Corticosteroid resistance in rheumatoid arthritis: molecular and cellular perspectives

Corticosteroids are often used to treat a range of chronic autoimmune inflammatory diseases such as asthma, inflammatory bowel disease and rheumatoid arthritis (RA). RA is the most prevalent autoimmune chronic inflammatory rheumatic disorder with a prevalence of 1% in developed nations. It is more common in women than men, suggesting that perturbations of the hormonal systems may be involved in disease pathophysiology. The aetiology of the disease is unknown, but the physiological mechanisms of inflammation involved in this disease share common pathways with other inflammatory situations [1, 2]. However, the reasons why inflammation persists in RA remain unknown but might relate in part to a dysregulation of the interactions between neuroendocrine and immune systems at the onset of acute inflammation [2–6]. Acute inflammation can be initiated by a number of inflammatory triggers. This results in a programmed sequence of physiological mechanisms which begin with the release of tumour necrosis factor alpha (TNFα), interleukin-1β (IL-1β) and IL-6 [3]. These cytokines activate a cascade of reciprocal local and systemic responses which result in increased secretion of corticotrophin-releasing hormone (CRH) and arginine vasopressin by the hypothalamus and production of adrenocorticotrophic hormone (ACTH), prolactin and macrophage migration inhibitory factor (MIF) by the pituitary gland and cortisol by the adrenal glands. Cortisol dampens inflammation by down-regulating the release of TNFα, IL-1β and IL-6 whilst MIF and prolactin counteract the effects of cortisol resulting in a balanced inflammatory/immune response [3–5]. If acute inflammation is not restrained, it enters a chronic phase, a central feature of many chronic autoimmune inflammatory diseases [6]. Neuroendocrine regulation of immune function is essential for survival during stress or infection and to modulate immune responses in inflammatory disease. Corticosteroids are the main effector endpoint of the neuroendocrine immune response to inflammation. At the molecular level, IL-1β, IL-6 and TNFα initiate a number of pro-inflammatory intracellular signalling events which include the activation of the transcriptional activities of activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) by a phosphorylation-dependent dissociation and/or degradation of I-κβ by specific kinases (I-κβ kinase 1 and 2) in the case of NF-I-κβ [7, 8]. These in turn enhance the production of a whole range of pro-inflammatory cytokines. These transcription factors are targets of action by cortisol and other corticosteroid type drugs [9]. NF-κβ is involved in the pathogenesis of inflammation in RA [10]. In addition IL-lβ and TNFα also activate the mitogen-activated protein kinase (MAPK) p38 pathway [11]. MAPK p38 activates the kinases MAPKAPK-2, which in turn targets adenosine/uridine-rich elements (AREs) of pro-inflammatory messenger ribonucleic acids (mRNAs) to bring about their stabilization [9, 12, 13]. IL-lβ and TNFα activate the second wave of cytokine release mechanisms [IL-8, IL-12, IL-15, IL-17, IL-18, interferon-α and -β, granulocyte–macrophage colony-stimulating factor (GM-CSF), fibroblast growth factor etc.] that augment the homeostatic signals necessary for the subsequent complex cellular/cytokine cascades of reactions, endothelial activation and enhanced cell adhesion. The body attempts to down-regulate inflammation by increasing corticosteroid production [3]. Synthetic corticosteroid analogues such as prednisolone have been made and are often used to treat chronic autoimmune inflammatory disease such as RA, asthma and inflammatory bowel disease. They can effectively reduce the parameters of inflammation such as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) and induce disease remission. However, in clinical practice, a proportion of patients fail to respond adequately to corticosteroid therapy [14–16]. On this basis, patients can be divided into corticosteroid sensitive (SS) and corticosteroid resistant (SR) subgroups. The underlying mechanisms involved in the SS and SR phenomenon in patients with RA remain unknown but are of considerable therapeutic interest. The mechanisms of action of corticosteroid can be subdivided into genomic and non-genomic effects [17]. The non-genomic effects which occur very rapidly are either specific or non-specific. The exact mechanisms involved in the non-genomic effects are unknown at the moment but may be related to alterations in the functional status of the cell membrane which may include lipid rafts and/or are mediated via some unknown membrane-bound receptors. These effects include analgesia and inhibition of adhesion molecule expression. The genomic effects are mediated via the corticosteroid receptor (CR) whose principal functions of transactivation, DNA binding and ligand binding are localized to specific DNA domains [9]. The gene for CR (NR3C1) is located on chromosome 5 (5q31). Alternative splicing of mRNA results in a number of isoforms (Fig. 1). CRα, the only functional receptor, is