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1
Lenter, M., A. Levinovitz, S. Isenmann, and D. Vestweber. "Monospecific and common glycoprotein ligands for E- and P-selectin on myeloid cells." Journal of Cell Biology 125, no. 2 (1994): 471–81. http://dx.doi.org/10.1083/jcb.125.2.471.
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E- and P-selectin are inducible cell adhesion molecules on endothelial cells, which function as Ca(2+)-dependent lectins and mediate the binding of neutrophils and monocytes. We have recently identified a 150-kD glycoprotein ligand for E-selectin on mouse myeloid cells, using a recombinant antibody-like form of mouse E-selectin. Here, we report that this ligand does not bind to an analogous P-selectin fusion protein. Instead, the chimeric P-selectin-IgG protein recognizes a 160-kD glycoprotein on the mouse neutrophil progenitor 32D cl 3, on mature mouse neutrophils and on human HL60 cells. The binding is Ca(2+)-dependent and requires the presence of sialic acid on the ligand. This P-selectin-ligand is not recognized by E-selectin. Removal of N-linked carbohydrate side chains from the 150-kD and the 160-kD monospecific selectin ligands abolishes the binding of both ligands to the respective selectin. Treatment of HL60 cells with Peptide: N-glycosidase F inhibited cell binding to P- and E-selectin. In addition, glycoproteins of 230 and 130 kD were found on mature mouse neutrophils, which bound both to E- and P-selectin in a Ca(2+)-dependent fashion. The signals detected for these ligands were 15-20-fold weaker than those for the monospecific ligands. Both proteins were heavily sialylated and selectin-binding was blocked by removal of sialic acid, but not by removal of N-linked carbohydrates. Our data reveal that E- and P-selectin recognize two categories of glycoprotein ligands: one type requires N-linked carbohydrates for binding and is monospecific for each of the two selectins and the other type binds independent of N-linked carbohydrates and is common for both endothelial selectins.
2
Frenette, Paul S., Cécile V. Denis, Linnea Weiss, et al. "P-Selectin Glycoprotein Ligand 1 (Psgl-1) Is Expressed on Platelets and Can Mediate Platelet–Endothelial Interactions in Vivo." Journal of Experimental Medicine 191, no. 8 (2000): 1413–22. http://dx.doi.org/10.1084/jem.191.8.1413.
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The platelet plays a pivotal role in maintaining vascular integrity. In a manner similar to leukocytes, platelets interact with selectins expressed on activated endothelium. P-selectin glycoprotein ligand 1 (PSGL-1) is the main P-selectin ligand expressed on leukocytes. Searching for platelet ligand(s), we used a P-selectin–immunoglobulin G (IgG) chimera to affinity purify surface-biotinylated proteins from platelet lysates. P-selectin–bound ligands were eluted with ethylenediaminetetraacetic acid. An ∼210-kD biotinylated protein was isolated from both human neutrophil and platelet preparations. A band of the same size was also immunopurified from human platelets using a monoclonal anti–human PSGL-1 antibody and could be blotted with P-selectin–IgG. Under reducing conditions, both the predicted PSGL-1 ∼210-kD dimer and the ∼120-kD monomer were isolated from platelets. Comparative immunoelectron microscopy and Western blotting experiments suggested that platelet PSGL-1 expression is 25–100-fold lower than that of leukocytes. However, patients with chronic idiopathic thrombocytopenic purpura who harbor predominantly young platelets displayed greater expression, indicating that PSGL-1 expression may be decreased during platelet aging. By flow cytometry, thrombin-activated platelets from normal individuals exhibited greater expression than those unstimulated. An inhibitory anti–PSGL-1 antibody significantly reduced platelet rolling in mesenteric venules, as observed by intravital microscopy. Our results indicate that functional PSGL-1 is expressed on platelets, and suggest an additional mechanism by which selectins and their ligands participate in inflammatory and/or hemostatic responses.
3
HARMS, Gesche, Regine KRAFT, Gerlinde GRELLE, Bärbel VOLZ, Jens DERNEDDE, and Rudolf TAUBER. "Identification of nucleolin as a new L-selectin ligand." Biochemical Journal 360, no. 3 (2001): 531–38. http://dx.doi.org/10.1042/bj3600531.
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Apart from leucocyte–endothelial interactions, the adhesion molecule L-selectin mediates the homotypic adhesion of leucocytes during recruitment at sites of acute inflammation, as well as intercellular adhesion of haematopoietic progenitor cells during haematopoiesis. There is evidence that, in addition to P-selectin glycoprotein ligand-1, other as-yet-unidentified proteins function as L-selectin ligands on human leucocytes and haematopoietic progenitor cells. In the present study, we show: (i) by affinity chromatography on L-selectin–agarose; (ii) by protein identification using MS; and (iii) by covalent cell-surface labelling with sulphosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate that the multifunctional nuclear protein nucleolin is partly exposed on the cell surface, and is a ligand of L-selectin in human leucocytes and haematopoietic progenitor cells.
4
Thomas, Susan N., Ronald L. Schnaar, and Konstantinos Konstantopoulos. "Podocalyxin-like protein is an E-/L-selectin ligand on colon carcinoma cells: comparative biochemical properties of selectin ligands in host and tumor cells." American Journal of Physiology-Cell Physiology 296, no. 3 (2009): C505—C513. http://dx.doi.org/10.1152/ajpcell.00472.2008.
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Selectins facilitate metastasis and tumor cell arrest in the microvasculature by mediating binding of selectin-expressing host cells to ligands on tumor cells. We recently identified CD44 variant isoforms as functional P-, but not E-/L-, selectin ligands on colon carcinoma cells. Furthermore, a ∼180-kDa sialofucosylated glycoprotein(s) mediated selectin binding in CD44-knockdown cells. Using immunoaffinity chromatography and tandem mass spectrometry, we identify podocalyxin-like protein (PCLP) as an alternative selectin ligand. Blot rolling and cell-free flow-based adhesion assays disclose that PCLP on LS174T colon carcinoma cells possesses E-/L-, but not P-, selectin binding activity. The selectin-binding determinants on LS174T PCLP are non-MECA-79-reactive sialofucosylated structures displayed on O-linked glycans, distinct from the MECA-79-reactive O-glycans on PCLP expressed by high endothelial venules, which is an L-selectin ligand. PCLP on CD44-knockdown LS174T cells exhibits higher HECA-452 immunoreactivity than PCLP on wild-type cells, suggesting that PCLP functions as an alternative acceptor for selectin-binding glycans. The enhanced expression of HECA-452 reactivity on PCLP from CD44-knockdown cells correlates with the increased avidity of PCLP for E- but not L-selectin. The novel finding that PCLP is an E-/L-selectin ligand on carcinoma cells offers a unifying perspective on the apparent enhanced metastatic potential associated with tumor cell PCLP overexpression and the role of selectins in metastasis.
5
Guyer, DA, KL Moore, EB Lynam, et al. "P-selectin glycoprotein ligand-1 (PSGL-1) is a ligand for L-selectin in neutrophil aggregation." Blood 88, no. 7 (1996): 2415–21. http://dx.doi.org/10.1182/blood.v88.7.2415.bloodjournal8872415.
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In inflammation, activated neutrophils adhere to endothelial cells and aggregate with one another. While beta 2-integrin and L-selectin are essential for aggregation, their ligands remain to be identified. We have previously shown that L-selectin mediates a carbohydrate-dependent interaction in aggregation (Simon et al: J Immunol 149:2765, 1992; Rochon et al: J Immunol 152:1385, 1994). We have suggested that the L-selectin counter-structure is a mucinlike protein and proposed that aggregation occurs through a two-step process involving L-selectin, beta 2-integrin, and their distinct counter-structures (Bennett et al: J Leuk Biol 58:510, 1995). A candidate ligand for L-selectin is P-selectin glycoprotein ligand-1 (PSGL-1), a mucinlike protein on neutrophils that binds P-and E-selectin. Using flow cytometry we show that the number and size of neutrophil aggregates is reduced with Fab fragments of PL1, an anti-PSGL-1 monoclonal antibody that blocks the interaction between P-selectin and PSGL-1 (Moore et al: J Cell Biol 128:661, 1995). In addition, monoclonal antibodies to L-selectin and PSGL-1 were used simultaneously to modulate the availability of these adhesion molecules on individual cell populations. The inhibition of aggregation by these antibodies is consistent with L-selectin and PSGL-1 being counter-structures. We suggest that L-selectin and PSGL-1 support a collisional cell-cell interaction that represents the first step in neutrophil aggregation.
6
Moore, K. L., N. L. Stults, S. Diaz, et al. "Identification of a specific glycoprotein ligand for P-selectin (CD62) on myeloid cells." Journal of Cell Biology 118, no. 2 (1992): 445–56. http://dx.doi.org/10.1083/jcb.118.2.445.
