RESUMEN
The spatial distribution of defect related deep band emission has been studied in zinc oxide (ZnO) nano- and microwires using depth resolved cathodoluminescence spectroscopy (DRCLS) in a hyperspectral imaging (HSI) mode within a UHV scanning electron microscope (SEM). Three sets of wires were examined that had been grown by pulsed laser deposition or vapor transport methods and ranged in diameter from 200 nm-2.7 µm. This data was analyzed by developing a 3D DRCLS simulation and using it to estimate the segregation depth and decay profile of the near surface defects. We observed different dominant defects from each growth process as well as diameter-dependent defect segregation behavior.
RESUMEN
The synchrotron x-ray absorption near edge structures (XANES) technique was used in conjunction with first-principles calculations to characterize Al-doped ZnO films. Standard characterizations revealed that the amount of carrier concentration and mobility depend on the growth conditions, i.e. H(2) (or O(2))/Ar gas ratio and Al concentration. First-principles calculations showed that Al energetically prefers to substitute on the Zn site, forming a donor Al(Zn), over being an interstitial (Al(i)). The measured Al K-edge XANES spectra are in good agreement with the simulated spectra of Al(Zn), indicating that the majority of Al atoms are substituting for Zn. The reduction in carrier concentration or mobility in some samples can be attributed to the Al(Zn)-V(Zn) and 2Al(Zn)-V(Zn) complex formations that have similar XANES features. In addition, XANES of some samples showed additional features that are the indication of some α-Al(2)O(3) or nAl(Zn)-O(i) formation, explaining their poorer conductivity.
RESUMEN
ZnO nanorods were grown on a variety of substrates such as Si, SiO(2)/Si and sapphire in a large-area pulsed laser deposition chamber designed for sensor device fabrication. Processing conditions were optimized to grow ZnO nanorods with or without seed layers. Au, Cr and BaSrTiO(3) (BST) seed layers were investigated to compare their effects on the diameter and orientation of ZnO nanorods. ZnO nanorods were observed to align better when grown on sapphire, Cr or BST seed layers as compared to Au or Si layers. The highest quality nanorods were those grown on BST seed layers, as shown by 4 K photoluminescence donor-bound-exciton linewidths of only 0.5 meV.
RESUMEN
Recent theory has found that native defects such as the O vacancy V(O) and Zn interstitial Zn(I) have high formation energies in n-type ZnO and, thus, are not important donors, especially in comparison to impurities such as H. In contrast, we use both theory and experiment to show that, under N ambient, the complex Zn(I)-N(O) is a stronger candidate than H or any other known impurity for a 30 meV donor commonly found in bulk ZnO grown from the vapor phase. Since the Zn vacancy is also the dominant acceptor in such material, we must conclude that native defects are important donors and acceptors in ZnO.
RESUMEN
We have used positron annihilation spectroscopy to determine the nature and the concentrations of the open volume defects in as-grown and electron irradiated (E(el)=2 MeV, fluence 6 x 10(17) cm(-2)) ZnO samples. The Zn vacancies are identified at concentrations of [V(Zn)] approximately 2 x 10(15) cm(-3) in the as-grown material and [V(Zn)] approximately 2 x 10(16) cm(-3) in the irradiated ZnO. These concentrations are in very good agreement with the total acceptor density determined by temperature dependent Hall experiments. Thus, the Zn vacancies are dominant acceptors in both as-grown and irradiated ZnO.
