A functional epitope on P-selectin that supports binding of P-selectin to P-selectin glycoprotein ligand-1 but not to sialyl Lewis X oligosaccharides

P-selectin mediates the adhesion of leukocytes to activated platelets and endothelial cells. To characterize the functional domains of P- selectin for ligand recognition, we established nine hybridoma cell lines secreting anti-rat P-selectin mAb. Among them, the mAb C215 bound both rat and human P-selectins, and inhibited binding of rat and human P-selectins to P-selectin glycoprotein ligand-1 (PSGL-1) from HL-60 cells. In contrast, mAb C215 failed to inhibit the binding of rat and human P-selectin-IgG to sialyl Lewis X (sLe(x)) oligosaccharides. Epitope mapping of mAb C215 using synthetic decapeptides revealed that mAb C215 binds specifically to an eight-residue epitope that spans amino acids 76-83 of rat P-selectin, a region completely conserved by human P-selectin. Synthetic peptides containing the mAb C215 epitope inhibited binding of P-selectin to PSGL-1, but not to sLe(x) oligosaccharides, suggesting that the C215 epitope on P-selectin may directly interact with a particular site on the PSGL-1 core protein essential for interaction with P-selectin, such as sulfated tyrosine residues. Our results suggest the presence of two ligand recognition sites on P-selectin necessary for binding to PSGL-1--one recognizes sLe(x), while the other recognises the PSGL-1 core protein.      

Platelet binding to monocytes increases the adhesive properties of monocytes by up-regulating the expression and functionality of beta1 and beta2 integrins

Human monocytes adhere to activated platelets, resulting in the formation of platelet-monocyte complexes (PMC). Complex formation depends on the interaction between platelet-displayed P-selectin and the specific ligand for P-selectin on leukocytes, P-selectin glycoprotein ligand-1 (PSGL-1). We have recently shown that monocytes within PMC have increased adhesive capacity to the activated endothelium. To better understand the effect of platelet binding on the capacity of monocytes to adhere to activated endothelium, the P-selectin-PSGL-1 interaction-induced changes in integrin functionality were studied. The binding of platelets to monocytes via P-selectin-PSGL-1 interactions was shown to increase expression and activity of alpha4beta1 and alphaMbeta2integrin, with a concomitant decrease in L-selectin expression. Furthermore, the binding of platelets to monocytes resulted in increased monocyte adhesion to intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and fibronectin. Platelet binding was also responsible for an increase in monocyte transendothelial migration. Similar effects were observed after engagement of PSGL-1 with specific antibodies or with P-selectin immunoglobulin protein. Our data suggest that platelets, by binding via P-selectin to PSGL-1 on monocytes, induce up-regulation and activation of beta1 and beta2integrins and increased adhesion of monocytes to activated endothelium. Hence, monocytes within PMC are in a higher state of activation and may have, therefore, an increased atherogenic capacity.     

PSGL-1-mediated adhesion of human hematopoietic progenitors to P-selectin results in suppression of hematopoiesis.

Cellular interactions are critical for the regulation of hematopoiesis. The sialomucin PSGL-1/CD162 mediates the attachment of mature leukocytes to P-selectin. We now show that PSGL-1 also functions as the sole receptor for P-selectin on primitive human CD34+ hematopoietic progenitor cells (HPC). More importantly, ligation of PSGL-1 by immobilized or soluble ligand or anti-PSGL-1 antibody results in a profound suppression of HPC proliferation stimulated by potent combinations of early acting hematopoietic growth factors. These data demonstrate an unanticipated but extremely marked growth-inhibitory effect of P-selectin on hematopoiesis and provide direct evidence that PSGL-1, in addition to its well-documented role as an adhesion molecule on mature leukocytes, is a potent negative regulator of human hematopoietic progenitors.     

The metalloprotease ADAM8 is associated with and regulates the function of the adhesion receptor PSGL-1 through ERM proteins

The P-selectin glycoprotein ligand-1 (PSGL-1) is involved in the initial contact of leukocytes with activated endothelium, and its adhesive function is regulated through its proteolytic processing. We have found that the metalloprotease ADAM8 is both associated with PSGL-1 through the ezrin–radixin–moesin actin-binding proteins and able to cause the proteolytic cleavage of this adhesion receptor. Accordingly, ADAM8 knockdown increases PSGL-1 expression, and functional assays show that ADAM8 is able to reduce leukocyte rolling on P-selectin and hence on activated endothelial cells. We conclude that ADAM8 modulates the expression and function of PSGL-1.     

