Molecular mechanisms of corticosteroids in all_ergic diseases (2001)

Corticosteroids are by far the most effective therapy currently available for all_ergic diseases, such as asthma. We now have a much better understanding of the molecular mechanisms whereby corticosteroids suppress inflammation in all_ergic diseases. Corticosteroids are a highly effective anti-inflammatory therapy in all_ergy, and the molecular mechanisms involved in the suppression of all_ergic inflammation are now better understood (1). Corticosteroids are effective clinicall_y because they block many of the inflammatory pathways that are abnormall_y activated in all_ergic diseases, and they have a very wide spectrum of anti-inflammatory actions. Corticosteroids bind to a single class of glucocorticoid receptor (GR) that is localized to the cytoplasm of target cells. Corticosteroids bind at the C-terminal end of the receptor, whereas the N-terminal end of the receptor is involved in gene transcription. Between these domains is the DNA-binding domain, which has two finger-like projections, formed by a zinc molecule bound to four cysteine residues, that bind to the DNA double helix. The inactive GR is bound to a protein complex that includes two molecules of the 90-kDa heat-shock protein (hsp90) and various other proteins that act as “molecular chaperones” to prevent the unoccupied GR from moving into the nuclear compartment. Once corticosteroids bind to GR, conformational changes in the receptor structure result in dissociation of these chaperone molecules, thereby exposing nuclear localization signals on GR, and resulting in rapid nuclear localization of the activated GR-corticosteroid complex and its binding to DNA (Fig. 1). Two GR molecules bind to DNA as a dimer, resulting in changed transcription. A splice variant of GR, termed GR-β, has been identified that does not bind corticosteroids, but does bind to DNA, and theoreticall_y may interfere with the action of corticosteroids by blocking GRE binding (2). Classical model of corticosteroid action. Corticosteroids enter the cell and bind to cytoplasmic glucocorticoid receptors (GR) that are complexed with two molecules of a 90-kDa heat-shock protein (hsp90). GR translocates to the nucleus, where, as a dimer, it binds to a glucocorticoid recognition element (GRE) on the 5′-upstream promoter sequence of steroid-responsive genes. GREs increase transcription, whereas negative GREs (nGREs) may decrease transcription, resulting in increased or decreased messenger RNA (mRNA) and protein synthesis. An isoform of GR, GR-β, binds to DNA but is not activated by corticosteroids. Corticosteroids produce their effect on responsive cells by activating GR to regulate directly or indirectly the transcription of certain target genes (3). The number of genes per cell directly regulated by corticosteroids is estimated to be between 10 and 100, but many genes are indirectly regulated through an interaction with other transcription factors. GR dimers bind to DNA at consensus sites termed “glucocorticoid response elements” (GREs) in the 5′-upstream promoter region of steroid-responsive genes. This interaction changes the rate of transcription, resulting in either induction or repression of the gene. Interaction of the activated GR homodimer with GRE usuall_y increases transcription, resulting in increased protein synthesis. GR may increase transcription by interacting with a large coactivator molecule, CREB-binding protein (CBP). CBP is bound at the start site of transcription, and this leads via a series of linking proteins to the binding and activation of RNA polymerase II, resulting in the formation of messenger RNA (mRNA) and then synthesis of protein. Binding of activated GR to CBP results in increased acetylation of core histones around which DNA is wound within the chromosomal structure (4), and this is critical for the subsequent activation of RNA polymerase II. For example, high concentrations of corticosteroids increase the secretion of the antiprotease secretory leukoprotease inhibitor (SLPI) from epithelial cells. This is associated with a selective acetylation of lysine residues 5 and 16 on histone 4, resulting in increased gene transcription. In controlling inflammation, the major effect of corticosteroids is to inhibit the synthesis of inflammatory proteins. This was originall_y believed to be through the interaction of GR with negative GREs, resulting in repression of transcription. However, negative GREs have only very rarely been demonstrated and are not a feature of the promoter region of inflammatory genes that are suppressed by steroids in the treatment of all_ergic diseases. Activated GRs may bind directly to several other activated transcription factors as a protein–protein interaction. This