RESUMEN
To elucidate molecular mechanisms underlying the association between respiratory viral infection and predisposition to subsequent bacterial infection, we used in vivo and in vitro models and human samples to characterize respiratory virus-induced changes in airway epithelial cell morphology, gene expression, and mucociliary function. Mouse paramyxoviral bronchitis resulted in airway epithelial cell infection and a distinct pattern of epithelial cell morphology changes and altered expression of the differentiation markers beta-tubulin-IV, Clara cell secretory protein, and Foxj1. Furthermore, changes in gene expression were recapitulated using an in vitro epithelial cell culture system and progressed independent of the host inflammatory response. Restoration of mature airway epithelium occurred in a pattern similar to epithelial cell differentiation and ciliogenesis in embryonic lung development characterized by sequential proliferation of undifferentiated cells, basal body production, Foxj1 expression, and beta-tubulin-IV expression. The effects of virus-induced alterations in morphology and gene expression on epithelial cell function were illustrated by decreased airway mucociliary velocity and impaired bacterial clearance. Similar changes in epithelial cell Foxj1 expression were also observed in human paramyxoviral respiratory infection. Taken together, these model systems of paramyxoviral respiratory infection mimic human pathology and identify epithelial cell Foxj1 expression as an early marker of epithelial cell differentiation, recovery, and function.
Asunto(s)
Cilios/fisiología , Proteínas de Unión al ADN , Células Epiteliales/metabolismo , Pulmón/metabolismo , Depuración Mucociliar/fisiología , Infecciones por Respirovirus/fisiopatología , Transactivadores/genética , Uteroglobina , Animales , División Celular/fisiología , Línea Celular , Células Cultivadas , Células Epiteliales/ultraestructura , Células Epiteliales/virología , Factores de Transcripción Forkhead , Regulación de la Expresión Génica , Humanos , Pulmón/citología , Pulmón/fisiopatología , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Proteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/fisiopatología , Mucosa Respiratoria/virología , Respirovirus , Infecciones por Respirovirus/metabolismo , Infecciones por Respirovirus/virología , Tráquea/citología , Tráquea/metabolismo , Tráquea/fisiopatología , Tubulina (Proteína)/genéticaRESUMEN
Adenoviral evolution has generated strategies to resist host cell antiviral systems, but molecular mechanisms for evasion of interferon (IFN) effects by adenoviruses during late-phase infection are poorly defined. In this study, we examined adenovirus type 5 (AdV) effects on IFN-gamma-dependent gene expression and Janus family kinase-signal transducer and activator of transcription signaling components in human tracheobronchial epithelial cells. We found that AdV infection specifically inhibited IFN-gamma-dependent gene expression in airway epithelial cells without evidence of epithelial cell injury or generation of a soluble extracellular inhibitor. Furthermore, infection with AdV for 18-24 h blocked phosphorylation/activation of the Stat1 transcription factor that regulates IFN-gamma-dependent genes. Although AdV also inhibited IFN-alpha-dependent phosphorylation of Stat1 and Stat2, interleukin-4-dependent phosphorylation of the related transcription factor Stat6 was not affected, indicating that the virus selectively affected specific signaling pathways. Our results indicate that AdV inhibition of the IFN-gamma signal transduction cascade occurs through loss of ligand-induced receptor complex assembly and consequent component phosphorylation and suggest that lack of complex assembly is due to decreased expression of the IFN-gammaR2 chain of the IFN-gamma receptor. IFN-gammaR2 is required at an early step in Janus family kinase-signal transducer and activator of transcription pathway activation and is expressed at low levels in airway epithelial cells, supporting the concept that adenoviral down-regulation of the level of this IFN-gamma receptor component allows for persistent modulation of IFN-gamma-dependent gene expression.