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P-selectin (CD62, GMP-140, PADGEM), a Ca(2+)-dependent lectin on activated platelets and endothelium, functions as a receptor for myeloid cells by interacting with sialylated, fucosylated lactosaminoglycans. P-selectin binds to a limited number of protease-sensitive sites on myeloid cells, but the protein(s) that carry the glycans recognized by P-selectin are unknown. Blotting of neutrophil or HL-60 cell membrane extracts with [125I]P-selectin and affinity chromatography of [3H]glucosamine-labeled HL-60 cell extracts were used to identify P-selectin ligands. A major ligand was identified with an approximately 250,000 M(r) under nonreducing conditions and approximately 120,000 under reducing conditions. Binding of P-selectin to the ligand was Ca2+ dependent and was blocked by mAbs to P-selectin. Brief sialidase digestion of the ligand increased its apparent molecular weight; however, prolonged digestion abolished binding of P-selectin. Peptide:N-glycosidase F treatment reduced the apparent molecular weight of the ligand by approximately 3,000 but did not affect P-selectin binding. Western blot and immunodepletion experiments indicated that the ligand was not lamp-1, lamp-2, or L-selectin, which carry sialyl Le(x), nor was it leukosialin, a heavily sialylated glycoprotein of similar molecular weight. The preferential interaction of the ligand with P-selectin suggests that it may play a role in adhesion of myeloid cells to activated platelets and endothelial cells.
7
Zöllner, Olaf, Martin C. Lenter, James E. Blanks, et al. "L-Selectin from Human, but Not from Mouse Neutrophils Binds Directly to E-Selectin." Journal of Cell Biology 136, no. 3 (1997): 707–16. http://dx.doi.org/10.1083/jcb.136.3.707.
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L-Selectin on neutrophils as well as inducible E- and P-selectin on endothelium are involved in the recruitment of neutrophils into inflamed tissue. Based on cell attachment assays, L-selectin was suggested to function as a carbohydrate presenting ligand for E- and P-selectin. However, previous affinity isolation experiments with an E-selectin–Ig fusion protein had failed to detect L-selectin among the isolated E-selectin ligands from mouse neutrophils. We show here that L-selectin from human neutrophils, in contrast to mouse neutrophils, can be affinity-isolated as a major ligand from total cell extracts using E-selectin–Ig as affinity probe. Binding of human L-selectin to E-selectin was direct, since purified L-selectin could be reprecipitated with E-selectin–Ig. Recognition of L-selectin was abolished by sialidase-treatment, required Ca2+, and was resistant to treatment with endoglycosidase F. Binding of L-selectin to a P-selectin–Ig fusion protein was not observed. In agreement with the biochemical data, the anti–Lselectin mAb DREG56 inhibited rolling of human neutrophils on immobilized E-selectin–Ig but not on P-selectin–Ig. No such inhibitory effect was seen with the anti–mouse L-selectin mAb MEL14 on mouse neutrophils. Rolling of E-selectin transfectants on purified and immobilized human L-selectin was inhibited by mAb DREG56. We conclude that L-selectin on human neutrophils is a major glycoprotein ligand among very few glycoproteins that can be isolated by an E-selectin affinity matrix. The clear difference between human and mouse L-selectin suggests that E-selectin–binding carbohydrate moieties are attached to different protein scaffolds in different species.
8
Moore, K. L., K. D. Patel, R. E. Bruehl, et al. "P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin." Journal of Cell Biology 128, no. 4 (1995): 661–71. http://dx.doi.org/10.1083/jcb.128.4.661.
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Neutrophils roll on P-selectin expressed by activated platelets or endothelial cells under the shear stresses in the microcirculation. P-selectin glycoprotein ligand-1 (PSGL-1) is a high affinity ligand for P-selectin on myeloid cells. However, it has not been demonstrated that PSGL-1 contributes to the rolling of neutrophils on P-selectin. We developed two IgG mAbs, PL1 and PL2, that appear to recognize protein-dependent epitopes on human PSGL-1. The mAbs bound to PSGL-1 on all leukocytes as well as on heterologous cells transfected with PSGL-1 cDNA. PL1, but not PL2, blocked binding of 125-I-PSGL-1 to immobilized P-selectin, binding of fluid-phase P-selectin to myeloid and lymphoid leukocytes, adhesion of neutrophils to immobilized P-selectin under static conditions, and rolling of neutrophils on P-selectin-expressing CHO cells under a range of shear stresses. PSGL-1 was localized to microvilli on neutrophils, a topography that may facilitate its adhesive function. These data indicate that (a) PSGL-1 accounts for the high affinity binding sites for P-selectin on leukocytes, and (b) PSGL-1 must interact with P-selectin in order for neutrophils to roll on P-selectin at physiological shear stresses.
9
Spertini, O., A. S. Cordey, N. Monai, L. Giuffrè, and M. Schapira. "P-selectin glycoprotein ligand 1 is a ligand for L-selectin on neutrophils, monocytes, and CD34+ hematopoietic progenitor cells." Journal of Cell Biology 135, no. 2 (1996): 523–31. http://dx.doi.org/10.1083/jcb.135.2.523.
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Selectins play a critical role in initiating leukocyte binding to vascular endothelium. In addition, in vitro experiments have shown that neutrophils use L-selectin to roll on adherent neutrophils, suggesting that they express a nonvascular L-selectin ligand. Using a L-selectin/IgM heavy chain (mu) chimeric protein as an immunocytological probe, we show here that L-selectin can bind to neutrophils, monocytes, CD34+ hematopoietic progenitors, and HL-60 and KG-1 myeloid cells. The interaction between L-selectin and leukocytes was protease sensitive and calcium dependent, and abolished by cell treatment with neuraminidase, chlorate, or O-sialoglycoprotein endopeptidase. These results revealed common features between leukocyte L-selectin ligand and the mucin-like P-selectin glycoprotein ligand 1 (PSGL-1), which mediates neutrophil rolling on P- and E-selectin. The possibility that PSGL-1 could be a ligand for L-selectin was further supported by the ability of P-selectin/mu chimera to inhibit L-selectin/mu binding to leukocytes and by the complete inhibition of both selectin interactions with myeloid cells treated with mocarhagin, a cobra venom metalloproteinase that cleaves the amino terminus of PSGL-1 at Tyr-51. Finally, the abrogation of L- and P-selectin binding to myeloid cells treated with a polyclonal antibody, raised against a peptide corresponding to the amino acid residues 42-56 of PSGL-1, indicated that L- and P-selectin interact with a domain located at the amino-terminal end of PSGL-1. The ability of the anti-PSGL-1 mAb PL-1 to inhibit L- and P-selectin binding to KG-1 cells further supported that possibility. Thus, apart from being involved in neutrophil rolling on P- and E-selectin, PSGL-1 also plays a critical role in mediating neutrophil attachment to adherent neutrophils. Interaction between L-selectin and PSGL-1 may be of major importance for increasing leukocyte recruitment at inflammatory sites.
10
Evangelista, Virgilio, Stefano Manarini, Rita Sideri, et al. "Platelet/Polymorphonuclear Leukocyte Interaction: P-Selectin Triggers Protein-Tyrosine Phosphorylation–Dependent CD11b/CD18 Adhesion: Role of PSGL-1 as a Signaling Molecule." Blood 93, no. 3 (1999): 876–85. http://dx.doi.org/10.1182/blood.v93.3.876.
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Abstract Polymorphonuclear leukocyte (PMN) adhesion to activated platelets is important for the recruitment of PMN at sites of vascular damage and thrombus formation. We have recently shown that binding of activated platelets to PMN in mixed cell suspensions under shear involves P-selectin and the activated β2-integrin CD11b/CD18. Integrin activation required signaling mechanisms that were sensitive to tyrosine kinase inhibitors.1 Here we show that mixing activated, paraformaldehyde (PFA)-fixed platelets with PMNs under shear conditions leads to rapid and fully reversible tyrosine phosphorylation of a prominent protein of 110 kD (P∼110). Phosphorylation was both Ca2+ and Mg2+ dependent and was blocked by antibodies against P-selectin or CD11b/CD18, suggesting that both adhesion molecules need to engage with their respective ligands to trigger phosphorylation of P∼110. The inhibition of P∼110 phosphorylation by tyrosine kinase inhibitors correlates with the inhibition of platelet/PMN aggregation. Similar effects were observed when platelets were substituted by P-selectin–transfected Chinese hamster ovary (CHO-P) cells or when PMN were stimulated with P-selectin–IgG fusion protein. CHO-P/PMN mixed-cell aggregation and P-selectin–IgG–triggered PMN/PMN aggregation as well as P∼110 phosphorylation were all blocked by antibodies against P-selectin or CD18. In each case PMN adhesion was sensitive to the tyrosine kinase inhibitor genistein. The antibody PL-1 against P-selectin glycoprotein ligand-1 (PSGL-1) blocked platelet/PMN aggregation, indicating that PSGL-1 was the major tethering ligand for P-selectin in this experimental system. Moreover, engagement of PSGL-1 with a nonadhesion blocking antibody triggered β2-integrin–dependent genistein-sensitive aggregation as well as tyrosine phosphorylation in PMN. This study shows that binding of P-selectin to PSGL-1 triggers tyrosine kinase–dependent mechanisms that lead to CD11b/CD18 activation in PMN. The availability of the β2-integrin to engage with its ligands on the neighboring cells is necessary for the tyrosine phosphorylation of P∼110.
