Lymphatic neoangiogenesis in human neoplasia and transplantation as experiments of nature

A plexus of lymphatic vessels guides interstitial fluid, passenger leukocytes, and tumor cells toward regional lymph nodes. Microvascular endothelial cells of lymph channels (LECs) are difficult to distinguish from those of blood vessels (BECs) because both express a similar set of markers, such as CD31, CD34, podocalyxin, von Willebrand factor. Analysis of the specific properties of LECs was hampered so far by lack of tools to isolate LECs. Recently, the 38 kD mucoprotein podoplanin was found to be expressed by microvascular LECs but not BECs in vivo. We have isolated podoplanin+ LECs and podoplanin- BECs from dermal microvascular endothelial cell suspensions by multicolor flow cytometry. Both endothelial cell types were propagated and stably expressed VE-cadherin, CD31, and von Willebrand factor. Molecules selectively displayed by LECs in vivo, i.e., podoplanin, the hyaluronate receptor LYVE-1, and the vascular endothelial cell growth factor (VEGF)-C receptor, Flt-4/VEGFR-3, were strongly expressed by expanded LECs, but not BECs. Conversely, BECs but not LECs expressed VEGF-C. This endothelial cell specialization extends to the secretion of biologically relevant chemotactic factors. LECs, but not BECs, constitutively secrete the CCR7 ligand SLC/CCL21 at their basal side, while both subsets, upon activation, release MIP-3α/CCL20 apically. Renal transplant rejection is caused by a lymphocyte rich inflammatory infiltrate that attacks cortical tubules and endothelial cells. Immunosupressive therapy reduces the number of infiltrating cells; however, their exit routes are not known. Here we demonstrate by immunohistochemistry on human renal transplant biopsies, a >50-fold increase of lymphatic vessel density over normal kidneys in grafts with nodular mononuclear infiltrates, using antibodies to the lymphatic endothelial marker protein podoplanin. Nodular infiltrates are constantly associated with newly formed, Ki-67 expressing lymphatic vessels and contain the entire repertoire of T and B lymphocytes to provide specific cellular and humoral alloantigenic immune responses, including Ki-67+, CD4+, and CD8+ T lymphocytes, S100+ dendritic cells, as well as Ki-67+ CD20+ B lymphocytes and λ and κ chain expressing plasmacytoid cells. Numerous chemokine receptor CCR7+ cells within the nodular infiltrates are apparently attracted by secondary lymphatic chemokine (SLC/CCL21) that is produced and released by lymphatic endothelial cells in a complex with podoplanin. Thus, lymphatic neoangiogenesis contributes not only to the export of the rejection infiltrate, but is also involved in the maintenance of a potentially detrimental alloreactive immune response in renal transplants, and provides a novel therapeutic target