In the lymphatic sinuses of draining lymph nodes, soluble lymph-borne antigens enter the reticular conduits in a size-selective manner and lymphocytes transmigrate to the parenchyma. The molecular mechanisms that control these processes are unknown. Here we unexpectedly found that PLVAP, a prototypic endothelial protein of blood vessels, was synthesized in the sinus-lining lymphatic endothelial cells covering the distal conduits. In PLVAP-deficient mice, both small antigens and large antigens entered the conduit system, and the transmigration of lymphocytes through the sinus floor was augmented. Mechanistically, the filtering function of the lymphatic sinus endothelium was dependent on diaphragms formed by PLVAP fibrils in transendothelial channels. Thus, in the lymphatic sinus, PLVAP forms a physical sieve that regulates the parenchymal entry of lymphocytes and soluble antigens.

Tyrosine kinase inhibitors (TKIs) have potent effects on malignant cells, and they also target kinases in normal cells, which may have therapeutic implications. Using a collection of 55 leukemia patients treated with TKI therapy (chronic myeloid leukemia, n=47; acute lymphoblastic leukemia, n=8), we found that dasatinib, a second-generation broad-spectrum TKI, induced a rapid, dose-dependent and substantial mobilization of non-leukemic lymphocytes and monocytes in blood peaking 1-2 h after an oral intake and the blood counts closely mirrored drug plasma concentration. A preferential mobilization was observed for natural killer (NK), NK T, B and γδ+ T cells. Mobilization was coupled with a more effective transmigration of leukocytes through an endothelial cell layer and improved cytotoxicity of NK cells. Platelet numbers decreased markedly after the drug intake in a proportion of patients. Similar effects on blood cell dynamics and function were not observed with any other TKI (imatinib, nilotinib and bosutinib). Thus, dasatinib induces a unique, rapid mobilization and activation of cytotoxic, extravasation-competent lymphocytes, which may not only enhance antileukemia immune responses but can also be causally related to the side-effect profile of the drug (pleural effusions, thrombocytopenia).

IntroductionDiscrimination between vascular and lymphatic endothelium is crucial but challenging in cell biology and pathology. An antigen defined by the monoclonal antibody Pathologische Anatomie Leiden-endothelium (PAL-E) has been a "gold standard" to define vascular endothelium. In contrast to the other established markers for vascular endothelium, such as factor VIII, CD31, and endoglin, PAL-E is completely absent from lymphatic endothelial cells. It is now known that the PAL-E antibody recognizes plasmalemma vesicle-associated protein-1 (PV-1). 1 PV-1 was originally discovered in rat lung endothelium. 2 The protein has a long extracellular domain of 380 amino acids, a single span transmembrane domain and a short 27 amino acids long intracellular N-terminal domain. The amino acid sequence of any of the domains gives no hints about the possible function of this protein. PV-1 forms homodimers in situ, and its extracellular domain contains 4 N-glycosylation sites. 3 In human, the protein has a molecular weight of approximately 60 kDa depending on the glycosylation. The subcellular localization of PV-1 is restricted to stomatal diaphragms of endothelial caveolae, containing high concentrations of caveolar proteins, such as caveolin-1. Furthermore, PV-1 was found in transendothelial channels and diaphragms of fenestrae in rat lung, 2 all structures that have been implicated in the transcellular exchange of liquids and macromolecules. 4,5 The migration of leukocytes from the bloodstream through the endothelium into the tissue is one of the central paradigms of inflammation and immunity. This process requires a sequence of events, starting with the retardation of leukocyte flow and leading via firm adhesion to transendothelial migration. 6,7 Leukocytes have been shown to cross endothelia via the paracellular and transcellular pathway. 8,9 Even though in vivo studies in the 1960s collected evidence that leukocytes can indeed extravasate from blood vessels via a transcellular pathway, 10,11 this view was largely disregarded. Only in the past few years have new in vitro studies revived this concept. 8,[12][13][14] Thus, although the recruitment of leukocytes from the bloodstream and the paracellular migration are well understood, the molecular mechanisms of transcellular migration are still incompletely resolved. Because the expression pattern of PV-1 would be compatible with its involvement in cell migration, this work was designed to analyze the behavior and role of PV-1 in the transmigration process both in vitro and in vivo. Methods Cell isolation and cultureHuman umbilical vein endothelial cells (HUVECs) were isolated and cultured as previously described. 15 Human dermal microvascular endothelial cells (HDMECs) were purchased from PromoCell. For isolation of lymphocytes, peripheral blood was collected from healthy volunteers and cells were isolated using Ficoll-Paque Plus (GE Healthcare) according to the manufacturer's instructions. The use of human material was approved by the Ethical Board of Turku University H...

Macrophages are required for normal embryogenesis, tissue homeostasis and immunity against microorganisms and tumours. Adult tissue-resident macrophages largely originate from long-lived, self-renewing embryonic precursors and not from haematopoietic stem-cell activity in the bone marrow. Although fate-mapping studies have uncovered a great amount of detail on the origin and kinetics of fetal macrophage development in the yolk sac and liver, the molecules that govern the tissue-specific migration of these cells remain completely unknown. Here we show that an endothelium-specific molecule, plasmalemma vesicle-associated protein (PLVAP), regulates the seeding of fetal monocyte-derived macrophages to tissues in mice. We found that PLVAP-deficient mice have completely normal levels of both yolk-sac- and bone-marrow-derived macrophages, but that fetal liver monocyte-derived macrophage populations were practically missing from tissues. Adult PLVAP-deficient mice show major alterations in macrophage-dependent iron recycling and mammary branching morphogenesis. PLVAP forms diaphragms in the fenestrae of liver sinusoidal endothelium during embryogenesis, interacts with chemoattractants and adhesion molecules and regulates the egress of fetal liver monocytes to the systemic vasculature. Thus, PLVAP selectively controls the exit of macrophage precursors from the fetal liver and, to our knowledge, is the first molecule identified in any organ as regulating the migratory events during embryonic macrophage ontogeny.