11
Evangelista, Virgilio, Stefano Manarini, Rita Sideri, et al. "Platelet/Polymorphonuclear Leukocyte Interaction: P-Selectin Triggers Protein-Tyrosine Phosphorylation–Dependent CD11b/CD18 Adhesion: Role of PSGL-1 as a Signaling Molecule." Blood 93, no. 3 (1999): 876–85. http://dx.doi.org/10.1182/blood.v93.3.876.403k25_876_885.
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Polymorphonuclear leukocyte (PMN) adhesion to activated platelets is important for the recruitment of PMN at sites of vascular damage and thrombus formation. We have recently shown that binding of activated platelets to PMN in mixed cell suspensions under shear involves P-selectin and the activated β2-integrin CD11b/CD18. Integrin activation required signaling mechanisms that were sensitive to tyrosine kinase inhibitors.1 Here we show that mixing activated, paraformaldehyde (PFA)-fixed platelets with PMNs under shear conditions leads to rapid and fully reversible tyrosine phosphorylation of a prominent protein of 110 kD (P∼110). Phosphorylation was both Ca2+ and Mg2+ dependent and was blocked by antibodies against P-selectin or CD11b/CD18, suggesting that both adhesion molecules need to engage with their respective ligands to trigger phosphorylation of P∼110. The inhibition of P∼110 phosphorylation by tyrosine kinase inhibitors correlates with the inhibition of platelet/PMN aggregation. Similar effects were observed when platelets were substituted by P-selectin–transfected Chinese hamster ovary (CHO-P) cells or when PMN were stimulated with P-selectin–IgG fusion protein. CHO-P/PMN mixed-cell aggregation and P-selectin–IgG–triggered PMN/PMN aggregation as well as P∼110 phosphorylation were all blocked by antibodies against P-selectin or CD18. In each case PMN adhesion was sensitive to the tyrosine kinase inhibitor genistein. The antibody PL-1 against P-selectin glycoprotein ligand-1 (PSGL-1) blocked platelet/PMN aggregation, indicating that PSGL-1 was the major tethering ligand for P-selectin in this experimental system. Moreover, engagement of PSGL-1 with a nonadhesion blocking antibody triggered β2-integrin–dependent genistein-sensitive aggregation as well as tyrosine phosphorylation in PMN. This study shows that binding of P-selectin to PSGL-1 triggers tyrosine kinase–dependent mechanisms that lead to CD11b/CD18 activation in PMN. The availability of the β2-integrin to engage with its ligands on the neighboring cells is necessary for the tyrosine phosphorylation of P∼110.
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Snapp, Karen R., Amy J. Wagers, Ron Craig, Lloyd M. Stoolman, and Geoffrey S. Kansas. "P-Selectin Glycoprotein Ligand-1 Is Essential for Adhesion to P-Selectin But Not E-Selectin in Stably Transfected Hematopoietic Cell Lines." Blood 89, no. 3 (1997): 896–901. http://dx.doi.org/10.1182/blood.v89.3.896.
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Abstract P-selectin (CD62P) is a member of the selectin family of adhesion molecules involved in the regulation of leukocyte traffic. P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like molecule that is thought to be a primary ligand for P-selectin. The interaction of P-selectin with PSGL-1 results in leukocyte rolling and recruitment of leukocytes to sites of inflammation and tissue injury. However, expression of PSGL-1 protein alone is insufficient for binding to P-selectin. Several posttranslational modifications of PSGL-1, including sialylation, sulfation, and fucosylation by α1,3-fucosyltransferase(s) (FucT), are required for functional interaction with P-selectin. Recently, several groups have reported that PSGL-1 might also serve as a ligand for E-selectin. Differential posttranslational modifications of PSGL-1 may determine whether it can interact with either P- or E-selectin or both. To determine whether PSGL-1 is essential for adhesion to P- or E-selectin, we have constructed and analyzed a panel of stably transfected K562 cells. K562 cells express FucT-IV but not FucT-VII or PSGL-1, and do not bind to either E- or P-selectin. K562 cells transfected with PSGL-1 cDNA also did not bind to either P- or E-selectin. Binding to P-selectin occurred only when K562 cells were cotransfected with both FucT-VII and PSGL-1. In contrast, expression of FucT-VII alone was sufficient for E-selectin binding. These data demonstrate that expression of PSGL-1 is not required for adhesion of a stably transfected hematopoietic cell line to E-selectin, and suggest that FucT-IV alone cannot properly modify PSGL-1, expressed in transfected K562 cells, to bind P-selectin.
13
Kappelmayer, János, and Béla Nagy. "The Interaction of Selectins and PSGL-1 as a Key Component in Thrombus Formation and Cancer Progression." BioMed Research International 2017 (2017): 1–18. http://dx.doi.org/10.1155/2017/6138145.
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Cellular interaction is inevitable in the pathomechanism of human disease. Formation of heterotypic cellular aggregates, between distinct cells of hematopoietic and nonhematopoietic origin, may be involved in events leading to inflammation and the complex process of cancer progression. Among adhesion receptors, the family of selectins with their ligands have been considered as one of the major contributors to cell-cell interactions. Consequently, the inhibition of the interplay between selectins and their ligands may have potential therapeutic benefits. In this review, we focus on the current evidence on the selectins as crucial modulators of inflammatory, thrombotic, and malignant disorders. Knowing that there is promiscuity in selectin binding, we outline the importance of a key protein that serves as a ligand for all selectins. This dimeric mucin, the P-selectin glycoprotein ligand 1 (PSGL-1), has emerged as a major player in inflammation, thrombus, and cancer development. We discuss the interaction of PSGL-1 with various selectins in physiological and pathological processes with particular emphasis on mechanisms that lead to severe disease.
14
MALHOTRA, Rajneesh, Malcolm WARD, Robert B. SIM, and Michael I. BIRD. "Identification of human complement Factor H as a ligand for L-selectin." Biochemical Journal 341, no. 1 (1999): 61–69. http://dx.doi.org/10.1042/bj3410061.
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The selectin family of adhesion molecules (E-, P- and L-selectins) is involved in leukocyte recruitment to sites of inflammation and tissue damage. Recently it has been shown that L-selectin is involved not only in leukocyte tethering and rolling, but also plays an important role in leukocyte activation. For example, glycosylation-dependent cell-adhesion molecule 1 (GlyCAM-1), a known ligand for L-selectin, has been shown to enhance β2-integrin function. GlyCAM-1 is a secreted protein and is present in mouse serum at a concentration of approx. 1.5 μg/ml. There is no obvious GlyCAM-1 homologue in man and, to date, L-selectin ligand(s) from human serum have not been characterized. Therefore we have used L-selectin affinity chromatography, followed by ion-exchange chromatography, to isolate specific ligand(s) for L-selectin. Using this procedure, we have isolated three major glycoproteins of apparent molecular masses 170 kDa, 70 kDa and 50 kDa. The 170 kDa protein band was digested with trypsin and peptides were analysed by delayed extraction matrix-assisted laser desorption ionization MS and protein database searching. The 170 kDa protein was identified as the human complement protein Factor H. Human Factor H, isolated by a different method, was shown to bind specifically to L-selectin in the presence of CaCl2, and binding was inhibited by anti-L-selectin antibodies, fucoidan and lipopolysaccharide. Only a part of the purified Factor H preparation bound to immobilized L-selectin. The interaction of Factor H with leukocyte L-selectin was shown to induce the secretion of tumour necrosis factor-α (TNF-α). Pretreatment of Factor H with sialidase reduced both the binding of L-selectin to Factor H and the Factor H-induced L-selectin-mediated TNF-α secretion by leukocytes. Taken together, these results demonstrate that a post-translationally modified form of human plasma Factor H is a potential physiological ligand for L-selectin.
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Geng, Jian-Guo, Hai-Bo Wang, Ming Chen, et al. "P-Selectin Activates Leukocyte Integrin alphaMbeta2 by Recruiting Phosphatidylinositol-3 Kinase to P-Selectin Glycoprotein Ligand-1 Cytoplasmic Domain through Adaptor Protein Naf1." Blood 104, no. 11 (2004): 652. http://dx.doi.org/10.1182/blood.v104.11.652.652.