Expression and function of P-selectin glycoprotein ligand 1 (CD162) on human basophils

BACKGROUND: The endothelial cell adhesion molecule P-selectin may contribute to selective leukocyte migration in allergic diseases by binding to its ligand, P-selectin glycoprotein ligand 1 (PSGL-1), on eosinophils and other leukocytes. Although expression of PSGL-1 on basophils has been detected in leukocyte typing workshops, its function on basophils has not been explored. OBJECTIVE: We sought to characterize the expression and function of PSGL-1 on human basophils and a basophil-like cell line (KU812) and to compare these characteristics with those for PSGL-1 on eosinophils and neutrophils. METHODS: Basophils, eosinophils, and neutrophils were enriched from peripheral blood by using density gradient centrifugation and immunomagnetic negative selection. KU812 cells were cultured by using standard techniques. Indirect immunofluorescence and flow cytometry were used to determine surface PSGL-1 expression under various conditions, and Western blotting was used to analyze the molecular forms of PSGL-1 on each cell type. Static adhesion assays were performed by using immobilized recombinant P-selectin and relevant blocking antibodies. Histamine release assays were done by using adherent and nonadherent basophils to determine whether adhesion by means of PSGL-1 altered basophil releasability. RESULTS: The expression of PSGL-1 on basophils was similar to that on neutrophils but was approximately 30% less bright than levels on eosinophils. Levels on basophils were 10-fold higher than on KU812 cells. Basophil activation by means of IgE cross-linking resulted in reductions in surface expression of PSGL-1 and L-selectin, as well as increased CD11b expression. Western blot analysis of PSGL-1 revealed that the molecular weights of the bands for neutrophils and basophils were similar, whereas those for eosinophils were of greater molecular weights. Static adhesion assays demonstrated that basophils bound well to P-selectin, whereas KU812 cells bound poorly. Adhesion of basophils to P-selectin was completely blocked by antibodies to either P-selectin or PSGL-1. Finally, adhesion to P-selectin did not alter the magnitude or kinetics of anti-IgE-induced histamine release. CONCLUSION: Expression of PSGL-1 on basophils is more similar to that on neutrophils than that on eosinophils. KU812 cells express much lower levels of this molecule but, like basophils and other cells, bind to P-selectin by means of PSGL-1. P-selectin expression at sites of allergic inflammation is likely to play an important role in human basophil recruitment, but adhesion by means of PSGL-1 does not alter IgE-dependent basophil histamine release.     

Stimulation of P-selectin glycoprotein ligand-1 on mouse neutrophils activates β2 -integrin mediated cell attachment to ICAM-1

The entry of neutrophils into inflamed tissues is initiated by cell rolling on the blood vessel wall followed by arrest and transendothelial migration. Rolling is mediated by the selectins, while the two subsequent steps require activated β₂ -integrins. We have investigated whether the binding of P-selectin to mouse neutrophils could trigger the activation of β₂ -integrins. We show that cross-linking of P-selectin glycoprotein ligand-1 (PSGL-1) on mouse neutrophils with an antibody-like recombinant form of P-selectin or with monoclonal antibodies stimulated the production of reactive oxygen intermediates and enhanced neutrophil attachment to intercellular adhesion molecule 1 (ICAM-1)-expressing CHO cells. This effect was independent of whether complete antibodies or F(ab ′ )₂ fragments were used. The adhesion-stimulating effect of P-selectin could be blocked by monoclonal antibodies against PSGL-1. Increase of cell attachment was dependent on lymphocyte function-associated antigen 1 (LFA-1) and on Mac-1, since it could be blocked with antibodies against both respective integrin α-chains. Moreover, cell surface expression of Mac-1 increased upon cross-linking of PSGL-1. In agreement with published data, treatment of human neutrophils with P-selectin-IgG did not enhance attachment to ICAM-1. Our data suggest that ligation of PSGL-1 on mouse neutrophils, but not on human neutrophils, activates β₂ -integrin mediated cell attachment to ICAM-1.     

