Adenosine regulates the IL-1ß-induced cellular functions of human gingival fibroblasts

Shinya Murakami, Tomoko Hashikawa, Teruyuki Saho, Masahide Takedachi, Takenori Nozaki, Yoshio Shimabukuro and Hiroshi Okada

Department of Periodontology, Division of Oral Biology and Disease Control, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan

Correspondence to: S. Murakami


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study we examined the influence of adenosine on the cellular functions of human gingival fibroblasts (HGF), such as the production of inflammatory cytokines and extracellular matrices (ECM), and the expression and function of adhesion molecules. Concerning the expression of adenosine receptors, RT-PCR analysis revealed that HGF expressed adenosine receptor A1, A2a and A2b, but not A3 mRNA. Ligation of adenosine receptors by adenosine or its related analogue, 2-chloroadenosine (2-CADO), N6-cyclopentyladenosine (CPA) or CGS21680 synergistically increased IL-1ß-induced IL-6 and IL-8 production. In terms of ECM expression, adenosine and the adenosine receptor agonists, 2-CADO and CPA, enhanced constitutive and IL-1ß-induced expression of hyaluronate synthase mRNA, but not the mRNA levels of other ECM, such as collagen type I, III and fibronectin. Moreover, the adherence of IL-1ß-stimulated HGF to activated lymphocytes was also inhibited by adenosine, which is in part explained by the fact that adenosine down-regulated the IL-1ß-induced expression of ICAM-1 on HGF. These results provide new evidence for the possible involvement of adenosine in the regulation of inflammatory responses in periodontal tissues.

Keywords: adenosine, cytokines, fibroblasts, human, immunomodulators, inflammation


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Periodontal disease is a chronic disease characterized by inflammatory cell accumulation in the extravascular gingival connective tissue. In such sites, inappropriate activation of inflammatory and resident cells becomes self-perpetuating, and can lead to chronic periodontal tissue destruction. Recently, it has been clarified that the functions of fibroblasts extend well beyond their primary function of producing the structural connective tissue proteins that comprise the ground substance in gingival connective tissue. Recent studies have demonstrated that gingival fibroblasts can produce cytokines and chemical mediators when they are activated with physiological and pathophysiological stimuli (16). Furthermore, gingival fibroblasts can regulate the retention and lodging of lymphocytes in periodontitis lesions by actively adhering with the lymphocytes via adhesion molecules such as VLA integrins, LFA-1/ICAM-1 and CD44 (7–12). Thus, it is certain that human gingival fibroblasts (HGF) regulate local inflammatory responses in inflamed periodontal tissues.

Adenosine, an endogenous nucleoside, is generated intracellularly and extracellularly through dephosphorylation of AMP by ecto 5'-nucleotidase, and partially deaminated to inosine, which is mediated by adenosine deaminase followed by conversion to uric acid (13–15). A series of reports have revealed that adenosine has a plethora of biological actions on a large variety of cells (13,14,16–21) and can modulate the various functions of cells involved in inflammatory responses (13,14,16–21). In fact, it has been documented that the concentration of adenosine increases up to 500–600 nM in inflammatory lesions, while that of adenosine under normal conditions is <300 nM (17). Thus, it appears to be possible that adenosine may modulate numerous inflammatory responses in mammalian systems. For example, administration of adenosine or an adenosine receptor agonist into rats, which had adjuvant-induced arthritis induced by an intradermal injection of Mycobacterium butyricum as a chronic inflammatory disease model, resulted in a reduction of the experimental arthritis (20). Sulfasalazine or methotrexate, commonly used anti-inflammatory agents for the treatment of rheumatoid arthritis, reduced the number of accumulated inflammatory cells in air pouches induced on the back of mice. Interestingly, adenosine deaminase and an adenosine receptor antagonist abrogate the anti-inflammatory effects of the agents, suggesting that adenosine is involved in their action (13, 14, 16–19,21,22).

