RESUMEN
Interleukin-5 (IL-5) production is necessary for eosinophilia associated with allergic conditions and parasitic infection. IL-5 mRNA is transiently expressed by activated T-lymphocytes. In this report, we have analysed DNA regulatory regions associated with inducible IL-5 expression in the human HSB-2 T-cell line. Only low levels of transcriptional activity were induced in cells transfected with up to 1.2 kb of DNA upstream of the IL-5 gene. DNase I hypersensitivity analysis was employed to identify additional regulatory sequences located outside this region. Two hypersensitivity sites (HS) were identified, one 2.5 kb 5' and the other 1.6 kb 3' from the gene, that were induced on activation of HSB-2 cells by stimuli that induced IL-5 expression. The 5' site, but not the 3' site, was found in primary human T-cells. The presence of the 5' HS did not always coincide with IL-5 expression. Inclusion of the region encompassing the 5' HS in promoter studies mediated a moderate increase in transcriptional activity, suggesting that enhancer elements essential for induction of maximal IL-5 transcription reside at a greater distance from the IL-5 gene.
Asunto(s)
Interleucina-5/genética , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Línea Celular , Desoxirribonucleasa I , Genes Reporteros , Humanos , Interleucina-5/biosíntesis , Células Jurkat , Luciferasas/biosíntesis , Luciferasas/genética , Proteínas Recombinantes de Fusión/biosíntesis , Mapeo Restrictivo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
PCR products were characterized by electrophoresis, blotting and hybridization. In addition to the bands of expected size, bands of slower electrophoretic mobility were often detected. The slower bands completely disappeared when the PCR products were subjected to slow cooling, treated with S1 nuclease or run on an alkaline gel, whereas the bands of expected size were unaffected. The slower bands are therefore likely to contain single-stranded DNA.
Asunto(s)
ADN de Cadena Simple/análisis , Electroforesis en Gel de Agar , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Línea Celular Transformada , ADN/genética , Humanos , Datos de Secuencia Molecular , Receptores de IgG/genéticaRESUMEN
Normal T cells secrete cytokines which have important effects on a wide range of other cells. In the present study, the expression of IL-2, IL-4, IL-5, IL-10 and IFN-gamma was assessed in PBMC from 3 cases of T cell leukemia, using reverse transcription and polymerase chain reaction (RT-PCR). The major difference between the leukemia cases and control PBMC samples was that, without in vitro stimulation, IFN-gamma was much more readily detected in 2 of the leukemia cases. IL-2 and IL-5 mRNA were also detected in 2 of the patients but not in the other samples. IL-4 mRNA was not detected in any unstimulated sample, whereas IL-10 was always present. After polyclonal stimulation in vitro, mRNA for all these cytokines was detected in all samples. Thus cytokine expression, particularly of IFN-gamma, may be more prominent in PBMC from adult T cell leukemia cases than in controls.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/fisiología , Interferón gamma/genética , Interleucinas/genética , Leucemia de Células T/genética , Adulto , Células Cultivadas , Humanos , Inmunofenotipificación , Leucocitos Mononucleares/fisiología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa/métodos , ARN/genética , ARN Mensajero/biosíntesis , Transcripción GenéticaRESUMEN
T cell cytokine expression may be induced by the cytokine IL-2 or via the TCR complex. The comparative effects of cytokine- and TCR-mediated signalling on the induction of human IL-5 mRNA were examined. Cytokine mRNA expression was analysed by RT-PCR in fresh peripheral blood mononuclear cells (PBMC) from normal individuals and in populations of activated T lymphocytes, derived from phytohaemagglutinin (PHA)-stimulated PBMC. rIL-2 induced IL-5 expression in PBMC, the kinetics of which were similar to the effects of PHA. rIL-4 induced IL-5 mRNA expression in activated T lymphocytes. IL-5 expression induced by either IL-2 or PHA was completely abolished by the protein synthesis inhibitor cycloheximide. rIL-2-induced IL-5 expression was resistant to cyclosporin A (CsA), whereas IL-5 expression elicited by PHA was inhibited by CsA, at doses as low as 10 ng/ml. Rapamycin (RAP) had no effect on rIL-2-stimulated IL-5 expression, but suppressed IL-5 expression induced by PHA. The inhibitory effect of RAP on PHA-induced IL-5 expression was more apparent at 12 and 24 h after stimulation than at earlier times. The resistance of IL-2 receptor (IL-2R) signalling to CsA and RAP indicates that the IL-2R and the TCR are associated with different pathways regulating IL-5 expression.