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Abstract The interaction of endothelial P-selectin (CD62P) with neutrophil PSGL-1 (P-selectin glycoprotein ligand-1, CD162) mediates neutrophil rolling, which acts in concert with cytokines and chemoattractants for integrin-mediated firm adhesion of neutrophils to vascular endothelial cells. Numerous studies have established that this sequence of events is critical to the extravasation of neutrophils during an inflammatory response. We have recently shown that cross-linking of PSGL-1 with P-selectin acts cooperatively with extracellular stimuli, such as cytokines and chemoattractants released from and displayed on the activated endothelial lining of the vessel walls, for optimal activation of beta2-integrins, an increase in their apparent affinity/avidity for cognate ligands, which in turn supports firm adhesion and transendothelial migration. To identify cellular proteins which interact with human PSGL-1 and trigger this cascade of events, we used the cytoplasmic domain of human PSGL-1 as the bait for screening of human leukocyte yeast two-hybrid library. During this genetic screening, we identified Naf-1 (Nef-associated factor 1; also called ABIN, A20 binding inhibitor of NF-kB, or VAN, virion-associated nuclear shuttle protein) as a binding partner to the cytoplasmic domain of PSGL-1. The specific interaction of Naf1 with PSGL-1 was confirmed by GST-fusion protein pull-down and co-immunoprecipitation experiments. As YPPM at 552–555 of Naf1a amino acid sequence is a known binding motif for p85 subunit of phosphatidylinositol-3 kinase (PI3K; YXXM in which phosphorylated Y is required for p85 binding), we performed co-immunoprecipitation experiments to show that Naf1a actually acted as an adaptor protein for PSGL-1 and p85 subunit of PI3K. In addition, we found that P-selectin activated the enzyme activity of PI3K in human neutrophils and PI3K specific inhibitors, wortmannin and LY294002, inhibited alphaMbeta2 activation and clustering induced by P-selectin. Our data thus delineate a novel PSGL-1 signal transduction pathway essential for transactivation of beta2-integrins.
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Gardiner, Elizabeth E., Mariagrazia De Luca, Tracy McNally, Alan D. Michelson, Robert K. Andrews, and Michael C. Berndt. "Regulation of P-selectin binding to the neutrophil P-selectin counter-receptor P-selectin glycoprotein ligand-1 by neutrophil elastase and cathepsin G." Blood 98, no. 5 (2001): 1440–47. http://dx.doi.org/10.1182/blood.v98.5.1440.
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In the inflammatory response, leukocyte rolling before adhesion and transmigration through the blood vessel wall is mediated by specific cell surface adhesion receptors. Neutrophil rolling involves the interaction of P-selectin expressed on activated endothelium and its counter-receptor on neutrophils, P-selectin glycoprotein ligand-1 (PSGL-1). Here, it is reported that P-selectin binding to neutrophils is lost under conditions that cause the release of proteinases from neutrophil primary granules. Treatment of neutrophils with the purified neutrophil granule proteinases, cathepsin G and elastase, rapidly abolished their capacity to bind P-selectin. This inactivation corresponded to loss of the N-terminal domain of PSGL-1, as assessed by Western blot analysis. A loss of intact PSGL-1 protein from the surfaces of neutrophils after the induction of degranulation was also detected by Western blot analysis. Cathepsin G initially cleaved near the PSGL-1 N-terminus, whereas neutrophil elastase predominantly cleaved at a more C-terminal site within the protein mucin core. Consistent with this, cathepsin G cleaved a synthetic peptide based on the PSGL-1 N-terminus between Tyr-7/Leu-8. Under conditions producing neutrophil degranulation in incubations containing mixtures of platelets and neutrophils, the loss of PSGL-1, but not P-selectin, from platelet-neutrophil lysates was detected. Cathepsin G- or neutrophil elastase-mediated PSGL-1 proteolysis may constitute a potential autocrine mechanism for down-regulation of neutrophil adhesion to P-selectin.
17
Lo, Chi Y., Aristotelis Antonopoulos, Rohitesh Gupta, et al. "Competition between Core-2 GlcNAc-transferase and ST6GalNAc-transferase Regulates the Synthesis of the Leukocyte Selectin Ligand on Human P-selectin Glycoprotein Ligand-1." Journal of Biological Chemistry 288, no. 20 (2013): 13974–87. http://dx.doi.org/10.1074/jbc.m113.463653.
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The binding of selectins to carbohydrate ligands expressed on leukocytes regulates immunity and inflammation. Among the human selectin ligands, the O-linked glycans at the N-terminus of the leukocyte cell-surface molecule P-selectin glycoprotein ligand-1 (PSGL-1, CD162) are important because they bind all selectins (L-, E-, and P-selectin) with high affinity under hydrodynamic shear conditions. Analysis of glycan microheterogeneity at this site is complicated by the presence of 72 additional potential O-linked glycosylation sites on this mucinous protein. To overcome this limitation, truncated forms of PSGL-1, called “PSGL-1 peptide probes,” were developed. Ultra-high sensitivity mass spectrometry analysis of glycans released from such probes along with glycoproteomic analysis demonstrate the presence of both the sialyl Lewis-X (sLeX) and the di-sialylated T-antigen (NeuAcα2,3Galβ1,3(NeuAcα2,6)GalNAc) at the PSGL-1 N-terminus. Overexpression of glycoprotein-specific ST6GalNAc-transferases (ST6GalNAc1, -2, or -4) in human promyelocytic HL-60 cells altered glycan structures and cell adhesion properties. In particular, ST6GalNAc2 overexpression abrogated cell surface HECA-452/CLA expression, reduced the number of rolling leukocytes on P- and L-selectin-bearing substrates by ∼85%, and increased median rolling velocity of remaining cells by 80–150%. Cell rolling on E-selectin was unaltered although the number of adherent cells was reduced by 60%. ST6GalNAc2 partially co-localizes in the Golgi with the core-2 β(1,6)GlcNAc-transferase C2GnT-1. Overall, the data describe the glycan microheterogeneity at the PSGL-1 N-terminus. They suggest that a competition between ST6GalNAc2 and C2GnT-1 for the core-1/Galβ1,3GalNAc glycan may regulate leukocyte adhesion under fluid shear.
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Thomas, Wendy. "For catch bonds, it all hinges on the interdomain region." Journal of Cell Biology 174, no. 7 (2006): 911–13. http://dx.doi.org/10.1083/jcb.200609029.
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Tensile mechanical force was long assumed to increase the detachment rates of biological adhesive bonds (Bell, 1978). However, in the last few years, several receptor–ligand pairs were shown to form “catch bonds,” whose lifetimes are enhanced by moderate amounts of force. These include the bacterial adhesive protein FimH binding to its ligand mannose (Thomas et al., 2002; Thomas et al., 2006), blood cell adhesion proteins P- and L-selectin binding to sialyl Lewis X (sLeX)–containing ligands (Marshall et al., 2003; Evans et al., 2004; Sarangapani et al., 2004), and the myosin–actin motor protein interaction (Guo and Guilford, 2006). The structural mechanism behind this counterintuitive force–enhanced catch bond behavior is of great interest.
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Bestebroer, Jovanka, Miriam J. J. G. Poppelier, Laurien H. Ulfman, et al. "Staphylococcal superantigen-like 5 binds PSGL-1 and inhibits P-selectin–mediated neutrophil rolling." Blood 109, no. 7 (2006): 2936–43. http://dx.doi.org/10.1182/blood-2006-06-015461.
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Abstract Staphylococcus aureus secretes several virulence factors interfering with host-cell functions. Staphylococcal superantigen-like (SSL) proteins are a family of 11 exotoxins with structural homology to superantigens but with generally unknown functions. Recently, we described that chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS31-121), a potent inhibitor of C5a-induced responses, is structurally homologous to the C-terminal domain of SSL5. Here, we identify P-selectin glycoprotein ligand-1 (PSGL-1), involved in the initial rolling of neutrophils along the endothelium, as a target for SSL5. SSL5 specifically bound to Chinese hamster ovary cells stably expressing PSGL-1 (CHO–PSGL-1), which was dependent of sulfation and sialylation. Furthermore, SSL5 bound to PSGL-1/Ig fusion protein immobilized on a biosensor chip. SSL5 affected binding of soluble P-selectin/Fc chimera, the principle ligand of PSGL-1, to CHO–PSGL-1 cells and inhibited adhesion of neutrophils to immobilized P-selectin under static conditions. Under flow conditions SSL5 strongly decreased neutrophil rolling on immobilized P-selectin/Fc and activated human endothelial cells. In conclusion, SSL5 interferes with the interaction between PSGL-1 and P-selectin, suggesting that S aureus uses SSL5 to prevent neutrophil extravasation toward the site of infection. This makes SSL5 a potential lead for the development of new anti-inflammatory compounds for disorders characterized by excessive recruitment of leukocytes.
