HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Figures Only Full Text (PDF) Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (42) Citing Articles via Google Scholar Google Scholar Articles by Mogi, M. Articles by Togari, A. Search for Related Content PubMed PubMed Citation Articles by Mogi, M. Articles by Togari, A.

Differential Expression of RANKL and Osteoprotegerin in Gingival Crevicular Fluid of Patients with Periodontitis

¹ Department of Pharmacology, School of Dentistry, Aichi-Gakuin University, Nagoya 464-8650, Japan; and
² Department of Periodontology, Matsumoto Dental University School of Dentistry, Shiojiri, Nagano 399-0781, Japan;

^* corresponding author, makio{at}dpc.aichi-gakuin.ac.jp

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
The receptor activator for NF-B ligand (RANKL) plays an important role in osteoclast formation. A recent study with animal models suggests the involvement of RANKL in the pathogenesis of this periodontal disease. However, no one has examined the level of RANKL in the body fluid of human subjects. This communication reports on the in vivo concentrations of RANKL and the RANKL decoy receptor osteoprotegerin (OPG) in the gingival crevicular fluid (GCF) of periodontal subjects with severe, moderate, and mild forms of the disease. An increased concentration of RANKL and a decreased concentration of OPG were detected in GCF from patients with periodontitis (*p < 0.05 vs. control subjects). The ratio of the concentration of RANKL to that of OPG in the GCF was significantly higher for periodontal disease patients than for healthy subjects (*p < 0.01). Taken together, these data suggest that RANKL and OPG contribute to osteoclastic bone destruction in periodontal disease. Abbreviations: GCF, gingival crevicular fluid; IL, interleukin; OPG, osteoprotegerin; RANKL, receptor activator for NF-B ligand.

KEY WORDS: RANKL • osteoprotegerin • gingival crevicular fluid • periodontal disease

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
The role of cytokines in periodontal disease has been evaluated in several studies (Genco, 1992; Roberts et al., 1997; Mogi et al., 1999). Recent studies have delineated many of the molecules, especially cytokines, required for osteoclast formation. In particular, osteoclast formation from precursor cells, as well as osteoclast activation, requires the receptor activator of NF-B ligand (RANKL; Yasuda et al., 1998). In vivo treatment of mice with RANKL activates osteoclasts, promotes bone loss, and causes severe hypercalcemia (Lacey et al., 1998). Osteoprotegerin (OPG), a secreted glycoprotein, is a decoy receptor for RANKL (Simonet et al., 1997). When OPG is present to bind to RANKL, the cell-to-cell signaling between marrow stromal cells and osteoclast precursors is inhibited, and osteoclasts are not formed (Simonet et al., 1997; Yasuda et al., 1998). Thus, RANKL and decoy receptors OPG expressed by bone-associated cells play important roles during osteoclast formation by balancing induction and inhibition.

Recent evidence indicates that a soluble form of RANKL is secreted by activated T-lymphocytes and osteoblasts (Wong et al., 1997; Kong et al., 1999; Nakashima et al., 2000). More importantly, RANKL secreted by activated T-cells promoted joint inflammation and bone and cartilage destruction in the chronic inflammatory disease rheumatoid arthritis (Kong et al., 1999). In addition to the increase in the level of RANKL protein in the inflamed synovium of rheumatoid arthritis patients, OPG concentrations in the synovial fluid were lower in patients with rheumatoid arthritis (as compared with concentrations in patients with other forms of arthritis), which resulted in an increased local and systemic RANKL:OPG ratio (Kotake et al., 2001). A recent study with animal models suggests the involvement of RANKL and OPG in the pathogenesis of periodontal disease (Teng et al., 2000). Further, several investigators have examined gingival crevicular fluid (GCF) in periodontal disease for cellular immune-response indicators (Cimasoni, 1983; Offenbacher et al., 1986; Reinhardt et al., 1993; Mogi et al., 1999); but thus far, no one has examined the level of RANKL or OPG in human GCF. We hypothesize that imbalances in the RANKL/OPG system may be related to the pathogenesis of periodontitis. To test this hypothesis, we conducted in vivo studies, and found an increased concentration of RANKL and a decreased concentration of OPG in the GCF from patients with periodontitis.

