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J Dent Res 82(11): 888-892, 2003
© 2003 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Involvement of CD73 (ecto-5'-nucleotidase) in Adenosine Generation by Human Gingival Fibroblasts

T. Hashikawa1, M. Takedachi1, M. Terakura1, T. Saho1, S. Yamada1, L.F. Thompson2, Y. Shimabukuro1, and S. Murakami1,*

1 Department of Periodontology, Division of Oral Biology and Disease Control, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan; and
2 Oklahoma Medical Research Foundation, Immunobiology and Cancer Program, Oklahoma City, OK 73104, USA;

* corresponding author, ipshinya{at}dent.osaka-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adenosine has various biological effects on human gingival fibroblasts (HGF) and epithelial cells closely associated with inflammation, such as cytokine production and cell adhesion. However, the mechanism of adenosine formation in periodontal tissues is not yet defined. In this study, we examined the involvement of CD73 (ecto-5'-nucleotidase) in adenosine generation by HGF. CD73 was detected on in vitro-maintained HGF by immunocytochemistry and flow cytometric analysis. Adenosine production was observed following the addition of 5'-AMP, the substrate of CD73-associated ecto-5'-nucleotidase. Moreover, the addition of 5'-AMP to cultured HGF resulted in the elevation of cyclic adenosine monophosphate (cAMP). The 5'-AMP-induced increase in intracellular cAMP level was inhibited markedly by xanthine amine congener, an adenosine receptor antagonist, and partially by {alpha},ß-methylene adenosine 5'-diphosphate, an ecto-5'-nucleotidase inhibitor. These results suggest that CD73 on HGF is a critical enzyme responsible for the generation of adenosine, an immunomodulator that activates adenosine receptors.

KEY WORDS: periodontal disease • gingival fibroblast • inflammation • adenosine • CD73


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Extracellular purines, mainly adenosine and ATP, have diverse biological effects on cellular functions and are associated with physiological and pathophysiological processes including apoptosis, ischemia, hypoxia, inflammation, and wound healing, during which purine nucleotides and nucleosides are released (Gordon, 1986). Specific extracellular receptors, P1 (adenosine receptors) and P2 receptors, mediate purinergic signaling and subsequent biological functions (Ralevic and Burnstock, 1998). Interestingly, adenosine has been found to possess numerous anti-inflammatory activities (Cronstein, 1994). Stimulation of adenosine receptors down-regulated pro-inflammatory cytokine production by macrophages (Bouma et al., 1994), neutrophil adhesion to endothelium (Cronstein et al., 1986), and lymphocyte binding to fibroblasts (Murakami et al., 2001). Furthermore, the adenosine A2a receptor has been reported to engage in the attenuation of inflammation and protection from tissue damage (Ohta and Sitkovsky, 2001).

CD39 (ATPDase) hydrolyzes both ATP and ADP to AMP, which is subsequently converted to adenosine through CD73 (ecto-5'-nucleotidase) (Zimmermann, 1992), a glycosyl phosphatidylinositol (GPI)-anchored molecule. This ecto-enzyme is distributed in various types of cells, such as epithelial cells (Strohmeier et al., 1997), muscle cells (Heidemann et al., 1985), endothelial cells (Nacimiento and Kreutzberg, 1990), neutrophils (Robinson and Karnovsky, 1983), lymphocytes (Resta et al., 1998), and fibroblasts (Widnell et al., 1982). CD73 expression and its enzyme activity are reported to be regulated by inflammatory mediators, such as TNF-{alpha} (Savic et al., 1990), IL-1ß (Savic et al., 1990), PGE2 (Savic et al., 1990), and nitric oxide donors (Siegfried et al., 1996). CD73 activation in response to such stimuli leads to the release of adenosine, which modulates inflammation via interaction with adenosine receptors. In periodontal tissues, ligation of adenosine receptors has been reported to induce the activation of human gingival fibroblasts (HGF) and epithelial cells (HGEC), which modifies their cellular functions, such as cytokine/nitric oxide production and cell adhesion to lymphocytes (Murakami et al., 2000, 2001). However, the mechanism by which adenosine is formed in inflamed periodontal tissue is not yet defined. In this study, we examined the expression of CD73 on HGF, the involvement of its ecto-enzyme activity in the production of extracellular adenosine, and the possible interaction of adenosine with adenosine receptors expressed on HGF.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Adenosine, adenosine 5'-monophosphate (5'-AMP), {alpha},ß-methylene adenosine 5'-diphosphate (AOPCP), 4-(3-butoxy-4-methoxy-benzyl) imidazolidin-2-one, and xanthine amine congener (XAC) were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Human Gingival Fibroblasts (HGF)
All human subjects participated in this study after providing informed consent to a protocol that was reviewed and approved by the Institutional Review Board of the Osaka University Graduate School of Dentistry. HGF were obtained from biopsies of healthy gingiva from healthy volunteers. All biopsies were explanted into an {alpha}-modification of Eagle’s medium ({alpha}-MEM, Nikken Biomedical laboratory, Kyoto, Japan) supplemented with 150 U/mL penicillin G, 150 µg/mL streptomycin, 300 µg/mL kanamycin sulfate, and 2.5 µg/mL fungizone, and then cultured with {alpha}-MEM supplemented with 10% fetal calf serum (FCS, Hazleton Research Products, Lenexa, KS, USA) at 37°C in a humidified atmosphere of 5% CO2/95% air. The cells which grew from the explants were detached by 0.05% trypsin-0.02% EDTA (Life Technologies, Grand Island, NY, USA) in PBS (Nikken Biomedical laboratory) and subcultured in plastic flasks (Corning, Corning, NY, USA). HGF were passed by trypsinization and used for experiments at passages 4-10.

