ATP Degradation Through Ectonucleotidase Impairs IL-1β-Induced IL-8 in Gingival Fibroblasts

Objectives: Adenosine tri-phosphate (ATP) is involved in inflammation not only by acting on cells of the immune system but also through stromal cells of the microenvironment. During inflammation, stressed cells can release ATP to the extracellular medium, which can be hydrolyzed to adenosine by the action of ectonucleotidases, such as CD39 and CD73. It was previously reported that ATP inhibits IL-1β-induced matrix metalloproteinases through the action of CD39 in primary human gingival fibroblasts (HGF). Also, it has been documented that CD73 expression in HGF is involved in adenosine production. However, little information is available on the influence of ectonucleotidases in the modulation of pro-inflammatory mediators such as IL-8. We hypothesize that ATP degradation through the action of ectonucleotidases can influence IL-1β-induced IL-8 by HGF.
Methods: HGF were isolated from gingival explants of healthy patients after obtaining informed consent. Gingival tissue was sectioned and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% heat-inactivated fetal bovine serum and antibiotics. IL-1β-induced IL-8 levels were evaluated after 100μM of ATP pretreatment with and without CD73 inhibitor. Also, after 2h, 4h, or 6h of IL-1β stimulation, we evaluated mRNA expression of CD39, CD73, adenosine receptors 2a and 2b by RT-qPCR.
Results: ATP pretreatment impaired IL-1β-induced IL-8 secretion. Even though neither CD39 or CD73 were significantly changed after IL-1β stimulation, adenosine receptors showed increased mRNA levels after 4h. Also, the inhibition of CD73 cancelled the ATP effect on IL-8 secretion.
Conclusions: ATP degradation through CD73 impairs IL-1β-induced IL-8 in HGF. These results provide a pre-clinical evidence on the role of the breakdown of ATP through the action of ectonucleotidases in the inflammatory environment. It is expected that these results will contribute to a better understanding of how purinergic signaling can potentially be used as a target for future therapeutic strategies.