	MATERIALS & METHODS

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Subject Selection
One hundred and thirty-two patients with chronic adult periodontitis around single-rooted teeth were selected from those patients referred to the Department of Periodontology of Matsumoto Dental University. Informed consent was obtained from all participants at the first visit, and the protocol for human participants was reviewed and approved by our institutional panel. Subjects selected were those without systemic disease, treatment for periodontal disease, juvenile periodontitis, acute gingival inflammation, or trauma from occlusion. All were in good general health with no history of antimicrobial or anti-inflammatory therapy or periodontal treatment for 6 mos before the start of the study. The patients were diagnosed based on the status of their entire dentition. Clinical recordings (probing pocket depth, bleeding on probing, clinical attachment level) were made at the mesiobuccal, mesiolingual, distobuccal, and middle of both buccal and lingual surfaces of the teeth examined. Two teeth per patient were in the dentition at baseline. During a preliminary screening, all periodontitis and control group patients met the following criteria: (1) a minimum of 20 yrs of age, (2) a minimum of 20 natural teeth excluding third molars, and (3) more than 70% of teeth in the same classification (see below) in each subject. Patients were classified according to pocket depth, attachment level, and bleeding on probing. The distance from the cemento-enamel junction to the bottom of the gingival pocket was measured for attachment level, and the distance from the gingival margin to the bottom of the pocket for probing depth. One examiner did all recordings. Patients included in the severe periodontitis group (47 patients; mean age, 42.5 yrs; collection site, n = 94) had pocket depths (8.47 ± 1.35 mm) and attachment loss greater than 7 mm, with bleeding on probing at each tooth. All patients having pocket depths (5.22 ± 0.71) and attachment loss of less than 6 and 4 mm, respectively, with bleeding on probing were defined as having moderate periodontitis (58 patients; mean age, 45.7 yrs; collection site, n = 116), and those having pocket depths (2.51 ± 0.47) and attachment loss of less than 3 mm with bleeding on probing were defined as having mild periodontitis (27 patients; mean age, 37.5 yrs; collection site, n = 54). Individuals in the control group (28 persons; mean age, 37.8 yrs; collection site, n = 56) were healthy, with pocket depths of less than 2 mm and no attachment loss, bleeding on probing, or radiographic bone loss.

Sample Collection
The GCF was sampled by the method of Offenbacher et al.(1986) with slight modification (Uematsu et al., 1996). We carefully removed all clinically detectable supragingival plaque without touching the gingiva, to minimize contamination of the paper strips by the plaque. The teeth with a single root were gently washed with water, and the sites under study were isolated with cotton rolls and gently dried with an air syringe. One paper strip was used for each collection site. Two sites were selected in each of the diseased participants and the healthy controls. Paper strips (Periopaper, Harco, Tustin, CA, USA) were carefully inserted 1 mm into the gingival crevice and left there for 30 sec. The volume of GCF in the periopaper was measured with a Periotron (Harco, Tustin, CA, USA). The paper strips from the individual sites were stored at -80°C for later processing. Paper strips for each participant were pooled, and the GCF was extracted and assayed for the content of RANKL and OPG. GCF was extracted from the paper strips with buffer (50 mM phosphate buffer, pH 7.2, containing protease inhibitors: 0.1 mM phenylmethylsulphonylfluoride and 5 µg/mL each of leupeptin, pepstatin, amastatin, chemostatin, and antipain), and collected following centrifugation.

RANKL and OPG Determination
RANKL was measured by a two-site ELISA (Kinpara et al., 2000); and OPG, by use of a commercially available two-site sandwich ELISA kit (R&D System, Minneapolis, MN, USA). All samples and standards were assayed twice. Data were reported as the concentrations of cytokine (pg/µL of GCF). Data were presented as the means ± SEM.

Statistical Analysis
Statistical analysis of differences in values between periodontal and control subjects was performed by ANOVA. The Spearman rank order correlation coefficient (r) was determined for the relationship between modulators and periodontal disease severity. The Mann-Whitney U-test was used to assess bleeding on probing.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
RANKL concentrations in the GCF of control subjects were below the limit of detection except in one case (Fig., A). Concentrations of RANKL in GCF were significantly higher in patients with each degree of periodontitis than in the healthy controls. There was a significant difference between mild periodontitis and moderate or severe periodontitis groups in terms of the RANKL concentration (p < 0.05; Fig., A).

