μM Dexamethasone Research Articles

Identification and Sequencing of Bcl and Apaf1 Genes in the Common Carp, Cyprinus Carpio.

INTRODUCTION. The role of apoptosis in the immune system is being studied in cultured carp, Cyprinus carpio an economically important farmed fish. In intensive culturing systems, stress is suspected of reducing the immune competence of farm animals. Our initial studies investigated the effect of dexamethasone, a corticosteroid analogue, on the different immunocompetent cells in the pronephros and thymus. This is currently being extended to identify and sequence an apoptotic regulator (Bcl-x) and an apoptotic activator gene (APAF1) from the carp cDNA library. METHOD. Cells from the pronephros and thymus were maintained in vitro, in L-15 medium (Sigma) supplemented with 2mM L- glutamine and containing penicillin (100i.u./ml), streptomycin (100µg/ml), and 5% heat- inactivated foetal calf serum, at 20 o C. They were then exposed to different concentrations of dexamethasone (0.1µM to10.0µM 2 ) for up to 48h in time. Control cells were those maintained in the culture medium lacking in dexamethasone. Cell viability and percentage of apoptotic cell death at times 0h, 24h and 48h were determined using nigrosine dye exclusion and acridine orange stain respectively. DNA from approximately 10 6 cells was isolated from each of the above samples and subjected to agarose gel electrophoresis. In the differential experiment, the different types of immunocompetent cells were labelled with monoclonal antibodies WCI04, WCI09 and WCI12 and their responses to 1.0µM dexamethasone over 6 hours was recorded. Degenerate primers for the Bcl-xL equivalent in carp were designed following a homology study on the sequences of Homo sapiens (human) and Xenopus laevis (South African clawed frog). The homology between human APAF1 and that of Danio rerio (Zebrafish) was examined, and primers constructed, for the comparable sequence in the carp. These were used in PCR to obtain sequences from the carp cDNA library. The sequences obtained will now be used as probes to sequence the rest of the gene(s). RESULTS. Statistical analysis reveals that there is a significant difference between the number of control cells undergoing apoptotic cell death and that of cells exposed to the different concentrations of dexamethasone (p<0.001) (Fig.1). This effect is greatest between the 0h and 24h time periods and appears to decrease after this time period, and is similar in the pronephros and the thymus.

Production of plasminogen activator inhibitor-1 by human mast cells and its possible role in asthma.

The plasminogen activator inhibitor type 1 (PAI-1) has an essential role in tissue remodeling. The PAI-1 gene was induced by a combination of phorbol ester and calcium ionophore at the highest level among the inducible human mast cell genes that we have analyzed on a DNA microarray. PAI-1 was secreted by both a human mast cell line (HMC)-1 and primary cultured human mast cells upon stimulation, whereas PAI-1 was undetectable in either group of unstimulated cells. The secretion of PAI-1 was due to de novo synthesis of PAI-1 rather than secretion of preformed PAI-1. The functional significance of PAI-1 secretion was demonstrated by complete inhibition of tissue-type plasminogen activator activity with supernatants of stimulated HMC-1 cells. Furthermore, we were able to regulate PAI-1 gene expression in HMC-1 cells by known therapeutic agents. High-dose (1 microM) dexamethasone induced PAI-1 mRNA expression. Cyclosporin down-regulated the expression of the PAI-1 gene. Cycloheximide abrogated PAI-1 mRNA expression, suggesting that transcription of the PAI-1 gene requires de novo synthesis of early gene products, including transcription factors. Finally, we demonstrated PAI-1 in lung mast cells from a patient with asthmatic attack by double-immunofluorescence study. This is the first report demonstrating that activated human mast cells release a striking amount of functionally active PAI-1. These results suggest that PAI-1 could play an important role in airway remodeling of asthma, and inhibition of PAI-1 activity could represent a novel therapeutic approach in the management of airway remodeling.