In addition, recent work has demonstrated that adenosine and an adenosine receptor agonist inhibit Escherichia coli. Lipopolysaccharide (LPS) induced tumor necrosis factor (TNF)-{alpha} production by monocytes/macrophages (23–27). It was also shown that adenosine and an adenosine receptor agonist affected the adherence of neutrophils to vascular endothelial cells (17–19,28), suggesting that adenosine may contribute to regulation of the adhesive interaction of immunocompetent cells to other cell types. However, very little is known about the regulatory effects of adenosine on inflammatory responses in inflamed periodontal lesions. Since HGF play a critical role in tissue homeostasis and the development of periodontal disease status as described above, investigation of how adenosine affects the biological functions of HGF is expected to provide crucial information for understanding the possible involvement of adenosine in inflammatory responses in periodontal diseases and for possible topical application of an adenosine-related analogue as a host modulating medicine for periodontal diseases.

In this study, in order to reveal how adenosine alters the cellular functions of HGF, we examined for the first time the effects of adenosine on expression of cytokine and extracellular matrices (ECM), and on the adhesive interactions between HGF and lymphocytes. Furthermore, we investigated the expression of adenosine receptor subtypes in HGF since it has been documented that there are at least four kinds of adenosine receptor subtypes through which adenosine transduces signals (29,30).


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Reagents
Recombinant human IL-1ß (Genzyme, Cambridge, MA), adenosine [Research Biochemicals International (RBI), Natick, MA], adenosine receptor agonist, 2-chloroadenosine (2-CADO) (RBI), N6-cyclopentyladenosine (CPA) (RBI), CGS-21680 hydrochloride (RBI) and adenosine receptor antagonist, xanthine amine congener (XAC) (RBI), were commercially obtained.

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 Faculty 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 supplemented with 150 U/ml penicillin G, 150 µg/ml streptomycin and 2.5 µg/ml amphotericin B, and then cultured with {alpha}-modified Eagle's medium supplemented with 10% FCS (Hazleton Research Products, Lenexa, KS) 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) in PBS and subcultured in plastic flasks (Corning, Corning, NY). HGF were passed by trypsinization and used for experiments at passages 4–13. The responsiveness of each HGF to adenosine or IL-1ß was basically similar.

Purification of peripheral blood mononuclear cells (PBMC) and polymorphonuclear leukocytes (PMNL)
PBMC and PMNL were collected from healthy donors by density-gradient centrifugation on Histopaque 1077 (density of 1.077 g/ml; Sigma, St Louis, MO). After being washed, the isolated PBMC were rosetted with neuraminidase-treated sheep red blood cells. After separation on a Histopaque gradient, rosetted cells were treated with pH 7.2 Tris-buffered ammonium chloride to lyse sheep red blood cells and the recovered cells were used as purified T lymphocytes. This cell population contained >95% CD3+ cells. Similarly, PMNL were separated by density-gradient centrifugation over Histopaque 1077. The interphase containing the mononuclear leukocytes and Histopaque were removed, and the remaining mixture of PMNL and erythrocytes was treated for 30 min with an ice-cold isotonic NH4Cl solution (155 mM NH4Cl, 10 mM KHCO3 and 0.1 mM EDTA, pH 7.4) to lyse the erythrocytes. The remaining PMNL were washed twice. The PMNL had a purity of >98% and consisted mainly of neutrophils (>95%).

mAb
84H10 (anti-ICAM-1) mAb was kindly provided by Dr S. Shaw (NIH) and TS1/22 (anti-LFA-1) mAb was purchased from ATCC (Rockville, MD). 4-145 (7) (anti-VLA ß1 mAb) and OS/37 (10) (anti-CD44 mAb) were previously described.

FACS analyses
Immunofluorescent cell staining was analyzed with a Flow cell sorter (FCS-1; Japan Spectroscopic, Tokyo, Japan) (8). A laser power of 500 mW at 488 nm was used for excitation. The fluorescence of FITC and propidium iodide were measured at 510 and 530 nm respectively.

Cell adhesion assay
T lymphocytes which had been stimulated with 0.5 µg/ml phorbol myristate acetate (PMA; Sigma) for 16 h were radiolabeled by incubation of 1x107 cells in 1 ml complete medium with 7.4 Bq of 51Cr for 1 h at 37°C and washed 3 times with complete medium (8). The labeled cells (5x105/well) were then added to 24-well plates (Corning, Corning, NY) containing confluent HGF monolayers. The plates were incubated for 30 min at 37°C and non-adherent cells were removed by three cycles of washing in prewarmed complete medium with vigorous agitation. Adherent cells were solubilized with Triton X-100 and the released 51Cr was counted with a {gamma}-counter. Percentages of adherent cells were determined by dividing c.p.m. from bound cells by the input cell-associated c.p.m. from which the spontaneous release was subtracted and multiplied by 100%

Detection of mRNA of cytokine, ECM and adenosine receptor subtypes by RT-PCR
Total RNA was isolated from each cell by RNAzol (Cinna/Biotecx, Friendswood, TX) according to the manufacturer's instructions.

