Lymphatic endothelium of the human tongue expresses multiple leukocyte adhesion molecules.

The expression of leukocyte adhesion molecules on lymphatic vessels of the human tongue was examined using histochemical and immunohistochemical methods. Three different types of lymphatic vessels were distinguished: type I vessels expressed intercellular adhesion molecule-1 (ICAM-1), platelet-endothelial cell adhesion molecule-1 (PECAM-1), and endothelial cell-selectin (ELAM-1); type II vessels expressed ICAM-1 and PECAM-1; and type III vessels expressed PECAM-1 only. The lymphatic vessels located very close to the oral epithelium (lymphatic capillaries) and the other lymphatic vessels near the oral epithelium were type I. The lymphatic vessels in the submucosal connective tissue (collecting lymphatic vessels) were type II and type III. The results suggest that there may be functional differences in the lymphatic endothelium, where lymphatic capillaries are more active than collecting lymphatic vessels in lymphocyte migration from tissue into the lymphatic vessels.     

Immunoelectron microscopic study of PECAM-1 expression on lymphatic endothelium of the human tongue

The expression of platelet-endothelial cell adhesion molecule-1 (PECAM-1) on lymphatic and blood vessels of the human tongue was examined with fluorescence and transmission electron microscopy (TEM). The study used anti-desmoplakins antiserum for light microscopic identification of the lymphatic vessels, plus a pre-embedding immunogold electron microscopic technique for TEM observations. Before making TEM observations, cryostat serial sections were immunostained with anti-desmoplakins or anti-PECAM-1 and then embedded. Semithin sections from each cryostat section were photographed under a light microscope and compared in order to identify the lymphatic vessels expressing PECAM-1. In fluorescence microscopy, PECAM-1 expression on lymphatic vessels was weaker than that on blood vessels. TEM observations showed that PECAM-1 expression on the blood vessels was observed only on the luminal surface of the endothelium. In lymphatic vessels, PECAM-1 expression was found both on the luminal and abluminal surfaces of the endothelium. The density of the PECAM-1 reaction products was lower in lymphatic vessels than in blood vessels. The density of PECAM-1 reaction products on the luminal surface of lymphatic vessels was higher than on the abluminal surfaces. The results suggest that blood vessels are more active than lymphatic vessels in leukocyte migration. The expression of PECAM-1 on the abluminal surface of lymphatic endothelium may allow leukocytes to adhere to the endothelium and interact in their migration from tissue into lymphatic vessels.     

Effects of TNF-alpha on leukocyte adhesion molecule expressions in cultured human lymphatic endothelium.

TNF-alpha alters leukocyte adhesion molecule expression of cultured endothelial cells like human umbilical vein endothelial cells (HUVEC). This study was designed to investigate the changes in vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and platelet endothelial cell adhesion molecule-1 (PECAM-1) expression with TNF-alpha stimulation in cultured human neonatal dermal lymphatic endothelial cells (HNDLEC). The real-time quantitative PCR analysis on HNDLEC showed that TNF-alpha treatment leads to increases of VCAM-1 and ICAM-1 mRNAs to the 10.8- and 48.2-fold levels of untreated cells and leads to a reduction of PECAM-1 mRNA to the 0.42-fold level of untreated cells. Western blot and immunohistochemical analysis showed that TNF-alpha leads to VCAM-1 and ICAM-1 expressions that were inhibited by antiserum to human TNF receptor or by AP-1 inhibitor nobiletin. In flow cytometry analysis, the number of VCAM-1- and ICAM-1-positive cells increased, and PECAM-1-positive cells decreased with TNF-alpha treatment. Regarding protein amounts produced in cells and amounts expressed on the cell surface, VCAM-1 and ICAM-1 increased in HNDLEC and HUVEC, and PECAM-1 decreased in HNDLEC in a TNF-alpha concentration-dependent manner. VCAM-1, ICAM-1, and PECAM-1 protein amounts in TNF-alpha-stimulated cells were lower in HNDLEC than in HUVEC. This suggests that the lymphatic endothelium has the TNF-alpha-induced signaling pathway, resulting in increased VCAM-1 and ICAM-1 expression to a weaker extent than blood endothelium and PECAM-1 reduction to a stronger extent than blood endothelium.     

