Absence of functional lymphatics within a murine sarcoma: a molecular and functional evaluation.
(17/274)
Despite a clinically recognized association between the lymphatics and metastasis, the biology of tumor-lymphatic interaction is not clearly understood. We report here that functional lymphatic capillaries are absent from the interior of a solid tumor, despite the presence within the tumor of the lymphangiogenic molecule vascular endothelial growth factor (VEGF)-C and endothelial cells bearing its receptor, VEGF receptor 3. Functional lymphatics, enlarged and VEGF receptor 3 positive, were detected in some tumors only at the tumor periphery (within 100 microm of the interface with normal tissue). We conclude that although lymphangiogenic factors are present, formation of functional lymphatic vessels is prevented, possibly due to collapse by the solid stress exerted by growing cancer cells. (+info)
ICAM-1-coupled cytoskeletal rearrangements and transendothelial lymphocyte migration involve intracellular calcium signaling in brain endothelial cell lines.
(18/274)
Endothelium of the cerebral blood vessels, which constitutes the blood-brain barrier, controls adhesion and trafficking of leukocytes into the brain. Investigating signaling pathways triggered by the engagement of adhesion molecules expressed on brain endothelial cells using two rat brain endothelial cell lines (RBE4 and GP8), we report in this paper that ICAM-1 cross-linking induces a sustained tyrosine phosphorylation of the phosphatidylinositol-phospholipase C (PLC)gamma1, with a concomitant increase in both inositol phosphate production and intracellular calcium concentration. Our results suggest that PLC are responsible, via a calcium- and protein kinase C (PKC)-dependent pathway, for p60Src activation and tyrosine phosphorylation of the p60Src substrate, cortactin. PKCs are also required for tyrosine phosphorylation of the cytoskeleton-associated proteins, focal adhesion kinase and paxillin, but not for ICAM-1-coupled p130Cas phosphorylation. PKC's activation is also necessary for stress fiber formation induced by ICAM-1 cross-linking. Finally, cell pretreatment with intracellular calcium chelator or PKC inhibitors significantly diminishes transmonolayer migration of activated T lymphocytes, without affecting their adhesion to brain endothelial cells. In summary, our data demonstrate that ICAM-1 cross-linking induces calcium signaling which, via PKCs, mediates phosphorylation of actin-associated proteins and cytoskeletal rearrangement in brain endothelial cell lines. Our results also indicate that these calcium-mediated intracellular events are essential for lymphocyte migration through the blood-brain barrier. (+info)
Experimentally induced recruitment of plasmacytoid (CD123high) dendritic cells in human nasal allergy.
(19/274)
Recent evidence suggests that the previously enigmatic cell type designated plasmacytoid monocytes can function as dendritic cells and contribute substantially to both innate and adaptive immunity. This cell type has previously been described only in bone marrow, blood, and organized lymphoid tissue, but not at effector sites with direct Ag exposure such as the mucosae. Plasmacytoid dendritic cells (P-DCs) matured in vitro can induce T cells to produce allergy-promoting Th2 cytokines; therefore, their possible occurrence in nasal mucosa during experimentally elicited allergic rhinitis was examined. Patients with silent nasal allergy were challenged topically with relevant allergen daily for 7 days. Biopsy specimens as well as blood samples were obtained before and during such provocation, and P-DCs were identified by their high expression of CD123 (IL-3R alpha-chain), together with CD45RA. Our results showed that P-DCs were present in low and variable numbers in normal nasal mucosa but increased dramatically during the allergic reaction. This accumulation concurred with the expression of the L-selectin ligand peripheral lymph node addressin on the mucosal vascular endothelium. The latter observation was particularly interesting in view of the high levels of L-selectin on circulating P-DC precursors and of previous reports suggesting that these cells can enter organized lymphoid tissue via high endothelial venules (which express peripheral lymph node addressin constitutively). Together, our findings suggested that P-DCs are involved in the triggering of airway allergy and that they are directed to allergic lesions by adhesion molecules that normally mediate leukocyte extravasation in organized lymphoid tissue. (+info)
JAM-2, a novel immunoglobulin superfamily molecule, expressed by endothelial and lymphatic cells.
