Inhibitory Action Of Glucocorticoids Research Articles

SAT-419 Mechanisms Underlying Glucocorticoid-Induced Suppression of Pulsatile LH: Insights from Immortalized Kisspeptin Cells

Stress inhibits fertility at least in part by inhibiting gonadotropin secretion. Part of the stress response is an increase in circulating glucocorticoid concentrations, specifically corticosterone in mice. We have demonstrated that continuously elevated corticosterone concentrations can inhibit ovarian cyclicity and suppress luteinizing hormone (LH) pulse frequency in female mice (ICN 2018, 310). Since the gonadotropin releasing hormone (GnRH) and LH pulse generator is likely formed by the kisspeptin neuron population in the arcuate nucleus of the hypothalamus, this suppression by corticosterone may be mediated by kisspeptin cells. Our immunohistochemical data indicating that arcuate kisspeptin neurons contain glucocorticoid receptor (GR) in mice (ICN 2018, 312), provides evidence in support of this hypothesis. To investigate direct actions of glucocorticoids in kisspeptin neurons, we utilized an immortalized mouse arcuate kisspeptin neuron cell line (KTaR cells). RT-PCR data indicated the presence of mRNA for GR in KTaR cells. KTaR cells were subsequently transfected with a mouse mamillary tumor virus (MMTV)-luciferase plasmid, which contains a positive glucocorticoid response element, and treated with varying concentrations of a synthetic glucocorticoid, dexamethasone. The MMTV promoter exhibited a dose-dependent increase in luciferase activity, with doses in the μM to nM range producing a significant increase in promoter activity compared to vehicle treated samples (n = 3 independent experiments). Together, these data indicate that functional GR is present in KTaR cells and that this cell type could be valuable in understanding glucocorticoid effects on LH pulse dynamics. Arcuate kisspeptin cells in mice (and other mammals) also contain neurokinin B and dynorphin, and are termed KNDy cells. The KNDy hypothesis for GnRH/LH pulse generation posits that each pulse of GnRH is caused by a pulse of kisspeptin secretion and that each pulse of kisspeptin is turned on (stimulated) by neurokinin B (NKB; encoded by Tac2) and terminated (inhibited) by dynorphin (encoded by pDyn). To determine if these genes are regulated by glucocorticoids, we treated KTaR cells with 1μM dexamethasone for 24 hrs and quantified transcript abundance with qPCR. Dexamethasone treatment did not alter mRNA abundance of Kiss1 or pDyn themselves or the receptors for NKB or dynorphin (Tacr1, Tacr2, Tacr3, and Oprk1). However, dexamethasone treatment caused a significant decrease in Tac2 mRNA abundance compared to vehicle treatment (51.7 ± 10.0% reduced). These experiments demonstrate that KTaR cells have functional GR and that dexamethasone treatment suppresses NKB transcript abundance. These data raise the possibility that the inhibitory actions of glucocorticoids on LH pulse frequency that we recently demonstrated in mice are mediated by an inhibition of NKB, a factor implicated in GnRH pulse onset.

Glucocorticoids Suppress Activation of Mast Cells through induction of Inhibitory Regulators of Signaling Pathways (94.13)

Abstract Glucocorticoids suppress production of inflammatory cytokines via the glucocorticoid receptor (GR) by inhibiting activity of relevant transcription factors, a process known as transrepression. Here we provide evidence that dexamethasone and other glucocorticoids also suppress activation of mast cells through induction of synthesis of inhibitory regulators of signaling pathways by a process referred to as transactivation. The glucocorticoid-inducible regulators identified so far include Src-like adaptor protein (SLAP), downstream of tyrosine kinase (Dok)-1, MAP kinase phosphatase-1 (MPK-1) (Mol. Pharmacol. 67:598, 2005; J. Immunology 177:2047, 2006),. Suppressor of cytokine signaling 1 (Socs1), and cytokine inducible SH2-containing protein (Cish) (unpublished data). We have found that the induction of these inhibitory regulators account for the inhibition of key antigen-mediated signaling events such as the activation of the tyrosine kinase Syk, MAP kinase pathways, and downstream transcription factors. The effects of overexpressed wild type GR, and a dimerization-negative mutant GR with minimal transactivation activity, further suggest that the inhibitory actions of glucocorticoids are dependent on gene transactivation as well as cytokine-gene transrepression. These findings have important implications in the design and use of glucocorticoids that exhibit selectivity towards transrepression.

