Role of extracellular adenosine triphosphate (eATP) on immunomodulatory function of human periodontal ligament cells

Abstract

Background: Adenosine triphosphate, a nucleotide that acts as an important extracellular messenger, is released into the extracellular environment during various physiologic and pathological conditions. Human periodontal ligament cells (hPDLCs) can release adenosine triphosphate into the extracellular environment in response to mechanical stress. Extracellular adenosine triphosphate (eATP) plays role in both inflammation and osteogenic differentiation processes. eATP also participates in immunosuppressive action on immune cells. However, the role of eATP on the immunomodulatory function of hPDLCs is still unclear. This study aims to investigate the effects of eATP on the immunomodulatory function of hPDLCs and the participation of specific purinergic P2 receptors in this phenomenon. Methods: hPDLCs were treated with various concentrations of eATP (0-500mM) for 24 hours. To examine the effect of eATP on pro-inflammatory cytokine release, mRNA expression of IL6 and IL8 was analyzed by RT-PCR. Specific P2X7 receptor inhibitors (BBG and KN62) were applied to confirm the involvement of the P2X7 receptor on IL6 and IL8 expression by eATP. To study the effect of eATP on immunosuppressive molecule release, mRNA, and protein expression of indoleamine-pyrrole 2,3-dioxygenase (IDO) and interferon-gamma (IFNg) expression was analyzed using RT-PCR, IDO enzymatic activity assay, and ELISA,respectively. The role of the purinergic P2 receptor was determined using calcium chelator (EGTA) and PKC inhibitor (PKCi). Chemical inhibitors (KN62 and BBG), small interfering RNA (siRNA), and P2X7 receptor agonist (BzATP) were used to confirm the involvement of P2X7 receptors on IDO and IFNg induction by hPDLCs. Results: eATP significantly induced IL6 and IL8 expression in a dose-dependent manner. Specific P2X7receptor inhibitors (BBG and KN62) significantly inhibited eATP-induced IL6 and IL8 expression. eATP significantly enhanced IDO and IFNg expression at both mRNA and protein levels. EGTA and PKCi reduced eATP-induced IDO and IFNg expressions by hPDLCs, confirming the role of calcium signaling. Chemical P2X7 inhibitors (KN62 and BBG) and siRNA targeting P2X7 receptors significantly inhibited the eATP-induced IDO and IFNg production. Correspondingly, BzATP markedly increased IDO and IFNg mRNA and protein expression levels. Conclusion: eATP induced both inflammation and immunosuppression of hPDLCs depending on the concentrations. P2X7 receptor signaling is involved in this eATP induced inflammation and immunosuppression phenomenon. eATP may become a promising target for periodontal regeneration by modulating immune response and further triggering tissue healing.