ImmunosuppressionSince the discovery of glucocorticoids in the s and the recognition of their anti-inflammatory effects, they have been amongst the most widely used and effective treatments to control inflammatory and autoimmune diseases. In recent years, a great deal of effort has been invested in identifying compounds that separate the beneficial anti-inflammatory effects from the adverse metabolic effects of glucocorticoids, with limited effect. It is clear that for these efforts to be immunosuppressive mechanism of action of corticosteroids, a greater understanding is required of the mechanisms by which glucocorticoids anavar test e and eq cycle immunosuppressive mechanism of action of corticosteroids anti-inflammatory and immunosuppressive actions. Recent research is shedding new light on some of these mechanisms and has produced some surprising new findings. Some of these recent developments are reviewed here.
Immunosuppression: Overview, History, Drugs
Inflammatory diseases such as asthma and rheumatoid arthritis are characterised at the molecular level by chronically increased expression of multiple cytokines, chemokines, kinins and their receptors, adhesion molecules, and inflammatory enzymes such as inducible nitric oxide synthase iNOS and the inducible cyclooxygenase COX However, these parameters of inflammation are effectively reduced by treatment with glucocorticoids by both direct and indirect mechanisms.
Similarly, glucocorticoids reduce T cell proliferation and increase T cell apoptosis via mechanisms that are at least partly the result of inhibition of the T cell growth factor, IL Therapeutically, the ability to suppress a number of inflammatory indices makes glucocorticoids among the most potent anti-inflammatory agents currently available for the treatment of chronic inflammatory diseases such as asthma.
Bodily insults, including inflammation, pain, infection or even mental stress, lead to activation of the hypothalamic-pituitary-adrenal HPA axis. These stimuli cause excitation of the hypothalamus, which responds by releasing corticotropin releasing hormone CRH also known as corticotropin releasing factor, CRF.
ACTH in turn stimulates the adrenal cortex to release glucocorticoids such as cortisol. Once within the blood, cortisol is transported to target organs where it elicits numerous metabolic effects including increased blood glucose levels, stimulation of gluconeogenesis in the liver, and the mobilisation of both amino and fatty acids fig 1. However, in addition to these metabolic effects, glucocorticoids are also potent endogenous immunological suppressors.
Thus, whilst the anti-inflammatory power of synthetic glucocorticoids derives from endogenous anti-inflammatory mechanisms, the clinical usefulness of these drugs is limited by HPA insufficiency and effects on bone metabolism in addition to the metabolic effects listed above. In this respect, it is often stated that the metabolic effects of glucocorticoids result from increased transcription of genes such as tyrosine aminotransferase TAT and phoshoenolpyruvate carboxykinase PEPCK , whereas the anti-inflammatory properties are attributed to negative transcriptional effects on inflammatory gene expression.
Effects of glucocorticoids on the hypothalamic-pituitary-adrenal HPA axis. This scheme shows the sites of synthesis and action of the main HPA hormones and the targets of glucocorticoid action see text for details. Based on analysis of dimerisation defective mice many of the effects of glucocorticoids are labelled as either dependent on D or independent of I GR DNA binding. Question marks indicate uncertainty as to the mechanism of action.
Abbreviations are to be found in the text. Adapted from Reichardt and Schutz. It is generally believed that most, if not all, the effects of glucocorticoids on cells are mediated via the glucocorticoid receptor GR. This amino acid protein was cloned in humans in and is a member of the superfamily of ligand regulated nuclear receptors.
Structure of the glucocorticoid receptor. A Linear representation of the amino acid glucocorticoid receptor showing the principal domains. B Enlargement of part of the DBD showing the amino acid sequence single letter codes of the two zinc fingers and the dimerisation loop in bold. Numbering of both the human and rat receptors is given.
The A to T mutation at position that gives rise to the dimerisation defective receptor is shown. In the absence of ligand, GR is predominantly maintained in the cytoplasm as an inactive multi-protein complex. This consists of two hsp90 molecules plus a number of other proteins including the immunophilins p59 and calreticulin. This causes dissociation of the multi-protein complex and allows nuclear translocation of GR by virtue of the nuclear localisation sequence within the DNA binding domain DBD.
Once within the nucleus, GR binds DNA sequences known as glucocorticoid response elements GREs to activate transcription of responsive genes referred to as transactivation table 1 , fig 3 A. This requires interactions between a group of five amino acids, known as the dimerisation or D loop, which is located within the DNA binding domain of each GR molecule and is essential for dimerisation and transcriptional activation fig 2 B. Models of glucocorticoid receptor transcriptional modulation.