Secreted Frizzled-Related Protein 1 Modulates Glucocorticoid Attenuation of Osteogenic Activities and Bone Mass

Prolonged glucocorticoid treatment is known to cause osteoporosis or aseptic necrosis. Secreted frizzled-related proteins 1 (SFRP1) and low-density lipoprotein-related protein 5 (LRP5), a Wnt protein antagonist and a coreceptor, have been found to regulate skeletogenesis. Whereas recent studies have reported that excess glucocorticoid promotes bone loss, the biological role of SFRP1 and LRP5 in regulating glucocorticoid attenuation of bone formation is not fully understood. We showed that a supraphysiological level of glucocorticoid enhanced SFRP1 but not LRP5 expression of primary mesenchymal cell cultures in vitro and osteoblasts at metaphyseal trabecular endosteum and chondrocytes at calcified cartilage in vivo. Glucocorticoid augmentation of SFRP1 expression was transcriptionally mediated. The inhibitory action of glucocorticoid on osteogenic differentiation appeared to be regulated by SFRP1 mediation of beta-catenin destabilization because knocking down SFRP1 by RNA interference abrogated the supraphysiological level of glucocorticoid attenuation of osteogenesis. Recombinant human SFRP1 reduced the promoting effect of physiological level of glucocorticoid on cytosolic beta-catenin accumulation, runt-related transcription factor-2 activation, and osteogenic activities. Glucocorticoid and recombinant human SFRP1 significantly increased osteochondral cell apoptosis associated with reduced mineral density, biomechanical properties, trabecular bone volume, and midshaft cortical bone areas in rat femurs. These findings suggest that SFRP1 modulates glucocorticoid-induced bone loss. Regulation of Wnt/SFRP signal transduction can be used in the future as an alternative strategy for the prevention of glucocorticoid-induced osteoporosis.

Glucocorticoids selectively inhibit paclitaxel-induced apoptosis: mechanisms and its clinical impact.

Paclitaxel (Taxol), a naturally occurring antimitotic agent, has shown significant cell-killing activity against tumor cells through induction of apoptosis. The mechanism by which paclitaxel induces cell death is not entirely clear. Recent studies in our laboratory discovered that glucocorticoids selectively inhibited paclitaxel-induced apoptosis without affecting the ability of paclitaxel to induce microtubule bundling and mitotic arrest. This finding implies that apoptotic cell death induced by paclitaxel may occur via a pathway independent of mitotic arrest. Through analyses of a number of apoptosis-associated genes or regulatory proteins, we found that glucocorticoids and paclitaxel possess opposite regulatory role in the NF-kappa B/Ikappa Balpha signaling pathway. Further studies indicate that paclitaxel activates Ikappa B Kinase (IKK), which in turn causes degradation of Ikappa Balpha and activation of NF-kappa B, whereas glucocorticoids antagonize paclitaxel-mediated NF-kappa B activation through induction of Ikappa Balpha synthesis. These results suggest that the NF-kappa B/Ikappa Balpha signaling pathway might play a critical role in the mediation or regulation of paclitaxel-induced cell death. On the other hand, since glucocorticoids (such as dexamethasone) are routinely used in the clinical application of paclitaxel to prevent hypersensitivity reactions and other adverse effects, the inhibitory action of glucocorticoids on paclitaxel-induced apoptosis also raises a clinically relevant question as to whether the pretreatment with glucocorticoids might interfere with the therapeutic efficacy of paclitaxel.

Glucocorticoid receptor gene polymorphisms in ACTH‐secreting pituitary tumours

The inhibitory action of glucocorticoids on the hypothalamic-pituitary axis is disrupted in ACTH-secreting pituitary tumours. The molecular events leading to the development of these tumours and their relative resistance to glucocorticoids are unknown. We investigated the presence of mutations and polymorphisms of the glucocorticoid receptor (GR) gene in corticotropinoma and their possible relationship with the tissue-specific resistance to glucocorticoids. DNA or RNA was extracted from 18 corticotropinomas and the GR gene was amplified by the polymerase chain reaction (PCR) or reverse transcriptase-PCR followed by automated direct sequencing. We did not identify any mutation in the coding region and the exon-intron boundary regions of the GR gene. The polymorphism AAT > AGT at codon 363 (N363S) was found in 17% and the polymorphism AAT > AAC at codon 766 (N766N) in 11% of tumours, both in heterozygous state. The polymorphisms at codons 22 and 23, at introns 3 and 4, and at codon 618, previously described in normal population, were not observed. Our results show that GR gene mutations are rare and unlikely to contribute to the glucocorticoid resistance observed in corticotropinomas. Polymorphisms in the GR gene might confer a selective advantage to tumorigenesis in corticotropinoma. However, there was no relationship between GR gene polymorphism and clinical presentation, tumour size or surgery outcome, suggesting that tumour growth may not be directly related to alterations of the GR gene structure.