Asunto(s)
Adenoviridae/genética , Interferones/metabolismo , Transducción de Señal , Animales , Apoptosis , Línea Celular , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Células Epiteliales/metabolismo , Células Epiteliales/virología , Citometría de Flujo , Regulación de la Expresión Génica , Humanos , Immunoblotting , Interferón gamma/metabolismo , Interferones/antagonistas & inhibidores , Interleucina-4/metabolismo , Janus Quinasa 1 , Ratones , Fosforilación , Pruebas de Precipitina , Proteínas Tirosina Quinasas/metabolismo , ARN Mensajero/metabolismo , Factor de Transcripción STAT1 , Factor de Transcripción STAT2 , Factor de Transcripción STAT6 , Factores de Tiempo , Tráquea/metabolismo , Tráquea/virología , Transactivadores/metabolismoAsunto(s)
Fibrosis Quística/complicaciones , Neumonía Bacteriana/complicaciones , Infecciones por Pseudomonas/complicaciones , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Humanos , Neumonía Bacteriana/metabolismo , Infecciones por Pseudomonas/metabolismoRESUMEN
The thin layer of airway surface liquid (ASL) contains antimicrobial substances that kill the small numbers of bacteria that are constantly being deposited in the lungs. An increase in ASL salt concentration inhibits the activity of airway antimicrobial factors and may partially explain the pathogenesis of cystic fibrosis (CF). We tested the hypothesis that an osmolyte with a low transepithelial permeability may lower the ASL salt concentration, thereby enhancing innate immunity. We found that the five-carbon sugar xylitol has a low transepithelial permeability, is poorly metabolized by several bacteria, and can lower the ASL salt concentration in both CF and non-CF airway epithelia in vitro. Furthermore, in a double-blind, randomized, crossover study, xylitol sprayed for 4 days into each nostril of normal volunteers significantly decreased the number of nasal coagulase-negative Staphylococcus compared with saline control. Xylitol may be of value in decreasing ASL salt concentration and enhancing the innate antimicrobial defense at the airway surface.
Asunto(s)
Bacterias/efectos de los fármacos , Bronquios/efectos de los fármacos , Sales (Química)/química , Tráquea/efectos de los fármacos , Xilitol/farmacología , Adulto , Bronquios/química , Bronquios/microbiología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Cloruros/metabolismo , Recuento de Colonia Microbiana , Fibrosis Quística/metabolismo , Fibrosis Quística/microbiología , Células Epiteliales/química , Células Epiteliales/efectos de los fármacos , Células Epiteliales/microbiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mucosa Nasal/efectos de los fármacos , Mucosa Nasal/microbiología , Concentración Osmolar , Tráquea/química , Tráquea/microbiología , Xilitol/químicaRESUMEN
Epithelial cells interact directly with bacteria in the environment and play a critical role in airway defense against microbial pathogens. In this study, we examined the response of respiratory epithelial cells to infection with nontypable Haemophilus influenzae. Using an in vitro cell culture model, we found that epithelial cell monolayers released significant quantities of IL-8 and expressed increased levels of ICAM-1 mRNA and surface protein in response to H. influenzae. In contrast, levels of IL-1beta, TNF-alpha, and MHC class I were not significantly affected, suggesting preferential activation of a specific subset of epithelial genes directed toward defense against bacteria. Induction of ICAM-1 required direct bacterial interaction with the epithelial cell surface and was not reproduced by purified H. influenzae lipooligosaccharide. Consistent with a functional role for this response, induction of ICAM-1 by H. influenzae mediated increased neutrophil adherence to the epithelial cell surface. Furthermore, in an in vivo murine model of airway infection with H. influenzae, increased epithelial cell ICAM-1 expression coincided with increased chemokine levels and neutrophil recruitment in the airway. These results indicate that ICAM-1 expression on human respiratory epithelial cells is induced by epithelial cell interaction with H. influenzae and suggest that an ICAM-1-dependent mechanism can mediate neutrophil adherence to these cells independent of inflammatory mediator release by other cell types. Direct induction of specific epithelial cell genes (such as ICAM-1 and IL-8) by bacterial infection may allow for rapid and efficient innate defense in the airway.
