RESUMEN
Bruton tyrosine kinase (BTK) is a cytoplasmic protein tyrosine kinase which controls crucial steps of differentiation of B lymphocytes. Mutations affecting either the PH, SH3, SH2 or kinase domain of BTK all give rise to X linked agammaglobulinaemia (XLA) in humans. In this study, the authors report that the BTK-SH3 domain binds to a set of proteins expressed in pro-B, pre-B and B cell lines. Three of them were characterized as Vav, Sam68 and EWS. The authors show that a Pro-->Leu substitution in a region of the SH3 domain, which is deleted in an XLA patient, is sufficient to abolish BTK-SH3 binding potential. The authors also report that several of the BTK-SH3 binding proteins, including Sam68, EWS and Vav, are tyrosine phosphorylated in conditions that also promote BTK kinase activity. For EWS and Sam68 this tyrosine phosphorylation was cell cycle dependent.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas de Unión al ADN/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Dominios Homologos src/inmunología , Proteínas Adaptadoras Transductoras de Señales , Agammaglobulinemia Tirosina Quinasa , Secuencia de Aminoácidos , Linfocitos B/enzimología , Ciclo Celular/inmunología , Línea Celular , Ribonucleoproteínas Nucleares Heterogéneas , Humanos , Datos de Secuencia Molecular , Mutación/inmunología , Fosforilación , Unión Proteica/inmunología , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-vav , Proteína EWS de Unión a ARN , Ribonucleoproteínas/biosíntesis , Sarcoma de Ewing/enzimología , Tirosina/metabolismoRESUMEN
Prior to the expression of the B cell antigen receptor, the mu heavy chain associates with two non-polymorphic polypeptides, lambda like and VpreB, which form a pseudo-light chain complex in pre-B cells and pre-B cell lines. Surface expression of the so-called pre-B cell receptor (pre-BCR) occurs only in the presence of Ig alpha and Ig beta, known to be involved both in B cell antigen receptor (BCR) signaling and trafficking. Although the pre-BCR organization is consistent with an efficient transport to the cell surface, most of the newly synthesized receptor remains within the cells, and so far, no data are available concerning the rate of exit from the endoplasmic reticulum. Using the human pre-B cell line Nalm-6, we found that only a small fraction (2%) of newly synthesized pre-BCR is transported to the cell surface within 4-6 h after synthesis, where it is constitutively re-internalized. Membrane Ig-heavy chain cross-linking induced internalization of surface pre-BCR within a few minutes, and the mechanisms underlying endocytosis were analyzed by immunofluorescence and confocal microscopy. Preincubation of the cells with either genistein or orthovanadate, which inhibit, respectively, tyrosine kinases and tyrosine phosphatases, blocked pre-BCR internalization in a dose-dependent manner, indicating that both activities are required for endocytosis. BCR internalization was also inhibited in a reversible manner by the drugs. In contrast, neither drug affected the size of the steady-state pool of internalized transferrin receptors. Thus, our data show that tyrosine phosphorylation and dephosphorylation are both required for cross-linking-induced pre-BCR and BCR internalization.