could be an important determinant of corticosteroid responsiveness, and it is a key mechanism whereby corticosteroids switch off inflammatory genes. Most of the inflammatory genes that are activated in asthma do not appear to have GREs in their promoter regions, and yet they are repressed by corticosteroids. There is persuasive evidence that corticosteroids inhibit the effects of the transcription factors that regulate the expression of genes that code for inflammatory proteins, such as cytokines, inflammatory enzymes, adhesion molecules, and inflammatory receptors. These “inflammatory” transcription factors include activator protein-1 (AP-1) and nuclear factor-κB (NF-κB), which may regulate many of the inflammatory genes that are switched on in asthmatic airways (5, 6). It was once believed that the activated GR interacted directly with activated transcription factors through a protein–protein interaction, but thus may be a feature of transfected cells, rather than of primary cells. Thus, in a chronicall_y transfected epithelial cell line with a NF-κB-driven reporter gene, there is relatively little effect of corticosteroids on transcription (7). Furthermore, treatment of asthmatic patients with high doses of inhaled corticosteroids that suppress airway inflammation is not associated with any reduction in NF-κB binding to DNA (8). This suggests that corticosteroids act downstream of the binding of proinflammatory transcription factors to DNA, and attention has now focused on their effects on chromatin structure and histone acetylation. There is increasing evidence that corticosteroids may have effects on the chromatin structure. DNA in chromosomes is wound around histone molecules in the form of nucleosomes (9, 10). Several transcription factors interact with large coactivator molecules, such as CBP and the related molecule p300, which bind to the basal transcription factor apparatus. Several transcription factors bind directly to CBP, including AP-1, NF-κB, STATs, and GR (11, 12) (Fig. 2). At the microscopic level, the chromatin may become dense or opaque due to the winding or unwinding of DNA around the histone core. Coactivator molecules, including CBP and the related p300, have histone acetylation activity that is stimulated by the binding of transcription factors, such as AP-1 and NF-κB. Acetylation of lysine residues in the N-terminal tails of core histones results in the unwinding of DNA that is tightly coiled around the histone core of the resting gene, thus opening up the chromatin structure. This all_ows transcription factors and RNA polymerase to bind more readily, thereby switching on or increasing transcription. Effect of corticosteroids on chromatin structure. Transcription factors, such as STATs, AP-1, and NF-κB, bind to coactivator molecules, such as CREB-binding protein (CBP) or p300, which have intrinsic histone acetyltransferase (HAT) activity, resulting in acetylation (-Ac) of histone residues. This leads to unwinding of DNA and all_ows increased binding of transcription factors, resulting in increased gene transcription. After activation by corticosteroids, glucocorticoid receptors (GR) bind to a glucocorticoid receptor coactivator, which is bound to the CBP. This results in deacetylation of histone, with increased coiling of DNA around histone, thus preventing transcription factor binding that leads to gene repression. The repression of genes reverses this process by deacetylation of the acetylated histone residues (13). Deacetylation of histones increases the winding of DNA around histone residues, resulting in dense chromatin structure and reduced access of transcription factors and RNA polymerase to their binding sites, and thereby leading to repressed transcription of inflammatory genes. Activated GR may bind to several transcription corepressor molecules that associate with proteins that have histone deacetylase (HDAC) activity, resulting in deacetylation of histone, increased winding of DNA around histone residues, and thus reduced access of transcription factors to their binding sites and therefore repression of inflammatory genes. In addition, activated GR recruits HDACs to the transcription start site, resulting in deacetylation of histones, and a decrease in inflammatory gene transcription (4). Several distinct HDACs are now recognized, and these are differentiall_y expressed and regulated in different cell types (14). This may contribute to the differences in responsiveness to corticosteroids between different genes and cells. It is increasingly recognized that GR may also affect the synthesis of some proteins by reducing the stability of mRNA, through effects on the ribonucleases that break down mRNA. Some inflammatory genes, such as the gene encoding GM-CSF, produce