Expression of CD73/ecto-5′-nucleotidase on human gingival fibroblasts and contribution to the inhibition of interleukin-1α-induced granulocyte-macrophage colony stimulating factor production

Background and objectives: CD73/5′-nucleotidase (5′-NT) is an ectoenzyme that participates in immune/inflammatory reactions. We examined the possible expression of CD73/5′-NT on human gingival fibroblasts (hGF), which are important to the immune/inflammatory system in periodontal tissue. Methods and results: We demonstrated that CD73/5′-NT was expressed on hGF by flow cytometry. We found that pre-treatment of hGF with 5′-AMP induced marked inhibition of granulocyte-macrophage colony-stimulating factor (GM-CSF) production from hGF upon stimulation with interleukin-1α (IL-1α) by enzyme-linked immunosorbent assay (ELISA). A specific inhibitor of 5′-NT, adenosine 5′-[α,β-methylene] diphosphate blocked the inhibition of GM-CSF production, suggesting that adenosine converted from 5′-AMP acts on the inhibitory effects. The GM-CSF inhibition suggested that A3 receptor might be involved. The rank order of agonists was found to be (N ⁶-benzyl-5′-N-ethylcarboxamidoadenosine) A3 receptor agonist ≥ (2-chloroadenosine) non-selective agonist > (CGS-21680) A2_A receptor agonist > adenosine ≥ (N⁶-cyclohexyladenosine) A1 agonist. Further support for the main role of A3 receptor was the binding A3 antagonist [9-chloro-2-(2-furanyl)-5-([phenyl-acetyl]amino)[1,2,4]-triazolo[1,5- c]quinazdine] reversed the effect of adenosine, but no significant reverse was observed by A1 (1,3-dipropyl-8-cyclopentylxanthine). A2 [3,7-dimethyl-1-(2- propargyl)xanthine], A2_A [8-(3-chlorostyryl)caffeine], and A2 _B (alloxazine) antagonists. The CD73/5′-NT expression was increased upon stimulation with gamma-interferon, but not other stimulants such as tumor necrosis factor-alpha, IL-4, lipopolysaccharide from Porphyromonas gingivalis and Escherichia coli, and fimbriae from P. gingivalis, and this increase was correlated with the enhanced GM-CSF inhibition by 5′-AMP but not adenosine. Conclusions: These findings suggested that CD73/5′-NT on hGF exerts an anti-inflammatory effects in periodontal disease by conversion from 5′-AMP to adenosine.