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Rodger P. Mcever - One of the best experts on this subject based on the ideXlab platform.
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p Selectin Glycoprotein ligand 1 forms dimeric interactions with e Selectin but monomeric interactions with l Selectin on cell surfaces
PLOS ONE, 2013Co-Authors: Yan Zhang, Rodger P. Mcever, Ning Jiang, Veronika I Zarnitsyna, Arkadiusz G Klopocki, Cheng ZhuAbstract:Interactions of Selectins with cell surface glycoconjugates mediate the first step of the adhesion and signaling cascade that recruits circulating leukocytes to sites of infection or injury. P-Selectin dimerizes on the surface of endothelial cells and forms dimeric bonds with P-Selectin Glycoprotein ligand-1 (PSGL-1), a homodimeric sialomucin on leukocytes. It is not known whether leukocyte L-Selectin or endothelial cell E-Selectin are monomeric or oligomeric. Here we used the micropipette technique to analyze two-dimensional binding of monomeric or dimeric L- and E-Selectin with monomeric or dimeric PSGL-1. Adhesion frequency analysis demonstrated that E-Selectin on human aortic endothelial cells supported dimeric interactions with dimeric PSGL-1 and monomeric interactions with monomeric PSGL-1. In contrast, L-Selectin on human neutrophils supported monomeric interactions with dimeric or monomeric PSGL-1. Our work provides a new method to analyze oligomeric cross-junctional molecular binding at the interface of two interacting cells.
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signal dependent slow leukocyte rolling does not require cytoskeletal anchorage of p Selectin Glycoprotein ligand 1 psgl 1 or integrin αlβ2
Journal of Biological Chemistry, 2012Co-Authors: Bojing Shao, Rodger P. Mcever, Arkadiusz G Klopocki, Tadayuki Yago, Phillip A Coghill, Padmaja Mehtadsouza, David W Schmidtke, William H RodgersAbstract:In inflamed venules, neutrophils roll on P- or E-Selectin, engage P-Selectin Glycoprotein ligand-1 (PSGL-1), and signal extension of integrin αLβ2 in a low affinity state to slow rolling on intercellular adhesion molecule-1 (ICAM-1). Cytoskeleton-dependent receptor clustering often triggers signaling, and it has been hypothesized that the cytoplasmic domain links PSGL-1 to the cytoskeleton. Chemokines cause rolling neutrophils to fully activate αLβ2, leading to arrest on ICAM-1. Cytoskeletal anchorage of αLβ2 has been linked to chemokine-triggered extension and force-regulated conversion to the high affinity state. We asked whether PSGL-1 must interact with the cytoskeleton to initiate signaling and whether αLβ2 must interact with the cytoskeleton to extend. Fluorescence recovery after photobleaching of transfected cells documented cytoskeletal restraint of PSGL-1. The lateral mobility of PSGL-1 similarly increased by depolymerizing actin filaments with latrunculin B or by mutating the cytoplasmic tail to impair binding to the cytoskeleton. Converting dimeric PSGL-1 to a monomer by replacing its transmembrane domain did not alter its mobility. By transducing retroviruses expressing WT or mutant PSGL-1 into bone marrow-derived macrophages from PSGL-1-deficient mice, we show that PSGL-1 required neither dimerization nor cytoskeletal anchorage to signal β2 integrin-dependent slow rolling on P-Selectin and ICAM-1. Depolymerizing actin filaments or decreasing actomyosin tension in neutrophils did not impair PSGL-1- or chemokine-mediated integrin extension. Unlike chemokines, PSGL-1 did not signal cytoskeleton-dependent swing out of the β2-hybrid domain associated with the high affinity state. The cytoskeletal independence of PSGL-1-initiated, αLβ2-mediated slow rolling differs markedly from the cytoskeletal dependence of chemokine-initiated, αLβ2-mediated arrest.
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replacing a lectin domain residue in l Selectin enhances binding to p Selectin Glycoprotein ligand 1 but not to 6 sulfo sialyl lewis x
Journal of Biological Chemistry, 2008Co-Authors: Arkadiusz G Klopocki, Cheng Zhu, Richard D Cummings, Padmaja Mehta, Tadayuki Yago, Jun Yang, Anne Leppanen, Nicolai V Bovin, Rodger P. MceverAbstract:Selectin-ligand interactions (bonds) mediate leukocyte rolling on vascular surfaces. The molecular basis for differential ligand recognition by Selectins is poorly understood. Here, we show that substituting one residue (A108H) in the lectin domain of L-Selectin increased its force-free affinity for a glycosulfopeptide binding site (2-GSP-6) on P-Selectin Glycoprotein ligand-1 (PSGL-1) but not for a sulfated-glycan binding site (6-sulfo-sialyl Lewis x) on peripheral node addressin. The increased affinity of L-SelectinA108H for 2-GSP-6 was due to a faster on-rate and to a slower off-rate that increased bond lifetimes in the absence of force. Rather than first prolonging (catching) and then shortening (slipping) bond lifetimes, increasing force monotonically shortened lifetimes of L-SelectinA108H bonds with 2-GSP-6. When compared with microspheres bearing L-Selectin, L-SelectinA108H microspheres rolled more slowly and regularly on 2-GSP-6 at low flow rates. A reciprocal substitution in P-Selectin (H108A) caused faster microsphere rolling on 2-GSP-6. These results distinguish molecular mechanisms for L-Selectin to bind to PSGL-1 and peripheral node addressin and explain in part the shorter lifetimes of PSGL-1 bonds with L-Selectin than P-Selectin.
