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Open Access

Peer-reviewed

Research Article

A2A Adenosine Receptors Are Differentially Modulated by Pharmacological Treatments in Rheumatoid Arthritis Patients and Their Stimulation Ameliorates Adjuvant-Induced Arthritis in Rats

  • Fabrizio Vincenzi,

    Affiliation Department of Clinical and Experimental Medicine, Pharmacology Section, University of Ferrara, Ferrara, Italy

    ⨯
  • Melissa Padovan,

    Affiliation Department of Clinical and Experimental Medicine, Rheumatology Section, University of Ferrara, Ferrara, Italy

    ⨯
  • Martina Targa,

    Affiliation Department of Clinical and Experimental Medicine, Pharmacology Section, University of Ferrara, Ferrara, Italy

    ⨯
  • Carmen Corciulo,

    Affiliation Department of Clinical and Experimental Medicine, Pharmacology Section, University of Ferrara, Ferrara, Italy

    ⨯
  • Sarah Giacuzzo,

    Affiliation Department of Clinical and Experimental Medicine, Rheumatology Section, University of Ferrara, Ferrara, Italy

    ⨯
  • Stefania Merighi,

    Affiliation Department of Clinical and Experimental Medicine, Pharmacology Section, University of Ferrara, Ferrara, Italy

    ⨯
  • Stefania Gessi,

    Affiliation Department of Clinical and Experimental Medicine, Pharmacology Section, University of Ferrara, Ferrara, Italy

    ⨯
  • Marcello Govoni,

    Affiliation Department of Clinical and Experimental Medicine, Rheumatology Section, University of Ferrara, Ferrara, Italy

    ⨯
  • Pier Andrea Borea,

    Affiliation Department of Clinical and Experimental Medicine, Pharmacology Section, University of Ferrara, Ferrara, Italy

    ⨯
  • Katia Varani

    vrk@unife.it

    Affiliation Department of Clinical and Experimental Medicine, Pharmacology Section, University of Ferrara, Ferrara, Italy

    ⨯

Abstract

A2A adenosine receptors (ARs) play a key role in the inhibition of the inflammatory process. The purpose of this study was to evaluate the modulation of A2AARs in rheumatoid arthritis (RA) patients after different pharmacological treatments and to investigate the effect of A2AAR stimulation in a rat model of arthritis. We investigated A2AAR density and functionality in RA progression by using a longitudinal study in RA patients before and after methotrexate (MTX), anti-TNFα agents or rituximab treatments. A2AARs were analyzed by saturation binding assays in lymphocytes from RA patients throughout the 24-month study timeframe. In an adjuvant-induced arthritis model in rats we showed the efficacy of the A2AAR agonist, CGS 21680 in comparison with standard therapies by means of paw volume assessment, radiographic and ultrasonographic imaging. Arthritic-associated pain was investigated in mechanical allodynia and thermal hyperalgesia tests. IL-10 release following A2AAR stimulation in lymphocytes from RA patients and in serum from arthritic rats was measured. In lymphocytes obtained from RA patients, the A2AAR up-regulation was gradually reduced in function of the treatment time and the stimulation of these receptors mediated a significant increase of IL-10 production. In the same cells, CGS 21680 did not affected cell viability and did not produced cytotoxic effects. The A2AAR agonist CGS 21680 was highly effective, as suggested by the marked reduction of clinical signs, in rat adjuvant-induced arthritis and associated pain. This study highlighted that A2AAR agonists represent a physiological-like therapeutic alternative for RA treatment as suggested by the anti-inflammatory role of A2AARs in lymphocytes from RA patients. The effectiveness of A2AAR stimulation in a rat model of arthritis supported the role of A2AAR agonists as potential pharmacological treatment for RA.

Citation: Vincenzi F, Padovan M, Targa M, Corciulo C, Giacuzzo S, Merighi S, et al. (2013) A2A Adenosine Receptors Are Differentially Modulated by Pharmacological Treatments in Rheumatoid Arthritis Patients and Their Stimulation Ameliorates Adjuvant-Induced Arthritis in Rats. PLoS ONE 8(1): e54195. https://doi.org/10.1371/journal.pone.0054195

Editor: Victor Sanchez-Margalet, Virgen Macarena University Hospital, School of Medicine, Spain

Received: October 18, 2012; Accepted: December 11, 2012; Published: January 11, 2013

Copyright: © 2013 Vincenzi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by grants from the Italian Ministry of Education, University and Research (MIUR-2009EE3SWA). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Rheumatoid Arthritis (RA) is a chronic autoimmune disease that primarily affects joints and causes pain, stiffness, swelling and limited motion [1], [2]. In RA the inflammatory process leads to progressive cartilage degradation with synovial hyperplasia, change in underlying bone with erosions and high levels of pro-inflammatory mediators [3], [4]. It is widely accepted that cytokines such as tumor necrosis factor α (TNFα) and interleukin (IL) family mediate a large variety of effector functions in the context of RA pathogenesis [5]. Conversely, the anti-inflammatory cytokine IL-10 is relatively unique in its ability to down-regulate the production of multiple pro-inflammatory cytokines, leading to the notion that IL-10 may modulate the disease expression in RA [6]. Early diagnosis and therapy are crucial in order to prevent unfavourable outcome avoiding joint deterioration and functional disability [7]. Conventional disease modifying anti-rheumatic drugs (DMARDs) such as methotrexate (MTX) in monotherapy or in combination are currently the first medications usually prescribed in RA patients [8], [9]. More recently, the introduction of anti-TNFα agents (e.g. adalimumab, etanercept) or the use of anti-CD20 B cell targeted therapy (rituximab, RTX) have provided a marked improvement in RA even if some patients do not respond or fail to maintain adequate response to these treatments [10], [11]. Therefore the availability of alternative therapeutic options, targeting the pathways associated to inflammatory processes in RA, could be very useful to assist clinicians in making alternative pharmacological treatment choices [12], [13].

Adenosine, a purine nucleoside, regulates the mechanisms of inflammation acting with four cell surface receptors named as A1, A2A, A2B and A3ARs which are coupled to different G proteins [14], [15]. In particular, A2AARs are targeted to be anti-inflammatory receptors and their activation suppress the elevated levels of pro-inflammatory cytokines [16]. It has been shown that A2AAR agonists are able to increase the production of the anti-inflammatory cytokine IL-10 in various in vitro and in vivo models [17], [18]. In human synoviocytes A2AAR stimulation decreased TNFα, IL-6 and IL-8 production and increased IL-10 release showing a marked down-regulation of the inflammatory status [19], [20].

