Peripheral lymph nodes (PLN) have several important physiologic functions. They are the principal sites of lymphocyte encounter with antigens (Ag) that penetrate the body's surfaces. It is critical for this process that lymphocytes enter PLN via high endothelial venules (REV) in the nodal paracortex. Here, pathogen-derived Ag is presented by dendritic cells (DC), which transport the Ag from peripheral tissues via afferent lymph vessels. A second function of PLN is the filtration of lymph fluid that percolates through PLN. This may prevent microorganisms or tumor cells in lymph fluid from reaching the systemic circulation. The mechanisms of lymph filtration are poorly understood, but are likely to involve phagocytes that may be recruited from the blood. An intravital microscopy model was recently established for this project that permits real-time analysis of cell migration and lymph flow in murine subiliac lymph nodes. The venular tree in subiliac PLN consists of five branching orders. The order I venule drains blood from higher order venules out of the PLN; upstream order IV and V venules are paracortical HEy. During the continuation of this project, the hypothesis will be tested that endothelial cells in different segments of this venular tree have specialized properties which result in a branching order-specific adhesive phenotype and topographic differences in leukocyte recruitment to PLN. Preliminary data suggest that medullary (order I and II) venules express trafficking molecules that are distinct from those in order IV, V and most order III Hey.
Aim1 will characterize the venular topography of lymphocyte traffic molecules involved in L-selectin-mediated rolling (glycosyltransferases and sulfotransferases, subaim 1.1) and subsequent integrin-dependent arrest (ICAM-l and -2). The transportation and segmental presentation of the CC chemokine TCA-4, which is critical for T cell and DC recruitment to PLN, will also be examined (subaim 1.2).
Aim 2 will characterize the topography, kinetics and molecular mechanisms of monocyte (subaim 2.1) and DC (subaim 2.2) recruitment in the venular tree of normal and inflamed PLN. The role of lymph-borne inflammatory chemokines such as MCP-1 will be examined. Preliminary results indicate that in vitro generated Ag-laden DC can be induced to home to PLN via HEV by retroviral transduction of a chimeric selectin. The molecular mechanisms of transformed DC recruitment to PLN and their applicability for DC vaccination will be examined (subaim 2.3). This project will dissect the molecular and microvascular mechanisms that regulate lymphocyte, monocyte and DC homing to PLN. This may lead to a better understanding of physiologic and pathologic phenomena in PLN and result in improved vaccination protocols against tumors, infections and inflammatory diseases.
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