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Torgersen, Dawn, Nicholas P. Mullin, and Kurt Drickamer. "Mechanism of Ligand Binding to E- and P-selectin Analyzed Using Selectin/Mannose-binding Protein Chimeras." Journal of Biological Chemistry 273, no. 11 (1998): 6254–61. http://dx.doi.org/10.1074/jbc.273.11.6254.
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Musser, John H., Mark B. Anderson, and Daniel E. Levy. "Glycomimetics as Selectin Inhibitors." Current Pharmaceutical Design 1, no. 2 (1995): 221–32. http://dx.doi.org/10.2174/1381612801666220918090305.
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The selectins are a family of cell adhesion molecules involved in the rolling stage of the transendothelial migration of leukocytes and their principal interaction is with cell-surface carbohydrates such as sialyl Lewis X (sLex). The selectin family currently consists of E-selectin, found on cytokine-activated endothelial cells, L selectin, identified by reaction with the monoclonal antibody MEL-14, and P-selectin, characterized utilizing antibodies differentiating between activated and resting platelets. The structural motifs of the selectins consist of a lectin binding domain succeeded by an epidermal growth factor (EGF) region. The oligosaccharide, sLeX, binds to E-selectin while L- and P-selectin recognize sLex or Lex related carbohydrates via the lectin binding domain. Since the extravasation of leukocytes is associated with disorders including immunoinflammatory diseases, reperfusion injury and adult respiratory distress syndrome, agents that block the initial process of leukocyte adhesion to the endothelium may have therapeutic utility. The potential therapeutic agents based on selectin binding inhibition are either large molecules related to selectins such as protein receptors/chimeras, monoclonal antibodies or peptidic/peptidomimetic compounds, or ligand-based small molecules such as oligosaccharides or carbohydrate mimics (Glycomimetics). An alternative approach to selectin-mediated leukocyte trafficking other than selectin binding inhibition involves the modulation of sLex biosynthesis via the inhibition of glycosyltransferases. Since sLex is an oligosaccharide, mimics of this selectin ligand may be thought of as a new class of glycomimetics.
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Yang, J., J. Galipeau, CA Kozak, BC Furie, and B. Furie. "Mouse P-selectin glycoprotein ligand-1: molecular cloning, chromosomal localization, and expression of a functional P-selectin receptor." Blood 87, no. 10 (1996): 4176–86. http://dx.doi.org/10.1182/blood.v87.10.4176.bloodjournal87104176.
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A mouse homolog of P-selectin glycoprotein ligand-1 (PSGL-1), a P- selectin receptor on myeloid cells, has been cloned using the human cDNA sequence to probe a cDNA library prepared from the mouse WEHI-3 monocytic cell line and a genomic DNA library prepared from 129/SvJ mouse tissue. The gene flanking the entire open reading frame of 397 amino acids is composed of a single exon. Mouse and human PSGL-1 show an overall similarity of 67% and an identity of 50% and contain a similar domain organization. However, there are 10 threonine/serine- rich decameric repeats in mouse PSGL-1 as compared with 15 threonine- rich repeats in human PSGL-1. When the mouse PSGL-1 cDNA is coexpressed with an alpha 1,3/1,4 fucosyltransferase cDNA in COS cells, a functional protein is expressed on the COS cell surface mediating binding to human P-selectin. The mouse PSGL-1 gene, Selpl, was mapped to a position on mouse chromosome 5 (Chr 5). Northern blot analyses of mouse tissues showed moderate expression of a PSGL-1 mRNA species in most tissues including heart, kidney, liver, muscle, ovary, and stomach and high levels of expression in blood, bone marrow, brain, adipose tissue, spleen, and thymus. Whereas certain mouse myeloid cell lines including PU5–1.8, WEHI-3B, and 32DC13 express high levels of PSGL-1 mRNA, only WEHI-3B and 32DC13 bind to P-selectin; this interaction is blocked by anti-PSGL-1 antibody. WEHI-3B cells bind significantly better to P-selectin than to E-selectin. Although comparable P-selectin binding is observed in 32DC13 cells, these cells bind better to E- selectin. Binding of 32DC13 cells to E-selectin is not blocked by anti- PSGL-1 antibody. Treatment of WEHI-3B cells with trypsin or neuraminidase abolished their ability to interact with P-selectin. These results indicate that mouse PSGL-1 has structural and functional homology to human PSGL-1 but is characterized by differences in the composition and number of the decameric repeats. PSGL-1 on mouse myeloid cells is critical for high-affinity binding to P-selectin but not E-selectin.
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Rivera-Nieves, Jesús, Tracy L. Burcin, Timothy S. Olson, et al. "Critical role of endothelial P-selectin glycoprotein ligand 1 in chronic murine ileitis." Journal of Experimental Medicine 203, no. 4 (2006): 907–17. http://dx.doi.org/10.1084/jem.20052530.
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L-selectin ligands might be relevant for inflammatory cell trafficking into the small intestine in a spontaneous model of chronic ileitis (i.e., SAMP1/YitFc mice). Immunoblockade of peripheral node addressin or mucosal addressin cell adhesion molecule 1 failed to ameliorate ileitis, whereas P-selectin glycoprotein ligand 1 (PSGL-1) neutralization attenuated both the adoptively transferred and spontaneous disease. PSGL-1 was detected in venules of mesenteric lymph node and small intestine by immunohistochemistry and confirmed by real-time reverse transcription polymerase chain reaction and flow cytometry. In addition, reconstitution of wild-type mice with PSGL-1−/− bone marrow demonstrated that PSGL-1 messenger RNA and PSGL-1 protein expression remained on endothelium, localized within mesenteric lymph node and small intestine. Endothelial PSGL-1 bound P-selectin–IgG and its blockade or genetic deletion altered the recruitment of lymphocytes to the small intestine, as revealed by intravital microscopy and homing studies. Endothelial expression of PSGL-1 adds a new dimension to the various cellular interactions involved in small intestinal recruitment. Thus, the multiple roles of PSGL-1 may explain why targeting this single adhesion molecule results in attenuation of chronic murine ileitis, a disease previously resistant to antiadhesion molecule strategies.
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Snapp, Karen R., Christine E. Heitzig, and Geoffrey S. Kansas. "Attachment of the PSGL-1 cytoplasmic domain to the actin cytoskeleton is essential for leukocyte rolling on P-selectin." Blood 99, no. 12 (2002): 4494–502. http://dx.doi.org/10.1182/blood.v99.12.4494.
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P-selectin glycoprotein ligand–1 (PSGL-1) serves as the leukocyte ligand for P-selectin, and many of the structural features of its ectodomain required for interactions with P-selectin have been uncovered. In contrast, the function of the highly conserved PSGL-1 cytoplasmic domain has not been explored. Stable transfectants expressing similar levels of either wild-type PSGL-1 or truncated PSGL-1 in which only 4 cytoplasmic residues were retained (designated PSGL-1Δcyto), were analyzed. Transfectants expressing full-length PSGL-1 rolled well on P-selectin. In contrast, rolling was almost completely absent in cells transfected with PSGL-1Δcyto, even at low shear. Importantly, cells expressing truncated PSGL-1 were able to bind soluble P-selectin and to bind COS cells overexpressing P-selectin, demonstrating that the P-selectin binding site on the PSGL-1Δcyto transfectants was intact and was capable of recognizing P-selectin. Impaired rolling by PSGL-1Δcyto transfectants was not due to alterations in subcellular localization because both wild-type and truncated PSGL-1 had similar surface distributions on K562 transfectants. Treatment of cells expressing native PSGL-1 with actin cytoskeletal toxins inhibited adhesion in a dose-dependent way. PSGL-1 was associated with the actin cytoskeleton, and this interaction was greatly impaired in PSGL-1Δcyto– expressing cells. The PSGL-1 cytoplasmic domain interacted selectively with the ezrin/radixin/moesin (ERM) protein moesin, but not with other ERM proteins or several other cytoskeletal linker proteins. Pharmacologic disruption of interactions between moesin and F-actin in cells expressing PSGL-1 resulted in a dose-dependent inhibition of rolling on P-selectin. Thus, attachment of PSGL-1 to the leukocyte cortical cytoskeleton is essential for leukocyte rolling on P-selectin.
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Senczuk, Anna M., John C. Reeder, Magda M. Kosmala, and May Ho. "Plasmodium falciparum erythrocyte membrane protein 1 functions as a ligand for P-selectin." Blood 98, no. 10 (2001): 3132–35. http://dx.doi.org/10.1182/blood.v98.10.3132.