The anti-inflammatory and tissue-protective effects elicited by A2AARs have been reported in several in vivo models of inflammatory diseases [21]. At present, different animal models of arthritis have been used to test novel therapeutic hypothesis, their efficacy and the presence of adverse effects [22], [23]. It has been shown that A2AAR stimulation ameliorated clinical signs and improved histological damage in murine collagen-induced arthritis model [24]. The treatment with A2AAR agonists decreased the expression of inflammatory cytokines and the degree of oxidative and nitrosative damage [25]. Adjuvant-induced arthritis is a commonly used model of inflammatory arthritis, with an incidence of around 90%, making it an ideal model in which to investigate arthritic changes and to evaluate compounds that might be of potential use as drugs for RA treatment [26], [27]. Recently, A2AAR agonists have been approved for clinical trials based on their anti-inflammatory and wound-healing properties [15], [28], [29]. In clinical evaluation, the chronic A2AAR activation was able to reduce the inflammatory status in specific diseases as diabetic nephropathy and to promote healing of diabetic foot ulcers [30].

In a previous study our group showed that A2A and A3ARs are up-regulated in early RA patients and after MTX treatment but not in RA patients treated with anti-TNFα agents [31]. An inverse correlation between A2A and A3AR density with disease activity score (DAS) was found in RA patients. Moreover, A2A and A3AR stimulation mediated a reduction of pro-inflammatory cytokines and matrix metalloproteinases production suggesting a role of adenosine in the inhibition of inflammation and cartilage degradation [32].

The aim of the present study was to evaluate in RA patients the influence of different biologic therapies as anti-TNFα drugs or RTX in comparison with MTX treatment on A2AAR density at various time points of treatment (from 0 to 24 months). In RA patients treated with RTX the relationship between A2AAR density and DAS 28 values was investigated. The changes of A2AAR expression after different pharmacological treatments in function of the time and the agonist-induced IL-10 production have suggested the potential use of A2AAR stimulation as RA therapeutic approach. As a consequence, in a complete Freund’s adjuvant (CFA)-induced arthritic rat model, the effect of a well known A2AAR agonist named 2-[p-[2-carboxyethyl)-phenetyl-amino]-5′-N-ethyl-carboxamido-adenosine (CGS 21680) was evaluated in controlling rat adjuvant arthritis and associated pain.

Materials and Methods

Ethics Statement

The study was approved by the local Ethic Committee of the University Hospital of Ferrara (Italy) and written informed consent was obtained from each participant in accordance with the principles outlined in the Declaration of Helsinki.

All animal procedures were conformed to the Italian guidelines for the use of animals in biomedical research (authorization from the Italian Ministry for Health 122/2011-B).

Sample Collection and Human Lymphocyte Preparation

Lymphocytes were isolated and prepared as previously described from the peripheral blood of control subjects and RA patients [31], [32]. The isolation of blood cells started no later than 3 to 4 hours after the samples had been taken. The blood was supplemented with 6% (by weight) Dextran T500 solution (Sigma-Aldrich, St Louis, MO, USA) and erythrocytes were allowed to settle down for 60 min. Leukocytes were pelleted by centrifugation for 15 min at 100×g and the remaining erythrocytes were lyzed in distilled water at 4°C rapidly restoring the isotonicity by NaCl solution. Then, the cells were pelleted by centrifugation for 5 min at 250×g, suspended in Krebs-Ringer phosphate buffer and layered onto 10 ml of Fycoll-Hypaque (GE Healthcare, Little Chalfont, UK). After centrifugation, mononuclear cells were washed in 0.02 M phosphate-buffered saline (PBS) at pH 7.2 containing 5 mM MgCl2 and 0.15 mM CaCl2. Finally, they were decanted into a culture flask and placed in a humidified incubator (5% CO2) for 2 hours at 37°C. This procedure, aimed at removing monocytes which adhere to the culture flasks, resulted in a purified lymphocyte preparation containing at least 99% small lymphocytes identified by morphological criteria. To obtain membrane suspensions, cell fractions were centrifuged in hypothonic buffer at 20000×g for 10 min. The resulting pellet was resuspended in tris HCl 50 mM buffer pH 7.4 containing 2 UI/ml adenosine deaminase (Sigma-Aldrich) and incubated for 30 min at 37°C. After the incubation the suspension was centrifuged again at 40000×g for 10 min and the final pellet was used for radioligand binding assays. The protein concentration was determined by a Bio-Rad method with bovine albumine as reference standard [19].

Saturation Binding Experiments to A2AARs

Saturation binding to A2AARs was carried out with the use of [3H]-4-(2-[7-amino-2-(2-furyl) [1], [2], [4]-triazolo[2,3-a] [1], [3], [5] triazin-5-ylamino] ethyl) phenol ([3H]-ZM 241385, specific activity 27 Ci/mmol, Biotrend, Cologne, Germany), as radioligand [31], [32]. Cell membranes (60 µg of protein) were incubated for 60 min at 4°C with various concentrations (0.1–20 nM) of [3H]-ZM 241385. Non specific binding was determined in the presence of 5-amino-7-(phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH 58261, 1 µM, Tocris, Bristol, UK) and was always <25% of the total binding. Bound and free radioactivity were separated by filtering the assay mixture through Whatman GF/B glass fiber filters by using a Brandel cell harvester. The filter bound radioactivity was counted in a 2810 TR liquid scintillation counter (Perkin-Elmer, Boston, MA, USA).

IL-10 Release in Cultured Lymphocytes

Isolated lymphocytes from untreated RA patients were suspended at a density of 106 cells/ml in RPMI 1640 medium supplemented with 2% fetal bovine serum (Euroclone, Milan, Italy) and seeded into 24-well plates. Lymphocytes were incubated for 24 hours in the absence or in the presence of CGS 21680 (1 nM–10 µM). A selective A2AAR antagonist SCH 58261 (1 µM) was also used to verify the specific involvement of these receptors in IL-10 release. At the end of incubation, the cell suspension was collected and centrifuged at 1000×g for 10 min at 4°C. The anti-inflammatory cytokine IL-10 levels were determined with a quantitative sandwich ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer instructions [33]. The reaction was developed with streptavidin-horseradish peroxidase and optical density was read at 450 nm wavelength.

Cell Viability and Cytotoxicity Assays

MTT and lactate dehydrogenase (LDH) assays were performed in lymphocytes from untreated RA patients after 24 hours of incubation with different concentration of CGS 21680 (1 nM–10 µM). In the MTT assay, the yellow tetrazolium salt is reduced in metabolically active cells to form insoluble purple formazan crystals, which are solubilized by the addition of a detergent. Briefly, 100 µl of cell suspension was placed in one well of a 96-well tissue culture plate, and 10 µl of MTT solution (2.5 mg/ml; Sigma-Aldrich) was added. After incubation for 4 hours at 37°C, 100 µl of acid-isopropanol (0.04 N HCl in isopropanol) was added and mixed by gentle pipetting to solubilize the cells. The optical density of the solution was read at 550 nm using a microplate reader [34].