Effects of oxidized fibrinogen on the functions of blood cells,blood clotting,and rheology

Oxidatively-modified fibrinogen induces platelet aggregation and potentiates ADP-induced platelet aggregation and production of active oxygen forms in zymosan-stimulated leukocytes. Fibrinogen induces IL-8 production in primary culture of endothelial cells from human umbilical vein; the oxidized form of fibrinogen is more active, similarly as during induction of the expression cell adhesion molecules (P-selectin and ICAM-1). Oxidized fibrinogen (10 and 20% oxidation degree) impairs microrheological properties of the blood, sharply reduces erythrocyte deformability, modifies blood viscosity, and reduces suspension stability of the blood. Oxidized fibrinogen modified blood clotting parameters and ADP-, ristocetin-, and collagen-induced platelet aggregation in whole blood. Oxidized fibrinogen disordered the formation of fibrin clot and blood clotting process. Platelet aggregation was activated in response to ADP, but not to ristocetin and collagen, the degree of activation increased in direct proportion to the degree of fibrinogen oxidation. This indicates the “dysregulatory” effect of oxidized fibrinogen on platelets. The formation of platelet complexes with polymorphonuclear leukocytes was intensified in the presence of oxidized fibrinogen; polymorphonuclear leukocyte luminol-dependent fluorescence intensity in the presence of platelets increased after incubation with oxidized fibrinogen in comparison with native fibrinogen. Hence, oxidized fibrinogen plays an important role in the development of atherosclerosis and its complications (thromboses). __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Supplement 2, pp. 21–32, April, 2007     

Tonsillar B cells do not express PSGL-1, but a significant fraction displays the cutaneous lymphocyte antigen and exhibits effective E- and P-selectin ligand activity

Skin-homing T cells are defined by the expression of the cutaneous lymphocyte-associated antigen (CLA) which enables the cells to selectively bind to vascular endothelial E-selectin close to sites of cutaneous inflammation, an initial step in the effective extravasation from blood into the inflamed tissue. Essentially all CLA on T cells decorates the backbone of the P-selectin glycoprotein ligand-1 (PSGL-1). In this study we show that human peripheral blood B cells (PBBC) and tonsillar B cells (TBC) do not display PSGL-1 in fluorescence-activated cell sorter analysis using different murine monoclonal antibodies and polyclonal rabbit anti-PSGL-1 antiserum. A significant population of TBC, however, expresses a HECA-452-reactive epitope. These cells represent nonactivated IgM(+)/IgG(-) mature B lymphocytes. Up to 50% of the TBC in a given preparation strongly bind to E- and up to 79% to P-selectin. The shear stress resistance in a parallel-plate flow chamber system was high. Neuraminidase treatment of TBC totally and O-sialoglycoprotein endopeptidase partially diminished HECA-452 reactivity and reduced E- but not P-selectin ligand activities. Mocarhagin had no effect in the assays. The data suggest a different ligand for P-selectin and a distinct glycoprotein carrier for the E-selectin ligand as compared to T cells or other leukocytes. Adhesion to P-selectin, however, still required sulfation of the ligand for function. Western blots of TBC cell lysates detected a >240-kD HECA-452-reactive material that was resistant to reducing conditions. Anti-PSGL-1 did not reveal immunoreactive material in these cell lysates. B cell activation did neither significantly change HECA positivity nor induce PSGL-1 expression. Cultured, activated TBC, however, maintained expression of the integrin alpha4beta7. Human peripheral blood B cells had similar cell surface characteristics to TBC. Our observations suggest that several adhesion molecules may be involved in B cell homing which include CLA, the P-selectin ligand, and structures such as alpha4beta7.     

A human colon carcinoma cell line exhibits adhesive interactions with P-selectin under fluid flow via a PSGL-1-independent mechanism.