Protein Kinase C Evokes Quantal Catecholamine Release from PC12 Cells via Activation of L-type Ca2+ Channels

Protein Kinase C Evokes Quantal Catecholamine Release from PC12 Cells via Activation of L-type Ca2+ Channels

Substance P induces tumor necrosis factor-α release from human skin via mitogen-activated protein kinase

Substance P plays an important role in neurogenic inflammation with granulocyte infiltration. To investigate cytokines involved in the substance P-induced inflammation and the mechanism of cell activation, we studied the release of TNF (tumor necrosis factor)-α and histamine from human skin slices in response to substance P and antigen. Substance P induced the release of histamine and TNF-α in a dose-dependent manner at concentrations from 0.8 to 100 μM. PD 098059 (2′-amino-3′-methoxyflavone) selectively inhibited the release of TNF-α, but not the release of histamine induced by either substance P or antigen. SB 203580 ([4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1 H-imidazole]) slightly inhibited TNF-α release induced by antigen, but not that induced by substance P, and slightly enhanced histamine release induced by either stimulation. The release of TNF-α in response to either stimulation was inhibited by 1 nM–1 μM dexamethasone, but histamine release was not affected. These results suggest that substance P, in addition to antigen, induced TNF-α release from human skin by a mitogen-activated protein (MAP) kinase, predominantly extracellular signaling-regulated protein kinase (ERK)-dependent, and dexamethasone-sensitive pathway, which is separate from that for histamine release from mast cells.

Protective Effects of Glucocorticoids on Nitric Oxide-induced Apoptosis in Leukaemia HL-60 Cells in Man

Inappropriate release of nitric oxide (NO) has been linked to the pathogenesis of several disease states. Glucocorticoids inhibit the synthesis of NO and could be effective in the treatment of NO-mediated diseases. We show the protective effect of dexamethasone on NO-induced apoptosis in leukaemia HL-60 cells in man. Agarose electrophoresis of the DNA extracted from HL-60 cells resulted in a ladder-like pattern of fragmentation after 6-and 9-h exposure of the cells to 1 mM S-nitrosoglutathione, a physiologically relevant NO donor. Pre-incubation of the cells with 1 μM dexamethasone for 6 h blocked NO-induced DNA fragmentation. This effect was abolished by RU-486, a steroid receptor antagonist, and cycloheximide, a protein synthesis inhibitor, suggesting that the protective effect of dexamethasone might require glucocorticoid receptor binding and protein synthesis. Concerning NO signal transduction, we found that dexamethasone efficiently prevented NO-induced activation of caspase-3 proteases, suggesting an inhibitory mechanism upstream of the terminal death pathway. Collectively, these findings highlight a novel protective effect of dexamethasone on NO-mediated cellular injury.

RU486 Increases Radiosensitivity and Restores Apoptosis through Modulation of HPV E6/E7 in Dexamethasone-Treated Cervical Carcinoma Cells

Objective.Cervical carcinoma tumors containing radioresistant cells are associated with decreased local control and survival. Therefore, strategies to increase cell kill during radiotherapy have a clear rationale. It was previously determined that treatment with the corticosteroid dexamethasone increased radioresistance and decreased apoptosis in C4-1 cervical carcinoma cells. The goal of this study was to determine whether hormone antagonists, specifically Mifepristone (RU486), could reverse the effects of dexamethasone on clonogenic survival and apoptosis following γ-irradiation.Methods. Cervical carcinoma cell line C4-1 cells were exposed to 1 μM dexamethasone in the presence or absence of 1 μM Mifepristone (RU486), a hormone antagonist, and irradiation. Cells were analyzed for steroid-dependent HPV E6/E7 mRNA expression (by Northern blot analysis), clonogenic survival, and apoptosis (by Annexin V staining and the DNA fragmentation assay). In addition, p53 protein levels were determined by Western blot analysis.Results. The hormone antagonist RU486 reversed dexamethasone-dependent upregulation of E6/E7 mRNA and restored radiation-induced p53 expression, apoptosis, and clonogenic survival to levels similar to those observed following irradiation alone.Conclusion. RU486 reverses glucocorticoid-dependent upregulation of HPV E6/E7, which corresponds to restoration of p53 expression, and restores radiosensitivity and apoptosis following γ-irradiation. Therefore, it appears that along with radiation, RU486 may be a beneficial agent in the treatment of hormone-reactive cervical tumors.