The precipitated RNA was redissolved in 0.1% diethylpyrocarbonate-treated distilled water.

cDNA synthesis and amplification via PCR were performed according to the methods described by Murakami et al. (10). To generate cDNA for PCR analysis, a 40 µl cDNA synthesis reaction mixture per RNA sample was prepared and incubated at 37°C for 60 min. A 40 µl cDNA synthesis reaction mixture contains: 5.2 µl of 0.1% diethylpyrocarbonate-treated distilled water, 4 µl 10xPCR buffer II (100 mM Tris–HCl, pH8.3, 500 mM KCl; Perkin-Elmer Cetus, Norwalk, CT); 6 µl 25mM MgCl2, 4 µl of each 10 mM deoxynucleotide triphosphates (Takara Shuzo, Shiga, Japan), 0.4 µl 20 U/ml RNase inhibitor (Perkin-Elmer Cetus), 1 µl 50 U/ml MMLV reverse transcriptase and 4 µl 0.25 µg/ml RNA sample. After incubation, all samples were heated to 94°C for 5 min to inactivate the reverse transcriptase.

Oligonucleotide PCR primers specific for IL-6, IL-8, hyaluronate (HA) synthase, collagen type I, III, fibronectin, laminin, adenosine A1, A2a, A2b and A3 receptor subtype, and GAPDH mRNA were synthesized by Clontech (Palo Alto, CA). The primer sequences are listed in Table 1Go. The cDNA samples prepared above were amplified by adding to a PCR reaction mixture which included 10 mM Tris–HCl buffer (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 0.15 mM dNTP mixture, 1.25 U AmpliTaq Gold (Perkin-Elmer, Foster City, CA), and 0.2 µM sense and antisense oligonucleotide primers. The PCR reaction mixture was overlaid with mineral oil (Aldrich, Milwaukee, WI) and subjected to amplification for two or three different numbers of cycles using a DNA thermal cycler 480 (Perkin-Elmer Cetus, Emeryville, CA). After initial denaturation at 94°C for 4 min, each cycle consisted of 94°C for 45 s, 60°C for 45 s and 72°C for 2 min. PCR products were analyzed by electrophoresis at 100 V for 30 min on 1.5% TAE agarose gel (Nippon Gene, Toyama, Japan) containing 0.5 µg/ml ethidium bromide.


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Table 1. PCR primer sequences
 
A3 receptor nested PCR
The PCR products from the A3 receptor reaction were re-amplified using nested amplification primers (Table 1Go). The PCR conditions were not changed for the re-amplification.

Determination of IL-6 and IL-8 production
HGF were placed onto 48-well culture plates (Corning) at 1x104 cells/ml in complete media. When the monolayers were confluent, the cells were rinsed twice with HBSS and cultured with 2-CADO (5x10–5 M), CPA (5x10–5 M) and CGS21680 (5x10–5 M) in the presence or absence of IL-1ß (25 U/ml) for 24 h. At the end of the incubation periods, the supernatants were collected, and stored at –20°C until determination of IL-6 and IL-8 levels. The IL-6 and IL-8 levels in culture supernatants were measured by using an ELISA kit (Genzyme, Cambridge, MA) according to the manufacturer's instructions.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Expression of adenosine A1, A2a and A2b, but not A3 receptor mRNA was detected in HGF
To examine the mRNA expression of adenosine receptor subtypes in HGF, RT-PCR was performed. As shown in Fig. 1Go, RNA transcripts for A1, A2a and A2b were present, but A3 receptor subtype mRNA was not detected. mRNA corresponding to all four receptors was observed in PBMC and PMNL. To further confirm that A3 receptor mRNA was not detected in HGF, a second round of PCR amplification using nested primers was carried out. Even though a more sensitive method to visualize the A3 messenger was used, A3 receptor mRNA was not detected in HGF, in spite of the detection of A3 mRNA in PBMC and PMNL (data not shown).



