Study of Host Defense Mechanism by Calprotectin in Periodontal Disease

钙卫蛋白在牙周病中宿主防御机制的研究

• 批准号: 13672188
• 负责人: KIDO Jun-ichi
• 金额: $ 1.98万
• 依托单位: The University of Tokushima
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672188/
• 关键词: Calprotectin; Perio dontopathic bacteria; Lipopolysaccharide; Neutrophil; Periodontal disease; Host defense

The existence of calprotectim (CPT), a leukocyte protein, in gingival tissue with periodontal disease and the effect of lipopolysaccharide (LPS) from periodontopahic bacteria on CPT expression were investigated to elucidate the regulatory mechanism of CPT in periodontal diseases. Gingival tissue was obtained from a periodontitis parient and immunostained using anti-CPT antibody (Ab). Neutrophils were separated from human healthy donors and incubated with LPS from P. gingivalis. CPT in cell and medium fractions was identified by immunobolotting analysis, and its amount was determined by ELISA. CPT was detected in the connective tissue with inflammatory cells and epithelium. P-LPS increased CPT release from neutrophils after 30 min incubation in a dose-dependent manner (0.1-1,000 ng/ml), and the released amount was increased to about 16 times that of control level by 1,000 ng/ml P-LPS. LPSs from A. actinomycelemcomitans, P. intermedia, F. nucleatum also induced calprotectin release, and P-LPS produced the maximal effect. It is known that CD14, Toll-like receptor (TLR) 2&4 and NF-_KB play roles in LPS signal transduction. To study the mechanism of P-LPS-induced CPT release, the effects of anti-CD14 and TLR Abs and NF-_KB inhibitors on P-LPS-induced CPT release were investigated. The anti-CD14 and TLR2 Abs significantly inhibited P-LPS-induced CPT release, but anti-TLR4 Abs had no effect. Five NF-_KB inhibitors blocked P-LPS-induced NF-_KB binding activity (Gel shift assay) and CPT release from neutrophils. Futhermore, P-LPS did not increase the expression of MRP8/14, subunits of CRT, mRNAs when they were investigated by RT-PCR. From these results, it is suggested that LPSs from periodontopahic bacteria including P. gingivalis induce CTP release from neutrophils and that CPT release is induced by P-LPS via the CD14-TLR2-NF_KB signal pathway

本研究旨在探讨白细胞蛋白钙卫蛋白（calprotectim，CPT）在牙周病牙龈组织中的存在及牙周炎细菌脂多糖（lipopolysaccharide，LPS）对CPT表达的影响，以阐明CPT在牙周病中的调控机制。牙龈组织从牙周炎患者获得，并使用抗CPT抗体（Ab）进行免疫染色。从人类健康供体中分离中性粒细胞，并与牙龈卟啉单胞菌的LPS一起孵育。通过免疫印迹分析鉴定细胞和培养基组分中的CPT，并通过ELISA测定其量。CPT主要分布于结缔组织、炎性细胞和上皮细胞。P-LPS以剂量依赖性方式（0.1 - 1，000 ng/ml）增加中性粒细胞孵育30 min后CPT的释放，1，000 ng/ml P-LPS使CPT的释放量增加至对照水平的约16倍。从A. actinomycelemcomitans、中间青霉（P.intermedia）、F.核质也能诱导钙卫蛋白的释放，其中P-LPS的作用最大。已知CD14、Toll样受体（TLR）2和4以及NF-κ B在LPS信号转导中起作用。为了研究P-LPS诱导CPT释放的机制，研究了抗CD14抗体、TLR抗体和NF-κ B抑制剂对P-LPS诱导CPT释放的影响。抗CD14和TLR2抗体能显著抑制P-LPS诱导的CPT释放，而抗TLR4抗体对CPT释放无影响。五种NF-κ B抑制剂阻断P-LPS诱导的NF-κ B结合活性（凝胶迁移试验）和中性粒细胞释放CPT。RT-PCR结果显示，LPS、Fuelin对MRP8/14、CRT亚单位mRNA的表达无明显影响。提示牙周炎细菌（包括牙龈卟啉单胞菌）的LPS可诱导中性粒细胞释放CTP，而P-LPS通过CD14-TLR2-NF_KB信号通路诱导CPT的释放