RESUMEN
Cell death is a characteristic consequence of cellular infection by influenza virus. Mounting evidence indicates the critical involvement of host-mediated cellular death pathways in promoting efficient influenza virus replication. Furthermore, it appears that many signaling pathways, such as NF-κB, formerly suspected to solely promote cell survival, can also be manipulated to induce cell death. Current understanding of the cell death pathways involved in influenza virus-mediated cytopathology and in virus replication is limited. This study was designed to identify host genes that are required for influenza-induced cell death. The approach was to perform genome-wide lentiviral-mediated human gene silencing in A549 cells and determine which genes could be silenced to provide resistance to influenza-induced cell death. The assay proved to be highly reproducible with 138 genes being identified in independent screens. The results were independently validated using siRNA to each of these candidates. Graded protection was observed in this screen with the silencing of any of 19 genes, each providing > 85% protection. Three gene products, TNFSF13 (APRIL), TNFSF12-TNFSF13 (TWE-PRIL) and USP47, were selected because of the high levels of protection conferred by their silencing. Protein and mRNA silencing and protection from influenza-induced cell death was confirmed using multiple shRNA clones and siRNA, indicating the specificity of the effects. USP47 knockdown prevented proper viral entry into the host cell, whereas TNFSF12-13/TNFSF13 knockdown blocked a late stage in viral replication. This screening approach offers the means to identify a large number of potential candidates for the analysis of viral-induced cell death. These results may also have much broader applicability in defining regulatory mechanisms involved in cell survival.
Asunto(s)
Citoprotección/genética , Técnicas de Silenciamiento del Gen , Factores Celulares Derivados del Huésped/genética , Orthomyxoviridae/fisiología , Muerte Celular/genética , Línea Celular Tumoral , Estudios de Asociación Genética , Factores Celulares Derivados del Huésped/metabolismo , Humanos , Complejos Multiproteicos/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Reproducibilidad de los Resultados , Internalización del Virus , Replicación Viral/genéticaRESUMEN
The effects of leptin-replacement therapy on the plasma proteome of three unique adults with genetically based leptin deficiency were studied longitudinally during the course of recombinant human leptin-replacement treatment. Quantitative proteomics analysis was performed in plasma samples collected during four stages: before leptin treatment was initiated, after 1.5 and 6 years of leptin-replacement treatment, and after 7 weeks of temporary interruption of leptin-replacement therapy. Of 500 proteins reliably identified and quantitated in those four stages, about 100 were differentially abundant twofold or more in one or more stages. Synchronous dynamics of abundances of about 90 proteins was observed reflecting both short- and long-term effects of leptin-replacement therapy. Pathways and processes enriched with overabundant synchronous proteins were cell adhesion, cytoskeleton remodeling, cell cycle, blood coagulation, glycolysis, and gluconeogenesis. Plausible common regulators of the above synchronous proteins were identified using transcription regulation network analysis. The generated network included two transcription factors (c-Myc and androgen receptor) that are known to activate each other through a double-positive feedback loop, which may represent a potential molecular mechanism for the long-term effects of leptin-replacement therapy. Our findings may help to elucidate the effects of leptin on insulin resistance.
