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Background and objectives: Aminopeptidase N (APN)/CD13 is a multifunctional ectoenzyme that is involved in anti-inflammatory reactions, control of immune reactions and differentiation of many cellular systems. Here, we hypothesized that CD13/APN would be expressed on human gingival fibroblasts (hGF) and would contribute to the regulation of immune responses in periodontal tissue. Methods and results: CD13/APN was expressed on hGF at the mRNA and protein levels as determined by reverse transcriptase–polymerase chain reaction (RT–PCR) and flow cytometry, respectively. Enzymatic activities accompanying the expression were assessed by colorimetrical analysis using the synthetic substrate Leu-p-nitroanilide. We examined the possible regulation of CD13/APN expression on hGF in response to T cell-derived cytokines. T helper (Th) 2 cell type cytokines such as interleukin-4 and interleukin-13, but not interleukin-2 or interleukin-15, preferentially increased the expression of proteins as well as the enzymatic activities of CD13/APN in a dose-dependent manner. Receptors for these cytokines, the interleukin-4 receptor α chain, interleukin-13 receptor α1 chain, and interleukin-2R common γ chain, were expressed on hGF assessed by RT–PCR or flow cytometry. hGF exhibited inhibitory effects for formyl-methionyl-leucyl-phenylalanine (FMLP)-induced polymorphonuclear leukocyte-activation that was evaluated by Mac-1 expression, and this inhibitory effect was partially recovered by pre-treatment with the APN-specific inhibitor bestatin. Conclusions: These findings suggested that CD13/APN expressed by hGF could contribute to the anti-inflammatory response in periodontal tissue, and may be involved in disease processes mediated by Th2 cells.

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-1alpha (IL-1alpha) by enzyme-linked immunosorbent assay (ELISA). A specific inhibitor of 5'-NT, adenosine 5'-[alpha,beta-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 (N6-benzyl-5'-N-ethylcarboxamidoadenosine) A3 receptor agonist > or = (2-chloroadenosine) non-selective agonist > (CGS-21680) A2A receptor agonist > adenosine > or = (N6-cyclohexyladenosine) A1 agonist. Further support for the main role of A3 receptor was the binding A3 antagonist [9-chloro-2-(2-furanyl)-5-([phenylacetyl]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], A2A[8-(3-chlorostyryl)caffeine], and A2B (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.

CD13/aminopeptidase N (APN) is a multifunctional ectoenzyme that is involved in anti-inflammatory reactions, control of immune reactions and differentiation of many cellular systems. In this study, we first demonstrated that CD13/APN was expressed on human gingival fibroblast (hGF). T helper (Th) 2 cell type cytokines such as IL-4 and IL-13, but not IL-2 or IL-15, preferentially increased the expression of CD13/APN, resulting in the inhibition of FMLP-mediated neutrophil activation through the degradation by CD13/APN on HGF. Our data suggested that CD13/APN expressed by hGF could contribute to the anti-inflammatory response in periodontal tissue, and may be involved in disease processes mediated by Th2 cells.