In human adults, chronic periodontitis is a major cause of tooth loss. There is now abundant evidence to support the concept that accumulation of bacteria plays an important role in periodontal disease. Human polymorphonuclear leukocytes (PMN) are vital to host defenses by virtue of their ability to migrate in a directed fashion, to attach and ingest invading microorganism, to release a portion of their lysosmal constituents and to exhibit a burst of oxidative metabolism. All of these functions are mediated by binding of stimuli to specific receptors on the PMN membrane. The objective of the proposed research is to find an explanation for the apparent dysfunction of PMN observed in patients with Localized Juvenile Periodontitis (LJP) and Generalized Juvenile Periodontitis (GJP). Experiments will be performed to determine the ability of well characterized and relevant chemotactic factors to specifically bind to LJP/GJP PMN, as well as to induce the phenomenon of stimulus-response coupling in these cells. Using highly purified human C5a and C5a des Arg, as well as the synthetic chemotactic peptide, N-formyl-methionyl-leucyl- phenylalanine (FMLP), we will examine the ability of LJP and GJP PMN to bind these peptides in a specific, saturable and reversible fashion. We determine binding affinities and number of binding sites per cell under varying experimental conditions. By using pulse-chase techniques with labeled and unlabeled peptides, we will examine internalization and reexpression of chemotactic receptors by LJP and GJP PMN. Similar experiments will be performed using fluoresceinated chemotactic peptides to determine, by real time analysis, receptor occupancy and kinetics to determine the effects of C5a, C5a des Arg and PMLP on early events of stimulus-response coupling (i.e., membrane depolarization, changes in cytosolic free calcium) and on other PMN functions (i.e., polarization, right angle scattering, chemotaxis, extracellular release of lysosomal constituents, generation of superoxide anion and hydrogen peroxide, adherence to human umbilical vein endothelial cells in vitro). These studies should elucidate, at least in part, the mechanisms responsible for defective PMN function in LJP and GJP.
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