**5.2 Genetic and acquired glucoccorticoid resistance**

There is a rare but well described familial or sporadic mutation of the GCR gene which results in GC resistance. This leads to activation of the HPA axis and compensatory elevations in circulating adrenocorticotropic hormone (ACTH) and cortisol. Patients with this disorder can develop adrenal hyperplasia as a result of the excess ACTH. Subsequent increase in mineralocorticoid and androgen release leads to a broad clinical spectrum whose manifestations depend on the severity of the disorder (Charmandari et al., 2008). This group of patients represents only a very small minority of GC resistance cases whilst acquired GC resistance in inflammatory conditions is quite common. In the last 10 years, more research effort has been focused on the problem of acquired GC resistance and several competing theories behind the underlying mechanism have emerged.

#### **5.3 Glucocrticoid receptor β expression**

14 Rheumatoid Arthritis – Treatment

Another interesting approach in the use of GC has been their use in combination with drugs that can amplify their intracellular effects at low doses. One approach looked at a preparation which combines low dose prednisolone in combination with dipyridamole (Kvien et al., 2008). This combination seemed to enhance the ability of GC to suppress the pro-inflammatory NFκB pathway (as mentioned in section 3.1) while sparing the genetranscription element of GC action which is associated with adverse effects. Other novel strategies involve targeting of the GC to the site of inflammation by encapsulating them in long-circulating liposomes (Schiffelers et al., 2006). This approach has shown promise in animal models of inflammatory arthritis but clinical studies are still lacking. These advances represent a potential new dawn for the use of GC in rheumatoid arthritis and have

GC resistance has been observed in the clinical setting for a long time and represents a challenge for clinicians as treatment requires larger doses of GC associated with an increased risk of adverse events. GC resistance may occur in a quarter to a third of RA patients. The emergence of this subgroup was first reported in a paper by Van Schaardenburg et al in 1995 (Van Schaardenburg et al., 1995). This study looked at elderly onset RA patients treated with oral prednisolone and a 30% discontinuation rate due to lack of efficacy was reported. Further confirmation of this phenomenon came in a study by Sliwinska-Stanczyk et al (Sliwinska-Stanczyk et al., 2007) who showed a 25% resistance rate in their 44 patients who had moderately active RA and who were not taking other disease modifying agents. This group went on to show that clinical GC resistance seemed to correlate with a failure of GC to adequately suppress in vitro peripheral blood mononuclear

The problem of GC-resistance is not unique to RA and has been observed in a range of inflammatory conditions including ulcerative colitis (UC), asthma and uveitis (Creed & Probert, 2007; Lee et al., 2009; Sousa et al., 2000; Barnes & Adcock, 2009). The proportion of 25-33% GC resistance seems to be preserved across the various diseases and the possible

There is a rare but well described familial or sporadic mutation of the GCR gene which results in GC resistance. This leads to activation of the HPA axis and compensatory elevations in circulating adrenocorticotropic hormone (ACTH) and cortisol. Patients with this disorder can develop adrenal hyperplasia as a result of the excess ACTH. Subsequent increase in mineralocorticoid and androgen release leads to a broad clinical spectrum whose manifestations depend on the severity of the disorder (Charmandari et al., 2008). This group of patients represents only a very small minority of GC resistance cases whilst acquired GC resistance in inflammatory conditions is quite common. In the last 10 years, more research effort has been focused on the problem of acquired GC resistance and several competing

**4.3 Other therapeutic advances** 

**5. Glucocorticoid resistance** 

implications for a number of other inflammatory diseases.

**5.1 The problem of glucocorticoid resistance** 

cell (PBMC) proliferation in the affected individuals.

**5.2 Genetic and acquired glucoccorticoid resistance** 

theories behind the underlying mechanism have emerged.

mechanisms underlying this are explored in the following sections.

GCR-β is an alternatively spliced form of the GC receptor and its over-expression has been linked with GC resistance in asthma, RA and inflammatory bowel disease (Hamid et al., 1999; Sousa et al., 2000; Kozaci et al., 2007; Orii et al., 2002). GCR-β does not bind GC and in fact its natural ligand (if it has one) remains unknown (Lewis-Tuffin, 2006). However, it does compete with GCR-α for the GRE binding sites on DNA, thus acting as a dominant negative inhibitor. Another anti-GC mechanism may be the disruption of active GCR-α translocation to the nucleus since the down regulation of GCR-β in the alveolar macrophages of patients with asthma leads to enhanced GCR-α localization and a greater response to GC. Moreover, it has been shown that various pro-inflammatory cytokines can up regulate the expression of GCR-β and this may explain why patients seem to develop clinical GC resistance with worsening of their inflammatory disease (Webster et al., 2001).

