Impaired stress response and reduced anxiety in mice lacking a functional corticotropin-releasing hormone receptor 1.

Corticotropin-releasing hormone (CRH) is a potent mediator of endocrine, autonomic, behavioural and immune responses to stress, and has been implicated in the stress-like and other aversive consequences of drug abuse, such as withdrawal from alcohol. Two CRH receptors, Crhr1 and Crhr2, have been identified in the mouse. Crhr1 is highly expressed in the anterior pituitary, neocortex, hippocampus, amygdala and cerebellum, and activation of this receptor stimulates adenylate cyclase. Here we show that in mice lacking Crhr1, the medulla of the adrenal gland is atrophied and stress-induced release of adrenocorticotropic hormone (ACTH) and corticosterone is reduced. The homozygous mutants exhibit increased exploratory activity and reduced anxiety-related behaviour under both basal conditions and following alcohol withdrawal. Our results demonstrate a key role of the Crhr1 receptor in mediating the stress response and anxiety-related behaviour.

AAV-mediated gene transfer of a checkpoint inhibitor in combination with HER2-targeted CAR-NK cells as experimental therapy for glioblastoma.

Glioblastoma (GB) is the most common primary brain tumor, which is characterized by low immunogenicity of tumor cells and prevalent immunosuppression in the tumor microenvironment (TME). Targeted local combination immunotherapy is a promising strategy to overcome these obstacles. Here, we evaluated tumor-cell specific delivery of an anti-PD-1 immunoadhesin (aPD-1) via a targeted adeno-associated viral vector (AAV) as well as HER2-specific NK-92/5.28.z (anti-HER2.CAR/NK-92) cells as components for a combination immunotherapy. In co-culture experiments, target-activated anti-HER2.CAR/NK-92 cells modified surrounding tumor cells and bystander immune cells by triggering the release of inflammatory cytokines and upregulation of PD-L1. Tumor cell-specific delivery of aPD-1 was achieved by displaying a HER2-specific designed ankyrin repeat protein (DARPin) on the AAV surface. HER2-AAV mediated gene transfer into GB cells correlated with HER2 expression levels, without inducing anti-viral responses in transduced cells. Furthermore, AAV-transduction did not interfere with anti-HER2.CAR/NK-92 cell-mediated tumor cell lysis. After selective transduction of HER2+ cells, aPD-1 expression was detected at the mRNA and protein level. The aPD-1 immunoadhesin was secreted in a time-dependent manner, bound its target on PD-1-expressing cells and was able to re-activate T cells by efficiently disrupting the PD-1/PD-L1 axis. Moreover, high intratumoral and low systemic aPD-1 concentrations were achieved following local injection of HER2-AAV into orthotopic tumor grafts in vivo. aPD-1 was selectively produced in tumor tissue and could be detected up to 10 days after a single HER2-AAV injection. In subcutaneous GL261-HER2 and Tu2449-HER2 immunocompetent mouse models, administration of the combination therapy significantly prolonged survival, including complete tumor control in several animals in the GL261-HER2 model. In summary, local therapy with aPD-1 encoding HER2-AAVs in combination with anti-HER2.CAR/NK-92 cells may be a promising novel strategy for GB immunotherapy with the potential to enhance efficacy and reduce systemic side effects of immune-checkpoint inhibitors.

Heterodimerization between mineralocorticoid and glucocorticoid receptor: a new principle of glucocorticoid action in the CNS.

In the mammalian central nervous system, responsiveness to glucocorticoids is mediated by both the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). These pharmacologically distinct receptors are believed to bind to common response elements as homodimers. We provide evidence that MR and GR can form a heterodimeric complex with DNA-binding and transactivation properties different from those of the respective homodimers. There was a high degree of cooperativity of MR and GR in binding to a glucocorticoid response element. Transient transfection of a neuroblastoma cell line revealed a transcriptional response pattern of coexpressed MR and GR distinct from that obtained by MR or GR alone. Our findings demonstrate that heterodimerization of MR and GR is a hitherto unrecognized principle for the transcriptional regulation of glucocorticoid-responsive genes in tissue coexpressing these receptors.

Progesterone receptor-mediated effects of neuroactive steroids.