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structurally distinct requirements for binding of p Selectin Glycoprotein ligand 1 and sialyl lewis x to anaplasma phagocytophilum and p Selectin
Journal of Biological Chemistry, 2003Co-Authors: Tadayuki Yago, Richard D Cummings, Anne Leppanen, Jason A Carlyon, Mustafa Akkoyunlu, Sougata Karmakar, Erol Fikrig, Rodger P. MceverAbstract:Colonization of neutrophils by the bacterium Anaplasma phagocytophilum causes the disease human granulocytic ehrlichiosis. The pathogen also infects mice, its natural host. Like binding of P-Selectin, binding of A. phagocytophilum to human neutrophils requires expression of P-Selectin Glycoprotein ligand-1 (PSGL-1) and alpha1-3-fucosyltransferases that construct the glycan determinant sialyl Lewis x (sLex). Binding of A. phagocytophilum to murine neutrophils, however, requires expression of alpha1-3-fucosyltransferases but not PSGL-1. To further characterize the molecular features that A. phagocytophilum recognizes, we measured bacterial binding to microspheres bearing specific glycoconjugates or to cells expressing human PSGL-1 and particular glycosyltransferases. Like P-Selectin, A. phagocytophilum bound to purified human PSGL-1 and to glycopeptides modeled after the N terminus of human PSGL-1 that presented sLex on an O-glycan. Unlike P-Selectin, A. phagocytophilum bound to glycopeptides that contained sLex but lacked tyrosine sulfation or a specific core-2 orientation of sLex on the O-glycan. A. phagocytophilum bound only to glycopeptides that contained a short amino acid sequence found in the N-terminal region of human but not murine PSGL-1. Unlike P-Selectin, A. phagocytophilum bound to cells expressing PSGL-1 in cooperation with sLex on both N-and O-glycans. Moreover, bacteria bound to microspheres coupled independently with glycopeptide lacking sLex and with sLex lacking peptide. These results demonstrate that, unlike P-Selectin, A. phagocytophilum binds cooperatively to a nonsulfated N-terminal peptide in human PSGL-1 and to sLex expressed on PSGL-1 or other Glycoproteins. Distinct bacterial adhesins may mediate these cooperative interactions.
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p Selectin Glycoprotein ligand 1 mediates l Selectin dependent leukocyte rolling in venules
Journal of Experimental Medicine, 2003Co-Authors: Rodger P. Mcever, Lijun Xia, Timothy S. Olson, Markus Sperandio, Michael L Smith, Bradley S Forlow, Klaus LeyAbstract:Leukocyte rolling in postcapillary venules of inflamed tissues is reduced in L-Selectin–deficient mice and mice treated with L-Selectin blocking antibodies, but the Glycoprotein ligand for L-Selectin in inflamed venules is unknown. Here, we show that L-Selectin–dependent rolling after P-Selectin blockade is completely absent in P-Selectin Glycoprotein ligand-1 (PSGL-1)−/− mice or wild-type mice treated with a PSGL-1 blocking monoclonal antibody. Immunohistochemistry and flow cytometry failed to show PSGL-1 expression on resting or inflamed endothelium or on platelets. To investigate whether leukocyte-expressed PSGL-1 is mediating L-Selectin–dependent rolling, we reconstituted lethally irradiated wild-type mice with PSGL-1−/− bone marrow cells. These chimeric mice showed no L-Selectin–dependent rolling, suggesting that leukocyte-expressed PSGL-1 mediates L-Selectin–dependent rolling. Frame-to-frame video analysis of L-Selectin–dependent rolling in wild-type mice showed that the majority of observed L-Selectin–dependent leukocyte rolling was between free flowing leukocytes and already adherent leukocytes or possibly leukocyte fragments, followed by E-Selectin–dependent leukocyte rolling along the endothelium. Leukocyte rolling was significantly slower for leukocyte–endothelial than leukocyte–leukocyte interactions. We conclude that leukocyte-expressed PSGL-1 serves as the main L-Selectin ligand in inflamed postcapillary venules. L-Selectin binding to PSGL-1 initiates tethering events that enable L-Selectin–independent leukocyte-endothelial interactions. These findings provide a molecular mechanism for the inflammatory defects seen in L-Selectin–deficient mice.
Richard D Cummings - One of the best experts on this subject based on the ideXlab platform.