Activation of HSF and selective increase in heat-shock proteins by acute dexamethasone treatment

Heat-shock proteins (HSPs) are an important family of endogenous protective proteins, which increase in response to myocardial ischemia and other stresses. Overexpression of HSP72 is cardioprotective. We were interested in the regulation of heat-shock factor (HSF), the transcription factor for HSP genes. Previously we have observed that the inflammatory cytokine tumor necrosis factor-alpha increases HSP72 levels and postulated that dexamethasone might effect the heat shock response. In the adult rat cardiac myocyte we found that treatment with either low (10 microM)- or high (100 microM)-dose dexamethasone activated HSF by 2-6 h as determined by gel shift assay without evidence of cytotoxicity. Although HSF activation is a key step in expression of HSP72, this may not result in an increase in HSP72. We found that 10 microM dexamethasone increased HSP72 38%, and 100 microM dexamethasone increased HSP72 62% (P < 0.05). HSP27 and HSP60 were unchanged. The selective increase in HSP72 was associated with protection of the cardiac myocytes from hypoxia and reoxygenation. We conclude that dexamethasone is a novel inducer of the heat shock response.

Effect of dietary vitamin E on apoptosis and proliferation of murine splenocytes

Vitamin E (Vit E) is one of the most potent natural lipophilic antioxidants shown to inhibit proliferation of cancer cells and influence apoptosis in in-vitro systems. In the present investigation, we studied the effect of dietary Vit E on splenocyte proliferation, apoptosis, and necrosis. Two-month old male ICR mice were fed an AIN tm 76 formula diet for three months with 10% (W/W) corn oil, supplemented with Vit E at normal (75 IU/kg) or higher (500 IU/kg) levels, or without Vit E as zero Vit E group. In vivo analysis revealed both serum lipid peroxides (MDA) and splenocyte peroxides (dichlorofluorescein staining) were found to be reduced with increased dietary Vit E levels in a dose-dependent manner; whereas, serum Vit E levels were found to be augmented with increased levels of dietary Vit E. Also, apoptosis and necrosis were measured in splenocytes incubated with and without 1 μM dexamethasone (DEX) in RPMI media for eight and 21 hours. Cells were stained with Annexin V and Propidium Iodide (PI), and both apoptotic and necrotic cells were analyzed simultaneously by a FACScan desktop flow cytometer using the Cell Quest Program. High and normal levels of Vit E increased necrosis by 29 and 18% in the cells placed in media, and 67 and 34% in cells incubated with DEX for eight hours, respectively, in comparison to that in the zero Vit E-fed group. However, in splenocytes from the high Vit E group apoptosis was decreased by 32 and 49.5% in cells incubated for eight hours in media alone or with DEX, respectively. Furthermore, in the high Vit E group necrosis was increased by 25% and apoptosis was decreased by 17% in cells incubated for 21 hr in media alone, compared to that of the zero Vit E group. In addition, proliferation of splenocytes, either stimulated or unstimulated with mitogens, was also found to be decreased with increasing levels of dietary Vit E. In conclusion, our results indicate that dietary Vit E appears to alter both cell proliferation and programmed cell death (apoptosis vs. necrosis), by lowering lipid peroxides.

Unchanged cytochrome P450 3A (CYP3A) expression and metabolism of midazolam, triazolam, and dexamethasone in mdr(−/−) mouse liver microsomes

P-Glycoprotein (P-gp) and cytochrome P450 3A (CYP3A) share common substrates and expression properties, but the relationship of mdr1 deficiency to CYP3A-mediated metabolism and protein expression is not established. The in vitro kinetic parameters of CYP3A-mediated metabolism of midazolam (MDZ), triazolam (TRZ), and dexamethasone (DEX) were studied in liver microsomes from three mdr1a(−/−) mice, one mdr1a/b(−/−) mouse, and mdr1a/b(+/+) controls. The kinetic profiles of CYP3A-mediated MDZ 4-hydroxylation were not significantly different between mdr1-deficient animals and controls. Overall mean (± SEM, N = 8) values were: V max, 0.74 ± 0.05 nmol/min/mg protein; K m , 28.2 ± 2.7 μM; and estimated intrinsic clearance, 0.026 ± 0.003 mL/min/mg protein. Likewise, rates of formation of α-OH- and 4-OH-TRZ (from 500 μM TRZ), and of DEX metabolites sensitive to ketoconazole inhibition, M1 and M5 (from 20 μM DEX), did not differ between mdr1-deficient and control animals. Immunoquantified microsomal CYP3A protein levels in mdr1a(−/−), mdr1a/b(−/−), and mdr1a/b(+/+) mice were not different, with overall mean immunoreactive protein levels of 2.68 ± 0.09 pmol/μg protein. Although CYP3A and P-gp share aspects of activity and expression, disruption of the mdr1 genes does not affect CYP3A-mediated metabolism or protein expression in the mouse.

