IntroductionThe 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).MethodsARs 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.ResultsIn 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.ConclusionsTaken 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.

Adenosine receptors (ARs) have an important role in the regulation of inflammation and their activation is involved in the inhibition of pro-inflammatory cytokine release. The effects of pulsed electromagnetic fields (PEMFs) on inflammation have been reported and we have demonstrated that PEMFs increased A2A and A3AR density and functionality in different cell lines. Chondrocytes and osteoblasts are two key cell types in the skeletal system that play important role in cartilage and bone metabolism representing an interesting target to study the effect of PEMFs. The primary aim of the present study was to evaluate if PEMF exposure potentiated the anti-inflammatory effect of A2A and/or A3ARs in T/C-28a2 chondrocytes and hFOB 1.19 osteoblasts. Immunofluorescence, mRNA analysis and saturation binding assays revealed that PEMF exposure up-regulated A2A and A3AR expression. A2A and A3ARs were able to modulate cAMP production and cell proliferation. The activation of A2A and A3ARs resulted in the decrease of some of the most relevant pro-inflammatory cytokine release such as interleukin (IL)-6 and IL-8, following the treatment with IL-1β as an inflammatory stimuli. In human chondrocyte and osteoblast cell lines, the inhibitory effect of A2A and A3AR stimulation on the release of prostaglandin E2 (PGE2), an important lipid inflammatory mediator, was observed. In addition, in T/C-28a2 cells, the activation of A2A or A3ARs elicited an inhibition of vascular endothelial growth factor (VEGF) secretion. In hFOB 1.19 osteoblasts, PEMF exposure determined an increase of osteoprotegerin (OPG) production. The effect of the A2A or A3AR agonists in the examined cells was enhanced in the presence of PEMFs and completely blocked by using well-known selective antagonists. These results demonstrated that PEMF exposure significantly increase the anti-inflammatory effect of A2A or A3ARs suggesting their potential therapeutic use in the therapy of inflammatory bone and joint disorders.

Background and purpose:Adenosine is an endogenous modulator, interacting with four G-protein coupled receptors (A1, A2A, A2B and A3) and acts as a potent inhibitor of inflammatory processes in several tissues. So far, the functional effects modulated by adenosine receptors on human synoviocytes have not been investigated in detail. We evaluated mRNA, the protein levels, the functional role of adenosine receptors and their pharmacological modulation in human synoviocytes. Experimental approach: mRNA, Western blotting, saturation and competition binding experiments, cyclic AMP, p38 mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-kB activation, tumour necrosis factor a (TNF-a) and interleukin-8 (IL-8) release were assessed in human synoviocytes isolated from patients with osteoarthritis. Key results: mRNA and protein for A1, A2A, A2B and A3 adenosine receptors are expressed in human synoviocytes. Standard adenosine agonists and antagonists showed affinity values in the nanomolar range and were coupled to stimulation or inhibition of adenylyl cyclase. Activation of A2A and A3 adenosine receptors inhibited p38 MAPK and NF-kB pathways, an effect abolished by selective adenosine antagonists. A2A and A3 receptor agonists decreased TNF-a and IL-8 production. The phosphoinositide 3-kinase or Gs pathways were involved in the functional responses of A3 or A2A adenosine receptors. Synoviocyte A1 and A2B adenosine receptors were not implicated in the inflammatory process whereas stimulation of A2A and A3 adenosine receptors was closely associated with a down-regulation of the inflammatory status. Conclusions and implications:These results indicate that A2A and A3 adenosine receptors may represent a potential target in therapeutic modulation of joint inflammation.

Cannabinoid CB(2) receptor activation by selective agonists has been shown to produce analgesic effects in preclinical models of inflammatory, neuropathic, and bone cancer pain. In this study the effect of a novel CB(2)agonist (MT178) was evaluated in different animal models of pain. First of all, in vitro competition binding experiments performed on rat, mouse, or human CB receptors revealed a high affinity, selectivity, and potency of MT178. The analgesic properties of the novel CB(2) agonist were evaluated in various in vivo experiments, such as writhing and formalin assays, showing a good efficacy comparable with that produced by the nonselective CB agonist WIN 55,212-2. A dose-dependent antiallodynic effect of the novel CB(2) compound in the streptozotocin-induced diabetic neuropathy was found. In a bone cancer pain model and in the acid-induced muscle pain model, MT178 was able to significantly reduce mechanical hyperalgesia in a dose-related manner. Notably, MT178 failed to provoke locomotor disturbance and catalepsy, which were observed following the administration of WIN 55,212-2. CB(2) receptor mechanism of action was investigated in dorsal root ganglia where MT178 mediated a reduction of [(3)H]-d-aspartate release. MT178 was also able to inhibit capsaicin-induced substance P release and NF-κB activation. These results demonstrate that systemic administration of MT178 produced a robust analgesia in different pain models via CB(2) receptors, providing an interesting approach to analgesic therapy in inflammatory and chronic pain without CB(1)-mediated central side effects.

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