Several 3 alpha-hydroxysteroids accumulate in the brain after local synthesis or after metabolization of steroids that are provided by the adrenals. The 3 alpha-hydroxy ring A-reduced pregnane steroids allopregnanolone and tetrahydrodeoxycorticosterone are believed not to interact with intracellular receptors, but enhance GABA-mediated chloride currents. The present study shows that these neuroactive steroids can regulate gene expression via the progesterone receptor. The induction of DNA binding and transcriptional activation of the progesterone receptor requires intracellular oxidation of the neuroactive steroids into progesterone receptor active 5 alpha-pregnane steroids. Thus, at physiological concentrations, these neuroactive steroids regulate neuronal function through their effects on both transmitter-gated ion channels and steroid receptor-regulated gene expression.

Prenatal immune challenge alters the hypothalamic-pituitary-adrenocortical axis in adult rats.

We investigated whether non-abortive maternal infections would compromise fetal brain development and alter hypothalamic-pituitary-adrenocortical (HPA) axis functioning when adult. To study putative teratogenic effects of a T cell-mediated immune response versus an endotoxic challenge, 10-d-pregnant rats received a single intraperitoneal injection of 5 x 10(8) human red blood cells (HRBC) or gram-negative bacterial endotoxin (Escherichia coli LPS: 30 micrograms/kg). The adult male progeny (3 mo old) of both experimental groups showed increased basal plasma corticosterone levels. In addition, after novelty stress the HRBC group, but not the LPS group, showed increased ACTH and corticosterone levels. Both groups showed substantial decreases in mineralocorticoid (MR) and glucocorticoid receptor (GR) levels in the hippocampus, a limbic brain structure critical for HPA axis regulation, whereas GR concentrations in the hypothalamus were unchanged and in anterior pituitary were slightly increased. HRBC and LPS indeed stimulated the maternal immune system as revealed by specific anti-HRBC antibody production and enhanced IL-1 beta mRNA expression in splenocytes, respectively. This study demonstrates that a T cell-mediated immune response as well as an endotoxic challenge during pregnancy can induce anomalies in HPA axis function in adulthood. Clinically, it may be postulated that disturbed fetal brain development due to prenatal immune challenge increases the vulnerability to develop mental illness involving inadequate responses to stress.

Molecular and functional evidence for in vitro cytokine enhancement of human and murine target cell sensitivity to glucocorticoids. TNF-alpha priming increases glucocorticoid inhibition of TNF-alpha-induced cytotoxicity/apoptosis.

Cytokine-induced glucocorticoid secretion and glucocorticoid inhibition of cytokine synthesis and pleiotropic actions act as important safeguards in preventing cytokine overreaction. We found that TNF-alpha increased glucocorticoid-induced transcriptional activity of the glucocorticoid receptor (GR) via the glucocorticoid response elements (GRE) in L-929 mouse fibroblasts transfected with a glucocorticoid-inducible reporter plasmid. In addition, TNF-alpha also enhanced GR number. The TNF-alpha effect on transcriptional activity was absent in other cell lines that express TNF-alpha receptors but not GRs, and became manifest when a GR expression vector was cotransfected, indicating that TNF-alpha, independent of any effect it may have on GR number, has a stimulatory effect on the glucocorticoid-induced transcriptional activity of the GR. Moreover, TNF-alpha increased GR binding to GRE. As a functional biological correlate of this mechanism, priming of L-929 cells with a low (noncytotoxic) dose of TNF-alpha significantly increased the sensitivity to glucocorticoid inhibition of TNF-alpha-induced cytotoxicity/apoptosis. TNF-alpha and IL-1 beta had the same stimulatory action on glucocorticoid-induced transcriptional activity of the GR via the GRE, in different types of cytokine/glucocorticoid target cells (glioma, pituitary, epithelioid). The phenomenon may therefore reflect a general molecular mechanism whereby cytokines modulate the transcriptional activity of the GR, thus potentiating the counterregulation by glucocorticoids at the level of their target cells.

Stress and hypothalamic-pituitary-adrenal axis function in experimental autoimmune encephalomyelitis and multiple sclerosis - a review.