Calreticulin is an interleukin‐3‐sensitive calcium‐binding protein in human basophil leukocytes

IL-3 enhances basophil histamine release upon stimulation with any known secretagogue. The molecular mechanism behind this regulation is not known, although some observations suggest that IL-3 modulates the calcium part of the signal transduction mechanism. The inhibitory action of glucocorticoids on basophils can be reversed by stimulation with IL-3. Calcium-binding proteins in the basophil cell line KU812 were identified by two-dimensional gel electrophoresis, Calcium-overlay assay, N-terminal sequence analysis, and mass spectometry. The presence of the same proteins in purified human basophil leukocytes was established by comigration of KU812 and human basophil proteins on the two-dimensional gels. The expression of the calcium-binding proteins in the absence and presence of IL-3 and/or anti-IgE was determined by densitometric measurement of the spots on the two-dimensional gels. Calreticulin was identified on the two-dimensional gel of KU812 proteins. A protein with exactly the same migration pattern was found on the gels of proteins from purified human basophils. Immunoblotting with a specific antihuman calreticulin antibody confirmed that this protein was calreticulin. Subsequent analysis showed that the expression of calreticulin in the basophils is upregulated twofold upon stimulation with rhIL-3, even in doses below those needed for enhancement of histamine release. The expression of calreticulin in human basophil leukocytes is regulated by IL-3. Calreticulin is known to modulate IP3-dependent Ca2+ influx in different cell systems, and calreticulin overexpression inhibits steroid-induced transcriptional activation. Therefore, modulation of calreticulin expression may be one mechanism by which IL-3 exerts its effects on human basophils.

Editorial: Inhibitory Actions of Glucocorticoids on Skeletal Growth. Is Local Insulin-Like Growth Factor I to Blame?

Editorial: Inhibitory Actions of Glucocorticoids on Skeletal Growth. Is Local Insulin-Like Growth Factor I to Blame?

Pulsatile thyrotropin secretion in patients with Addison's disease during variable glucocorticoid therapy.

The inhibitory action of physiological to pathophysiological serum cortisol levels on TSH secretion were investigated in 12 patients with Addison's disease on 3 occasions. 1) In continuation of the conventional hydrocortisone (HC) substitution, a medium dose of HC (0.5 mg/kg) was infused over 23 h. 2) After 24-h withdrawal of HC, the patients had placebo infusion over 23 h. 3) After 5 days of dexamethasone (1.5 mg/day), a high dose of HC (2.0 mg/kg) was infused over 23 h. Blood sampling was performed every 10 min during the last 10 h of the study period, followed by a TRH test (10 micrograms, iv), To mimic the normal diurnal rhythm for serum cortisol, HC was infused in graduated doses, and during medium dose infusion, the serum cortisol level and the TSH pulsatility pattern were similar to those seen in normal controls. The TSH mean level was 1.0 +/- 0.5 mU/L during medium doses of HC, increasing significantly (P < 0.05) to 2.0 +/- 1.6 mU/L during the low cortisol state and was significantly (P < 0.05) suppressed to 0.4 +/- 0.2 mU/L during high doses of glucocorticoids, when the pulse frequency was also significantly reduced (P < 0.01). Together with a dose-dependent inhibitory action of glucocorticoids on the TSH response to TRH, our data indicate that even physiological serum levels of cortisol have an influence on endogenous TSH secretion, probably caused by regulation of the pituitary sensitivity to TRH.

Effects of bacterial endotoxin on the glucocorticoid feedback regulation of adrenocortical response to stress.