It has been postulated that endothelial cell adhesion molecules involved in leukocyte recruitment play a role in metastasis. Using an in vitro flow model, we studied the adhesion of the human colon carcinoma cell line KM12-L4 to P-selectin, an inducible endothelial-expressed adhesion molecule involved in leukocyte recruitment. Recombinant forms of P-selectin and Chinese hamster ovary cells stably expressing P-selectin supported attachment and rolling of KM12-L4 cells at 1 to 2 dynes/cm2. The adhesive interactions to P-selectin were abolished by pretreatment of the KM12-L4 cells with neuraminidase but were unaltered by pretreatment of the KM12-L4 cells with O-sialoglycoprotein endopeptidase, an enzyme that cleaves mucin type glycoproteins such as P-selectin glycoprotein ligand-1 (PSGL-1). PSGL-1 is the only counter-receptor for P-selectin known to mediate myeloid cell adhesion to P-selectin under flow. Flow cytometric and Northern blot analyses revealed that KM12-L4 cells did not express PSGL-1 and monoclonal antibody PL1, a function-blocking monoclonal antibody to PSGL-1, had no inhibitory effect on KM12-L4 adhesion to P-selectin under flow. Compared with HL-60 cells, which express PSGL-1, the KM12-L4 cells exhibited a slightly lower rate of attachment to P-selectin and rolled at a significantly higher velocity. In summary, KM12-L4 human colon carcinoma cells interact with P-selectin, under flow, through a PSGL-1-independent adhesion pathway.     

Platelets Control Leukocyte Recruitment in a Murine Model of Cutaneous Arthus Reaction