Flow Cytometric Analysis
Cell-surface antigens were detected by flow cytometry (FCM) with the use of a Becton-Dickinson FACSCalibur. Cells were stained as previously described (Murakami et al., 1993), with use of an anti-human CD73 monoclonal antibody (mAb) (Alexis Biochemicals, San Diego, CA, USA) or isotype-matched murine myeloma protein (Mouse IgG1, MOPC-21) as a control, followed by the addition of FITC-goat anti-mouse IgG (Zymed Laboratories, San Francisco, CA, USA). Data were collected on 10,000 cells for single-color staining and analyzed with the use of CellQuest software (Becton-Dickinson, Mountain View, CA, USA).

Immunocytochemical Staining
Monolayers of HGF grown to confluence in 60 mm Poly-L-Lysine-coated glass-bottomed dish (Matsunami Glass Ind., Ltd., Osaka, Japan) were incubated with anti-CD73 mAb (AD2, Pharmingen, San Diego, CA, USA) or isotype-matched murine myeloma protein (Mouse IgG1, MOPC-21) as a control at 4°C for 15 min. The cells were then fixed in 4% paraformaldehyde for 10 min at room temperature and incubated with Alexa Fluor 594 goat anti-mouse immunogloblin G (Molecular Probes, Engene, OR, USA) for 15 min at room temperature. Finally, the cells were counterstained with DAPI (0.1 µg/mL, SIGMA). The cells were washed 3 times with PBS after each step.

Radioimmunoassay for Adenosine Measurements
Monolayers of HGF grown to confluence in 12-well plates (Corning) were washed twice with Hanks’ balanced salt solution (HBSS, Sigma Chemical Co., St. Louis, MO, USA), and 1 mL of serum-free {alpha}-MEM was added to each well. Plates were then incubated for 30 min at 37°C in 5% CO2. 5'-AMP (300 nM) was added to the cells in a final volume of 1 mL, and the incubation was allowed to proceed for various time periods. In some experiments, HGF were treated with 5'-AMP (20 µM) in the presence or absence of AOPCP (0.2 µM, 2 µM, 20 µM, and 200 µM) for 10 min. The concentration of adenosine was determined with the use of a radioimmunoassay kit (Yamasa Shoyu Co., Ltd. Chiba, Japan) according to manufacturer’s instructions.

Measurements of Cyclic Adenosine Monophosphate (cAMP) Responses
Monolayers of HGF grown to confluence in six-well plates (Corning) were washed twice with HBSS. Two mL of serum-free {alpha}-MEM were added to each well, and the plates were then incubated for 2 hrs at 37°C in 5% CO2. Cells were pre-incubated for 10 min at 37°C in the same medium containing the cAMP phosphodiesterase inhibitor, 4-(3-butoxy-4-methoxy-benzyl) imidazolidin-2-one, at 10 µM. Incubations of cells with various agents were performed in the same plates, containing media alone, adenosine (20 µM), or AMP (20 µM), in the presence or absence of AOPCP (0.2 µM, 2 µM, 20 µM, and 200 µM) or XAC (10 µM) for 5 min at 37°C in 5% CO2. cAMP levels were determined by means of a cAMP enzyme immunoassay (EIA) kit (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA) according to manufacturer’s instructions. All assays were performed in quadruplicate with 50 µL of cell extracts. cAMP levels in unstimulated cells were subtracted from the values shown.

