A plexus of lymphatic vessels guides interstitial fluid, passenger leukocytes, and tumor cells toward regional lymph nodes. Microvascular endothelial cells (ECs) 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 (vWF), etc. 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. Here we isolated for the first time podoplanin+ LECs and podoplanin− BECs from dermal cell suspensions by multicolor flow cytometry. Both EC types were propagated and stably expressed VE-cadherin, CD31, and vWF. 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, fms-like tyrosine kinase 4 (Flt-4)/VEGFR-3, were strongly expressed by expanded LECs, but not BECs. Conversely, BECs but not LECs expressed VEGF-C. LECs as well as BECs formed junctional contacts with similar molecular composition and ultrastructural features. Nevertheless, the two EC types assembled in vitro in vascular tubes in a strictly homotypic fashion. This EC specialization extends to the secretion of biologically relevant chemotactic factors: LECs, but not BECs, constitutively secrete the CC chemokine receptor (CCR)7 ligand secondary lymphoid tissue chemokine (SLC)/CCL21 at their basal side, while both subsets, upon activation, release macrophage inflammatory protein (MIP)-3α/CCL20 apically. These results demonstrate that LECs and BECs constitute stable and specialized EC lineages equipped with the potential to navigate leukocytes and, perhaps also, tumor cells into and out of the tissues.

The lymphatic vessels (lymphatics) play an important role in channeling fluid and leukocytes from the tissues to the secondary lymphoid organs. In addition to driving leukocyte egress from blood, chemokines have been suggested to contribute to leukocyte recirculation via the lymphatics. Previously, we have demonstrated that binding sites for several pro-inflammatory beta-chemokines are found on the endothelial cells (ECs) of lymphatics in human dermis. Here, using the MIP-1alpha isoform MIP-1alphaP, we have extended these studies to further support the contention that the in situ chemokine binding to afferent lymphatics exhibits specificity akin to that observed in vitro with the promiscuous beta-chemokine receptor D6. We have generated monoclonal antibodies to human D6 and showed D6 immunoreactivity on the ECs lining afferent lymphatics, confirmed as such by staining serial skin sections with antibodies against podoplanin, a known lymphatic EC marker. In parallel, in situ hybridization on skin with antisense D6 probes demonstrated the expression of D6 mRNA by lymphatic ECs. D6-immunoreactive lymphatics were also abundant in mucosa and submucosa of small and large intestine and appendix, but not observed in several other organs tested. In lymph nodes, D6 immunoreactivity was present on the afferent lymphatics and also in subcapsular and medullary sinuses. Tonsilar lymphatic sinuses were also D6-positive. Peripheral blood cells and the ECs of blood vessels and high endothelial venules were consistently nonreactive with anti-D6 antibodies. Additionally, we have demonstrated that D6 immunoreactivity is detectable in some malignant vascular tumors suggesting they may be derived from, or phenotypically similar to, lymphatic ECs. This is the first demonstration of chemokine receptor expression by lymphatic ECs, and suggests that D6 may influence the chemokine-driven recirculation of leukocytes through the lymphatics and modify the putative chemokine effects on the development and growth of vascular tumors.

SununarySuggestive evidence indicates that immunoglobulin E (IgE)-dependent activation of mononuclear phagocytes plays an important pathogenic role in allergic tissue inflammation. Prevailing opinion holds that low affinity IgE receptors are the relevant IgE-binding structures on monocytes/ macrophages and that functional events occurring after cross-linking of membrane-bound IgE on these cells are mediated by these receptors. Here we demonstrate that Ix-ripheral blood monocytes can bind monomeric IgE via the high affinity IgE receptor (FceRI) and that FceRI expression on these ceils is upregulated in atopic persons. Further, we demonstrate that, upon monocyte adherence to substrate, bridging of monocyte FcdLl is followed by cell activation. We propose that direct interaction of multivalent allergen with Fc~RI+-bound IgE on mononuclear phagocytes results in cell signaling via Fcd~I and that the biological consequences of this event may critically influence the outcome of allergic reactions.

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Certain types of dendritic cells (DCs) appear in inflammatory lesions of various etiologies, whereas other DCs, e.g., Langerhans cells (LCs), populate peripheral organs constitutively. Until now, the molecular mechanism behind such differential behavior has not been elucidated. Here, we show that CD1a+ LC precursors respond selectively and specifically to the CC chemokine macrophage inflammatory protein (MIP)-3α. In contrast, CD14+ precursors of DC and monocytes are not attracted by MIP-3α. LCs lose the migratory responsiveness to MIP-3α during their maturation, and non-LC DCs do not acquire MIP-3α sensitivity. The notion that MIP-3α may be responsible for selective LC recruitment into the epidermis is further supported by the following observations: (a) MIP-3α is expressed by keratinocytes and venular endothelial cells in clinically normal appearing human skin; (b) LCs express CC chemokine receptor (CCR)6, the sole MIP-3α receptor both in situ and in vitro; and (c) non-LC DCs that are not found in normal epidermis lack CCR6. The mature forms of LCs and non-LC DCs display comparable sensitivity for MIP-3β, a CCR7 ligand, suggesting that DC subtype–specific chemokine responses are restricted to the committed precursor stage. Although LC precursors express primarily CCR6, non-LC DC precursors display a broad chemokine receptor repertoire. These findings reflect a scenario where the differential expression of chemokine receptors by two different subpopulations of DCs determines their functional behavior. One type, the LC, responds to MIP-3α and enters skin to screen the epidermis constitutively, whereas the other type, the “inflammatory” DC, migrates in response to a wide array of different chemokines and is involved in the amplification and modulation of the inflammatory tissue response.