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replacing a lectin domain residue in l Selectin enhances binding to p Selectin Glycoprotein ligand 1 but not to 6 sulfo sialyl lewis x
Journal of Biological Chemistry, 2008Co-Authors: Arkadiusz G Klopocki, Cheng Zhu, Richard D Cummings, Padmaja Mehta, Tadayuki Yago, Jun Yang, Anne Leppanen, Nicolai V Bovin, Rodger P. MceverAbstract:Selectin-ligand interactions (bonds) mediate leukocyte rolling on vascular surfaces. The molecular basis for differential ligand recognition by Selectins is poorly understood. Here, we show that substituting one residue (A108H) in the lectin domain of L-Selectin increased its force-free affinity for a glycosulfopeptide binding site (2-GSP-6) on P-Selectin Glycoprotein ligand-1 (PSGL-1) but not for a sulfated-glycan binding site (6-sulfo-sialyl Lewis x) on peripheral node addressin. The increased affinity of L-SelectinA108H for 2-GSP-6 was due to a faster on-rate and to a slower off-rate that increased bond lifetimes in the absence of force. Rather than first prolonging (catching) and then shortening (slipping) bond lifetimes, increasing force monotonically shortened lifetimes of L-SelectinA108H bonds with 2-GSP-6. When compared with microspheres bearing L-Selectin, L-SelectinA108H microspheres rolled more slowly and regularly on 2-GSP-6 at low flow rates. A reciprocal substitution in P-Selectin (H108A) caused faster microsphere rolling on 2-GSP-6. These results distinguish molecular mechanisms for L-Selectin to bind to PSGL-1 and peripheral node addressin and explain in part the shorter lifetimes of PSGL-1 bonds with L-Selectin than P-Selectin.
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structurally distinct requirements for binding of p Selectin Glycoprotein ligand 1 and sialyl lewis x to anaplasma phagocytophilum and p Selectin
Journal of Biological Chemistry, 2003Co-Authors: Tadayuki Yago, Richard D Cummings, Anne Leppanen, Jason A Carlyon, Mustafa Akkoyunlu, Sougata Karmakar, Erol Fikrig, Rodger P. MceverAbstract:Colonization of neutrophils by the bacterium Anaplasma phagocytophilum causes the disease human granulocytic ehrlichiosis. The pathogen also infects mice, its natural host. Like binding of P-Selectin, binding of A. phagocytophilum to human neutrophils requires expression of P-Selectin Glycoprotein ligand-1 (PSGL-1) and alpha1-3-fucosyltransferases that construct the glycan determinant sialyl Lewis x (sLex). Binding of A. phagocytophilum to murine neutrophils, however, requires expression of alpha1-3-fucosyltransferases but not PSGL-1. To further characterize the molecular features that A. phagocytophilum recognizes, we measured bacterial binding to microspheres bearing specific glycoconjugates or to cells expressing human PSGL-1 and particular glycosyltransferases. Like P-Selectin, A. phagocytophilum bound to purified human PSGL-1 and to glycopeptides modeled after the N terminus of human PSGL-1 that presented sLex on an O-glycan. Unlike P-Selectin, A. phagocytophilum bound to glycopeptides that contained sLex but lacked tyrosine sulfation or a specific core-2 orientation of sLex on the O-glycan. A. phagocytophilum bound only to glycopeptides that contained a short amino acid sequence found in the N-terminal region of human but not murine PSGL-1. Unlike P-Selectin, A. phagocytophilum bound to cells expressing PSGL-1 in cooperation with sLex on both N-and O-glycans. Moreover, bacteria bound to microspheres coupled independently with glycopeptide lacking sLex and with sLex lacking peptide. These results demonstrate that, unlike P-Selectin, A. phagocytophilum binds cooperatively to a nonsulfated N-terminal peptide in human PSGL-1 and to sLex expressed on PSGL-1 or other Glycoproteins. Distinct bacterial adhesins may mediate these cooperative interactions.
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N-terminal residues in murine P-Selectin Glycoprotein ligand-1 required for binding to murine P-Selectin
Blood, 2002Co-Authors: Lijun Xia, J. Michael Mcdaniel, Vishwanath Ramachandran, Kiem N. Nguyen, Richard D CummingsAbstract:P-Selectin binds to the N-terminal region of human P-Selectin Glycoprotein ligand-1 (PSGL-1). For optimal binding, this region requires sulfation on 3 tyrosines and specific core-2 O-glycosylation on a threonine. P-Selectin is also thought to bind to the N terminus of murine PSGL-1, although it has a very different amino acid sequence than human PSGL-1. Murine PSGL-1 has potential sites for sulfation at Tyr13 and Tyr15 and for O-glycosylation at Thr14 and Thr17. We expressed murine PSGL-1 or constructs with substitutions of these residues in transfected Chinese hamster ovary cells that coexpressed the glycosyltransferases required for binding to P-Selectin. The cells were assayed for binding to fluid-phase P-Selectin and for tethering and rolling on P-Selectin under flow. In both assays, substitution of Tyr13 or Thr17 markedly diminished, but did not eliminate, binding to P-Selectin. In contrast, substitution of Tyr15 or Thr14 did not affect binding. Substitution of all 4 residues eliminated binding. Treatment of cells with chlorate, an inhibitor of sulfation, markedly reduced binding of wild-type PSGL-1 to P-Selectin but did not further decrease binding of PSGL-1 with substitutions of both tyrosines. These data suggest that sulfation of Tyr13 and O-glycosylation of Thr17 are necessary for murine PSGL-1 to bind optimally to P-Selectin. Because it uses only one tyrosine, murine PSGL-1 may rely more on other peptide components and O-glycosylation to bind to P-Selectin than does human PSGL-1.
