RESUMEN

BACKGROUND: Normal skin contains no epidermal calprotectin. In biopsies from various inflammatory skin disorders, however, this antimicrobial protein occurs in the cytoplasm of keratinocytes. OBJECTIVES: To exclude the possibility of epidermal uptake of calprotectin from granulocytes and macrophages in diseased skin, we investigated whether cytokine-stimulated human keratinocytes can express calprotectin in vitro. METHODS: Keratinocytes from healthy individuals were cultured in serum-free keratinocyte medium. The cells were stimulated with different cytokines [interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-10 and IL-13], both separately and in various combinations. Cytoplasmic protein levels of calprotectin were measured by an enzyme-linked immunosorbent assay performed on fixed adherent keratinocytes, and mRNA expression was determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Calprotectin was produced by cytokine-stimulated keratinocytes, especially in response to combinations of the proinflammatory cytokines, which showed an additive upregulatory effect. When expression of mRNA for the light (MRP-8) and heavy (MRP-14) calprotectin chain was determined by RT-PCR, their respective levels were shown to be increased four- to ninefold and three- to fivefold after 24 h of combined stimulation with IFN-gamma and TNF-alpha. The time course of calprotectin production showed no significant elevation for the first 16 h but then increased and peaked after 36 h. CONCLUSIONS: Cultured human keratinocytes stimulated with proinflammatory cytokines produce calprotectin, suggesting that epidermal expression of this antimicrobial protein in diseased skin reflects compartmentalized synthesis rather than uptake from dermal inflammatory cells.

Asunto(s)

Citocinas/farmacología , Queratinocitos/metabolismo , Complejo de Antígeno L1 de Leucocito/metabolismo , Adulto , Células Cultivadas , Sinergismo Farmacológico , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Interferón gamma/farmacología , Interleucina-1/farmacología , ARN/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia Arriba

RESUMEN

Secretory Abs constitute the first line of specific immune defense at mucosal surfaces. Such Abs are generated by the active transport of polymeric Ig (pIg) across secretory epithelia mediated by the pIgR, also known as transmembrane secretory component (SC). The proinflammatory cytokine TNF-alpha is a key mediator of host responses to infections, and it can stimulate protein synthesis-dependent transcriptional up-regulation of pIgR/SC in the HT-29 intestinal adenocarcinoma cell line. By reporter gene assay we identified a novel TNF-alpha-responsive region located within a 748-bp fragment in intron 1 of the human pIgR/SC gene which depended on an NF-kappaB/Rel site for full responsiveness. EMSAs demonstrated preferential binding of the NF-kappaB/Rel family member p65 (RelA) to this DNA element after TNF-alpha stimulation, with weaker and more delayed binding of p50. Furthermore, the TNF-alpha-responsive region in intron 1 required cooperation with DNA elements located in the proximal promoter region of the gene. Mutational analysis demonstrated that an IFN-stimulated response element near the transcriptional start site in exon 1 was involved in the TNF-alpha responsiveness. Thus, DNA elements located >4 kb apart were found to cooperate in TNF-alpha-induced pIgR/SC up-regulation. The intronic TNF-alpha-responsive enhancer overlapped with a recently identified IL-4-responsive enhancer. Several intronic DNA elements found to be functionally important in the human gene are highly conserved between the human and mouse pIgR/SC genes, suggesting the presence of a conserved cytokine-responsive enhancer region.

Asunto(s)

Intrones/inmunología , FN-kappa B/genética , Regiones Promotoras Genéticas/inmunología , Proteínas Proto-Oncogénicas c-rel/genética , Receptores de Inmunoglobulina Polimérica/genética , Transcripción Genética/inmunología , Factor de Necrosis Tumoral alfa/fisiología , Composición de Base , Sitios de Unión/genética , Sitios de Unión/inmunología , Secuencia de Consenso , Regulación de la Expresión Génica/inmunología , Células HT29 , Humanos , Interferón gamma/genética , Intrones/fisiología , FN-kappa B/metabolismo , FN-kappa B/fisiología , Subunidad p50 de NF-kappa B , Regiones Promotoras Genéticas/fisiología , Proteínas Proto-Oncogénicas c-rel/metabolismo , Proteínas Proto-Oncogénicas c-rel/fisiología , Receptores de Inmunoglobulina Polimérica/metabolismo , Elementos de Respuesta/inmunología , Componente Secretorio/genética , Eliminación de Secuencia , Factor de Transcripción ReIA , Transfección

RESUMEN

The polymeric IgR (pIgR) mediates transport of dimeric IgA and pentameric IgM across mucosal epithelia, thereby generating secretory Abs. Its expression is up-regulated at the transcriptional level by IL-4 in HT-29 cells. In this study, we demonstrate that IL-4 mediates up-regulation of human pIgR through a 554-bp IL-4-responsive enhancer in intron 1. Mutation of a binding site for STAT-6 within this region abolished IL-4-induced enhancement, while an adjacent putative C/EBP site was dispensable. IL-4 treatment induced binding of STAT6 to the intronic STAT6 site, but cooperation with nearby upstream and downstream DNA elements was required for IL-4 responsiveness. Furthermore, IL-4-mediated increased transcription of the pIgR-derived enhancer, like the endogenous pIgR gene, required de novo protein synthesis. Interestingly, a conditionally active form of STAT6 sufficed to activate a pIgR-derived enhancer in HT-29 cells, but not in Cos-1 cells, suggesting a requirement for cell type-specific factors. Thus, STAT6 activation mediates a delayed transcriptional enhancement of pIgR by induction of a de novo synthesized protein that cooperates with STAT6 itself bound to its cognate DNA element in intron 1. This mechanism may represent a general strategy for how pleiotropic cytokines elicit cell type-specific transcriptional responses.

