The anti-inflammatory and immunosuppressive effects of glucocorticoids, recent developments and mechanistic insights

1Natural and synthetic glucocorticoids remain at the forefront of anti-inflammatory and immunosuppressive therapies. They are widely used to treat both acute and chronic inflammations, including rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, psoriasis and eczema, as well as being used in treatment of certain leukaemias and in immunosuppressive regimes following organ transplant. At any one time, an estimated ∼1% of the total adult population of the UK receives oral glucocorticoid therapy. However, long-term use of oral glucocorticoids is associated with serious side effects, including osteoporosis, metabolic disease and increased risk of cardiovascular disease—in themselves, somewhat paradoxically, inflammatory conditions. Over the 60 years since the discovery of glucocorticoids, much has been learnt of the molecular mechanisms by which they act (recently reviewed;,,,,,,, and ), with milestones being the characterisation of the glucocorticoid receptor (GR) as a DNA binding protein that regulates transcription initiation (reviewed, ), the cloning of GR and the discovery that many of the immunosuppressive actions of glucocorticoids are mediated by interference with signalling by the key inflammatory transcriptional regulators; NF-κB and AP-1 (reviewed, ). However, much also remains unknown and the past few years have provided crucial mechanistic insights into the dynamic nature of GR interactions with DNA and higher order chromatin structures, into the subtleties of ligand effects on GR function, the regulation of endogenous ligand access to GR, glucocorticoid effects on leukocyte differentiation and function and the mechanistic basis for some of the repressive actions of glucocorticoids, critical for their anti-inflammatory effects.

2The host inflammatory response is a primary defence mechanism engaged immediately following injury or infection which is necessary to restore homeostasis following successful elimination of the injurious agent, ultimately leading to resolution and tissue repair. Although categorically distinct, the innate (the relatively non-specific immediate host defence system that provides a rapid reaction to infection and tissue damage) and adaptive (the more slowly acquired, highly antigen-specific response) immune systems interact and often overlap during an inflammatory response. Indeed, although acute inflammation is largely mediated by the innate immune system, the adaptive immune system often plays a major role in chronic inflammatory disease, with dysregulated lymphocyte responses.

3Inflammation is initiated at the site of injury by resident cells, particularly mast cells and resident macrophages, which release pro-inflammatory mediators including bioactive amines, lipid mediators and cytokines—typically TNF-α and IL-1. These cause vasodilation, increased capillary permeability (humoral response) and leukocyte emigration into injured tissues (cellular response), resulting in the hallmark pain, heat, redness and swelling of inflammation as well as generating a chemotactic gradient to guide and activate recruited cells to the site of injury. Although specific characteristics depend on the immune exposure (e.g. irritant vs pathogen), the recruitment process and activation of inflammatory cells are common. Activated granulocytes, crucial to contain microbial infection, are rapidly attracted to the inflamed site, followed by monocyte emigration from blood vessels and subsequent maturation into macrophages. Once at the inflamed site, neutrophils undergo constitutive apoptosis, functionally isolating them from the inflammatory environment by loss of stimulated chemotaxis, phagocytosis, degranulation and respiratory burst, whilst at the same time, facilitating safe removal of their potentially histotoxic contents by macrophages. Foreign antigens are taken up by antigen presenting cells; particularly dendritic cells, but also macrophages, that then migrate to draining lymph nodes where they instruct the adaptive immune system (T and B lymphocytes), shaping the subsequent immune response. As the inflammatory response progresses and evolves, mononuclear cells predominate and resolution normally ensues. Successful resolution of acute inflammation is an active and highly regulated process and dependent on mechanisms engaged early in the inflammatory response that programme the trajectory and form of the subsequent resolution (reviewed, and ). Persistence of the initiating stimulus invariably leads to chronic inflammation, with the typical dysregulation between destructive inflammatory and excessive healing responses seen in diseases such as arthritis, atherosclerosis and asthma.

4Glucocorticoids inhibit many of the initial events in an inflammatory response. They also promote the resolution of inflammation although the mechanisms by which they do so have received less attention than those associated with suppression of the initial response. Acutely, glucocorticoids inhibit the vasodilation and increased vascular permeability that occurs following inflammatory insult and they decrease leukocyte emigration into inflamed sites, effects that require new protein synthesis (reviewed ). They also alter leukocyte distribution/trafficking, death/survival and, importantly, alter cellular differentiation programmes, thus shaping the subsequent response (see below).