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p Selectin Glycoprotein ligand 1 deficient mice have impaired leukocyte tethering to e Selectin under flow
Journal of Clinical Investigation, 2002Co-Authors: Lijun Xia, Richard D Cummings, Markus Sperandio, Tadayuki Yago, Michael J Mcdaniel, Sonia Pearsonwhite, Klaus LeyAbstract:P-Selectin Glycoprotein ligand-1 (PSGL-1) mediates rolling of leukocytes on P-Selectin under flow. The Glycoproteins that enable leukocyte tethering to or rolling on E-Selectin are not known. We used gene targeting to prepare PSGL-1–deficient (PSGL-1–/–) mice, which were healthy but had moderately elevated total blood leukocytes. Fluid-phase E-Selectin bound to approximately 70% fewer sites on PSGL-1–/– than PSGL-1+/+ neutrophils. Compared with PSGL-1+/+ leukocytes, significantly fewer PSGL-1–/– leukocytes rolled on E-Selectin in vitro, because their initial tethering to E-Selectin was impaired. The residual cells that tethered rolled with the same shear resistance and velocities as PSGL-1+/+ leukocytes. Compared with PSGL-1+/+ mice, significantly fewer PSGL-1–/– leukocytes rolled on E-Selectin in TNF-α–treated venules of cremaster muscle in which P-Selectin function was blocked by an mAb. The residual PSGL-1–/– leukocytes that tethered rolled with slow velocities equivalent to those of PSGL-1+/+ leukocytes. These results reveal a novel function for PSGL-1 in tethering leukocytes to E-Selectin under flow.
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noncovalent association of p Selectin Glycoprotein ligand 1 and minimal determinants for binding to p Selectin
Journal of Biological Chemistry, 2000Co-Authors: Terry Kay Epperson, Rodger P. Mcever, K D Patel, Richard D CummingsAbstract:P-Selectin Glycoprotein ligand-1 (PSGL-1) is a disulfide-bonded, homodimeric mucin ( approximately 250 kDa) on leukocytes that binds to P-Selectin on platelets and endothelial cells during the initial steps in inflammation. Because it has been proposed that only covalently dimerized PSGL-1 can bind P-Selectin, we investigated the factors controlling dimerization of PSGL-1 and re-examined whether covalent dimers are required for binding its P-Selectin. Recombinant forms of PSGL-1 were created in which the single extracellular Cys (Cys(320)) was replaced with either Ser (C320S-PSGL-1) or Ala (C320A-PSGL-1). Both recombinants migrated as monomeric species of approximately 120 kDa under both nonreducing and reducing conditions on SDS-polyacrylamide gel electrophoresis. P-Selectin bound similarly to cells expressing either wild type or mutated forms of PSGL-1 in both flow cytometric and rolling adhesion assays. Unexpectedly, chemical cross-linking studies revealed that both C320S- and C320A-PSGL-1 noncovalently associate in the plasma membrane and cross-linking generates dimeric species. Chimeric recombinants of PSGL-1 in which the transmembrane domain in PSGL-1 was replaced with the transmembrane domain of CD43 (CD43TMD-PSGL-1) could not be chemically cross-linked, suggesting that residues within the transmembrane domain of PSGL-1 are required for noncovalent association. Cells expressing CD43TMD-PSGL-1 bound P-Selectin. To further address the ability of P-Selectin to bind monomeric derivatives of PSGL-1, intact HL-60 cells were trypsin-treated, which generated a soluble approximately 25-kDa NH(2)-terminal fragment of PSGL-1 that bound to immobilized P-Selectin. Because N-glycosylation of PSGL-1 hinders trypsin cleavage, a recombinant form of PSGL-1 was generated in which all three potential N-glycosylation sites were mutated (DeltaN-PSGL-1). Cells expressing DeltaN-PSGL-1 bound P-Selectin, and trypsin treatment of the cells generated NH(2)-terminal monomeric fragments (<10 kDa) of PSGL-1 that bound to P-Selectin. These results demonstrate that Cys(320)-dependent dimerization of PSGL-1 is not required for binding to P-Selectin and that a small monomeric fragment of PSGL-1 is sufficient for P-Selectin recognition.
Takako Hirata - One of the best experts on this subject based on the ideXlab platform.