Asunto(s)
Linfocitos B/metabolismo , Endocitosis , Glicoproteínas de Membrana/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Fosfatasas/fisiología , Proteínas Tirosina Quinasas/fisiología , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal/fisiología , Linfocitos B/inmunología , Transporte Biológico , Línea Celular , Retículo Endoplásmico/metabolismo , Genisteína , Humanos , Cadenas Ligeras de Inmunoglobulina , Inmunoglobulina de Cadenas Ligeras Subrogadas , Cadenas mu de Inmunoglobulina/inmunología , Cadenas mu de Inmunoglobulina/metabolismo , Recubrimiento Inmunológico , Isoflavonas/farmacología , Microscopía Confocal , Microscopía Fluorescente , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Vanadatos/farmacologíaRESUMEN
IL-7 was identified originally as a specific pre-B cell growth factor. We have investigated its signal transduction mechanism by using the human pre-B cell line Nalm-6, and have found that it stimulates tyrosine phosphorylation of various proteins: pp27, pp43, pp54, pp64, pp78, pp90, pp105, and pp120. Antiphosphotyrosine immunoprecipitates from IL-7-stimulated Nalm-6 showed two major proteins of M(r) = 60,000 and 55,000, capable of autophosphorylation. Autophosphorylation was maximal 10 min after the cells were challenged with the cytokine. Antiphosphotyrosine immunoprecipitates from IL-7-stimulated cells also increased tyrosine phosphorylation of the exogenously added substrate histone H2B. Furthermore, by using a polyclonal anti-IL-7 receptor (IL-7R) Ab in Western blotting analysis, we observed that antiphosphotyrosine immunoprecipitates were associated with the IL-7R in a transient manner. These data indicate that the IL-7R associates with tyrosine-phosphorylated proteins as its amino acid sequence is devoid of a putative site of tyrosine phosphorylation. These results were confirmed as several 32P-labeled proteins were visualized after immunoprecipitation by using anti-IL-7R Ab. Anti-IL-7R immunoprecipitates from IL-7-stimulated cells revealed a unique band of M(r) = 60,000 associated with the receptor able to autophosphorylate in the presence of ATP and Mn2+. Hence, we identified p59fyn and p53/56lyn to be stimulated by IL-7. In contrast to p53/56lyn, p59fyn was found to be associated constitutively with the cloned IL-7R. These data emphasize the role of the src family in hematopoiesis.
Asunto(s)
Linfocitos B/enzimología , Interleucina-7/farmacología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Interleucina/metabolismo , Familia-src Quinasas , Secuencia de Aminoácidos , Linfocitos B/citología , Diferenciación Celular , Activación Enzimática , Hematopoyesis , Humanos , Datos de Secuencia Molecular , Péptidos/química , Fosfoproteínas/metabolismo , Fosfotirosina , Proteínas Proto-Oncogénicas c-fyn , Receptores de Interleucina/química , Receptores de Interleucina-7 , Transducción de Señal , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
Interleukin-7 (IL-7) was originally identified as a pre-B cell growth factor whose proliferating activity has been extended to numerous target cells including T lymphocytes. We investigated c-myc mRNA expression, an oncogene associated with proliferation, in the murine T cell line D10 G4.1 and freshly isolated thymocytes since both target cells proliferate in response to IL-7. We find that blockade of the tyrosine kinase pathway by genistein, a potent tyrosine kinase inhibitor, inhibits both IL-7-dependent D10 G4.1 cell proliferation and c-myc mRNA expression which appears to involve de novo mRNA synthesis and to be under the control of short-lived protein repressor(s). We have also examined possible signal transduction pathways which might regulate c-myc mRNA expression in the murine T cell line. IL-7 biological activity is not affected by stimulation of the protein kinase C pathway by phorbol esters. Thus, IL-7 regulates c-myc mRNA expression in a protein kinase C-independent manner and these data are strengthened by protein kinase C depletion which does not modify IL-7 c-myc mRNA responsiveness. In contrast and independent of protein kinase C activation, intracellular calcium mobilization by means of ionomycin reduces IL-7 induction of c-myc mRNA expression and may represent a physiological mechanism whereby IL-7 bioactivity is regulated. The activity of IL-7 on c-myc mRNA expression has been extended to freshly isolated thymocytes and we find a synergistic effect of IL-7 with concanavalin A. Taken together our results illuminate the molecular mechanism of IL-7 c-myc induction in the T lineage by ascribing a role for tyrosine kinase and increase in intracellular calcium in both IL-7 induced gene induction and cell proliferation.