Multiple sclerosis (MS) is an inflammatory and degenerative disease of the CNS with an assumed autoimmune-mediated pathogenesis. Stressful life events have been hypothesized as potential triggers of disease exacerbation. Animal studies using experimental autoimmune encephalomyelitis (EAE), as a model for MS, suggest that decreased hypothalamic-pituitary-adrenal (HPA) function may play a role in the increased susceptibility and severity of the disease. Histopathological studies of the hypothalamus point to disturbances in corticotropin-releasing hormone (CRH) regulation as a result of MS lesions in this area. Functional endocrine tests (e.g., the combined Dexamethasone-CRH test) showed a disturbed negative feedback after steroid application in MS patients. Hyper- and hypoactivity of the HPA axis, have been described to be associated with more severe courses. This paper presents an overview of the evidence for a role of HPA dysfunction in EAE and MS based on stress-experimental studies.

Do antidepressants stabilize mood through actions on the hypothalamic-pituitary-adrenocortical system?

Patients suffering from severe depression often show an increased activity of the hypothalamic-pituitary-adrenocortical (HPA) system, a premature escape from the cortisol suppressant action of dexamethasone, and a number of other neuroendocrine changes. This might be explained by defective glucocorticoid feedback inhibition. Normalization of the hyperactive HPA system occurs during successful antidepressant pharmacotherapy of depressive illness, and this could be achieved by antidepressant-induced increases in the cellular corticosteroid receptors, rendering the HPA system more susceptible to feedback inhibition by cortisol. Both mineralocorticoid- and glucocorticoid-receptor mRNA levels and hormone-binding activities are found to be increased following treatment of different cell lines or animals with antidepressants. Since the timecourse of antidepressant actions on corticosteroid receptors follows more closely that of clinical improvement of depression, antidepressants might elevate mood in depressives through their long-term effects on HPA regulation.

Long-term voluntary exercise and the mouse hypothalamic-pituitary-adrenocortical axis: impact of concurrent treatment with the antidepressant drug tianeptine.

We investigated whether voluntary exercise and concurrent antidepressant treatment (tianeptine; 20 mg/kg/day; 4 weeks) exert synergistic effects on the mouse hypothalamic-pituitary-adrenocortical (HPA) axis. Animals had access to a running wheel, were treated with the antidepressant, or received both conditions combined. Control mice received no running wheel and no drug treatment. Exercise resulted in asymmetric changes in the adrenal glands. Whereas sedentary mice had larger left adrenals than right ones, this situation was abolished in exercising animals, mainly due to enlargement of the right adrenal cortex. However, antidepressant treatment alone was ineffective whereas the combination of antidepressant treatment and exercise resulted in an enlargement of both adrenal cortices. In these respective conditions, the levels of tyrosine hydroxylase (TH) mRNA expression in the left and right adrenal medullas varied greatly in parallel to the changes observed in the adrenal cortex sizes. TH mRNA expression in the locus coeruleus of exercising mice was significantly increased irrespective of concomitant tianeptine treatment. Corticotrophin-releasing factor mRNA levels in the hypothalamic paraventricular nucleus were decreased after voluntary exercise but were unaffected by tianeptine. Exercise, particularly in combination with tianeptine treatment, resulted in decreased early morning baseline plasma levels of corticosterone. If animals were exposed to novelty (i.e. a mild psychological stressor), a decreased response in plasma corticosterone levels was observed in the exercising mice. By contrast, after restraint, a mixed physical and psychological stressor, exercising mice showed an enhanced response in plasma corticosterone compared to the controls; a response which was even further boosted in exercising mice concomitantly treated with tianeptine. Under either condition, plasma adrenocorticotrophic hormone levels were not different between groups. Thus, voluntary exercise impacts substantially on HPA axis regulation. Concurrent tianeptine treatment results in synergistic actions, mainly at the adrenal level, affecting both its structure and function.

Psychological stress increases hippocampal mineralocorticoid receptor levels: involvement of corticotropin-releasing hormone.

We investigated whether acute stressors regulate functional properties of the hippocampal mineralocorticoid receptor (MR), which acts inhibitory on hypothalamic-pituitary-adrenocortical activity. Exposure of rats to forced swimming or novelty evoked a significant rise in density of MR immunoreactivity in all hippocampal subfields after 24 hr, whereas exposure to a cold environment was ineffective. Time course analysis revealed that the effect of forced swimming on MR peaked at 24 hr and returned to control levels between 24 and 48 hr. In pyramidal neurons of CA2 and CA3, marked rises were already observed after 8 hr. Radioligand binding assays showed that corticotropin-releasing hormone (CRH) injected intracerebroventricularly into adrenalectomized rats also produced a rise in hippocampal MR levels; an effect for which the presence of corticosterone, but not dexamethasone, at the time of injection was a prerequisite. Moreover, pretreatment with the CRH receptor antagonist (d-Phe(12),Nle(21,38),alpha-Me-Leu(37))-CRH(12-41) blocked the effect of forced swimming on hippocampal MR levels. To investigate whether the rise in MR levels had any functional consequences for HPA regulation, 24 hr after forced swimming, a challenge test with the MR antagonist RU 28318 was conducted. The forced swimming exposed rats showed an enhanced MR-mediated inhibition of HPA activity. This study identifies CRH as an important regulator of MR, a pathway with marked consequence for HPA axis regulation. We conclude that the interaction between CRH and MR presents a novel mechanism involved in the adaptation of the brain to psychologically stressful events.

