CD73-dependent adenosine dampens interleukin-1β-induced CXCL8 production in gingival fibroblasts: Association with heme oxygenase-1 and adenosine monophosphate-activated protein kinase

Abstract

Background: During inflammation, stressed or infected cells can release adenosine triphosphate (ATP) to the extracellular medium, which can be hydrolyzed to adenosine by ectonucleotidases such as ectonucleoside triphosphate diphosphohydrolase 1 (CD39) and 5'-nucleotidase (CD73). The role of CD73 in the modulation of cytokine release by human gingival fibroblasts (HGFs) remains underexplored. Here, we investigated whether CD73-mediated hydrolysis of extracellular ATP (eATP) could affect interleukin (IL)-1β-induced CXCL8 secretion.

Methods: The levels of mRNA expression of adenosine receptors, CD39 and CD73 of periodontitis samples were retrieved from a public database. Moreover, HGF mRNA levels were measured by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) after 3, 6, or 24 hours of IL-1β stimulation. IL-1β-induced CXCL8 protein levels were measured after pretreatment with 100-µM eATP in the presence or absence of CD73 inhibitor. The effect of eATP degradation to adenosine on CXCL8 levels was investigated using agonist and antagonist of adenosine receptors.

Results: Levels of CD39, CD73, and adenosine receptor mRNA were differentially modulated by IL-1β. ATP pretreatment impaired IL-1β-induced CXCL8 secretion and required activation of heme oxygenase-1 (HO-1) and phosphorylated adenosine monophosphate-activated protein kinase (pAMPK). The inhibition of CD73 or the inhibition of adenosine receptors abrogated the ATP effect on CXCL8 secretion.

Conclusions: CD73-generated adenosine dampens IL-1β-induced CXCL8 in HGFs and involves HO-1 and pAMPK signaling. These results imply that CD73 is a negative regulator of the inflammatory microenvironment, suggesting that this ectoenzyme could be involved in the generation of deficient CXCL8 gradient in chronic inflammation.

Keywords: 5′-nucleotidase; adenosine; adenosine triphosphate; fibroblasts; gingiva; interleukin-1 beta.

Figure 1.

Ectonucleotidases (A, B) and adenosine receptor (C-F) mRNA expression in samples from healthy and periodontitis tissues from public expression database NCBI GEO by microarray analysis [dataset GSE10334] of 64 samples per group. *Statistically significant differences, p < 0.05.

Figure 2.

Expression of adenosine receptors and ectonucleotidase by IL-1β-stimulated human gingival fibroblasts (HGF). HGF were stimulated with 1 ng/mL IL-1β, and mRNA expression of ENTPD1 (CD39) (A) and NT5E (CD73) (B) was evaluated after 3, 6, or 24 h. Protein levels of the ectonucleotidases were evaluated after 24 h of IL-1β challenge (C). Quantification of protein expression was performed by densitometric analysis and is presented as fold increase compared with control (non-stimulated cells) (D). Adenosine receptor mRNA expression for ADORA 1 (E), ADORA 2a (F), ADORA2b (G) and ADORA 3 (H). The GAPDH gene was used as the reference gene for RT-qPCR and actin protein was used as a loading control for Western Blot. Data are representative of three independent experiments. *Statistically significant differences, p < 0.05 compared with control.

Figure 3.

Extracellular ATP attenuates IL-1β-induced CXCL8 secretion and increases the levels of HO-1 and pAMPK in HGF. Cells were pre-treated with 100 μM ATP for 1 hour prior to the 1 ng/mL IL-1β stimulation, and were incubated for a total of 24 h. Protein levels of CXCL8 (A) were measured by ELISA in cell supernatants. HO-1 (B), pAMPK and total AMPK (C) production were measured by Western blot. Protein quantification of HO-1 (D) and pAMPK (E) was performed by densitometric analysis and is presented as fold increase compared with control (non-stimulated cells). Data are representative of three independent experiments. *Statistically significant differences, p < 0.05.

Figure 4.

ATP impairment of IL-1β-induced CXCL8 secretion is via CD73-generated adenosine and involves upregulation of HO-1 and pAMPK. Cells were pre-treated with (+) or without (−) 100 μM ATP, 0.1 μM CGS-15943, 50 nM NECA or 100 μM α,β Methyleneadenosine 5′-diphosphate sodium salt (AMP-CP) ^, as indicated in each graph, 1 hour prior to stimulation with1 ng/mL IL-1β, and were incubated for a total of 24 h. CXCL8 levels (A,B and C) were measured in cell supernatants by ELISA. Protein levels of pAMPK, total AMPK (D) and HO-1 (F) were measured by Western blot. Protein quantification of p-AMPK (E) and HO-1 (G) was performed by densitometric analysis and is presented as fold increase compared with control (non-stimulated cells). Data are representative of three independent experiments. *Statistically significant differences, p < 0.05. ^†p <0.01.

Figure 5.

Adenosine generated from eATP breakdown through the action of CD73 can dampen IL-1β-induced CXCL8 secretion. This process involves the upregulation of HO-1 and pAMPK. Cell stimulation with IL-1β promotes CXCL8 expression and protein synthesis. Extracellular ATP, which has been shown to be released during cellular stress can be hydrolyzed to ADP/AMP by CD39. AMP can be further degraded by CD73, resulting in adenosine generation. Interaction of extracellular adenosine or its agonist NECA with adenosine receptors (A1, A2a, A2b or A3) dampens IL-1β-induced CXCL8 secretion via upregulation of HO-1 and pAMPK. CGS-15943 (Adenosine receptor blocker); AMP-CP (CD73 inhibitor).