Asunto(s)
Calcio/metabolismo , Regulación de la Expresión Génica , Genes myc , Interleucina-7/administración & dosificación , Proteínas Tirosina Quinasas/metabolismo , Linfocitos T/metabolismo , Timo/metabolismo , Animales , Concanavalina A/administración & dosificación , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-7/fisiología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Fosfotirosina , Proteína Quinasa C/fisiología , ARN Mensajero/genética , Transducción de Señal , Factores de Tiempo , Activación Transcripcional , Tirosina/análogos & derivados , Tirosina/metabolismoAsunto(s)
Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Proteínas Portadoras/farmacología , Citotoxicidad Inmunológica/efectos de los fármacos , Regulación hacia Abajo , Humanos , Ratas , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/fisiología , Receptores del Factor de Necrosis Tumoral , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Substitution of glycine for arginine at position 127 of the mature human interleukin 1 beta protein generates a mutant IL-1 beta protein (IL-1 beta R----G) which binds cellular IL-1 receptors with high affinity but fails to elicit significant proliferation of T-helper cells (Gehrke, L., Jobling, S. A., Paik, L. S. K., McDonald, B., Rosenwasser, L. J., and Auron, P. E. (1990) J. Biol. Chem. 265, 5922-5925). Although both IL-1 beta and the IL-1 beta R----G mutein stimulate transcription of fibroblast immediate early (fos and jun) and early (IL-1 beta and IL-6) genes, the IL-1 beta R----G mutein, in contrast to the wild-type IL-1 beta protein, induces minimal or no transcription of late genes such as procollagenase and prostromelysin. The effect of the naturally occurring IL-1 receptor antagonist protein (IL-1ra) on fibroblast transcription is distinct from that of the IL-1 beta R----G mutein, for the IL-1ra fails to stimulate not only late (procollagenase and prostromelysin) but also immediate early (fos and jun) gene expression. These data suggest that the IL-I beta R----G mutein triggers an incomplete or defective signal transduction cascade and demonstrate that fibroblast fos and jun expression is not necessarily accompanied by increased transcription of genes containing the AP-1 binding site. These data also suggest that at least two events are required for IL-1-mediated late gene induction in fibroblasts.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Interleucina-1/genética , Metaloendopeptidasas/biosíntesis , Mutación , Proteínas Proto-Oncogénicas/biosíntesis , Factores de Transcripción/biosíntesis , Northern Blotting , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Fibroblastos/citología , Fibroblastos/enzimología , Regulación Enzimológica de la Expresión Génica , Humanos , Interleucina-1/metabolismo , Cinética , Metaloendopeptidasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-fos , Proteínas Proto-Oncogénicas c-jun , Factores de Transcripción/genética , Transcripción Genética , Activación TranscripcionalRESUMEN
Tumour necrosis factor alpha (TNF-alpha) plays an important role in microbial defence and tissue damage by activating neutrophils. Therefore the ability of natural molecules to regulate the activity of TNF-alpha is likely to be of major importance in our understanding of the mechanisms of inflammation. We have examined the effects of a highly purified urine-derived TNF inhibitor (TNF inh) on the TNF-alpha-induced respiratory burst in human neutrophils. TNF-alpha inh-treated TNF-alpha was unable to stimulate a neutrophil lucigenin-dependent chemiluminescence response and superoxide formation. Treatment of TNF with the inhibitor also significantly reduced the priming ability of TNF-alpha for a response to the peptide f-met-leu-phe. These results show that the ability of TNF-alpha to induce a key neutrophil response is amenable to regulation by the TNF-alpha inh.
Asunto(s)
Neutrófilos/inmunología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Células Cultivadas , Humanos , Mediciones Luminiscentes , Neutrófilos/metabolismo , Superóxidos/sangre , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Interleukin-1 (IL-1) has been shown to regulate glycosaminoglycan (GAG) synthesis. We therefore investigated whether an IL-1 inhibitor or IL-6 modulates IL-1 biologic activities in human synovial cells and cultured articular cartilage. We found that in the presence of a constant amount of IL-1 beta, stimulation of hyaluronic acid (HA) synthesis by the IL-1 inhibitor was inhibited in a dose-dependent manner. Similarly, the decrease in sulfated GAG synthesis induced by IL-1 was reversed by the addition of the IL-1 inhibitor. In contrast, IL-6 did not affect the production of HA, prostaglandin E2, or collagenase in synovial cells, nor did it affect GAG in organ cultures when tested in the presence or absence of IL-1 beta. Hence, IL-6 was ineffective in modulating IL-1 bioactivities on HA or sulfated GAG synthesis. These results emphasize the importance of IL-1 and IL-1 inhibitor in connective tissue destruction and raise questions concerning the role of IL-6 in this pathogenesis.
