Permanent CNI treatment for prevention of renal allograft rejection in sensitized hosts can be replaced by regulatory T cells.

Recent data suggest that donor-specific memory T cells (T(mem)) are an independent risk factor for rejection and poor graft function in patients and a major challenge for immunosuppression minimizing strategies. Many tolerance induction protocols successfully proven in small animal models e.g. costimulatory blockade, T cell depletion failed in patients. Consequently, there is a need for more predictive transplant models to evaluate novel promising strategies, such as adoptive transfer of regulatory T cells (Treg). We established a clinically more relevant, life-supporting rat kidney transplant model using a high responder (DA to LEW) recipients that received donor-specific CD4(+)/ 8(+) GFP(+) T(mem) before transplantation to achieve similar pre-transplant frequencies of donor-specific T(mem) as seen in many patients. T cell depletion alone induced long-term graft survival in naïve recipients but could not prevent acute rejection in T(mem)(+) rats, like in patients. Only if T cell depletion was combined with permanent CNI-treatment, the intragraft inflammation, and acute/chronic allograft rejection could be controlled long-term. Remarkably, combining 10 days CNI treatment and adoptive transfer of Tregs (day 3) but not Treg alone also induced long-term graft survival and an intragraft tolerance profile (e.g. high TOAG-1) in T(mem)(+) rats. Our model allows evaluation of novel therapies under clinically relevant conditions.

The DNA binding-independent function of the glucocorticoid receptor mediates repression of AP-1-dependent genes in skin.

The glucocorticoid receptor (GR) mediates the biological effects of glucocorticoids (GCs) through activation or repression of gene expression, either by DNA binding or via interaction with other transcription factors, such as AP-1. Work in tissue culture cells on the regulation of AP-1-dependent genes, such as collagenase (MMP-13) and stromelysin (MMP-3) has suggested that the antitumor and antiinflammatory activity of GCs is mediated, at least in part, by GR-mediated downmodulation of AP-1. Here, we have identified phorbol ester-induced expression of MMP-3 and MMP-13 in mouse skin as the first example of an in vivo system to measure negative interference between AP-1 and GR in the animal. Cell type-specific induction of these genes by tumor promoters is abolished by GCs. Importantly, this is also the case in GR(dim) mice expressing a DNA binding-defective mutant version of GR. In contrast, the newly identified target genes in skin, plasma glutathione peroxidase and HSP-27, were induced by GC in wild-type, but not in GR(dim) mice. Thus, these data suggest that the DNA binding-independent function of the GR is dispensable for repression of AP-1 activity in vivo and responsible for the antitumor promoting activity of GCs.

Resetting of circadian time in peripheral tissues by glucocorticoid signaling.

In mammals, circadian oscillators reside not only in the suprachiasmatic nucleus of the brain, which harbors the central pacemaker, but also in most peripheral tissues. Here, we show that the glucocorticoid hormone analog dexamethasone induces circadian gene expression in cultured rat-1 fibroblasts and transiently changes the phase of circadian gene expression in liver, kidney, and heart. However, dexamethasone does not affect cyclic gene expression in neurons of the suprachiasmatic nucleus. This enabled us to establish an apparent phase-shift response curve specifically for peripheral clocks in intact animals. In contrast to the central clock, circadian oscillators in peripheral tissues appear to remain responsive to phase resetting throughout the day.

Genetic dissection of glucocorticoid receptor function in mice.

Upon hormone binding, the activated glucocorticoid receptor (GR) functions as a transcription factor via different modes of action to control gene expression. Recent gene-targeting studies in mice provide new insight into the role of GR in vivo and are helping decipher the molecular mechanisms underlying its actions.

Corticosteroid actions in hippocampus require DNA binding of glucocorticoid receptor homodimers.

Glucocorticoids are secreted from the adrenal gland in very high amounts after stress. In the brain, these stress hormones potently modulate ionic currents, monoaminergic transmission, synaptic plasticity and cellular viability, most notably in the hippocampus where corticosteroid receptors are highly enriched. Here we show that at least some of these actions require DNA binding of glucocorticoid receptor (GR) homodimers.