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Fig. 1. Expression of adenosine receptor subtype mRNA in HGF. RT-PCR was performed to examine the adenosine receptor A1, A2a, A2b and A3 subtype mRNA in HGF, PBMC and PMNL (neutrophil). Results of one representative experiment among three identical experiments are shown. The number of PCR cycles is shown above each lane.

 
2-CADO enhanced the expression of IL-1 ß-induced IL-6 and IL-8 mRNA in HGF
Fibroblasts can express various cytokine mRNAs (1,2,5,6,31–33). To determine whether the adenosine receptor agonist has a regulatory effect on the mRNA expression of the cytokines in HGF, we examined the influence of the adenosine receptor agonist, 2-CADO, on IL-1ß-induced cytokine mRNA expression by HGF. As previously described, treatment of HGF with IL-1ß led to an increase of IL-6 and IL-8 mRNA expression (Fig. 2Go). Interestingly, the IL-1ß-induced IL-6 and IL-8 mRNA expression were increased by 2-CADO (Fig. 2Go), while IL-6 and IL-8 mRNA expression was not dramatically altered by addition of 2-CADO alone. The up-regulation of IL-1ß-induced IL-6 and IL-8 mRNA expression by 2-CADO was reversed by XAC (data not shown).



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Fig. 2. Effects of 2-CADO on IL-6 and IL-8 mRNA expression by HGF stimulated with IL-1ß. HGF were cultured in the presence or absence of IL-1ß (25 U/ml) with or without 2-CADO (100 µM) for 3.5 h, and then RT-PCR was carried out to detect IL-6 and IL-8 mRNA expression in HGF. Results of one representative experiment among three identical experiments are shown. The number of PCR cycles is shown above each lane.

 
Adenosine receptor agonists synergistically increased IL-6 and IL-8 production by HGF
We then examined the effects of adenosine receptor agonists on IL-1ß-induced IL-6 and IL-8 production by HGF at the protein level. The IL-6 and IL-8 levels in culture supernatants were determined by ELISA as described in Methods. Both IL-6 and IL-8 productions were not dramatically altered by addition of 2-CADO, CPA (A1 adenosine receptor agonist) or CGS21680 (A2a adenosine receptor agonist) alone (Fig. 3Go). On the other hand, IL-1ß stimulation clearly increased IL-6 and IL-8 production by HGF (Fig. 3Go). When HGF were stimulated with IL-1ß in the presence of 2-CADO, both IL-6 and IL-8 productions were synergistically enhanced. Furthermore, the IL-1ß-induced IL-6 and IL-8 productions were also synergistically increased by CPA and by CGS21680. Similar data were also observed for adenosine instead of adenosine receptor agonists described above (data not shown). However, the action of adenosine was less effective than that of 2-CADO.



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Fig. 3. Effects of 2-CADO, CPA and CGS21680 on IL-6 and IL-8 productions by HGF stimulated with IL-1ß. HGF were cultured in the presence or absence of IL-1ß (25 U/ml) with or without 2-CADO (50 µM), CPA (50 µM) or CGS21680 (10 µM) for 24 h. After the incubation, the culture supernatants were collected and the IL-6 (A) and IL-8 (B) levels in the supernatants were measured by ELISA as described in Methods. Results of one representative experiment among five identical experiments are shown.

 
Adenosine and 2-CADO enhanced HA synthase mRNA, but not collagen type I, III, laminin or fibronectin mRNA
The production of ECM by HGF is known to be involved not only in periodontal tissue homeostasis, but also in the development of inflammation and tissue remodeling. In order to investigate whether the constitutive production of ECM by HGF would be affected by adenosine, an RT-PCR analysis was performed. As shown in Fig. 4Go, treatment of HGF with 2-CADO led to an increased expression of HA synthase mRNA, whereas no changes in the expression of collagen type I, III, fibronectin or laminin mRNA were observed. Furthermore, CPA but not CGS21680 induced HA synthase mRNA expression in HGF (Fig. 5Go). Adenosine also induced HA synthase mRNA expression in HGF and the expression was markedly abrogated by XAC (Fig. 6Go). We then examined the effect of 2-CADO on the IL-1ß-induced HA synthase mRNA expression. As shown in Fig. 7Go, stimulation with IL-1ß increased HA synthase mRNA. When HGF were stimulated with IL-1ß in the presence of 2-CADO, IL-1ß-induced HA synthase mRNA expression was further up-regulated (Fig. 7Go).