(20/274)
Cell-cell contacts are essential for morphogenesis and tissue function and play a vital role in mediating endothelial cohesion within the vascular system during vessel growth and organization. We identified a novel junctional adhesion molecule, named JAM-2, by a selective RNA display method, which allowed identification of transcripts encoding immunoglobulin superfamily molecules regulated during coculture of endothelial cells with tumor cells. The JAM-2 transcript is highly expressed during embryogenesis and is detected in lymph node and Peyer's patches RNA of adult mice. Accordingly, antibodies specific for JAM-2 stain high endothelial venules and lymphatic vessels in lymphoid organs, and vascular structures in the kidney. Using real time video microscopy, we show that JAM-2 is localized within minutes to the newly formed cell-cell contact. The role of the protein in the sealing of cell-cell contact is further suggested by the reduced paracellular permeability of cell monolayer transfected with JAM-2 cDNA, and by the localization of JAM-2 to tight junctional complexes of polarized cells. Taken together, our results suggest that JAM-2 is a novel vascular molecule, which participates in interendothelial junctional complexes. (+info)
The beta-chemokine receptor D6 is expressed by lymphatic endothelium and a subset of vascular tumors.
(21/274)
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. (+info)
Compartmentalization of Peyer's patch anlagen before lymphocyte entry.
(22/274)
We have shown that Peyer's patch (PP) first develops as a simple and even cell aggregation during embryogenesis. To investigate when and how such a simple cell aggregation forms the complex PP architecture, we analyzed the distribution of cells expressing IL-7R alpha (PP inducer cells), VCAM-1 (mesenchymal cells), CD11c (dendritic cells), and mature lymphocytes by whole-mount immunostaining of 17.5 days post coitus to 2 days postpartum mouse gut. Our results show that compartmentalization of PP anlagen commences at day 18.5 of gestation by clustering and subsequent follicle formation of IL-7R alpha(+), VCAM-1(+), and CD11c(+) cells. This process adds the primitive architecture of PP anlage with several follicles in which IL-7R alpha(+) cells localize in the center, while VCAM-1(+) and CD11c(+) cells localize at the fringe. This follicle formation is accompanied by the establishment of PP-specific vascular network expressing mucosal addressin cellular adhesion molecule-1. Mature B and T lymphocytes entering in the PP anlage are distributed promptly to their own target zones; B cells to the follicle and T cells to nonfollicular zones. Our analysis of scid/scid mouse indicate that the initial processes including formation of PP-specific vascular network occur in the absence of lymphocytes. These observations indicate that the basic architecture of PP is formed by a set of cell lineages assembled during the initial phase of induction of PP anlagen before entry of mature lymphocytes. (+info)
Targeting of lymphotoxin-alpha to the tumor elicits an efficient immune response associated with induction of peripheral lymphoid-like tissue.
(23/274)
A recombinant antibody-lymphotoxin-alpha fusion protein induced an adaptive immune response protecting mice from melanoma. Importantly, this fusion protein elicited the formation of a lymphoid-like tissue in the tumor microenvironment containing L-selectin+ T cells and MHC class II+ antigen-presenting cells, as well as B and T cell aggregates. Furthermore, PNAd+/TCA4+ high endothelial venules were observed within the tumor, suggesting entry channels for naive T cell infiltrates. Over the course of therapy, a marked clonal expansion of certain TCR specificities occurred among tumor-infiltrating lymphocytes that displayed reactivity against melanoma cells and the TRP-2(180-188) peptide. Consequently, naive T cells may have been recruited to as well as primed and expanded in the lymphoid-like tissue induced by the lymphotoxin-alpha fusion protein at the tumor site. (+info)
Adenoviral expression of vascular endothelial growth factor-C induces lymphangiogenesis in the skin.
(24/274)
The growth of blood and lymphatic vasculature is mediated in part by secreted polypeptides of the vascular endothelial growth factor (VEGF) family. The prototype VEGF binds VEGF receptor (VEGFR)-1 and VEGFR-2 and is angiogenic, whereas VEGF-C, which binds to VEGFR-2 and VEGFR-3, is either angiogenic or lymphangiogenic in different assays. We used an adenoviral gene transfer approach to compare the effects of these growth factors in adult mice. Recombinant adenoviruses encoding human VEGF-C or VEGF were injected subcutaneously into C57Bl6 mice or into the ears of nude mice. Immunohistochemical analysis showed that VEGF-C upregulated VEGFR-2 and VEGFR-3 expression and VEGF upregulated VEGFR-2 expression at 4 days after injection. After 2 weeks, histochemical and immunohistochemical analysis, including staining for the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), the vascular endothelial marker platelet-endothelial cell adhesion molecule-1 (PECAM-1), and the proliferating cell nuclear antigen (PCNA) revealed that VEGF-C induced mainly lymphangiogenesis in contrast to VEGF, which induced only angiogenesis. These results have significant implications in the planning of gene therapy using these growth factors. (+info)