Mice with an increased glucocorticoid receptor gene dosage show enhanced resistance to stress and endotoxic shock.

Targeted mutagenesis of the glucocorticoid receptor has revealed an essential function for survival and the regulation of multiple physiological processes. To investigate the effects of an increased gene dosage of the receptor, we have generated transgenic mice carrying two additional copies of the glucocorticoid receptor gene by using a yeast artificial chromosome. Interestingly, overexpression of the glucocorticoid receptor alters the basal regulation of the hypothalamo-pituitary-adrenal axis, resulting in reduced expression of corticotropin-releasing hormone and adrenocorticotrope hormone and a fourfold reduction in the level of circulating glucocorticoids. In addition, primary thymocytes obtained from transgenic mice show an enhanced sensitivity to glucocorticoid-induced apoptosis. Finally, analysis of these mice under challenge conditions revealed that expression of the glucocorticoid receptor above wild-type levels leads to a weaker response to restraint stress and a strongly increased resistance to lipopolysaccharide-induced endotoxic shock. These results underscore the importance of tight regulation of glucocorticoid receptor expression for the control of physiological and pathological processes. Furthermore, they may explain differences in the susceptibility of humans to inflammatory diseases and stress, depending on individual prenatal and postnatal experiences known to influence the expression of the glucocorticoid receptor.

Immunomodulatory activities of glucocorticoids: insights from transgenesis and gene targeting.

The glucocorticoid receptor (GR) is involved in the regulation of numerous physiological processes. In the immune system, it is thought to participate in lymphocyte apoptosis, T cell development and inflammatory responses. The extensive use of synthetic glucocorticoids as anti-inflammatory, immunosuppressive and anti-neoplastic drugs underscores the importance of the GR in immunomodulation. However, notwithstanding the long history of GR research and the clinical use of glucocorticoids, many questions regarding their mode of action remain. The following review summarizes the molecular genetic approaches that were taken during the last decade to answer some of these questions. The use of transgenic and knock-out mice has enabled gain-of-function and loss-of-function mutations in the GR to be generated even in restricted cell-types. Furthermore, these techniques possess the great advantage of allowing GR activities to be studied in the living animal. Many new and exciting findings have thereby been generated but despite the enormous efforts of several laboratories, a complete picture on the role of GR in the immune system is only just emerging.

Glucocorticoid signalling--multiple variations of a common theme.

Recent advances in molecular genetics have brought us closer to answering the question as to which mechanisms are used by steroid hormone receptors to control transcription. While binding to specific response elements in the promoter and enhancer regions of many genes has for a long time been considered as the major mode of action, a growing number of alternative transcriptional control mechanisms have been identified in the last couple of years. With the recent finding that DNA binding of the glucocorticoid receptor is not essential for survival, mechanisms depending on cross-talk with other transcription factors through protein-protein interactions have gained attention. In this mini-review, we will discuss the roles of DNA binding-dependent and -independent transcriptional modes of action in development and physiology as exemplified by the analysis of glucocorticoid receptor function.

Mammary gland development and lactation are controlled by different glucocorticoid receptor activities.