Asunto(s)
Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Leptina/deficiencia , Leptina/genética , Adulto , Proteínas Sanguíneas/análisis , Adhesión Celular/genética , Citoesqueleto/genética , Citoesqueleto/metabolismo , Femenino , Terapia Genética , Gluconeogénesis/genética , Glucólisis/genética , Terapia de Reemplazo de Hormonas , Humanos , Resistencia a la Insulina , Masculino , Redes y Vías Metabólicas/genética , Proteoma/análisis , Proteoma/metabolismoRESUMEN
The proposed model is based on the measurement of the retention times of 346 tryptic peptides in the 560- to 4,000-Da mass range, derived from a mixture of 17 protein digests. These peptides were measured in HPLC-MALDI MS runs, with peptide identities confirmed by MS/MS. The model relies on summation of the retention coefficients of the individual amino acids, as in previous approaches, but additional terms are introduced that depend on the retention coefficients for amino acids at the N-terminal of the peptide. In the 17-protein mixture, optimization of two sets of coefficients, along with additional compensation for peptide length and hydrophobicity, yielded a linear dependence of retention time on hydrophobicity, with an R2 value about 0.94. The predictive capability of the model was used to distinguish peptides with close m/z values and for detailed peptide mapping of selected proteins. Its applicability was tested on columns of different sizes, from nano- to narrow-bore, and for direct sample injection, or injection via a pre-column. It can be used for accurate prediction of retention times for tryptic peptides on reversed-phase (300-A pore size) columns of different sizes with a linear water-ACN gradient and with TFA as the ion-pairing modifier.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Fragmentos de Péptidos/aislamiento & purificación , Mapeo Peptídico/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Aminoácidos , Animales , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Teóricos , Datos de Secuencia Molecular , Peso Molecular , Redes Neurales de la Computación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Mapeo Peptídico/estadística & datos numéricos , Proteómica/métodos , Proteómica/estadística & datos numéricos , TripsinaRESUMEN
To identify inhibitors of interleukin-8 (IL-8) production, a high throughput assay was developed using the QuantiGene nucleic acid quantification kit that employs branched-chain DNA (bDNA) technology to measure the mRNA directly from cells. Unlike polymerase chain reaction and other technologies that employ target amplification, the QuantiGene system uses signal amplification. To perform the assay, various molecular probes capable of hybridizing with IL-8 mRNA were designed and synthesized. A human lung epithelial cell line was treated with interleukin-1alpha (IL-1alpha) to stimulate the IL-8 gene expression and the mRNA was measured using the QuantiGene system. The QuantiGene assay was sensitive, flexible, and reproducible and achieved equivalent or better sensitivity than promoter-reporter assays, and eliminated the time required for constructing a promoter-reporter system. Our data show that bDNA technology has the potential to be used as a high throughput screening assay.
Asunto(s)
Ensayo de Amplificación de Señal de ADN Ramificado/métodos , Evaluación Preclínica de Medicamentos/métodos , Interleucina-8/genética , ARN Mensajero/análisis , Juego de Reactivos para Diagnóstico , Recuento de Células , Línea Celular , Sondas de ADN/genética , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Inmunoensayo , Interleucina-1/farmacología , Interleucina-8/análisis , Interleucina-8/biosíntesis , Mediciones Luminiscentes , ARN Mensajero/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
The assembly of signaling molecules surrounding the integrin family of adhesion receptors remains poorly understood. Recently, the membrane protein caveolin was found in complexes with beta1 integrins. Caveolin binds cholesterol and several signaling molecules potentially linked to integrin function, e.g., Src family kinases, although caveolin has not been directly implicated in integrin-dependent adhesion. Here we report that depletion of caveolin by antisense methodology in kidney 293 cells disrupts the association of Src kinases with beta1 integrins resulting in loss of focal adhesion sites, ligand-induced focal adhesion kinase (FAK) phosphorylation, and adhesion. The nonintegrin urokinase receptor (uPAR) associates with and stabilizes beta1 integrin/caveolin complexes. Depletion of caveolin in uPAR-expressing 293 cells also disrupts uPAR/integrin complexes and uPAR-dependent adhesion. Further, beta1 integrin/caveolin complexes could be disassociated by uPAR-binding peptides in both uPAR-transfected 293 cells and human vascular smooth muscle cells. Disruption of complexes by peptides in intact smooth muscle cells blocks the association of Src family kinases with beta1 integrins and markedly impairs their migration on fibronectin. We conclude that ligand-induced signaling necessary for normal beta1 integrin function requires caveolin and is regulated by uPAR. Caveolin and uPAR may operate within adhesion sites to organize kinase-rich lipid domains in proximity to integrins, promoting efficient signal transduction.