Platelets have been shown to be important in inflammation, but their role in the cutaneous Arthus reaction remains unclear. To assess the role of platelets in this pathogenetic process, the cutaneous Arthus reaction was examined in wild-type mice and mice lacking E-selectin, P-selectin, or P-selectin glycoprotein ligand-1 (PSGL-1) with or without platelet depletion by busulfan, a bone marrow precursor cell-specific toxin. Edema and hemorrhage induced by immune complex challenge significantly decreased in busulfan-treated wild-type mice compared with untreated mice. Busulfan treatment did not affect edema and hemorrhage in P-selectin- or PSGL-1-deficient mice, suggesting that the effect by busulfan is dependent on P-selectin and PSGL-1 expression. The inhibited edema and hemorrhage paralleled reduced infiltration of neutrophils and mast cells and reduced levels of circulating platelets. Increased cutaneous production of interleukin-6, tumor necrosis factor-α, and platelet-derived chemokines during Arthus reaction was inhibited in busulfan-treated wild-type mice relative to untreated mice, which paralleled the reduction in cutaneous inflammation. Flow cytometric analysis showed that immune complex challenge generated blood platelet-leukocyte aggregates that decreased by busulfan treatment. In thrombocytopenic mice, the cutaneous inflammation after immune complex challenge was restored by platelet infusion. These results suggest that platelets induce leukocyte recruitment into skin by forming platelet-leukocyte aggregates and secreting chemokines at inflamed sites, mainly through the interaction of P-selectin on platelets with PSGL-1 on leukocytes.The pathogenesis of autoimmune diseases frequently involves the formation of IgG-containing immune complexes (ICs) inducing inflammatory responses with significant tissue injury, commonly referred to as type III hypersensitivity reaction. This IC injury has been implicated in the pathogenesis of vasculitis syndrome, systemic lupus erythematosus, rheumatoid arthritis, and cryoglobulinemia.¹ The mechanisms by which the immune system controls effector responses to ICs are of central importance for developing therapeutic strategies. The standard animal model for the inflammatory response in these IC-mediated diseases is the Arthus reaction.² Analyses using gene knockout mice have revealed that activation of the complement system, especially C5a and its interaction with C5a receptor, and of Fc receptors for IgG on inflammatory cells, particularly mast cells, are both required to initiate the Arthus reaction.^3–8 In addition, accumulation of neutrophils and mast cells is necessary for the progression of the IC-mediated vascular tissue damage, which results in edema and hemorrhage.^3–8Leukocyte recruitment from the circulation to a site of inflammation is an essential process in the inflammatory response. Leukocytes first tether and roll on vascular endothelial cells, before they are activated to adhere firmly and subsequently immigrate into the extravascular space. This multistep process is highly regulated by multiple cell-surface adhesion molecules.^9,10 The selectins cooperate to support leukocyte tethering and rolling along inflamed vascular walls by mediating leukocyte interactions with glycoconjugated counter-receptors expressed by endothelium, adherent platelets, or leukocytes. The selectin family consists of three cell-surface molecules expressed by leukocytes (L-selectin), vascular endothelium (E- and P-selectins), and platelets (P-selectin).¹¹ Although the adhesive mechanisms underlying the capture and immobilization of circulating leukocytes in inflamed blood vessels have been well described, factors triggering and controlling the leukocyte recruitment into inflamed sites are poorly understood.The multistep process of leukocyte tethering and rolling, followed by leukocyte activation and firm adhesion, also occurs on activated platelets.¹² Platelets are essential for primary hemostasis, but they also play an important pro-inflammatory role.^13,14 Platelets normally circulate in a quiescent state, protected from untimely activation by inhibitory mediators released from intact endothelial cells. Endothelial dysfunction and changes in release of antiplatelet factors lead to increased platelet activation followed by their interaction with leukocytes, and increased platelet adhesion and aggregation.^15,16 On activation, platelets can change their shapes as well as the expression pattern of adhesion molecules, and secrete neutrophil and endothelial activators inducing production of pro-inflammatory cytokines.¹⁷ These changes are associated with the adhesion of platelets to leukocytes and endothelium.¹⁴ Thus, platelets are important amplifiers of acute inflammation.Platelets accumulate in inflammatory lesions concomitantly with leukocytes and regulate a variety of inflammatory responses by secreting or activating adhesion proteins, growth factors, and coagulation factors.^18,19 These proteins induce widely differing biological activities, including cell adhesion, chemotaxis, cell survival, and proliferation, all of which accelerate the inflammatory process.²⁰ In vitro and in vivo studies have shown that platelets bind to leukocytes through their surface protein.^12,14,20,21 Indeed, previous studies have reported that platelet-leukocyte aggregates are formed in circulating blood of asthmatic patients.²² Platelets express much amounts of P-selectin than endothelium and also bind endothelium via selectin dependent and independent mechanisms.^23–25 In addition to classical leukocyte recruitment process, platelets bound to activated endothelial cells can interact with leukocytes, which results in secondary capture that induces interactions of leukocytes with platelets first, followed by leukocyte-endothelial cell interaction.²⁶ Leukocytes within platelet-leukocyte complexes have increased adhesive capacity to the activated endothelium.²⁷ Therefore, platelet can function as a bridge between the circulating leukocyte and endothelium.We previously showed that mice lacking P-selectin (P-selectin^−/−) or mice treated with anti- P-selectin glycoprotein ligand-1 (PSGL-1) antibody (Ab) exhibited reduced Arthus reaction that is associated with decreased infiltration of neutrophils and mast cells.^28,29 In addition to interacting with selectins and selectin ligands on endothelial cells, leukocytes can also interact with selectins and selectin ligands presented by platelets or their microparticle fragments, which are all found at sites of inflammation.³⁰ This indicates that observations of altered leukocyte recruitment in selectin- and selectin ligand-deficient mice must be discussed in light of altered selectin and selectin-ligand expression not only by endothelial cells, but also by platelets. Recently, involvement of platelets has been demonstrated in the pathogenesis of inflammatory disorders, including asthma,^22,31 arthritis,¹⁸ inflammatory bowel disease,³² and chronic allergic dermatitis.³³ Although the role of platelets in inflammatory process is being increasingly recognized, it remains unknown how platelets induce leukocyte recruitment in the cutaneous Arthus reaction. A recent report has identified a role of platelets in promoting IC-induced leukocyte recruitment to the cremaster muscle in a murine model of reverse passive Arthus reaction.³⁴ However, the relative role of each leukocyte and adhesion molecule in the inflammation varies according to the tissue site and the nature of inflammatory stimuli.²⁹ Therefore, to clarify the importance of platelets, their surface adhesion molecule expression, and platelet-derived chemokines on leukocyte recruitment, we examined the cutaneous Arthus reaction in wild-type, P-selectin^−/−, E-selectin^−/−, and PSGL-1^−/− mice, with or without treatment with busulfan, a bone marrow precursor cell-specific toxin.     