5Most of the anti-inflammatory and immunosuppressive actions of glucocorticoids are attributable either directly or indirectly to the transcriptional effects of GR agonism which alters transcription of numerous genes in leukocytes, both up and down. Although expression of mineralocorticoid receptor (MR), which binds glucocorticoids with high affinity in the absence of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2; see below), has been reported in immune cells (which do not normally express 11β-HSD2, ), it appears of little consequence to the anti-inflammatory effects of glucocorticoids, and may even have pro-inflammatory effects. It should also be noted that some anti-inflammatory effects of glucocorticoids are apparent within minutes and a number are independent of the transcriptional effects of GR (see for example, and ). Notably, glucocorticoids repress transcription of many genes encoding pro-inflammatory cytokines and chemokines, cell adhesion molecules and key enzymes involved in the initiation and/or maintenance of the host inflammatory response (reviewed, and ). Many of these genes are commonly over-expressed during chronic non-resolving inflammation. Intriguingly, recent data have shown that whereas glucocorticoid administration 1 h following endotoxin (lipopolysaccharide, LPS) challenge is immunosuppressive, administration of the same glucocorticoid dose prior to LPS challenge augments immune responses. This was true in both the brain and the liver, raising the possibility that chronic hypothalamic–pituitary–adrenal (HPA) axis activation (as may occur during chronic inflammation for example) exaccerbates inflammation in the brain and elsewhere (reviewed in ).

6Until recently, it was widely believed that the repressive, anti-inflammatory effects of glucocorticoids were dependent on the ability of GR to inhibit the activity of crucial transcriptional regulators of pro-inflammatory genes, including NF-κB and AP-1, by a mechanism termed “transrepression”. This contrasted with the metabolic actions of glucocorticoids which require gene activation by GR. This view has recently been revised, with the discovery that key anti-inflammatory actions of glucocorticoids are brought about through gene activation (and see below).

7The ability of GR to repress the activity of NF-κB and AP-1 as well as other key immunomodulatory transcription factors has been a major focus of research into the mechanisms underlying the anti-inflammatory effects of glucocorticoids. Although a role for transactivation by GR in repression of NF-κB was implicated – inducing expression of the NF-κB inhibitor, IκBα – this was restricted to certain cell types and did not appear to be a universal mechanism. Biochemical and genetic evidence suggested that negative regulation by a transrepression (or “tethering”) mechanism independent of DNA binding by GR underpinned the repressive effects of glucocorticoids (reviewed and ) and this, by extension, was thought to be responsible for their clinical efficacy. In contrast to gene activation by GR, which at the time was believed to occur exclusively through homodimers of GR binding to palindromic glucocorticoid response elements (GREs), transrepression involves direct protein-protein binding of GR to other transcription factors and interference with their mechanism of action. The interaction requires the DNA binding domain of GR (but not DNA binding per se) and does not require conventional homodimerisation of GR. Transrepression was not affected by mutation of alanine 458 to threonine (A458T) within the dimerisation interface of the DNA binding domain that dramatically reduces GR transactivation of the MMTV-LTR promoter and the tyrosine aminotransferase (TAT) gene promoter, both classically activated by GR through well-characterised palindromic GREs. Considerable support for the transrepression hypothesis came from in vivo experiments in mice in which the wild-type GR was replaced by the dimerisation (dim) mutant (GRdim mice). Glucocorticoid-mediated repression of AP-1-dependent transcription and other pro-inflammatory genes was intact in these mice, in contrast to defective GRE-dependent transactivation. However, transactivation of some genes, including that encoding phenylethanolamine N-methyltrasferase (PNMT), remained intact. It has since emerged that the GRE in the PNMT gene belongs to a different class of GRE to the palindromic consensus, and comprises a series of repeated half sites to which GR directly binds to activate transcription from the PNMT promoter. Dual specificity phosphatase (DUSP) 1, also known as MKP-1 (mitogen activated protein kinase [MAPK] phosphatase 1), a crucial anti-inflammatory gene, also remains fully inducible in GRdim mice, offering an alternative interpretation of their phenotype. Glucocorticoid induction of DUSP1 may have some similarity to that of PNMT, as recent work has shown the glucocorticoid responsive region in the DUSP1 gene contains 3 closely spaced half sites, although a tethering mechanism of activation dependent on C/EBPβ has also been implicated. Thus, at least some, but not all, of the dimerisation-independent repressive actions of glucocorticoids may be wholly or partly dependent on induction of DUSP1. Consistent with this, glucocorticoids failed to suppress zymosan-induced inflammation in DUSP1−/− mice, whereas inflammation was suppressed in control mice. However, DUSP1−/− mice remain sensitive to the suppressive effects of glucocorticoids in mast cell-dependent anaphylaxis, implicating other mechanisms in the anti-inflammatory action of glucocorticoids in this model of inflammation. Intriguingly, DUSP1−/− mice are resistant to diet-induced obesity, raising the possibility that DUSP1 may also be involved in some of the metabolic effects of glucocorticoids.