Asunto(s)
Glicosaminoglicanos/biosíntesis , Interleucina-1/farmacología , Interleucina-6/farmacología , Sialoglicoproteínas/farmacología , Cartílago Articular/citología , Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Células Cultivadas , Dinoprostona/biosíntesis , Relación Dosis-Respuesta a Droga , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Ácido Hialurónico/biosíntesis , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/antagonistas & inhibidores , Colagenasa Microbiana/biosíntesis , Proteínas Recombinantes/farmacología , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/metabolismoRESUMEN
Inhibitory factors towards IL-1 have been identified in the urine and in the supernatants of human monocyte cultures and have been shown to act as receptor antagonists. We have investigated whether a natural inhibitor purified from human urine (uIL-1ra) and a recombinant molecule expressed using the gene for an IL-1 antagonist isolated from monocytes (rIL-1ra) can alter responses to human rIL-1 alpha in organ cultures of fetal rat long bones and neonatal mouse calvariae. The two preparations probably contained similar or identical molecules, because an antibody to rIL-1ra reacted with uIL-1ra by Western blot analysis. uIL-1ra and rIL-1ra specifically blocked stimulation of bone resorption by rIL-1 in both culture systems, as well as the increase in PGE2 production in cultured calvariae. Resorptive effects of parathyroid hormone and TNF-alpha were not blocked. The uIL-1ra preparation had some intrinsic resorbing activity, but on gel chromatography this appeared in fractions that eluted earlier than uIL-1ra. Concentration ratios of rIL-1ra to rIL-1 as low as 10 could block the resorptive response of fetal rat long bones, whereas concentration ratios of 100 to 1000 were required to block IL-1 action on neonatal mouse calvariae. The inhibitory effects appeared to be competitive, because increasing concentrations of IL-1 overcame the block of bone resorption in both systems and the inhibition of PGE2 production in calvariae.
Asunto(s)
Resorción Ósea , Dinoprostona/biosíntesis , Interleucina-1/antagonistas & inhibidores , Proteínas/farmacología , Receptores Inmunológicos/antagonistas & inhibidores , Sialoglicoproteínas/farmacología , Animales , Western Blotting , Calcio/metabolismo , Humanos , Técnicas In Vitro , Proteína Antagonista del Receptor de Interleucina 1 , Peso Molecular , Hormona Paratiroidea/antagonistas & inhibidores , Proteínas/genética , Proteínas/inmunología , Ratas , Receptores de Interleucina-1 , Proteínas Recombinantes/farmacología , Sialoglicoproteínas/genética , Sialoglicoproteínas/inmunología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
Previous studies have shown that urine of febrile patients contains a tumor necrosis factor alpha inhibiting activity (TNF-alpha Inh) when tested in a cytotoxicity assay using the tumor necrosis factor alpha (TNF-alpha)-susceptible cell line L929. In the present study, we investigated the relationship between the TNF-alpha Inh and a potential soluble form of the receptor, as the former has been shown to block TNF-alpha activities by binding to the ligand. We demonstrate that human TNF-alpha is affected to a greater extent than is murine TNF-alpha. This species specificity of the inhibitor correlates with the binding studies of TNF receptor interactions already reported. We raised a polyclonal antibody to TNF-alpha Inh that neutralizes its activity and does not recognize TNF-alpha. Solubilized cross-linked 125I-labeled TNF-alpha receptor complex could be immunoprecipitated by using either anti-TNF-alpha or anti-TNF-alpha Inh antibody, suggesting immunological cross-reactivity between the receptor and the inhibitor. By using fluorescein isothiocyanate-coupled TNF-alpha, it was possible to visualize by fluorescence-activated cell sorter analysis the TNF-alpha receptor on phytohemagglutinin/interleukin 2-activated T cells. A similar increase of immunofluorescence intensity of the activated T cells was observed by using anti-TNF-alpha Inh antibody revealed with a fluorescein isothiocyanate-coupled goat anti-rabbit IgG1 conjugate, suggesting that the TNF-alpha Inh is also expressed as a membrane protein. Taken together, our results suggest that the TNF-alpha Inh originally described might be a soluble form of the TNF receptor itself.