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P-Selectin Glycoprotein Ligand-1 Negatively Regulates T-Cell Immune Responses
Journal of immunology (Baltimore Md. : 1950), 2009Co-Authors: Masanori Matsumoto, Masayuki Miyasaka, Takako HirataAbstract:Cell surface sialomucins often act as antiadhesive molecules by virtue of their extended structure and negative charge. CD43 is one such sialomucin, expressed on most leukocytes. P-Selectin Glycoprotein ligand-1 (PSGL-1) is another sialomucin expressed by leukocytes. It serves as a major Selectin ligand, but no antiadhesive role for it has been described. In this study, we showed that PSGL-1-deficient T cells, like CD43-deficient T cells, exhibited increased adhesion and proliferation compared with wild-type cells. The loss of both PSGL-1 and CD43 led to a further increase in T cell adhesion and proliferation. The reexpression of full-length PSGL-1 or CD43 in double-deficient CD4 + T cells reversed their increased adhesion and proliferation phenotype. Using chimeric constructs of human CD8 and either PSGL-1 or CD43, we demonstrated that the intracellular domain of PSGL-1 or CD43 is required for suppressing proliferation but not adhesion. Furthermore, in a mouse model of inflammatory bowel disease induced by the adoptive transfer of naive T cells into RAG-deficient hosts, a PSGL-1 deficiency exacerbated the development of inflammation. These results reveal a novel regulatory role for PSGL-1 in T cell adhesion and proliferation and suggest that PSGL-1 negatively regulates T cell immune responses in vivo.
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an l Selectin ligand distinct from p Selectin Glycoprotein ligand 1 is expressed on endothelial cells and promotes neutrophil rolling in inflammation
Blood, 2008Co-Authors: Akiko Shigeta, Masanori Matsumoto, Masayuki Miyasaka, Thomas F Tedder, John B Lowe, Takako HirataAbstract:Neutrophils recruited from the blood are key players in the innate immune response. Selectins play critical roles in neutrophil recruitment by mediating their tethering and rolling in inflamed venules. While the roles of P- and E-Selectin in this process are well established, the mechanisms of L-Selectin–mediated neutrophil recruitment remain elusive. One proposal is that tethering is mediated by L-Selectin on flowing neutrophils interacting with P-Selectin Glycoprotein ligand-1 (PSGL-1) on adherent neutrophils. To clarify whether L-Selectin–mediated neutrophil recruitment depends entirely on PSGL-1, we examined the impact of L-Selectin deficiency in mice with a PSGL-1–deficient background. L-Selectin and PSGL-1 double-knockout mice exhibited a higher increase in their peripheral blood neutrophil count and a worse defect in neutrophil recruitment into the inflamed peritoneum than PSGL-1–deficient mice. Intravital microscopy of inflamed cremaster muscle venules showed that L-Selectindeficiency or antibody blockade of L-Selectin reduced the residual leukocyte rolling in PSGL-1–deficient mice. Flow cytometric analyses showed that the endothelial cells from the cremaster muscle bound L-Selectin in a PSGL-1–independent manner. These results provide evidence for the existence of an L-Selectin ligand distinct from PSGL-1 in inflammation and indicate that such a ligand is expressed on endothelial cells, promoting neutrophil rolling in vivo.
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p Selectin Glycoprotein ligand 1 mediates l Selectin independent leukocyte rolling in high endothelial venules of peripheral lymph nodes
International Immunology, 2007Co-Authors: Nari Harakawa, Masayuki Miyasaka, Barbara C. Furie, Akiko Shigeta, Bruce Furie, Glenn Merrillskoloff, Masahiro Wato, Toshiro Okazaki, Naochika Domae, Takako HirataAbstract:Lymphocyte homing to peripheral lymph nodes (LNs) requires L-Selectin. Previous studies, however, suggest that there are L-Selectin-independent mechanisms of lymphocyte homing. P-Selectin Glycoprotein ligand-1 (PSGL-1) is a major ligand for P-Selectin expressed in a Selectin-binding form on myeloid cells and subsets of lymphoid cells. To discover whether PSGL-1 plays a role in lymphocyte homing, we examined leukocyte rolling and adhesion in the high endothelial venules (HEVs) of the subiliac LNs of wild-type and PSGL-1-deficient mice by intravital microscopy. There were no significant differences in blood velocity or wall shear stress between wild-type and PSGL-1-deficient mice. Although the leukocyte rolling fraction was not altered in PSGL-1-deficient mice, infusion of an anti-L-Selectin mAb into these mice completely abolished leukocyte rolling, while the same treatment in wild-type mice inhibited 90% of the leukocyte rolling. This residual rolling in wild-type mice appears to depend on the PSGL-1-P-Selectin interaction, since infusion of an anti-L-Selectin mAb together with an anti-PSGL-1 mAb or anti-P-Selectin mAb almost completely abolished the rolling. PSGL-1 deficiency also led to a higher rolling velocity, suggesting that PSGL-1 mediates leukocyte rolling at low velocities. P-Selectin was found to be expressed on the HEVs of subiliac LNs under the conditions of intravital microscopy. Taken together, these results indicate that the interaction of PSGL-1 with P-Selectin constitutes a second mechanism of leukocyte rolling in the HEVs of peripheral LNs.