LDH activity can be used as an indicator of cell membrane integrity and serves as general meaning to assess cytotoxicity resulting from chemical compounds or environmental toxic factors. LDH assay was performed in human lymphocytes from untreated RA patients as previously described [35]. At the end of incubation with CGS 21680 for 24 hours, 100 µl of supernatant per well was harvested and transferred into a new 96-well, flat-bottom plate. LDH substrate (100 µl) was added to each well and incubated for 30 min at room temperature protected from light. The absorbance of the samples was measured at 490 mm on spectrophotomer.

Animals and Treatments

Male Sprague-Dawley rats with a mean weight of 150−180 g were obtained from Charles River Laboratories (Milan, Italy) and maintained in a climate-controlled environment under 12 hours light/12 hours dark conditions. The animals were fed standard rodent chow and water ad libitum. Adjuvant-induced arthritis was elicited in rats according to established methods [36]. Briefly, rats were injected subcutaneously in the plantar surface of the left hind paw with 100 µl of CFA (1 mg/ml) containing heat killed Mycobacterium Tubercolosis suspended in paraffin oil (Sigma-Aldrich). The rats (n = 30) were subsequently divided into five groups of six rats each. A stock solution of CGS 21680 were prepared in DMSO and further diluted in PBS for an injection volume of 0.3 ml per rat. Group 1 was injected with CFA but not pharmacologically treated. Group 2 represented the sham group where the rats were injected with PBS solution. Group 3 was treated with MTX (2 mg/kg) administered subcutaneously (s.c.) in a single dose a week. Group 4 was treated with CGS 21680 at the dose of 0.1 mg/kg intraperitoneally (i.p.) three times a week. Group 5 was treated with etanercept at a single dose of 5 mg/kg a week, s.c. The pharmacological treatments started 1 week after CFA injection. Clinical evaluation of arthritis was performed throughout the study and animals were sacrificed on day 28.

Mechanical Allodynia Evaluation

Paw withdrawal thresholds were determined using the Dynamic Plantar Aesthesiometer (Ugo Basile, Milan, Italy), an apparatus that generates a mechanical force linearly increasing with time [37]. Rats were placed individually in plastic cages with a wire mesh bottom and allowed to acclimatize for at least 2 hours. Increasing mechanical stimulation (2.5 g/s, cut-off force: 50 g) was applied to the plantar surface of a hind paw. The nociceptive threshold was defined as the force, in grams, at which the rat withdraw its paw. When a withdrawal response occurred, the stimulus was terminated and the response threshold electronically measured.

Measurement of Thermal Hyperalgesia

The response to noxious thermal stimulus was determined using a Plantar Test Apparatus (Ugo Basile) [38]. Briefly, animals were placed into a Plexiglas chamber for 2 hours acclimatization period followed by testing. A movable infrared radiant heat source was placed directly under the plantar surface of the hind paw and the time taken for hind paw withdrawal was monitored (withdrawal latency). A cut-off latency of 30 s was imposed to avoid tissue damage and the mean of three values was used for data analysis.

Assessment of Paw Volume

The rat pawvolume was measured before CFA injection and at day 1, 7, 14, 21, and 28 after CFA injection using a plethysmometer (Ugo Basile) [39]. The plethysmometer consists of 2 vertical interconnected water-filled Perspex cells, the larger of which is used to measure volume displacement produced by dipping the rat paw in.

Radiographic Analysis

On day 28, rats were anesthetized and placed on Fuji Medical X-ray and exposed to an X-ray source for 125 ms at 50 kVp, 125 mA (15.6 mA/s). The X-ray film was developed in a one in five dilution of Agfa G150 Developer for 2.5 min and fixed using a one in four dilution of Agfa G354 Fixing bath for 2.5 min. Radiographs were placed in slide mounts and projected [36]. The radiological alterations were arbitrarily graded between 0 and 3 according to the severity of the swelling of the soft tissue around the joints of the hind paws, osteoporosis, periarticular bone erosion and narrowing of the joint space. The radiographic score was: grade 0 =  normal; grade 1 =  mild change compared to normal; grade 2 =  moderate change compared to normal; grade 3 =  marked change compared to normal.

Ultrasonographic Assessment

On days 7, 21 and 28 joint ultrasound (US) assessment was performed by using an Aplio XG (Toshiba, Tokyo, Japan) scanner with 12 MHz linear transducer. The US assessment included transverse and longitudinal scanning of left paw, lateral dorsal view. Synovial power Doppler (PD) was evaluated selecting a region that included bony margins, joint space and a variable view of surrounding tissues such as ankle joint. Moreover, joint effusion, synovitis and bone erosions was assessed by grey-scale scan and PD activity. Pulse repetition frequency was adjusted at the lowest permissible to maximize sensitivity. Color gain was set just below the level that causes the appearance of noise artefacts. A total US score was assigned semiquantitatively (grade 0–3) by a combination of grey-scale scan and PD signals [40], [41]. In particular, effusion/synovitis was graded as: 0 =  absent; 1 =  mild; 2 =  moderate; 3 =  marked. Bone erosion was graded as: 0 =  smooth bone surface; 1 =  irregularities of the bone surface, in the absence of discontinuities displayable on two scanning planes; 2 =  discontinuity shown by two scanning planes; 3 =  marked bone destruction. Power Doppler was graded as: 0 =  normal, absence of flow signals; 1 =  mild, isolated flow signals; 2 =  moderate, vases confluent; 3 =  marked, multiple signals over half of the intra-articular surface.

Western Blotting Analysis and IL-10 Levels

Rats were killed by decapitation 28 days after CFA injection, the spleen was removed and a cell suspension of splenocytes was prepared by using sterilized nylon mesh gauze in the presence of sterile saline. Mononuclear cells were isolated by centrifugation for 15 min at 500×g of the cell suspension through Fycoll-Hypaque [42]. Purified lymphocyte preparation was primarily obtained as described above for human lymphocytes. The cells were lysed in Triton lysis buffer [32] and aliquots of total protein samples (50 µg) were analysed using a specific rat A2AAR antibody (Alpha Diagnostic, San Antonio, TX, USA). Filters were washed and incubated for 1 hour at room temperature with peroxidase-conjugated secondary antibody (1∶2000 dilution). Specific reaction were revealed with enhanced chemiluminescence Western blotting detection reagent (GE Healthcare). Western blotting assays were also normalized against the housekeeping protein GAPDH.

Rat blood were collected, centrifuged at 3000×g for 10 min, and serumwas separated and stored at −20°C until assay.IL-10 levels in rat serum samples were evaluated by using an ELISA kit following manufacturer’s instruction (R&D System).