OBJECTIVE: Regulation of physiological processes by glucocorticoids is achieved by binding to the glucocorticoid receptor (GR) and subsequent modulation of gene expression, either by DNA binding-dependent mechanisms or via protein-protein interaction with other transcription factors. The purpose of this study was to define the molecular mechanism of GR underlying the control of mammary gland development and lactation. DESIGN: To dissect the mechanism of GR action in the mammary gland, we used genetically modified mice carrying a DNA binding-defective GR. These mice retain the ability to regulate transcription by protein-protein interaction but fail to control gene expression by DNA binding-dependent mechanisms. Thus, they allow the study of the mode of GR action in vivo. METHODS: The development of the mammary gland and milk protein synthesis during lactation were studied using histological and biochemical methods. RESULTS: Our findings demonstrated that the lack of the DNA binding function of GR impairs the ductal development of the mammary gland in virgin females and that this can presumably be accounted for by reduced proliferation of epithelial cells. In contrast, lactating females have normally differentiated mammary glands and are fully capable of milk protein production. This is in good agreement with the demonstration that the DNA binding-defective GR is still able to interact with phosphorylated Stat5 proteins, suggesting that transcriptional regulation by protein-protein interaction forms the basis of glucocorticoid action in this process. CONCLUSIONS: The present study has demonstrated that GR plays an important role in the mammary gland and that it uses different molecular modes of action to control development and milk protein synthesis.

Mutagenesis of the glucocorticoid receptor in mice.

The glucocorticoid receptor is an ubiquitously expressed transcription factor involved in the regulation of many different physiological processes. Activated by glucocorticoids the receptor regulates transcription positively or negatively either by direct binding to DNA or by protein protein interactions. In order to define the role of the receptor during development and in physiology several mutations have been generated in the mouse. Mice with a disrupted glucocorticoid receptor gene die shortly after birth due to respiratory failure indicating an important role of the receptor in lung function. Transcription of genes encoding gluconeogenic enzymes in the liver is decreased, proliferation of erythroid progenitors is impaired and the HPA axis is strongly upregulated. To analyze molecular mechanisms of glucocorticoid receptor action in vivo a point mutation has been introduced into the mouse genome which allows to separate DNA-binding-dependent from DNA-binding-independent actions of the receptor. Mice homozygous for the point mutation survive indicating that DNA-binding of the receptor is not required for survival. Induction of glucoconegenic enzymes and proliferation of erythroid progenitors however is impaired. Interestingly, repression of corticotropin releasing factor (CRF) synthesis is maintained, whereas proopiomelanocortin (POMC) expression is upregulated. Since mice with a disrupted glucocorticoid receptor gene die shortly after birth attempts using the Cre/loxP-recombination system are made to bypass early lethality and to study the function of the receptor in defined cell types of adult animals.

Analysis of glucocorticoid signalling by gene targeting.

Glucocorticoids are involved in the regulation of numerous physiological processes. The majority of these effects are thought to be mediated by the glucocorticoid receptor (GR) via activation and repression of gene expression. In most cases activation requires binding of a receptor-dimer to DNA while repression is mediated by protein-protein-interaction of GR-monomers with other transcription factors. To analyse the molecular mechanisms that underlie glucocorticoid effects, mouse mutations in the GR gene were generated and analysed. In order to address the role of glucocorticoid receptor signalling during development and in physiology, the gene was disrupted by gene targeting. Most of the mice homozygous for the mutation die shortly after birth due to severe lung atelectasis. Additional defects were found in the adrenals, liver, brain, bone marrow and thymus as well as in the feedback-regulation of the HPA-axis. To approach the question which functions of the GR are regulated by DNA-binding and which by protein-protein-interaction, a point mutation was introduced into the dimerization domain of the GR which is located in the DNA-binding domain. By homologous recombination in ES-cells using the Cre/loxP-system, mice carrying this mutation were generated [GR(dim) mice]. The mice are fully viable although they show impaired inducibility of gluconeogenetic enzymes in liver, defects in longterm renewal of erythroid progenitors and increased expression of POMC and ACTH in the pituitary. However neither in the lung nor the adrenals were any histological abnormalities found. In conclusion GR(dim)-mice represent a valuable tool to further analyse mechanisms of physiological effects of the GR.

Corticotropin-releasing hormone expression is the major target for glucocorticoid feedback-control at the hypothalamic level.

