RESUMEN
Inducible nitric-oxide synthase (iNOS) is an important signaling protein involved in the regulation of biological processes (e.g. vasodilation, inflammation) and is subject to transcriptional regulation by cytokines and lipopolysaccharide (LPS). Full activation of the human iNOS (hiNOS) promoter by cytokines (i.e., tumor necrosis factor-alpha, interleukin-1beta, interferon-gamma (IFN-gamma)) required downstream and upstream nuclear factor-kappaB (-115, -8283) and activator protein-1 (AP-1) (-5115, -5301) transcription factor binding sites. Human lung epithelial (A549) cells were transiently transfected with luciferase reporter plasmids containing an 8.3-kilobase human iNOS promoter to examine the molecular signaling events necessary for hiNOS transcriptional activation. The combination of LPS and IFN-gamma, but neither alone, increased hiNOS promoter activity 28-fold, in a reaction requiring two critical AP-1 (JunD-Fra-2) promoter binding sites. Mitogen-activated protein kinases (MAPKs) were assessed as potential activators of AP-1 and the hiNOS promoter. Both pharmacological and molecular inhibitors of the extracellular signal-related kinase (ERK) and p38 pathways reduced cytokine mixture (CM)- and LPS/IFN-gamma-induced promoter activation. By gel retardation analysis, the addition of MAP/ERK kinase-1 and p38 inhibitors significantly diminished AP-1 binding in both CM- and LPS/IFN-gamma-stimulated cells. Thus, p38- and ERK-dependent pathways, through effects on the AP-1 complex, activate the hiNOS promoter in cells stimulated with CM or LPS/IFN-gamma.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/fisiología , Óxido Nítrico Sintasa/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas , Factor de Transcripción AP-1/fisiología , Sitios de Unión , Flavonoides/farmacología , Humanos , Imidazoles/farmacología , Interferón gamma/farmacología , Janus Quinasa 2 , Lipopolisacáridos/farmacología , MAP Quinasa Quinasa 1 , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Tirosina Quinasas/fisiología , Piridinas/farmacología , Células Tumorales Cultivadas , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
A transgenic (Tg) mouse expressing human IL-15 was generated to define the role of IL-15 in the normal immune response. Overexpression of IL-15 resulted in an increase of NK, CD44(hi)CD8 memory T cells, and gammadelta T cells. Additionally, we observed the emergence of a novel type of NK-T cells with CD8alphaalpha' expression. Due to the expansion and activation of NK cells, the IL-15Tg mouse showed enhanced innate immunity. In adaptive T cell immunity, the roles of IL-15 contrasted with those of IL-2. IL-15 inhibited IL-2-induced T cell death, which plays a role in the maintenance of peripheral self-tolerance. IL-15 thus seems to contribute to enhanced immune memory by selectively propagating memory T cells and by blocking T cell death mediated by IL-2.
Asunto(s)
Muerte Celular/fisiología , Interleucina-15/fisiología , Interleucina-2/fisiología , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Ciclo Celular , Citocinas/biosíntesis , Humanos , Interleucina-15/genética , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Recuento de Linfocitos , Ratones , Ratones Transgénicos , TransgenesRESUMEN
The involvement of AP-1 and NF-kappaB transcription factors in cytokine-mediated induction of human inducible nitric oxide synthase (hiNOS) promoter activity was examined. Luciferase reporter plasmids, containing mutations in AP-1 and NF-kappaB sites, in a hiNOS promoter extending from -8.3 kilobase pairs (kb) to +168, were transiently expressed in A549 cells, and promoter activity was determined after treatment with a cytokine mixture (CM) containing interleukin 1-beta, interferon-gamma, and tumor necrosis factor-alpha. Mutation of the AP-1 heptad located -5301 base pairs upstream decreased gene activation by 90% in a -8.3-kb promoter and a shorter -5.574-kb promoter. Disruption of AP-1 (at -5115) or NF-kappaB (at -115 and -8283) sites reduced promoter activity by 45, 67, and 52%, respectively. Responsiveness to CM was decreased by 85% in constructs mutated in both NF-kappaB sites. By gel retardation analyses, CM increased AP-1- and NF-kappaB binding. Supershift analysis identified Jun D and Fra-2 as components of AP-1 complexes. Each kappaB site bound different complements of NF-kappaB/Rel family members (downstream site, Rel A/p50; upstream site, Rel A/Rel A). Rel A was maximally, whereas IkappaB-alpha was minimally, expressed in nuclei after 1 h of CM treatment, corresponding with the peak in NF-kappaB inding activity. Thus, AP-1 and NF-kappaB are important cis-elements for induction of hiNOS gene transcription.