Asunto(s)
Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Haemophilus influenzae/inmunología , Molécula 1 de Adhesión Intercelular/biosíntesis , Pulmón/metabolismo , Pulmón/microbiología , Adhesinas Bacterianas/fisiología , Animales , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Antígenos de Superficie/metabolismo , Adhesión Celular/inmunología , Comunicación Celular/inmunología , Línea Celular , Movimiento Celular/inmunología , Células Epiteliales/inmunología , Infecciones por Haemophilus/inmunología , Infecciones por Haemophilus/metabolismo , Infecciones por Haemophilus/patología , Haemophilus influenzae/fisiología , Humanos , Molécula 1 de Adhesión Intercelular/fisiología , Interleucina-8/metabolismo , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Pulmón/inmunología , Ratones , Ratones Endogámicos C57BL , Pseudomonas aeruginosa/inmunologíaRESUMEN
Analysis of the host response to viral infection generally has focused on the capacity of viruses to activate or repress transcription of cellular genes, and this approach is also characteristic of work on RNA viruses such as respiratory syncytial virus (RSV). In the present study, it appeared initially that RSV-driven expression of a critical immune regulator, the beta-chemokine RANTES (regulated upon activation, normal T cell expressed and secreted), in primary-culture airway epithelial cells also depended on inducible gene transcription because expression was accompanied by coordinate increases in transcriptional initiation rate and gene promoter activity. However, RSV-driven increases in RANTES gene transcription and promoter activity were small and transient relative to RANTES expression, and they were no different in size and duration than for inactivated RSV that was incapable of fully inducing RANTES expression. These findings suggested that the increase in RANTES gene transcription was not sufficient for inducible expression and that critical regulatory effects occurred at a posttranscriptional level. This type of mechanism for virus-inducible expression of RANTES was established when we found that replicating (but not inactivated) RSV markedly increased RANTES mRNA half-life (from 0.8 to 6.8 h). In addition, RNase protection assays of heterologous promoter/reporter plasmids indicate that basal instability of RANTES mRNA is mediated at least in part by nucleotides 11-389 of the RANTES gene, and this region is also the target for induction by virus. The distinct pathway for production of RANTES (in combination with cytokine-dependent expression of RANTES and related immune-response genes) may more effectively coordinate immune cell interaction with epithelial barrier cells to mediate host defense.
Asunto(s)
Quimiocina CCL5/genética , ARN Mensajero/genética , Virus Sincitiales Respiratorios/genética , Replicación Viral/genética , Bronquios/inmunología , Bronquios/metabolismo , Células Cultivadas , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Cinética , Regiones Promotoras Genéticas , Virus Sincitiales Respiratorios/inmunología , Tráquea/inmunología , Tráquea/metabolismo , Transcripción GenéticaRESUMEN
Cytokine effects on immunity and inflammation often depend on the transcription factors termed signal transducers and activators of transcription (STATs), so STAT signaling pathways are candidates for influencing inflammatory disease. We reasoned that selective IFN responsiveness of the first STAT family member (Stat1) and Stat1-dependent immune-response genes such as intercellular adhesion molecule-1 (ICAM-1), IFN regulatory factor-1 (IRF-1), and Stat1 itself in airway epithelial cells provides a basis for detecting cytokine signaling abnormalities in inflammatory airway disease. On the basis of nuclear localization and phosphorylation, we found that epithelial Stat1 (but not other control transcription factors) was invariably activated in asthmatic compared with normal control or chronic bronchitis subjects. Furthermore, epithelial levels of activated Stat1 correlated with levels of expression for epithelial ICAM-1, IRF-1, and Stat1, and in turn, ICAM-1 levels correlated with T-cell accumulation in tissue. However, only low levels of IFN-gamma or IFN-gamma-producing cells were detected in airway tissue in all subjects. The results therefore provide initial evidence linking abnormal behavior of STAT pathways for cytokine signaling to the development of an inflammatory disease. In that context, the results also change the current scheme for asthma pathogenesis to one that must include a localized gain in transcriptional signal ordinarily used for a T helper 1-type cytokine (IFN-gamma) in combination with allergy-driven overproduction of T helper 2-type cytokines.
