RESUMEN
Reactive oxygen intermediates (ROIs), such as hydrogen peroxide (H2O2), have been implicated as second messengers in the activation of NF-kappaB by a variety of stimuli, including tumour necrosis factor-alpha (TNF-alpha). The aim of the present study was to examine the effects of ROIs on NF-kappaB activation in primary human CD3+ T lymphocytes and human peripheral blood mononuclear cells (PBMCs). For comparison purposes, Jurkat T cells (subclones JR and JE6.1) were also investigated. Cells were incubated in the presence of either H2O2 or TNF-alpha and nuclear proteins were extracted. NF-kappaB binding was assessed by electrophoretic mobility shift assays (EMSAs). The concentration of H2O2 required to activate NF-kappaB in human primary CD3+ T lymphocytes was as low as 1 microM. In contrast, much higher concentrations of H2O2 were required to activate NF-kappaB in PBMCs and in the JR subclone of Jurkat T cells. H2O2-induced NF-kappaB activation was not observed in the JE6.1 subclone of Jurkat T cells. NF-kappaB was activated by TNF-alpha in all four cell types tested. In PBMCs and Jurkat T cells (subclones JR and JE6.1), this activation could be inhibited by pre-treatment with the antioxidants, pyrrolidine dithiocarbamate (PDTC) and N-acetyl-L-cysteine (NAC). Our results support a role for ROIs in NF-kappaB-DNA binding in human primary T lymphocytes.
Asunto(s)
ADN/metabolismo , Peróxido de Hidrógeno/farmacología , FN-kappa B/metabolismo , Linfocitos T/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ensayo de Cambio de Movilidad Electroforética , Humanos , Células Jurkat , Unión Proteica/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
OBJECTIVE: To investigate the activation of NF-kappaB in the carrageenan rat air pouch model of inflammation in a time course experiment, and the effect of dexamethasone on NF-kappaB activation. METHODS: Air pouch tissue treated with carrageenan (inflamed tissue) was obtained from rats killed at days 1, 2, 3, 6, 14, 21, 28 and 35 after carrageenan challenge. Tissue was also taken from non-carrageenan treated pouches (non-inflamed tissue) at day 3, and from inflamed tissues treated with dexamethasone. Tissue sections were wax embedded and stained with an "activity specific" monoclonal antibody raised against the nuclear localisation signal (NLS) of the p65 sub-unit of NF-kappaB. RESULTS: Cells containing activated NF-kappaB were detected in the intimal and sub-intimal regions of the air pouches as early as day 1. There was a significant increase in cells staining for activated NF-kappaB as the inflammation progressed. Initially cells staining were more prominent in the intimal versus sub-intimal region (p<0.001 for day 1) and at later time points the pattern was reversed (p<0.001 for day 6). There was a significant reduction in the number of cells staining for activated NF-kappaB in tissue taken from dexamethasone treated rats, compared with inflamed pouches alone (p<0.001). At no time point was positive staining for activated NF-kappaB observed in blood vessels. CONCLUSIONS: Activated NF-kappaB is present in the inflamed air pouch and the activation is associated with the inflammatory response to carrageenan. Treatment with dexamethasone resulted in reduced numbers of cells staining for activated NF-kappaB.
Asunto(s)
Inflamación/metabolismo , FN-kappa B/metabolismo , Animales , Antiinflamatorios/uso terapéutico , Carragenina , Dexametasona/uso terapéutico , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Técnicas para Inmunoenzimas , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Masculino , Ratas , Ratas WistarRESUMEN
Several bone resorptive stimuli affect osteoclasts indirectly by modulating the production and release of osteoblastic factors. Using electrophoretic mobility shift assays, we found that not only tumour necrosis factor-alpha (TNF-alpha) but also interleukin-1beta and parathyroid hormone (PTH) caused dose and time-related increases in nuclear factor kappaB (NF-kappaB)-DNA binding in Saos-2 human osteoblastic (hOB) cells. Activation of NF-kappaB by TNF-alpha was reproduced in primary hOBs. In contrast, consistent with their previously reported lack of response to steroid hormones, Saos-2 cells did not respond to 1,25-dihydroxyvitamin D(3). We suggest that NF-kappaB activation in osteoblastic cells constitutes an important pathway in osteoblast-mediated resorptive signalling.