Asunto(s)
Citocinas/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , FN-kappa B/metabolismo , Óxido Nítrico Sintasa/genética , Factor de Transcripción AP-1/metabolismo , Transcripción Genética/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Western Blotting , Cartilla de ADN , Humanos , Óxido Nítrico Sintasa de Tipo II , Regiones Promotoras Genéticas , ARN Mensajero/genética , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
Expression of human inducible nitric oxide synthase (hiNOS) is under cytokine control and is transcriptionally regulated. The hiNOS and mouse iNOS (miNOS) genes are regulated differently by cytokines. To understand better the transcriptional regulation of the hiNOS gene, the 8.3-kb hiNOS promoter was characterized. Promoter activity was evaluated by transient transfection of hiNOS luciferase constructs in A549 human alveolar type II epithelium-like cells in the presence and absence of cytokines (IFN-gamma, IL-1 beta, and TNF-alpha). Important cytokine-responsive elements are located at -3665 to -5574 bp (containing two perfectly matched AP-1 sites which are not present in miNOS promoter) and -8093 to -8296 bp (one perfectly matched NF-kappa B site) of the hiNOS promoter region. Likely, these two AP-1 sites and the upstream NF-kappa B site are important in the transcriptional induction of hiNOS by cytokines. Our data demonstrate the molecular basis for the different cytokine-stimulated characteristics of hiNOS and miNOS genes.
Asunto(s)
Citocinas/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa/genética , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Células Epiteliales , Humanos , Interferón gamma/farmacología , Interleucina-1/farmacología , Luciferasas/biosíntesis , Ratones , Datos de Secuencia Molecular , Óxido Nítrico Sintasa de Tipo II , Alveolos Pulmonares , ARN Mensajero/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Transcripción Genética , Transfección , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Superoxide dismutases play an important protective role in the lung defense against the pro-oxidative effect of fibrous dusts (e.g. crocidolite fibers). Particularly crocidolite, but also other asbestos fibers, are known to induce cellular antioxidant defense. Although rockwool, a man-made fiber made from rock, is used widely for insulation purposes, its effects on the superoxide dismutases in bronchoepithelial cells have not been investigated. Thus, the purpose of this study was to determine whether human bronchoepithelial cells (BEAS 2B) respond to rockwool fibers (115-4 experimental rockwool fiber) by induction of MnSOD mRNA and an increase of MnSOD activity levels. The results were compared with BEAS 2B cells exposed to silica (alpha-quartz: DQ12; SiO2) and UICC (Union Internationale Contre le Cancer) crocidolite (concentrations of all dusts: 0, 2, 5, 10, 25, 50 micrograms/cm2 = 0, 2.4, 6, 12, 30, 60 micrograms/ml; 24-h exposure) as control fibers. Scanning electron microscopy confirmed close dust cell contact under all experimental settings. Very low MnSOD mRNA baseline levels rose significantly (p < 0.001) in BEAS 2B cells exposed to all three dusts at 2 micrograms/cm2. However, at > 25 micrograms/cm2 MnSOD mRNA levels in silica- and crocidolite- but not in rockwool-exposed cells decreased. Slight (no significance) increases of MnSOD activity were observed which decreased at higher dust (> 5 micrograms/cm2) concentrations. These results suggest that: (1) like crocidolite and silica, rockwool accelerates MnSOD gene expression in bronchoepithelial cells; (2) an increase of MnSOD mRNA levels is not accompanied by MnSOD activity elevation; (3) in contrast to rockwool, high concentrations (> or = 25 micrograms/cm2) of crocidolite and silica reduced MnSOD activity and MnSOD mRNA levels. Because oxidants (H2O2) and crocidolite fibers were shown to reduce SOD activity, lack of active MnSOD protein may be caused by inactivation on a post-translational level. Furthermore, the decline of MnSOD mRNA and MnSOD activity levels coincides with increasing cytotoxicity. In conclusion, rockwool was demonstrated to induce MnSOD gene expression, perhaps because of its pro-oxidative effect in bronchoepithelial cells. In contrast to crocidolite and silica, rockwool fibers are not cytotoxic in this experimental setting.