B Interaction of GR with a second transcription factor can activate transcription from composite binding sites in a manner that involves DNA binding of both factors. E At a competitive nGRE, binding of GR to the GRE site prevents binding of factors that are required for transcriptional activation and therefore causes transcriptional repression.
G Interaction of GR with a second transcription factor can repress transcription from composite binding sites in a manner that involves DNA binding of both factors. Thus, positive GR-dependent transcriptional mechanisms are not generally considered to explain the more rapid 0—12 hours repressive effects of glucocorticoids on inflammatory genes.
Following on from the characterisation of positive GRE sequences was the postulation that the negative regulation of transcription referred to as transrepression by glucocorticoids occurred via negative GRE sites nGRE. However, in addition to conferring glucocorticoid dependent repression, this region is also necessary for basal POMC transcription and also overlaps with a site that is involved in promoter activation.
It is therefore likely that negative regulation of POMC expression is, in fact, achieved by multiple transrepressive mechanisms see below. However, as this site shows only modest homology to a consensus GRE, it is possible that GR binding is weak and the repressive mechanism more truly resembles that for AP-1 below.
Similarly, a region in the bovine prolactin gene that binds GR and confers nGRE activity also acts as constitutive positive enhancer. One such mechanism is thought to arise from interaction between GR and transcriptional activators. This phenomenon was first described for AP-1 and was thought to involve direct protein-protein interactions between GR and AP However, a number of problems exist with this explanation.
In considering the various repressive mechanisms involving standard, competitive, tethering, or other nGRE sites, it is worth noting that these schemes could be viewed as variations or continuations of essentially similar mechanisms fig 3 D—G.
In a given experimental context, the investigator will generally only detect the predominant mechanism involved. Thus, for example, in the case of competition for binding sites fig 3 E , GR directly binds DNA and prevents binding of factors that are necessary for transcriptional activation.
However, this binding will undoubtedly involve steric or other hindrance of additional unknown factors, which may also contribute to the repression in a manner depicted in fig 3 D. In addition, binding of GR to a competitive nGRE or any other site is also to some extent stabilised by interactions with surrounding factors that are themselves in contact with the DNA. The repressive interaction will therefore also show characteristics of a tethering or composite type nGRE fig 3 F and G. Furthermore, it is possible to view the composite nGRE as an intermediate between these two extremes.
Thus, at nGRE sites, GR prevents the positive transcriptional response via mechanisms that are likely to involve multiple protein-protein interactions that prevent activation of the basal transcription complex by activating transcription factors or co-factors.
In any given case it is likely that a combination of these mechanisms will contribute to the overall trans- repressive effect fig 3 D—G.
This would lead to transcriptional silencing and would provide a novel mechanism whereby glucocorticoids can repress transcription. Deletion studies with mutant GR constructs have shown that the steroid binding domain, the activation domains, and a functional DNA binding domain are necessary for efficient hormone inducible transcription from GRE containing promoters. The above studies have clearly shown that some of the repressive and activating functions of GR may be dissociated at the protein level.
The question that then arises is whether these functions can be differentially activated by steroid ligands. From a therapeutic point of view, this could have great significance if, as mentioned above, the repressive functions of GR mediate the anti-inflammatory effects whilst gene activation is responsible for the metabolic, and therefore undesirable, effects of glucocorticoids.
Some degree of functional separation is achieved by steroid antagonists such as RU RU This compound shows little ability to transactivate GR dependent transcription. However, other undesirable effects on, for example, bone metabolism may also be mediated via transrepressive mechanisms involving negatively regulated genes such as osteocalcin fig 1.
One powerful tool for studying genetically engineered proteins in an in vivo context is the use of transgenic mice. To gain further insights into the mechanisms of GR action, a line of transgenic mice was established in which the wild type GR was replaced with a receptor containing the AT mutation in the D loop fig 2 B. Furthermore, it appears that transrepression involving tethering mechanisms remains intact, at least in respect of AP-1 dependent genes. Further analyses are now required to examine the ability of these mice to suppress various inflammatory responses in response to glucocorticoids as well as to determine the extent of any other undesirable effects.
Such studies will shed light on the mechanisms of gene repression and, in particular, the relative contribution of DNA binding dependent and independent effects of GR action. In addition, these studies will act as a validation exercise for the possible therapeutic benefits of second generation dissociating glucocorticoids.