Asunto(s)
Caveolinas , Adhesión Celular/fisiología , Integrina beta1/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal/fisiología , Caveolina 1 , Línea Celular , Humanos , Proteínas de la Membrana/genética , Modelos Biológicos , Músculo Liso Vascular/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa , TransfecciónRESUMEN
Integrins play an important role in lymphocyte adhesion to cellular and extracellular components of their microenvironment. The regulation of such adhesion often involves changes in the functional state of the integrins rather than alterations in their expression levels. Although the functional basis for such transitions is unknown, a possible role for disulfide exchange might be postulated based on the observations that integrin function can be activated by bifunctional reducing agents or by Abs that react with areas adjacent to predicted long-range disulfide bonds in integrins. Recently, it has been reported that enzymes that catalyze disulfide exchanges such as protein disulfide isomerase (PDI) are present on the surface of lymphoid cells, raising the possibility that such enzymes might be involved in the control of lymphocyte adhesion. A number of inhibitors of PDI function were examined for their effects on integrin-mediated adherence of T cells. The results did not support role for PDI in the regulation of integrin function, as the inhibitors somatostatin A, tocinoic acid, dithiobisnitrobenzoic acid, and anti-PDI mAb did not interfere with adherence. However, one of the PDI inhibitors, bacitracin, selectively interfered with the beta1 integrin-mediated adherence of lymphoid cells to collagen, fibronectin, laminin, and VCAM-1, and with alpha4beta7-dependent adherence to fibronectin and to VCAM-1. In contrast, alpha(v)beta3- and alpha(L)beta2-mediated adherence were not inhibited. Thus, it appears that bacitracin may be a selective inhibitor of beta1 and beta7 integrin functions by an as yet unknown mechanism.
Asunto(s)
Bacitracina/farmacología , Cadenas beta de Integrinas , Integrina beta1/fisiología , Integrinas/fisiología , Linfocitos/fisiología , Linfocitos B/efectos de los fármacos , Linfocitos B/enzimología , Linfocitos B/fisiología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/inmunología , Agregación Celular/efectos de los fármacos , Agregación Celular/inmunología , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Humanos , Integrinas/antagonistas & inhibidores , Células Jurkat , Linfocitos/efectos de los fármacos , Linfocitos/enzimología , Proteína Disulfuro Isomerasas/antagonistas & inhibidores , Linfocitos T/efectos de los fármacos , Linfocitos T/enzimología , Linfocitos T/fisiologíaRESUMEN
Members of the beta 1 integrin family mediate cellular adherence to a wide range of extracellular and cell surface associated ligands. Conformational changes have been shown to be associated with integrin activation and ligand binding. Some studies suggest that there may be a restricted region of the beta 1 integrin that serves as the target for regulatory antibodies which can inhibit or stimulate integrin function. Here we identify an inhibitory epitope that is located at a distinct sight from that suggested for other inhibitory antibodies. Three different adhesion blocking antibodies, JB1A, C30B, and D11B bind to a peptide corresponding to residues 82-87 of the mature beta 1 chain. Mn++ inhibited the binding of JB1A to purified beta 1 integrin. In contrast the binding of several other antibodies to beta 1 were not influenced by these conditions. JB1A binding to purified peptide was also inhibited by Mn++ suggesting that it related to interference with the antibody function rather than a cation dependent change in the epitope. Our data 1) directly demonstrates the peptide sequence recognised by three adhesion blocking antibodies to the human beta 1 integrin chain 2) identifies a novel epitope located at residues 82-87, distinct from that of previously described regulatory epitopes 3) characterises a Mn++ sensitive antibody integrin interaction. Collectively, these results indicate the existence of multiple regulatory sites on the beta 1 integrin molecule.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Mapeo Epitopo , Integrina beta1/análisis , Integrina beta1/genética , Secuencia de Aminoácidos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Clonación Molecular , Biblioteca de Genes , Humanos , Integrina beta1/inmunología , Células Jurkat/química , Células Jurkat/citología , Células Jurkat/fisiología , Magnesio/farmacología , Datos de Secuencia Molecular , Fragmentos de Péptidos/farmacología , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Integrins can be expressed in at least three functional states (i.e. latent, active, and ligand-occupied). However, the molecular bases for the transitions between these states are unknown. In the present study, changes in the accessibility of several beta1 epitopes (e.g. N29, B44, and B3B11) were used to probe activation-related conformational changes. Dithiothreitol or Mn2+ activation of integrin-mediated adhesion in the human B cell line, IM9, resulted in a marked increase in the exposure of the B44 epitope, while N29 expression levels were most sensitive to dithiothreitol treatment. These results contrasted with the epitope expression patterns of spontaneously adherent K562 cells, where N29 was almost fully accessible and B44 was low. Addition of a soluble ligand resulted in a marked increase in B44 levels, suggesting that this antibody detected a ligand-induced binding site. The N29 epitope was mapped to a cysteine-rich region near the NH2 terminus of the integrin chain, thus defining a novel regulatory site. These studies indicate that the activation of integrin function by different stimuli may involve related but nonidentical conformations. Both Mn2+ and dithiothreitol appear to induce localized conformational changes that mimic a ligand-occupied receptor. This differs from the "physiologically" activated integrins on K562 cells that display a marked increase in overall epitope accessibility without exposure of the ligand-induced binding site epitopes. The increased exposure of the N29 site on K562 cells may indicate a role for this region in the regulation of integrin function.