Lymphatic endothelial murine chloride channel calcium-activated 1 is a ligand for leukocyte LFA-1 and Mac-1

The lymphatic circulation mediates drainage of fluid and cells from the periphery through lymph nodes, facilitating immune detection of lymph-borne foreign Ags. The 10.1.1 mAb recognizes a lymphatic endothelial Ag, in this study purified by Ab-affinity chromatography. SDS-PAGE and mass spectrometry identified murine chloride channel calcium-activated 1 (mCLCA1) as the 10.1.1 Ag, a 90-kDa cell-surface protein expressed in lymphatic endothelium and stromal cells of spleen and thymus. The 10.1.1 Ab-affinity chromatography also purified LFA-1, an integrin that mediates leukocyte adhesion to endothelium. This mCLCA1-LFA-1 interaction has functional consequences, as lymphocyte adhesion to lymphatic endothelium was blocked by 10.1.1 Ab bound to endotheliumor by LFA-1 Ab bound to lymphocytes. Lymphocyte adhesion was increased by cytokine treatment of lymphatic endothelium in association with increased expression of ICAM-1, an endothelial surface protein that is also a ligand for LFA-1. By contrast, mCLCA1 expression and the relative contribution of mCLCA1 to lymphocyte adhesion were unaffected by cytokine activation, demonstrating that mCLCA1 and ICAM-1 interactions with LFA-1 are differentially regulated. mCLCA1 also bound to the LFA-1-related Mac-1 integrin that is preferentially expressed on leukocytes. mCLCA1-mediated adhesion of Mac-1- or LFA-1-expressing leukocytes to lymphatic vessels and lymph node lymphatic sinuses provides a target for investigation of lymphatic involvement in leukocyte adhesion and trafficking during the immune response.     

Tissue-specific distributions of alternatively spliced human PECAM-1 isoforms

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell adhesion molecule that is highly expressed on the surface of endothelial cells and some hematopoietic cells. Its cytoplasmic domain is encoded by multiple exons, which undergo alternative splicing. Here, we demonstrate that the human PECAM-1 cytoplasmic domain undergoes alternative splicing, generating six different isoforms. RT-PCR cloning and DNA sequence analysis indicated that human tissue and endothelial cells express multiple isoforms of PECAM-1, including the full-length PECAM-1 and five other isoforms, which lack exon 12, 13, 14, or 15 or exons 14 and 15. The full-length PECAM-1 is the predominant isoform detected in human tissue and endothelial cells. This is in contrast to murine endothelium, in which the PECAM-1 isoform lacking exons 14 and 15 is the predominant isoform. The PECAM-1 isoform lacking exon 13 detected in human tissue and endothelial cells is absent in murine endothelium. The expression pattern of PECAM-1 isoforms changes during tube formation of endothelial cells on Matrigel, which may indicate specialized roles for specific isoforms of PECAM-1 during angiogenesis. The data presented here demonstrate that human PECAM-1 undergoes alternative splicing, generating multiple isoforms in vascular beds of various tissues. Therefore, the regulated expression of these isoforms may influence endothelial cell adhesive properties during angiogenesis and/or vasculogenesis.     

Statin-inhibited endothelial permeability could be associated with its effect on PECAM-1 in endothelial cells

The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are known to inhibit leukocyte recruitment to endothelium but the mechanism is less understood. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is an endothelial junction protein involved in leukocyte diapedesis. We hypothesize that in endothelial cells, statins may well recruit PECAM-1 to exert their inhibitory effect on leukocyte trans-endothelial migration (TEM). In lovastatin-treated resting human umbilical vein endothelial cells (HUVECs), increased levels of mRNA and protein of PECAM-1 as well as its bio-synthesis (all approximately 2-fold) were observed by real-time PCR, Western blotting and 35S-labeled methionine incorporation assay, respectively. Moreover, in lovastatin treated resting cells as well as TNF-alpha activated endothelial cells, unanimously decreased Triton X-100 insoluble and soluble PECAM-1 ratio was observed. Such changes were accompanied by decreased TEM of U-937 cells (a promonocyte cell line). All lovastatin's effects were abrogated by mevalonic acid. In resting HUVECs, geranylgeranyl pyrophosphate (GGPP), but not farnesyl pyrophosphate (FPP) (both are isoprenoid intermediates in the cholesterol biosynthesis pathway) compromised the effect of lovastatin on PECAM-1 expression, whereas C3 toxin, an inhibitor of small G proteins, exerted statin-like effect. CONCLUSION: Statin-reduced endothelial permeability could be attributed to altered intracellular distribution of PECAM-1 in endothelial cells. We speculate that lovastatin regulates PECAM-1 expression in HUVECs through the mevalonate-GGPP pathway by inhibiting of Rho small GTPase.     