Induction of cytokine receptors by glucocorticoids: functional and pathological significance.

Current concepts on the role of glucocorticoid hormones in the regulation of inflammatory and immune responses depict this role as being inhibitory. Over the past decade, however, a large variety of studies have shown that glucocorticoids also exert stimulatory effects on immune function, suggesting that the present concept of the role of glucocorticoids in the immune system in not sufficient and needs to be extended. Here, Jan Wiegers and Hans Reul ask how these apparently paradoxical effects fit together and what their functional and pathological significance might be.

Type I and type II corticosteroid receptor gene expression in the rat: effect of adrenalectomy and dexamethasone administration.

We have used 32P-labeled cRNA probes directed against Type I (mineralocorticoid, high affinity glucocorticoid) and Type II (classical glucocorticoid) receptor mRNA to screen various tissues, and have investigated the effect of adrenalectomy (ADX) and dexamethasone (DM) administration on their levels in hippocampus. Both Northern blot and S1 nuclease analysis showed Type I mRNA to be high in hippocampus, colon, and heart; low in liver; and undetectable in thymus. Type II mRNA was high in liver, thymus, and brain; and low in testis and parotid. A transient increase in both hippocampal Type I and Type II mRNA was noted at 1-3 days post ADX. DM similarly elicited a rise in hippocampal Type I mRNA at 2-4 days after ADX, but prevented the ADX-induced increment in Type II mRNA. In contrast to the transient increase in Type I receptor mRNA levels, hippocampal levels of Type I receptors measured by [3H]aldosterone binding were constant 1-16 days post ADX. DM administration caused a doubling in Type I receptor levels over 4 days, with plateau levels at 4-16 days; previously, DM has been shown to lower Type II receptor levels in the hippocampus.(ABSTRACT TRUNCATED AT 250 WORDS)

Transactivation and synergistic properties of the mineralocorticoid receptor: relationship to the glucocorticoid receptor.

The human mineralocorticoid (hMR) and glucocorticoid (hGR) receptors mediate biological responses to adrenal corticosteroids and synthetic ligands. In transient transfection studies, corticosteroid-responsive promoters were used to monitor the hormone-dependent transcriptional regulatory properties of both receptors. The hMR mediates a lower stimulation of the transcription rate than the hGR and does not show cooperative activity on promoters containing multiple palindromic glucocorticoid-responsive elements. The functional importance of the amino-terminus in this differential response was demonstrated by hMR/hGR hybrid receptors in which this region was exchanged or deleted. These experiments revealed that the hMR amino-terminus does not provide the strong transactivation function present in the equivalent hGR domain and, in contrast to the hGR amino-terminus, interferes with the synergistic activity mediated by the DNA- and ligand-binding domains of both receptors.

Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation.