Asunto(s)
Ditiotreitol/farmacología , Integrina beta1/química , Magnesio/farmacología , Conformación Proteica/efectos de los fármacos , Reactivos de Sulfhidrilo/farmacología , Linfocitos B/metabolismo , Línea Celular , Humanos , Integrina beta1/efectos de los fármacos , LigandosRESUMEN
Proteolipid protein (PLP), a transmembrane protein expressed only in the central nervous system (CNS), is a candidate target autoantigen for autoimmune-mediated demyelination. We have evaluated the effect of a recombinant form of the PLP protein, delta PLP4, in a murine model of experimental autoimmune encephalomyelitis (EAE). PLP-specific T-cell responses were observed following immunization of SJL/J, PL/J and SWR mice with delta PLP4, demonstrating processing of the protein to several distinct antigenic epitopes. Clinical EAE associated with inflammation and demyelination in the CNS also developed after sensitization of mice with delta PLP4 in adjuvant. Conversely, tolerance to delta PLP4 in adult mice and prevention of PLP peptide 139-151-induced EAE was induced by intravenous injection of soluble delta PLP4. The prevention of disease onset was paralleled by a significant reduction in demyelination and CNS inflammatory cell infiltration and diminished PLP139-151-specific T-cell proliferative responses. These results are consistent with the establishment of peripheral T-cell tolerance and reinforce the notion that recombinant myelin antigens and intravenous tolerance induction may prove useful in the modulation of the human demyelinating disease, multiple sclerosis (MS).
Asunto(s)
Encefalomielitis Autoinmune Experimental/prevención & control , Tolerancia Inmunológica/fisiología , Proteína Proteolipídica de la Mielina/metabolismo , Animales , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Humanos , Inmunización , Inyecciones Intravenosas , Ratones , Ratones Endogámicos , Proteína Proteolipídica de la Mielina/inmunología , Proteína Proteolipídica de la Mielina/farmacología , Fragmentos de Péptidos/farmacología , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/fisiologíaRESUMEN
It has been shown that peripheral T cell tolerance can be induced by systemic antigen administration. We have been interested in using this phenomenon to develop antigen-specific immunotherapies for T cell-mediated autoimmune diseases. In patients with the demyelinating disease multiple sclerosis (MS), multiple potentially autoantigenic epitopes have been identified on the two major proteins of the myelin sheath, myelin basic protein (MBP) and proteolipid protein (PLP). To generate a tolerogenic protein for the therapy of patients with MS, we have produced a protein fusion between the 21.5-kD isoform of MBP (MBP21.5) and a genetically engineered form of PLP (deltaPLP4). In this report, we describe the effects of treatment with this agent (MP4) on clinical disease in a murine model of demyelinating disease, experimental autoimmune encephalomyelitis (EAE). Treatment of SJL/J mice with MP4 after induction of EAE either by active immunization or by adoptive transfer of activated T cells completely prevented subsequent clinical paralysis. Importantly, the administration of MP4 completely suppressed the development of EAE initiated by the cotransfer of both MBP- and PLP-activated T cells. Prevention of clinical disease after the intravenous injection of MP4 was paralleled by the formation of long-lived functional peptide-MHC complexes in vivo, as well as by a significant reduction in both MBP- and PLP-specific T cell proliferative responses. Mice treated with MP4 were resistant to disease when rechallenged with an encephalitogenic PLP peptide emulsified in CFA, indicating that MP4 administration had a prolonged effect in vivo. Administration of MP4 was also found to markedly ameliorate the course of established clinical disease. Finally, MP4 therapy was equally efficacious in mice defective in Fas expression. These results support the conclusion that MP4 protein is highly effective in suppressing disease caused by multiple neuroantigen epitopes in experimentally induced demyelinating disease.
