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: 6104731
• 负责人: Suzanne M. Michalek
• 金额: $ 6.85万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-12-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6104731
• 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.

牙周炎是一种慢性牙周组织炎症性疾病 其中黑色素拟杆菌，特别是 牙龈卟啉单胞菌（拟杆菌），已被牵连的病因 剂. 拟杆菌属的微生物组分，包括其 脂多糖（LPS）与最初的浸润有关 淋巴细胞、单核细胞/巨噬细胞和中性粒细胞， 这种疾病的特征。 然而，微生物和宿主相关的 所涉及的机制尚未完全了解。 细菌LPS具有 激活宿主细胞产生炎症因子的能力 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是中性粒细胞的化学引诱物， T淋巴细胞及其产生由TNF-α、IL-1和LPS诱导。 因此，IL-8沿着其它细胞因子和微生物LPS可能是 参与自我放大循环，促进细胞事件相关 炎症性疾病。 很可能细胞的调节 功能是由基因表达的变化介导的， 通过LPS和/或通过细胞产物如IL-1、TNF-α的直接刺激， α或IFN-γ。 经典LPS（例如，大肠杆菌LPS 大肠杆菌）是细胞因子，特别是IL-1的有效诱导剂，但仅 最近，我们开始了解其中的分子机制， 在LPS激活细胞和诱导和调节 细胞因子产生。 拟杆菌的LPS在结构上是 与经典LPS不同并且效力低于经典LPS。 因此 这些不同的LPS分子与细胞相互作用的方式， 激活可能是不同的。 的总体目标 本申请中提出的研究证明拟杆菌属 LPS刺激宿主细胞产生炎性细胞因子， 刺激机制与经典LPS所涉及的机制不同; 发育阶段和细胞来源影响细胞的发育， 可以产生的细胞因子的概况。 具体而言，我们将（1） 确定拟杆菌LPS与 经典LPS刺激人单核细胞/巨噬细胞和牙龈， 真皮成纤维细胞产生炎性细胞因子IL-1，TNF-α， IL-6和IL-8以及IL-1抑制剂。 剂量反应和时间过程 将进行研究，以确定信息的诱导序列， 和蛋白质以及体外培养后产生的每种因子的量 用LPS或LPS和细胞因子孵育细胞。 细胞因子特异性 将通过反向PCR方法鉴定mRNA，并测定其水平。 产生的细胞因子将在功能测定中和通过ELISA进行评估。 我们还将（2）确定是否存在差异的概况， 牙龈成纤维细胞产生的细胞因子， 肉芽肿组织和牙周膜， 用拟杆菌LPS刺激。 最后，我们将（3）确定 IL-1、IL-1抑制剂、TNF-α、IL-6和IL-8的细胞产生 牙龈组织中精氨酸特异性mRNA原位杂交检测 从牙周炎患者中获得，并确定是否检测到 细胞因子mRNA与细胞中检测到的每种细胞因子的水平相关， 从这些受试者获得的龈沟液样本。 免疫组织 技术将用于确定组织的细胞组成 活组织检查 这些研究将通过以下方式为机制提供证据： 哪种拟杆菌LPS刺激宿主细胞， 这种微生物LPS、细胞因子及其抑制剂在 介导炎症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活动， 发生在发炎的组织，这将有助于发展， 治疗和预防牙周炎的更好方法， 其他炎症性疾病。