Cornelia M. Spies

Membrane glucocorticoid receptors (mGCR) are expressed in normal human peripheral blood mononuclear cells and up-regulated after in vitro stimulation and in patients with rheumatoid arthritis

Glucocorticoids mediate their therapeutic actions mostly by genomic effects via cytosolic receptors, but some effects are too rapid to be mediated by changes at the genomic level. The detailed mechanisms of these nongenomic actions are still unclear. Membrane‐bound glucocorticoid receptors (mGCR) have been suggested to be involved, although their physiological existence in humans so far is hypothetical. For the first time we demonstrate the existence of mGCR on monocytes and B cells obtained from healthy blood donors using high‐sensitivity immunofluorescent staining. Immunostimulation with lipopolysaccharide increases the percentage of mGCR‐positive monocytes, which can be prevented by inhibiting the secretory pathway. Overexpression of the human glucocorticoid receptor α alone is not sufficient to enhance mGCR expression. These in vitro findings are consistent with our clinical observation that in patients with rheumatoid arthritis the frequency of mGCR positive monocytes is increased and positively correlated with disease activity. We conclude that mGCR are 1) indeed physiologically present in healthy blood donors, but remained unidentified by conventional techniques due to their small number per cell and 2) actively up‐regulated and transported through the cell after immunostimulation. These receptors may reflect a feedback mechanism of the organism upon immunostimulation and/or play a role in pathogenesis.—Bartholome, B., Spies, C. M., Gaber, T., Schuchmann, S., Berki, T., Kunkel, D., Bienert, M., Radbruch, A., Burmester, G.‐R., Lauster, R., Scheffold, A., Buttgereit, F. Membrane glucocorticoid receptors (mGCR) are expressed in normal human peripheral blood mononuclear cells and up‐regulated after in vitro stimulation and in patients with rheumatoid arthritis. FASEB J. 18, 70–80 (2004)

Defining conditions where long-term glucocorticoid treatment has an acceptably low level of harm to facilitate implementation of existing recommendations: viewpoints from an EULAR task force

There is convincing evidence for the known and unambiguously accepted beneficial effects of glucocorticoids at low dosages. However, the implementation of existing recommendations and guidelines on the management of glucocorticoid therapy in rheumatic diseases is lagging behind. As a first step to improve implementation, we aimed at defining conditions under which long-term glucocorticoid therapy may have an acceptably low level of harm. A multidisciplinary European League Against Rheumatism task force group of experts including patients with rheumatic diseases was assembled. After a systematic literature search, breakout groups critically reviewed the evidence on the four most worrisome adverse effects of glucocorticoid therapy (osteoporosis, hyperglycaemia/diabetes mellitus, cardiovascular diseases and infections) and presented their results to the other group members following a structured questionnaire for final discussion and consensus finding. Robust evidence on the risk of harm of long-term glucocorticoid therapy was often lacking since relevant study results were often either missing, contradictory or carried a high risk of bias. The group agreed that the risk of harm is low for the majority of patients at long-term dosages of ≤5 mg prednisone equivalent per day, whereas at dosages of >10 mg/day the risk of harm is elevated. At dosages between >5 and ≤10 mg/day, patient-specific characteristics (protective and risk factors) determine the risk of harm. The level of harm of glucocorticoids depends on both dose and patient-specific parameters. General and glucocorticoid-associated risk factors and protective factors such as a healthy lifestyle should be taken into account when evaluating the actual and future risk.

Origin and Functional Activity of the Membrane-Bound Glucocorticoid Receptor

OBJECTIVE Glucocorticoids (GCs) exert their antiinflammatory and immunosuppressive effects in humans primarily via the cytosolic GC receptor (cGR) but also via rapid, nongenomic mechanisms. Most likely, membrane-bound GRs (mGR) are involved in nongenomic GC signaling. The aim of this study was to investigate the origin and functional activity of mGR. METHODS We analyzed the origin of mGR using mGR-expressing HEK 293T cells, by transient and stable RNA interference-mediated GR reduction. GR messenger RNA (mRNA) and cGR and mGR protein levels were analyzed by real-time quantitative polymerase chain reaction, immunoblotting, and high-sensitivity immunofluorescence staining. Furthermore, we analyzed the functional activity of mGR, using membrane-impermeable bovine serum albumin (BSA)-bound dexamethasone (DEX-BSA) in human monocytes. Membrane-bound GR-expressing monocytes were treated with DEX, DEX-BSA, or BSA. Cell lysates were analyzed using PepChip arrays in order to identify kinases triggered by DEX-BSA, with validation using Bio-Plex assays and immunoblotting. RESULTS Our data showed that transient reduction of GR mRNA in HEK 293T cells decreased cGR protein levels but not mGR protein levels. However, stably transfected cells showed reduced cGR protein expression and significantly reduced mGR protein expression. Furthermore, 51 kinase substrates were identified for which phosphorylation was either reduced or increased. We observed p38 MAP kinase (MAPK) as one possible upstream kinase. Validation of these data by Bio-Plex phosphoprotein assay and immunoblotting showed increased phosphorylation of p38 MAPK after treatment with DEX-BSA. CONCLUSION Our data demonstrate that the human GR gene encodes for both cGR and mGR. Membrane-bound GR retains functional activity, as indicated by induced phosphorylation of p38 MAPK due to DEX-BSA treatment. Membrane-bound GR-mediated cellular signaling needs to be investigated further in order to clarify its therapeutic potential.