Statistical Analysis
The data were analyzed by one-way ANOVA with repeated measures followed by Fisher’s multiple-range test for the determination of differences between groups.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Surface Expression of CD73 on HGF
The expression of CD73 on HGF was first examined. FCM analysis revealed that cultured HGF expressed CD73 constitutively (Fig. 1AGo). Similarly, immunocytochemical analysis clearly demonstrated the expression of CD73 on HGF (Fig. 1BGo). Furthermore, we examined the enzyme activity of ecto-5'-nucleotidase (CD73) by measuring the conversion of [8-14C]-IMP to [8-14C]-inosine. We found that the ecto-5'-nucleotidase enzyme activity on HGF was proportional to the cell number and was abrogated by the addition of the competitive inhibitor of 5'-nucleotidase, AOPCP (data not shown).



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  		Figure 1. Surface expression of CD73 on HGF. (A) Analysis of surface expression of CD73 on HGF by FCM. HGF were stained with anti-human CD73 Ab (solid line) plus FITC-goat anti-mouse IgG. Staining with the isotype-matched control is shown in the shaded peak. The data shown are representative of three separate experiments. (B) Cytochemical localization of CD73 on HGF. HGF cultured in the glass-bottomed dish were stained with anti-human CD73 mAb, fixed in 4% paraformaldehyde, incubated with Alexa Fluor 594 goat anti-mouse IgG, and counterstained with DAPI. The data shown are representative of three separate experiments. Scale bars = 100 µm.

 
Conversion of 5'-AMP to Adenosine by CD73 on HGF
To confirm that adenosine could be converted from 5'-AMP by CD73 on HGF, we examined a time course of adenosine concentration following addition of 5'-AMP to the culture supernatant. Cells were treated with 300 nM 5'-AMP and examined for adenosine concentration after various time periods (Fig. 2AGo). An increased adenosine concentration was observed in a time-dependent manner up to 20 min. The adenosine concentration reached a maximum value at 20 min and remained constant up to 60 min (Fig. 2AGo). At longer time periods, the concentration of adenosine decreased. To confirm whether adenosine could be generated from 5'-AMP via CD73, we treated cells with 5'-AMP (20 µM) in the presence or absence of AOPCP (0.2 µM, 2 µM, 20 µM, or 200 µM) and examined them for adenosine concentration. As shown in Fig. 2BGo, AOPCP reduced the production of adenosine in a dose-dependent manner.



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  		Figure 2. Adenosine levels converted from 5'-AMP by ecto-5'-nucleotidase (CD73) on HGF. (A) Kinetics of extracellular adenosine levels. HGF were treated with 300 nM 5'-AMP for different time intervals. The data shown are representative of three separate experiments. (B) Effects of AOPCP on extracellular adenosine levels. HGF were treated with 20 µM 5'-AMP in the presence or absence of 0.2 µM, 2 µM, 20 µM, and 200 µM AOPCP for 10 min. The data shown are the mean ± SD of 4 adenosine concentration determinations and are representative of three separate experiments. *p < 0.05 compared with group of AMP.

 
cAMP Responses to Adenosine Converted from 5'-AMP in HGF
We have already reported that adenosine can enhance the intracellular cAMP level of HGF through the activation of adenosine receptors (Murakami et al., 2000). Thus, to examine whether adenosine produced from 5'-AMP by the enzyme activity of CD73 could participate in the activation of adenosine receptors on HGF, we treated the cells with 5'-AMP and then examined the intracellular cAMP levels. Treatment of HGF with 5'-AMP as well as adenosine for 5 min resulted in an increase in intracellular cAMP (Fig. 3AGo), and these effects were inhibited by treatment with the adenosine receptor antagonist, XAC. Furthermore, treatment with AOPCP decreased the cAMP response to adenosine converted from 5'-AMP by CD73 in a dose-dependent manner (Fig. 3BGo). In contrast, the treatment with AOPCP did not alter the cAMP response by adenosine itself (data not shown).



