STEREOCHEMICALLY CONSTRAINED LIGANDS TO DEFINE PMN RECEPTOR BINDING SITES

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Localized juvenile periodontitis (LJP) is an adolescent disease characterized by severe alveolar bone loss confined largely to the first molars and incisors. Immunologic evaluation of these patients has revealed an intrinsic neutrophil chemotactic defect in approximately 70% of the patients studied. The chemotactic defect of LJP neutrophils appears to be correlated with reduced numbers of membrane receptors for chemotactic substances. Nevertheless, a molecular basis of the chemotactic defect characteristic of LJP neutrophils has not been defined. Neutrophils are dynamic cells capable of altering their metabolic disposition in response to diverse external stimuli. Exposure of these cells to chemotaxins augments their expression of C3 receptors. Upon subsequent exposure to connective tissue proteins such as fibronectin, C3 receptors become "activated" such that they mediate ingestion of C3-coated particles. Whether the presence of reduced numbers of chemotaxin receptors limits the capacity of LJP neutrophils to increase expression of C3 receptors following chemotaxin stimulation or the capacity of these receptors to become activated subsequent to fibronectin exposure has not been addressed. It has long been assumed that peripheral blood neutrophils are homogeneous. Recent studies indicate, however, that these cells are functionally quite heterogeneous. Moreover, the presence of antigenically distinct subpopulations of neutrophils has been demonstrated using monoclonal antibodies and flow cytometric techniques. It has been suggested that neutrophil heterogeneity may have implications in certain pathologic states. A primary focus of the proposed studies will be, therefore, to utilize flow cytometric methods in assessing the extent of functional heterogeneity of LJP neutrophils with respect to expression of receptors for biologically active fragments of the third and fifth components of complement. Taking advantage of the fact that, unlike terminally differentiated neutrophils, immature neutrophils express membrane receptors for C3d (termed CR2 receptors), we propose to study binding of a monoclonal anti-CR2 antibody to LJP neutrophils using both flow cytometric methods and conventional binding assays employing radiolabeled antibody. Further, flow cytometric methods will be utilized to assess C3 receptor expression on untreated and chemotaxin-treated neutrophils. A further specific aim will be to define the capacity of fibronectin to activate C3 receptors on chemotaxin-treated LJP neutrophils, as determined by phagocytosis of C3-coated erythrocytes. The results of the proposed studies will provide fundamental information regarding the nature and functional consequences of chemotactic factor hyporesponsiveness characteristics of LJP neutrophils.