mRNA that has a sequence rich in AU nucleotides at the 3′-untranslated end. It is this region that interacts with the ribonucleases that break down mRNA, thus switching off protein synthesis (15). Corticosteroids may control inflammation by inhibiting many aspects of the inflammatory process in all_ergy through increasing the transcription of anti-inflammatory genes and decreasing the transcription of inflammatory genes (Table 1). Corticosteroids may suppress inflammation by increasing the synthesis of anti-inflammatory proteins. For example, corticosteroids increase the synthesis of lipocortin-1, a 37-kDa protein that has an inhibitory effect on phospholipase A2 (PLA2), and therefore may inhibit the production of lipid mediators. Corticosteroids induce the formation of lipocortin-1 in several cells, and recombinant lipocortin-1 has acute anti-inflammatory properties. However, lipocortin-1 does not appear to be increased by inhaled corticosteroid treatment in asthma (16). Corticosteroids increase the expression of other potentiall_y anti-inflammatory proteins, such as interleukin (IL)-1 receptor antagonist (which inhibits the binding of IL-1 to its receptor), SLPI (which inhibits proteases, such as tryptase), neutral endopeptidase (which degrades bronchoactive peptides such as kinins), CC-10 (an immunomodulatory protein), the inhibitor of NF-κB (IκB-α), and IL-10 (an anti-inflammatory cytokine). The expression of IL-10 in macrophages from asthmatic patients is decreased, and this may increase the expression of several inflammatory genes. Corticosteroids increase secretion of IL-10 and may therefore overcome this defect (17) Corticosteroids increase the expression of β2-adrenoceptors by increasing the rate of transcription and the human β2-receptor gene has three potential GREs (18). Corticosteroids double the rate of β2-receptor gene transcription, in human lung in vitro, resulting in increased expression of β2-receptors (19). This also occurs in vivo in nasal mucosa after treatment with topical corticosteroids (20). This may be relevant in asthma, as corticosteroids may prevent downregulation of β-receptors in response to prolonged treatment with β2-agonists. In rats, corticosteroids prevent downregulation and reduced transcription of β2-receptors in response to chronic exposure to β-agonists (21). The inhibitory effect of corticosteroids on cytokine synthesis is likely to be of particular importance in the control of inflammation in all_ergic inflammation, as cytokines play a critical role in the chronic inflammatory process (22). Corticosteroids inhibit the transcription of many cytokines and chemokines that are relevant in all_ergy (Table 1). These inhibitory effects are due, at least in part, to an inhibitory effect on the transcription factors that regulate induction of these cytokine genes, including AP-1 and NF-κB. For example, eotaxin, which is important in selective attraction of eosinophils from the circulation into the airways, is regulated in part by NF-κB, and its expression in airway epithelial cells is inhibited by corticosteroids (23). Many transcription factors, in addition to AP-1 and NF-κB, are likely to be involved in the regulation of inflammatory genes in asthma. IL-4 and IL-5 expression in T cells plays a critical role in all_ergic inflammation, but NF-κB does not play a role, whereas the transcription factor nuclear factor of activated T cells (NF-AT) is important (24). AP-1 is a component of the NF-AT transcription complex, so that corticosteroids inhibit IL-5, at least in part, by inhibiting the AP-1 component of NF-AT. There may be marked differences in the response of different cells and of different cytokines to the inhibitory action of corticosteroids, and these differences may depend on the relative abundance of transcription factors within different cell types. Thus, in alveolar macrophages and peripheral blood monocytes, GM-CSF secretion is more potently inhibited by corticosteroids than IL-1β or IL-6 secretion (25). Nitric oxide (NO) synthase may be induced by proinflammatory cytokines, resulting in NO production. NO may amplify asthmatic inflammation and contribute to epithelial shedding and airway hyperresponsiveness (AHR) through the formation of peroxynitrite. The induction of the inducible form of NOS (iNOS) is inhibited by corticosteroids. In cultured human pulmonary epithelial cells, proinflammatory cytokines result in increased expression of iNOS and increased NO formation, due to increased transcription of the iNOS gene, and this is inhibited by corticosteroids acting partly through inhibition of NF-κB (26). Corticosteroids inhibit the synthesis of several other inflammatory mediators implicated in asthma through an inhibitory effect on the induction of