Data and Statistical Analysis

Dissociation equilibrium constants for saturation binding, affinity or KD values, as well as the maximum densities of specific binding sites, Bmax were calculated for a system of one or two-binding site populations by non-linear curve fitting using the program Ligand purchased from Kell Biosoft [19]. All data are reported as mean ± SD except for radiographic and ultrasonographic scores that are expressed as median and interquartile range. Statistical analysis of the data was performed by repeated measures analysis of variance (ANOVA) followed by Dunnett’s t-test or unpaired two-sided Student’s t-test for comparison of two samples. In the case of radiological or US scoring, Wilcoxon’s nonparametric analysis of variance for comparison of two samples was performed. All analysis were carried out using GraphPad Prism 5.0 statistical software package and differences were considered statistically significant with a p value less than 0.05 (Graph Pad Software, San Diego, CA, USA).

Results

Patients and Control Subjects

All patients enrolled in this study were recruited from the Rheumatology Section, Department of Clinical and Experimental Medicine, University of Ferrara, Italy. A total of 60 patients were included and divided in RA patients treated with MTX (n = 24), with anti TNFα agents (adalimumab or etanercept, n = 12) or with RTX (n = 24). RA patients with established disease fulfilled the American College of Rheumatology (ACR) 1987 criteria for RA [43]. The demographic, clinical and pharmacological details are listed in Table 1. All patients have continued to be regularly monitored during the timeframe of the study. Clinical information about disease characteristics such as disease onset, previous and current therapy, rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (ACPA) presence were reported (Table 1). Disease Activity Score evaluated in 28 joints (DAS 28) and Health Assessment Questionnaire (HAQ) were completed for all patients [44], [45]. The statistical analysis of the clinic parameters such as DAS 28 and HAQ reveals a significant difference between the groups that is considered quite common within a cohort of RA patients. Healthy controls (n = 60), matched for similar age to RA patients, were volunteers from Ferrara University Hospital Blood Bank.

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Table 1. Clinical features and pharmacological treatments in RA patients.

https://doi.org/10.1371/journal.pone.0054195.t001

Binding Parameters of A2AARs in Lymphocytes of RA Patients

Affinity (KD) and density (Bmax) of A2AARs were evaluated in lymphocyte membranes obtained from RA patients at different time points of treatment with MTX, anti-TNFα agents or RTX (Table 2). Before the treatment start, RA patients revealed an up-regulation of A2AAR density and a lower affinity if compared with age matched healthy controls (KD = 1.34±0.85 nM; Bmax = 57±46 fmol/mg protein). In MTX-treated patients, A2AAR affinity and density were found to be significantly different from control subjects in the time points investigated, even if a significant reduction of KD and Bmax values after 24 months of treatment respect to the beginning of the study was observed. In lymphocytes from RA patients treated with anti-TNFα drugs, a statistically significant decrease of A2AAR density was obtained after 9 months of treatment. In the same patients the A2AAR Bmax values were similar to control subjects after 24 months from the beginning of the treatment. Interestingly, RTX-treated patients showed a significant reduction of Bmax and KD after 3 months of treatments reaching values similar to control subjects at the subsequent time points investigated (from 6 to 24 months). Fig. 1A summarises the A2AAR density values obtained in lymphocytes from RA patients treated with MTX, anti-TNFα agents or RTX after 0, 3, 6, 9, 12 and 24 months from the treatment start.

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Figure 1. A2AAR upregulation in RA patients and in vitro stimulation by CGS 21680 increased IL-10 production.

(A) A2AAR Bmax values in lymphocytes from RA patients evaluated at different time points of treatment with MTX, anti-TNFα drugs or RTX. Data are expressed as mean ± SD. *, p<0.01 vs healthy controls (white bar, Bmax = 57±46 fmol/mg protein); #, p<0.01 vs t = 0. (B) Effect of CGS 21680 (1 nM–10 µM) and SCH 58261 (1 µM) on IL-10 production in cultured lymphocytes from untreated RA patients. Data are expressed as mean ± SD. *, p<0.05vs basal; **, p<0.01 vs basal; #, p<0.01 vs CGS 21680 (1 µM).

https://doi.org/10.1371/journal.pone.0054195.g001

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Table 2. A2AAR affinity (KD, nM) and density (Bmax, fmol/mg protein) in lymphocytes from RA patients at different time points of treatment.

https://doi.org/10.1371/journal.pone.0054195.t002

Effect of A2AAR Stimulation on IL-10 Release from Lymphocytes of RA Patients

In cultured lymphocytes obtained from untreated RA patients (n = 30), the A2AAR agonist CGS 21680 was able to induce a dose-dependent significant increase of the anti-inflammatory cytokine IL-10 levels (Fig. 1B). In particular, CGS 21680 at 1 µM and at 10 µM concentration determined a marked release of IL-10 with an increase of 3.3 and 4.0 fold versus basal condition (p<0.01), respectively. The effect of A2AAR stimulation on IL-10 release was completely blocked by the A2AAR selective antagonist, SCH 58261(Fig. 1B). These data strongly support the potential use of A2AAR agonist in RA as anti-inflammatory agent and, consequently, a series of in vivo experiments were performed to test this hypothesis.

CGS 21680 did not Affect Cell Viability and did not have Cytotoxic Effects on Human Lymphocytes

The cell viability was assessed in human lymphocytes obtained from untreated RA patients exposed for 24 hours to different concentrations (1 nM –10 µM) of CGS 21680. The A2AAR agonist did not affect cell viability at all the tested concentrations respect to the control condition. In particular, at the highest concentration tested (10 µM) CGS 21680 showed a mean absorbance value of 0.63±0.12 that was not statistically different from the mean value obtained in control condition (0.59±0.10).

The A2AAR agonist CGS 21680 did not modify LDH release in human lymphocytes at the different tested concentrations (1 nM –10 µM). The mean absorbance value obtained with CGS 21680 at the 10 µM concentration was 0.32±0.06 and it did not differ from control condition (0.35±0.08). These results suggested that the treatment with GCS 21680 was not cytotoxic for human lymphocytes.

The A2AAR Density and DAS 28 Gradually Reduced in RA Patients Treated with RTX

To date the 28 joint disease activity score (DAS 28) represents a validated composite index to assess RA disease activity. Fig. 2A reports the DAS 28 values in RA patients evaluated at different time points of treatment with MTX, anti-TNFα drugs or RTX. To investigate the involvement of A2AARs in RA progression we have studied the association between these receptor subtypes and DAS 28 in function of the time of treatment. Interestingly, in RTX-treated patients the DAS 28 reduction (from 5.57 to 2.11) respect to the time of treatment was closely associated to a significant A2AAR decrease of 3.5 fold after 12 months respect to RA untreated patients (Fig. 2B, Table 2). In particular, the time-dependent relationship (from 0 to 12 months) between A2AARs and DAS 28 in lymphocytes from RA patients treated with RTX revealed a significant reduction of these parameters (Fig. 2B).

