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

Abstract

SUMMARYPeriodontitis 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-k B 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.

B and T Lymphocytes Are the Primary Sources of RANKL in the Bone Resorptive Lesion of Periodontal Disease

“…19 RANKL protein expression has been demonstrated by T cells isolated from periodontitis gingival tissue by flow cytometry. 38 Consistent with our findings that diseased gingival tissue contains much more sRANKL than healthy tissue ( Figure 1A) and that the concentration of sRANKL is greater in deeper gingival crevices ( Figure 1B) is the recent report that the ratio of sRANKL to OPG in gingival crevice fluid (GCF) is elevated in periodontitis patients compared to healthy patients. 39 Therefore, the findings presented herein that RANKL is expressed by T cells and also B cells and that a higher percentage of B cells express RANKL than T cells in periodontitis tissue provides information quite relevant to the elucidation of bone resorptive factors in periodontal disease.…”

“…17 Sakata and colleagues 41 reported that dental mesenchymal cells produce OPG and enhance OPG production in response to proinflammatory factors such as IL-1␤ or tumor necrosis factor (TNF)-␣. Nagasawa and colleagues 38 showed that lipopolysaccharide stimulation of gingival fibroblasts induced OPG expression and inhibited differentiation of monocytes to osteoclast cells. Although the ELISA system in the present study detected OPG protein production in both healthy and diseased gingival tissues, mRNA message for OPG was not detected in healthy gingival tissue as measured by RT-PCR.…”

MicroRNA‐31a‐5p from aging BMSCs links bone formation and resorption in the aged bone marrow microenvironment

“…Once the BMSCs reached 80%–90% confluence, cells were cultured with fresh α‐MEM for 24 hr for further investigation. Next, the supernatant was collected and stored at −20°C (Nagasawa et al., 2002). To induce osteogenic differentiation, cells were cultured in an osteogenic medium containing complete medium supplemented with 50 μM ascorbic acid, 10 mM β‐glycerophosphate, and 10 −7  M dexamethasone.…”

“…Then, nonadherent cells in the supernatant were collected and seeded in 12‐well plates at a density of 1 × 10 6 and cultured in complete medium with M‐CSF (20 ng/ml) for the first 3 days. Three days later, these cells were refreshed with supernatant supplemented with 15% FBS and 1% penicillin/streptomycin, M‐CSF (20 ng/ml) and RANKL (20 ng/ml) every 3 days (Nagasawa et al., 2002). …”

RANKL expression is differentially modulated by TLR2 and TLR4 signaling in fibroblasts and osteoblasts

“…LPS stimulation may have a positive role in the modulation of osteoclastogenesis by inducing RANKL and/or inhibiting OPG expression or a negative role by reducing the expression of RANK or M-CSF receptor on osteoclast precursor cells [18]. In human gingival fibroblasts, LPS had a bone-protective effect by inducing the production of OPG [19]. In this study we present data on the differential regulation of RANKL and OPG gene expression after activation of TLR2, -4 and IL1R in fibroblasts and osteoblasts, providing insight into the relative contribution of p38 MAPK and NF-kB signaling to the expression of these genes in each cell type.…”