Our objective is to characterize leukocyte surface receptors for endothelium (LRE), and to define their role in lymphocyte traffic and leukocyte extravasation in sites of inflammation. 1) We will examine the expression of LRE by defined human lymphocyte subsets using a) a functional in vitro assay of lymphocyte binding to high endothelial venules (HEV, specialized venules that mediate the migration of lymphocytes from the blood); and b) FACS analyses of cells stained with a monoclonal antibody (Hermes-1) against human LRE. 2) Human lymphocyte LRE will be characterized structurally in immunoprecipitation/gel analyses, and functionally by their mimicry of or their effect on lymphocyte-HEV interaction in vitro. 3) We have found that mouse neutrophils utilize molecules similar if not identical to lymphocyte LRE to interact with endothelial cells in vitro (in the frozen section model) and in vivo during migration into acute inflammatory sites. By using fluorescence and immunoprecipitation analyses with Hermes-1 and MEL-14 (anti-mouse lymphocyte receptor for lymph node HEV), in combination with functional in vitro assays of binding to endothelial cells, we will ask whether monocytes, natural killer cells (LGL), mast cells and other leukocytes (or cell lines) utilize similar molecules to interact with endothelium. FACS analyses will be used to study the regulation of LRE during leukocyte maturation, differentiation, and activation. 4) To understand the site-specific extravasation of neutrophils versus lymphocytes, we will a) explore the role of leukocyte and/or endothelial cell activation in lymphocyte and neutrophil interactions with endothelium; b) study functionally defined factor(s) that enhance lymphocyte-HEV interaction; and c) seek structural and functional differences between lymphocyte and neutrophil LRE. 5) To define mechanisms controlling lymphocyte traffic into inflamed extralymphoid tissues, we will use in vitro and in vivo assays to examine the function and specificity of HEV in sites of chronic inflammation in pathologic specimens (inflamed skin, rheumatoid synovium) and in CFA-induced granulomas in mice. Preliminary studies indicate that a novel receptor class (distinct from organ-specific receptor systems operating in lymph nodes and mucosal lymphoid tissues) may direct lymphocyte traffic into inflamed synovium, if so we will attempt to define these receptors by producing monoclonal antibodies capable of blocking lymphocyte interactions with synovial HEV. These studies will improve our understanding of normal and pathologic inflammatory responses. They have the potential to lead to site-selective means for controlling localized inflammatory/autoimmune disease processes. In addition, they may shed light on molecular and evolutionary aspects of cell-cell recognition mechanisms in other interacting cellular systems.
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