Asunto(s)
Receptores de Superficie Celular/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Anticuerpos , Western Blotting , Supervivencia Celular/efectos de los fármacos , Reacciones Cruzadas , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Cinética , Células L/citología , Células L/efectos de los fármacos , Células L/metabolismo , Ratones , Peso Molecular , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/aislamiento & purificación , Receptores del Factor de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Lipopolysaccharide (LPS) induces cell-associated interleukin 1 (IL 1) production in the human promonocytic cell line U937. Demonstration of cell-associated IL 1 activity was based on the ability of LPS-treated U937 cells, subsequently fixed with paraformaldehyde, to stimulate thymocyte proliferation in the presence of phytohemagglutinin. Like soluble IL 1 (sIL 1), cell-associated IL 1 is capable of inducing PGE2 and/or collagenase production by dermal fibroblasts and human synovial cells in a dose-dependent manner. It is thus a mediator of the inflammatory response owing to a direct intercellular contact located at the membrane level, where bound molecules may trigger inflammation at a local site of action. We reported that the natural (approximately 23 kDa) IL 1 inhibitor (IL 1 INH) from the urine of febrile patients inhibited all the sIL-1-induced biologic activities under investigation and that it acted by binding to the IL 1 receptor, thus blocking the interaction of the monokine with the receptor. Data demonstrate that the IL 1 INH also blocks cell-associated IL 1-induced T cell proliferation and PGE2 production by both dermal fibroblasts and synovial cells as well as collagenase production by the latter cell type. Thus, as for the sIL 1, a feedback mechanism exists for cell-associated IL 1-induced bioactivities.
Asunto(s)
Dinoprostona/biosíntesis , Interleucina-1/antagonistas & inhibidores , Colagenasa Microbiana/biosíntesis , Sialoglicoproteínas/fisiología , Linfocitos T/fisiología , Línea Celular , Fibroblastos/metabolismo , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/metabolismo , Activación de Linfocitos , Piel/metabolismo , Membrana Sinovial/metabolismoRESUMEN
The urine of some febrile patients has been shown to contain a tumor necrosis factor-alpha-inhibiting activity (TNF-alpha INH) when tested in a cytotoxicity assay using the TNF-susceptible cell line L-929. The inhibitor was purified to homogeneity using a simple three-step procedure which included a TNF-alpha affinity column, cation exchange and reverse-phase chromatography. The NH2-terminal amino acid sequence of the inhibitor showed no sequence similarity with proteins in the data bases used. Using gel filtration, it was shown that TNF-alpha and the inhibitor form a stable complex which eluted with a molecular weight of about 75,000. This value corresponds to the sum of the inhibitor (approximately 30,000) and TNF-alpha (approximately 45,000-50,000) molecular weight. The TNF-alpha INH blocked prostaglandin E2 production by dermal fibroblasts in a dose-dependent manner, providing evidence for antiinflammatory activity. TNF-alpha INH also blocked class I antigen expression in a dose-dependent manner as measured using the human Colo 205 tumor cell line. Furthermore, TNF-alpha INH affected TNF-alpha synergism with IFN-gamma-induced HLA-DR antigen expression but had no effect on IFN-gamma activity. The data presented demonstrate that TNF-alpha bioactivity can be regulated at the protein level.
Asunto(s)
Adyuvantes Inmunológicos/aislamiento & purificación , Antiinflamatorios no Esteroideos/aislamiento & purificación , Proteínas/aislamiento & purificación , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Secuencia de Aminoácidos , Línea Celular , Dinoprostona/biosíntesis , Fibroblastos/metabolismo , Antígenos HLA/biosíntesis , Antígenos HLA-D/biosíntesis , Humanos , Interferón gamma/fisiología , Datos de Secuencia Molecular , Proteínas/fisiología , Piel/metabolismoRESUMEN
The urine of patients with fever above 39 degrees C contains an inhibitor of interleukin 1. Herein we describe the purification of the interleukin 1 inhibitor by ion-exchange chromatography, hydrophobic chromatography, gel filtration and negative immunosorption. The purified protein has a molecular weight of 26,000; its size is reduced to 24,000 upon treatment with endoglycosidase F. The IL 1 inhibitor is a competitive inhibitor and acts by binding to the IL 1 receptor. The inhibitor recognizes the murine and human IL 1 receptor with similar affinity. The purified IL 1 inhibitor has a specific activity of 3 x 10(7) to 4.5 x 10(7) units/mg as tested on the human astrocytoma (U-373) cell proliferation bioassay and murine EL4.6.1 cell IL 1 receptor-binding assay.