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Figure 2. A2AAR Bmax and DAS 28 values gradually reduced in function of the time of treatment.

(A) DAS 28 values in RA patients evaluated at different time points of treatment with MTX, anti-TNFα drugs or RTX. Data are expressed as mean ± SD.*, p<0.01 vs t = 0 months. (B) Time-dependent relationship between A2AAR Bmax and DAS 28 in lymphocytes from RTX treated RA patients.

https://doi.org/10.1371/journal.pone.0054195.g002

Effect of CGS 21680 on CFA-induced Arthritis in Comparison with MTX and Etanercept

Fig. 3A shows representative image ofthe left hind paws of sham and CFA-injected rats on day 28. The injection of CFA evoked a marked paw swelling that peaked within 7 days and was sustained for 28 days. The left hind paw volume of CFA-injected rats increased from 1.28±0.24 ml to 2.65±0.29 ml, when evaluated 7 days after CFA-injection (Fig. 3C). The treatment of CFA-injected rats with the A2AAR agonist CGS 21680 resulted in a statistically significant decrease of paw swelling 14 days after CFA injection compared with untreated arthritic rats. This effect was comparable to those obtained treating the rats with a classic drug as MTX or with a biologic drug as etanercept. Interestingly, after 21 days an higher inhibition of paw swelling was observed in A2AAR agonist-treated rats respect to the rats treated with MTX or etanercept, with paw volume being 40% less than that in untreated CFA-injected rats. This major effect of CGS 21680 was still evident 28 days after CFA injection, with a paw volume not statistically different from sham rats. Fig. 3B shows the radiographic images of the left hind paws on day 28. In particular, CFA-injected rats developed definite joint space narrowing of the intertarsal joints, diffuse soft tissue swelling that included the digits, marked periosteal thickening, cystic enlargement of bone, and extensive erosions. The animal treated with MTX, etanercept or CGS 21680 exhibited a marked decrease of radiographic scores in comparison with untreated rats (Fig. 3D). Interestingly, rats treated with CGS 21680 showed less radiological osteoporosis and less destructive changes than those treated with MTX or etanercept. In addition, the animals that were treated with CGS 21680 maintained good health condition and were not significantly different in body weight from the rats of the sham group (data not shown).

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Figure 3. A significant decrease of paw swelling and radiographic damage in CFA-injected rats treated with CGS 21680 was detected.

Representative photographs (A) and radiographs (B) of left hind paw from sham rats and CFA-injected rats in the absence or in the presence of chronic treatment with etanercept (ETA), methotrexate (MTX) or CGS 21680 at 28 days after CFA-injection. (C) Paw volume measurements of sham rats and CFA-injected rats untreated or treated with ETA, MTX or CGS 21680 at 0, 1, 7, 14, 21 and 28 days after CFA-injection. The results are presented as mean ± SD (n = 6 for each group). *, p<0.05 and **, p<0.01 vs CFA injected rats. (D) Radiographic score (grade 0–3) of the examined rats at 28 days after CFA-injection. The results are presented as median and interquartile range. *, p<0.01 vs sham rats; #, p<0.01 vs CFA-injected rats.

https://doi.org/10.1371/journal.pone.0054195.g003

Ultrasonographic Assessment of Arthritis Progression in CFA-injected Rats

The US assessment showed the persistence of effusion and active synovitis in the CFA-injected rats at all the time points examined in comparison with sham rats. Notably, the animals treated with MTX, etanerceptor CGS 21680 exhibited a gradual reduction of the synovitis (Fig. 4A). The total US score decreased over the study period in rats treated with MTX or etanercept reaching a median value of 1.50 [0.88–2.13] or 1.75 [1.38–2.00] in the evaluation performed 28 days after CFA injection, respectively (Fig. 4B). At the same time point, CGS 21680-treated rats had an overall median score of 0.75 [0.50–1.63], showing a significant reduction as compared with untreated CFA-injected rats (overall US median score: 3.00 [2.38–3.00]) (Fig. 4B). In addition, rats treated with CGS 21680 showed low level US activity (power Doppler, PD ≤1) while rats treated with MTX or etanercept exhibited higher signs of destructive changes and erosion in comparison with rats treated with CGS 21680.

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Figure 4. A reduction of the synovitis was present in CFA-injected rats after chronic treatment with CGS 21680.

(A) Representative ultrasonographic (US) pictures of left hind paw andlongitudinal lateral view from sham rats and CFA-injected rats in the absence or in the presence of chronic treatment with etanercept (ETA), methotrexate (MTX) or CGS 21680 at 7, 21 and 28 days after CFA-injection. Total US score of the examined rats 28 days after CFA-injection. (B) Grey-scale and power doppler signals were assigned semiquantitatively and summarized in a total US score (grade 0–3). The results are presented as median and interquartile range (n = 6 for each group). *, p<0.01 vs sham rats; #, p<0.01 vs CFA-injected rats.

https://doi.org/10.1371/journal.pone.0054195.g004

Pain Responses in CFA-injected Rats After Different Pharmacological Treatments

The CFA-induced arthritic rat model is an extensively used laboratory model for the study of arthritic pain since the animals exhibit mechanical allodynia, thermal hyperalgesia and pain on joint movement which are the prominent features of arthritic pain in man [38]. In the present study, CFA-injected rats showed a significant reduction in paw withdrawal threshold compared to sham rats from 1 to 28 days after CFA injection (Fig. 5). In mechanical allodynia, the rats treated with MTX or etanercept (2 mg/kg or 5 mg/kg, respectively) increased the withdrawal threshold compared to CFA-injected rats from 14 to 28 days after arthritis induction. CGS 21680 treatment significantly increased (p<0.01) withdrawal threshold respect to CFA-injected rats showing a higher effect than MTX or etanercept on day 21 (Fig. 5A). Similarly, the treatment with MTX, etanercept or CGS 21680 decreased thermal hyperalgesia as compared to CFA control group (Fig. 5B). Interestingly, the anti-allodynic and anti-hyperalgesic effects of CGS 21680 confirmed the involvement of A2AAR stimulation in the reduction of arthritis-associated pain.

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Figure 5. The treatment with CGS 21680 decreased the mechanical allodynia and thermal hyperalgesia in CFA-injected rats.