Asunto(s)
Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Proteínas Recombinantes de Fusión/uso terapéutico , Vacunación , Vacunas Sintéticas/uso terapéutico , Traslado Adoptivo , Secuencia de Aminoácidos , Animales , Apoptosis , Femenino , Antígenos de Histocompatibilidad , Tolerancia Inmunológica , Activación de Linfocitos , Ratones , Ratones Endogámicos MRL lpr , Datos de Secuencia Molecular , Proteína Básica de Mielina , Proteína Proteolipídica de la Mielina , Péptidos , Ingeniería de Proteínas , Linfocitos T/inmunología , Receptor fas/biosíntesisRESUMEN
Haemophilus ducreyi is a major cause of genital ulcer disease in many developing countries and is associated with augmented transmission of human immunodeficiency virus (HIV). However, the mechanisms through which H. ducreyi produces ulceration are poorly understood. The characteristics of the host response to H. ducreyi and the pathobiology of its potential contribution to increased HIV susceptibility are not known. Chancroid ulcer biopsies from 8 patients were analyzed histologically and immunohistochemically. All biopsies had perivascular and interstitial mononuclear cell infiltrates that extended deep into the dermis. The infiltrate, which contained macrophages and CD4 and CD8 lymphocytes, was consistent with a delayed hypersensitivity type cell-mediated immune response. The recruitment of CD4 T lymphocytes and macrophages may in part explain the facilitation of HIV transmission in patients with chancroid.
Asunto(s)
Chancroide/patología , Biopsia , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Chancroide/inmunología , Humanos , Inmunohistoquímica , Inflamación/patología , Kenia/epidemiología , Macrófagos , Masculino , Piel/patología , Úlcera/patologíaRESUMEN
The beta1 integrins can be expressed on the surface of cells in a latent form, which is activated by a variety of stimuli. As an approach to examining the transition to an active receptor, a panel of stimulatory antibodies to beta1 were produced and characterized. These antibodies induced adherence of the T-leukemic cell line Jurkat to collagen and fibronectin. Competitive antibody binding assays indicated the existence of at least three distinct epitope clusters A (B3B11, JB1B, 21C8), B (B44, 13B9), and C(N29) defined by the indicated antibodies. Two antibodies to the A site, JB1B and B3B11, were shown to localize to positions 671-703 and 657-670, respectively, of the beta1. This region is located in an area encompassing a predicted disulfide bond between linearly distant cysteines in beta1 (Cys415-Cys671). The homologous region of the beta3 integrin (490 690 and 602 690) has been shown to be one of the sites recognized by stimulatory antibodies to ligand-induced binding sites. The present results indicate the existence of multiple stimulatory regions and suggest considerable homology between the locations of beta1 and beta3 regulatory sites.
Asunto(s)
Integrina beta1/fisiología , Secuencia de Aminoácidos , Animales , Anticuerpos , Especificidad de Anticuerpos , Unión Competitiva , Células CHO , Adhesión Celular , Línea Celular , Clonación Molecular , Colágeno , Cricetinae , Epítopos , Fibronectinas , Humanos , Integrina beta1/química , Integrina beta1/inmunología , Leucemia de Células T , Ratones , Datos de Secuencia Molecular , Mutagénesis Insercional , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Transfección , Células Tumorales CultivadasRESUMEN
Expression of the 17.5-kDa truncated form of human recombinant macrophage colony stimulating factor (rM-CSF, 4-153) in Escherichia coli is complicated by the replacement of methionine residues by norleucine. In order to detect and quantitate this mistranslational event, the intact and the S-carboxyamidomethylated proteins were analyzed by amino acid analysis, automated Edman amino acid sequencing, and electrospray mass spectrometry. In addition, the endoproteinase Glu-C generated peptides were subjected to amino acid sequencing, high-performance liquid chromatography, and electrospray ionization mass spectrometry. The extent of norleucine substitution in different batches of rM-CSF varied between 0% and 20%. The relative instability of methionine residues needs to be considered when calculating the extent of norleucine substitution at methionine positions. The mass spectrometry of the intact rM-CSF allowed for examination of the distribution of multiply substituted methionine to norleucine species, and it enabled detection and quantitation of the norleucine incorporation down to the approximately 3% level. Selective ion chromatograms of molecular ions of interest obtained in reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry of proteolytic fragments offered a reliable and fast method of detection and quantitation of norleucine-containing peptides. Norleucine residues were uniformly distributed among all four methionine positions (10, 27, 61, and 65). A substitution of methionine by its structural norleucine analog does not have any effect on the activity of the refolded rM-CSF dimers.