8The anti-inflammatory actions of glucocorticoid-induced genes have been recently reviewed. Briefly, as well as DUSP1 and IκB, this class of genes includes IL-10, a potent immunomodulatory and anti-inflammatory cytokine, Glucocorticoid-induced leucine zipper (GILZ), a protein whose mechanism of action is unclear but which interacts with, and inhibits the function of, NFκB and AP-1 and annexin AI (AnxA1), a calcium-dependent phospholipid binding protein. GILZ knockout mice have not been reported, but AnxA1-deficient mice show defective glucocorticoid suppression of inflammation in carrageenin-induced oedema, zymosan-induced peritonitis and antigen-induced arthritis. IL-10-deficient mice develop autoimmune disease and chronic inflammation, but effects of glucocorticoids in these mice have not been reported. However, IL-10 has been implicated in negative regulation of corticosterone synthesis, acting at the adrenal gland, providing a plausible homeostatic mechanism to terminate HPA axis activation once inflammation is resolving. Like IL-10, administration of AnxA1 can mimic a subset of the effects of glucocorticoids (although in T cells, AnxA1 effects may be opposite to those of glucocorticoids). Similarly, ectopic expression of GILZ in T cells and dendritic cells can mimic some of the effects of glucocorticoid. Indeed, some of the effects of both IL-10 and AnxA1 may even be mediated by GILZ, although as IL-10, AnxaA1 and GILZ all alter differentiation or activation state of immune cells, such conclusions remain tentative.

9Glucocorticoids induce GILZ in a variety of cell types, both immune and non-immune, through palindromic GREs in the 5′-flanking region of the GILZ gene, dependent on dimerisation of GR. How they regulate IL-10 and AnxA1 remains unclear. In vitro, glucocorticoids increase IL-10 expression in monocytes, macrophages and, dependent on antigen presenting cells, in T cells, but whether this is related to glucocorticoid-driven alterations in cellular differentiation (see below) or whether glucocorticoid-induced IL-10 indeed mediates some of the effects of glucocorticoids on differentiation as suggested remains unknown. Glucocorticoids increase AnxA1 expression on human monocytes and neutrophils in vivo and in pituitary folliculostellate cells in vitro and whilst the underlying mechanisms remain unclear, they appear to involve both non-genomic and genomic actions of glucocorticoids, the latter both modest and relatively slow. Moreover, glucocorticoids decrease AnxA1 mRNA in T cells, raising the possibility that glucocorticoid regulation in adaptive and innate immune cells may be related to differentiation/activation state rather than being a direct regulation.

10Transcriptional repression by GR has always been the subject of debate, as alluded to above, including the extent to which it is dependent or independent of direct GR DNA binding. However, it is agreed that gene activation requires DNA binding by GR. Much of the early work on GR transcriptional activation was based around a consensus GR binding site, comprising two 6 bp “half sites” arranged in an inverted repeat (palindrome) separated by a 3 bp spacer, derived from comparisons of around 20 GR binding sites in promoters including the MMTV-LTR. Subsequent work has confirmed this but additionally shown GR-mediated gene regulation to be much more complex (reviewed in detail in,,, and ). A recent unbiased screen of GR binding sites coupled with transcriptome analysis showed that genes activated by glucocorticoid had GR bound within a median distance of 11 kb from the transcription start site whereas repressed genes had GR bound a median of 146 kb from the transcription start site, suggesting that repression occurs independently of promoter-proximal GR binding. In silico prediction, genome scanning, chemically directed sequence-specific disruption of GR binding and chromatin immunoprecipitation experiments have shown that sequences that match the GR consensus do not necessarily bind GR in cells and that disruption of GR binding to the conserved half site sequence 5′-WGWWCW-3′ (where W = A/T) only affects a minority of glucocorticoid-regulated genes, both repressed and activated. It has long been clear that many GR binding sites (core sites) are embedded in “composite” glucocorticoid responsive units (e.g., and ). Core GR binding sites vary considerably around the consensus although the precise sequence in and immediately around the core GR binding site in a gene (the glucocorticoid responsive unit) is highly conserved between species. Moreover, actual occupancy by GR is influenced by post-translational modification and depends on cell-specific factors. Thus, efficient glucocorticoid regulation depends on concomitant binding by other transcription factors at composite elements, to the extent that GR may “tether” at some promoters, retained principally by protein–protein interactions rather than direct interactions with DNA. Given that GR homodimerisation, at least of the isolated DNA binding domain, only occurs on DNA binding, then monomers of GR may bind to divergent sequences at composite response elements or by tethering to other transcription factors. Thus, the context of the GR binding site is crucial with the outcome – repression, activation or even specificity (MR vs GR) – dependent on the cell-specific complement of transcription factors. Whether and how GR contacts DNA might be critical. The GR DNA binding sequence itself acts as an allosteric regulator of GR function, dictating the pattern of regulation that ensues following GR binding. DNA binding induces conformational changes in the dimerisation interface that expose otherwise silent transcriptional activation surfaces. These conformational changes are exquisitely sensitive to the DNA sequence, with single base pair differences differentially affecting GR conformation and transcriptional regulation. Further complexity is revealed at the level of chromatin, where GR binding is highly dynamic and invariably occurs at either consititutive or hormone inducible nuclease accessible sites (regions of “open” chromatin) at which the requirement for chromatin remodelling complexes differs. These dynamic and gene-specific differences in chromatin remodelling by GR are likely to be highly cell-specific and could underlie the complex kinetics of glucocorticoid responses, where glucocorticoid responsive genes may exhibit alternate activation and repression, with poor correlation in some cases between GR binding to response elements and target gene response. Elucidating the nature of GR interactions with target genes, especially in the immune system, will be crucial to understanding their anti-inflammatory effects, but the challenge will be to establish these actions in physiologically relevant settings.