Previous studies have shown that LPS and cytokines modulate the binding of glucocorticoids (GCs) in the CNS, and therefore may affect the negative feedback exerted by GCs. In this study, we investigated the effect of lipopolysaccharide (LPS) on the inhibitory action of GCs upon the adrenocortical response to a neural stressful stimulus. Male rats were treated with either LPS (50 micrograms/kg) or saline for 5 consecutive days. Two days later, the LPS- and saline-treated rats were injected intraperitoneally with either dexamethasone (20 micrograms/kg) or saline and sacrificed 3.5 h later, after exposure to acute stressful photic stimulation. In saline-pretreated rats, photic stimulation caused a 5-fold increase in serum corticosterone levels compared to basal levels, and pretreatment with dexamethasone completely abolished this response. In LPS-pretreated rats, corticosterone levels following photic stimulation increased 20-fold, and dexamethasone was ineffective. Additional experiments were conducted to examine whether the impairment in the negative feedback was specific to the prolonged LPS treatment, rather than to LPS-induced hypersecretion of GCs. In groups of rats which were exposed to either daily acoustic stress or daily administration of corticosterone (5 mg, twice daily) for 5 days, the pattern of corticosterone secretion mimicked the corticosterone secretion induced by LPS. In these groups, the adrenocortical response to acute photic stimulation and the effect of dexamethasone were similar to saline-pretreated controls. These results suggest that LPS impairs the negative feedback of either endogenous or exogenous GC upon the adrenocortical response to stress. This finding may be relevant to the enhanced adrenocortical activity associated with sepsis and major depression.

Interactions of glucocorticoids and cyclic AMP in the tissue-specific regulation of angiotensinogen

Angiotensinogen (Ao) is the obligatory glycoprotein substrate for the enzymatic generation of the angiotensin peptides. There are high concentrations of Ao in plasma, originating mostly from hepatic biosynthesis. Numerous additional tissues which, among others, include the heart, adrenal, kidney, pituitary and brain, also express Ao [1], The Ao synthesized by these tissues is available for the local generation of angiotensins. Consequently, changes in the availability of Ao via regulation of its synthesis and secretion is likely to directly affect the activity of local angiotensin-generating systems. Regulation of Ao by glucocorticoids (GCs) has been investigated mostly for liver where secretion and mRNA content increase several-fold [2–4]. However, sensitivity to GCs is tissue-specific and has been particularly difficult to show in brain tissue [3, 5, 6]. In addition, it has been demonstrated in the liver that GCs interact additively with cAMP and permissively with interleukins [2, 4]. Recently we reported an inhibitory action of GC in C6 glioma cells which could be prevented by cAMP and isoprenaline [7]. Since GCs and cAMP are known to interact negatively or positively in gene expression, depending on tissue [8, 9], it is possible that the range of GC sensitivities reported for Ao results from tissue specific interactions with cAMP or with some other factor. We report recent data in which cell cultures of H4 (rat hepatoma) and primary cultures of pituitary, astrocyte and neuronal cells were used to investigate the interaction of the glucocorticoid, dexamethasone (DEX) with cAMP.

Arginine normalizes the growth hormone (GH) response to GH-releasing hormone in adult patients receiving chronic daily immunosuppressive glucocorticoid therapy.

Glucocorticoids are thought to inhibit GH secretion through an enhancement of endogenous somatostatin tone. The aim of our study was to evaluate the effect of arginine, a secretagogue that increases GH secretion acting at the hypothalamic level, probably by decreasing somatostatin tone, on GH-releasing hormone (GHRH)-induced GH secretion in three male and five female adult patients with nonendocrine disease who were receiving daily immunosuppressive glucocorticoid therapy. Six normal subjects (four males and two females) served as controls. GHRH-induced GH secretion was evaluated after 30-min iv infusion of saline (100 mL) or arginine (30 g) in 100 mL saline. After saline administration, steroid-treated patients showed a blunted GH response to GHRH (GH peak, 8.7 +/- 2.4 micrograms/L) compared to that of normal subjects (GH peak, 23.8 +/- 3.9 micrograms/L). The GH responses to GHRH increased (P less than 0.05) after pretreatment with arginine compared to saline pretreatment in both normal subjects (GH peak, 36.6 +/- 4.0 micrograms/L) and steroid-treated patients (GH peak, 28.4 +/- 5.5 micrograms/L). The GH responses to GHRH plus arginine were not significantly different in steroid-treated and normal subjects. Thus, arginine is able to normalize the GH response to GHRH in patients receiving chronic glucocorticoid treatment. Our data are evidence that the stimulatory action of arginine and the inhibitory action of glucocorticoids on GH secretion are mediated by opposite effects on hypothalamic somatostatin tone.

