A(2A) adenosine receptors are differentially modulated by pharmacological treatments in rheumatoid arthritis patients and their stimulation ameliorates adjuvant-induced arthritis in rats

Abstract

A(2A) 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 A(2A)ARs in rheumatoid arthritis (RA) patients after different pharmacological treatments and to investigate the effect of A(2A)AR stimulation in a rat model of arthritis. We investigated A(2A)AR 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. A(2A)ARs 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 A(2A)AR 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 A(2A)AR stimulation in lymphocytes from RA patients and in serum from arthritic rats was measured. In lymphocytes obtained from RA patients, the A(2A)AR 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 A(2A)AR 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 A(2A)AR agonists represent a physiological-like therapeutic alternative for RA treatment as suggested by the anti-inflammatory role of A(2A)ARs in lymphocytes from RA patients. The effectiveness of A(2A)AR stimulation in a rat model of arthritis supported the role of A(2A)AR agonists as potential pharmacological treatment for RA.

Figure 1. A_2AAR upregulation in RA patients and in vitro stimulation by CGS 21680 increased IL-10 production.

(A) A_2AAR 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).

Figure 2. A_2AAR 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 A_2AAR Bmax and DAS 28 in lymphocytes from RTX treated RA patients.

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.

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.

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.

Figure 6. A_2AARs 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 A_2AARs 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.