encoded for by exons 2–9α [18]. The CRβ isoform is the alternative splice variant of exon 9β instead of 9α and does not bind corticosteroids. CRβ accounts for 0.2–1% of the total CR expression [19, 20]. CRβ is thought to have a dominant role as a negative inhibitor of CRα [21, 22]. However, there are conflicting data refuting this role [23, 24]. The insertion of arginine in the DNA-binding domain at exon 4 results in another receptor splice variant CRγ, which has decreased transactivation activity [25]. The CR-P isoform is encoded for by exons 2–7 plus several base pairs from the subsequent intron region [26]. This isoform lacks the ligand-binding domain and therefore cannot bind corticosteroids. Its function is unknown. The CR-P transcripts account for 10–20% CR mRNA and are reported to be up-regulated in some haematological malignancies [27–29]. CR-A results from excision of exons 5–7, resulting in juxtaposition of exons 8 to 4; its function is unknown [26]. CRα is maintained in the cytoplasm as an inactive multi-complex protein consisting amongst other proteins of two Hsp90 molecules, Hsp70 and a number of other proteins such as immunophillin p59 and calreticulin which act as chaperones and co-chaperones. The binding of corticosteroid (CS) to the CRα to form the corticosteroid/receptor complex (CS/CR), induces a conformational change leading to dissociation of the receptor from the multi-protein complex. The CRα then dimerizes, transactivates and the CS/CR complex translocates to the nucleus to bind to the specific DNA motifs, the glucocorticosteroid response elements (GRE) (transactivation) [30]. GRE can mediate both positive and negative corticosteroid effects [30, 31] (Fig. 2). Human glucocorticosteroid receptor gene and alternatively spliced isoforms. After being internalized into cytoplasm, corticosteroid (CS) binds to the receptors to form the corticosteroid/receptor complex (CS/CR). Receptor dissociates from the inactive, multi-protein complex. The CS/CR complex translocates to the nucleus to bind to the specific DNA motifs, the glucocorticosteroid response elements (GRE). GRE can mediate both positive and negative corticosteroid effects. It also interferes with the transcriptional activity of AP-1 and NF-κB. Corticosteroids up-regulate I-κB production which binds to NF-κB and sequesters it in the cytoplasm by inhibiting its nuclear translocation. The CS/CR complex also interferes with the transcriptional activity of AP-1 by inhibiting its binding to its DNA site by interfering with the protein to protein interactions (transrepression) [9]. Transrepression has also been demonstrated with other transcription factors such as NF-κB [32–34] and signal transducer and activator of transcription (STAT) proteins such as STAT3 [35], STAT5 [36] and STAT 6 [37]. CRα has been shown to block the c-Jun N-terminal kinase (JNK) signalling cascade by abrogating the c-Jun phosphorylation on Ser-63/73 and thus inhibit AP-1 activation [38–40]. Corticosteroids up-regulate I-κβα production [7, 78]. I-κβα binds to NF-κB and masks the nuclear localizing sequence, thus sequestering it in the cytoplasm by inhibiting its nuclear translocation [7, 8]. In the cytoplasm, NF-κB exists as a heterodimer of mainly p50 and p65 subunits that are bound I-κBs which exists in a number of isoforms of which I-κβα is the most well characterized [41]. Cellular activation leads to the phosphorylation and degradation of I-κBs. NF-κB is then released as the active form, which then translocates to the nucleus to initiate the transcription of a number of target genes [42]. CS/CR complex also interferes with the binding of NF-κB to its site on DNA (Fig. 2). The inhibition of the protein to protein interactions of AP-1 and NF-κB respectively by corticosteroids involves deacetylation of acetylated core histones [43, 44]. Acetylation of histones allows unwinding of the local DNA chromatin structure and enables RNA polymerase II to enhance gene transcription. Histone acetylation is regulated by a balance between the activity of histone acetyltransferases (HATs) and histone deacetylases (HDACs) which reverses the process, leading to gene repression [44]. CRα acts as a direct inhibitor of the NF-κB-induced HAT activity and also recruits HDACs to the NF-κB/HAT complex [45]. The net effect will be in increased tightening of DNA around histone residues leading to reduced access to the DNA by transcription factors such as AP-1 and NF-κB and repression of pro-inflammatory genes [45]. One feature that is common to the repression of both AP-1- and NF-κB-dependent transcription mediated by CRα is that the effect is mutual. CRα is not only capable of repressing AP-1 and NF-κB, but that both transcription factors may repress CRα-dependent transcription. Co-activator molecules such as cAMP response element binding protein (CREB)-binding protein (CBP) [46] and the steroid receptor co-activator 1 (SRC-1) [47] both interact with CRα to modulate its activity. For instance CBP is associated with HAT activity and may account for the synergistic interactions between NF-κB and AP-1. The CR/CS complex also interacts with phosphorylated