Asunto(s)
Factor Estimulante de Colonias de Macrófagos/química , Metionina/química , Norleucina/química , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Clonación Molecular , Escherichia coli , Humanos , Hidrólisis , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/farmacología , Espectrometría de Masas , Datos de Secuencia Molecular , Norleucina/análisis , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/farmacología , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologíaRESUMEN
The control of lymphocyte adhesion is critical for proper cellular functions. The alpha 2 beta 1 integrin complex serves as a receptor for collagen on lymphocytes. The T cell leukemia Jurkat expresses alpha 2 beta 1 in a latent form on the cell surface. Three types of stimuli, antibody to the alpha 2 chain (JBS2), antibody to the beta 1 chain (JB1B), and phorbol ester (PMA), each induce activation of alpha 2 beta 1-dependent Jurkat binding to collagen. A comparison of the JBS2, JB1B, and PMA induction requirements indicated that the JB1B and the JBS2 effects are not sensitive to staurosporine while the PMA response is completely inhibited. Combinations of functionally saturating concentrations of the stimuli displayed an additive effect. Collectively these results suggest that several factors contribute to the generation of full integrin functionality and cellular adhesion, thus providing a possible basis for incrementally controlling the adhesive potential of lymphoid cells.
Asunto(s)
Antígenos CD/metabolismo , Adhesión Celular/fisiología , Colágeno/metabolismo , Integrinas/metabolismo , Linfocitos T/metabolismo , Alcaloides/farmacología , Anticuerpos Monoclonales/farmacología , Especificidad de Anticuerpos , Antígenos CD/inmunología , Adhesión Celular/efectos de los fármacos , Humanos , Integrina alfa2 , Integrina beta1 , Integrinas/inmunología , Proteína Quinasa C/antagonistas & inhibidores , Estaurosporina , Linfocitos T/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
The folding pathway for a 150-amino acid recombinant form of the dimeric cytokine human macrophage colony-stimulating factor (M-CSF) has been studied. All 14 cysteine residues in the biologically active homodimer are involved in disulfide linkages. The structural characteristics of folding intermediates blocked with iodoacetamide reveal a rapid formation of a small amount of a non-native dimeric intermediate species followed by a slow progression via both monomeric and dimeric intermediates to the native dimer. The transition from monomer to fully folded dimer is complete within 25 h at room temperature at pH 9.0. The blocked intermediates are stable under conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thus represent various dimeric and folded monomeric species of the protein with different numbers of disulfide bridges. Peptide mapping and electrospray ionization mass spectrometry revealed that a folded monomeric species of M-CSF contained three of the four native disulfide bridges, and this folded monomer also showed some biological activity in a cell-based assay. The results presented here strongly suggest that M-CSF can fold via two different pathways, one involving monomeric intermediates and another involving only dimeric intermediates.