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  		Figure 3. cAMP responses to adenosine converted from 5'-AMP by CD73. (A) Cells were pre-incubated with 10 µM 4-(3-butoxy-4-methoxy-benzyl)imidazolidin-2-one for 10 min at 37°C and stimulated with 20 µM adenosine (open bar) or 5'-AMP (closed bar) for 5 min at 37°C in the presence or absence of 10 µM XAC. The data shown are the mean ± SD of 4 cAMP determinations and are representative of three separate experiments. *p < 0.05 compared with the adenosine group; **p < 0.05 compared with the AMP group. (B) Effects of AOPCP on cAMP responses. HGF were treated with 20 µM 5'-AMP in the presence or absence of 0.2 µM, 2 µM, 20 µM, and 200 µM AOPCP for 10 min. The data shown are the mean ± SD of 4 cAMP determinations and are representative of three separate experiments. *p < 0.05 compared with the AMP group.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It has been documented that adenosine regulates the cellular function, such as cytokine production and cell adhesion, of gingival epithelial cells and fibroblasts (Murakami et al., 2001, 2002). However, the mechanism of adenosine formation in periodontal tissues has not yet been identified. This study reports, for the first time, CD73 expression in HGF and its involvement in the production of adenosine by HGF (Fig. 1Go). Moreover, this study indicates that CD73, ecto-5'-nucleotidase, plays a critical role in the activation of adenosine receptors expressed on HGF.

Adenosine concentrations are elevated at inflammatory sites (Cronstein, 1994), and the immunomodulating effects of adenosine have been shown by in vitro (Green et al., 1991; Cronstein et al., 1993) and in vivo (Bouma et al., 1994) studies. CD73 expression and its enzyme activity were regulated by several inflammatory mediators (Savic et al., 1990; Siegfried et al., 1996). An increase in ecto-5'-nucleotidase activity has been shown to be modulated through interaction of mesangial cells with macrophages or fibroblasts (Stefanovic et al., 1995) and stimulation of cells with extracellular matrices (Mehul et al., 1993). Therefore, it is certainly plausible that CD73-dependent adenosine generation may be regulated by several factors during inflammatory and healing processes.

Adenosine generation is dependent not only on ecto-5'-nucleotidase activity but also on the concentration of 5'-AMP, the substrate of the enzyme. 5'-AMP can be generated by CD39 (ATPDase) from ATP or ADP released from activated cells and damaged tissue (Burnstock, 1990). However, little CD39 is expressed on unstimulated or IL-1ß-stimulated HGF (data not shown), making it unlikely that CD39 on HGF contributes substantially to the generation of 5'-AMP. Instead, 5'-AMP production may be attributed to cells that express high levels of CD39, such as platelets, endothelial cells, and leukocytes (Koziak et al., 1999). Indeed, stimulated neutrophils have been reported to release 5'-AMP during inflammatory processes (Barrett et al., 1990).

Adenosine receptors are coupled to G-proteins that regulate adenylate cyclase and, in turn, intracellular cAMP concentrations (Ralevic and Burnstock, 1998). Following the addition of 5'-AMP, cAMP elevation was observed in HGF and was clearly inhibited by XAC, an adenosine receptor antagonist (Fig. 3AGo). These results strongly suggest that adenosine generated by CD73 on HGF could subsequently not only bind to, but also activate, G-protein-coupled adenosine receptors. As expected, treatment with AOPCP, an ecto-5'-nucleotidase inhibitor, decreased the cAMP response following the addition of 5'-AMP (Fig. 3BGo), but not adenosine (data not shown). It is interesting to note that nearly equivalent amounts of cAMP were generated from both adenosine and 5'-AMP at 20 µM. Analysis of these data suggests that the conversion of 5'-AMP to adenosine is very rapid and that CD73 and adenosine receptors may be co-localized on the surface of HGF, as was reported by Matsuoka et al.(2002).

Elevation of endogenous adenosine at inflammatory sites has been demonstrated to attenuate inflammation (Cronstein, 1994) and promote wound healing (Montesinos et al., 2002). Although the mechanism by which adenosine modulates inflammatory responses in periodontitis is not fully understood, it has been shown that engagement of adenosine receptors on HGEC and HGF leads to the alteration of various cellular functions (Murakami et al., 2001, 2002). In this study, we demonstrated for the first time that HGF can generate adenosine through CD73. Since adenosine can be metabolized by adenosine deaminase and adenosine kinase, inhibition of these enzymes can increase local adenosine concentrations. Adenosine-up-regulating agents that influence adenosine generation and degradation, as well as adenosine receptor agonists, may be useful as anti-inflammatory therapeutic agents for the treatment of periodontitis. Further studies will be required to explore the efficacy of drugs controlling adenosine receptor activation for the treatment of periodontal diseases.


   ACKNOWLEDGMENTS
 
This work was supported by Grants-in-Aid from the Japan Society for the Promotion of Science (JSPS) (Nos. 13307061, 11470461, 14370709, 14370617, and 15659498), by the 21st century COE program for JSPS, and by an NIH grant (AI18220).

Received March 7, 2003; Last revision August 18, 2003; Accepted August 27, 2003


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 DISCUSSION
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