Regulation of 15-lipoxygenase expression and mucus secretion by IL-4 in human bronchial epithelial cells.

Our laboratory has recently shown that mucus differentiation of cultured normal human tracheobronchial epithelial (NHTBE) cells is accompanied by the increased expression of 15-lipoxygenase (15-LO). We used differentiated NHTBE cells to investigate the regulation of 15-LO expression and mucus secretion by inflammatory cytokines. Interleukin (IL)-4 and IL-13 dramatically enhanced the expression of 15-LO, whereas tumor necrosis factor-alpha, IL-1beta, and interferon (IFN)-gamma had no effect. These cytokines did not increase the expression of cyclooxygenase-2, with the exception of a modest induction by IL-1beta. The IL-4-induced 15-LO expression was concentration dependent, and mRNA and protein expression increased within 3 and 6 h, respectively, after IL-4 treatment. In metabolism studies with intact cells, 15-hydroxyeicosatetraenoic acid (15-HETE) and 13-hydroxyoctadecadienoic acid (13-HODE) were the major metabolites formed from exogenous arachidonic acid and linoleic acid. No prostaglandins were detected. IL-4 treatment dramatically increased the formation of 13-HODE and 15-HETE compared with that in untreated NHTBE cells, and several additional 15-LO metabolites were observed. Pretreatment of NHTBE cells with IFN-gamma or dexamethasone did not inhibit the IL-4-induced expression of 15-LO except at high concentrations (100 ng/ml of IFN-gamma and 10 microM dexamethasone). IL-4 treatment inhibited mucus secretion and attenuated the expression of the mucin genes MUC5AC and MUC5B at 12-24 h after treatment. Addition of 15-HETE precursor and 13-HODE precursor to the cultures did not alter mucin secretion or mucin gene expression. On the basis of the data presented, we conclude that the increase in 15-LO expression by IL-4 and attenuation of mucus secretion may be independent biological events.

Differential Immediate Early Gene Expression in Conditional Hepatitis B Virus pX-transforming VersusNontransforming Hepatocyte Cell Lines

We report construction and characterization of tetracycline-controlled hepatitis B virus pX-expressing hepatocyte (AML12) cell lines. These cell lines were constructed in AML12 clonal isolates (clones 3 and 4), which express constitutively the tetracycline-controlled transactivator. Since pX is implicated in HCC, this immortalized hepatocyte model system was used to investigate the mechanism of pX in transformation. Clonal isolates of 3pX and 4pX lineages display conditional synthesis of pX mRNA and protein and a 2-fold increase in growth saturation density following tetracycline removal, implicating pX in monolayer overgrowth. Interestingly, only 3pX clones display pX-dependent anchorage independence. Clone 3 lineages express hepatocyte nuclear factor-1alpha and hepatocyte-specific marker genes; clone 4 lineages express hepatocyte nuclear factor-1beta and reduced levels of hepatocyte-specific marker genes, suggesting the importance of the differentiated hepatocyte in pX-mediated oncogenic transformation. Importantly, 3pX and 4pX lineages display differential expression of immediate early genes c-fos and ATF3. The pX-transforming 3pX lineage displays early, pX-dependent induction of ATF3 and prolonged induction of c-fos. The nontransforming 4pX cells display an absence of pX-dependent ATF3 induction and transient induction of c-fos. Our results support the direct link of pX expression to oncogenic transformation in 3pX lineage clones and underscore the advantage of this conditional cellular model system for studying mechanisms of pX-mediated oncogenesis.