Asunto(s)

Interleucina-4/fisiología , Receptores de Inmunoglobulina Polimérica/biosíntesis , Elementos de Respuesta/inmunología , Transactivadores/fisiología , Transcripción Genética/inmunología , Regulación hacia Arriba/inmunología , Animales , Sitios de Unión/genética , Sitios de Unión/inmunología , Células COS , Chlorocebus aethiops , Cicloheximida/farmacología , Elementos de Facilitación Genéticos/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Genes Reporteros/inmunología , Vectores Genéticos/síntesis química , Vectores Genéticos/inmunología , Células HT29 , Humanos , Interleucina-4/antagonistas & inhibidores , Intrones/inmunología , Cinética , Luciferasas/antagonistas & inhibidores , Luciferasas/genética , Datos de Secuencia Molecular , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , Receptores de Inmunoglobulina Polimérica/antagonistas & inhibidores , Receptores de Inmunoglobulina Polimérica/genética , Secuencias Reguladoras de Ácidos Nucleicos/inmunología , Factor de Transcripción STAT6 , Factores de Tiempo , Transactivadores/metabolismo , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

The industrially important polysaccharide alginate is a linear copolymer of beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G). It is produced commercially by extraction from brown seaweeds, although some of the bacteria belonging to the genera Azotobacter and Pseudomonas also synthesize alginates. Alginates are synthesized as mannuronan, and varying amounts of the M residues in the polymer are then epimerized to G residues by mannuronan C-5-epimerases. The gel-forming, water-binding, and immunogenic properties of the polymer are dependent on the relative amount and sequence distribution of M and G residues. A family of seven calcium-dependent, secreted epimerases (AlgE1-7) from Azotobacter vinelandii have now been characterized, and in this paper the properties of all these enzymes are described. AlgE4 introduces alternating M and G residues into its substrate, while the remaining six enzymes introduce a mixture of continuous stretches of G residues and alternating sequences. Two of the enzymes, AlgE1 and AlgE3, are composed of two catalytically active domains, each introducing different G residue sequence patterns in alginate. These results indicate that the enzymes can be used for production of alginates with specialized properties.

Asunto(s)

Alginatos/química , Alginatos/metabolismo , Carbohidrato Epimerasas/metabolismo , Azotobacter vinelandii/enzimología , Azotobacter vinelandii/genética , Secuencia de Bases , Biotecnología , Carbohidrato Epimerasas/genética , Secuencia de Carbohidratos , Cartilla de ADN/genética , Diseño de Fármacos , Evolución Molecular , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular

RESUMEN

Secretory IgA (SIgA) is the best defined effector component of the mucosal immune system. Generation of SIgA and secretory IgM (SIgM) in exocrine glands and mucous membranes depends on a fascinating cooperation between local plasma cells that produce polymeric IgA (pIgA, mainly dimers and some larger polymers) and pentameric IgM, and secretory epithelial cells that express the polymeric Ig receptor (pIgR)--also known as transmembrane secretory component. After release from the local plasma cells and diffusion through the stroma, pIgA and pentameric IgM become readily bound to pIgR, and are then actively transported across secretory epithelial cells for extrusion into external secretions after cleavage of pIgR. Much knowledge has recently been obtained at the molecular level about the regulation of pIgR-mediated transport of antibodies. This mechanism is of considerable biological interest because SIgA and SIgM form the first line of specific immunological defense against infectious agents and other harmful substances that may enter the body through the mucosae.

Asunto(s)

Inmunoglobulina A Secretora/biosíntesis , Inmunoglobulina M/biosíntesis , Receptores de Inmunoglobulina Polimérica/fisiología , Animales , Células Productoras de Anticuerpos/fisiología , Citocinas/fisiología , Regulación de la Expresión Génica , Humanos , Inmunoglobulina A/clasificación , Inmunoglobulina A Secretora/química , Cadenas J de Inmunoglobulina/fisiología , Inmunoglobulina M/química , Receptores de Inmunoglobulina Polimérica/biosíntesis , Receptores de Inmunoglobulina Polimérica/química , Componente Secretorio/fisiología , Factores de Transcripción/fisiología

RESUMEN

Previous studies have indicated that the presence of an E2F site is not sufficient for G1/S phase transcriptional regulation. For example, the E2F sites in the E2F1 promoter are necessary, but not sufficient, to mediate differential promoter activity in G0 and S phase. We have now utilized the E2F1 minimal promoter to test several hypotheses that could account for these observations. To test the hypothesis that G1/S phase regulation is achieved via E2F-mediated repression of a strong promoter, a variety of transactivation domains were brought to the E2F1 minimal promoter. Although many of these factors caused increased promoter activity, growth regulation was not observed, suggesting that a general repression model is incorrect. However, constructs having CCAAT or YY1 sites or certain GC boxes cloned upstream of the E2F1 minimal promoter displayed E2F site-dependent regulation. Further analysis of the promoter activity suggested that E2F requires cooperation with another factor to activate transcription in S phase. However, we found that the requirement for E2F to cooperate with additional factors to achieve growth regulation could be relieved by bringing the E2F1 activation domain to the promoter via a Gal4 DNA binding domain. Our results suggest a model that explains why some, but not all, promoters that contain E2F sites display growth regulation.

Asunto(s)

Proteínas Portadoras , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Proteínas de Unión al ADN , Factores de Transcripción/metabolismo , Células 3T3 , Animales , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Genes Reporteros , Homeostasis , Ratones , Modelos Biológicos , Oligodesoxirribonucleótidos , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Fase de Descanso del Ciclo Celular , Proteína 1 de Unión a Retinoblastoma , Fase S , Factor de Transcripción DP1 , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Activación Transcripcional , Transfección