Asunto(s)
Ciclosporina/farmacología , Inmunosupresores/farmacología , Interleucina-2/farmacología , Interleucina-5/biosíntesis , Leucocitos Mononucleares/efectos de los fármacos , Polienos/farmacología , Adyuvantes Inmunológicos/farmacología , Células Cultivadas , Cicloheximida/farmacología , Resistencia a Medicamentos , Humanos , Interleucina-4/farmacología , Interleucina-5/genética , Leucocitos Mononucleares/metabolismo , Fitohemaglutininas/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/efectos de los fármacos , Proteínas Recombinantes/farmacología , SirolimusRESUMEN
The cytokine interleukin-5 (IL-5) selectively induces the proliferation, differentiation, and activation of mature eosinophils. The immunosuppressive agents cyclosporin A (CsA) and FK506 ameliorate the influx of eosinophils seen in allergic conditions such as asthma. We investigated the mechanisms controlling IL-5 messenger RNA (mRNA) expression in human T-lymphocytes in the presence of CsA or FK506. Fresh human peripheral blood mononuclear cells (PBMC); 7-day cultured PBMC, which represent a population of activated T-lymphocytes derived from PBMC; and the T-cell line HSB-2 were used. A novel polymerase chain reaction (PCR)-based nuclear run-on assay was employed to investigate the rate of IL-5 gene transcription. IL-5 mRNA degradation was measured by quantitative reverse transcriptase (RT)-PCR. CsA and FK506 strongly inhibited cellular IL-5 mRNA expression in response to phytohemagglutinin (PHA), or to phorbol myristate acetate (PMA), and/or calcium ionophore. Marked inhibition was observed in PBMC, 7-day cultured PBMC, and HSB-2 cells. Nuclear run-on assays done with either 7-day cultured PBMC or HSB-2 cells demonstrated striking inhibition of IL-5 gene transcription by both CsA and FK506 at levels reflecting the degree of reduction of total cellular IL-5 mRNA abundance. Neither CsA or FK506 had any detectable effect on the stability of IL-5 mRNA. Thus, the inhibitory effect of CsA and FK506 on cellular IL-5 mRNA expression can be explained by inhibition of the rate of IL-5 gene transcription.
Asunto(s)
Ciclosporina/farmacología , Inmunosupresores/farmacología , Interleucina-5/metabolismo , Tacrolimus/farmacología , Transcripción Genética/efectos de los fármacos , Células Cultivadas , Humanos , Interleucina-5/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Células Tumorales CultivadasRESUMEN
The expression of IL5 by CD4+CD45RA+, CD4+CD45R0+ and CD3+CD8+ subsets of human peripheral blood mononuclear cells was assessed. Interleukin 5 expression was detected by RNA extraction, reverse transcription and polymerase chain reaction. Populations of highly purified cells were obtained by a protocol of sequential plastic adherence, magnetic bead separation and flow cytometric cell sorting. IL5 was clearly expressed in the CD4+CD45R0+ subset from 3 to 48 hr after activation. The CD4+CD45RA+ and CD3+CD8+ subsets expressed very much less IL5. By contrast, IL2 expression was readily detected in all sorted populations. Thus, in activated CD4+ cells, IL5 was predominantly expressed in the CD4+CD45R0+ subset, a pattern of expression corresponding to that reported for a number of other cytokines, and differing from that of IL2.