A link between cell cycle and cell death: Bax and Bcl-2 modulate Cdk2 activation during thymocyte apoptosis.

Resting thymocytes undergoing apoptosis in response to specific stimuli degrade the cdk inhibitor p27(Kip1) and upregulate Cdk2 kinase activity. Inhibition of Cdk2 kinase activity efficiently blocks cell death via certain apoptosis pathways whereas overexpression of Cdk2 accelerates such cell death, suggesting its involvement in the signal transduction pathways activated by certain apoptotic stimuli. We found that Cdk2 activation during thymocyte apoptosis can be regulated by p53, Bax and Bcl-2. The highly elevated Cdk2 kinase activity in the apoptosing thymocytes is not associated with its canonical cyclins, cyclin E and cyclin A, and requires de novo synthesis of proteins for activation to take place. We therefore propose Cdk2 activation to be a crucial event in distinct pathways of apoptosis and the point at which the cell cycle and cell death pathways interact.

Receptor-mediated induction of human dermal fibroblast ectoaminopeptidase N by glucocorticoids.

. Aminopeptidase N was demonstrated in human dermal fibroblasts as an ectoenzyme. The enzyme has wide substrate specificity, with a K m of 0.63 mM and V max of 338 nmol min−1 mg−1. Addition of fetal calf serum to the culture medium increased aminopeptidase N activity up to 63% by 10% serum in a 48-h culture. Treatment of fibroblasts by dexamethasone increased ectoaminopeptidase N activity in a dose- and time-dependent manner. Maximal increase of aminopeptidase N occurred after treatment with 1 μM dexamethasone for 3 days. Actinomycin D, a blocker of RNA synthesis, and cycloheximide, an inhibitor of protein synthesis, did not alter basal aminopeptidase N activity. However, they prevented stimulation by dexamethasone. RU 38486, a glucocorticoid receptor antagonist, suppressed the dexamethasone-induced increase in aminopeptidase N activity. This study shows that human dermal fibroblasts contain ectoaminopeptidase N controlled by glucocorticoids through a receptor-mediated mechanism.

Dexamethasone regulates basic fibroblast growth factor, nerve growth factor and S100 β expression in cultured hippocampal astrocytes

Glucocorticoids regulate hippocampal neuron survival during fetal development, in the adult, and during aging; however, the mechanisms underlying the effects are unclear. Since astrocytes contain adrenocortical receptors and synthesize and release a wide variety of growth factors, we hypothesized that glucocorticoids may alter neuron–astrocyte interactions by regulating the expression of growth factors in hippocampal astrocytes. In this study, three growth factors, which are important for hippocampal neuron development and survival, were investigated: basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and S100 β. Enriched type I astrocyte cultures were treated with 1 μM dexamethasone (DEX), a synthetic glucocorticoid, for up to 120 h. Cells and culture medium were collected and total RNA and protein were measured at 6, 12, 24, 48, 72, 96 and 120 h after the initiation of hormone treatment. Growth factor mRNA levels were measured and quantified using solution hybridization–RNase protection assays and protein levels were quantified using ELISA methods. We report that DEX stimulates the bFGF mRNA levels over the 120-h treatment. In contrast, DEX suppresses NGF mRNA continuously over the same period of treatment. DEX induces a biphasic response in S100 β mRNA levels. In addition, some of the changes in gene expression are translated into parallel changes in protein levels of these growth factors. Our results demonstrate that dexamethasone can differentially regulate the expression of growth factors in hippocampal astrocytes in vitro. This suggests that one of the mechanisms through which glucocorticoids affect hippocampal functions may be by regulating the expression of astrocyte-derived growth factors.