Two receptor systems for corticosterone (CORT) can be distinguished in rat brain: mineralocorticoid-like or CORT receptors (CR) and glucocorticoid receptors (GR). The microdistribution and extent of occupation of each receptor population by CORT were studied. The CR system is restricted predominantly to the lateral septum and hippocampus. Within the hippocampus, the highest density occurs in the subiculum +/- CA1 cell field (144 fmol/mg protein) and the dentate gyrus (104 fmol/mg protein). Affinity of CR for CORT was very high (Kd, approximately 0.5 nM). The GR system has a more widespread distribution in the brain. The highest density for GR is in the lateral septum (195 fmol/mg protein), the dentate gyrus (133 fmol/mg protein), the nucleus tractus solitarii and central amygdala. Substantial amounts of GR are present in the paraventricular nucleus and locus coeruleus and low amounts in the raphe area and the subiculum + CA1 cell field. The affinity of GR for CORT (Kd, approximately 2.5-5 nM) was 6- to 10-fold lower than that of CR. Occupation of CR by endogenous ligand was 89.5% during morning trough levels of pituitary-adrenal activity (plasma CORT, 1.4 micrograms/100 ml). Similar levels of occupation (88.7% and 97.6%) were observed at the diurnal peak (plasma CORT, 27 micrograms/100 ml) and after 1 h of restraint stress (plasma CORT, 25 micrograms/100 ml), respectively. Furthermore, a dose of 1 microgram CORT/100 g BW, sc, resulted in 80% CORT receptor occupation, whereas GR were not occupied. For 50% occupation of GR, doses needed to be increased to 50-100 micrograms/100 g BW, and for 95% occupation, a dose of 1 mg CORT was required. The plasma CORT level at the time of half-maximal GR occupation was about 25 micrograms/100 ml, which is in the range of levels attained after stress or during the diurnal peak of pituitary-adrenal activity. Thus, CR are extensively filled (greater than 90%) with endogenous CORT under most circumstances, while GR become occupied concurrent with increasing plasma CORT concentrations due to stress or diurnal rhythm. We conclude that CORT action via CR may be involved in a tonic (permissive) influence on brain function with the septohippocampal complex as a primary target. In view of the almost complete occupation of CR by endogenous hormones, the regulation of the CORT signal via CR will, most likely, be by alterations in the number of such receptors. In contrast, CORT action via GR is involved in its feedback action on stress-activated brain mechanisms, and GR occur widely in the brain.(ABSTRACT TRUNCATED AT 400 WORDS)

Chronic treatment of rats with the antidepressant amitriptyline attenuates the activity of the hypothalamic-pituitary-adrenocortical system.

The effects of the tricyclic antidepressant amitriptyline on the rat hypothalamic-pituitary-adrenocortical (HPA) system were studied. The time-course experiments showed that amitriptyline, given via the drinking water (4.5 mg/kg.day), produces significant decreases (P < 0.05) in adrenal weight after 5 (-20%) and 7 weeks (-21%) of treatment. Hippocampal mineralocorticoid receptor (MR) levels were down-regulated at days 3 (-27%) and 7 (-20%), and transiently up-regulated at 2 (+40%), 5 (+74%), and 7 (+18%) weeks of treatment. Hippocampal glucocorticoid receptor (GR) levels were slightly down-regulated at days 3 (-8%) and 7 (-17%), transiently up-regulated by 26% at 5 weeks, and indistinguishable from controls after 7 weeks of treatment. MR levels were unchanged in the hypothalamus and neocortex, whereas hypothalamic GR concentrations were elevated and neocortical receptor levels were not altered. Dose-response experiments showed significant decreases in adrenal weight when rats were treated with 4.5 (-14%), 8.8 (-16%) and 14.5 (-13%) mg/kg.day antidepressant, but this applied only for the 4.5- (-14%) and 8.8- (-12%) mg/kg.day doses when the ratio of adrenal weight to body weight was considered. The dose-response relationship regarding hippocampal GR content displayed an inverted U-shaped curve, whereas this was less marked for MR levels. A dose of 4.5 mg/kg.day appeared to be optimal for the rise in MR as well as GR. Concerning the neuroendocrine implications of chronic antidepressant treatment, amitriptyline (5 weeks, 4.5 mg/kg.day) produced significant decreases in basal (ACTH, -47%; corticosterone, -31%) as well as stress (30 min novel environment)-induced plasma ACTH (-38%) and corticosterone (-57%) levels. Previous experiments have forwarded a role of limbic MR in the tonic control of basal HPA activity. Based on the present data, we hypothesize that during amitriptyline treatment a rise in limbic MR may be the initial phenomenon in a successively adjusting HPA system, as evidenced by the decreasing plasma hormone concentrations, declining adrenal size, and up-regulation of GR in particular brain regions.

Local administration of recombinant human interleukin-1 beta in the rat hippocampus increases serotonergic neurotransmission, hypothalamic-pituitary-adrenocortical axis activity, and body temperature.

