ENDOTHELIAL CELL BIOLOGY IN INFLAMMATION

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This proposal is directed at understanding the biology of endothelial cells (EC) in inflammation, and in particular the role of EC in controlling leukocyte extravasation. We have developed a panel of monoclonal antibodies (MAbs) defining segmental and pan-EC antigens; inflammation- induced antigens expressed by postcapillary venules (PCV) involved in leukocyte extravasation; and tissue-specific antigens including "vascular addressins", organ-specific EC molecules that direct the homing of lymphocytes. Our studies will focus primarily on the addressins; and on neutrophil-, monocyte-, and lymphocyte-specific adhesion mechanisms induced on EC during inflammation. The peripheral lymph node and mucosal vascular addressins (PNAd and MAd) will be characterized structurally in conventional biochemical and lectin-binding analyses; and functionally in assays of lymphocyte binding to immunoisolated PNAd. The expression of the addressins and of other EC differentiation antigens during embryogenesis, and during the development of lymphoid tissues, will be explored immunohistologically. The expression and role of the addressins in chronic inflammation will be studied in pathologic or autoimmune inflammatory models in the mouse (NOD mice, EAE, murine cerebral malaria, arthritis); and in normal and pathologically inflamed tissues in man. MAbs produced against inflamed mouse tissues (including several MAbs already identified), or against cytokine- or LPS-activated cultured EC, will be used to identify and characterize inflammation-induced antigens expressed by PCV that support leukocyte extravasation. The involvement of these antigens in leukocyte-EC interactions will be assessed in antibody inhibition studies using a) ex vivo assays of neutrophil-, monocyte-, or lymphocyte binding to postcapillary venules (PCV) in inflamed tissues; b) conventional leukocyte homing/localization studies; and c) intravital videomicroscopy of in vivo leukocyte-EC adhesion and diapedesis. The regulation of leukocyte-specific adhesion mechanisms and of the addressins in response to cytokines and/or tissue-specific factors will be studied in vitro using cultured mouse EC or human umbilical vein endothelium. Biochemical, serologic, and functional approaches will be used to characterize and determine the functional significance of two additional EC antigens, defined by Mabs MECA-32 and HECA-452, that may be involved in lymphocyte extravasation. A long-term goal will be to identify and characterize the synovial vascular addressin. The remarkable diversity and specificity of leukocyte-EC interactions renders this an exciting model for the study of basic mechanisms of cell- cell recognition and cellular positioning in vivo. Furthermore, definition of the mechanisms by which EC's control leukocyte extravasation is critical to an understanding of inflammatory and immune responses, and may permit highly selective intervention in these processes.