Glucocorticoids inhibit fructose 2,6-bisphosphate synthesis in rat thymocytes. Opposite effect of cycloheximide

The content of fructose 2,6-bisphosphate (Fru(2,6) P 2) and lactate production in triamcinolone acetonide-treated rats thymocytes was studied. The effect in vitro of corticosterone and dexamethasone on normal thymocytes was also examined. Glucocorticoids produced a marked decrease in Fru(2,5) P 2 content and lactate production. The largest effect was observed with triamcinolone acetonide (7.5 mg per kg body weight), which after 20 h of treatment produced over 90% of inhibition. This change was accompanied by the decrease of both phosphofructokinase-1 and -2 activities and ATP levels, without modifications of hexoses phosphate content. The inhibitory actions of glucocorticoids were abolished by cycloheximide, an inhibitor of protein synthesis. Furthermore this drug, by itself, increased Fru(2,6) P 2 content by more than 50% compared with the controls.

Steroid control of central neuronal interactions and function

Steroids have potent actions on the brain which can be categorized as; (i) fast (∼ms-s), (ii) intermediate (h-days), (iii) long-term reversible (days-weeks) and (iv) long-term irreversible. Here attention is focussed on the intermediate and long-term reversible effects of steroids with emphasis on glucocorticoids and oestrogen. Glucocorticoid negative feedback is generally classified as fast, delayed and long-term. Fast negative feedback would appear to depend mainly on a reduction in pituitary responsiveness to corticotrophin releasing factor-41 (CRF-41) and possibly arginine vasopressin (AVP). Delayed feedback is mediated by reduced AVP release into hypophysial portal blood and blockade of the ACTH response to CRF-41. Long-term negative feedback is a consequence of reduced CRF-41 and AVP release into portal blood. Lesion and electrical stimulation studies pinpoint the paraventricular nuclei as the main site at which glucocorticoids act to control ACTH release. Oestrogen at physiologically low plasma concentrations inhibits gonadotrophin secretion. At physiologically high plasma concentrations, such as those that occur during the preovulatory surge, oestradiol-17β stimulates the biosynthesis of LHRH mRNA and LHRH and the release of LHRH into hypophysial portal blood. Oestradiol also increases pituitary responsiveness to LHRH. The action of oestrogen on LHRH neurons is probably mediated by interneurons and may involve disinhibition; this view is supported by our in situ hybridization studies which show that oestrogen, in its positive feedback mode, significantly reduces the synthesis of proopiomelanocortin mRNA in arcuate neurons which when active are likely to inhibit LHRH neurons. The mechanism of action of oestrogen on the pituitary gland is not yet established, but clues from the action of the priming effect of LHRH suggests that oestrogen may potentiate phosphoinositide second messenger cascades. LHRH priming involves the synthesis of a 70 kDa protein the N-terminus of which is identical to an oestrogen-induced protein in the ventromedial hypothalamic nucleus involved in lordosis, and to that of phospholipase Cα. Attention is drawn to the remarkable economy of the system by which a single steroid, oestrogen, has effects on the brain and pituitary gland which result in a co-ordinated sequence of amplifier cascades which lead first to the ovulatory surge of luteinizing hormone and then to mating behaviour, both of which are obviously essential for continuation of the species. Long-term reversible effects of steroids are exemplified by (i) the action of testosterone, and its metabolite, oestrogen, in stimulating the synthesis of AVP in neurons of the bed nucleus of the stria terminalis, and (ii) the action of oestrogen in reducing by four-fold the amount of the steroid anaesthetic, alphaxalone, required to induce anaesthesia in the male. These stimulatory and inhibitory actions of glucocorticoids and gonadal steroids on the brain and pituitary gland provide excellent models by means of which the molecular mechanisms of central neurotransmission in mammalian brain can be established.