In this study, we equipped rats with a microdialysis probe in the hippocampus, which enabled stress-free intrahippocampal administration of recombinant human IL-1 beta (hIL-1 beta). Perfusion of the probes was conducted with a Ringer's solution containing 0.1 or 1.0 microM hIL-1 beta or without hIL-1 beta, usually for 6 h. Time-dependent changes in serotonergic neurotransmission and hypothalamic-pituitary-adrenocortical activity were simultaneously monitored by measuring serotonin [5-hydroxytryptamine (5-HT)], 5-hydroxyindoleacetic acid, and corticosterone concentrations in the dialysates. In control rats, there was a clear relationship between extracellular 5-HT concentrations in the hippocampus and behavioral activity. Extracellular 5-HT levels were up to twice as high in behaviorally active rats compared to those in resting or sleeping animals. Intrahippocampal administration of hIL-1 beta markedly increased extracellular 5-HT concentrations in the hippocampus and induced a significant decrease in behavioral activity, thereby uncoupling the parallelism between changes in 5-HT and changes in behavioral activity observed in control rats. Perfusion with 0.1 microM hIL-1 beta, but not with 1 microM hIL-1 beta, produced a decrease in 5-hydroxyindoleacetic acid levels, followed by a return to preinfusion levels. Moreover, intrahippocampal administration of hIL-1 beta increased hypothalamic-pituitary-adrenocortical axis activity, as evidenced by marked increases in both plasma ACTH and plasma and dialysate corticosterone levels. In addition, a rise in body temperature by approximately 2 C was observed at time points at which the effects of hIL-1 beta on 5-HT and corticosterone levels were (near-)maximal. hIL-1 beta-treated rats displayed typical characteristics of sickness behavior, such as immobility, piloerection, and a curled-up body posture. Most importantly, no effects were found either with heat-inactivated hIL-1 beta or when hIL-1 beta was administered via a probe implanted in the neocortex. Based on these results, we postulate that the hippocampal IL-1 system may play an important role in the coordination of neuroendocrine, autonomic, and behavioral responses after an immune challenge.

Susceptibility and resistance to experimental allergic encephalomyelitis: relationship with hypothalamic-pituitary-adrenocortical axis responsiveness in the rat.

Susceptibility to experimental allergic encephalomyelitis (EAE) may be influenced by variations in the production of endogenous glucocorticoids. We investigated whether this concept is consistent across different genotypes and paradigms of EAE. In the major histocompatibility complex-disparate rat strains, Lewis (LEW), Brown Norway (BN), and Dark Agouti (DA), inflammatory and inflammatory-demyelinating variants of EAE were induced by immunization with myelin basic protein and myelin oligodendrocyte glycoprotein, respectively. We analyzed hormone production in EAE and after exposure to novel environment. DA and BN rats showed a robust hypothalamic-pituitary-adrenocortical (HPA) axis response to novelty stress and produced significantly higher ACTH and corticosterone plasma levels compared with LEW rats. However, HPA axis responsiveness was not associated with a generalized resistance to EAE, as both DA and LEW rats were susceptible to myelin basic protein-induced EAE. Moreover, both robust HPA responder strains, DA and the EAE-resistant BN rat, were highly susceptible to myelin oligodendrocyte glycoprotein-induced EAE. In animals of all strains, clinical disease was associated with significantly elevated plasma levels of corticosterone, and no differences in brain glucocorticoid-binding receptors were detected. Therefore, HPA axis characteristics are not a predictor of disease susceptibility in EAE.

Long-term intracerebroventricular corticotropin-releasing hormone administration induces distinct changes in rat splenocyte activation and cytokine expression.

The effects of long-term corticotropin-releasing hormone (CRH) infusion in the lateral ventricle of the rat on hypothalamic-pituitary-adrenocortical (HPA) axis parameters and on the immune system function were studied. Compared with infusion of vehicle, the CRH treatment produced a sustained overactivity of the HPA axis, as evidenced by elevated plasma ACTH and corticosterone levels, increased anterior pituitary POMC messenger RNA (mRNA) expression, and adrenal enlargement. Long-term CRH treatment also inhibited body weight gain and reduced thymus and spleen weight. In the CRH-treated animals, both Concanavalin A (Con A)-induced T lymphocyte proliferation and lipopolysaccharide (LPS)-induced B lymphocyte mitogenesis was largely suppressed. Surprisingly, interleukin-2 (IL-2) levels were higher in supernatants of splenocyte cultures from CRH-treated rats than in those of control animals. However, IL-2 receptor alpha chain (IL-2R alpha) mRNA expression after Con A stimulation was highly suppressed in the CRH-treated animals. In addition, Northern blot analysis of RNA from splenocytes isolated from spleens of CRH-treated rats revealed a marked expression of IL-1 beta mRNA, in contrast to the barely detectable levels of this cytokine in control animals. Moreover, incubation of total splenocytes and spleen macrophages with LPS resulted in an enhanced induction of IL-1 beta mRNA in cells of CRH-treated rats compared with that of control animals. When adrenalectomized rats were treated with CRH or vehicle, the effects of the CRH treatment on T and B cell proliferation, IL-2 production, and IL-1 beta mRNA expression were abolished. Thus, a continuously increased HPA axis drive results in disparate changes in immune system function. Whether the observed changes in cytokine expression should be regarded as physiologically adaptive adjustments in support of immune function or as potentially pathological anomalies remains to be elucidated.

