BACTEROIDES LPS/CYTOKINE REGULATION IN INFLAMED GINGIVAL TISSUE

发炎牙龈组织中拟杆菌脂多糖/细胞因子的调节

• 批准号: 6270281
• 负责人: Suzanne M. Michalek
• 金额: $ 20.82万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-12-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6270281
• 关键词: Bacteroides; Bacteroides gingivalis; bactericidal immunity; biopsy; cytokine; enzyme linked immunosorbent assay; fibroblasts; gene expression; gingiva; human tissue; immunocytochemistry; immunopathology; in situ hybridization; inflammation; interleukin 1; interleukin 6; interleukin 8; laboratory mouse; leukocyte activation /transformation; lipopolysaccharides; macrophage; messenger RNA; monocyte; neutralizing antibody; oral bacteria; periodontitis; polymerase chain reaction; tissue /cell culture; tumor necrosis factor alpha

Periodontitis is a chronic inflammatory disease of periodontal tissues in which the black-pigmented Bacteroides species, particularly Porphyromonas (Bacteroides) gingivalis, have been implicated as etiologic agents. Microbial components of the Bacteroides, including their lipopolysaccharide (LPS), have been implicated in the initial infiltrate of lymphocytes, monocytes/macrophages and neutrophils which is characteristic of this disease. However, the microbial and host related mechanisms involved are not fully understood. Bacterial LPS has the ability to activate host cells for the production of inflammatory factors such as interleukin-1 (IL-1), tumor-necrosis factor-alpha (TNF-alpha), IL-6, IL-8 and IL-1 inhibitor. IL-8 is a chemoattractant for PMNs and T lymphocytes and its production is induced by TNF-alpha, IL-1 and LPS. Therefore, IL-8 along with other cytokines and microbial LPS may be involved in self-amplifying loops in promoting cellular events associated with inflammatory disease. It is likely that modulation of cellular function is mediated by changes in gene expression which can result from direct stimulation by LPS and/or by cellular products such as IL-1, TNF- alpha or IFN-gamma. Classical LPS (e.g., LPS derived from Escherichia coli) is a potent inducer of cytokines, especially IL-1, but only recently have we begun to understand the molecular mechanisms involved in LPS activation of cells and in the induction and regulation of cytokine production. The LPS of the Bacteroides is structurally different from and is less potent than classical LPS. Therefore, the manner in which these different LPS molecules interact with cells for activation is likely to be different. the overall objectives of the studies proposed in this application are to demonstrate that Bacteroides LPS stimulates host cells to produce inflammatory cytokines; that the mechanisms of stimulation differ from those involved with classical LPS; and that eh developmental stage and the source of cells influence the profile of cytokines which can be produced. Specifically, we will (1) determine the differences in the ability of Bacteroides LPS compared to classical LPS to stimulate human monocytes/macrophages and gingival and dermal fibroblasts to produce the inflammatory cytokines IL-1, TNF-alpha, IL-6 and IL-8, as well as IL-1 inhibitor. Dose response and time course studies will be performed to determine the induction sequence of message and protein and the amount of each factor produced following in vitro incubation of cells with LPS or LPS and cytokines. Cytokine-specific mRNA will be identified by the reverse PCR method and the levels of cytokines produced will be assessed in functional assays and by ELISA. We will also (2) determine whether differences exist in the profile of cytokines produced by gingival fibroblasts derived from healthy and granulomatous tissue and from the periodontal ligament following in vitro stimulation with Bacteroides LPS. Finally, we will (3) determine the cellular production of IL-1, IL-1 inhibitor, TNF-alpha, IL-6 and IL-8 by in situ hybridization for cytokine-specific mRNA in gingival biopsies obtained from patients with periodontitis and determine if detection of cytokine mRNA correlates with the level of each cytokine detected in crevicular fluid samples obtained from these subjects. Immunohistologic techniques will be used to define the cellular composition of tissue biopsies. These studies will provide evidence for the mechanism(s) by which Bacteroides LPS stimulates host cells and will define the contribution of this microbial LPS, cytokines and their inhibitors in mediating events occurring in inflamed periodontal tissues. Taken together, these studies should help us understand the cellular events that take place in inflamed tissue which will help in the development of better methods for treatment and prevention of periodontitis as well as other inflammatory diseases.