Asunto(s)
Glicoproteínas/orina , Interleucina-1/antagonistas & inhibidores , Receptores Inmunológicos/metabolismo , Unión Competitiva , Cromatografía , Cromatografía Líquida de Alta Presión , Fiebre/orina , Glicoproteínas/metabolismo , Glicosilación , Humanos , Técnicas de Inmunoadsorción , Activación de Linfocitos , Peso Molecular , Receptores de Interleucina-1RESUMEN
The purpose of the present investigation was to purify a urine-derived tumor necrosis factor alpha inhibitor (TNF alpha INH) and to characterize its mechanism of action. For the purification procedure, urine was concentrated and TNF alpha INH purified by ion-exchange chromatographies, gel filtration, TNF alpha affinity column, and reverse-phase chromatography. The TNF alpha INH migrates with an apparent Mr of approximately 33,000 when estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis run under both reducing and nonreducing conditions. Elution of TNF alpha INH activity from the gel yields also a approximately 33,000-Da inhibitory fraction. Besides inhibiting TNF alpha-induced cytotoxicity in L929 cells in the presence of actinomycin D, the TNF alpha INH impeded in a dose-dependent manner prostaglandin E2 production and expression of cell-associated interleukin-1 by human dermal fibroblasts. Therefore, TNF alpha INH is active on both actinomycin D-treated and untreated cells. In contrast to TNF alpha, TNF beta-induced cytotoxicity was only slightly affected by the inhibitor. This specificity was confirmed by the fact that it affected neither interleukin-1 alpha nor interleukin-1 beta biologic activities. The mechanism of action of TNF alpha INH involves blocking of 125I-TNF alpha binding to the promonocytic cell line U937. Moreover, preincubation of 125I-TNF alpha with TNF alpha INH increased binding inhibition, suggesting an interaction between TNF alpha and the inhibitor.
Asunto(s)
Sialoglicoproteínas , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Línea Celular , Supervivencia Celular , Cromatografía Liquida , Dactinomicina/farmacología , Dinoprostona/antagonistas & inhibidores , Dinoprostona/biosíntesis , Electroforesis en Gel de Poliacrilamida , Fibroblastos , Humanos , Hidrólisis , Proteína Antagonista del Receptor de Interleucina 1 , Proteínas/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores del Factor de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/aislamiento & purificación , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Interleukin 1 (IL 1) and tumor necrosis factor (TNF) increase the expression of a number of T lymphocyte-specific genes in the T-cell hybrid PC60. We show here that human IL 1 alpha and 1 beta, mouse IL 1 alpha and mouse TNF-alpha strongly enhance expression of reporter genes transcribed from the SV40 early promoter in this cell line. We found that IL 1 and TNF each induce up to a 30-fold increase in the rate of transcription, resulting in a proportional increase of mRNA and protein levels. Induction with IL 1 and TNF is detectable after 1 h and reaches a plateau after about 15 h. Removal of these factors from the culture medium results in complete reversion. Induction with IL 1, but not with TNF, can be inhibited with an inhibitor of IL 1 binding. The effects of both factors are not impaired by the protein synthesis inhibitor cycloheximide, suggesting that they stimulate expression from the SV40 early promoter by activation of preexisting transcription factors.
Asunto(s)
Interleucina-1/farmacología , Regiones Promotoras Genéticas , Virus 40 de los Simios/genética , Linfocitos T/fisiología , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Animales , Línea Celular , Cicloheximida/farmacología , Ratones , Receptores de Interleucina-2/biosíntesis , Receptores de Interleucina-2/genética , TransfecciónRESUMEN
Several lines of evidence suggest that autoimmune processes are involved in the pathogenesis of Type I diabetes mellitus. Monocyte-macrophages are among the first mononuclear cells to invade the islets of Langerhans in various murine diabetic syndromes, and blockade of monocyte-macrophage functions by injection of silica particles in these animals prevents the development of the disease. Monokines such as interleukin 1 (IL-1) are known to mediate tissue lesions by inducing collagenase and prostaglandin E2 (PGE2) production. In addition, IL-1 has been demonstrated to inhibit proinsulin biosynthesis and secretion in pancreatic islet cells. Using 3-d cultured rat islets we have found that (a) the lowering of insulin release induced by human recombinant IL-1 (rIL-1) is dose-dependent with a decrease to 21% of control value at the higher rIL-1 tested concentration (500 pg/ml), and about two times more pronounced than the decrease in cellular insulin content, which reached 44% of control value at the highest rIL-1 concentration; (b) rIL-1 stimulates islets to secrete PGE2 but the addition of indomethacin, which blocks PGE2 production, does not affect the decrease in insulin release and content caused by IL-1, suggesting a limited role of endogenous PGE2 as a mediator in this system; and (c) a specific, noncytotoxic IL-1 inhibitor, shown in other cell systems to block the binding of IL-1 to its receptor, prevents the rIL-1 lowering of insulin content and minimizes the decrease of insulin release.
