Expression and functional role of adenosine receptors in regulating inflammatory responses in human synoviocytes

Abstract

Background and purpose: Adenosine is an endogenous modulator, interacting with four G-protein coupled receptors (A(1), A(2A), A(2B) and A(3)) 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)-kappaB activation, tumour necrosis factor alpha (TNF-alpha) and interleukin-8 (IL-8) release were assessed in human synoviocytes isolated from patients with osteoarthritis.

Key results: mRNA and protein for A(1), A(2A), A(2B) and A(3) 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 A(2A) and A(3) adenosine receptors inhibited p38 MAPK and NF-kappaB pathways, an effect abolished by selective adenosine antagonists. A(2A) and A(3) receptor agonists decreased TNF-alpha and IL-8 production. The phosphoinositide 3-kinase or G(s) pathways were involved in the functional responses of A(3) or A(2A) adenosine receptors. Synoviocyte A(1) and A(2B) adenosine receptors were not implicated in the inflammatory process whereas stimulation of A(2A) and A(3) adenosine receptors was closely associated with a down-regulation of the inflammatory status.

Conclusions and implications: These results indicate that A(2A) and A(3) adenosine receptors may represent a potential target in therapeutic modulation of joint inflammation.

Figure 1

Culture of human synoviocytes. (A) phase contrast and (B) vimentin expression by immunofluorescence. Nuclei were counterstained in blue with DAPI. Original magnification, ×200. (C) CD14 and vWF mRNA expression in macrophages (MC), endothelial cells (EC) and in synoviocytes (S). One microgram of total RNA has been loaded and stained with ethidium bromide to confirm equal RNA quantity. M = DNA ladder marker (Biolabs, Ipswich, MA, USA).

Figure 2

(A) mRNA expression (n= 4) of A₁, A_2A, A_2B and A₃ adenosine receptors (AR) and (B) representative Western blotting analysis in human synoviocytes. Densitometric analysis (n= 4) for adenosine receptors were also shown (C).

Figure 4

Affinity values of selected A₁, A_2A, A_2B and A₃ adenosine receptor agonists and antagonists obtained from competition binding experiments for A₁ (A), A_2A (B), A_2B (C) and A₃ (D) adenosine receptors. Each value represents the mean ± SEM of four separate experiments performed in duplicate.

Figure 3

Saturation curves (A) and Scatchard plot (B) of [³H]DPCPX, [³H]ZM 241385, [³H]MRE 2029F20, [³H]MRE 3008F20 binding to A₁, A_2A, A_2B and A₃ adenosine receptors (AR) in human synoviocytes respectively. Each value represents the mean ± SEM of four separate experiments performed in duplicate.

Figure 5

Stimulatory effect on cAMP levels of CGS 21680 (1 µmol·L⁻¹) and NECA (1 µmol·L⁻¹) in the absence and in the presence of SCH 58261 (1 µmol·L⁻¹) and MRE 2029F20 (1 µmol·L⁻¹) respectively (A). Inhibitory effect of CHA (1 µmol·L⁻¹) and Cl-IB-MECA (1 µmol·L⁻¹) in the absence and in the presence of DPCPX (1 µmol·L⁻¹) and MRE 3008F20 (1 µmol·L⁻¹) respectively (B). Each value represents the mean ± SEM of three separate experiments performed in triplicate. *P < 0.01 versus control conditions.

Figure 6

Western blotting analysis of phosphorylated p38 (P-p38) in the absence and in the presence of LPS (10 µg·mL⁻¹). The effect of examined adenosine receptor agonists (1 µmol·L⁻¹) and antagonists (1 µmol·L⁻¹) was also evaluated (A). Densitometric analysis (n= 4) of the bands obtained were also shown (B). *P < 0.01 versus control conditions; **P < 0.01 versus LPS conditions; #P < 0.02 versus LPS conditions; $P < 0.01 versus CGS 21680; §P < 0.02 versus Cl-IB-MECA.

Figure 7

Effect of adenosine receptor agonists (1 µmol·L⁻¹) and antagonists (1 µmol·L⁻¹) in human synoviocytes on NF-κB activation which was evaluated by detecting phosphorylated p50 (A) and p65 (B) proteins in nuclear extracts (n= 4). *P < 0.01 versus control conditions. **P < 0.01 versus LPS conditions; #P < 0.01 versus CHA; $P < 0.01 versus CGS 21680; †P < 0.05 versus NECA; ‡P < 0.01 versus NECA; §P < 0.01 versus Cl-IB-MECA.

Figure 8

Tumour necrosis factor α (TNF-α) (A) and interleukin-8 (IL-8) (B) levels in human synoviocytes in control conditions and stimulated by LPS (10 µg·mL⁻¹). TNF-α and IL-8 levels were also calculated (n= 4) in the presence of adenosine receptor agonists (1 µmol·L⁻¹) and antagonists (1 µmol·L⁻¹). *P < 0.01 versus control conditions. **P < 0.01 versus LPS conditions; #P < 0.01 versus CHA; $P < 0.01 versus CGS 21680; †P < 0.05 versus NECA; ‡P < 0.01 versus NECA; §P < 0.01 versus Cl-IB-MECA.