#### **5.4 Defects in histone acetylation**

The role of defective histone acetylation in acquired GC resistance has emerged principally from studies on patients with asthma and chronic obstructive pulmonary disease (COPD).

As described previously, inflammatory stimuli ultimately lead to the activation of NFκB which binds to specific κB recognition sites on the promoter regions of inflammatory genes in addition coactivators which cause acetylation of core histones. This leads to their unravelling and opens up the genes for transcription. Activated GCRs inhibit this effect directly by binding the coactivators and recruiting histone deacetylase (HDAC) 2 which reverses the acetylation (Rhen & Cidlowski, 2005). GCRs themselves become acetylated upon ligand binding to allow them to bind GREs and can be targeted directly by HDAC2.

HDAC2 activity has been shown to be reduced in alveolar macrophages of GC resistant asthma patients and patients with COPD (Ito et al., 2005; Hew et al., 2006). This reduced activity is thought to be secondary to the oxidative stress resulting from smoking (Rahman & Adcock, 2006). Smoking and obesity, both causes of oxaditive stress, are both risk factors for developing rheumatoid arthritis (Symmons et al., 1997). In COPD it has been shown that low dose oral theophylline can reverse GC resistance by restoring HDAC2 activity (Ito et al., 2005). This effect is independent of phosphodiesterase inhibition and is mediated via the selective inhibition phospho-inositide-3-kinase-δ (PI3Kδ). This is an enzyme which is activated by oxidative stress in patients with COPD (To et al., 2010). This pathway has not been studied in rheumatoid arthritis and presents a novel way of reversing GC resistance.

#### **5.5 T Helper-17 cells and glucocorticoid resistance**

T helper (Th) cells differentiate into distinct phenotypes under the influence of the inflammatory cytokine milieu which is largely dictated by cytokines released from monocyte derived macrophages. IL-17 producing T-helper cells (TH-17 cells) have recently been identified as a distinct pro-inflammatory T-helper subset and their role in various autoimmune processes including RA (Kirkham et al., 2006), multiple sclerosis (Matusevicius et al., 1999), psoriasis and inflammatory bowel disease (Duerr, 2006) is becoming more apparent. They seem to have a reciprocal relationship with regulatory IL-10 secreting T helper cells (McGeachy, 2007) and drive an inflammatory response which is dominated by

The Clinical Role of Glucocorticoids in the Management of Rheumatoid Arthritis 17

Other work in this area has shown higher levels of IL-17 mRNA in the bronchial biopsies of asthmatic patients compared to controls with increased expression of GCR-β in response to IL-17. Dexamethasone was unable to decrease IL-17 induced IL-6 expression in these asthmatic patients (Vazquez-Tello et al., 2010). Conversely, the synthetic GC dexamethasone (Dex) normally induces the anti-inflammatory cytokine IL-10 in Th cells, and a deficiency in IL-10 up regulation in response to GC has been demonstrated in GC-resistant asthma (Xystrakis, 2005). Importantly IL-10 has been shown to enhance expression of GCR-α (Xystrakis, 2005). Very little work has been carried out in this field in the context of RA but these findings suggest that a disturbed balance of T cell derived cytokines may be causing

The emerging concept is that the human GC-resistant phenotype is disease independent, and observations of immune responses in GC-resistant individuals across medical specialities strongly supports this (Barnes & Adcock 2009; Schewitz et al., 2009; Norman & Hearing, 2002). Moreover, the data suggests that T helper cell responses in GC-resistant individuals are biased against IL-10 and in favour of IL-17. Importantly, there is also evidence that such a cytokine profile may be instrumental in regulating the ratio of glucocorticoid receptor (GCR) isoforms. IL-10 has been shown to enhance expression of GCR-α (Xystrakis, 2005), which augments GC-responses (Lewis-Tuffin, 2006), and IL-17 upregulates the level of GCR-β (Vazquez-Tello et al., 2010), which attenuates GC-responses. Consistent with this, PBMCs from GC-resistant patients with RA express higher levels of GCR-β (Kozaci et al., 2007) as do bronchoalveolar lavage washings from patients with GCresistant asthma (Vazquez-Tello et al., 2010). As mentioned earlier, monocyte derived macrophages and dendritic cells have a huge influence on T helper phenotype differentiation and their precise role in this model requires further research. Macrophages may well be the master regulators of this GC-resistant phenotype through their influencing

It is interesting to note that there seems to be no resistance to the action of GC in terms of adverse effects. The most likely reason for this is that adverse effects are predominantly mediated by the excess activation of the transcription pathways which mediate the physiological role of GC action. Therefore administered exogenous GC potentially acts on all cells while the anti-inflammatory effects of GC are only mediated via their action on activated pro-inflammatory cells. Thus if these pro-inflammatory cells become GC resistant, GC resistant inflammation will occur alongside GC mediated adverse events. One key weakness of this model is that it does not take into account the important findings relating to histone acetylation which Barnes and colleagues have elucidated over the last 20 years and it would be interesting to study the effects of T helper derived cytokines on HDAC2

Glucocorticoids have become an even more important therapeutic intervention in rheumatoid arthritis both for the control of acute disease flares and for the long term prevention of joint erosions. A better understanding of their mechanisms of action has

GC resistance by altering the balance of GCR subtype expression.