Asunto(s)
Asma/metabolismo , Proteínas de Unión al ADN/metabolismo , Transactivadores/metabolismo , Asma/inmunología , Secuencia de Bases , Bronquios/citología , Bronquios/metabolismo , Estudios de Casos y Controles , Cartilla de ADN , Células Epiteliales/metabolismo , Humanos , Interferón gamma/metabolismo , Factor de Transcripción STAT1 , Células TH1/inmunologíaRESUMEN
Immune cell migration into and through mucosal barrier sites in general and airway sites in particular is a critical feature of immune and inflammatory responses, but the determinants of transepithelial (unlike transendothelial) immune cell traffic are poorly defined. Accordingly, we used primary culture airway epithelial cells and peripheral blood mononuclear cells to develop a cell monolayer system that allows for apical-to-basal and basal-to-apical T cell transmigration that can be monitored with quantitative immunofluorescence flow cytometry. In this system, T cell adhesion and subsequent transmigration were blocked in both directions by monoclonal antibodies (mAbs) against lymphocyte function-associated antigen 1 (LFA-1) or intercellular adhesion molecule 1 (ICAM-1) (induced by interferon gamma [IFN-gamma] treatment of epithelial cells). The total number of adherent plus transmigrated T cells was also similar in both directions, and this pattern fit with uniform presentation of ICAM-1 along the apical and basolateral cell surfaces. However, the relative number of transmigrated to adherent T cells (i.e., the efficiency of transmigration) was increased in the basal-to-apical relative to the apical-to-basal direction, so an additional mechanism was needed to mediate directional movement towards the apical surface. Screening for epithelial-derived beta-chemokines indicated that IFN-gamma treatment caused selective expression of RANTES (regulated upon activation, normal T cell expressed and secreted), and the functional significance of this finding was demonstrated by inhibition of epithelial-T cell adhesion and transepithelial migration by anti-RANTES mAbs. In addition, we found that epithelial (but not endothelial) cells preferentially secreted RANTES through the apical cell surface thereby establishing a chemical gradient for chemotaxis across the epithelium to a site where they may be retained by high levels of RANTES and apical ICAM-1. These patterns for epithelial presentation of ICAM-1 and secretion of RANTES appear preserved in airway epithelial tissue studied either ex vivo with expression induced by IFN-gamma treatment or in vivo with endogenous expression induced by inflammatory disease (i.e., asthma). Taken together, the results define how the patterns for uniform presentation of ICAM-1 along the cell surface and specific apical sorting of RANTES may serve to mediate the level and directionality of T cell traffic through epithelium (distinct from endothelium) and provide a basis for how this process is precisely coordinated to route immune cells to the mucosal surface and maintain them there under normal and stimulated conditions.
Asunto(s)
Quimiocina CCL5/metabolismo , Quimiotaxis de Leucocito , Células Epiteliales/inmunología , Molécula 1 de Adhesión Intercelular/biosíntesis , Linfocitos T/inmunología , Tráquea/inmunología , Adhesión Celular , Polaridad Celular , Células Cultivadas , Células Epiteliales/citología , Humanos , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Membrana Mucosa/citología , Membrana Mucosa/inmunología , Tráquea/citologíaRESUMEN
A major goal of our research is to understand how immune cells (especially T cells) infiltrate the pulmonary airway during host defense and inflammatory disease (especially asthma). In that context, we have proposed that epithelial cells lining the airway provide critical biochemical signals for immune-cell influx and activation and that this epithelial-immune cell interaction is a critical feature of airway inflammation and hyperreactivity. In this brief report, we describe our progress in defining a subset of epithelial immune-response genes the expression of which is coordinated for viral defense both directly in response to replicating virus and indirectly under the control of a specific interferon-gamma signal transduction pathway featuring the Stat1 transcription factor as a critical relay signal between cytoplasm and nucleus. Unexpectedly, the same pathway is also activated during asthmatic airway inflammation in a setting where there is no apparent infection and no increase in interferon-gamma levels. The findings provide the first evidence of an overactive Stat1-dependent gene network in asthmatic airways and a novel molecular link between mucosal immunity and inflammation. The findings also offer the possibility that overactivity of Stat1-dependent genes might augment a subsequent T helper cell (Th1)-type response to virus or might combine with a heightened Th2-type response to allergen to account for more severe exacerbations of asthma.
