A2A and A3 adenosine receptor expression in rheumatoid arthritis: upregulation, inverse correlation with disease activity score and suppression of inflammatory cytokine and metalloproteinase release

Abstract

Introduction: The reduction of the inflammatory status represents one of the most important targets in rheumatoid arthritis (RA). A central role of A2A and A3 adenosine receptors (ARs) in mechanisms of inflammation has been reported in different pathologies. The primary aim of this study was to investigate the A2A and A3ARs and their involvement in RA progression measured by Disease Activity Score in 28 or 44 joints (DAS28 or DAS).

Methods: ARs were analyzed by saturation binding assays, mRNA and Western blotting analysis in lymphocytes from early and established RA patients. The effect of A2A and A3AR agonists in nuclear factor kB (NF-kB) pathway was evaluated. Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) release was carried out by A2A and A3AR activation. AR pharmacological regulation in matrix metalloproteinase-1 (MMP-1) and metalloproteinase-3 (MMP-3) release was also studied.

Results: In lymphocytes obtained from RA patients, A2A and A3ARs were up-regulated if compared with healthy controls. A2A and A3AR activation inhibited the NF-kB pathway and diminished inflammatory cytokines such as TNF-α, IL-1β and IL-6. A2A and A3AR agonists mediated a reduction of MMP-1 and MMP-3 release. A2A and A3AR density inversely correlated with DAS28 and DAS suggesting a direct role of the endogenous activation of these receptors in the control of RA joint inflammation.

Conclusions: Taken together these data demonstrate that the inflammatory and clinical responses in RA are regulated by A2A and A3ARs and support the use of A2A and/or A3AR agonists as novel and effective pharmacological treatment in RA patients.

Figure 1

A₁ A_2A A_2B and A₃ARs in RA patients. A) Relative AR mRNA levels determined by RT-PCR in human lymphocytes from ERA (n = 32), RA patients (n = 63) and control subjects (n = 90). B) Representative Western blotting analysis shows immunoblot signals of ARs in ERA, RA patients and controls. C) Densitometric analysis of AR expression in human lymphocytes from ERA (n = 32), RA patients (n = 63) and control subjects (n = 90) indicated as a ratio of β-actin (loading control). Data are expressed as a means ± SEM. *, P < 0.01 vs control group.

Figure 2

Saturation binding experiments of A_2A and A₃ARs in RA patients. Saturation curves (left) and Scatchard plots (right) showing the binding of (³H)-ZM 241385 to A_2AARs (A and B) as well as the binding of (³H)-MRE 3008F20 to A₃ARs (C and D) in lymphocyte membranes derived from 90 healthy controls (•), 32 ERA patients (■), 63 RA patients (▲). Saturation binding experiments were performed as described in the online supplementary material and the data are reported in Table 2.

Figure 3

Effect of A_2A and A₃AR stimulation in NF-kB, TNF-α, IL-1β, IL-6, MMP-1 and MMP-3. Effect of a well-known A_2AAR agonist and antagonist (CGS 21680, 100 nM; SCH 442416, 1 μM) or A₃AR agonist and antagonist (Cl-IB-MECA, 100 nM; MRS 1334, 1 μM) in cultured lymphocytes of ERA (n = 30), RA patients (n = 30) and healthy subjects (n = 30) on: NF-kB activation (A) which was evaluated by detecting phosphorylated p65 proteins in nuclear extracts. The effect of the same compounds was also established in TNF-α release in control conditions and stimulated by PMA 5 ng/ml (B) and in IL-1β (C) and IL-6 levels (D). The effect of the same compounds in monocytes from ERA, RA patients and healthy subjects in MMP-1 (E) and MMP-3 (F) activation was investigated. Functional experiments were carried out as described in the online supplementary material. Values are the mean and SEM. *, P < 0.01 versus controls (A); *, P < 0.01 versus PMA-treated cells (B-F).

Figure 4

Correlation between DAS28 or DAS and A_2A or A₃AR density. Linear regression analysis between DAS28 or DAS and the receptor density expressed as Bmax (fmol/mg protein) of A_2A (A, C) and A₃ARs (B, D) in lymphocytes from 32 ERA and 63 RA patients considered as a whole.