Asunto(s)
Factor Estimulante de Colonias de Macrófagos/metabolismo , Pliegue de Proteína , Secuencia de Aminoácidos , Cisteína , Disulfuros , Escherichia coli/genética , Humanos , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/aislamiento & purificación , Factor Estimulante de Colonias de Macrófagos/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Mapeo Peptídico , Conformación Proteica , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
beta 1 integrin containing complexes have been implicated as the primary adhesion structures in many lymphocyte extracellular matrix (ECM) interactions. However, many B lymphocytes lack surface expression of the beta 1 subunit, implying that this subpopulation of lymphoid cells must employ alternate adhesion structures if they are to maintain an interactive capacity with ECM. An examination of the adherence properties of the beta 1 integrin-negative B cell line JY indicated that these cells exhibit little or no basal adherence to any of the ECM components examined. However, these cells could be induced to adhere to the ECM components fibronectin, laminin, and vitronectin following treatment with PMA. Blocking studies with monoclonal antibodies indicated the alpha v beta 3 integrin complex was involved in the attachment to each of these ligands. However, the adherence to fibronectin displayed a complex pattern of inhibition suggesting the involvement of other ECM receptors. The utilization of the alpha v beta 3 complex was not unique to the JY cell line. Other B cell lines were observed to employ alpha v beta 3, and these lines similarly lacked expression of beta 1 integrin. These results indicate that alpha v beta 3 can act as a lymphoid ECM-adhesion structure which may provide an alternative means for lymphocytes to interact with ECM. Furthermore, these studies provide evidence for the presence of lymphoid-associated alpha v beta 3 integrins with regulatable activity, which contrasts with the constitutive adhesive potential of these complexes when present on other cell types.
Asunto(s)
Linfocitos B/fisiología , Adhesión Celular , Matriz Extracelular/metabolismo , Integrinas/metabolismo , Receptores de Citoadhesina/metabolismo , Anticuerpos Monoclonales , Linfocitos B/metabolismo , Sitios de Unión , Línea Celular , Colágeno/metabolismo , Fibronectinas/metabolismo , Glicoproteínas/metabolismo , Humanos , Integrina beta1 , Laminina/metabolismo , Manganeso/farmacología , Receptores de Citoadhesina/inmunología , Receptores de Vitronectina , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas , VitronectinaRESUMEN
The human tumour cell lines RPMI 8226 and Daudi are potent inducers of V gamma 9-expressing T cells. The inducing element of RPMI 8226 has not been defined but evidence suggests that a member of the GroEL heat shock protein (HSP) family (HSP 58) may have a role in the induction by Daudi cells. The present study examined the reactivity patterns of gamma delta T-cell clones generated in response to RPMI 8226 and addressed the possible role of HSP 58 in this process. RPMI 8226 induced a population of V gamma 9 TCR+ cells which were heterogeneous in terms of their cell surface markers, patterns of proliferation and cytotoxic responses. All clones expressed CD3, CD2, CD18 and CD29. They demonstrated variability in expression of CD56, CD8 and HLA-DR. RPMI 8226 stimulated proliferation in purified bulk gamma delta cultures and clones. Daudi was also capable of inducing these cells to proliferate while mycobacterial products were not effective. The clones demonstrated a limited non-MHC-restricted cytotoxicity pattern with some evidence of clonal heterogeneity. Although both Daudi and RPMI 8226 were sensitive to lysis by the clones, cold inhibition experiments indicated differential activity towards these targets. Anti-HSP 58 was inhibitory to gamma delta T-cell induction by RPMI 8226, Daudi and mycobacterial products. However, the anti-HSP 58 antibody appears to bind to the surface of at least six different tumour cell lines with no correlation to their ability to induce gamma delta T cells and the anti-HSP 58 inhibited non-gamma delta responses.