Asunto(s)
Bronquios/enzimología , Células Epiteliales/enzimología , Regulación de la Expresión Génica/efectos de los fármacos , Fibras Minerales/toxicidad , Dióxido de Silicio/toxicidad , Superóxido Dismutasa/biosíntesis , Superóxido Dismutasa/genética , Asbesto Crocidolita/análisis , Asbesto Crocidolita/toxicidad , Northern Blotting , Bronquios/efectos de los fármacos , Bronquios/ultraestructura , Línea Celular , Células Epiteliales/efectos de los fármacos , Células Epiteliales/ultraestructura , Humanos , Hibridación in Situ , Manganeso/metabolismo , Microscopía Electrónica de Rastreo , Fibras Minerales/análisis , Dióxido de Silicio/análisis , Superóxido Dismutasa/metabolismoRESUMEN
Paraquat (Pq) is a herbicide which is very toxic to all animals and to man. It generates free radicals and leads to acute or chronic lung injury and usually to death. So far, the role of lipid peroxidation of cell membranes in the mechanism of its toxicity has not been proved satisfactorily and therefore in the present study we examined the concentration of hydrogen peroxide (H2O2) and various lipid peroxidation products (LPP) such as conjugated dienes (CD), lipid hydroperoxides (LH), malonyldialdehyde (MDA) and Schiff bases in selected organs of the rat given a single intraperitoneal dose 35 mg kg-1 Pq. We also evaluated the influence of a mucolytic and probably antioxidant drug, ambroxol, on Pq-induced changes in the concentration of H2O2 and LPP. Paraquat increased the hepatic concentration of H2O2, CD, LH and MDA by approximately fourfold. Though the dose of Pq was nearly twice the LD50 dose, we did not notice any changes in the concentration of these substances in the critical organ, lung or heart and kidney. Ambroxol alleviated the increase of H2O2 in the liver but did not reduce the concentration of LPP. Moreover, the drug administered alone induced lipid peroxidation in the liver. Our results indicate that Pq dose not induce H2O2 production and lipid peroxidation in the lung but it increases the concentration of H2O2 and LPP in the liver. Ambroxol inhibits the Pq-induced increase in the concentration of H2O2 in the liver without protecting it against lipid peroxidation. Moreover, the drug alone may act as a pro-oxidant.
Asunto(s)
Ambroxol/uso terapéutico , Peróxido de Hidrógeno/análisis , Peróxido de Hidrógeno/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Paraquat/envenenamiento , Intoxicación/tratamiento farmacológico , Vísceras/metabolismo , Animales , Masculino , Especificidad de Órganos , Ratas , Ratas WistarRESUMEN
In 78 patients with pulmonary emphysema (49 smokers, 29 nonsmokers) urinary desmosine excretion (UDE) and neutrophil elastase activity (NE) were evaluated. UDE and NE were similar in smokers and nonsmokers and significantly higher than in age, sex-matched control groups. UDE correlated better with NE in nonsmokers than in smokers. Cigarette smoking did not effect UDE in patients with pulmonary emphysema. Obtained results indicate, that UDE measurement could be used in diagnosis and monitoring of lung elastolysis in pulmonary emphysema and suggest the key role of neutrophil elastase in pathogenesis of this disorder.