Glucocorticoid production is controlled via the hypothalamo-pituitary-adrenal (HPA) axis by a negative feedback mechanism involving the glucocorticoid receptor (GR). A major site of regulation is the hypothalamus, where the GR is thought to repress the expression of genes such as corticotropin-releasing hormone (CRH) and arginine-vasopressin (AVP). To define the role of the GR in this feedback loop in more detail, the content of CRH, AVP and neurophysin in the median eminence of mice carrying a targeted disruption of the GR gene was studied using immunohistochemistry. GR-deficient mice were found to contain five times more CRH in the median eminence than wild-type littermates. In contrast, no significant change in the content of AVP was observed in the outer layer of the median eminence and neurophysin was also only moderately increased. Our studies suggest that, at the hypothalamic level, CRH synthesis is the major target for feedback control by the GR and that transcriptional control of AVP and neurophysin plays only a supportive role in this process.

Mice with targeted mutations of glucocorticoid and mineralocorticoid receptors: models for depression and anxiety?

Impaired corticosteroid receptor signaling is a key mechanism in the pathogenesis of stress-related psychiatric disorders such as depression and anxiety. Since in vivo expression and functional studies of corticosteroid receptors are not feasible in the human central nervous system, such analyses have to be done in animal models. Transgenic mice with mutations of corticosteroid receptors are promising tools, which allow us to investigate the role of these proteins in the pathogenesis of symptoms characteristic for depression and anxiety. This review summarizes the neuroendocrinological and behavioral findings that have been obtained in six different mouse strains with specific mutations that influence the expression or the function of the glucocorticoid or the mineralocorticoid receptor (MR). The analyses of these mice helped to define molecular concepts of how corticosteroid receptors regulate the activity of the hypothalamic-pituitary-adrenal (HPA) system. Furthermore, some of these mutant mice exhibited characteristic alterations in behavioral tests for anxiety and despair. However, so far, none of the mouse strains described here can be viewed as an animal model of a specific psychiatric disease defined by common diagnostic criteria. Using high throughput technologies for the identification of genes regulated by glucocorticoid receptor (GR) and MR in brain areas responsible for specific symptoms of stress-related disorders will yield potential new drug targets for the treatment of depression and anxiety.

Mechanisms of glucocorticoids in the control of neuroinflammation.

Glucocorticoids (GCs) are widely used to treat inflammatory diseases such as multiple sclerosis (MS). They predominantly act through the GC receptor, a member of the nuclear receptor superfamily that controls transcription by several different mechanisms. Owing to its ubiquitous expression, there are a variety of cell types that could serve as GC targets in the pathogenesis and treatment of MS. This brings about a great diversity of mechanisms potentially involved in the modulation of neuroinflammation by GCs, including the induction of apoptosis, repression of pro-inflammatory mediators and the expansion of myeloid-derived suppressor cells. Nevertheless, it is not well understood which of these mechanisms are essential for therapeutic efficacy. In this review, we summarise findings made concerning the actions of GCs in MS and its animal model experimental autoimmune encephalomyelitis, and also elucidate current concepts and developments that pertain to this clinically highly relevant treatment regimen.

Aberrant expression of Notch1 interferes with the B-lymphoid phenotype of neoplastic B cells in classical Hodgkin lymphoma.

Plasticity of committed mouse B cells has been demonstrated by inactivation of the B-cell commitment transcription factor PAX5, resulting in loss of the B-cell phenotype and differentiation into various hematopoietic lineages. Furthermore, mature mouse B cells could be reprogrammed into macrophages by overexpression of myeloid-specific transcription factors. Here, we report that aberrant activity of the transmembrane receptor, Notch1, interferes with the B-lymphoid phenotype of mature human germinal center-derived B cells in Hodgkin lymphoma, so called Hodgkin and Reed-Sternberg cells. They have lost the B-cell phenotype despite their mature B-cell origin. Notch1 remodels the B-cell transcription factor network by antagonizing the key transcription factors E2A and early B-cell factor (EBF). Through this mechanism, B lineage-specific genes were suppressed and B lineage-inappropriate genes were induced. We provide evidence that absence of the Notch inhibitor Deltex1 contributes to deregulated Notch activity in Hodgkin and Reed-Sternberg cells. These data suggest that Notch activation interferes with dedifferentiation of neoplastic B cells in Hodgkin lymphoma.