enzymes, such as cyclooxygenase-2 and cytosolic PLA2 (27). Corticosteroids also decrease the transcription of genes coding for certain receptors. Thus, the gene for the NK1-receptor, which mediates the inflammatory effects of tachykinins in the airways, has an increased expression in asthma and is inhibited by corticosteroids, probably via an inhibitory effect on AP-1 (28). Corticosteroids also inhibit the transcription of the NK2-receptor, which mediates the bronchoconstrictor effects of tachykinins (29). Corticosteroids also inhibit the expression of the inducible bradykinin B1-receptor and bradykinin B2-receptor (30). Adhesion molecules play a key role in the trafficking of inflammatory cells to sites of inflammation. The expression of many adhesion molecules on endothelial cells is induced by cytokines, and corticosteroids may lead indirectly to a reduced expression via their inhibitory effects on cytokines such as IL-1β and TNF-α. Corticosteroids may also have a direct inhibitory effect on the expression of adhesion molecules, such as ICAM-1 and E-selectin, at the level of gene transcription. ICAM-1 and VCAM-1 expression in bronchial epithelial cell lines and monocytes is inhibited by corticosteroids (31). Corticosteroids markedly reduce the survival of certain inflammatory cells, such as eosinophils. Eosinophil survival is dependent on the presence of certain cytokines, such as IL-5 and GM-CSF. Exposure to corticosteroids blocks the effects of these cytokines and leads to programmed cell death, or apoptosis, although the corticosteroid-sensitive molecular pathways have not yet been defined (32). By contrast, corticosteroids decrease apoptosis in neutrophils and thus prolong their survival (33). This may contribute to the lack of anti-inflammatory effects of corticosteroids in chronic obstructive pulmonary disease (COPD), in which neutrophilic inflammation is predominant. Corticosteroids may have direct inhibitory actions on several inflammatory cells and structural cells that are implicated in asthma (Fig. 3). Cellular effect of corticosteroids. Corticosteroids inhibit the release of inflammatory mediators and cytokines from alveolar macrophages in vitro (25). Inhaled corticosteroids reduce the secretion of chemokines and proinflammatory cytokines from alveolar macrophages in asthmatic patients, whereas the secretion of IL-10 is increased (17). Corticosteroids have a direct inhibitory effect on mediator release from eosinophils, although they are only weakly effective in inhibiting the secretion of reactive oxygen species and eosinophil basic proteins. More importantly, corticosteroids induce apoptosis by inhibiting the prolonged survival due to IL-3, IL-5, and GM-CSF (32, 33), resulting in an increased number of apoptotic eosinophils in the induced sputum of asthmatic patients (34). In asthma, there is a delay in the apoptosis of eosinophils, which is reversed by treatment with corticosteroids (35). One of the best described actions of corticosteroids in asthma is a reduction in circulating eosinophils, an effect which may reflect an action on eosinophil production in the bone marrow. T helper 2 cells (Th2) play an important orchestrating role in asthma through the release of the cytokines IL-4, IL-5, IL-9, and IL-13, and they may be an important target for corticosteroids in asthma therapy. Corticosteroids also induce apoptosis in T cells. While corticosteroids do not appear to have a direct inhibitory effect on mediator release from lung mast cells, chronic corticosteroid treatment is associated with a marked reduction in the numbers of mucosal mast cells. This may be linked to a reduction in the production of IL-3 and stem-cell factor (SCF), which are necessary for mast-cell expression at mucosal surfaces. Mast cells also secrete various cytokines (TNF-α, IL-4, IL-5, IL-6, and IL-8), and this may also be inhibited by corticosteroids (36). Dendritic cells in the epithelium of the respiratory tract appear to play a critical role in antigen presentation in the lung, as they have the capacity to take up all_ergen, process it into peptides, and present it, via MHC molecules on the cell surface, to uncommitted T cells. In experimental animals, the number of dendritic cells is markedly reduced by systemic and inhaled corticosteroids, thus dampening the immune response in the airways (37). Neutrophils, which are not prominent in the of asthmatic patients, are not to the effects of corticosteroids. systemic corticosteroids increase peripheral a which may reflect an increased survival due to an inhibitory action on apoptosis (33). GR gene expression in the airways is most prominent in endothelial cells of the bronchial circulation and airway epithelial cells. 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