11GR is widely, almost ubiquitously, expressed. Thus, glucocorticoids affect virtually all immune cells and, moreover, precise effects depend upon differentiation and activation state of the cell, making interpretation of in vivo effects in specific cell populations difficult. Nevertheless, several lines of mice with global alteration of GR have provided vital new information about the functions of GR both in regulating the HPA axis and immunity and inflammation. Mice with hypomorphic (GRhypo) or null alleles of GR die neonatally, but haematopoietic progenitors from these mice have been used to reconstitute the immune system in lethally irradiated wild-type mice. Other models survive and include mice with increased GR density (yGR mice with 2 extra GR alleles), with decreased GR density (GR+/− mice and anti-sense rat GR transgenic mice, ) and mice with altered GR function; GRdim and GRM601L mice (a knock-in of a human GR mutant with increased glucocorticoid sensitivity). In all these models the HPA axis is affected because of the central feedback actions of glucocorticoids, compensating for altered GR function in all but the hypomorphic and null mice. All show inflammatory/immune phenotypes (see below). Recent experiments using conditional GR knockout mice as well as transgenic mice have also shed light on the cell-specific functions of GR during immune and inflammatory responses. Tissue-specific models reported include T cell-specific or myeloid cell knockout of GR as well as transgenic mice with thymus over- and under-expression of GR. Some of the immune and inflammatory phenotypes of these various mice have been reviewed in detail elsewhere and are discussed below.

12Glucocorticoid action in T cells and, in particular, in thymus, where naive T cells (that have yet to encounter antigen) develop, has been the subject of intensive research yet remains highly controversial. During T cell development, immature thymocytes progress from double negative (for the CD4 and CD8 T cell markers) to double positive cells (CD4+CD8+) which undergo positive selection (only thymocytes that bind MHC complexed with self-antigen survive) and negative selection (against cells that interact too strongly with self-antigen) to mature into either CD4+ or CD8+ single positive cells; the T cell repertoire. Double positive cells, the majority of the thymocyte population, are highly sensitive to glucocorticoid-induced apoptosis, effective at physiological levels of glucocorticoids. Much in vitro evidence points to a crucial role for glucocorticoids in regulating T cell number, repertoire and function, yet the in vivo evidence is discordant. This topic has been extensively reviewed and will only be briefly discussed here. Suffice it to say, complete lack of GR globally, in T cells alone or inability to dimerise (GRdim mice) does not appear to affect thymocyte number or subsets, although these thymocytes are completely glucocorticoid resistant (reviewed in ). Conversely, mice with a global increase in GR levels (yGR mice) or function (GRM604L mice) have normal thymocyte numbers and subsets, yet both lines of mice show increased glucocorticoid sensitivity of thymocytes in vitro. However, transgenic mice with increased GR in T cells (directed by the proximal Lck promoter or a doxycycline-inducible CD2 promoter) show reduced thymic cellularity with increased thymocyte sensitivity to glucocorticoids in vitro. Finally, in 2 of 3 models in which GR density is reduced by expression of antisense GR either globally (neurofilament promoter) or in T cells (Lck promoter), thymic cellularity is increased, albeit modestly. However, the 3rd antisense model (also using the proximal Lck promoter) showed the opposite effect, with reduced cellularity and altered T cell repertoire. Reconciling these discrepant results will no doubt inform on the cell-specific roles of GR in thymus and T cell selection.

13T cell GR is required to survive lethal activation of T cells. Mice with conditional deletion of GR in T cells (in which the Lck promoter was used to drive Cre recombinase-mediated excision of a “floxed” GR gene) showed increased mortality following activation of T cells either by antibody to CD3ɛ or following injection of bacterial superantigen (staphylococcal enterotoxin A), due to failure to repress cyclooxygenase (COX)-2 expression. Interestingly, these mice showed much worse tissue damage in the gastrointestinal tract than control mice following T cell activation, although damage in other tissues was similar, suggesting a particular role for GR in T cells within the gastrointestinal mucosa in controlling immune activation.