Effects of hypoxia and/or lack of glucose on cellular energy metabolism and cytokine production in stimulated human CD4+ T lymphocytes.

Oxidative phosphorylation and/or glycolysis provide energy, mainly in the form of ATP, which ensures proper functioning of immune cells such as CD4(+) T lymphocytes. However, the main substrates, namely oxygen and glucose, are known to remain for a relatively short time in the inflamed tissue and in other clinical situations where immune cells need to function properly. Therefore, we examined the effect of hypoxia and/or lack of glucose on cellular energy metabolism and on cytokine secretion in stimulated human CD4(+) T lymphocytes. Human CD4(+) T cells were MACS-isolated using peripheral blood obtained from healthy donors. Stimulated cells were incubated in medium with or without glucose for 6h in a sealed chamber which led to cumulative hypoxia. During this incubation period, (i) oxygen saturation was measured continuously using a Clark-type electrode, and (ii) samples were taken at different time points in order to quantify for each the viability of cells, intracellular reactive oxygen species (iROS), ATP levels, glycolytic enzyme activity, mRNA expression of hexokinase-1 and superoxide dismutase-1, and concentrations of several different cytokines. Stimulated CD4(+) T cells which were incubated under normoxic conditions served as controls. Under hypoxic conditions, lack of glucose exerted a biphasic effect on cellular oxygen consumption: initially higher but later lower respiration rates were measured when compared to conditions where glucose was available. Lack of glucose strongly increased the number of dead cells and the formation of iROS under normoxia but not under hypoxia. Under both normoxic and hypoxic conditions, intracellular ATP levels remained almost unchanged during the incubation period if glucose was present, but decreased significantly in the absence of glucose, despite the enhanced glycolytic enzyme activity. Measurements of stimulated cytokine production demonstrated (i) that cumulative hypoxia stimulates especially the secretion of IL-1beta, IL-10 and IL-8, and (ii) that lack of glucose results in lower cytokine concentrations. We demonstrate that CD4(+) T cells are highly adaptive in bioenergetic terms which ensure their proper function under extreme conditions of glucose and/or oxygen availability as found under physiological and pathophysiological conditions. Hypoxia seems to facilitate inflammatory reactions and angiogenesis.

Circadian rhythms in rheumatology--a glucocorticoid perspective.

The hypothalamic-pituitary-adrenal (HPA) axis plays an important role in regulating and controlling immune responses. Dysfunction of the HPA axis has been implicated in the pathogenesis of rheumatoid arthritis (RA) and other rheumatic diseases. The impact of glucocorticoid (GC) therapy on HPA axis function also remains a matter of concern, particularly for longer treatment duration. Knowledge of circadian rhythms and the influence of GC in rheumatology is important: on the one hand we aim for optimal treatment of the daily undulating inflammatory symptoms, for example morning stiffness and swelling; on the other, we wish to disturb the HPA axis as little as possible. This review describes circadian rhythms in RA and other chronic inflammatory diseases, dysfunction of the HPA axis in RA and other rheumatic diseases and the recent concept of the hepato-hypothalamic-pituitary-adrenal-renal axis, the problem of adrenal suppression by GC therapy and how it can be avoided, and evidence that chronotherapy with modified release prednisone effective at 02:00 a.m. can inhibit proinflammatory sequelae of nocturnal inflammation better compared with GC administration in the morning but does not increase the risk of HPA axis insufficiency in RA.

