Role and Function of A2A and A₃ Adenosine Receptors in Patients with Ankylosing Spondylitis, Psoriatic Arthritis and Rheumatoid Arthritis

Abstract

Rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA) are chronic inflammatory rheumatic diseases that affect joints, causing debilitating pain and disability. Adenosine receptors (ARs) play a key role in the mechanism of inflammation, and the activation of A_2A and A₃AR subtypes is often associated with a reduction of the inflammatory status. The aim of this study was to investigate the involvement of ARs in patients suffering from early-RA (ERA), RA, AS and PsA. Messenger RNA (mRNA) analysis and saturation binding experiments indicated an upregulation of A_2A and A₃ARs in lymphocytes obtained from patients when compared with healthy subjects. A_2A and A₃AR agonists inhibited nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activation and reduced inflammatory cytokines release, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6. Moreover, A_2A and A₃AR activation mediated a reduction of metalloproteinases (MMP)-1 and MMP-3. The effect of the agonists was abrogated by selective antagonists demonstrating the direct involvement of these receptor subtypes. Taken together, these data confirmed the involvement of ARs in chronic autoimmune rheumatic diseases highlighting the possibility to exploit A_2A and A₃ARs as therapeutic targets, with the aim to limit the inflammatory responses usually associated with RA, AS and PsA.

Keywords: adenosine receptors; ankylosing spondylitis; inflammation; psoriatic arthritis; rheumatoid arthritis.

Figure 1

Adenosine receptors (ARs) A_2A and A₃ARs are upregulated in patients’ lymphocytes with chronic inflammatory rheumatic diseases. (A) Relative ARs messenger RNA (mRNA) levels determined by real-time polymerase chain reaction (RT-PCR) in human lymphocytes from ERA (n = 26), RA (n = 30), AS (n = 18), PsA patients (n = 8) and control subjects (n = 80); (B) Affinity of A₁, A_2A, A_2B, and A₃ARs expressed as K_D values, in lymphocytes derived from ERA (n = 26), RA (n = 30), AS (n = 18) and PsA patients (n = 8) in comparison to control subjects (n = 80); (C) Density of A₁, A_2A, A_2B, and A₃ARs, expressed as the maximum specific binding (Bmax), in lymphocytes derived from ERA (n = 26), RA (n = 30), AS (n = 18) and PsA patients (n = 8) in comparison to control subjects (n = 80). Data are expressed as the mean ± SEM. * p < 0.01 vs. control group.

Figure 2

Saturation binding experiments in lymphocyte membranes from patients with chronic inflammatory rheumatic diseases. Saturation curves (A,C) and Scatchard plots (B,D) showing the binding of [³H]-ZM 241385 to A_2AARs (A,B) and the binding of [³H]-MRE 3008F20 to A₃ARs (C,D) in lymphocyte membranes derived from 80 controls, 26 ERA patients, 30 RA patients, 18 AS patients and 8 PsA patients. Data are expressed as the mean ± SEM.

Figure 3

Increased potency of A_2A and A₃AR agonists in patients’ lymphocytes with ERA, RA, AS and PsA diseases compared to control subjects. Concentration-response curves of CGS 21680 (A) or Cl-IB-MECA (B) on cyclic adenosine monophosphate (cAMP) assays in lymphocytes obtained from control subjects (n = 80), ERA (n = 26), RA (n = 30), AS (n = 18) and PsA patients (n = 8). Data are expressed as the mean ± SEM. * p < 0.01 vs. control group.

Figure 4

A_2A and A₃AR stimulation reduced nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activation and tumor necrosis factor-α (TNF-α) release. Effect of the A_2AAR agonist CGS 21680 (100 nM) and A₃AR agonist Cl-IB-MECA (100 nM) on NF-κB p65 subunit activation (A) or TNF-α release (B) in cultured lymphocytes from ERA (n = 26), RA (n = 30), AS (n = 18) and PsA patients (n = 8) in comparison to control subjects (n = 80). The A_2AAR antagonist SCH 442416 (1 μM) and the A₃AR antagonist MRS 1334 (1 μM) abrogated the effect of the agonists. Data are expressed as the mean ± SEM. * p < 0.01 vs. untreated cells (A); * p < 0.01 vs. phorbol myristate acetate (PMA)-stimulated cells (B).

Figure 5

A_2A and A₃AR activation reduced interleukin (IL)-1β and IL-6 release. Effect of the A_2AAR agonist CGS 21680 (100 nM) and A₃AR agonist Cl-IB-MECA (100 nM) on IL-1β (A) and IL-6 (B) release in cultured lymphocytes from ERA (n = 26), RA (n = 30), AS (n = 18) and PsA patients (n = 8) in comparison to control subjects (n = 80). The A_2AAR antagonist SCH 442416 (1 μM) and the A₃AR antagonist MRS 1334 (1 μM) blocked the effect of the agonists. Data are expressed as the mean ± SEM. * p < 0.01 vs. PMA-stimulated cells.

Figure 6

A_2A and A₃AR activation reduced matrix metalloproteinases MMP-1 and MMP-3 production. Effect of the A_2AAR agonist CGS 21680 (100 nM) and of the A₃AR agonist Cl-IB-MECA (100 nM) on MMP-1 (A) or MMP-3 (B) production in cultured monocytes from ERA (n = 26), RA (n = 30), AS (n = 18) and PsA patients (n = 8) in comparison to control subjects (n = 80). The A_2AAR antagonist SCH 442416 (1 μM) and the A₃AR antagonist MRS 1334 (1 μM) blocked the effect of the agonists. Data are expressed as the mean ± SEM. * p < 0.01 vs. PMA-stimulated cells.