (IL-6) were also low in LPS-stimulated monocytes of LR. Because expression of the LPS receptor CD14 was similar in LR and HR monocytes, our data suggest that differences in signal transduction account for the LR and HR phenotype.
Asunto(s)
Regulación de la Expresión Génica , Monocitos/metabolismo , Factor de Necrosis Tumoral alfa/genética , Northern Blotting , Cicloheximida/farmacología , Dactinomicina/farmacología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunidad , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Masculino , Monocitos/efectos de los fármacos , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Cells of the murine macrophage cell line P388D1 express cell surface CD14 and respond to LPS (lipopolysaccharide) stimulation with the production of TNF (tumor necrosis factor). When the cells are stimulated with LPS a second time then little TNF is produced, i.e. the cells are tolerant. Flow cytometry analysis demonstrates that this tolerance is not due to a downregulation of the CD14 cell surface receptor. Analysis of proteins binding to the -516 NF-kappa B motif of the murine TNF promoter reveals that constitutive p50p50 and LPS stimulation lead to mobilization of a heterodimer consisting of p65/c-rel. In tolerant cells less of the p65/c-rel heterodimer is mobilized but there is a strong upregulation of p50p50. These data show that tolerance to LPS in murine macrophages may involve a predominance of p50 homodimers.