Pyridostigmine Blocks the Inhibitory Effect of Glucocorticoids on Growth Hormone-Releasing Hormone Stimulated Growth Hormone Secretion in Normal Man

Glucocorticoids have been shown to inhibit GH secretion in normal man when acutely and chronically administered in pharmacological amounts. Pyridostigmine (PD), an acetylcholinesterase inhibitor, is able to elicit GH secretion when administered alone and to enhance the GH response to GHRH in normal subjects probably via a decrease in the hypothalamic release of somatostatin. The aim of the present study was to investigate the influence of glucocorticoids on the GH response to PD administered either alone or in combination with GHRH in normal adult subjects. Six healthy adult volunteers underwent six experimental protocols. They received 1) human (h) GHRH(1-29)NH2, 100 micrograms injected as an iv bolus; 2) cortisone acetate, 50 mg administered orally (po) 60 min before an hGHRH iv bolus injection; 3) PD, 120 mg administered po, 60 min before an hGHRH iv bolus injection; 4) PD and cortisone acetate, administered po 60 min before an hGHRH iv bolus injection; 5) PD, administered po 60 min before a saline iv bolus injection; 6) PD and cortisone acetate administered po 60 min before a saline iv bolus injection. Mean GH levels, peak GH levels, and GH area under the curves (AUCs) were significantly lower after GHRH + cortisone as compared to GHRH alone. However, these parameters were not significantly different after PD + GHRH + cortisone when compared to PD + GHRH and after PD + cortisone when compared to PD alone. We conclude that acute administration of pharmacological amounts of glucocorticoids cannot inhibit the GH response to PD alone or in combination with GHRH. Thus, we hypothesize that the inhibitory action of glucocorticoids on the GH response to GHRH in man may be mediated by an enhancement of either somatostatin release by the hypothalamus or somatostatin action on the pituitary.

Glucocorticoids: Potent Inhibitors and Stimulators of Growth Hormone Secretion*

Excessive glucocorticoid concentrations are well recognized inhibitors of linear growth, due in part to their suppression of GH secretion. The mechanism of this inhibition has been unclear, especially since glucocorticoids enhance the in vitro GH response of pituitary cells to GH-releasing hormone (GHRH). We investigated the possibility that hypothalamic somatostatin might be mediating these dichotomous observations by using passive immunization techniques. The GH response to GHRH was significantly blunted in rats pretreated with the synthetic glucocorticoid, dexamethasone, compared to that in normal animals. In marked contrast, the immunoneutralization of somatostatin resulted in a significantly enhanced GH response to GHRH in dexamethasone-treated animals. These results suggest that the previously described inhibitory action of glucocorticoids on GH secretion in vivo are mediated via altered hypothalamic somatostatin tone.

Dexamethasone effects on microvascular endothelial cell lipid composition

Previous studies have shown that treatment of cultured rabbit coronary microvessel endothelial (RCME) cells with dexamethasone results in a dose-, time-, and glucocorticoid-dependent inhibition of prostaglandin release. In the present study, the effects of dexamethasone on RCME membrane lipid composition and release of arachidonic acid were examined. This study demonstrated that dexamethasone treatment did not significantly alter the relative distribution of membrane phospholipids but did result in changes of fatty acid composition. There was an increase in saturated and monounsaturated fatty acids and a decrease in polyunsaturated fatty acids. Dexamethasone treatment did not reduce A23187-stimulated arachidonic acid release, despite inhibiting prostaglandin release by 50%. Studies with radiolabeled arachidonic acid suggest that dexamethasone may exert some actions on membrane remodeling, an effect that will require further investigation. Our data strongly suggest that the inhibitory actions of glucocorticoids on prostaglandin release in cultured RCME cells are not the result of a generalized inhibition of arachidonic acid release, and alternate mechanisms must therefore be considered.

The implication of opiates in the glucocorticoid-mediated inhibition of LH secretion in rats.

The present study was designed to test the effect of acute administration of dexamethasone on the postcastration and gonadotrophin-releasing hormone (GnRH)-induced rise of LH, and to examine whether the inhibitory action of glucocorticoids on LH secretion was mediated by the opioid system. Rats were castrated and injected 10-10.5 h later in a first set of experiments with saline, dexamethasone (250 micrograms/rat), nalmefene (2 mg/kg body weight) or nalmefene plus dexamethasone. The response to GnRH was tested 11 h after castration (time 0) in all groups. Dexamethasone caused a significant (P less than 0.005) decrease in basal serum concentrations of LH, while saline and nalmefene did not induce any change. The administration of dexamethasone preceded by nalmefene increased basal concentrations of LH (1.6 times), with an effect significantly greater than that of dexamethasone (P less than 0.001), nalmefene (P less than 0.05) or saline (P less than 0.005) alone. GnRH induced a significant (P less than 0.001) increase in serum concentrations of LH in all groups. In a second set of experiments, administration of naloxone (2 mg/kg body weight) increased LH levels (P less than 0.05) and similarly reversed the inhibitory effect of dexamethasone on LH.(ABSTRACT TRUNCATED AT 250 WORDS)

The mature, mitogen-responsive, human thymocyte population comprises two functionally distinct T-cell subsets differing in exogenous interleukin requirements for mitogenesis and in sensitivity to glucocorticoids.

