The role of complement in periodontal disease can be either protective or pathologic depending on the stage of the disease and the pathogens involved. Our hypothesis is that the humoral inflammatory factor C5a, the chemotatic cytokine IL-8 and/or formulated bacterial peptides (f-MLF) play a significant role in promoting gingival tissue injury resulting from bacterial infection. This process may be set in motion either by the anaerobic gram-negative bacteria or the proteases they elaborate. Although complement activation can be initiated directly by contact between plasma and bacterial particles, we propose that bacterial proteases are primarily responsible for prolonged generation of C5a in periodontal disease. The potent chemotactic factors C5a/f-MLF recruit neutrophils and macrophages which in turn release protases, IL-8 and other cellular cytokines that accelerate tissue degradation and promotes edema. Persistent edema due to leakage of plasma fluid from the microvasculature of the periodontium is a usual feature of periodontal diseases. Plasma protein filtrate contains C5 which may be degraded by bacterial proteases, with resultant release of the chemotaxin C5A. Prolongation of localized inflammatory cell sequestration is consistent with the hypothesis of ongoing C5a generation, which may contribute to the chronic inflammatory response that is the hallmark of periodontitis. The early events in periodontitis are likely driven by bacterial (i.e. formulated peptides) and/or humoral (i.e. C5a) chemotactic factors which initiate the cascade of cellular infiltration. The initial cellular influx may be amplified later in the process by cell-derived chemotactic factors such as PAF, IL-8 and LTB4. This proposal is designed to first demonstrate evidence for C5a/IL-8 at the injury site, examine effects of the bacterial products (i.e proteases) on the various activation factors and their receptors, and then test the hypothesis that C5a/IL-8 involvement actually occurs and contributes significantly in promoting the disease. Secondly, I propose to specifically evaluate processes such as """"""""priming"""""""" of the neutrophils/macrophages by bacterial endotoxin, which enhances responsiveness of the cells to chemotactic factors. C5a and IL-8 receptors were recently demonstrated on cultured epithelial cells (HEp-2 and KB) and on epithelial cells in human gingival tissue (see preliminary results section). Both anti-C5a/IL-8 and anti-C5a/IL-8 receptor antibodies, capable of neutralizing binding and in vivo cellular migration, have been developed. These reagents will be used to explore the actions of the proteases from P.gingivalis on the chemotaxins and their receptors. We have shown that both Arg- and Lys-gingipain degrade C3 and C5 and generate bioactive C5a (see preliminary results section). In addition, we observed that oxygen radical treatment of C5 significantly enhanced C5a generation by the gingipains (manuscript in preparation). We propose to explore possible C5a/IL-8 induced epithelial cell functions including the release of acute phase proteins and cytokines. It is proposed that early events in the inflammatory response of periodontal disease are mediated by the chemotaxins and that understanding these events may lead to therapeutically useful modes of intervention.
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