Asunto(s)
Insulina/biosíntesis , Interleucina-1/antagonistas & inhibidores , Islotes Pancreáticos/efectos de los fármacos , Sialoglicoproteínas/farmacología , Animales , Dinoprostona/biosíntesis , Humanos , Indometacina/farmacología , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/farmacología , Islotes Pancreáticos/metabolismo , Técnicas de Cultivo de Órganos , Ratas , Ratas Endogámicas , Receptores Inmunológicos/efectos de los fármacos , Receptores de Interleucina-1 , Proteínas Recombinantes/farmacologíaRESUMEN
Urine of some febrile patients exhibits a TNF-alpha inhibitory activity (TNF-alpha INH), sensitive to heat and trypsin, with an apparent mol wt of 40-60 X 10(3) and a pI range of 5.5-6.1. As for the Il-1 INH, the TNF INH activity involves a competitive mechanism of action suggesting the existence of a family of negative feedback-regulating molecules interfering with cytokines actions.
Asunto(s)
Fiebre/orina , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Carcinoma de Células Pequeñas/orina , Cromatografía en Gel , Fiebre/etiología , Sarcoma Histiocítico/orina , Humanos , Células L/efectos de los fármacos , Peso Molecular , Miositis/orina , Sepsis/orina , Factor de Necrosis Tumoral alfa/farmacología , Orina/análisisRESUMEN
Urine from monocytic leukemia and other febrile patients contains an inhibitor of interleukin 1 (IL-1), as measured by prostaglandin E2 and collagenase production by human fibroblasts and synovial cells. With the use of recombinant IL-1, the IL-1 inhibitor was partially purified by using ammonium sulfate precipitation, anion-exchange, and gel filtration chromatographies. IL-1 inhibitory activity elutes with an 18,000 to 25,000 apparent molecular size. The same fractions also inhibit IL-1 assayed by the proliferation of murine thymocytes and human fibroblasts. Both forms of human recombinant IL-1, IL-1 alpha and IL-1 beta, which show only 26% homology, but nevertheless bind to the same receptor, are affected by this natural inhibitor to the same extent. In contrast, human recombinant tumor necrosis factor, which shares some of the biologic activities of IL-1, is not inhibited by the urinary IL-1 inhibitor. This study shows that the various biologic activities of both forms of human recombinant IL-1 are inhibited by a partially purified natural urine-derived factor.
Asunto(s)
Fiebre/orina , Glicoproteínas/antagonistas & inhibidores , Interleucina-1/antagonistas & inhibidores , Proteínas/aislamiento & purificación , Sialoglicoproteínas , División Celular/efectos de los fármacos , Células Cultivadas , Cromatografía en Gel , Dinoprostona , Fibroblastos/análisis , Fibroblastos/efectos de los fármacos , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Colagenasa Microbiana/biosíntesis , Peso Molecular , Prostaglandinas E/biosíntesis , Proteínas/farmacología , Proteínas Recombinantes/antagonistas & inhibidores , Líquido Sinovial/patología , Factor de Necrosis Tumoral alfaRESUMEN
Urine from febrile patients was found to contain a novel inhibitor of interleukin 1 (IL-1) bioactivity that blocked the specific binding of radioiodinated IL-1 to its receptor in a dose-dependent fashion. Strong inhibition of IL-1 binding was still obtained when cells were preincubated with the inhibitor and washed, thus suggesting that the inhibitor binds to a surface structure (possibly the IL-1 receptor itself). The inhibitor was distinct from IL-1 as determined by both physical parameters (size and antigenicity) and receptor-binding characteristics (apparent affinity and dissociation rate). These data provide evidence for a physiologic regulator of IL-1 activity that functions in vivo via direct interference with ligand binding.