Paw withdrawal threshold in mechanical allodynia (A) and in thermal hyperalgesia (B) in sham rats and CFA-injected rats in the absence or in the presence of chronic treatment with etanercept (ETA), methotrexate (MTX) or CGS 21680 at 0, 1, 7, 14, 21 and 28 days after CFA-injection. The results are presented as mean ± SD (n = 6 for each group). *, p<0.05 and **, p<0.01 vs CFA-injected rats.

https://doi.org/10.1371/journal.pone.0054195.g005

A2AAR Expression in Lymphocytes and IL-10 Release in Serum from CFA-injected Rats

In lymphocytes from CFA-injected rats, Western blotting (Fig. 6A) and densitometric analysis (Fig. 6B) performed 28 days after CFA injection indicated a significant increase in A2AAR expression in comparison with sham rats. The treatment with MTX produced a significant reduction of A2AAR up-regulation although minor than those obtained after etanercept or CGS 21680 treatment. Moreover, IL-10 levels were measured in serum samples from sham and CFA-injected rats as reported in Fig. 6C. Reduced levels of IL-10 in CFA control group respect to sham rats were observed. In CFA-injected rats chronic treatment with CGS 21680 was able to restore IL-10 levels whereas MTX or etanercept had a weaker effect.

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Figure 6. A2AARs were up-regulated in lymphocytes from CFA-injected rats and were able to modulate IL-10 levels.

Western blotting (A) and densitometric analysis (B) of A2AARs in CFA-injected rats untreated or treated with etanercept (ETA), methotrexate (MTX) or CGS 21680 in comparison with sham rats. (C) ETA, MTX or CGS 21680 chronic treatment elicited an increase of IL-10 levels in serum of CFA-injected rats.The results are presented as mean ± SD (n = 6 for each group).*, p<0.01 vs sham rats; #, p<0.01 vs CFA-injected rats.

https://doi.org/10.1371/journal.pone.0054195.g006

Discussion

To date no clinical trials are present evaluating the effects of A2AAR agonists in RA patients since more preclinical studies are needed to better elucidate their pharmacological properties. RA treatment has progressed for the advent of biologic DMARDs even if the risks of infection, infusion-associated reactions or malignancy together to the high costs of these treatments could limit their clinical use [5].

In the present study, we have investigated the involvement of A2AARs in RA by evaluating their affinity and density in lymphocytes from RA patients at different time points after the treatment with classic or biologic DMARDs. We have found an up-regulation of A2AARs in untreated RA patients that was gradually reduced in function of the treatment time and in different ways depending on the type of drug used. These results complete our previous evidence regarding A2A and A3ARs in blood cells from early RA and RA patients where an alteration of these receptors was found [31], [32] and highlight important information on the effect of biological treatments. The longitudinal study (from 0 to 24 months) performed for the first time in this paper aimed to assess the time-dependent changes of A2AAR expression in differentially treated RA patients. In MTX-treated RA patients A2AARs were present in high levels at all the time points of treatment whilst in anti-TNFα-treated RA patients A2AAR density normalized to control values after 24 months of treatment. RA patients treated with RTX exhibited A2AAR binding characteristics similar to control values after 9 and until 24 months of treatment. It is tempting to speculate that the different behavior of examined drugs in RA patients on A2AAR density could be correlated with the several mechanisms of action involving the inflammatory process regulated by these heterogeneous therapies. DAS 28 values progressively reduced in function of the time of treatment and in different way respect to the type of drugs used with a significant reduction after 3 months of RTX treatment. Moreover, the time-dependent relationship between A2AARs and DAS 28 in lymphocytes from RTX treated RA patients strongly suggested the involvement of A2AARs in RA progression. Interestingly, high levels of DAS 28 indicating the presence of a marked inflammatory status are accompanied by an increased A2AAR density. These data suggest that A2AAR expression is sensibly affected by inflammation and tend to normalize with the disease remission. Thus, A2AARs could be an useful tool to monitor RA progression following conventional or biologic therapies. On the other hand, A2AAR upregulation could be interpreted as a possible compensatory mechanism that is needed to opposite the inflammatory status. For this reason we have investigated the effect of an A2AAR agonist CGS 21680 on the production of the anti-inflammatory cytokine IL-10 in lymphocytes from RA patients. IL-10, a major immunoregulatory cytokine, is mainly produced by lymphocytes or macrophages and plays an important role in RA pathogenesis mediating the down-regulation of the inflammatory response. In particular, it has been demonstrated that IL-10 suppresses joint swelling and deformation as well as necrosis of cartilage in RA animal models [46]. We have found that CGS 21680 mediates a significant increase in IL-10 levels in cultured lymphocytes from RA patients confirming the link between the use of A2AAR agonist and the reduction of the inflammation. Moreover, CGS 21680 did not reduce cell viability and was not able to exert cytotoxic effects in human lymphocytes.

Based on these relevant results we have investigated the effect of A2AAR stimulation in a model of experimental arthritis represented by CFA-induced arthritic rats. Interestingly, CGS 21680 treatment was able to reduce the severity of arthritis being at least as efficacious as MTX or etanercept. In particular, CGS 21680 strongly decreased the clinical signs of arthritis induced by CFA injection in rats as demonstrated by radiological and US evaluation. It is well reported that the latter technique is more accurate than standard clinical examination at detecting synovitis in human RA [47], [48]. In this study we have applied for the first time, to our knowledge, the US evaluation in the adjuvant-induced arthritis model showing a marked reduction of total US score in rats treated with CGS 21680. The data from US assessment are in agreement with radiographic analysis suggesting the relevance of these technical approaches for the examination of arthritis damage in animal models.

It is well reported that enhanced pain perception is common among patients with RA [49]. The identification of novel pharmacological strategy that reduce the severity of the disease, could contribute to decrease the associated pain, improving general clinical and functional outcomes. In CFA-induced arthritis, A2AAR stimulation was able to decrease arthritis-associated pain as observed by the threshold increase in mechanical allodynia and in thermal hyperalgesia. These results demonstrated the effectiveness of CGS 21680 in adjuvant-induced model of arthritis in rats and are in agreement with those found using different experimental approaches in collagen-induced arthritis (CIA) in mice [25]. Recently, a phosphorylated A2AAR agonist was demonstrated to be a potent immunosuppressant in a model of arthritis acting by an up-regulation of CD73 and A2AAR expression [50].

To complete our study, we have verified in CFA-injected rats an A2AAR up-regulation similar to that found in RA patients confirming the relevance of this animal model to study the involvement of A2AARs in the disease. Moreover, the anti-inflammatory effect of CGS 21680 was also demonstrated by the increase of IL-10 levels in serum from CFA-injected rats reflecting the data reported in lymphocytes from RA patients. This experimental evidence confirmed previous results obtained in human chondrocytes from RA patients where the stimulation of A2AARs increased IL-10 release [24]. In addition, in a murine calvaria model of wear particle induced bone resorption, the treatment with CGS 21680 mediated a decrease of pro-inflammatory cytokines secretion, whereas IL-10 was markedly increased in bone [51]. These in vivo evidence suggested that the beneficial effect of CGS 21680 in experimental arthritis could be due, at least in part, to the increase of IL-10 which counterbalance the overexpression of inflammatory mediators. It is worth noting that the reduction of the inflammation could underlie the normalization of A2AARs in lymphocytes from arthritic rats after CGS 21680 chronic treatment.