Role of lateral cell-cell border location and extracellular/transmembrane domains in PECAM/CD31 mechanosensation

Phosphorylation of tyrosine residues on platelet-endothelial cell adhesion molecule-1 (PECAM-1), followed by signal transduction events, has been described in endothelial cells following exposure to hyperosmotic and fluid shear stress. However, it is unclear whether PECAM-1 functions as a primary mechanosensor in this process. Utilizing a PECAM-1-null EC-like cell line, we examined the importance of cellular localization and the extracellular and transmembrane domains in PECAM-1 phosphorylation responses to mechanical stress. Tyrosine phosphorylation of PECAM-1 was stimulated in response to mechanical stress in null cells transfected either with full length PECAM-1 or with PECAM-1 mutants that do not localize to the lateral cell-cell adhesion site and that do not support homophilic binding between PECAM-1 molecules. Furthermore, null cells transfected with a construct that contains the intact cytoplasmic domain of PECAM-1 fused to the extracellular and transmembrane domains of the interleukin-2 receptor also underwent mechanical stress-induced PECAM-1 tyrosine phosphorylation. These findings suggest that mechanosensitive PECAM-1 may lie downstream of a primary mechanosensor that activates a tyrosine kinase.     

Identification of select lymphocyte homing molecules and vascular addressins in lymphotoxin-alpha deficient mice

The transmigration of lymphocytes across vascular endothelium is a critical step for the localization of lymphocytes to lymph nodes in both naive and immune reactive states. Mice deficient in lymphotoxin-alpha (LT-alpha) lack peripheral and gut associated lymph nodes. Lymphocyte function and homing ability are reported to be normal in these mice yet information regarding cell adhesion molecules and counterpart vascular addressins is lacking. The phenotype of peripheral lymphocytes from LT-alpha deficient mice was investigated by the use of fluorescent activated cell sorting and immunohistochemistry. No difference was detected in the splenocyte and tissue expression of L-selectin, alpha4beta7 or its individual integrin components, mucosal addressin cell adhesion molecule (MAdCAM-1), intracellular adhesion molecule (ICAM-1), peripheral node addressin (PNAd), or platelet/endothelial cell adhesion molecule (PECAM-1) between wild-type and LT-alpha deficient mice. Therefore, impaired expression of these lymphocyte homing and vascular addressin molecules is apparently not included in the phenotype of the LT-alpha deficient mouse.     

PECAM-1 isoform-specific activation of MAPK/ERKs and small GTPases: implications in inflammation and angiogenesis

Platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) is expressed on the surface of endothelial cells (EC) and leukocytes. PECAM-1 plays an important role in endothelial-leukocyte and endothelial-endothelial cell-cell interactions. The anti-PECAM-1 antibody-mediated blockade of these interactions inhibits transendothelial migration (TEM) of leukocytes and angiogenesis. PECAM-1 may accommodate these processes through the regulation of cell adhesive and migratory mechanisms. How PECAM-1 regulates these dynamic processes remain unknown. Here we show that PECAM-1 transduces outside-in signals, which activate MAPK/ERKs and small GTPases. This occurs through PECAM-1-mediated formation of intracellular-signaling complexes, Shc/Grb2/SOS1 and/or Crkl/C3G, which is initiated by PECAM-1 engagement on the surface of leukocytes and/or EC. Src, SHP2, and alternative PECAM-1 pre-mRNA splicing play a regulatory role in these signaling events. Our findings reveal that PECAM-1 engagement on the cell surface can transduce "outside-in" signals and activate MAPK/ERKs and small GTPases, impacting both cadherin-mediated cell-cell and integrin-mediated cell-matrix interactions. Thus, we propose PECAM-1 is an important mediator of vascular barrier and regulator of leukocyte and EC adhesion and migration.     

Dissociation between alveolar transmigration of neutrophils and lung injury in hyperoxia

