Respiratory syncytial virus (RSV) is a common cause of bronchiolitis in infants. Although antiinflammatory in nature, glucocorticoids have been shown to be ineffective in the treatment of RSV-induced bronchiolitis and wheezing. In addition, the effectiveness of glucocorticoids at inhibiting RSV-induced proinflammatory cytokine production in cell culture has been questioned. In this study, we have investigated the effect of RSV infection on glucocorticoid-induced gene activation in lung epithelium-derived cells. We show that RSV infection inhibits dexamethasone induction of three glucocorticoid receptor (GR)-regulated genes (glucocorticoid-inducible leucine zipper, FK506 binding protein, and MAPK phosphatase 1) in A549, BEAS-2B cells, and primary small airway epithelial cells. UV irradiation of the virus prevents this repression, suggesting that viral replication is required. RSV is known to activate the nuclear factor ?B (NF?B) pathway, which is mutually antagonistic towards the GR pathway. However, specific inhibition of NF?B had no effect on the repression of GR-induced genes by RSV infection, indicating that RSV repression of GR is independent of NF?B. RSV infection of A549 cells does not alter GR protein levels or GR nuclear translocation but does reduce GR binding to the promoters of the glucocorticoid responsive genes analyzed in this study. Repression of GR by RSV infection may account for the apparent clinical ineffectiveness of glucocorticoids in RSV bronchiolitis therapy. In addition, this data adds to our previously published data suggesting that GR may be a general target for infectious agents. Identifying the mechanisms through which this suppression occurs may lead to the development of novel therapeutics.
The use of cyclodextrins as tools to establish the role of cholesterol rafts in cellular functions has become a widely accepted procedure. However, the adverse effects of cyclodextrins as the cholesterol-depleting agents on cellular structure and functions are not reported in detail. Therefore, in the current study, we investigated the membrane-perturbing actions and cytotoxicity of the two widely used cellular cholesterol-depleting cyclodextrins methyl-beta-cyclodextrin (MbetaCD) and hydroxypropyl-beta-cyclodextrin (HPCD) in our well-established bovine pulmonary artery endothelial cell (BPAEC) in vitro model system. BPAECs treated with different concentrations of MbetaCD and HPCD (2% and 5%, wt/vol.) for 15-180 min showed significant loss of membrane cholesterol, cytotoxicity, cell morphology alterations, actin cytoskeletal reorganization, alterations in cellular proteins and membrane fatty acid composition, and decrease in trans-endothelial electrical resistance (TER). MbetaCD induced a marked loss of cellular proteins, as compared to that caused by HPCD under identical conditions. More noticeably, MbetaCD caused a drastic loss of membrane lipid fatty acids in BPAECs, as compared to HPCD which failed to cause such alteration. Removal of cholesterol by cyclodextrin (especially MbetaCD) treatment apparently caused loss of fluidity of the cell membrane and leakage of vital cellular molecules including proteins and fatty acids, and thus caused cytotoxicity and loss of cell morphology in BPAECs. Replenishment of cells with cholesterol following its depletion by MbetaCD treatment significantly attenuated the depletion of cellular cholesterol, cytotoxicity and morphological alterations in BPAECs, indicating the importance of membrane cholesterol in vascular EC integrity. Also, the current study offered a safer method of cholesterol removal from membranes and lipid rafts by HPCD, suggesting its use in studies to investigate the role of lipid raft-associated cholesterol in cellular functions.
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