In conclusion, we have demonstrated the involvement of A2AARs in RA pathogenesis based on the modulation of their expression in function of the time after different pharmacological treatments. Moreover, our in vitro and in vivo results highlight the role of A2AAR signalling as a protective anti-inflammatory system making A2AAR agonists an ideal and more physiological-like therapeutic alternative for the treatment of chronic autoimmune joint diseases such as RA. As a matter of fact, A2AAR agonists, mimicking an endogenous protective system, could have less limitation and side effects than DMARDs or biologic drugs although with a comparable effectiveness. These experimental results support the role of A2AAR as therapeutic target and strongly suggest the potential use of novel pharmacological approaches based on A2AAR stimulation in RA treatment.

Author Contributions

Conceived and designed the experiments: FV MG PAB KV. Performed the experiments: MP MT CC SG SM SG. Analyzed the data: FV MP KV. Contributed reagents/materials/analysis tools: FV MP MT CC SG. Wrote the paper: FV MG PAB KV.

References

  1. 1. McInnes IB, Schett G (2011) The pathogenesis of rheumatoid arthritis. N Engl J Med 365: 2205–2219.
  2. 2. Joseph A, Brasington R, Kahl L, Ranganathan P, Cheng TP, et al. (2010) Immunologic rheumatic disorders. J Allergy Clin Immunol 125: 204–215.
  3. 3. Belavic JM (2010) Annual Drug Update 2010 in review. Nurse Pract 36: 10–22.
  4. 4. Varani K, Padovan M, Govoni M, Vincenzi F, Trotta F, et al. (2010) The role of adenosine receptors in rheumatoid arthritis. Autoimmun Rev 10: 61–64.
  5. 5. Emery P, Dörner T (2011) Optimising treatment in rheumatoid arthritis: a review of potential biological markers of response. Ann Rheum Dis 70: 2063–2070.
  6. 6. MacKay K, Milicic A, Lee D, Tikly M, Laval S, et al. (2003) Rheumatoid arthritis susceptibility and interleukin 10: a study of two ethnically diverse populations. Rheumatology(Oxford) 42: 149–153.
  7. 7. Shaver TS, Anderson JD, Weidensaul DN, Shadhouri SS, Busch RE, et al. (2008) The problem of rheumatoid arthritis disease activity and remission in clinical practice. J Rheumatol 35: 1015–1022.
  8. 8. Cronstein BN (2010) How does methotrexate suppress inflammation? Clin Exp Rheumatol 28: 21–23.
  9. 9. Ishaq M, Muhammad JS, Hameed K, Mirza AI (2011) Leflunomide or methotrexate? Comparison of clinical efficacy and safety in low socio-economic rheumatoid arthritis patients. Mod Rheumatol 21: 375–380.
  10. 10. Furst DE, Keystone EC, Fleischmann R, Breedveld FC, Burmester GR, et al. (2012) Updated consensus statement on biological agents for the treatment of rheumatic diseases. Ann Rheum Dis 71: i2–45.
  11. 11. Scott DL (2012) Biologics-based therapy for the treatment of rheumatoid arthritis. Clin Pharmacol Ther 91: 30–43.
  12. 12. Dörner T, Kinnan N, Tak P (2010) Targeting B cells in immune-mediated inflammatory disease: a comprehensive review of mechanisms of action and identification of biomarkers. Pharmacol & Therapeutics 125: 464–475.
  13. 13. Singh JA, Wells GA, Christensen R, Tanjong Ghogomu E, Maxwell L, et al. (2011) Adverse effects of biologics: a network meta-analysis and Cochrane overview. Cochrane Database Syst Rev 2: CD008794.
  14. 14. Fredholm BB, IJzerman AP, Jacobson KA, Linden J, Müller CE (2011) International Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature and classification of adenosine receptors -an update. Pharmacol Rev 63: 1–34.
  15. 15. Gessi S, Merighi S, Fazzi D, Stefanelli A, Varani K, et al. (2011) Adenosine receptor targeting in health and disease. Expert Opin Investig Drugs 20: 1591–1609.
  16. 16. Forrest CM, Harman G, McMillan RB, Stoy N, Stone TW, et al. (2005) Modulation of cytokine release by purine receptors in patients with rheumatoid arthritis. Clin Exp Rheumatol 23: 89–92.
  17. 17. Moore CC, Martin EN, Lee GH, Obrig T, Linden J, et al. (2008) An A2A adenosine receptor agonist, ATL313, reduces inflammation and improves survival in murine sepsis models. BMC Infect Dis 8: 141.
  18. 18. Nowak M, Lynch L, Yue S, Ohta A, Sitkovsky M, et al. (2010) The A2A adenosine receptor controls cytokine production in iNKT cells. Eur J Immunol 40: 682–687.
  19. 19. Varani K, Vincenzi F, Tosi A, Targa M, Masieri FF, et al. (2010) Expression and functional role of adenosine receptors in regulating inflammatory responses in human synoviocytes. Br J Pharmacol 160: 101–115.
  20. 20. Ongaro A, Varani K, Masieri FF, Pellati A, Massari L, et al. (2012) Electromagnetic fields (EMFs) and adenosine receptors modulate prostaglandin E(2) and cytokine release in human osteoarthritic synovial fibroblasts regulating inflammatory responses in human synoviocytes. J Cell Physiol 227: 2461–2469.
  21. 21. Milne GR, Palmer TM (2011) Anti-inflammatory and immunosuppressive effects of the A2A adenosine receptor. Scientific World Journal 11: 320–339.
  22. 22. Yang T, Wang Z, Wu F, Tan J, Shen Y, et al. (2010) A variant of TNFR2-Fc fusion protein exhibits improved efficacy in treating experimental rheumatoid arthritis. PLOS computational biology 6: 1–7.
  23. 23. Kollias G, Papadaki P, Apparailly F, Vervoordeldonk MJ, Holmdahl R, et al. (2011) Animal models for arthritis: innovative tools for prevention and treatment. Ann Rheum Dis 70: 1357–1362.
  24. 24. Bitto A, Polito F, Irrera N, D’Ascola A, Avenoso A, et al. (2011) Polydeoxyribonucleotide reduces cytokine production and the severity of collagen-induced arthritis by stimulation of adenosine A2A receptor. Arthritis Rheum 63: 3364–3371.
  25. 25. Mazzon E, Esposito E, Impellizzeri D, Di Paola R, Melani A, et al. (2011) CGS 21680, an agonist of the adenosine A2A receptor, reduces progression of murine type II collagen-induced arthritis. J Rheumatol 38: 2119–2129.