Progressive loss of endothelial P-selectin expression with increasing malignancy in colorectal cancer

Adhesion of inflammatory cells to vascular endothelium is mediated by specific cell adhesion receptors on both leukocytes and endothelial cells. One of the adhesion molecules on the endothelium is P-selectin. Decreased vascular P-selectin expression has been associated with tumor progression in melanoma patients. We now report on the expression of endothelial P-selectin in colorectal cancer (CRC). We studied a colorectal tissue specimen series ranging from normal colorectal tissue via unmetastasized primary tumors to tumors with the same depth of invasion at the primary site but with liver metastases. Moreover, P-selectin expression levels in liver metastases were determined. The number of P-selectin positive vessels as a fraction of the total number of vessels, both intra- and peritumorally, was determined by staining for CD62P and CD34, respectively. Furthermore, by immunostaining for leukocytes (CD45) and macrophages (CD68), it was evaluated whether levels of P-selectin expression influenced infiltrate density and composition. The results showed that levels of peritumoral P-selectin expression were reciprocal to the degree of progression in CRC. This relation was even more pronounced intratumorally: in metastasized primary tumors and in the metastatic lesions, P-selectin expression was virtually absent. This distribution pattern was reflected in the numbers of leukocytes that accumulated in the various tissues, since in the primary tumors with metastases, and in the metastatic lesions, hardly any infiltrating cells were observed. In these lesions, leukocytes were present in the peritumoral zone, but seemed unable to enter the tumor tissue. In primary tumors without metastasis, the intratumoral leukocyte infiltration density was significantly higher. Recruitment levels of macrophages remained constant throughout the different tissues. We suggest that downregulation of endothelial P-selectin expression is a mechanism by which CRC lesions evade inflammatory regression and, thereby, progress to a more advanced stage of malignancy.     

Transgene expression of alpha(1,2)-fucosyltransferase-I (FUT1) in tumor cells selectively inhibits sialyl-Lewis x expression and binding to E-selectin without affecting synthesis of sialyl-Lewis a or binding to P-selectin

During inflammation, E- and P-selectins appear on activated endothelial cells to interact with leukocytes through sialyl-Lewis x and sialyl-Lewis a antigens (sLe(x/a)). These selectins can also interact with tumor cells in a sialyl-Lewis-dependent manner and for this reason, they are thought to play a key role in metastasis. Diverting the biosynthesis of sialyl-Lewis antigens toward nonadhesive structures is an attractive gene therapy for preventing the hematogenous metastatic spread of cancers. We have previously shown that transfection of alpha(1,2)-fucosyltransferase-I (FUT1) in Chinese hamster ovary (CHO) cells had a slight effect on the overall sialylation while the synthesis of sLE(x) was dramatically prevented. We herein delivered the gene of FUT1 by a human immunodeficiency virus-derived lentiviral vector to three human cancer cell lines including pancreatic (BxPC3), hepatic (HepG2), and colonic (HT-29) cancer cells. We found that on FUT1 transduction, all cells exhibited a dramatic decrease in sLe(x) synthesis with a concomitant increase in Le(y) and Le(b) expression, without any detectable effect on the level of cell surface sLe(a) antigens. In parallel, FUT1-transduced HT-29 and HepG2 cells, but not BxPC3 cells, failed to interact with E-selectin as assessed by E-selectin-binding assay or dynamic adhesion to activated endothelial cells. We show also that transduced FUT1 efficiently fucosylates the P-selectin ligand PSGL-1 without altering P-selectin binding. These results have important implications for understanding cell-specific reactions underlying the synthesis of selectin ligands in cancer cells and may provide a basis for the development of anti-metastatic gene therapy.     