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Fig. 4. Effects of 2-CADO on mRNA expression of ECM and HA synthase in HGF. HGF were cultured in the presence or absence of 2-CADO (100 µM) for 3.5 h, and then RT-PCR was carried out to detect collagen type I, III, fibronectin, laminin and HA synthase mRNA expression in HGF. Results of one representative experiment among three identical experiments are shown. The number of PCR cycles is shown above each lane.

 


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Fig. 5. Effects of CPA and CGS21680 on HA synthase mRNA in HGF. HGF were cultured in the presence or absence of 2-CADO (100 µM), CPA (50 µM) or CGS21680 (10 µM) for 3.5 h and then RT-PCR was carried out to detect HA synthase mRNA expression in HGF. Results of one representative experiment among three identical experiments are shown. The number of PCR cycles is shown above each lane.

 


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Fig. 6. Inhibitory effects of XAC on adenosine-induced mRNA expression of HA synthase in HGF. HGF were cultured in the presence or absence of XAC (5x10–5 M) with or without adenosine (100 µM) and then RT-PCR was carried out to detect HA synthase mRNA expression in HGF. Results of one representative experiment among three identical experiments are shown. The number of PCR cycles is shown above each lane.

 


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Fig. 7. Effects of 2-CADO on mRNA expression of HA synthase in HGF stimulated with IL-1ß. HGF were cultured in the presence or absence of IL-1ß (25 U/ml) with or without 2-CADO (100 µM) for 3.5 h and then RT-PCR was carried out to detect HA synthase mRNA expression in HGF. Results of one representative experiment among three identical experiments are shown. The number of PCR cycles is shown above each lane.

 
2-CADO prevented IL-1 ß-induced ICAM-1 expression on HGF
To evaluate the effects of the adenosine receptor agonist, 2-CADO, on the expression of cell adhesion molecules on HGF, which are responsible for the binding of lymphocytes to HGF, FACS analysis was carried out as described in Methods. HGF were cultured with or without IL-1ß in the presence or absence of adenosine, and then the expression of both ICAM-1 and CD44 on HGF was analyzed. HGF constitutively expressed a very low level of ICAM-1 and a high level of CD44, and IL-1ß increased the expression of ICAM-1 by HGF (Fig. 8Go). Interestingly, adenosine completely abolished the IL-1ß-enhanced expression of ICAM-1 (Fig. 8Go). However, adenosine did not alter the constitutive expression of ICAM-1 or CD44 expression by HGF (Fig. 8Go). Similar to CD44, adenosine did not affect the HGF expression of integrin ß1 which was recognized by 4-145 mAb (data not shown).



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Fig. 8. Inhibitory effects of adenosine on IL-1ß-induced ICAM-1 expression on HGF. HGF which had been cultured with or without IL-1ß (25 U/ml) in the presence of adenosine (100 µM) for 15 h at 37°C were harvested and then incubated with 84H10 or OS/37 at 4°C for 30 min, followed by washing and incubating with FITC-labeled goat ant-mouse IgG at 4°C for 30 min. FACS analysis was performed as described in Methods. The dashed line in each panel represents FACS profiles of HGF stained with FITC-labeled goat anti-mouse IgG used only as a negative control.

 
Lymphocyte adherence to HGF was partially suppressed by adenosine
The fact that adenosine down-regulated the IL-1ß-induced ICAM-1 expression by HGF suggests that adenosine may influence the adhesive interaction between lymphocytes and HGF. To examine this possibility, a cell adhesion assay using T lymphocytes and HGF monolayers was performed in the presence or absence of adenosine. Activation of HGF with IL-1ß increased the adhesion of activated T lymphocytes to HGF monolayers, and the IL-1ß-increased binding was partially but significantly blocked by 84H10 (IgG1, {kappa}) and adenosine (Fig. 9Go). It was confirmed that subclass-matched control mAb did not influence the binding (data not shown). In addition, the inhibition by adenosine was reversed by addition of the adenosine receptor antagonist, XAC (data not shown).