Human thymocytes were separated into 10 fractions by continuous Percoll density gradient centrifugation. Almost all the cells able to proliferate in response to phytohaemagglutinin (PHA) and about 80% of the thymocytes carrying the mature T3 marker were contained within the first two lightest fractions (Fr1 and Fr2). However, cells in Fr1 and in Fr2 were clearly different in terms of their interleukin requirement for mitogenesis, i.e. in the presence of interleukin-1 (IL-1), Fr1 thymocytes showed proliferative responses to PHA that were three to five times greater than those of the Fr2 cells, whereas in the presence of exogenous IL-2 both Fr1 and Fr2 cells had mitogenic responses of a similar magnitude. These differences could not be explained by accessory cell contamination or by different kinetics of the proliferative responses. Furthermore, in the presence of exogenous IL-1, Fr1 thymocytes showed a greater capacity for producing IL-2. In contrast, there was no difference in the expression of functional IL-2 receptors between the two fractions. It can be concluded that the mature thymocyte subpopulation contains at least two functionally distinct subsets, which differ in density, and that during intrathymic maturation the capacities to produce and to respond to IL-2 do not develop simultaneously. Although both the number and the binding characteristics of glucocorticoid (GC) receptor sites did not differ significantly between Fr1 and Fr2 thymocytes, dexamethasone was more effective in inhibiting the PHA-induced mitogenesis of Fr2 than of Fr1 cells. Thus, the GC sensitivity of T-cell mitogenesis is not directly correlated with receptor-related variables. In contrast with observations made on peripheral T cells, the inhibitory activity of GC on thymocyte mitogenesis was only partially reversed by the addition of exogenous IL-2. Thus, the inhibitory action of GC on thymocyte mitogenesis cannot be explained only by the steroid-induced inhibition of IL-1--IL-2 production; it must also be due to a GC-inhibitory effect on the IL-2-responsive cell proliferation.

Regulation of hydrocortisone binding sites by hydrocortisone in human bone marrow fibroblasts

The incubation of human bone marrow fibroblasts with hydrocortisone (10(-7) M) produces a time-dependent depletion of hydrocortisone-binding sites. This effect is glucocorticoid-specific, since glucocorticoid agonists cause depletion to the same extent as hydrocortisone. After withdrawal of the hormone from the incubation medium, cells are able to replenish their complement of receptor. The existence of this down-regulation process may represent a protective mechanism against the partial proliferative inhibitory action of glucocorticoids for bone marrow fibroblasts and also a stimulus for differentiation to adipocytes.

Mechanisms of glucocorticoid-induced immunosuppression: Inhibitory effects on expression of fc receptors and production of T-cell growth factor

T-cell growth factor, produced by mitogen or antigen-stimulated spleen cells, is necessary for the proliferation of T-lymphocytes in culture, and may be responsible for clonal expansion of antigen-responsive T-cells. We have found that treatment of Con A-stimulated rat spleen cells or human peripheral mononuclear cells with 100 n M dexamethasone inhibited the production of the growth factor by 95%. This effect was specific for glucocorticoids at physiologic concentrations. Addition of T-cell growth factor completely reversed the inhibition by glucocorticoid of mitogen-induced blastogenesis. We propose that the inhibitory actions of glucocorticoids on antigen- or mitogen-induced T-cell blastogenesis are related to inhibition of production of T-cell growth factor. These inhibitory effects may be responsible for the delay in the primary immune response produced by glucocorticoids. Fc receptors play important roles in phagocytosis and other aspects of immune responses. We have studied the effects of glucocorticoids on Fc receptors on the human progranulocytic cell line HL-60. Treatment of HL-60 cells with dexamethasone for 72 h reduced by 35–50% the number of Fc receptors per cell (normally about 20.000) with no effect on cell viability or proliferation. Overall rates of leucine incorporation were stimulated by 10–20%. A half-maximal response was obtained with 10nM dexamethasone. The effect was specific for glucocorticoids. These findings suggest that an important component in glucocorticoid-induced immunosuppression may be a reduction in Fc receptors.