The objective of this study was to quantitatively assess changes in cell adhesion molecule (CAM) expression on the pulmonary endothelial surface during hyperoxia and to assess the functional significance of those changes on cellular trafficking and development of oxygen-induced lung injury. Mice were placed in >95% O(2) for 0-72 h, and pulmonary injury and neutrophil (PMN) sequestration were assessed. Specific pulmonary CAM expression was quantified with a dual-radiolabeled MAb technique. To test the role of CAMs in PMN trafficking during hyperoxia, blocking MAbs to murine P-selectin, ICAM-1, or platelet-endothelial cell adhesion molecule-1 (PECAM-1) were injected in wild-type mice. Mice genetically deficient in these CAMs and PMN-depleted mice were also evaluated. PMN sequestration occurred within 8 h of hyperoxia, although alveolar emigration occurred later (between 48 and 72 h), coincident with rapid escalation of the lung injury. Hyperoxia significantly increased pulmonary uptake of radiolabeled antibodies to P-selectin, ICAM-1, and PECAM-1, reflecting an increase in their level on pulmonary endothelium and possibly sequestered blood cells. Although both anti-PECAM-1 and anti-ICAM-1 antibodies suppressed PMN alveolar influx in wild-type mice, only mice genetically deficient in PECAM-1 showed PMN influx suppression. Neither CAM blockade, nor genetic deficiency, nor PMN depletion attenuated lung injury. We conclude that early pulmonary PMN retention during hyperoxia is not temporally associated with an increase in endothelial CAMs; however, subsequent PMN emigration into the alveolar space may be supported by PECAM-1 and ICAM-1. Blocking PMN recruitment did not prevent lung injury, supporting dissociation between PMN infiltration and lung injury during hyperoxia in mice.     

Expression pattern of alternatively spliced PECAM-1 isoforms in hematopoietic cells and platelets

PECAM-1 (CD31) is a cell adhesion molecule that is highly expressed in the endothelium. Hematopoietic cells including platelets, monocytes, neutrophils, and some T cells also express moderate levels of PECAM-1. PECAM-1 undergoes alternative splicing generating a number of isoforms in the endothelium. However, the expression of PECAM-1 isoforms in hematopoietic cells and platelets has not been determined. Here, we examined the expression pattern of PECAM-1 isoforms in human and rodent hematopoietic cells and platelets by RT-PCR and DNA sequencing analysis. Our results showed that multiple PECAM-1 isoforms are expressed in a cell-type and species-specific pattern. We identified seven human PECAM-1 isoforms, six murine PECAM-1 isoforms, and four rat PECAM-1 isoforms. The full-length PECAM-1 was the predominant isoform detected in human cells. The PECAM-1 isoforms that lack exon 14 and 15 (delta14&15) or delta12,14&15 were the predominant isoform in rodent cells. In addition, we identified a novel PECAM-1 isoform, delta13&14, in human hematopoietic cells. Thus, hematopoietic cells express multiple isoforms of PECAM-1 in a pattern similar to that observed in the endothelium of the same species. The regulated expression of these isoforms may be important during hematopoiesis and transendothelial migration.     

Nitration of PECAM-1 ITIM tyrosines abrogates phosphorylation and SHP-2 binding

Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is a cell adhesion molecule with a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) that, when phosphorylated, binds Src homology 2 domain-containing protein-tyrosine phosphatase (SHP-2). PECAM-1 is expressed at endothelial cell junctions where exposure to inflammatory intermediates may result in post-translational amino acid modifications that affect protein structure and function. Reactive nitrogen species (RNS), which are produced at sites of inflammation, nitrate tyrosine residues, and several proteins modified by tyrosine nitration have been found in diseased tissue. We show here that the RNS, peroxynitrite, induced nitration of both full-length cellular PECAM-1 and a purified recombinant PECAM-1 cytoplasmic domain. Mass spectrometric analysis of tryptic fragments revealed quantitative nitration of ITIM tyrosine 686. A synthetic peptide containing 3-nitrotyrosine at position 686 could not be phosphorylated nor bind SHP-2. These data suggest that ITIM tyrosine nitration may represent a mechanism for modulating phosphotyrosine-dependent signal transduction pathways.     

A novel monoclonal antibody specific for lymphatic endothelium

The difficulty of identifying and differentiating lymphatic and blood microvessels in tissue sections can be overcome by a monoclonal antibody specific for lymphatic endothelium. Unfortunately, the only known antibody also reacts with the endothelium of some blood vessels. The technique of double immunization (passive, with an antiserum to blood endothelium, and active, with a suspension of lymphatic endothelial cells) was, therefore, used to increase the chances of recognizing specific lymphatic antigens by the mouse immune system. The monoclonal antibody obtained, LyMAb, a G₁ immunoglobulin, reacted strongly with the endothelium of bovine thoracic duct, mesenteric collecting vessels and lymphatic vessels of gall-bladder and lymph nodes and moderately with those of the intestinal wall. Blood vessels (intercostal arteries, azygos vein and blood microvessels of all organs tested) were consistently negative. The antibody was species-specific and did not react with formalin-fixed, paraffin-embedded sections. Cross-reactivity was limited to some connective tissue fibres and scattered cells in the lymph node parenchyma, intestinal villi and hepatic lobules.     