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Fig. 9. Inhibitory effects of adenosine pretreatment on the binding of IL-1ß-stimulated HGF to activated T lymphocytes. HGF were cultured in the presence or absence of IL-1ß (25 U/ml) with or without adenosine (100 µM) for 15 h at 37°C. T lymphocytes, which had been stimulated with PMA (0.5 µg/ml) for 15 h and subsequently labeled with 51Cr, were incubated on the monolayers of the IL-1ß-activated HGF in the presence or absence of 84H10 (5 µg/ml) for 30 min at 37°C. Percent binding of each well was calculated as described in Methods. Data are shown as the mean ± SE percent of three wells. Results of one representative experiment among five identical experiments are shown. Asterisks indicate a statistically significant difference (P < 0.05, by Student's t-test) compared with the culture of IL-1ß-stimulated HGF without adenosine or 84H10.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study, we found that adenosine or its related analogue up-regulates both IL-1ß-induced cytokine production and HA synthase expression by HGF and down-regulates IL-1ß-induced ICAM-1 expression on HGF, resulting in suppression of lymphocyte adherence to HGF.

We observed that 2-CADO, CPA (an A1-specific agonist) and CGS21680 (an A2a-specific agonist) synergistically enhanced the IL-1ß-induced IL-6 and IL-8 production by HGF (Figs 2 and 3GoGo). These findings suggest that signal(s) via both A1 and A2a adenosine receptor subtypes may play a central role for the up-regulation of IL-6 and IL-8 expression by the activated HGF. Interestingly, however, it was shown that adenosine or adenosine receptor agonists suppressed pro-inflammatory cytokine production by LPS- or IL-1ß-activated human monocytes and endothelial cells (23–27). These findings, along with this study, suggest that the effects of adenosine on cytokine production vary between different cell types and between different stimuli.

Recent work has shown that at least four distinct adenosine receptor subtypes were identified and that each receptor subtype can transduce different signals intracellularly (17). The present study demonstrated that HGF expressed A1, A2a and A2b receptor subtype mRNA, but not A3 mRNA (Fig. 1Go). In contrast, mRNA expression of all four receptor subtypes (A1, A2a, A2b and A3) was detected in endothelial cells and human monocytes (17,29,30,34–36). In addition to the difference in receptor subtype(s), the expression ratio between the subtypes may be distinct (Fig. 1Go). Furthermore, our preliminary experiments showed that treatments of HGF with LPS, PMA, IL-1ß, IFN-{gamma} or adhesive interactions with lymphocytes did not have an influence on the expression of A1 or A2b subtype mRNA, while stimulation of HGF with LPS, PMA or IL-1ß increased the expression of A2a subtype mRNA. The expression of A3 receptor mRNA could not be detected even following activation of HGF with LPS, PMA, IL-1ß, IFN-{gamma} or adhesion with lymphocytes. These findings suggest that the pattern of expression in adenosine receptor subtypes depends on each cell type and its activation status, which may in part explain the disparity of effects of adenosine on different target cells.

So far, it has been demonstrated that activation of adenosine receptors affects the intracellular levels of cAMP and inositol triphosphate, and the activation of phospholipase C, protein kinase A and C. In terms of inflammatory cytokine production by HGF, we have previously demonstrated that adenosine regulates the production of IL-6 by HGF probably via the cAMP/protein kinase A pathway (37). On the other hand, it was recently reported that IL-1ß-induced ICAM-1 expression on HGF was down-regulated by intracellular cAMP elevation (38). Thus, it can be speculated that ligation of adenosine receptors with adenosine differentially regulates the expression of IL-6, IL-8 and ICAM-1 by HGF probably through the up-regulation of intracellular cAMP level.

In this study, we investigated the effect of adenosine on expression of ECM for the first time. Production of ECM is one of the critical functions of HGF. The matrices products are not only constituents of intercellular space, but also play multifunctional roles in the growth and differentiation of cells during the course of inflammatory reactions and process of wound healing. This study demonstrated that adenosine induced the expression of HA synthase mRNA, which is responsible for HA production in HGF (Figs 4 and 6GoGo), but did not influence the constitutive mRNA expression of collagen type I, III, laminin or fibronectin. In addition, IL-1ß-induced HA synthase mRNA expression was further up-regulated when the gingival fibroblasts were stimulated with IL-1ß in the presence of 2-CADO (Fig. 7Go). The fact that 2-CADO and CPA but not CGS21680 induced the expression of HA synthase mRNA (Fig. 5Go) suggests that A1 adenosine receptor may be responsible for the up-regulation of HA synthase mRNA expression by HGF.