View larger version (23K): [in this window] [in a new window]   Figure. Plots depicting RANKL (pg/µL; A) and OPG (pg/µL; B) concentrations and RANKL/OPG ratio (C) in the subgingival crevices of control, mild, moderate, and severely diseased subjects. The content of RANKL in the GCF was determined by an ELISA, and that of OPG also by ELISA, a commercially available one. To improve the display of data in the Fig., we made the scales in ‘A’ and ‘B’ the same. Data are expressed as the mean ± SEM. Control (28), mild (27), moderate (58), and severe (47) subjects were analyzed. * P < 0.05, ** P < 0.01, *** P < 0.005 (compared with control)), and # P < 0.05 as indicated by the brackets.

To provide a better definition of the relationship between the alteration of modulators and the conditions of periodontitis, we examined the association of these modulator concentrations with specific indicators of disease severity (such as pocket probing depth and attachment level) and gingival inflammation (bleeding). The concentration of RANKL decreased as the pocket probing depth increased. Also, there was no association between the RANKL concentration and gingival inflammation. Furthermore, OPG concentrations were also not associated significantly with clinical measures of disease severity.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
This study clearly demonstrated an increase in RANKL levels and a significant decrease in OPG levels in the GCF of patients with periodontitis. What is the reason for these alterations in RANKL and OPG levels in the GCF? Most of the soluble form of RANKL in the GCF originates from activated T-lymphocytes, but some is released from other cells (especially osteoblasts) in the periodontal tissues. OPG mRNA is found in osteoblasts, gingival fibroblasts, and periodontal ligament cells (Sakata et al., 1999). Various pro-inflammatory and pro-resorptive cytokines have been detected in the GCF from patients with periodontitis (Cimasoni, 1983; Offenbacher et al., 1986; Reinhardt et al., 1993; Mogi et al., 1999). Interestingly, tumor necrosis factor-, IL (interleukin)-1ß, IL-6, and prostaglandin E₂ have all been demonstrated to enhance RANKL mRNA and protein levels, whereas these cytokines and prostaglandin E₂ were shown to inhibit, concurrently, OPG mRNA and protein levels in osteoblasts (Hofbauer et al., 2000; Nakashima et al., 2000). Therefore, we speculate that the alteration of RANKL and OPG levels in the GCF reflects the comprehensive biological responses that occur during the process of periodontitis.

Regarding periodontitis, what is the biological role of RANKL and OPG? Since OPG is locally present in excess amounts over RANKL under physiological conditions, the RANKL level may be determined by OPG, which traps RANKL at local sites, and all the RANKL complexed with OPG may be released into the circulation. Therefore, an increase in local and systemic RANKL:OPG ratios is important for RANKL action in vivo. We previously demonstrated an increase in the RANKL:OPG ratio in the synovial fluid in patients with rheumatoid arthritis (Kotake et al., 2001). Interestingly, the RANKL:OPG ratio for the GCF of periodontitis patients in the current study is higher than that for the synovial fluid of patients with rheumatoid arthritis. The RANKL/OPG ratio was also increased and correlated with markers of bone resorption, osteolytic lesions, and markers of disease activity in multiple myeloma (Terpos et al., 2003). Although we could not determine the bioactivities for RANKL and OPG because of the small amount of these cytokines in the GCF, these results suggest that excess production of RANKL and the decrease in OPG in the periodontium of patients with periodontitis contributed to osteoclastic bone resorption there.

We could not demonstrate that the RANKL and/or OPG concentrations were associated significantly with clinical measurements of disease severity (e.g., pocket depth and attachment level) and inflammation (e.g., % of sites bleeding when probed). The patients with mild periodontitis had a higher concentration of RANKL in their GCF than those with moderate or severe periodontitis (Fig.). OPG in the GCF from periodontal patients also decreased as disease severity increased (Fig.). These findings are perhaps not surprising, because the patients with moderate and/or severe periodontitis had fewer bone-associated cells and less bone-surrounding tissue because of bone resorption; therefore, the cells supplying RANKL and OPG would also be fewer, thus resulting in a smaller quantity of these modulators being released into the GCF.