The neutrophil-specific antigen CD177 is a counter-receptor for platelet endothelial cell adhesion molecule-1 (CD31)

Human neutrophil-specific CD177 (NB1 and PRV-1) has been reported to be up-regulated in a number of inflammatory settings, including bacterial infection and granulocyte-colony-stimulating factor application. Little is known about its function. By flow cytometry and immunoprecipitation studies, we identified platelet endothelial cell adhesion molecule-1 (PECAM-1) as a binding partner of CD177. Real-time protein-protein analysis using surface plasmon resonance confirmed a cation-dependent, specific interaction between CD177 and the heterophilic domains of PECAM-1. Monoclonal antibodies against CD177 and against PECAM-1 domain 6 inhibited adhesion of U937 cells stably expressing CD177 to immobilized PECAM-1. Transendothelial migration of human neutrophils was also inhibited by these antibodies. Our findings provide direct evidence that neutrophil-specific CD177 is a heterophilic binding partner of PECAM-1. This interaction may constitute a new pathway that participates in neutrophil transmigration.     

Deletions in the cytoplasmic domain of platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31) result in changes in ligand binding properties

Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a member of the immunoglobulin superfamily present on platelets, endothelial cells, and leukocytes that may function as a vascular cell adhesion molecule. The purpose of this study was to examine the role of the cytoplasmic domain in PECAM-1 function. To accomplish this, wild- type and mutated forms of PECAM-1 cDNA were transfected into murine fibroblasts and the functional characteristics of the cells analyzed. Wild-type PECAM-1 localized to the cell-cell borders of adjacently transfected cells and mediated heterophilic, calcium-dependent L-cell aggregation that was inhibitable by a polyclonal and two monoclonal anti-PECAM-1 antibodies. A mutant protein lacking the entire cytoplasmic domain did not support aggregation or move to cell-cell borders. In contrast, both forms of PECAM-1 with partially truncated cytoplasmic domains (missing either the COOH-terminal third or two thirds of the cytoplasmic domain) localized to cell-cell borders in 3T3 cells in a manner analogous to the distribution seen in cultured endothelial cells. L-cells expressing these mutants demonstrated homophilic, calcium-independent aggregation that was blocked by the polyclonal anti-PECAM-1 antibody, but not by the two bioactive monoclonal antibodies. Although changes in the cytoplasmic domain of other receptors have been shown to alter ligand-binding affinity, to our knowledge, PECAM-1 is the first example of a cell adhesion molecule where changes in the cytoplasmic domain result in a switch in the basic mechanism of adhesion leading to different ligand-binding specificity. Variations in the cytoplasmic domain could thus be a potential mechanism for regulating PECAM-1 activity in vivo.     

Role of immunoreceptor tyrosine-based inhibitory motifs of PECAM-1 in PECAM-1-dependent cell migration

Platelet endothelial cell adhesion molecule (PECAM-1), a transmembrane glycoprotein, has been implicated in angiogenesis, with recent evidence indicating the involvement of PECAM-1 in endothelial cell motility. The cytoplasmic domain of PECAM-1 contains two tyrosine residues, Y663 and Y686, that each fall within an immunoreceptor tyrosine-based inhibitory motif (ITIM). When phosphorylated, these residues together mediate the binding of the protein tyrosine phosphatase SHP-2. Because SHP-2 has been shown to be involved in the turnover of focal adhesions, a phenomenon required for efficient cell motility, the association of this phosphatase with PECAM-1 via its ITIMs may represent a mechanism by which PECAM-1 might facilitate cell migration. Studies were therefore done with cell transfectants expressing wild-type PECAM or mutant PECAM-1 in which residues Y663 and Y686 were mutated. These mutations eliminated PECAM-1 tyrosine phosphorylation and the association of PECAM-1 with SHP-2 but did not impair the ability of the molecule to localize at intercellular junctions or to bind homophilically. However, in vitro cell motility and tube formation stimulated by the expression of wild-type PECAM-1 were abrogated by the mutation of these tyrosine residues. Importantly, during wound-induced migration, the number of focal adhesions as well as the level of tyrosine phosphorylated paxillin detected in cells expressing wild-type PECAM-1 were markedly reduced compared with control cells or transfectants with mutant PECAM-1. These data suggest that, in vivo, the binding of SHP-2 to PECAM-1, via PECAM-1’s ITIM domains, promotes the turnover of focal adhesions and, hence, endothelial cell motility. platelet endothelial cell adhesion molecule-1; endothelial cells; angiogenesis