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.
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