Watanabe A, Takeshita A, Kitano S, Hanazawa S
Department of Oral Microbiology, Meikai University School of Dentistry, Sakado City, Saitama, Japan.
Infect Immun. 1996 Nov;64(11):4488-94. doi: 10.1128/iai.64.11.4488-4494.1996.
Lipopolysaccharide (LPS) induces expression of inflammatory cytokines in monocytes/macrophages via CD14, one of the LPS receptors, which is expressed predominantly in these cells. It has been demonstrated that Porphyromonas gingivalis LPS (P-LPS) also is able to induce inflammatory cytokines in human gingival fibroblasts. Therefore, it is important to determine whether CD14 is expressed in gingival fibroblasts and to define the P-LPS-mediated signal-transducing mechanism in the cells. In this study, we observed unexpectedly by immunohistochemical, Western blotting (immunoblotting), and Northern (RNA) blotting assays that CD14 is expressed at high density in human gingival fibroblasts. P-LPS-induced expression of the monocyte chemoattractant protein 1 (MCP-1) gene in the cells was inhibited markedly by treatment with anti-human CD14 antibody and was completely inhibited by herbimycin A, a potent inhibitor of tyrosine kinase. The inhibitor also dramatically inhibited monocyte chemotactic activity of and MCP-1 production by the cells. Furthermore, P-LPS-induced expression of the MCP-1 gene in the cells also was blocked by inhibitors of two transcription factors, i.e., curcumin, an inhibitor of AP-1, and pyrolidine dithiocarbamate, an inhibitor of NF-kappaB. Both inhibitors inhibited monocyte chemotactic activity in the culture supernatant of P-LPS-treated cells. Gel shift mobility assay showed stimulation of the AP-1 and NF-kappaB contents in P-LPS-treated cells. This study is the first to demonstrate the expression of CD14 in human gingival fibroblasts and to show that the signal-transducing pathway of P-LPS in the cells is mediated by CD14.
脂多糖（LPS）通过CD14（LPS受体之一，主要在单核细胞/巨噬细胞中表达）诱导单核细胞/巨噬细胞中炎性细胞因子的表达。已证明牙龈卟啉单胞菌脂多糖（P-LPS）也能够诱导人牙龈成纤维细胞中炎性细胞因子的产生。因此，确定CD14是否在牙龈成纤维细胞中表达以及确定P-LPS介导的细胞内信号转导机制非常重要。在本研究中，我们通过免疫组织化学、蛋白质印迹法（免疫印迹）和RNA印迹分析意外观察到，CD14在人牙龈成纤维细胞中高表达。用抗人CD14抗体处理可显著抑制细胞中P-LPS诱导的单核细胞趋化蛋白1（MCP-1）基因的表达，而酪氨酸激酶的强效抑制剂赫比霉素A可完全抑制该表达。该抑制剂还显著抑制细胞的单核细胞趋化活性和MCP-1的产生。此外，细胞中P-LPS诱导的MCP-1基因表达也被两种转录因子抑制剂所阻断，即AP-1抑制剂姜黄素和NF-κB抑制剂吡咯烷二硫代氨基甲酸盐。这两种抑制剂均抑制P-LPS处理细胞培养上清液中的单核细胞趋化活性。凝胶迁移率变动分析显示P-LPS处理细胞中AP-1和NF-κB含量增加。本研究首次证明了CD14在人牙龈成纤维细胞中的表达，并表明细胞中P-LPS的信号转导途径由CD14介导。