14GR is also required in peripheral T cells for the immunosuppressive effects of glucocorticoid therapy in experimental autoimmune encephalomyelitis (EAE; a mouse model of multiple sclerosis). Like chimeric mice with GR−/− haemotopoietic cells (following lethal irradiation of wild-type mice and reconstitution with GR−/− haemotopoietic cell progenitors), GRLckCre mice (a second line, distinct from the mice mentioned above) showed an earlier onset of disease than controls (indicating a critical role for endogenous glucocorticoid) which was refractory to dexamethasone treatment. In control mice, but not in GRLckCre mice, glucocorticoid therapy induced apoptosis of T cells in peripheral lymphoid organs and down-regulated adhesion molecules, thus reducing migration of T cells to the site of inflammation. Importantly, mice heterozygous for a null mutation of GR (GR+/− mice), with half the normal level of GR, showed a more severe EAE disease course with greater inflammation in the spinal cord than control mice and were resistant to the suppressive effects of a sub-maximal dose of dexamethasone (4 mg/kg) that was effective in normal mice. This suggests that GR density in peripheral T cells is a critical determinant of sensitivity and that despite the presence of functional GR, clinical glucocorticoid resistance can arise. However, in sepsis, another model of inflammation, GR in T cells is not required for glucocorticoid suppression (cited as unpublished data in ).

15The phenotype of mice with a conditional deletion of GR in myeloid cells has been reported by 2 groups. In both cases, the LysM promoter was used to drive Cre recombinase-mediated excision of a floxed GR gene. This results in efficient excision in macrophages and granulocytes in GRLsyMCre mice, with variable excision in other myeloid cell types including dendritic cells and mast cells. Similar to adrenalectomised mice, GRLsyMCre mice showed greater mortality following LPS challenge than control mice. This was attributed to failure to induce DUSP1, required to inhibit p38 MAPK activated by engagement of toll-like receptor (TLR) 4 by LPS. However, given that GRdim mice show normal glucocorticoid induction of DUSP1 in macrophages, yet are susceptible to lethal sepsis (cited in ) as unpublished data), it is likely that other important myeloid cell anti-inflammatory actions of glucocorticoids are also required in this model of inflammation. In contrast to endotoxaemia, mice with deletion of GR in myeloid cells remained fully sensitive to therapeutic suppression of EAE by glucocorticoid, although they did show exacerbated disease.

16GR in myeloid cells, but not T cells, are required for glucocorticoid suppression of contact allergy (a T cell-dependent delayed-type hypersensitivity response, such as occurs in response to metals or poison ivy). Whereas T cell deletion of GR (GRLckCre) had no effect, deletion of GR in myeloid cells (GRLysMCre) abolished glucocorticoid suppression of inflammation, allowed persistent leukocyte infiltration into the inflamed area and impaired glucocorticoid suppression of macrophage IL-1β, MCP-1 and MIP-2 secretion. Interestingly, in contrast to irritant-induced skin inflammation, glucocorticoid repression of T cell-dependent contact allergy did not occur in GRdim mice, and although TNFα remained glucocorticoid-suppressible in GRdim macrophages, MCP-1 and MIP-2 were not repressed. Thus, the immunosuppressive effects of GR are likely to result from multiple mechanisms, which are cell-type and stimulus-type dependent.

17The elucidation of the cell-specific roles of GR within other leukocyte populations and also within T cell subsets will be informed by future conditional knockouts. Knockout of GR in mast cells, a major target in glucocorticoid suppression of allergic responses, will be revealing, as will GR disruption in B cells. Like T cells, glucocorticoids also reduce circulating B cell numbers. Consistent with this, blood lymphocyte levels (T and B cells) as well as monocyte/neutrophils (CD11b+ cells) were markedly reduced following reconstitution of irradiated wild-type mice with GRM604L glucocorticoid-hypersensitive haematopoietic progenitor cells. Elucidation of the underlying mechanisms may be helpful in the treatment of some early T and B cell leukaemias that respond to glucocorticoids.

18As well as profoundly affecting the function of immune cells, glucocorticoids also alter differentiation programmes of progenitor cells. Thus, chronically stressful conditions (when endogenous glucocorticoid production is high) or glucocorticoid pharmacotherapy may alter immune cell differentiation and indeed, probably shape the immune response as it develops. This may be of relevance in the induction of peripheral tolerance to allergenic stimuli, a major clinical application of glucocorticoids.