Glucocorticoids in T cell apoptosis and function.

Glucocorticoids (GCs) are a class of steroid hormones which regulate a variety of essential biological functions. The profound anti-inflammatory and immunosuppressive activity of synthetic GCs, combined with their power to induce lymphocyte apoptosis place them among the most commonly prescribed drugs worldwide. Endogenous GCs also exert a wide range of immunomodulatory activities, including the control of T cell homeostasis. Most, if not all of these effects are mediated through the glucocorticoid receptor, a member of the nuclear receptor superfamily. However, the signaling pathways and their cell type specificity remain poorly defined. In this review, we summarize our present knowledge on GC action, the mechanisms employed to induce apoptosis and the currently discussed models of how they may participate in thymocyte development. Although our knowledge in this field has substantially increased during recent years, we are still far from a comprehensive picture of the role that GCs play in T lymphocytes.

CD28-mediated costimulation impacts on the differentiation of DC vaccination-induced T cell responses.

Costimulatory signals such as the ones elicited by CD28/B7 receptor ligation are essential for efficient T cell activation but their role in anti-tumour immune responses remains controversial. In the present study we compared the efficacy of DC vaccination-induced melanoma specific T cell responses to control the development of subcutaneous tumours and pulmonary metastases in CD28-deficient mice. Lack of CD28-mediated costimulatory signals accelerated tumour development in both model systems and also the load of pulmonary metastases was strongly increased by the end of the observation period. To scrutinize whether lack of CD28 signalling influences priming, homing or effector function of Trp-2(180-188)/K(b)-reactive T cells we investigated the characteristics of circulating and tumour infiltrating T cells. No difference in the frequency of Trp-2(180-188)/K(b)-reactive CD8+ T cells could be demonstrated among the cellular infiltrate of subcutaneous tumours after DC vaccination between both genotypes. However, the number of IFN-gamma-producing Trp-2-reactive cells was substantially lower in CD28-deficient mice and also their cytotoxicity was reduced. This suggests that CD28-mediated costimulatory signals are essential for differentiation of functional tumour-specific CD8+ T-effector cells despite having no impact on the homing of primed CD8+ T cells.

Feedback control of glucocorticoid production is established during fetal development.

BACKGROUND: Glucocorticoids are involved in the regulation of metabolic, immunological, and developmental processes. Their synthesis is tightly controlled by feedback regulation through the hypothalamus-pituitary-adrenal (HPA) axis, allowing the organism to respond to stress in an adequate manner and to adapt to new situations. Disturbance of these regulatory mechanisms leads to major human diseases. By generating mice with a targeted mutation in the glucocorticoid receptor (GR) locus, it was possible to analyze the mechanism by which glucocorticoids control the HPA axis, under conditions where at least part of the feedback control was absent early in development. MATERIALS AND METHODS: RNase-protection and in situ hybridization assays were used to compare messenger RNA (mRNA) levels of genes involved in the control of the HPA axis in both GR-mutant and wild-type animals. RESULTS: Negative feedback regulation of the HPA axis by glucocorticoids, which is established around Day E16.5 of embryonic development in wild-type mice, does not occur in GR-mutants, resulting in an increased expression of proopiomelanocortin mRNA in the anterior lobe of the pituitary and of corticotropin-releasing hormone mRNA in the paraventricular nucleus of the hypothalamus. However, the expression of both arginine vasopressin and mineralocorticoid receptor in the brain is not affected. In the neurointermediate lobe of the pituitary, expression of the proopiomelanocortin gene was inversely regulated, compared with its expression in the anterior lobe. CONCLUSIONS: GR-dependent regulation of the HPA axis is established during fetal development, suggesting that maternal factors have an important role in influencing the HPA axis of the adult offspring.