Stimulus-secretion coupling in porcine adrenal chromaffin cells: Effect of dexamethasone

Recent studies from this laboratory established that dexamethasone (DEX) potentiates Ca2+ current via voltage-gated Ca2+ channels (VGCC), and as a consequence potentiates agonist-induced cytosolic Ca2+ transients in rat adrenal chromaffin cells. The present study examined whether DEX can also modulate VGCC activity and agonist-induced cytosolic Ca2+ transients in porcine adrenal medullary chromaffin (PAMC) cells, and if so whether this results in alterations in catecholamine secretion. Forty-eight-hr exposure to 1 microM DEX significantly increased peak Ca2+ current (delta + 138%; n = 6; P < 0.05) in PAMC cells. DEX treatment also significantly potentiated the increase in cytosolic Ca2+ in response to membrane depolarization with KCl (delta + 20%; n = 29; P < 0.05), but did not affect the amplitude of Ca2+ transients elicited by nicotine or acetylcholine. Despite the potentiation of intracellular Ca2+, DEX treatment had no effect on KCl-induced secretion of either norepinephrine or epinephrine. These data demonstrate that as in the rat chromaffin cell, DEX can also increase VGCC activity in PAMC cells. However, the subsequent potentiation of selected agonist-induced increases in intracellular Ca2+ does not appear to be sufficient to alter catecholamine secretion.

DEXAMETHASONE PREVENTS INTERLEUKIN-1β-MEDIATED INHIBITION OF RAT ISLET INSULIN SECRETION WITHOUT DECREASING NITRIC OXIDE PRODUCTION

The deleterious effects of interleukin 1 (IL-1) on insulin-producing β-cells are partly mediated by the generation of the free radical nitric oxide (NO). We aimed to assess the effect of several steroidal hormones on IL-1β-induced inhibition of rat islet insulin secretion in vitro, and their possible regulatory effects on NO production. Incubation of newborn rat islets for 24 h in the presence of 150 pg/ml IL-1β revealed that dexamethasone dose-dependently attenuated the inhibitory effect of IL-1β on insulin release in response to a 2-h glucose challenge. Physiological and supraphysiological concentrations of testosterone, 17β-estradiol, progesterone, 1,25-dihydroxyvitamin-D3and vitamin D analogues (KH1060 and MC1288) were ineffective. Dexamethasone (1 μM) increased the production of NO in IL-1β-treated rat islets, as measured by the concentration of nitrite in the media. However, 1–5 μM dexamethasone inhibited IL-1β-induced NO production by RIN cells. Dexamethasone (1 μM) did not affect the inhibitory action of the NO donor S-nitroso penicillamine (500 μM) on rat islet insulin secretion. We conclude that dexamethasone partially protects against IL-1β-induced inhibition of rat islet insulin secretion, an effect which is not mediated through modulation of the NO pathway.

Evidence for the shuttle model for G Sα activation of adenylyl cyclase

Knowledge of the nature of the interaction between the stimulatory G protein (G s) and the adenylyl cyclase catalytic unit (C) is essential for interpreting the effects of G s mutations and expression levels on cellular response to a wide variety of hormones, drugs, and neurotransmitters. It has been proposed that ß-adrenergic receptor activation of adenylyl cyclase occurs either by a two-step “shuttle” mechanism where the receptor activates G s independently of cyclase followed by G sα activation of cyclase independent of the receptor; or the receptor activates a “precoupled” G s—C complex in a single step. Simulations of the two models revealed that the two forms of activation are distinguishable by the effect of G s levels on epinephrine-stimulated EC 50 values for cyclase activation; specifically, the shuttle model predicts an increased potency of epinephrine stimulation as levels of G sα increase. To address this problem, S49 cyc − cells were stably transfected with the gene for G sα long regulated by the MMTV LTR promoter, which allowed for an induction of G sα long expression levels over a 40-fold range by incubation of the cells for various times with 5 μM dexamethasone. Expression of G sα was strongly correlated to the appearance of GTP shifts in the competitive binding of epinephrine with [ 125I]iodocyanopindolol to the β-adrenergic receptors and epinephrine-stimulated adenylyl cyclase activity. Most importantly, high expression of G sα resulted in lower EC 50 values for epinephrine and prostaglandin E 1 stimulation of adenylyl cyclase activity. The decrease in EC 50 did not occur as a result of a change in β 2-adrenergic receptor, G iα, Gβα, or adenylyl cyclase levels. These novel findings demonstrate that a change in the level of a protein downstream of a plasma membrane receptor can influence hormone potency. We explain these results by using kinetic arguments to suggest that some fraction of hormone-activated adenylyl cyclase occurs via a shuttle mechanism, and not a purely precoupled mechanism.