Although we examined whether a reduction in a protective factor (OPG) is directly related to the risk for periodontitis, we have no definite answer at present. In vitro administration of OPG prevented osteoclastogenesis by synovial fibroblasts derived from patients with rheumatoid arthritis, proving the principle that blocking RANKL through administration of OPG may provide a novel therapeutic strategy in rheumatoid arthritis (Kong et al., 1999). Teng et al.(2000) also demonstrated that stimulation of CD4⁺ T-cells by A. actinomycetemcomitans induced production of RANKL and osteoclast activation and that in vivo inhibition of RANKL function with OPG diminished alveolar bone destruction and reduced the number of periodontal osteoclasts. Analysis of these data therefore suggests that RANKL is an important cytokine that mediates bone destruction in human rheumatoid arthritis and periodontitis, and that OPG, as a decoy receptor of RANKL, has a protective role in bone-destructive diseases. Previous data, taken together with our current findings, suggest that manipulation of the expression of RANKL and/or OPG in patients with periodontitis may be a promising new treatment strategy for the inhibition of bone destruction.

	ACKNOWLEDGMENTS

 
This work was supported by a grant-in-aid from the program Scientific Frontier Promoted Research and by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan (nos. 12671821 and 15591986 to M.M.).

Received January 2, 2003; Last revision August 29, 2003; Accepted December 3, 2003

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Cimasoni G (1983). Crevicular fluid updated. Monogr Oral Sci 12:1–152.

Genco RJ (1992). Host responses in periodontal diseases: current concepts. J Periodontol 63:338–355.[ISI][Medline]

Hofbauer LC, Khosla S, Dunstan CR, Lacey DL, Boyle WJ, Riggs BL (2000). The roles of osteoprotegerin and osteoprotegerin ligand in the paracrine regulation of bone resorption. J Bone Miner Res 15:2–12.[ISI][Medline]

Kinpara K, Mogi M, Kuzushima M, Togari A (2000). Osteoclast differentiation factor in human osteosarcoma cell line. J Immunoassay 21:327–340.[ISI][Medline]

Kong YY, Feige U, Sarosi I, Bolon B, Tafuri A, Morony S, et al. (1999). Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand. Nature 402:304–309.[Medline]

Kotake S, Udagawa N, Hakoda M, Mogi M, Yano K, Tsuda E, et al. (2001). Activated human T cells directly induce osteoclastogenesis from human monocytes: possible role of T cells in bone destruction in rheumatoid arthritis patients. Arthritis Rheum 44:1003–1012.[ISI][Medline]

Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, et al. (1998). Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93:165–176.[ISI][Medline]

Mogi M, Otogoto J, Ota N, Inagaki H, Minami M, Kojima K (1999). Interleukin 1beta, interleukin 6, beta2-microglobulin, and transforming growth factor-alpha in gingival crevicular fluid from human periodontal disease. Arch Oral Biol 44:535–539.[ISI][Medline]

Nakashima T, Kobayashi Y, Yamasaki S, Kawakami A, Eguchi K, Sasaki H, et al. (2000). Protein expression and functional difference of membrane-bound and soluble receptor activator of NF-kappaB ligand: modulation of the expression by osteotropic factors and cytokines. Biochem Biophys Res Commun 275:768–775.[ISI][Medline]

Offenbacher S, Odle BM, Van Dyke TE (1986). The use of crevicular fluid prostaglandin E₂ levels as a predictor of periodontal attachment loss. J Periodontal Res 21:101–112.[ISI][Medline]

Reinhardt RA, Masada MP, Kaldahl WB, DuBois LM, Kornman KS, Choi JI, et al. (1993). Gingival fluid IL-1 and IL-6 levels in refractory periodontitis. J Clin Periodontol 20:225–231.[ISI][Medline]

Roberts FA, McCaffery KA, Michalek SM (1997). Profile of cytokine mRNA expression in chronic adult periodontitis. J Dent Res 76:1833–1839.[Abstract/Free Full Text]

Sakata M, Shiba H, Komatsuzawa H, Fujita T, Ohta K, Sugai M, et al. (1999). Expression of osteoprotegerin (osteoclastogenesis inhibitory factor) in cultures of human dental mesenchymal cells and epithelial cells. J Bone Miner Res 14:1486–1492.[ISI][Medline]

Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, et al. (1997). Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 89:309–319.[ISI][Medline]

Teng YT, Nguyen H, Gao X, Kong YY, Gorczynski RM, Singh B, et al. (2000). Functional human T-cell immunity and osteoprotegerin ligand control alveolar bone destruction in periodontal infection. J Clin Invest 106:R59–R67.