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Abstract The malarial protein Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a parasite protein that is exported to the surface of the infected erythrocyte, where it is inserted into the red cell cytoskeleton in the second half of the parasite life cycle. The surface expression of PfEMP1 coincides with the occurrence of the adhesion of infected erythrocytes to vascular endothelium. This protein has been shown to interact with CD36, intercellular adhesion molecule-1 (ICAM-1) and chondroitin sulfate A (CSA). In this study, it is demonstrated by affinity purification and western blot analysis that PfEMP1 also functions as a cell surface ligand for P-selectin, an adhesion molecule that has been shown to mediate the rolling of infected erythrocytes under physiologic flow conditions, leading to a significant increase in adhesion to CD36 on activated platelets and microvascular endothelium.
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von Andrian, UH, JD Chambers, EL Berg, et al. "L-selectin mediates neutrophil rolling in inflamed venules through sialyl LewisX-dependent and -independent recognition pathways." Blood 82, no. 1 (1993): 182–91. http://dx.doi.org/10.1182/blood.v82.1.182.bloodjournal821182.
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The glycoprotein (GP) L-selectin initiates adhesive interactions between leukocytes and endothelial cells (EC). It functions as a lymphocyte-lectin homing receptor recognizing carbohydrate determinants of the peripheral lymph node addressing on high endothelial venules. It also mediates neutrophil rolling, the earliest interaction of neutrophils with acutely inflamed venules. Neutrophil L-selectin presents sialyl-LewisX (sLe(X)) as a ligand to P- and E-selectin in vitro, and we have proposed that this is a major mechanism of L- selectin-mediated rolling in vivo. In contrast, the contribution of neutrophil L-selectin as a receptor protein recognizing one (or more) ligand(s) on inflamed EC is unclear. To address this question, an sLe(X)-negative murine pre-B cell line, L1–2, that can neither bind vascular selectins nor roll in inflamed rabbit venules, was transfected with human L-selectin cDNA. L-selectin expression in stable transfectants was sufficient to confer significant rolling in vivo. Rolling was unaffected by neuraminidase treatment but completely blocked by anti-L-selectin monoclonal antibody (MoAb) DREG-56. Thus, L- selectin can initiate leukocyte interactions with EC determinants potentially through recognition of endothelial carbohydrates. In contrast, when human neutrophils were tested, rolling was reduced, but not abolished, by MoAb DREG-56. Likewise, treatment with neuraminidase or anti-sLe(X) MoAbs decreased, but did not abrogate, neutrophil rolling, consistent with residual EC recognition via L-selectin. Combination of MoAb DREG-56 and neuraminidase resulted in almost complete loss of rolling, as did removal of glycosylated L-selectin by chymotrypsin. Together with the demonstrable rolling of L-selectin transfectants, our results support the concept of a bidirectional interaction between L-selectin bearing sLe(X) on neutrophils and activated EC in vivo. These findings also suggest that L-selectin may mediate rolling of lymphocytes that lack carbohydrate ligands for E- or P-selectin, although probably less efficiently than through bidirectional recognition.
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Borges, Eric, Wolfgang Tietz, Martin Steegmaier, et al. "P-Selectin Glycoprotein Ligand-1 (PSGL-1) on T Helper 1 but Not on T Helper 2 Cells Binds to P-Selectin and Supports Migration into Inflamed Skin." Journal of Experimental Medicine 185, no. 3 (1997): 573–78. http://dx.doi.org/10.1084/jem.185.3.573.
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We have shown recently that mouse Th1 cells but not Th2 cells are selectively recruited into inflamed sites of a delayed-type hypersensitivity (DTH) reaction of the skin. This migration was blocked by monoclonal antibodies (mAb) against P- and E-selectin. Here we show that Th1 cells bind to P-selectin via the P-selectin glycoprotein ligand-1 (PSGL-1). This is the only glycoprotein ligand that was detectable by affinity isolation with a P-selectin–Ig fusion protein. Binding of Th1 cells to P-selectin, as analyzed by flow cytometry and in cell adhesion assays, was completely blocked by antibodies against PSGL-1. The same antibodies blocked partially the migration of Th1 cells into cutaneous DTH reactions. This blocking activity, in combination with that of a mAb against E-selectin, was additive. PSGL-1 on Th2 cells, although expressed at similar levels as on Th1 cells, did not support binding to P-selectin. Thus, the P-selectin–binding form of PSGL-1 distinguishes Th1 cells from Th2 cells. Furthermore, PSGL-1 is relevant for the entry of Th1 cells into inflamed areas of the skin. This is the first demonstration for the importance of PSGL-1 for mouse leukocyte recruitment in vivo.
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Salmi, Marko, Sami Tohka, Ellen L. Berg, Eugene C. Butcher, and Sirpa Jalkanen. "Vascular Adhesion Protein 1 (VAP-1) Mediates Lymphocyte Subtype-specific, Selectin-independent Recognition of Vascular Endothelium in Human Lymph Nodes." Journal of Experimental Medicine 186, no. 4 (1997): 589–600. http://dx.doi.org/10.1084/jem.186.4.589.
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Interactions between lymphocyte surface receptors and their ligands on vascular endothelial cells regulate the exit of lymphocytes from the circulation. Distinct subsets of mononuclear cells bind to high endothelial venules (HEVs) in different lymphoid organs to a different extent, but the molecular mechanisms behind this selectivity have remained poorly characterized. Here we show that vascular adhesion protein-1 (VAP-1) mediates subtype-specific binding of CD8-positive T cells and natural killer cells to human endothelium. VAP-1–dependent, oligosaccharide-dependent peripheral lymph node (PLN) HEV adhesion under shear was independent of L-selectin, P-selectin glycoprotein ligand 1, and α4 integrins, the known lymphocyte receptors involved in the initial recognition of endothelial cells. PLN HEV adhesion was also critically dependent on peripheral lymph node vascular addressins (PNAds), but lymphocyte L-selectin was absolutely required for PNAd binding. Most lymphocytes relied on both PNAd and VAP-1 in HEV binding. The overlapping function of L-selectin ligands and VAP-1 in PLN introduces a new control point into the lymphocyte extravasation process. Finally, intravital microscopy revealed that VAP-1 is involved in initial interactions between human lymphocytes and endothelial cells in inflamed rabbit mesenterial venules in vivo. In conclusion, VAP-1 is a novel contact-initiating ligand that discriminates between different subpopulations of mononuclear cells and is an appealing target for selective modulation of adhesion of CD8- and CD16-positive effector cells.
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Mehta, Padmaja, Kamala D. Patel, Thomas M. Laue, Harold P. Erickson, and Rodger P. McEver. "Soluble Monomeric P-Selectin Containing Only the Lectin and Epidermal Growth Factor Domains Binds to P-Selectin Glycoprotein Ligand-1 on Leukocytes." Blood 90, no. 6 (1997): 2381–89. http://dx.doi.org/10.1182/blood.v90.6.2381.
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Abstract Under shear stress, leukocytes use P-selectin glycoprotein ligand-1 (PSGL-1) to tether to and roll on P-selectin expressed on activated platelets or endothelial cells. P-selectin has an NH2-terminal lectin domain, an epidermal growth factor (EGF)-like motif, nine consensus repeats (CRs), a transmembrane domain, and a cytoplasmic tail. To determine whether the CRs are required for P-selectin to bind PSGL-1, we expressed a soluble protein (Lec-EGF) that contained only the lectin and EGF domains, plus a short C-terminal epitope tag. Electron microscopy and hydrodynamic analysis confirmed that Lec-EGF was monomeric, as previously shown for soluble P-selectin (sPS) that contained the lectin and EGF domains plus all nine CRs. Fluid-phase Lec-EGF or sPS inhibited binding of oligomeric125I-labeled membrane-derived P-selectin (mPS) to PSGL-1 on neutrophils and binding of 125I-PSGL-1 to immobilized mPS. The IC50 for inhibiting binding of mPS to neutrophils was fivefold greater for Lec-EGF than for sPS, whereas the IC50 for inhibiting binding of mPS to purified PSGL-1 was indistinguishable for Lec-EGF and sPS. Under static or shear conditions, neutrophils used PSGL-1 to tether to or roll on Lec-EGF that was captured by an immobilized monoclonal antibody to the C-terminal epitope. These data show that P-selectin requires only the lectin and EGF domains to bind to PSGL-1.
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Mehta, Padmaja, Kamala D. Patel, Thomas M. Laue, Harold P. Erickson, and Rodger P. McEver. "Soluble Monomeric P-Selectin Containing Only the Lectin and Epidermal Growth Factor Domains Binds to P-Selectin Glycoprotein Ligand-1 on Leukocytes." Blood 90, no. 6 (1997): 2381–89. http://dx.doi.org/10.1182/blood.v90.6.2381.2381_2381_2389.