STAT5 [36]. This association, which does not involve the binding to the GRE, enhances the transcriptional activity of STAT5 to increase IL-2 receptor alpha (IL-2Rα) expression and β-casein production in mammary glands amongst other effects. Corticosteroids also induce apoptosis of lymphocytes and thymocytes, but these effects may be secondary to the inhibition of cytokine growth and proliferation factors. The activity of the pro-inflammatory kinase cascade systems, such as the extracellular regulated kinase (ERK) and JNK mitogen-activated kinases (MAPKs) [48–51] are modulated by corticosteroids. For instance, it has recently been shown that corticosteroids induce the sustained expression of MAPK phosphatase 1 (MKP-1) which inhibits the MAPK signalling pathways by dephosphorylating proteins [52, 53]. The mRNAs encoding immune mediators contain AREs within their 3′ untranslated regions (UTRs) [54, 55]. AREs and MAPK p38 signalling pathways are involved in the regulation of mRNA stability [53]. The MAPK p38 pathway is activated by pro-inflammatory cytokines and in turn activated/phosphorylated MAPK p38 activates the kinase MAPKAPK-2, which in turn targets the AREs of pro-inflammatory mRNAs to bring about their stabilization [56]. Corticosteroids induce the production of MKP-1 which potently inactivates phosphorylated MAPK p38 leading to the destabilization of pro-inflammatory mRNAs by corticosteroids [52, 53, 56, 57]. Finally, corticosteroids also induce the production of lipocortin, an anti-inflammatory protein made by peripheral blood mononuclear cells which mediates a number of corticosteroid effects. The regulation of gene expression also requires an orchestrated and coordinated control of the cross-talk between transcription factors to regulated transcriptional, post-transcriptional, translational and post-translational events. These mechanisms are all modulated by corticosteroids. The body needs to regulate the biological effects of corticosteroids. The mechanisms involved in the regulation of corticosteroid effects include alterations in the bioavailability of corticosteroids within the respective microenvironment of the target tissues and counter-regulation by pro-inflammatory cytokines and hormones. Factors such as route of administration may be important in determining corticosteroid bioavailability and bioactivity. For instance, malabsorption of orally administered steroid may cause a failure to respond to therapy in some patients with small bowel disease given enteric-coated corticosteroid preparations. Nevertheless, corticosteroids pass through the cell membrane well. Once in the cell, prior to binding to the CRα, the multidrug resistant pump (MDR1) could potentially extrude the steroids from the cell [58]. Corticosteroid bioactivity may be regulated in target tissues and cells by the enzyme 11-β-hydroxysteroid dehydrogenase (11-BHD). In humans, 11-BHD exists as two isoenzymes type 1 and type 2 [59]. The type 2 11-BHD inactivates cortisol by converting it to cortisone which is not bioactive, whilst the type 1 isoenzyme converts cortisone to cortisol, and thus may amplify the biological activity of corticosteroids [59]. The pituitary gland secretes MIF in parallel to the secretory circadian pattern of ACTH [5]. MIF is also locally secreted by macrophages in response to pro-inflammatory stimuli and low levels of corticosteroids (10−12–10−9 m) [5]. MIF down-regulates the immunosuppressive effects of corticosteroids [5]. The exact mechanisms have not been fully characterized. MIF activates the ERK1/ERK2–MAPK pathway signalling leading to enhanced phospholipase A2 (PLA2) production and cell proliferation [60]. PLA2 is a key target of the anti-inflammatory actions of corticosteroids. MIF regulates IL-2 secretion and T-cell proliferation [60], which may in part be mediated via increased cellular expression of prolactin [4]. Essentially, hormones may be divided into anti-inflammatory hormones, such as cortisol and melatonin, and pro-inflammatory hormones, such as CRH, prolactin, arginine vasopressin and substance P [3, 4, 61, 62–73]. The biological effects of prolactin have been studied more extensively. Prolactin, like MIF, is produced by the pituitary gland and peripheral blood mononuclear cells and antagonizes the effects of corticosteroids in vivo and in vitro [4, is related to the of the cytokine and which include and IL-2 to It is an essential for and cells and can activate cells and macrophages is essential for IL-2 and is an important for T-cell proliferation and enhances production At the molecular level, prolactin its effects via prolactin receptors on macrophages and and cells The of the hormone to the prolactin receptors activates the of kinases which in turn and activate STAT5 STAT5 then translocates to the nucleus to bind to the respective STAT5 response elements on DNA leading to transcription STAT5 is also activated by cytokines such as IL-2 CRα can complex with activated STAT5 [36]. The complex enhances transcription and potentially the levels of intracellular leading to reduced effects [42]. 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