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. 2002 Nov;130(2):338-44.
doi: 10.1046/j.1365-2249.2002.01990.x.

LPS-stimulated human gingival fibroblasts inhibit the differentiation of monocytes into osteoclasts through the production of osteoprotegerin

T Nagasawa  1 , H Kobayashi, M Kiji, M Aramaki, R Mahanonda, T Kojima, Y Murakami, M Saito, Y Morotome, I Ishikawa
Affiliations

LPS-stimulated human gingival fibroblasts inhibit the differentiation of monocytes into osteoclasts through the production of osteoprotegerin

T Nagasawa et al. Clin Exp Immunol. 2002 Nov.

Abstract

Periodontitis is an inflammatory bone disease caused by Gram-negative anaerobic bacteria, but the precise mechanism of bone destruction remains unknown. Activated T lymphocytes secrete receptor activator of NF-kappaB ligand (RANKL) and support the differentiation of monocytes into mature osteoclasts. The purpose of this study was to examine the expression of RANKL and its inhibitor, osteoprotegerin (OPG), in inflamed gingival tissue and to clarify the role of human gingival fibroblasts (HGFs) in osteoclastogenesis regulated by RANKL. HGFs and gingival mononuclear cells (GMCs) were obtained from chronic periodontitis patients during routine periodontal surgery. Expression of OPG and RANKL mRNA in gingival tissue and HGFs was examined with RT-PCR. OPG production was measured using ELISA. Expression of RANKL, CD4, CD8 and CD69 on GMCs was determined by flow-cytometry using RANK-Fc fusion protein and the respective monoclonal antibodies. Osteoclastogenesis by RANKL was assayed by counting the number of tartarate-resistant acid phosphatase (TRAP)-positive cells after culturing human peripheral blood monocytes with recombinant human RANKL and macrophage-colony stimulating factor (M-CSF) for 10 days. OPG and RANKL mRNA were expressed in 80% (16/20) and 25% (5/20) of periodontitis lesions, respectively. OPG, but not RANKL, mRNA was expressed within HGFs. OPG mRNA expression and production by HGFs was augmented by LPS stimulation. All GMC samples expressed CD69, and two of five GMC samples expressed RANKL. The culture supernatant of LPS-stimulated gingival fibroblasts significantly reduced the number of TRAP positive cells generated by culturing monocytes with RANKL and M-CSF. The present study suggests that LPS-stimulated HGFs inhibit monocyte differentiation into osteoclasts through the production of OPG.

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Figures

Fig. 1
Fig. 1
(a) Time-course of OPG and GAPDH mRNA expression in HGFs stimulated with E. coli LPS. HGFs were stimulated with E. coli LPS and collected 6, 12, 24 and 48 h after stimulation. RT-PCR analysis for OPG and GAPDH was carried out as described in the Materials and methods. The results were representative of five timed experiments. (b) OPG/GAPDH mRNA ratio in HGFs stimulated with E. coli LPS. Expression intensity of OPG and GAPDH mRNA was calculated with a SYNGENE Bio Imaging system and the OPG/GAPDH mRNA ratio was calculated. The results were representative of five experiments.
Fig. 2
Fig. 2
(a) Expression of OPG and GAPDH mRNA in different HGF lines following stimulation with E. coli LPS. HGFs were stimulated with E. coli LPS and collected 24 h after stimulation. RT-PCR analysis for OPG and GAPDH was carried out as described in the Materials and methods. The results were representative of five experiments. (b) Production of OPG in different HGF lines stimulated with E. coli LPS. HGFs were stimulated with E. coli LPS and their culture supernatant collected 24 h after stimulation. The relative amount of OPG within the supernatant of each culture was measured using ELISA as described in the Materials and methods. The results were representative of five experiments.
Fig. 3
Fig. 3
(a) Expression of CD69 in CD4+ and CD8+ GMCs. GMCs were prepared from inflamed gingival tissue and stained with anti-CD4, CD8 and CD69 antibodies. Representative results from five experiments are shown. (b) Expression of RANKL on GMCs. GMCs were prepared from inflamed gingival tissue and stained with RANK-Fc fusion protein, followed by FITC-conjugated protein A. Representative results from five experiments are shown.
Fig. 4
Fig. 4
(a) Effect of HGF culture supernatant on the differentiation of monocytes into TRAP-positive cells. Peripheral blood monocytes were cultured with RANKL and M-CSF in the presence or absence of supernatant from HGF culture (HGF sup) or supernatant from an E. coli LPS-stimulated HGF culture (LPS-HGF sup). The number of TRAP-positive cells in each sample was calculated 10 days after cultivation. The results were representative of five experiments. (b) Effect of anti-OPG neutralizing antibody on the inhibitory effect of HGF culture supernatant. Peripheral blood monocytes were cultured with RANKL, M-CSF and supernatant from E. coli LPS-stimulated HGF culture (LPS-HGF sup) in the presence or absence of anti-OPG neutralizing antibody. The number of TRAP-positive cells in each sample was calculated 10 days after cultivation. The results were representative of five experiments. *P < 0·05.
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