Periodontitis is a widespread disease in the United States and is the main for loss of teeth in adults. Although the cause is clearly bacterial plaque, most of the elements responsible for disease progression from inflammatory gingivitis to connective tissue destruction are unknown. Progression of the disease is defined by loss of connective tissue attachment to the tooth followed by migration of the junctional epithelium along the root surface apically creating an irreversible loss of connective tissue attachment and a periodontal pocket which will not resolve without therapy. This proposal draws from concepts of tissue interactions during embryological development, wound healing and regulatory phenomena in inflammatory diseases to create a hypothesis stating that epithelial and connective tissue cells signal each other locally to produce an environment resulting in matrix degradation and epithelial migration. It is proposed that the epithelium secretes cytokines that induce extracellular matrix-degrading enzymes in connective tissue fibroblasts, and that the fibroblasts in turn secrete other paracrine factors capable of inducing proliferation and migration in the junctional epithelium. This cycle is initiated by bacterial products, such as lipopolysaccaharride and is augmented by cytokines from inflammatory leukocytes in the connective tissue. The matrix adjacent to the migrating epithelium is also modified by proteinases and biosynthesis to enhance epithelial migration. This application addresses the hypothesis by using immunocytochemistry to determine the location of extra cellular matrix-degrading enzymes, regulatory cytokines and their receptors in the periodontium in normal and inflamed tissue. In situ hybridization will be used to confirm the cell sources of soluble molecules. This information will be used in the next phase of experiments to determine which cytokines are present in each gingival condition and which cell types produce them. The second phase of experiments will use more progressively complex in vitro models of keratinocytes and fibroblasts to test production and regulatory mechanisms for matrix-degrading enzymes and the action of cytokines found in the first phase to be present or altered in the tissue. Of specific interest will be the regulation by prostaglandins and LPSs of epithelial cytokine and proteinase biosynthesis and epithelial migration. Many growth factors and cytokines induce the release of prostaglandin-E2 and their effects may be mediated and modulated by this agent. The biosynthesis of metalloproteinases, plasminogen activators and the cytokines IL-1-alpha, IL-6, and TGF-beta will be analyzed by mRNA hybridization with specific cDNA probes, and by specific protein and by enzyme assays. Media conditioned by each cell type will be interchanged to look for novel factors influencing epithelial and fibroblast biosynthetic behavior. Qualitative changes in these molecules will serve as a sound basis for the design of experiments in controlled models of active periodontitis and in studying epithelial migration and regulation of biosynthesis in vitro.
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