Our previous work suggests that the adhesive interactions of HGF to lymphocytes are profoundly involved in the retention and accumulation of lymphocytes in inflamed periodontal tissues (7–12). Furthermore, it was also revealed that the adhesion between HGF and lymphocytes caused mutual cell activation. Thus, the continuous and/or excessive interactions between the cell types may be responsible for periodontal tissue destruction (7–9,11,12). This heterotypic cell–cell adhesion is mediated by a variety of adhesion molecules, such as VLA integrins, LFA-1/ICAM-1 and CD44 (7–9,11,12). In particular, the LFA-1/ICAM-1 pathway plays a critical role when HGF are activated with inflammatory cytokines such as IL-1ß, TNF-{alpha} and IFN-{gamma} (12). In this study, it was demonstrated that adenosine abrogated the IL-1ß-induced expression of ICAM-1 on HGF (Fig. 8Go), and partially inhibited the binding between HGF and activated T lymphocytes (Fig. 9Go). This suggests that locally secreted adenosine in inflamed periodontal tissue may down-regulate lodging and accumulation of lymphocytes. On the other hand, the constitutive expression of ICAM-1, CD44 and VLA integrins on HGF was not altered by adenosine (Fig. 8Go and data not shown). Furthermore, TNF-{alpha}-induced up-regulation of ICAM-1 expression on HGF was not altered by adenosine (data not shown), suggesting that adenosine may have an influence only on IL-1-induced phenomena. As previously demonstrated, the CD44/HA pathway is also involved in the adhesion between activated T lymphocytes and HGF (9). Since adenosine enhanced HA synthase expression by HGF (Fig. 7Go), it may be speculated that the role of the CD44/HA pathway would be increased when the gingival fibroblasts were stimulated with IL-1ß in the presence of 2-CADO. Interestingly, inhibitory effects of adenosine on IL-1ß-induced adhesion was less than that of 84H10 (Fig.8Go). This may be caused at least in part by the increased role of the CD44/HA pathway caused by adenosine treatment.

So far, implication of adenosine for inflammatory processes is not fully defined yet. In the case of periodontal diseases, IL-1ß is one of the key inflammatory cytokines and the increased expression of IL-1ß is closely associated with the disease activity or condition (11), so that HGF in inflamed periodontal lesions are thought to be seriously influenced by locally secreted IL-1ß. Although the present study revealed that occupation of the adenosine receptor modulates the IL-1ß-induced cellular functions of HGF, further investigations are still necessary to define whether adenosine-mediated functions are beneficial or harmful for inflammatory processes of periodontal diseases. As we have discussed above, the difference in the pattern of expression in adenosine receptor subtypes may in part explain the disparity of effects of adenosine on different target cells or tissues. In addition, it is suggested that adenosine may alter the effects on the local inflammatory responses during the course of the inflammatory process. This may be caused in part by the change in the local concentration of adenosine at the site of inflammation. For example, adenosine may promote the pro-inflammatory actions at sites of tissue injury or microbial invasion where relatively lower concentrations of adenosine were released but may be a feedback regulator of inflammation at sites of tissue damage where higher concentrations of adenosine were released and/or accumulated. Thus, in order to pharmacologically regulate the inflammatory responses by adenosine-related analogues, it is very important to determine which adenosine receptor subtype(s) should be activated or inactivated and what concentration of the reagents should be locally achieved.

Further investigation with regard to the functions of adenosine on HGF through distinct receptor subtypes, which may transduce different intracellular messages, will provide greater insight into the biology and pharmacology of adenosine and its related analogues in inflammatory responses in periodontal disease.


    Acknowledgments
 
The authors thank Dr Linda Thompson for her critical review of this manuscript. This work was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture, and from the Japan Society for the Promotion of Science (09307043, 10670965, 11470461, 12672034 and 13307061). T. H. is a recipient of the Fellowship Grant of the KATO Memorial Bioscience Foundation.


    Abbreviations
 
2-CADO 2-chloroadenosine
CPA N6-cyclopentyladenosine
ECM extracellular matrices
HA hyaluronate
HGF human gingival fibroblasts
LPS lipopolysaccharide
PBMC peripheral blood mononuclear cells
PMA phorbol myristate acetate
PMNL polymorphonuclear leukocytes
TNF tumor necrosis factor
XAC xanthine amine congener

    Notes
 
Transmitting editor: T. Hamaoka

Received 7 July 2001, accepted 3 September 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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