Effects of nicotinic acid on endothelial cells and platelets

BackgroundInteractions between platelets and endothelial cells under inflammatory conditions lead to an increased expression of various activity markers of atherosclerosis in the vessel wall. The purpose of this study was to investigate possible protective effects of nicotinic acid in an in vitro endothelial cell model.MethodsAfter a 24-hour incubation period with nicotinic acid (1 mmol/l), human umbilical vein endothelial cells were stimulated for 1 h with lipopolysaccharide and were then incubated in direct contact with activated platelets. Following this incubation, the expression of CD40L and CD62P on platelets and the expression of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, uPAR, and MT1-MMP on endothelial cells were measured by flow cytometry. Supernatants were analyzed by ELISA for soluble MCP-1 and MMP-1.ResultsThe increased expression of VCAM-1 on endothelial cells by proinflammatory stimulation with activated platelets was significantly reduced through preincubation with nicotinic acid (P<.05). Furthermore, platelets in direct contact with preincubated endothelial cells showed a significant reduction in their CD62P and CD40L expression when compared to platelets incubated with untreated endothelial cells (P<.05). Treatment with nicotinic acid did not have a significant effect on ICAM-1, uPAR, and MT1-MMP expression on endothelial cells. Levels of soluble MCP-1 and MMP-1 in supernatants were lower after preincubation with nicotinic acid.ConclusionNicotinic acid inhibits platelet activation after platelets contacted nicotinic acid treated endothelial cells and inhibits VCAM-1 expression on human endothelial cells under inflammatory conditions. These findings suggest a possible pleiotropic therapeutic relevance of nicotinic acid in atherosclerosis.     

Cutaneous lymphocyte-associated antigen (CLA) T cells up-regulate P-selectin ligand expression upon their activation

Memory T cells expressing CLA occur in humans and accumulate in normal and inflamed skin. These cells uniformly bind to the vascular adhesion molecule E-selectin, yet only a subset binds to P-selectin. The latter cells are distinguished by the mAb CHO-131, and are enriched in psoriasis lesions. Activated T cells up-regulate CLA expression, but little is currently known about their binding to P-selectin. We observed that CLA⁺ CD4⁺ T cells derived from stimulated naive T cells uniformly express the CHO-131 epitope. This occurred as well upon the restimulation of memory CLA⁺ CD4⁺ T cells. The latter cells also expressed higher levels of PSGL-1 modified by P-selectin glycan ligands; C2GlcNAcT-1 mRNA, a glycosyltransferase critical for such glycan synthesis; and more uniformly bound to P-selectin. Our findings thus indicate that unlike memory CLA⁺ CD4⁺ T cells, when activated these cells can broadly bind to P-selectin, suggesting a more diverse tissue trafficking capacity.     

Molecular and functional interactions among monocytes, platelets, and endothelial cells and their relevance for cardiovascular diseases

Platelets, monocytes, and endothelial cells are instrumental in the development and progression of cardiovascular diseases. Inflammation, a key process underlying cardiovascular disorders, is accompanied and amplified by activation of platelets and consequent binding of such platelets to the endothelium. There, platelet-derived chemokines, in conjunction with increased expression of adhesion molecules, promote the recruitment of circulating monocytes that will eventually migrate across the endothelial lining of the vessel into the tissues. Additionally, platelets may already become activated in the circulation and may form platelet-monocyte complexes, which show increased adhesive and migratory capacities themselves but also facilitate recruitment of noncomplexed leukocytes. They should therefore be considered as important mediators of inflammation. In molecular terms, these events are additionally governed by chemokines released and presented by the endothelium as well as the different classes of endothelial adhesion molecules that regulate the interactions among the various cell types. Most important in this respect are the selectins and their ligands, such as P-selectin glycoprotein (GP) ligand 1, and the integrins binding to Ig-like cell adhesion molecules as well as to GP, such as von Willebrand factor, present in the extracellular matrix or on activated endothelium. This review aims to provide an overview of these complex interactions and of their functional implications for inflammation and development of cardiovascular disease.     

Inhibition of leukocyte adhesion to endothelial cells of human umbilical vein by Y-24180, An antagonist of platelet-activating factor

Effect of Y-24180 (4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dimethyl-6H-thieno[3, 2-f] [1,2,4]triazolo[4,3-a][1,4]diazepine), an antagonist of platelet-activating factor (PAF), on adhesion of leukocytes to endothelial cells of human umbilical vein was examined. Y-24180 inhibited the adhesion of guinea-pig peritoneal exudate cells activated by leukotriene B₄ to endothelial cells in a concentration-dependent manner. WEB2086, another PAF antagonist, did not show the inhibitory effect. Inhibition by Y-24180 was not influenced by addition of PAF. Y-24180 had no effect on the expression of intercellular adhesion molecule-1 or endothelial-leukocyte adhesion molecule-1 on endothelial cells activated with interleukin-1β, while it suppressed the expression of CD18 antigen on human peripheral blood polymorphonuclear cells activated with leukotrien B₄, inhibiting their adhesion to endothelial cells activated with interleukin-1β. In oxazolone-induced ear edema in mice, which involves delayed-type hypersensitivity with infiltration by inflammatory cells, Y-24180 dose-dependently inhibited the increase in ear weights, but WEB2086 did not. These results indicate that Y-24180 inhibits not only the actions of PAF but also the adhesion of leukocytes by suppressing the expression of CD18 antigen, inducing the inhibition of infiltration by inflammatory cells.     