Asunto(s)
Asma/inmunología , Mucosa Nasal/inmunología , Animales , Células Epiteliales/inmunología , Regulación de la Expresión Génica , Humanos , Inmunidad Mucosa/genética , Inmunidad Mucosa/inmunología , Inflamación , Linfocitos T/inmunologíaRESUMEN
The action of adenoviral E1A oncoprotein on host immune-response genes has been attributed to interaction with p300/CBP-type transcriptional coactivators in competition with endogenous transcription factors such as signal transducer and activator of transcription (STAT) proteins. However, we show that mutant forms of E1A that no longer bind p300/CBP can still interact directly with Stat1 (via E1A N-terminal and Stat1 C-terminal residues) and block IFNgamma-driven, Stat1-dependent gene activation and consequent function during early-phase infection in the natural host cell. The results provide a distinct and more specific mechanism for E1A-mediated immune suppression and an alternative model of IFNgamma-driven enhanceosome formation that may allow for other adaptors (in addition to p300/CBP) to link Stat1 to the basal transcription complex.
Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , Proteínas de Unión al ADN/fisiología , Transactivadores/fisiología , Activación Transcripcional , Proteínas E1A de Adenovirus/genética , Adenovirus Humanos/fisiología , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interferón gamma/farmacología , Proteínas Nucleares/metabolismo , Factor de Transcripción STAT1 , Linfocitos T/efectos de los fármacos , Transactivadores/metabolismoRESUMEN
A subset of epithelial immune-response genes (including intercellular adhesion molecule-1 (ICAM-1)) depends on an IFN-gamma signal transduction pathway with the Stat1 transcription factor as a critical intermediate. Excessive local activation of this pathway may lead to airway inflammation, so we sought to selectively down-regulate the pathway using a dominant-negative strategy for inhibition of epithelial Stat1 in a primary culture airway epithelial cell model. Using a Stat1-deficient cell line, we demonstrated that transfection of wild-type Stat1 expression plasmid restored appropriate Stat1 expression and IFN-gamma-dependent phosphorylation as well as consequent IFN-gamma activation of cotransfected ICAM-1 promoter constructs and endogenous ICAM-1 gene expression. However, mutations of Stat1 at Tyr-701 (JAK kinase phosphorylation site), Glu-428/429 (putative DNA-binding site), His-713 (splice site resulting in Stat1beta formation), or Ser-727 (MAP kinase phosphorylation site) all decreased Stat1 capacity to activate the ICAM-1 promoter. The Tyr-701 mutant (followed by the His-713 mutant) were most effective in disabling Stat1 function and in overcoming the activating effect of cotransfected wild-type Stat1 in this cell system thereby highlighting the effectiveness of blocking Stat1 homo- and hetero-dimerization. In experiments using primary culture human tracheobronchial epithelial cells (hTBECs) and each of the four Stat1 mutant plasmids, transfection with the Tyr-701 and His-713 mutants again most effectively inhibited IFN-gamma activation of an ICAM-1 gene promoter construct. Then by transfecting hTBECs with wild-type or mutant Stat1 tagged with a Flag reporter sequence, we used dual immunofluorescence to show that hTBECs expressing the Tyr-701 or His-713 mutants were prevented from expressing endogenous ICAM-1 in response to IFN-gamma treatment. The capacity of a specific Stat1 mutations to exert a potent dominant-negative effect on IFN-gamma signal transduction provides for further definition of Stat1 structure function and a means for natural or engineered expression of mutant Stat1 to selectively down-regulate activity of this pathway in a cell type- or tissue-specific manner during immune and/or inflammatory responses.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Regulación de la Expresión Génica/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/biosíntesis , Interferón gamma/farmacología , Transactivadores/antagonistas & inhibidores , Sustitución de Aminoácidos , Proteínas de Unión al ADN/genética , Humanos , Molécula 1 de Adhesión Intercelular/genética , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas , Factor de Transcripción STAT1 , Relación Estructura-Actividad , Transactivadores/genéticaRESUMEN