So far this review has focused on the transcriptional mechanisms by which glucocorticoids regulate the expression of responsive genes. However, correctly regulated gene expression requires the coordinated control of transcriptional that is, the rate of gene transcription , post-transcriptional for example, mRNA stability , translational that is, protein synthesis , and post-translational for example, protein processing, modification or degradation events.
In addition, other post-translational processes involving, for example, intracellular localisation or, in the case of cytokines, secretion may also act as control points. Given this myriad of mechanisms involved in the regulation of gene expression, it is likely that a number, if not all, of these processes are also targets of glucocorticoid action.
In recent years it has become increasingly apparent that many genes are regulated to a substantial degree by post-transcriptional and translational mechanisms. In A cells this effect required ongoing RNA and protein synthesis, suggesting the need for dexamethasone inducible gene synthesis.
One further example of post-transcriptional downregulation by dexamethasone has been shown for monocyte chemoattractant protein 1 MCP Furthermore, the dependence on ongoing transcription or translation indicates that many of these effects involve transcriptional activation and therefore presumably the transactivation functions of GR.
A model for glucocorticoid dependent repression of proinflammatory genes. A generalised inflammatory cascade is shown. Cytokine binding to its cognate receptor localised in the plasma membrane pm leads to activation of a kinase cascade consisting of kinases 1, 2, and 3 K1, K2, and K3.
K3 translocates across the nuclear membrane nm and then phosphorylates the transcription factor TF which actives transcription of an inflammatory protein gene. This leads to mRNA synthesis transcription and protein synthesis translation of the inflammatory protein. Binding of glucocorticoid to the glucocorticoid receptor GE leads to dissociation of the heat shock proteins hsp90 and translocation of GR to the nucleus. It was noted above that glucocorticoids induce apoptosis of both T cells and eosinophils.
In this respect entry of the cell to the programmed cell death pathway is an active process and requires a variety of newly synthesised proteins. Unlike apoptosis, inhibition of T cell proliferation by glucocorticoids occurs at least in part by repression of cell cycle genes such as the G1 progression factor, cyclin D3.
In the preceding sections we have seen how glucocorticoids, acting via GR, can positively regulate transcription from GRE sites and negatively regulate transcription via a variety of mechanisms including nGRE and tethering sites. However, a number of other transcription modulating activities of GR exist which may result in biologically significant effects.
Furthermore, the functional outcome of factors binding to these sites depends very much on the cellular environment. Thus, dexamethasone may activate or repress transcription from these elements depending on the cell type. The existence of the above interactions raises the possibility that, in addition to effects in respect of acute phase genes in the liver, these types of response may also play a role in the anti-inflammatory actions of glucocorticoids.
At present these interactions have not been examined using dissociated steroids or the various transactivation and transrepression defective GR mutants.
Consequently, we do not currently understand the mechanisms behind these effects nor can we guess the role that these processes may play in the anti-inflammatory actions of glucocorticoids. However, it is tempting to speculate that putative glucocorticoid inducible genes, which are involved in the post-transcriptional repression of inflammatory genes, may display similar responses.
In recent years a number of glucocorticoid inducible signalling proteins such as the serum and glucocorticoid inducible kinase sgk , 25 the glucocorticoid induced diacylglycerol kinase, and the small Ras-like GTPase, dexras 1 have been identified. These and other similar proteins may be expected to elicit direct, albeit as yet uncharacterised, dexamethasone dependent signalling effects. Furthermore, it is likely that additional glucocorticoid inducible genes involved in signal transduction will be characterised and collectively these new signalling proteins will lead to novel glucocorticoid dependent responses.
Two events that are dramatically altering our ability to characterise changes in gene expression at the mRNA level are the revolution in microarray or gene chip technology and the imminent completion of the human genome project. In this respect the sequencing of human chromosome 22 was recently reported. This information can then be combined with array or chip technology to analyse thousands, tens of thousands or, ultimately, hundreds of thousand of genes for changes in mRNA expression following a given stimulus or in a given cell type.
Such analyses may allow the molecular characterisation of individuals who fail to respond to steroids and could lead to individual specific therapeutic approaches. In addition, the identification of genes involved in the glucocorticoid response will facilitate the identification of responses that are desirable for anti-inflammatory effects and those that are undesirable—for example, those that promote bone metabolism or give rise to Cushing's syndrome.
However, these mechanisms alone do not appear to explain the full ability of glucocorticoids to repress many inflammatory genes.