Terpos E, Szydlo R, Apperley JF, Hatjiharissi E, Politou M, Meletis J, et al. (2003). Soluble receptor activator of nuclear factor kappaB ligand-osteoprotegerin ratio predicts survival in multiple myeloma: proposal for a novel prognostic index. Blood 102:1064–1069.[Abstract/Free Full Text]

Uematsu S, Mogi M, Deguchi T (1996). Interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha, epidermal growth factor, and beta₂-microglobulin levels are elevated in gingival crevicular fluid during human orthodontic tooth movement. J Dent Res 75:562–567.[Abstract/Free Full Text]

Wong BR, Josien R, Lee SY, Sauter B, Li HL, Steinman RM, et al. (1997). TRANCE (tumor necrosis factor (TNF)-related activation-induced cytokine), a new TNF family member predominantly expressed in T cells, is a dendritic cell-specific survival factor. J Exp Med 186:2075–2080.[Abstract/Free Full Text]

Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki SI, et al. (1998). Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 95:3597–3602.[Abstract/Free Full Text]

This article has been cited by other articles:

				G.E. Wise and G.J. King Mechanisms of Tooth Eruption and Orthodontic Tooth Movement J. Dent. Res., May 1, 2008; 87(5): 414 - 434. [Abstract] [Full Text] [PDF]

				N. Bostanci, G. Emingil, B. Afacan, B. Han, T. Ilgenli, G. Atilla, F.J. Hughes, and G.N. Belibasakis Tumor Necrosis Factor-{alpha}-converting Enzyme (TACE) Levels in Periodontal Diseases J. Dent. Res., March 1, 2008; 87(3): 273 - 277. [Abstract] [Full Text] [PDF]

				T. Kawai, T. Matsuyama, Y. Hosokawa, S. Makihira, M. Seki, N. Y. Karimbux, R. B. Goncalves, P. Valverde, S. Dibart, Y.-P. Li, et al. B and T Lymphocytes Are the Primary Sources of RANKL in the Bone Resorptive Lesion of Periodontal Disease Am. J. Pathol., September 1, 2006; 169(3): 987 - 998. [Abstract] [Full Text] [PDF]

				U.H. Lerner Inflammation-induced Bone Remodeling in Periodontal Disease and the Influence of Post-menopausal Osteoporosis. J. Dent. Res., July 1, 2006; 85(7): 596 - 607. [Abstract] [Full Text] [PDF]

				T. Wakita, M. Mogi, K. Kurita, M. Kuzushima, and A. Togari Increase in RANKL: OPG Ratio in Synovia of Patients with Temporomandibular Joint Disorder. J. Dent. Res., July 1, 2006; 85(7): 627 - 632. [Abstract] [Full Text] [PDF]

				P. Kang, J. Korostoff, A. Volgina, W. Grzesik, and J. M DiRienzo Differential effect of the cytolethal distending toxin of Actinobacillus actinomycetemcomitans on co-cultures of human oral cells J. Med. Microbiol., August 1, 2005; 54(8): 785 - 794. [Abstract] [Full Text] [PDF]

				P. Valverde, T. Kawai, and M.A. Taubman Potassium Channel-blockers as Therapeutic Agents to Interfere with Bone Resorption of Periodontal Disease J. Dent. Res., June 1, 2005; 84(6): 488 - 499. [Abstract] [Full Text] [PDF]

				D.D. Bosshardt Are Cementoblasts a Subpopulation of Osteoblasts or a Unique Phenotype? J. Dent. Res., May 1, 2005; 84(5): 390 - 406. [Abstract] [Full Text] [PDF]

				G. N. Belibasakis, A. Johansson, Y. Wang, C. Chen, S. Kalfas, and U. H. Lerner The Cytolethal Distending Toxin Induces Receptor Activator of NF-{kappa}B Ligand Expression in Human Gingival Fibroblasts and Periodontal Ligament Cells Infect. Immun., January 1, 2005; 73(1): 342 - 351. [Abstract] [Full Text] [PDF]

This Article Abstract Figures Only Full Text (PDF) Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (42) Citing Articles via Google Scholar Google Scholar Articles by Mogi, M. Articles by Togari, A. Search for Related Content PubMed PubMed Citation Articles by Mogi, M. Articles by Togari, A.