Our objective is to characterize the structure and function of leukocyte surface molecules that mediate binding of circulating leukocytes to endothelium, and to define their role in lymphocyte traffic and leukocyte extravasation in sites of inflammation. 1) Functional properties of gp90 """"""""homing receptors"""""""" will be defined at the molecular level: The ability of affinity isolated gp90s to bind high endothelial cells will be tested using artificial lipid membrane techniques: and their capacity to bind vascular addressing, and to interact as lectins with PPME and related carbohydrate inhibitors of HEV recognition, will be studied using fluorescence energy transfer. 2) Antibody inhibition studies will be carried out to determine whether the integrin-related adhesion molecules LFA-1 and LPAM-1 are involved in lymphocyte binding to purified mucosal addressing. 3) The cellular and molecular mechanisms responsible for leukocyte-specific interactions with postcapillary venules (PCV) during extravasation into inflammatory sites will be explored. Ex vivo assays of leukocyte binding to PCV in frozen sections of inflamed tissues will define the specificity of neutrophil, monocyte, and eosinophil binding to endothelium during acute, chronic, and parasitic inflammation. Ex vivo assays, intravital microscopic observations of blood-borne fluorescent- labeled leukocytes interacting with inflamed PCV or HEV, and conventional in vivo homing/localization studies will be used to explore the cellular and molecular mechanisms involved in leukocyte-specific PCV interactions (margination and rolling, adhesion, cementing of binding, diapedesis: and inhibition of these events by antibodies to homing receptors, the Mac- 1/Lfa-1 family, and other adhesion molecules). 4) Anti-gp90 homing receptor antibodies will be produced in an attempt to a) define variable, function-associated epitopes on gp90 Mermes in the human b) identify hypothesized gp90 mucosal homing receptors in the mouse; and c) define common epitopes or domains on murine gp90 mucosal homing receptors in the mouse; and c) define common epitopes or domains on murine gp90 homing receptors of diverse specificities. 5) Ex vivo leukocyte-PCV binding and in vivo assays will be used to assess the role of specific inflamed PCV recognition in the traffic of activated T cells to delayed type hypersensitivity and allograft sites. and of """"""""gut homing"""""""" lymphocyte subsets to the mucosal lamina propria. 6) The effects of IFN-gamma on leukocyte expression of adhesion molecules in vivo, and on leukocyte interactions with PCV in inflammatory sites will be explored to help elucidate the mechanism or suppression of neutrophil infiltration into inflammatory sites during systemic IFN-gamma treatment. Preliminary studies demonstrate a critical role for tissue-and leukocyte subset-specific recognition of PCV in leukocyte extravasation, and hence in normal and pathologic inflammatory responses. The proposed studies should further define the cellular and molecular mechanisms of leukocyte- PCV recognition, potentially leading to strategies allowing specific manipulation of inflammatory and autoimmune disease processes.
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