19Dendritic cells are key antigen presenting cells that bridge the innate and adaptive immune systems. Immature dendritic cells are activated when they capture, process, then present antigens, maturing into immunostimulatory cells in the process. Activated dendritic cells migrate to draining lymph nodes where they interact with naive T cells to instruct the adaptive immune response. Suppression of dendritic cells maturation and function has been implicated in the immunosuppressive effects of glucocorticoids. However, glucocorticoids do not merely suppress dendritic cell activity, but reprogramme them to so-called “tolerogenic dendritic cells” which can elicit a state of hypo-responsiveness in T cells and induce formation of regulatory T (Treg) cells. Differentiation of tolerogenic dendritic cells is dependent, at least in part, on GILZ. Exactly how GILZ mediates anti-inflammatory effects currently remains unclear, although it is likely to depend on protein-protein interactions between GILZ and a variety of intracellular signalling proteins, including the inflammatory regulators NF-κB and activated Ras, the latter being a key protein in signalling to MAP kinases. Nevertheless, the active suppression of antigen-specific immunity through Treg induction by glucocorticoid-programmed dendritic cells undoubtedly contributes to their efficacy in allergic disease and probably other chronic inflammatory diseases. Interestingly, as well as promoting a tolerogenic phenotype in dendritic cells, glucocorticoids may also contribute to tolerance through direct effects on T cells. Stimulation of CD4+ cells in the presence of glucocorticoid (or even more effectively, together with vitamin D3) induced IL-10-secreting Treg cells, able to regulate Th1 responses and autoimmunity. A more complete understanding of these effects will improve clinical use of glucocorticoids.

20These dramatic effects of glucocorticoids on haematopoietic cell differentiation are not restricted to dendritic cells. Glucocorticoids induce a similar anti-inflammatory phenotype in macrophage differentiation. Work over the past decade has shown that a key mechanism to resolve inflammation is the recognition and phagocytosis of dying cells by monocytes/macrophages. Glucocorticoids increase macrophage phagocytosis of apoptotic cells, a strongly anti-inflammatory process, by at least 2 mechanisms. In already differentiated human monocyte-derived macrophages, they induce a protein S/Mer tyrosine kinase-dependent apoptotic cell clearance pathway. However, when included during the differentiation process, glucocorticoids also programme blood monocyte differentiation into a highly phagocytic “anti-inflammatory” macrophage phenotype, a phenomenon that can be modulated by local cytokine environment, may be mediated in part by Anxa1 and which also includes induction of Mer tyrosine kinase as well as other anti-inflammatory genes important in phagocytosis, chemotaxis and protection against oxidative stress. Whether GILZ plays a similar important role in the glucocorticoid-directed macrophage differentiation programme to that it plays in re-programming dendritic cells has not been addressed. Interestingly, these glucocorticoid-programmed macrophages show down-regulation of adhesion-related proteins suggesting they have increased migratory properties. Importantly, in mouse monocytes, glucocorticoids induced a phenotype with low adhesiveness, high migratory capacity and markers suggestive of a tumour-associated macrophage phenotype, believed to be important for tumour-associated immunosuppression. The authors further speculate that this may be a mechanism underlying the higher incidence and faster progression of tumours in patients on long-term glucocorticoid therapy. Intriguingly, in aged mice, elevated plasma glucocorticoid levels were associated with an increase in the proportion of suppressor macrophages in spleen, suggesting that endogenous glucocorticoids may regulate macrophage phenotype in vivo, similar to the potent synthetic glucocorticoids used in the in vitro studies. However, further clarification of the macrophage phenotypes involved in both the in vivo and in vitro studies is required before conclusions can be drawn concerning the involvement of glucocorticoids in suppressor macrophage differentiation and function.

21A variety of GR ligands are in use clinically which have different potency and differ in their biological efficacy. Different ligands clearly induce different conformations of GR that have different gene regulatory properties - indeed this has recently been exploited in the search for “dissociated” ligands which dissociate the beneficial anti-inflammatory (repressive) effects of GR agonism from the unwanted metabolic (activation) effects. Structural studies, including of dexamethasone, fluticasone fuorate and, more recently non-steroidal agonists bound in the GR ligand binding domain have shown that GR readily changes conformation to accommodate large moieties on the ligand and have illustrated new possibilities for mode of ligand binding to GR. Thus, different conformations of GR resulting from different ligand associations may have cell-specific and target gene-specific properties that offer potential for future pharmacological exploitation.

22Much of the research on the anti-inflammatory and immunosuppressive actions of glucocorticoids has been carried out with saturating levels of synthetic hormones. In humans, these include prednisolone and methylprednisolone, but in animal models and in vitro, dexamethasone is most commonly used. This has provided an over-simplified view of the immunomodulatory actions of glucocorticoids and may have overlooked important opportunities for therapeutic manipulation of endogenous glucocorticoid action. This subject has been previously reviewed, and the discussion here is restricted to more recent insights.