Full textAbstract:
Under shear stress, leukocytes use P-selectin glycoprotein ligand-1 (PSGL-1) to tether to and roll on P-selectin expressed on activated platelets or endothelial cells. P-selectin has an NH2-terminal lectin domain, an epidermal growth factor (EGF)-like motif, nine consensus repeats (CRs), a transmembrane domain, and a cytoplasmic tail. To determine whether the CRs are required for P-selectin to bind PSGL-1, we expressed a soluble protein (Lec-EGF) that contained only the lectin and EGF domains, plus a short C-terminal epitope tag. Electron microscopy and hydrodynamic analysis confirmed that Lec-EGF was monomeric, as previously shown for soluble P-selectin (sPS) that contained the lectin and EGF domains plus all nine CRs. Fluid-phase Lec-EGF or sPS inhibited binding of oligomeric125I-labeled membrane-derived P-selectin (mPS) to PSGL-1 on neutrophils and binding of 125I-PSGL-1 to immobilized mPS. The IC50 for inhibiting binding of mPS to neutrophils was fivefold greater for Lec-EGF than for sPS, whereas the IC50 for inhibiting binding of mPS to purified PSGL-1 was indistinguishable for Lec-EGF and sPS. Under static or shear conditions, neutrophils used PSGL-1 to tether to or roll on Lec-EGF that was captured by an immobilized monoclonal antibody to the C-terminal epitope. These data show that P-selectin requires only the lectin and EGF domains to bind to PSGL-1.
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Cappenberg, Anika, Marina Kardell, and Alexander Zarbock. "Selectin-Mediated Signaling—Shedding Light on the Regulation of Integrin Activity in Neutrophils." Cells 11, no. 8 (2022): 1310. http://dx.doi.org/10.3390/cells11081310.
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As a consequence of tissue injury or infection, neutrophils are recruited in a stepwise recruitment process from the bloodstream into the surrounding tissue. Selectins are a family of adhesion molecules comprised of L-, E-, and P-selectin. Differences in expression patterns, protein structure, and ligand binding characteristics mediate distinct functions of each selectin. Interactions of selectins and their counter-receptors mediate the first contact of neutrophils with the endothelium, as well as subsequent neutrophil rolling along the endothelial surface. For efficient neutrophil recruitment, activation of β2-integrins on the cell surface is essential. Integrin activation can be elicited via selectin- as well as chemokine-mediated inside-out signaling resulting in integrin conformational changes and clustering. Dysregulation of selectin-induced integrin activation on neutrophils is involved in the development of severe pathological disease conditions including leukocyte adhesion deficiency (LAD) syndromes in humans. Here, we review molecular mechanisms involved in selectin-mediated signaling pathways in neutrophils and their impact on integrin activation, neutrophil recruitment, and inflammatory diseases.
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Cano-Mendez, Alan, Nallely García-Larragoiti, Maria Damian-Vazquez, et al. "Platelet Reactivity and Inflammatory Phenotype Induced by Full-Length Spike SARS-CoV-2 Protein and Its RBD Domain." International Journal of Molecular Sciences 23, no. 23 (2022): 15191. http://dx.doi.org/10.3390/ijms232315191.
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A state of immunothrombosis has been reported in COVID-19. Platelets actively participate in this process. However, little is known about the ability of SARS-CoV-2 virus proteins to induce platelet activity. Platelet-rich plasma (PRP) was incubated with spike full-length protein and the RBD domain in independent assays. We evaluated platelet activation through the expression of P-selectin and activation of glicoprotein IIbIIIa (GP IIbIIIa), determined by flow cytometry and the ability of the proteins to induce platelet aggregation. We determined concentrations of immunothrombotic biomarkers in PRP supernatant treated with the proteins. We determined that the spike full-length proteins and the RBD domain induced an increase in P-selectin expression and GP IIbIIIa activation (p < 0.0001). We observed that the proteins did not induce platelet aggregation, but favored a pro-aggregating state that, in response to minimal doses of collagen, could re-establish the process (p < 0.0001). On the other hand, the viral proteins stimulated the release of interleukin 6, interleukin 8, P-selectin and the soluble fraction of CD40 ligand (sCD40L), molecules that favor an inflammatory state p < 0.05. These results indicate that the spike full-length protein and its RBD domain can induce platelet activation favoring an inflammatory phenotype that might contribute to the development of an immunothrombotic state.
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Maugeri, Norma, Patrizia Rovere-Querini, Virgilio Evangelista та ін. "Neutrophils phagocytose activated platelets in vivo: a phosphatidylserine, P-selectin, and β2 integrin–dependent cell clearance program". Blood 113, № 21 (2009): 5254–65. http://dx.doi.org/10.1182/blood-2008-09-180794.
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Abstract Activated platelets express ligands, which are recognized by counterreceptors on neutrophils. Here, we show that the ensuing cell-to-cell interaction programs neutrophil phagocytic function, resulting in activated platelet clearance. Neutrophils that have internalized platelets circulate in the blood of patients with acute myocardial infarction, and the extent of platelet clearance correlates with expression of platelet activation, including P-selectin. Activated platelets injected intravenously in experimental animals are detectable in circulating neutrophils 60 minutes after, and within 3 hours, more than 70% circulating neutrophils have internalized platelets. Platelet clearance comprises 2 events: adhesion to neutrophils, which requires divalent cations and depends on P-selectin, on the P-selectin glycoprotein ligand-1 (PSGL-1), and on the CD11b/CD18 β2 integrin; and internalization, which is abrogated by the phosphatidylserine-binding protein annexin A5. Adhesion to platelets causes neutrophil degranulation and is blocked by antibodies specific for P-selectin and PSGL-1, either in a synthetic medium in vitro or in the whole blood, therefore in the presence of a physiologic array of plasma cofactors and opsonins. The data suggest that the interaction between circulating platelets and neutrophils influences innate immune functions, possibly contributing to regulate vascular inflammation.
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Lu, Sydney X., Maria Lia Palomba, Il-Kang Na, et al. "P-Selectin in Recipients of Allogeneic Bone Marrow Transplantation Regulates Experimental Graft-Versus-Host-Disease." Blood 114, no. 22 (2009): 1335. http://dx.doi.org/10.1182/blood.v114.22.1335.1335.
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Abstract Abstract 1335 Poster Board I-357 Alloreactive T cells are crucial for graft-versus-host-disease (GVHD) pathophysiology, and we hypothesized that controlling their trafficking can ameliorate GVHD. P-selectin is a dimeric glycoprotein found on most inflamed endothelium, which interacts with multiple lectin-type molecules on leukocytes, including T cells. We used murine allogenienc BMT models to study GVHD and found that P-selectin−/− recipients exhibited significantly less GVHD mortality and morbidity, as well as decreased GVHD of the skin, liver and small bowels. However, WT and P-selectin−/− allo-BMT recipients had comparable large bowel GVHD. This decrease in target organ and systemic GVHD was associated with diminished infiltration of alloactivated T cells into the Peyer's Patches and small bowels, coupled with increased numbers of donor T cells in the spleen and secondary lymphoid organs (SLO) on day 14 and day 35 post-transplant. However, donor alloreactive T cells in WT and P-selectin−/− allo-BMT recipients had similar alloactivation and apoptosis, and donor alloactivated T cells from WT and P-selectin−/− allo-BMT recipients with GVHD showed similar proliferation in vitro in a mixed leukocyte reaction, suggesting that the inflammatory environment in WT and P-selectin−/− recipients was comparable. Finally, non-transplanted P-selectin−/− mice, and P-selectin−/− mice which had received the allo-BMT conditioning regimen but not a donor graft, had similar cellularity in the majority of tissues examined as corresponding WT controls. This suggests that the differential cellularity of donor alloactivated T cells in WT and P-selectin−/− allo-BMT recipients with GVHD is probably largely dependent on trafficking and tissue infiltration during inflammation. Since P-selectin glycoprotein ligand 1 (PSGL1) is the best-described P-selectin ligand, and all leukocytes constitutively bear high levels of membrane PSGL1, we next hypothesized that PSGL1−/− donor alloreactive T cells would be defective in trafficking into GVHD target organs, and that PSGL1−/− donor T cells would cause decreased target organ damage, systemic GVHD, and mortality. However, allo-BMT recipients of WT and PSGL1−/− donor T cells had comparable survival and clinical GVHD scores, and further analyses on day 14 post-transplant revealed that recipients of WT and PSGL1−/− donor T cells also had similar numbers of donor alloactivated T cells in the spleen, liver, mesenteric and peripheral lymph nodes, and Peyer's Patches. Additionally, WT and PSGL1−/− donor T cells had comparable proliferation as measured by CFSE dilution, and comparable alloactivation in vivo as determined by levels of CD25, CD44, and CD62L, suggesting similar T cell function. As PSGL1−/− and WT donor T cells appeared to have equal functionality and accumulated in GVHD target tissues and lymphoid tissues in a similar fashion, we asked whether PSGL1−/− T cells might display other P-selectin ligands. Flow cytometric analyses of T cells from non-transplanted PSGL1−/− mice, and analyses of PSGL1−/− alloactivated T cells on day 14 after allo-BMT, revealed that these cells displayed substantial levels of cell-surface P-selectin ligands as defined by positive staining with recombinant P-selectin-IgG-Fc fusion protein at levels similar to those found on WT T cells, suggesting that although absence of P-selectin on host tissues may ameliorate GVHD, multiple donor leukocyte P-selectin ligands interact meaningfully with P-selectin. Our studies suggest that P-selectin may be required for trafficking into inflamed tissues but not SLO, and that donor T cells may utilize multiple P-selectin ligands apart from PSGL1 to interact with P-selectin and traffic into inflamed tissues during GVHD. We conclude that targeting P-selectin may be a viable target for GVHD prophylaxis or treatment. Disclosures No relevant conflicts of interest to declare.