STAT (signal transducer and activator of transcription) proteins combine with cytokine receptors and receptor-associated kinases in distinct protein/protein interactions that are critical for STAT-dependent signal transduction events, but the nature of any subsequent STAT interactions with DNA-binding proteins in the nucleus is less certain. Based on assays of DNA/protein binding and activity of transfected reporter plasmids, we determined that occupation of contiguous DNA-binding sites for Stat1 (the first member of the STAT family) and the transcriptional activator Sp1 are both required for full activation of the intercellular adhesion molecule-1 gene by interferon-gamma. Thus, Stat1 binding to DNA cannot by itself be equated with biologic actions of Stat1. In co-immunoprecipitation experiments, we also obtained evidence of direct and selective Stat1/Sp1 interaction (in primary culture cells without overexpression), further indicating that Stat1/Sp1 synergy confers an element of specificity in the pathway leading to cytokine-activated transcription and cytokine-dependent immunity and inflammation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Molécula 1 de Adhesión Intercelular/biosíntesis , Transactivadores/metabolismo , Transcripción Genética , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Cloranfenicol O-Acetiltransferasa/análisis , Cloranfenicol O-Acetiltransferasa/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Expresión Génica/efectos de los fármacos , Humanos , Molécula 1 de Adhesión Intercelular/genética , Interferón gamma/farmacología , Luciferasas/análisis , Luciferasas/biosíntesis , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes , Secuencias Repetitivas de Ácidos Nucleicos , Factor de Transcripción STAT1 , Transducción de Señal , Transactivadores/biosíntesis , TransfecciónRESUMEN
To define the relationship between T cell phenotype and adhesiveness, we examined T cell adhesion to endothelial cell, fibroblast, and epithelial cell monolayers as well as extracellular matrix proteins (collagen and fibronectin) using a three-color flow cytometry-based adherence assay that minimizes basal adhesion levels and facilitates quantitative lymphocyte subtyping. Regardless of monolayer type, monolayer stimulation conditions, or T cell activation status, we found that the gamma delta-TCR-bearing T cells adhered more efficiently than alpha beta T cells. The difference was based predominantly on increased levels of activatable LFA-1 (and to a lesser degree VLA-4) because: 1) it correlated precisely with inhibitability by anti-LFA-1 (and VLA-4) mAbs and the levels of LFA-1 (and VLA-4) on the cell surface, and 2) it persisted after maximal LFA-1 (and VLA-4) activation with phorbol dibutyrate. In contrast to most cases of alpha beta T cell behavior, gamma delta T cell adhesion to cell monolayers was not linked to memory status, i.e., there was no difference between naive V delta 1+ and memory V delta 2+ populations in levels of LFA-1 (or VLA-4) expression or LFA-1- (or VLA-4-) dependent adhesion to cell monolayers. However, V delta 1+ cells exhibited higher levels of VLA-5 that correlated with an increased adhesiveness to fibronectin and to a 120-kDa fibronectin fragment (FN-120) that contains only the VLA-5-binding domain but not to type I collagen or to a fibronectin fragment (FN-40) that binds only VLA-4. Taken together, the results define a hierarchy for integrin (LFA-1, VLA-4, and VLA-5) expression and consequent adhesion among T cell subsets that is linked to TCR gene usage (but not necessarily linked to memory status) and may thereby help to explain the accumulation and retention of V delta 1+ gamma delta T cells in epithelial and connective tissues.