Asunto(s)
Proteínas Bacterianas/inmunología , Proteínas de Choque Térmico/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Subgrupos de Linfocitos T/inmunología , Células Tumorales Cultivadas/inmunología , Chaperonina 60 , Células Clonales , Citotoxicidad Inmunológica , Humanos , Técnicas In Vitro , Interleucina-2/farmacología , Activación de Linfocitos , Proteínas RecombinantesRESUMEN
CD45, the leukocyte common Ag, has been shown to characterize T cell development both within the thymus and among peripheral T cells. The work reported here demonstrates that human multinegative (MN) thymocytes, depleted of cells bearing CD3, CD4, CD8, and CD19, express predominantly the high molecular mass CD45RA isoform, and lack low molecular mass CD45RB isoforms and CD45R0 as detected by immunofluorescence. By immunoprecipitation of surface-labeled CD45 molecules from MN thymocytes, a proportion of the CD45 is in fact of low molecular mass but does not include epitopes recognized by CD45R0, nor by CD45RB mAb specific for the p190. This suggests either glycosylation variants of CD45RB/CD45R0 undetectable by our mAb, or underglycosylated CD45RA. MN thymocytes lack TCR-alpha beta mRNA confirming their early developmental stage. Upon culture with IL-2 or with mitogenic combinations of anti-CD2/CD28 mAb, MN thymocytes differentiate to acquire CD3, TCR-alpha beta, and in some cases CD4 and/or CD8. We have predicted that maintenance of CD45RA and lack of CD45R0 expression is fundamental to generative thymic development. If correct, this demands that unlike peripheral T cells, differentiation of MN thymocytes should be accompanied by prolonged expression of high molecular mass CD45 isoforms. Analysis of CD45 isoform expression during MN thymocyte development confirms this prediction and indicates that expression of CD45RA is maintained, at increasing density, for at least 8 to 12 days of culture. Unlike peripheral blood T cells, this is accompanied by the gradual acquisition of firstly the p190 isoforms of CD45RB and later by CD45R0, resulting in a population of CD3+TCR-alpha beta cells coexpressing CD45RA/RBp190/R0. Dot blot analysis of mRNA from differentiating MN thymocytes indicates prolonged expression of mRNA encoding CD45 exons a, b, and c, again in contrast to peripheral T cells which lose all mRNA for alternatively spliced CD45 exons within the first 24 h poststimulation. This is discussed in the context of negative selection during thymic development and interconversion of T cell subsets.
Asunto(s)
Antígenos CD/análisis , Antígenos de Diferenciación de Linfocitos T/análisis , Células Madre Hematopoyéticas/inmunología , Antígenos de Histocompatibilidad/análisis , Receptores de Antígenos de Linfocitos T/análisis , Linfocitos T/inmunología , Antígenos CD/genética , Complejo CD3 , Antígenos CD4/análisis , Antígenos CD8/análisis , Diferenciación Celular , Células Cultivadas , Exones , Antígenos de Histocompatibilidad/genética , Humanos , Antígenos Comunes de Leucocito , Peso Molecular , ARN Mensajero/análisis , Receptores de Antígenos de Linfocitos T alfa-beta/genéticaRESUMEN
Lymphocytes adhere to fibronectin (FN) via multiple receptors of the VLA (beta 1, CD29 integrin) family. The cellular requirement for the variety of FN receptors (FNR) which have been described is unclear, but they may be associated with differential signalling processes, cooperative effects which may stabilize cellular attachment, or cell homing and retention processes. The present study was undertaken to examine the FN adherence properties and receptor utilization patterns of human B cells. Of ten B-cell culture lines which were studied, six demonstrated a significant adherence to FN. Among these, four employed alpha 4 beta 1, (CD49d/29) and two employed alpha 4 beta 1/alpha 5 beta 1, (CD49d/29, CD49e/29). There was no apparent correlation between the differentiation status of the lines and their FNR utilization patterns. Furthermore, expression of FNR alone was not sufficient to confer FN binding potential. Freshly isolated tonsil B cells did not display significant adherence to FN. Following stimulation, a marked increase in VLA antigens was observed, and the capacity to attach to FN-coated surfaces was co-acquired. Analysis of the induced bulk B-cell population demonstrated that both alpha 4 beta 1 and alpha 5 beta 1 were used for adherence. These results clearly indicate that activated B cells, similar to T cells, may express and utilize alpha 5 beta 1 as a FNR.
Asunto(s)
Linfocitos B/química , Fibronectinas/metabolismo , Receptores Inmunológicos/análisis , Adhesión Celular , Línea Celular , Humanos , Receptores de Fibronectina , Receptores de Antígeno muy Tardío/análisis , Receptores de Antígeno muy Tardío/fisiologíaRESUMEN
The interaction of the human T cell leukemia, Jurkat, with the extracellular matrix components collagen and fibronectin was examined. These cells displayed constitutive binding to fibronectin and low levels of adherence to collagen which were enhanced following stimulation with phorbol esters. The relevant binding structures were identified as members of the CD29/beta 1 integrin family of adhesion molecules. Adherence to collagen and to fibronectin was mediated by alpha 2 beta 1 and alpha 5 beta 1, respectively. The enhancement of adherence by phorbol esters did not involve up-regulation of receptor expression but appeared to derive from the increased functionality of structures which were expressed on the cell surface.