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cd43 collaborates with p Selectin Glycoprotein ligand 1 to mediate e Selectin dependent t cell migration into inflamed skin
Journal of Immunology, 2007Co-Authors: Masanori Matsumoto, Masayuki Miyasaka, Akiko Shigeta, Yuko Furukawa, Toshiyuki Tanaka, Takako HirataAbstract:Activated T cell migration into nonlymphoid tissues is initiated by the interactions of P- and E-Selectin expressed on endothelial cells and their ligands on T cells. P-Selectin Glycoprotein ligand-1 (PSGL-1) has been the only E-Selectin ligand demonstrated to function during the in vivo migration of activated T cells. We show in this study that CD43-deficient Th1 cells, like PSGL-1-deficient cells, exhibited reduced E-Selectin-binding activity compared with wild-type cells. Th1 cells with a PSGL-1 and CD43 double deficiency showed even less E-Selectin-binding activity. In migration assays in which adoptively transferred cells migrate to inflamed skin P- and E-Selectin dependently, CD43 contributed significantly to PSGL-1-independent Th1 cell migration. In addition, in vivo activated T cells from the draining lymph nodes of sensitized mice deficient in PSGL-1 and/or CD43 showed significantly decreased E-Selectin-binding activity and migration efficiency, with T cells from double-deficient mice showing the most profound decrease. Collectively, these results demonstrate that the CD43 expressed on activated T cells functions as an E-Selectin ligand and thereby mediates T cell migration to inflamed sites, in collaboration with PSGL-1.
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rolling of th1 cells via p Selectin Glycoprotein ligand 1 stimulates lfa 1 mediated cell binding to icam 1
Journal of Immunology, 2005Co-Authors: Kazuyuki Atarashi, Masanori Matsumoto, Takako Hirata, Naotoshi Kanemitsu, Masayuki MiyasakaAbstract:Activated T cells migrate from the blood into nonlymphoid tissues through a multistep process that involves cell rolling, arrest, and transmigration. P-Selectin Glycoprotein ligand-1 (PSGL-1) is a major ligand for P-Selectin expressed on subsets of activated T cells such as Th1 cells and mediates cell rolling on vascular endothelium. Rolling cells are arrested through a firm adhesion step mediated by integrins. Although chemokines presented on the endothelium trigger integrin activation, a second mechanism has been proposed where signaling via rolling receptors directly activates integrins. In this study, we show that Ab-mediated cross-linking of the PSGL-1 on Th1 cells enhances LFA-1-dependent cell binding to ICAM-1. PSGL-1 cross-linking did not enhance soluble ICAM-1 binding but induced clustering of LFA-1 on the cell surface, suggesting that an increase in LFA-1 avidity may account for the enhanced binding to ICAM-1. Combined stimulation by PSGL-1 cross-linking and the Th1-stimulating chemokine CXCL10 or CCL5 showed a more than additive effect on LFA-1-mediated Th1 cell adhesion as well as on LFA-1 redistribution on the cell surface. Moreover, PSGL-1-mediated rolling on P-Selectin enhanced the Th1 cell accumulation on ICAM-1 under flow conditions. PSGL-1 cross-linking induced activation of protein kinase C isoforms, and the increased Th1 cell adhesion observed under flow and also static conditions was strongly inhibited by calphostin C, implicating protein kinase C in the intracellular signaling in PSGL-1-mediated LFA-1 activation. These results support the idea that PSGL-1-mediated rolling interactions induce intracellular signals leading to integrin activation, facilitating Th1 cell arrest and subsequent migration into target tissues.
J B Hansen - One of the best experts on this subject based on the ideXlab platform.
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elevated plasma levels of p Selectin Glycoprotein ligand 1 positive microvesicles in patients with unprovoked venous thromboembolism
Journal of Thrombosis and Haemostasis, 2018Co-Authors: Simin Jamaly, Manjunath Goolyam Basavaraj, Irina Starikova, Randi Olsen, Sigrid Kufaas Braekkan, J B HansenAbstract:Essentials PSGL-1+ microvesicles (MVs) may be important in venous thromboembolism (VTE). We measured plasma levels and parental origin of PSGL-1+ MVs in patients with unprovoked VTE. VTE patients had higher plasma levels of PSGL-1+ MVs than healthy controls. The PSGL-1+ MVs originated mainly from monocytes and endothelial cells. SUMMARY Background Microvesicles (MVs) express antigens from their parental cells and have a highly procoagulant surface. Animal studies suggest that P-Selectin Glycoprotein ligand-1-positive (PSGL-1+ ) MVs play a role in the pathogenesis of venous thromboembolism (VTE). Objective The aim of this study was to determine plasma levels, the cellular origin and the morphological characteristics of PSGL-1+ MVs in patients with unprovoked VTE. Methods We conducted a population-based case-control study in 20 patients with a history of unprovoked VTE and 20 age- and sex-matched healthy controls recruited from the general population. Plasma levels, the cellular origin and the morphological characteristics of PSGL-1+ MVs were evaluated using flow cytometry, electron microscopy and confocal microscopy. Results Plasma levels of PSGL-1+ MVs were associated with increased risk of VTE. The odds ratio per one standard deviation increase in PSGL-1+ MVs was 3.11 (95% confidence interval [CI], 1.41-6.88) after adjustment for age and sex, and 2.88 (95% CI, 1.29-6.41) after further adjustment for body mass index. The PSGL-1+ MVs originated mainly from monocytes and endothelial cells determined by double staining with markers of parental cells using flow cytometry and transmission electron microscopy. Scanning electron microscopy of PSGL-1-labeled plasma-derived MVs displayed dominantly spherical vesicles that varied between 50 and 300 nm in diameter. Conclusions Increased plasma levels of PSGL-1+ MVs are associated with the risk of unprovoked VTE. Large population-based prospective studies are required to validate our findings.