23Synthetic glucocorticoids, especially dexamethasone, have higher affinity, greater bioavailability (unlike the natural hormones, most bind poorly or not at all to corticosteroid binding globulin) and are poorly metabolised, thus they persist in plasma much longer than endogenous glucocorticoids (cortisol, corticosterone). Moreover, the endogenous hormones are released from the adrenal gland in both a circadian and a highly pulsatile (ultradian) manner (reviewed in). Recent work from the laboratories of Gordon Hager and Stafford Lightman has shown that this pulsatile release of glucocorticoids is coupled to a highly dynamic pattern of GR-mediated transcriptional bursts, driven by rapid recycling of GR occupancy of chromatin binding sites in response to the hormonal pulses in vivo as well as in vitro. This pulsatility did not occur with constant administration of hormone, nor did it happen with synthetic ligands, including dexamethasone, which failed to cause significant ultradian cycling of GR on chromatin and consequently failed to couple fluctuations in hormone levels with transcriptional response. Thus, transcriptional output can be profoundly altered by synthetic GR ligands or even with natural hormones if not administered in the natural pattern. Moreover, basal levels of glucocorticoids in vivo exert tonic effects. Thus, macrophages elicited by thioglycollate in the peritoneum of adrenalectomised rats behaved very differently to macrophages from sham operated rats, with much greater TNFα secretion and NO production in the unstimulated state, which could only be marginally increased by LPS/IFNγ stimulation. Cytokines themselves are potent activators of the HPA axis, and may permanently programme endogenous glucocorticoid secretion when elevated in early life. Importantly, when the HPA axis is activated, not only is plasma cortisol elevated (corticosterone in rodents), but so is plasma cortisone (11-dehydrocorticosterone in rodents), itself intrinsically inert due to poor binding to GR, but which is available in plasma (it shows negligible binding to corticosteroid binding globulin) and which can be readily enzymatically converted inside cells to the active steroid by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1).

24The last 2 decades have produced a wealth of information on the importance of pre-receptor steroid metabolism. By interconverting active glucocorticoids and inert 11-keto metabolites (cortisone, 11-dehydrocorticosterone), 11β-HSD modulates intracellular access of glucocorticoid to receptors. Type 2 11β-HSD (11β-HSD2) inactivates glucocorticoids in vivo, thus protecting the otherwise non-selective MR from occupation by glucocorticoids. In contrast, because 11β-HSD1 reactivates glucocorticoids, it increases intracellular glucocorticoid concentration. In addition to cortisone (the natural metabolite), certain synthetic steroids (notably prednisone/prednisolone) are also substrates for the 11β-HSD enzymes. The reaction direction of 11β-HSD1 is dictated by its association with hexose-6-phosphate dehydrogenase (H6PD), which couples glucose-6-phosphate oxidation to NADP reduction, generating NADPH co-factor to drive 11β-HSD1 reductase activity. 11β-HSD1 has attracted a lot of recent attention as a potential therapeutic target for metabolic disease, with inhibitors currently under clinical development. Overexpression of 11β-HSD1 in adipose tissue is associated with obesity in both humans and rodents and in transgenic mice, additionally causes hypertension and insulin resistance (reviewed in ). Conversely, inhibition of, or deficiency in 11β-HSD1 reduces hyperglycemia and improves insulin sensitivity in non-insulin dependent diabetes in humans and rodents. Selective inhibition of 11β-HSD1 also prevented progression of atherosclerosis in Apoe−/− mice and lowered levels of circulating MCP-1, a cytokine that recruits monocytes to sites of injury. It will be important to determine the extent to which these pro-inflammatory effects of 11β-HSD1 are due to its dysregulation in adipose tissue and possibly other tissues in metabolic disease.

2511β-HSD1 is widely expressed, with highest expression in classical glucocorticoid target tissues. Given the crucial immunomodulatory actions of glucocorticoids and the strong association between inflammation and insulin resistance/metabolic disease, the role of 11β-HSD1 in the inflammatory response is of great interest. This area has been recently reviewed and the discussion here is restricted to recent developments and questions.