Asunto(s)
Antígenos CD/análisis , Interleucina-5/biosíntesis , Subgrupos de Linfocitos T/metabolismo , Secuencia de Bases , Antígenos CD4/análisis , Separación Celular , Cartilla de ADN , Citometría de Flujo , Expresión Génica , Humanos , Interleucina-2/biosíntesis , Antígenos Comunes de Leucocito/análisis , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Subgrupos de Linfocitos T/inmunologíaRESUMEN
The estrogen receptor (ER) suppresses transcriptional activity of the RelA subunit of nuclear factor-kappaB in a hormone-dependent manner by a mechanism involving both the receptor DNA binding domain and ligand binding domain (LBD). In this study we examine the role of the ER LBD in mediating ligand-dependent RelA transrepression. Both ERalpha and ERbeta inhibit RelA in response to 17beta-estradiol but not in the presence of antihormones. We have identified residues within the ERalpha LBD that are responsible for receptor dimerization and show that dimerization is necessary for transactivation and transrepression. Moreover we have generated mutant receptors that have lost their ability to inhibit RelA but retain their capacity to stimulate transcription and conversely mutants that are transcriptionally defective but capable of antagonizing RelA. Overexpression of p160 and cAMP-response element-binding protein-binding protein/p300 co-activators failed to relieve repression of RelA, which is consistent with the demonstration that RelA inhibition can occur independently of these co-activators. These findings suggest it is unlikely that sequestration of these cofactors required for ER transcriptional activation can account for hormone-dependent antagonism of RelA. The identification of ER mutants that discriminate between transactivation and transrepression implies that distinct surfaces within the LBD are involved in mediating these two receptor functions.
Asunto(s)
Mutación , Receptores de Estrógenos/química , Receptores de Estrógenos/genética , Activación Transcripcional , Animales , Western Blotting , Células COS , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Dimerización , Estradiol/farmacología , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Ligandos , Ligasas/antagonistas & inhibidores , Ligasas/metabolismo , Mutagénesis Sitio-Dirigida , FN-kappa B/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Plásmidos/metabolismo , Mutación Puntual , Estructura Terciaria de Proteína , Transactivadores/metabolismo , Transcripción Genética , TransfecciónRESUMEN
Steroid receptor co-activator (SRC1) is one of a number of transcriptional co-activators that are capable of potentiating the activity of nuclear receptors including the oestrogen receptor (ER). Here we report that two isoforms, SRC1a and SRC1e, which diverge at their C-termini, are functionally distinct as they differ in their abilities to enhance the activity of the ER in intact cells. SRC1e enhanced the ability of the ER to stimulate transcription to a greater extent than SRC1a, which had negligible effects on certain promoters. To elucidate the basis of this functional difference, we compared the nuclear receptor-binding properties and mapped the transcriptional activation domains in the two SRC1 isoforms. Both isoforms share a triplet of nuclear receptor-binding motifs (LXXLL motifs) for binding to functional ER dimers, and an activation domain which co-localizes with the CBP-binding domain, while SRC1a contains a unique LXXLL motif in its C-terminus. Although this LXXLL motif increases the affinity for the ER in vitro, it does not appear to be responsible for the functional difference between the two isoforms. This difference is due to a second activation domain that is CBP independent and is suppressed in the SRC1a isoform. Thus, SRC1 exists as functionally distinct isoforms which are likely to play different roles in ER-mediated transcription.
Asunto(s)
Receptores de Estrógenos/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Animales , Células COS , Histona Acetiltransferasas , Humanos , Datos de Secuencia Molecular , Coactivador 1 de Receptor Nuclear , Unión Proteica , Receptores de Estrógenos/química , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
Polyclonal B cell activation is characteristic of HIV infection and occurs in the presence of severe CD4+ lymphocyte depletion. In contrast, CD4+ lymphocytes are the dominant T cell in the reactive lymphoid tissues of patients not infected with HIV. In this study, lymph node biopsies from eight HIV-infected patients with persistent generalized lymphadenopathy syndrome (PGL) were assessed for IL-1 beta, IL-2, IL-4, IL-6, IL-10, interferon-gamma (IFN-gamma) and tumour necrosis factor-beta (TNF-beta) gene expression using the polymerase chain reaction (PCR). The cytokine gene expression of two cases of reactive adenopathy in patients not infected with HIV was assessed for comparison. IFN-gamma was expressed much more strongly in the PGL samples than in control reactive lymphoid tissues, whereas the other cytokines were expressed to a similar extent in both types of tissues. IFN-gamma may have an important role in maintaining the adenopathy of HIV-infected patients. Expression of cytokines such as IL-2, IL-4 and IL-10 in HIV nodes may be adequate to allow the recruitment of naive B cells to the reactive process.