Masayuki Miyasaka - One of the best experts on this subject based on the ideXlab platform.
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P-Selectin Glycoprotein Ligand-1 Negatively Regulates T-Cell Immune Responses
Journal of immunology (Baltimore Md. : 1950), 2009Co-Authors: Masanori Matsumoto, Masayuki Miyasaka, Takako HirataAbstract:Cell surface sialomucins often act as antiadhesive molecules by virtue of their extended structure and negative charge. CD43 is one such sialomucin, expressed on most leukocytes. P-Selectin Glycoprotein ligand-1 (PSGL-1) is another sialomucin expressed by leukocytes. It serves as a major Selectin ligand, but no antiadhesive role for it has been described. In this study, we showed that PSGL-1-deficient T cells, like CD43-deficient T cells, exhibited increased adhesion and proliferation compared with wild-type cells. The loss of both PSGL-1 and CD43 led to a further increase in T cell adhesion and proliferation. The reexpression of full-length PSGL-1 or CD43 in double-deficient CD4 + T cells reversed their increased adhesion and proliferation phenotype. Using chimeric constructs of human CD8 and either PSGL-1 or CD43, we demonstrated that the intracellular domain of PSGL-1 or CD43 is required for suppressing proliferation but not adhesion. Furthermore, in a mouse model of inflammatory bowel disease induced by the adoptive transfer of naive T cells into RAG-deficient hosts, a PSGL-1 deficiency exacerbated the development of inflammation. These results reveal a novel regulatory role for PSGL-1 in T cell adhesion and proliferation and suggest that PSGL-1 negatively regulates T cell immune responses in vivo.
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an l Selectin ligand distinct from p Selectin Glycoprotein ligand 1 is expressed on endothelial cells and promotes neutrophil rolling in inflammation
Blood, 2008Co-Authors: Akiko Shigeta, Masanori Matsumoto, Masayuki Miyasaka, Thomas F Tedder, John B Lowe, Takako HirataAbstract:Neutrophils recruited from the blood are key players in the innate immune response. Selectins play critical roles in neutrophil recruitment by mediating their tethering and rolling in inflamed venules. While the roles of P- and E-Selectin in this process are well established, the mechanisms of L-Selectin–mediated neutrophil recruitment remain elusive. One proposal is that tethering is mediated by L-Selectin on flowing neutrophils interacting with P-Selectin Glycoprotein ligand-1 (PSGL-1) on adherent neutrophils. To clarify whether L-Selectin–mediated neutrophil recruitment depends entirely on PSGL-1, we examined the impact of L-Selectin deficiency in mice with a PSGL-1–deficient background. L-Selectin and PSGL-1 double-knockout mice exhibited a higher increase in their peripheral blood neutrophil count and a worse defect in neutrophil recruitment into the inflamed peritoneum than PSGL-1–deficient mice. Intravital microscopy of inflamed cremaster muscle venules showed that L-Selectindeficiency or antibody blockade of L-Selectin reduced the residual leukocyte rolling in PSGL-1–deficient mice. Flow cytometric analyses showed that the endothelial cells from the cremaster muscle bound L-Selectin in a PSGL-1–independent manner. These results provide evidence for the existence of an L-Selectin ligand distinct from PSGL-1 in inflammation and indicate that such a ligand is expressed on endothelial cells, promoting neutrophil rolling in vivo.