Dexamethasone inhibits cytokine-induced intercellular adhesion molecule-1 up-regulation on endothelial cell lines

Intercellular adhesion molecule-1 (ICAM-1) expression on three endothelial cell lines was differently modulated by pro-inflammatory cytokines, such as interleukin-1 β and tumour necrosis factor- α (TNF- α) and the glucocorticoid hormone dexamethasone. Incubation of EA.hy926 cells with 1 μM dexamethasone prior to addition of TNF- α consistently reduced ICAM-1 induction by approximately 40%. EA.hy926 cell responsiveness to the steroid was validated by detecting specific dexamethasone binding, with a calculated affinity constant of 1.3 nM and a maximal number of sites of 35×10 3 per cell. To establish the generality of dexamethasone inhibition upon ICAM-1 up-regulation, two other endothelial cell lines were assessed. Incubation of LT4 and ECV304 cells with interleukin-1 β or TNF- α produced a significant increase in ICAM-1 expression on their cell surface (ranging from a 2-fold increase for interleukin-1 β to a 5-fold increase for TNF- α). Addition of dexamethasone was again able to significantly reduced this induction. Finally, the effect of the steroid on cytokine-induced ICAM-1 up-regulation was functionally related to its ability to suppress in vitro neutrophil trans-endothelial passage. Overall these data indicate that ICAM-1 is a likely molecular target for the anti-inflammatory action exerted by dexamethasone. Inhibition of ICAM-1 up-regulation may, at least in part, mediate the potent anti-migratory action displayed by this class of anti-inflammatory drugs.

T-cell receptor ligation by peptide/MHC induces activation of a caspase in immature thymocytes: the molecular basis of negative selection.

T-cell receptors (TCRs) are created by a stochastic gene rearrangement process during thymocyte development, generating thymocytes bearing useful, as well as unwanted, specificities. Within the latter group, autoreactive thymocytes arise which are subsequently eliminated via a thymocyte-specific apoptotic mechanism, termed negative selection. The molecular basis of this deletion is unknown. Here, we show that TCR triggering by peptide/MHC ligands activates a caspase in double-positive (DP) CD4+ CD8+ thymocytes, resulting in their death. Inhibition of this enzymatic activity prevents antigen-induced death of DP thymocytes in fetal thymic organ culture (FTOC) from TCR transgenic mice as well as apoptosis induced by anti-CD3epsilon monoclonal antibody and corticosteroids in FTOC of normal C57BL/6 mice. Hence, a common caspase mediates immature thymocyte susceptibility to cell death.

Glucocorticoid‐xenobiotic interactions: Dexamethasone‐mediated potentiation of cytochrome P4501A induction by β‐naphthoflavone in a fish hepatoma cell line (PLHC‐1)

AbstractThe induction of CYP1A by the polycyclic aromatic hydrocarbon (PAH)‐type inducer β‐naphthoflavone (BNF) in the Poeciliopsis‐lucida hepatocellular carcinoma cell line (PLHC‐1), and the effects of the glucocorticoid receptor (GR) agonist dexamethasone (DEX) on this response were examined. Dose‐response studies revealed that BNF is three orders of magnitude less potent than the planar halogenated aromatic hydrocarbon 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) as an inducer of the CYP1A activity ethoxyresorufin‐O‐deethylase (EROD), and that the apparent efficacy for the induction by BNF is 50% of that obtained with TCDD. Addition of 10 μM DEX resulted in potentiation of CYP1A induction at all doses of BNF tested. The degree of that potentiation of induction of CYP1A protein levels and EROD activity differed substantially between doses of BNF and at different times of exposure. For example, the maximal degree of potentiation of EROD induction by DEX was 12‐fold in PLHC‐1 cells treated with 0.1 μM BNF, 19‐fold in cells treated with 1 μM BNF, and 8‐fold in cells treated with 10 μM BNF. These maximal degrees of potentiation of EROD induction were obtained after 30 h with 0.1 μM BNF, 48 h with 1 μM BNF, and 72 h with 10 μM BNF. These results demonstrate interactions between GR and aryl hydrocarbon receptor pathways that could influence the response of fish to xenobiotic exposure.