Energy metabolism and rheumatic diseases: from cell to organism

In rheumatic and other chronic inflammatory diseases, high amounts of energy for the activated immune system have to be provided and allocated by energy metabolism. In recent time many new insights have been gained into the control of the immune response through metabolic signals. Activation of immune cells as well as reduced nutrient supply and hypoxia in inflamed tissues cause stimulation of glycolysis and other cellular metabolic pathways. However, persistent cellular metabolic signals can promote ongoing chronic inflammation and loss of immune tolerance. On the organism level, the neuroendocrine immune response of the hypothalamic-pituitary adrenal axis and sympathetic nervous system, which is meant to overcome a transient inflammatory episode, can lead to metabolic disease sequelae if chronically activated. We conclude that, on cellular and organism levels, a prolonged energy appeal reaction is an important factor of chronic inflammatory disease etiology.

The supplementary therapeutic DMARD role of low-dose glucocorticoids in rheumatoid arthritis

The management of rheumatoid arthritis (RA) is primarily based on the use of disease-modifying antirheumatic drugs (DMARDs), mainly comprising synthetic chemical compounds (that is, methotrexate or leflunomide) and biological agents (tumor necrosis factor inhibitors or abatacept). On the other hand, glucocorticoids (GCs), used for decades in the treatment of RA, are effective in relieving signs and symptoms of the disease, but also interfere with radiographic progression, either as monotherapy or in combination with conventional synthetic DMARDs. GCs exert most of their biological effects through a genomic action, using the cytosolic GC receptor and then interacting with the target genes within target cells that can result in increased expression of regulatory - including anti-inflammatory - proteins (transactivation) or decreased production of proinflammatory proteins (transrepression). An inadequate secretion of GCs from the adrenal gland, in relation to stress and inflammation, seems to play an important role in the pathogenesis and disease progression of RA. At present there is clear evidence that GC therapy, especially long-term low-dose treatment, slows radiographic progression by at least 50% when given to patients with early RA, hence satisfying the conventional definition of a DMARD. In addition, long-term follow-up studies suggest that RA treatment strategies which include GC therapy may favorably alter the disease course even after their discontinuation. Finally, a low-dose, modified night-release formulation of prednisone, although administered in the evening (replacement therapy), has been developed to counteract the circadian (night) rise in proinflammatory cytokine levels that contributes to disease activity, and might represent the way to further optimize the DMARD activity exerted by GCs in RA.

Rimexolone inhibits proliferation, cytokine expression and signal transduction of human CD4+ T-cells.

Rimexolone is a lipophilic glucocorticoid drug used for local application. Only few data are available describing its effects on immune cell functions. In this study we investigated the effects of rimexolone on the proliferation of human CD4+ T-cells using dexamethasone as standard reference. Isolated CD4+ T-cells were pre-incubated with rimexolone or dexamethasone at different concentrations for 10 min (10(-11)/10(-8)/10(-5)M) and stimulated with anti-CD3/anti-CD28 for 96 h. Proliferation was determined by flow cytometry. The percentage of dividing cells was significantly reduced by 10(-5)M rimexolone and dexamethasone; however, the average number of cell divisions was unchanged. In addition, production of IL-2 and other cytokines was reduced by both glucocorticoids at 10(-5)M. Interestingly, we observed a rimexolone-induced down-regulation of CD4 expression in unstimulated and non-dividing cells. The inhibitory effects on proliferation and CD4 expression could be blocked by the glucocorticoid-antagonist RU486 and were not due to glucocorticoid-induced apoptosis. Rimexolone and dexamethasone showed a similar potential to induce IkappaBalpha gene expression. We demonstrate rimexolone and dexamethasone to impair T-cell signalling pathways by rapid non-genomic suppression of the phosphorylation of Akt, p38 and ERK. We conclude that rimexolone and dexamethasone inhibit T-cell proliferation as well as cytokine production of activated CD4+ T-cells in a similar manner. As these inhibitory effects predominantly occur at high concentrations, a relatively high occupation-rate of cytosolic glucocorticoid receptors is needed, but receptor-mediated non-genomic effects may also be involved. It is implied that these effects contribute to the well-known beneficial anti-inflammatory and immunomodulatory effects of glucocorticoid therapy.

High-sensitivity immunofluorescence staining: a comparison of the liposome procedure and the FASER technique on mGR detection.

