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.

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.

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.

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.

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.

Molecular and cellular mechanisms of T cell development.

The thymus is central to the establishment of a functioning immune system. Here is the place where T cells mature from hematopoietic progenitors, driven by mutual interactions of stromal cells and the developing thymocytes. As a result, different types of T cells are generated, all of which have been carefully selected for the ability to act in host defense towards non-self and against the potential to mount pathogenic self-reactive autoimmune responses. In this review we summarize our present knowledge on the lineage decisions taking place during this development, the selection processes responsible for shaping the T cell antigen-receptor repertoire, the interactions with the stromal components and the signal transduction pathways which transform the interactions with the thymic microenvironment into cellular responses of survival, proliferation, differentiation and, importantly, also of cell death.

Repression of inflammatory responses in the absence of DNA binding by the glucocorticoid receptor.

The glucocorticoid receptor (GR) acts both as a transcription factor itself on genes carrying GR response elements (GREs) and as a modulator of other transcription factors. Using mice with a mutation in the GR, which cannot activate GRE promoters, we examine whether the important anti-inflammatory and immune suppressive functions of glucocorticoids (GCs) can be established in this in vivo animal model. We find that most actions are indeed exerted in the absence of the DNA-binding ability of the GR: inhibition of the inflammatory response of locally irritated skin and of the systemic response to lipopolysaccharides. GCs repress the expression and release of numerous cytokines both in vivo and in isolated primary macrophages, thymocytes and CD4(+) splenocytes. A transgenic reporter gene controlled by NF-kappa B exclusively is also repressed, suggesting that protein- protein interaction with other transcription factors such as NF-kappa B forms the basis of the anti-inflammatory activity of GR. The only defect of immune suppression detected so far concerns the induced apoptosis of thymocytes and T lymphocytes.

Point mutation in the mouse glucocorticoid receptor preventing DNA binding impairs spatial memory.

Activation of central glucocorticoid receptors caused by the stress that is associated with a learning task facilitates storage of the acquired information. The molecular mechanism underlying this phenomenon is entirely unknown. Glucocorticoid receptors can influence transcription both through DNA binding-dependent and -independent mechanisms. To assess the importance of these two modes of action for spatial memory, we here used male mutant mice in which homodimerization and DNA binding of the glucocorticoid receptor is largely prevented (GR(dim/dim)) while protein-protein interactions still can take place. These mice showed a selective impairment of spatial memory in the water maze. Locomotion and anxiety-related parameters measured in an open field and a light/dark preference task were comparable for mutant and control mice. Mutant mice released more corticosterone than control mice under basal resting conditions and in response to swimming, which could have influenced memory processes of the mice. However, mimicking the task-related increase in corticosterone by supplementary injection of corticosterone (250 microg/kg, i.p.) in adrenalectomized mice, resulting in equal plasma corticosterone concentrations in both genotypes, improved spatial memory of control mice but had no effect on mutant mice. These findings suggest that task-related facilitating effects of corticosterone on spatial memory indeed depend on DNA binding of the glucocorticoid receptor rather than on protein-protein interactions of the receptor with other transcription factors. Although it cannot be excluded that both processes are involved in a coordinated way, interrupting the DNA-binding capacity of the receptor is sufficient to induce impairment.

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.

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.

Molecular genetic analysis of glucocorticoid signaling using the Cre/loxP system.

Glucocorticoids (GC) are involved in a plethora of physiological processes that range from the regulation of the stress response and the control of the immune system to modulation of behavior. Most GC effects are mediated by the glucocorticoid receptor (GR) via activation and repression of gene expression. Whereas in most cases activation requires DNA binding of the receptor, repression is usually mediated by protein-protein interaction with other transcription factors. To decipher the molecular mode of action of GR, mice were generated by gene targeting carrying a point mutation in one of the dimerization domains, thus abrogating DNA binding by GR. Analysis of these mice demonstrated that thymocyte apoptosis and stress erythropoiesis require the DNA binding-dependent function of GR, whereas lung development and the anti-inflammatory activity of GR are mediated by protein-protein interaction. Furthermore, to study the role of GC in the brain, mice were generated specifically lacking GR function in the nervous system. Using these mice we demonstrated that GR is essential for the regulation of the HPA-axis and the stress response, as well as for the control of emotional behavior. Taken together, gene targeting using the Cre/loxP system proved to be highly valuable for the analysis of both molecular mechanism and tissue-specific functions of the GR.

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.

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.

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.

Molecular genetic dissection of glucocorticoid receptor function in vivo.

Glucocorticoids are involved in numerous physiological processes. Most of their effects are mediated by the glucocorticoid receptor (GR) via activation and repression of gene expression. Whereas activation requires DNA binding of the receptor, repression is usually mediated by protein-protein interactions with other transcription factors. To decipher the molecular mode of action of GR, mice were generated by gene targeting, carrying a point mutation in one of the dimerization domains, thus abrogating DNA binding by GR. These GRdim mice survive to adulthood and thereby allowed analysis of the mechanism used by GR in the control of physiological processes. Specifically, stress erythropoiesis was found to require the DNA binding-dependent function of GR whereas the antitumor-promoting activity of GR in skin is mediated by interaction with the transcription factor AP-1. Furthermore, the immunosuppressive and anti-inflammatory activity of glucocorticoids is largely independent of GR DNA-binding, suggesting that GRdim mice might be useful in the future for the search of steroidal anti-inflammatory drugs with reduced side-effects.

Genetic disruption of mineralocorticoid receptor leads to impaired neurogenesis and granule cell degeneration in the hippocampus of adult mice.

To dissect the effects of corticosteroids mediated by the mineralocorticoid (MR) and the glucocorticoid receptor (GR) in the central nervous system, we compared MR-/- mice, whose salt loss syndrome was corrected by exogenous NaCI administration, with GR-/- mice having a brain-specific disruption of the GR gene generated by the Cre/loxP-recombination system. Neuropathological analyses revealed a decreased density of granule cells in the hippocampus of adult MR-/- mice but not in mice with disruption of GR. Furthermore, adult MR-/- mice exhibited a significant reduction of granule cell neurogenesis to 65% of control levels, possibly mediated by GR due to elevated corticosterone plasma levels. Neurogenesis was unaltered in adult mice with disruption of GR. Thus, we could attribute long-term trophic effects of adrenal steroids on dentate granule cells to MR. These MR-related alterations may participate in the pathogenesis of hippocampal changes observed in ageing, chronic stress and affective disorders.

The glucocorticoid receptor is required for stress erythropoiesis.

The glucocorticoid receptor (GR) coordinates a multitude of physiological responses in vivo. In vitro, glucocorticoids are required for sustained proliferation of erythroid progenitors (ebls). Here, we analyze the impact of the GR on erythropoiesis in vivo, using GR-deficient mice or mice expressing a GR defective for transactivation. In vitro, sustained proliferation of primary ebls requires an intact GR. In vivo, the GR is required for rapid expansion of ebls under stress situations like erythrolysis or hypoxia. A particular, GR-sensitive progenitor could be identified as being responsible for the stress response. Thus, GR-mediated regulation of ebl proliferation is essential for stress erythropoiesis in vivo.