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p Selectin Glycoprotein ligand 1 mediates l Selectin independent leukocyte rolling in high endothelial venules of peripheral lymph nodes
International Immunology, 2007Co-Authors: Nari Harakawa, Masayuki Miyasaka, Barbara C. Furie, Akiko Shigeta, Bruce Furie, Glenn Merrillskoloff, Masahiro Wato, Toshiro Okazaki, Naochika Domae, Takako HirataAbstract:Lymphocyte homing to peripheral lymph nodes (LNs) requires L-Selectin. Previous studies, however, suggest that there are L-Selectin-independent mechanisms of lymphocyte homing. P-Selectin Glycoprotein ligand-1 (PSGL-1) is a major ligand for P-Selectin expressed in a Selectin-binding form on myeloid cells and subsets of lymphoid cells. To discover whether PSGL-1 plays a role in lymphocyte homing, we examined leukocyte rolling and adhesion in the high endothelial venules (HEVs) of the subiliac LNs of wild-type and PSGL-1-deficient mice by intravital microscopy. There were no significant differences in blood velocity or wall shear stress between wild-type and PSGL-1-deficient mice. Although the leukocyte rolling fraction was not altered in PSGL-1-deficient mice, infusion of an anti-L-Selectin mAb into these mice completely abolished leukocyte rolling, while the same treatment in wild-type mice inhibited 90% of the leukocyte rolling. This residual rolling in wild-type mice appears to depend on the PSGL-1-P-Selectin interaction, since infusion of an anti-L-Selectin mAb together with an anti-PSGL-1 mAb or anti-P-Selectin mAb almost completely abolished the rolling. PSGL-1 deficiency also led to a higher rolling velocity, suggesting that PSGL-1 mediates leukocyte rolling at low velocities. P-Selectin was found to be expressed on the HEVs of subiliac LNs under the conditions of intravital microscopy. Taken together, these results indicate that the interaction of PSGL-1 with P-Selectin constitutes a second mechanism of leukocyte rolling in the HEVs of peripheral LNs.
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cd43 collaborates with p Selectin Glycoprotein ligand 1 to mediate e Selectin dependent t cell migration into inflamed skin
Journal of Immunology, 2007Co-Authors: Masanori Matsumoto, Masayuki Miyasaka, Akiko Shigeta, Yuko Furukawa, Toshiyuki Tanaka, Takako HirataAbstract:Activated T cell migration into nonlymphoid tissues is initiated by the interactions of P- and E-Selectin expressed on endothelial cells and their ligands on T cells. P-Selectin Glycoprotein ligand-1 (PSGL-1) has been the only E-Selectin ligand demonstrated to function during the in vivo migration of activated T cells. We show in this study that CD43-deficient Th1 cells, like PSGL-1-deficient cells, exhibited reduced E-Selectin-binding activity compared with wild-type cells. Th1 cells with a PSGL-1 and CD43 double deficiency showed even less E-Selectin-binding activity. In migration assays in which adoptively transferred cells migrate to inflamed skin P- and E-Selectin dependently, CD43 contributed significantly to PSGL-1-independent Th1 cell migration. In addition, in vivo activated T cells from the draining lymph nodes of sensitized mice deficient in PSGL-1 and/or CD43 showed significantly decreased E-Selectin-binding activity and migration efficiency, with T cells from double-deficient mice showing the most profound decrease. Collectively, these results demonstrate that the CD43 expressed on activated T cells functions as an E-Selectin ligand and thereby mediates T cell migration to inflamed sites, in collaboration with PSGL-1.
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rolling of th1 cells via p Selectin Glycoprotein ligand 1 stimulates lfa 1 mediated cell binding to icam 1
Journal of Immunology, 2005Co-Authors: Kazuyuki Atarashi, Masanori Matsumoto, Takako Hirata, Naotoshi Kanemitsu, Masayuki MiyasakaAbstract:Activated T cells migrate from the blood into nonlymphoid tissues through a multistep process that involves cell rolling, arrest, and transmigration. P-Selectin Glycoprotein ligand-1 (PSGL-1) is a major ligand for P-Selectin expressed on subsets of activated T cells such as Th1 cells and mediates cell rolling on vascular endothelium. Rolling cells are arrested through a firm adhesion step mediated by integrins. Although chemokines presented on the endothelium trigger integrin activation, a second mechanism has been proposed where signaling via rolling receptors directly activates integrins. In this study, we show that Ab-mediated cross-linking of the PSGL-1 on Th1 cells enhances LFA-1-dependent cell binding to ICAM-1. PSGL-1 cross-linking did not enhance soluble ICAM-1 binding but induced clustering of LFA-1 on the cell surface, suggesting that an increase in LFA-1 avidity may account for the enhanced binding to ICAM-1. Combined stimulation by PSGL-1 cross-linking and the Th1-stimulating chemokine CXCL10 or CCL5 showed a more than additive effect on LFA-1-mediated Th1 cell adhesion as well as on LFA-1 redistribution on the cell surface. Moreover, PSGL-1-mediated rolling on P-Selectin enhanced the Th1 cell accumulation on ICAM-1 under flow conditions. PSGL-1 cross-linking induced activation of protein kinase C isoforms, and the increased Th1 cell adhesion observed under flow and also static conditions was strongly inhibited by calphostin C, implicating protein kinase C in the intracellular signaling in PSGL-1-mediated LFA-1 activation. These results support the idea that PSGL-1-mediated rolling interactions induce intracellular signals leading to integrin activation, facilitating Th1 cell arrest and subsequent migration into target tissues.