LPS-stimulated human gingival fibroblasts inhibit the differentiation of monocytes into osteoclasts through the production of osteoprotegerin
LPS-stimulated human gingival fibroblasts inhibit the differentiation of monocytes into osteoclasts through the production of osteoprotegerin
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DOI：
10.1046/j.1365-2249.2002.01990.x
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发表时间：
2002-11-01
影响因子：
4.6
通讯作者：
Ishikawa, I
中科院分区：
文献类型：
--
作者：
Nagasawa, T;Kobayashi, H;Ishikawa, I
牙周炎是由革兰氏阴性厌氧菌引起的一种炎症性骨病，但其确切的骨质破坏机制尚不清楚。活化的T淋巴细胞分泌核因子-kappaB受体激活物(RANKL)，支持单核细胞向成熟破骨细胞分化。本研究的目的是检测RANKL及其抑制物骨保护素(OPG)在炎症牙龈组织中的表达，探讨人牙龈成纤维细胞(HGFs)在RANKL调节破骨细胞生成中的作用。从慢性牙周炎患者常规牙周手术中获取HGFs和牙周单个核细胞(GMC)。RT-PCR法检测牙周组织和HGFs中OPG和RANKL基因的表达。用双抗体夹心法测定OPG产量。用RANK-Fc融合蛋白和相应的单抗，用流式细胞仪检测GMC表面RANKL、CD4、CD8和CD69的表达。用重组人RANKL和巨噬细胞集落刺激因子(M-CSF)共同培养人外周血单核细胞10天后，通过计数抗酒石酸酸性磷酸酶(TRAP)阳性细胞数来检测RANKL的破骨细胞生成情况。OPG和RANKL在牙周炎病变中的表达分别为80%(16/20)和25%(5/20)。在HGFs中表达OPG，而不表达RANKL。脂多糖刺激后，HGFs的OPG mRNA表达和产量均增加。所有GMC标本均表达CD69，5例GMC中2例表达RANKL。牙周成纤维细胞培养上清可显著减少RANKL和M-CSF联合培养单核细胞所产生的TRAP阳性细胞。本研究提示，内毒素刺激的HGFs通过产生OPG抑制单核细胞向破骨细胞的分化。
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-kappa 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.