牙周炎是牙周组织的慢性炎症性疾病 其中黑色素拟杆菌属物种，特别是 牙龈卟啉单胞菌（拟杆菌）已被认为是病因 代理。 拟杆菌属的微生物成分，包括它们的 脂多糖（LPS），与初始渗透有关 淋巴细胞、单核细胞/巨噬细胞和中性粒细胞 本病的特点。 然而，微生物和宿主相关 所涉及的机制尚不完全清楚。 细菌脂多糖具有 激活宿主细胞产生炎症因子的能力 例如白细胞介素-1 (IL-1)、肿瘤坏死因子-α (TNF-α)、 IL-6、IL-8 和 IL-1 抑制剂。 IL-8 是 PMN 的化学引诱剂 T 淋巴细胞及其产生是由 TNF-α、IL-1 和 LPS 诱导的。 因此，IL-8 与其他细胞因子和微生物 LPS 可能是 参与自我放大循环，促进相关的细胞事件 患有炎症性疾病。 细胞的调节很可能 功能是由基因表达的变化介导的，而基因表达的变化可能是由于 LPS 和/或细胞产物（例如 IL-1、TNF-）的直接刺激 α 或 IFN-γ。 经典 LPS（例如，源自大肠杆菌的 LPS） 大肠杆菌）是细胞因子的有效诱导剂，尤其是 IL-1，但仅 最近我们开始了解所涉及的分子机制 细胞的 LPS 激活以及诱导和调节 细胞因子的产生。 拟杆菌的 LPS 在结构上是 与经典 LPS 不同，并且其效力较经典 LPS 低。 因此， 这些不同的 LPS 分子与细胞相互作用的方式 激活可能会有所不同。 总体目标 本申请中提出的研究旨在证明拟杆菌 LPS刺激宿主细胞产生炎症细胞因子；认为 刺激机制与经典 LPS 的刺激机制不同； 发育阶段和细胞来源影响 可以产生的细胞因子的概况。 具体来说，我们将 (1) 确定拟杆菌 LPS 与 经典 LPS 刺激人类单核细胞/巨噬细胞和牙龈 真皮成纤维细胞产生炎症细胞因子 IL-1、TNF-α、 IL-6和IL-8，以及IL-1抑制剂。 剂量反应和时程 将进行研究以确定消息的诱导序列 以及体外产生的蛋白质和每种因子的量 将细胞与 LPS 或 LPS 和细胞因子一起孵育。 细胞因子特异性 通过反向PCR方法鉴定mRNA，并检测mRNA的水平 产生的细胞因子将通过功能测定和 ELISA 进行评估。 我们还将 (2) 确定以下人员的概况是否存在差异： 源自健康和牙龈成纤维细胞产生的细胞因子 体外培养的肉芽肿组织和牙周膜 用拟杆菌 LPS 刺激。 最后，我们将（3）确定 细胞产生 IL-1、IL-1 抑制剂、TNF-α、IL-6 和 IL-8 牙龈活检中细胞因子特异性 mRNA 的原位杂交 从牙周炎患者身上获取并确定是否检测到 细胞因子 mRNA 与检测到的每种细胞因子的水平相关 从这些受试者获得的裂隙液样本。 免疫组织学 技术将用于定义组织的细胞组成 活检。 这些研究将为该机制提供证据 拟杆菌 LPS 刺激宿主细胞并定义 这种微生物脂多糖、细胞因子及其抑制剂在 介导发炎的牙周组织中发生的事件。 采取 总之，这些研究应该有助于我们了解细胞事件 发生在发炎组织中，这将有助于发展 更好的治疗和预防牙周炎的方法 其他炎症性疾病。