The long range goal of this investigation has been the furthering of our knowledge of the structural and functional relationships of the inflammatory process, by defining the complex interactions between the neutrophil and its environment. To date, this has been accomplished by the study of neutrophils from Localized Juvenile Periodontitis (LJP) patients which exhibit defects in measureable functions. During the previous six years of this grant, work has focused upon the neutrophil surface.
The aims of the previous proposals sought to elucidate the biochemical defects of the LJP neutrophil and how these variations translated into functional abnormalitites. As a logical extension of these studies, the currently proposed work will focus upon the next step in the process of cellular recognition, that of signal transduction. Again, the defective LJP neutrophil will serve as a disease model for investigation. To further elucidate the transduction of membrane signals in the human neutrophil, employing the LJP defect model as a probe of these pathways, the following experiments are proposed: 1. To determine the effects of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) on the chemotactic function of human polymorphonuclear leukocytes from LJP patients and normal controls. Based upon the putative roles of cAMP and cGMP as second messengers in receptor-ligand interaction, the cAMP and cGMP levels of LJP and controls will be determined before and after stimulation with FMLP and C5a. 2. Since the receptor density for FMLP and C5a is known to be reduced in resting LJP neutrophils, the next aim is to evaluate receptor number and density on neutrophils defective in chemotaxis before and after IBMX treatment. 3. In order to better understand the stages of signal transduction and to understand the role of phospholipase C and protein kinase C in LJP neutrophils, intracellular Calcium ion translocation will be monitored using fluorescent probes. Release of membrane bound Ca++ will be monitored by fluorescent changes of cells labelled with chlortetracycline and release of Ca++ into the cytotsol will be measured by fluorescent changes in cells prelabelled with Quin-2 or Fura-2. 4. Protein kinase C has been implicated in the activation sequence for normal human neutrophils. Based upon preliminary data implicating a protein kinase C abnormality in LJP, experiments will be performed to determine if the activation of protein kinase C is a requirement for initiation of chemotaxis in LJP neutrophils or if the protein kinase C response is altered in LJP. To begin, these determinations will be made by measurements of protein kinase C activity and translocation.
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