Flow cytometry has become a widely-used and powerful tool for the characterization of cells according to their expression of specific proteins. However, sensitivity of this method is still limited since conventionally labeled antibodies can be conjugated with at maximum 1–10 dye molecules. This fact resulted in the need to develop new techniques in order to identify molecules which are expressed in very low but functionally relevant amounts. In the past, we have successfully used a liposome-based high-sensitivity immunofluorescence technique to measure the expression of low abundant membrane bound glucocorticoid receptors (mGR) on different cell types. The use of this technique allows the detection of as few as 50–100 antigen molecules per cell which is due to a 100-fold to 1000-fold increase in fluorescence signal intensity compared with conventional methods. The higher sensitivity is achieved since thousands of dye molecules can be enclosed in liposomes. Another modern high-sensitivity immunofluorescence staining method is the purchasable Fluorescence Amplification by Sequential Employment of Reagents (FASER) procedure. Here, we aimed at comparing sensitivity and specificity of these two techniques for the detection of the mGR. Our data demonstrate the FASER technique to be more sensitive and also more specific for the detection of mGR as compared to the liposome technique. However, both methods have advantages and disadvantages which are discussed in detail.

OP0086 Alterations of immune cellular circadian rhythms in rheumatoid arthritis

Background The circadian variation of clinical symptoms and the underlying variation of cytokine and hormone levels in rheumatoid arthritis (RA) are well described and have already led to the successful application of chronotherapy with prednisone (Buttgereit et al., Lancet, 2008). Much less is known about the circadian rhythms of different immune cell populations in RA. Objectives In this pilot study we investigated molecular, cellular and humoral circadian parameters in postmenopausal female RA patients in comparison to healthy control subjects. Methods Blood samples from postmenopausal female patients with active RA (DAS 28 ≥4.2) (n=5) and postmenopausal female healthy controls (n=5) were collected every 2 hours for 24 hours and analysed by flow cytometry and multiplex suspension array of 28 cytokines. Clock gene expression of isolated CD14+ monocytes was analysed by quantitative RT-PCR. Endogenous circadian rhythm dynamics of macrophages were determined by means of a Bmal1-promotor driven luciferase reporter construct. COSINOR analysis was used for statistical analysis of the groups. Results Expression of the clock gene RevErbα in CD14+ monocytes showed a significant circadian expression pattern in both RA patients and healthy controls subjects, whereas the clock genes Per2 and Per3 were not expressed in a circadian manner in RA patients but in healthy controls only. The amplitude of the endogenous circadian rhythm of macrophages tended to be lower in RA patients than in healthy controls, whereas period length was not altered. In flow cytometric analysis of surface marker expression of blood cells we found a significant circadian rhythm in RA patients and healthy subjects for the frequency of CD3-CD56+ natural killer (NK) cells, Interleukin-8 Receptor (IL-8R) expressing CD4+ T helper and CD8+ cytotoxic T cells, and CXCR4 expressing CD4+ T helper and CD8+ cytotoxic cells. A significant circadian rhythm was not detectable in RA patients but in healthy controls only for CD3+CD56+ NK T cells. In contrast, a significant circadian expression of IL-8R+ monocytes was found in RA patients only but not in healthy subjects. Of note, CCR7 did not at all show a circadian expression. A significant circadian cytokine expression was detected only for MCP-1 in healthy controls. Conclusions This is the first indication of alterations of clock gene expression and endogenous circadian rhythms in immune cells of RA patients. Traffic of peripheral blood cells shows circadian variation in RA patients and healthy controls with characteristic peak phases, especially in NK cells and chemokine receptor expressing cells. NKT and other cells may lose their normal circadian rhythm in RA, whereas IL-8R expression on monocytes may be established as new “inflammatory” circadian rhythm in RA patients. These findings provide new aspects of RA chronobiology and may have therapeutic implications. Disclosure of Interest C. Spies: None Declared, T. Gaber: None Declared, P. Hoff: None Declared, J. Mazuch: None Declared, B. Maier: None Declared, M. Hahne: None Declared, C. Strehl: None Declared, C. Tran: None Declared, N. Soboleva: None Declared, A. Stoehr: None Declared, M. Wagegg: None Declared, M. Fangradt: None Declared, M. Jakstadt: None Declared, D. Huscher: None Declared, G.-R. Burmester: None Declared, J. Detert: None Declared, A. Kramer: None Declared, F. Buttgereit Grant/Research support from: This study was supported by Horizon Pharma AG, Reinach, Switzerland and Merck KGaA, Darmstadt, Germany. Dr. Buttgereit reports receiving consultancy fees, honoraria and travel expenses from Merck Serono, Horizon Pharma (formerly Nitec Pharma) Mundipharma Int Ltd and grant support from Merck Serono and Horizon Pharma., Consultant for: see above, Speakers Bureau: see above