Inhibitory effect of non-anticoagulant heparin (S-NACH) on pancreatic cancer cell adhesion and metastasis in human umbilical cord vessel segment and in mouse model

Metastasis is the most devastating aspect of cancer and it is the main cause of morbidity and mortality in cancer patients. Tumor cell adhesion to the vascular endothelial cell lining is an important step in metastatic progression and is prompted by platelets. Mucin 1 is over-expressed and aberrantly glycosylated in more than 60% of pancreatic ductal adeno-carcinomas, which mediate adhesion of pancreatic cancer cells to platelets via P-selectin. The anticoagulant low molecular weight heparins (LMWHs), which are commonly used in venous Thromboprophylaxis and treatment, appear to have an effect on cancer survival. The aim of this study is to investigate the effect of platelets on human pancreatic cancer MPanc96 cell adhesion to the endothelial cell vessel wall, and to examine the effect of heparin derivatives on MPanc96 adhesion using a novel, in vitro model of human umbilical cord vein. The modified heparin S-NACH (sulfated non-anticoagulant heparin), which is devoid of antithrombin (AT) binding and devoid of inhibition of systemic AT-dependent coagulation factors such as factor Xa and IIa, and the LMWH tinzaparin both potently reduced adhesion and invasion of fluorescence-labeled MPanc96 cancer cells to the endothelial layer of umbilical cord vein in a dose-dependent manner. S-NACH effectively inhibited P-selectin mediated MPanc96 cell adhesion, and inhibited cell adhesion and invasion similar to tinzaparin, indicating that systemic anticoagulation is not a necessary component for heparin attenuation of cancer cell adhesion, invasion, and metastasis. Also, S-NACH and tinzaparin versus unfractionated heparin, heparin derivatives enoxaparin, deltaparin, fraxiparin, and fondaparinux were evaluated for their effect on platelet-cancer cell adhesion. An in vivo anti-metastatic S-NACH-treated nude mouse model of MPanc96 pancreatic cancer cell metastasis demonstrated potent anti-metastasis efficacy as evidenced by IVIS imaging and histological staining.     

Endotoxin Down-Modulates P-Selectin Glycoprotein Ligand-1 (PSGL-1, CD162) on Neutrophils in Humans

P-selectin glycoprotein ligand-1 (PSGL-1, CD162), the counter-receptor for P-selectin and possibly E- and L-selectin, mediates rolling of leukocytes during inflammation and, thus, plays a pivotal role in hemostasis and inflammation. PSGL-1 is constitutively expressed on circulating leukocytes. Until recently, PSGL-1 has been considered not to be regulated upon cell activation. As modulation of PSGL-1 has only recently been reported for three proinflammatory substances, PSGL-1 regulation was examined during systemic inflammation in humans. Nine healthy human volunteers received a bolus of 2 ng/kg LPS i.v. Endotoxin infusion down-modulated PSGL-1 expression on neutrophils, with a maximum at 6-8 hr (-22%; P =0.001 vs. baseline and placebo), which correlated with peak neutrophilia. Similar PSGL-1 down-regulation was observed on monocytes. sPSGL-1 plasma levels increased trendwise after LPS infusion (+12% at 6 hr; P =0.10). In vitro LPS stimulation of whole blood significantly down-regulated PSGL-1 on neutrophils (-43%) and monocytes (-35%) as early as 2 hr ( P <0.05; n =5) in both EDTA and lepirudin anticoagulated samples. In summary, PSGL-1 is down-modulated on neutrophils and monocytes during endotoxemia in humans. PSGL-1 down-regulation could potentially facilitate the development of neutrophilia.