**5.6 A unifying model for glucocorticoid resistance** 

of the T helper cells (Fig 9).

expression.

**6. Conclusions** 

neutrophils (Miossec et al., 2009). There is increasing evidence to suggest they play a role in GC resistance in a variety of inflammatory diseases.

The earliest reports of TH-17 cell involvement in GC resistance emerged from the asthma research community. McKinley et al. showed in a mouse model of asthma that naive T cells which were polarized to the TH-17 phenotype during differentiation (by adding IL-23, IL-6 and TGF-β in vitro) were less sensitive to dexamethasone compared to cells which differentiated to the TH-2 phenotype (McKinley et al., 2008). Subsequent work has shown an expanded TH-17 subset within PBMC cultures of patients with UC and uveitis (Lee et al., 2009; Lee et al., 2007). The data from the uveitis and UC studies seems to suggest that the TH-17 phenotype is inherently GC resistant when tested using in-vitro stimulation assays. It seems that their number is expanded in patients with clinical GC resistance.

Fig. 9. The proposed model for GC resistance. Monocyte derived macrophages influence T helper cell phenotype differentiation through various cytokines. The balance of proinflammatory TH-17 cells and induced regulatory iTRegs alters the balance of IL-17, which increases GCR-β expression and hence reduces response to GC, and IL-10, which increases GCR-α expression and hence increases responsiveness to GC. The balance between these cytokines determines the balance between GC resistance and GC responsiveness.

Other work in this area has shown higher levels of IL-17 mRNA in the bronchial biopsies of asthmatic patients compared to controls with increased expression of GCR-β in response to IL-17. Dexamethasone was unable to decrease IL-17 induced IL-6 expression in these asthmatic patients (Vazquez-Tello et al., 2010). Conversely, the synthetic GC dexamethasone (Dex) normally induces the anti-inflammatory cytokine IL-10 in Th cells, and a deficiency in IL-10 up regulation in response to GC has been demonstrated in GC-resistant asthma (Xystrakis, 2005). Importantly IL-10 has been shown to enhance expression of GCR-α (Xystrakis, 2005). Very little work has been carried out in this field in the context of RA but these findings suggest that a disturbed balance of T cell derived cytokines may be causing GC resistance by altering the balance of GCR subtype expression.

#### **5.6 A unifying model for glucocorticoid resistance**

The emerging concept is that the human GC-resistant phenotype is disease independent, and observations of immune responses in GC-resistant individuals across medical specialities strongly supports this (Barnes & Adcock 2009; Schewitz et al., 2009; Norman & Hearing, 2002). Moreover, the data suggests that T helper cell responses in GC-resistant individuals are biased against IL-10 and in favour of IL-17. Importantly, there is also evidence that such a cytokine profile may be instrumental in regulating the ratio of glucocorticoid receptor (GCR) isoforms. IL-10 has been shown to enhance expression of GCR-α (Xystrakis, 2005), which augments GC-responses (Lewis-Tuffin, 2006), and IL-17 upregulates the level of GCR-β (Vazquez-Tello et al., 2010), which attenuates GC-responses. Consistent with this, PBMCs from GC-resistant patients with RA express higher levels of GCR-β (Kozaci et al., 2007) as do bronchoalveolar lavage washings from patients with GCresistant asthma (Vazquez-Tello et al., 2010). As mentioned earlier, monocyte derived macrophages and dendritic cells have a huge influence on T helper phenotype differentiation and their precise role in this model requires further research. Macrophages may well be the master regulators of this GC-resistant phenotype through their influencing of the T helper cells (Fig 9).

It is interesting to note that there seems to be no resistance to the action of GC in terms of adverse effects. The most likely reason for this is that adverse effects are predominantly mediated by the excess activation of the transcription pathways which mediate the physiological role of GC action. Therefore administered exogenous GC potentially acts on all cells while the anti-inflammatory effects of GC are only mediated via their action on activated pro-inflammatory cells. Thus if these pro-inflammatory cells become GC resistant, GC resistant inflammation will occur alongside GC mediated adverse events. One key weakness of this model is that it does not take into account the important findings relating to histone acetylation which Barnes and colleagues have elucidated over the last 20 years and it would be interesting to study the effects of T helper derived cytokines on HDAC2 expression.