Enhancement of rat splenic lymphocyte mitogenesis after short term preexposure to corticosteroids in vitro.

Numerous previous studies investigating the effects of corticosteroids on immune proliferation reported almost unanimously inhibitory effects. However, in many of these studies, high concentrations of corticosteroids (micromolar range) and long incubation times (days) were used. We have investigated whether corticosteroid hormones at low physiological (nanomolar) vs. high (micromolar) concentrations have distinct effects on immune cells after short term (minutes) as opposed to long term preexposure (hours). When rat splenocytes were preincubated with high concentrations (0.1-1 microM) of corticosterone (CORT) or with the specific glucocorticoid agonist RU 28362 (0.1 microM) for 1-6 h, washed, and then exposed to the T-cell mitogen Concanavalin-A, a time- and dose-dependent inhibition of lymphocyte mitogenesis over the next 3 days of culture was found. Preincubation with the glucocorticoid antagonist RU 486 completely blocked this inhibitory effect of CORT and RU 28362. Preexposure of splenocytes to CORT for 10-60 min did not alter mitogenesis. No differences were observed between intact and adrenalectomized (ADX) rats. However, if splenocytes from ADX rats were used in the presence of the glucocorticoid antagonist RU 486, the proliferative response over a 3-day period was stimulated by short term preexposure (10-30 min) to low concentrations (3-30 nM) of CORT. Under these incubation conditions, the mineralocorticoid aldosterone, in the presence of RU 486; also produced a stimulatory effect on ADX splenic lymphocyte mitogenesis, whereas RU 28362 was not effective. Corticosteroid receptor binding studies revealed the presence of mineralocorticoid (MR) as well as glucocorticoid (GR) receptors (45 and 600 fmol/mg protein, respectively) in the spleen. In conclusion, low physiological concentrations of CORT and aldosterone have novel stimulatory properties on mitogenesis of splenic lymphocytes from ADX rats. These effects become evident in the presence of GR antagonist and persist after short corticosteroid preexposure times. In contrast, after prolonged preincubation with high concentrations of corticosteroids, the well known immunosuppressive action is observed. We postulate that distinct MR- and GR-mediated effects may underlie these differential steroid actions on splenic lymphocyte proliferation.

Glucocorticoids suppress interleukin-1 receptor antagonist synthesis following induction by endotoxin.

Glucocorticoids, as part of their physiological role in the control of inflammatory and immune processes, suppress the expression of IL-1 and other cytokines. We have found a dose-dependent inhibition by dexamethasone (10 nM to 10 microM) of mRNA levels of the recently cloned IL-1 receptor antagonist (IL-1ra) in endotoxin-stimulated human monocytes. At the same concentrations, both dexamethasone and cortisol inhibited the secretion of IL-1ra. These inhibitory effects were reversed by blocking glucocorticoid receptors with the specific antagonist RU 38486, but not by adding exogenous IL-1, even up to 100 ng/ml, to the monocytes. A similar inhibition of IL-1ra mRNA and protein secretion was found in monocytes obtained after dexamethasone administration in vivo. In addition, we observed parallel increases in glucocorticoid and IL-1ra levels following endotoxin administration to normal volunteers. Our results show that glucocorticoids shut down not only IL-1 but also IL-1ra expression, ruling out induction of IL-1ra as part of the glucocorticoid antiinflammatory mechanism. The control of the delicate immunoregulatory balance of the IL-1/IL-1ra system during endotoxemia underscores the physiological importance of glucocorticoids in the final control of immune responses.