26Like GR, 11β-HSD1 is widely expressed in immune cells, but its expression is highly dependent on the differentiation and activation state of the cell. Circulating leukocytes show little or no 11β-HSD1 expression, consistent with negligible 11β-HSD1 activity in monocytes and low levels in lymphocytes and neutrophils. However, following differentiation of monocytes to macrophages or dendritic cells, 11β-HSD1 activity is dramatically increased and is further increased with polarisation to “M1” pro-inflammatory macrophages compared to “M2” anti-inflammatory macrophages. Amplification of endogenous glucocorticoids by 11β-HSD1 may influence macrophage state as thioglycollate-elicited peritoneal macrophages from 11β-HSD1-deficient mice showed increased pro-inflammatory cytokine production following LPS stimulation in vitro, in the absence of added 11β-HSD1 substrate as well as in vivo. Interestingly, macrophage 11β-HSD1 is down-regulated following phagocytosis of apoptotic neutrophils; a pro-resolution process. The down-regulation of 11β-HSD1 may be part of this mechanism to terminate inflammation. In the absence of 11β-HSD1, mice are more sensitive to endotoxaemia, acute inflammation is more severe, and pro-resolution mechanisms may be delayed. We have recently found 11β-HSD1 expression and activity in mast cells, key initiators of acute inflammation and critical targets of the anti-inflammatory actions of glucocorticoids in allergy. Mast cells from 11β-HSD1-deficient mice are hypersensitive to degranulating stimuli, suggesting that endogenous glucocorticoid amplification via 11β-HSD1 restrains their activity. This may be analagous to dendritic cells where it has also been suggested that 11β-HSD1 activity, which is set at high levels in immature dendritic cells and decreases following CD40 activation (a signal that supports cytotoxic T cell differentiation ), may raise the threshold for dendritic cell-induced immune activation, by increasing glucocorticoid action within immature dendritic cells.

27Within lymphoid cells, 11β-HSD1 activity is present at low levels but is increased following activation of CD4+ T cells through the T cell receptor or following polarisation into Th1 or Th2 cells, possibly enhancing the ability of endogenous glucocorticoids to suppress Th1 cytokines (in Th1 cells) as well as sparing or promoting Th2 cytokine production by Th2 cells. Thymocytes also express low levels of 11β-HSD1, which is markedly increased, in parallel with GR levels, by burn injury in mice, a strong stimulus to the HPA axis. The increase in 11β-HSD1 activity was associated with an increased rate of thymocyte apoptosis. The same may be true in human lymphocyte precursors. In a recent study in acute lymphoblastic leukaemia (ALL) patients, glucocorticoid treatment increased both 11β-HSD1 and GR mRNA levels in glucocorticoid-sensitive leukaemic cells, but decreased 11β-HSD1 mRNA levels in cells resistant to the pro-apoptotic effects of glucocorticoids. This combination of increased 11β-HSD1 and GR expression is likely to contribute very significantly to the increased glucocorticoid sensitivity of the leukaemic cells, though whether low 11β-HSD1 and GR expression is a cause or an effect of the glucocorticoid resistance in glucocorticoid-resistant leukaemia remains to be determined.

28Of great interest is the relevance of 11β-HSD1 to human inflammatory disease. 11β-HSD1 expression is increased in a cell-specific manner by pro-inflammatory cytokines, particularly IL-1 and TNFα (reviewed, ) and at sites of inflammation. However, it may be decreased in others. Recent data have shown decreased 11β-HSD1 mRNA levels in lung in a mouse model of tuberculosis, where, similar to other inflammatory conditions, 11β-HSD1 shows a reciprocal regulation to 11β-HSD2. An even further level of complexity is revealed by the findings that in human rheumatoid arthritis, 11β-HSD1 is increased in synovial fibroblasts, whereas synovial macrophages express 11β-HSD2; suggesting a complex control over glucocorticoid availability within the rheumatic joint. 11β-HSD2 is not normally expressed in macrophages, nor is it expressed here during acute inflammation, at least in mice. However, other groups have also reported 11β-HSD2 expression in rheumatoid arthritis patients in synovial macrophages, immortalised B cells and peripheral blood mononuclear cells. Whether this is part of an adaptive response to inflammation or contributes to glucocorticoid resistance will be important to establish.

29Finally, recent work is starting to elucidate the mechanisms that regulate 11β-HSD1 expression. Whilst the C/EBP family of transcription factors was implicated some years ago, recent data have shown the pivotal role of C/EBPβ in the adipocyte expression and tissue-specific regulation of 11β-HSD1, and importantly, in mediating the effects of pro-inflammatory mediators on transcription of the gene. C/EBPβ has also been implicated in the glucocorticoid-induction of 11β-HSD1 in adipose tissue, although this may be a tissue-specific mechanism. Given that C/EBPβ occupies a pivotal position in the integration of metabolic and inflammatory signals, is a critical component of some glucocorticoid responsive units (e.g., and ) and is also a glucocorticoid target gene itself, being regulated at both mRNA and post-translational levels by glucocorticoids in a tissue-specific manner, it will be extremely informative to unravel the associations between C/EBPβ, 11β-HSD1 and glucocorticoids in determination of immune cell differentiation and function.

30Many aspects of the anti-inflammatory actions of glucocorticoids have not been covered here. However, it is clear that the field is at an exciting stage. The next few years should provide a big step forward in our understanding of how these important hormones exert their effects, with concomitant advances in the clinical treatment of inflammatory disease.