  26. 26. Bush KA, Kirkham BW, Walker JS (2002) The in vivo effects of tumor necrosis factor blockade on the early cell mediated immune events and syndrome expression in rat adjuvant arthritis. ClinExpImmunol 127: 423–429.
  27. 27. Hegen M, Keith JC Jr, Collins M, Nickerson-Nutter CL (2008) Utility of animal models for identification of potential therapeutics for rheumatoid arthritis. Ann Rheum Dis 67: 1505–1515.
  28. 28. Gao ZG, Jacobson KA (2007) Emerging adenosine receptor agonists. Expert Opin Emerg Drugs 12: 479–492.
  29. 29. Valls MD, Cronstein BN, Montesinos MC (2009) Adenosine receptor agonists for promotion of dermal wound healing. Biochem Pharmacol 77: 1117–1124.
  30. 30. Manera C, Saccomanni G (2010) A2A receptor ligands: past, present and future trends. Curr Top Med Chem 10: 902–922.
  31. 31. Varani K, Massara A, Vincenzi F, Tosi A, Padovan M, et al. (2009) Normalization of A2A and A3 adenosine receptor up-regulation in rheumatoid arthritis patients by treatment with anti-tumor necrosis factor α but not methotrexate. Arthritis Rheum 60: 2880–2891.
  32. 32. Varani K, Padovan M, Vincenzi F, Targa M, Trotta F, et al. (2011) A2A and A3 adenosine receptor expression in rheumatoid arthritis: upregulation, inverse correlation with disease activity score and suppression of inflammatory cytokine and metalloproteinase release. Arthritis Res Ther13: R197.
  33. 33. Shi M, Wang A, Prescott D, Waterhouse CC, Zhang S, et al. (2011) Infection with an intestinal helminth parasite reduces Freund’s complete adjuvant-induced monoarthritis in mice. Arthritis Rheum 63: 434–444.
  34. 34. Himer L, Csóka B, Selmeczy Z, Koscsó B, Pócza T, et al. (2010) Adenosine A2A receptor activation protects CD4+ T lymphocytes against activation-induced cell death. FASEB J 24: 2631–2640.
  35. 35. Vincenzi F, Targa M, Corciulo C, Gessi S, Merighi S, et al. (2012) The anti-tumor effect of A3 adenosine receptors is potentiated by pulsed electromagnetic fields in cultured neural cancer cells. PLoS One 7: e39317.
  36. 36. Almarestani L, Fitzcharles MA, Bennett GJ, Ribeiro-da-Silva A (2011) Imaging studies in Freund’s complete adjuvant model of regional polyarthritis, a model suitablefor the study of pain mechanisms, in the rat. Arthritis Rheum 63: 1573–1581.
  37. 37. Wright DE, Jonson MS, Arnett MG, Smittkamp SE, Ryals JM (2007) Selective changes in nocifensive behavior despite normal cutaneous axon innervations in leptin receptor-null mutant (db/db) mice. J Peripher Nerv Syst 12: 250–261.
  38. 38. Kaur S, Bijjem KR, Sharma PL (2011) Anti-inflammatory and antihyperalgesic effects of the combination of ibuprofen and hemin in adjuvant-induced arthritis in the Wistar rat. Inflammopharmacology 19: 265–272.
  39. 39. Patel HB, Bombardieri M, Sampaio AL, D’Acquisto F, Gray M, et al. (2010) Anti-inflammatory and antiosteoclastogenesis properties of endogenous melanocortin receptor type 3 in experimental arthritis. FASEB J 24: 4835–4843.
  40. 40. Backhaus M, Burmester GR, Gerber T, Grassi W, Machold KP, et al. (2001) Working Group for Musculoskeletal Ultrasound in the EULAR Standing Committee on International Clinical Studies including Therapeutic Trials, Guidelines for musculoskeletal ultrasound in rheumatology. Ann Rheum Dis 60: 641–649.
  41. 41. Meenagh G, Filippucci E, Delle Sedie A, Riente L, Iagnocco A, et al. (2008) Ultrasound imaging for the rheumatologist. XVIII. Ultrasound measurements. Clin Exp Rheumatol 26 982–985.
  42. 42. Sakowicz-Burkiewicz M, Kocbuch K, Grden M, Szutowicz A, Pawelczyk T (2010) Regulation of adenosine receptors expression in rat B lymphocytes by insulin. J Cell Biochem 109: 396–405.
  43. 43. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, et al. (1988) The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 31: 315–324.
  44. 44. Prevoo ML, van’t Hof MA, Kuper HH, van Leeuwen MA, van de Putte LB, et al. (1995) Modifieddisease activity scores that include twenty eight-joint counts. Development and validation in aprospective longitudinal study of patients with rheumatoid arthritis. Arthritis Rheum 38: 44–48.
  45. 45. Soubrier M, Lukas C, Sibilia J, Fautrel B, Roux F, et al. (2011) Disease activity score-driven therapy versus routine care in patients with recent-onset active rheumatoid arthritis: data from the GUEPARD trial and ESPOIR cohort. Ann Rheum Dis 70: 611–615.
  46. 46. Zhang J, Zhang Y, Jin J, Li M, Xie K, et al. (2011) The 1082A/G polymorphism in the Interleukin-10 gene and the risk of rheumatoid arthritis: a meta-analysis. Cytokine 56: 351–355.
  47. 47. Naredo E, Bonilla G, Gamero F, Uson J, Carmona L, et al. (2005) Assessment of inflammatory activity in rheumatoid arthritis: a comparative study of clinical evaluation with grey scale and power Doppler ultrasonography. Ann Rheum Dis 64: 375–381.
  48. 48. Naredo E, Collado P, Cruz A, Palop MJ, Cabero F, et al. (2007) Longitudinal power Doppler ultrasonographic assessment of joint inflammatoryactivity in early rheumatoid arthritis: predictive value in disease activity and radiologic progression. Arthritis Rheum 57: 116–124.
  49. 49. Wood PB (2009) Enhanced pain perception in rheumatoid arthritis: novel considerations. Curr Pain Headache Rep 13: 434–439.
  50. 50. Flögel U, Burghoff S, van Lent PL, Temme S, Galbarz SL, et al. (2012) Selective activation of adenosine A2A receptors on immune cells by a CD73-dependent prodrug suppresses joint inflammation in experimental rheumatoid arthritis. Sci Transl Med 4: 146ra108.
  51. 51. Mediero A, Frenkel SR, Wilder T, He W, Mazumder A, et al. (2012) Adenosine A2A receptor activation prevents wear particle-induced osteolysis. Sci Transl Med 4: 135ra65.