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Chien, Sylvia S., Jin Dai, John L. Magnani, et al. "E-Selectin Ligand Expression By Leukemic Blasts Is Associated with Prognosis in Patients with AML." Blood 132, Supplement 1 (2018): 1513. http://dx.doi.org/10.1182/blood-2018-99-119449.
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Abstract Introduction. The E-selectin ligand is a carbohydrate structure (sialyl Lewis x) expressed on myeloid cells and plays a role in cell adhesion and activation by binding specifically to E-selectin on endothelial cells. Acute myeloid leukemia (AML) blasts express the E-selectin ligand on glycoforms of CD44, CD62L and CD65. Interaction of AML blasts in the marrow microenvironment is believed to involve a number of receptors, including CXCR4, VLA4, and CD44. E-selectin in the vascular niche regulates dormancy for hematopoietic stem cells (Winkler IG et al. 2012), and chemoresistance and survival in AML. E-selectin inhibitors are now being studied in clinical trials in combination with chemotherapy for hematologic malignancies. Here, we evaluate the clinical significance of the expression of the E-selectin ligand using a panel of AML patient blood and bone marrow samples. Methods. We studied 89 serially acquired AML patient samples obtained with informed consent on an IRB approved protocol, 40 from new diagnosis patients, and 49 from relapsed or refractory patients. Five patients had favorable, 30 intermediate, and 43 unfavorable risk cytogenetics. We used multicolor flow cytometry to examine binding of an E-selectin-Fc chimeric protein or staining by HECA452 antibody that recognizes sialyl Lewis a/x carbohydrate epitopes. The blast population was gated by forward scatter vs. side scatter, then CD45 vs. side scatter. Leukemia stem cells (LSCs) were defined as CD34+CD38-CD123+. Results. Percent E-selectin-Fc functional binding for the AML blast population ranged from 0.4 to 99.4%, mean 32.1% and median 20.9%. Percent staining by HECA452 antibody ranged from 0.9 to 99.6%, mean 54.3% and median 58%. These data suggest that the epitope recognized by the HECA452 antibody is present, but not always functionally active. The E-sel-Fc binding % was correlated with HECA452 % expression by Pearson's product-moment correlation, p=1.543e-06, correlation coefficient 0.50. We found a number of statistical correlations between expression of E-selectin ligand and clinical characteristics. There were statistically significant differences for mean fluorescence intensity (MFI) based on disease status, newly diagnosed vs. relapsed/refractory patients for E-sel-Fc binding, with values of 2884 vs. 11764 (p=0.0026 by Welch two sample t-test, p=0.000074 by Wilcoxon rank sum test with continuity correction) and HECA452 staining 3387 vs. 8994 (p=0.00038 by t-test, p=0.0012 by Wilcoxon rank sum test) as depicted in left side figure, respectively. By Pearson's product-moment correlation, the MFI for E-sel-Fc binding was correlated with the MFI for HECA452 staining, p=5.815e-08, correlation coefficient 0.55. There were also significant differences for E-sel-Fc binding % favorable/intermediate vs. unfavorable risk cytogenetics, 24% vs. 39%, p=0.019 (t-test), p=0.033 (Wilcoxon rank sum test) (middle figure), but no difference for MFI. There was a tight correlation between expression of E-selectin ligand by the blast population and the leukemia stem cell population defined as CD34+CD38-CD123+, with p< 2.2e-16 (Pearson's product-moment correlation) and correlation coefficient 0.90 for % expression by E-sel-Fc binding as depicted in right side figure, and by MFI for E-sel-Fc binding, p= 3.24e-09 and correlation coefficient 0.65. E-sel-Fc binding MFI correlated with Flt3 mutation status (ITD and TKD), with mutation 9919 vs. 3858 without, p=0.042, and with a trend toward significance by HECA staining MFI, with mutation 8733 vs. 5101 without, p=0.055. There were no statistically significant differences for other features such as age, gender, secondary vs de novo AML, or presenting WBC. Conclusions. E-selectin ligands are variably expressed by AML blasts and LSCs. Mean fluorescence intensity of E-selectin-Fc binding is 4-fold higher for relapsed/refractory patients than for newly diagnosed patients (p=0.0026), suggesting that sequestration in the vascular niche of the marrow is associated with chemotherapy resistance. Percent E-selectin-Fc binding is higher in patients with unfavorable than favorable/intermediate risk (p=0.019) and MFI correlated with Flt3 mutation status (p=0.042), again correlating with worse prognosis. Moreover, expression of E-selectin ligands by LSCs is correlated with blasts from the same patients, suggesting a role for E-selectin in leukemia survival and propagation. Figure. Figure. Disclosures Magnani: GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Fogler:GlycoMimetics: Employment, Equity Ownership. Thackray:GlycoMimetics: Employment, Equity Ownership. Becker:Novartis: Research Funding; BMS: Research Funding; CVS Caremark: Consultancy; Abbvie: Research Funding; Rocket Pharmaceuticals: Research Funding; Amgen: Research Funding; Pfizer: Consultancy; JW Pharmaceuticals: Research Funding; Trovagene: Research Funding; GlycoMimetics: Research Funding.
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Barkalow, Fern J., Kurt L. Barkalow, and Tanya N. Mayadas. "Dimerization of P-selectin in platelets and endothelial cells." Blood 96, no. 9 (2000): 3070–77. http://dx.doi.org/10.1182/blood.v96.9.3070.
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Abstract P-selectin is a leukocyte adhesion receptor stored in platelets and endothelial cells and is translocated to the surface upon cell activation. Purified P-selectin is oligomeric and has increased avidity for its ligand relative to the monomeric form, but whether P-selectin self-associates in the membrane of intact cells is not known. A chemical cross-linking approach was used to show that P-selectin is present as noncovalent dimers in resting platelets, human umbilical vein endothelial cells, and heterologous RIN5F cells expressing P-selectin. The results of 2-dimensional isoelectric focusing are consistent in showing P-selectin dimers as homodimers, but they are composed of a more basic subset of P-selectin than the monomers. This suggests that the dimers are a biochemically distinct subset of P-selectin. P-selectin dimers form in the endoplasmic reticulum and Golgi compartments of human umbilical vein endothelial cells only after synthesis of the mature P-selectin subunit, and are not preferentially stored in Weibel-Palade bodies as compared with the monomeric form. Platelet activation with thrombin receptor–activating peptide leads to the presence of P-selectin monomers and homodimers on the cell surface as well as P-selectin heterodimers, which are composed of P-selectin and an unidentified protein of approximately 81 kd molecular weight. In summary, these studies demonstrate that P-selectin is homodimeric in situ and that platelet activation leads to the formation of an additional activation-specific heterodimeric species. In addition, the homodimer has unique biochemical characteristics compared with the monomeric form, and dimerization occurs in the endoplasmic reticulum and Golgi compartments of endothelial cells.
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Barkalow, Fern J., Kurt L. Barkalow, and Tanya N. Mayadas. "Dimerization of P-selectin in platelets and endothelial cells." Blood 96, no. 9 (2000): 3070–77. http://dx.doi.org/10.1182/blood.v96.9.3070.h8003070_3070_3077.
Full textAbstract:
P-selectin is a leukocyte adhesion receptor stored in platelets and endothelial cells and is translocated to the surface upon cell activation. Purified P-selectin is oligomeric and has increased avidity for its ligand relative to the monomeric form, but whether P-selectin self-associates in the membrane of intact cells is not known. A chemical cross-linking approach was used to show that P-selectin is present as noncovalent dimers in resting platelets, human umbilical vein endothelial cells, and heterologous RIN5F cells expressing P-selectin. The results of 2-dimensional isoelectric focusing are consistent in showing P-selectin dimers as homodimers, but they are composed of a more basic subset of P-selectin than the monomers. This suggests that the dimers are a biochemically distinct subset of P-selectin. P-selectin dimers form in the endoplasmic reticulum and Golgi compartments of human umbilical vein endothelial cells only after synthesis of the mature P-selectin subunit, and are not preferentially stored in Weibel-Palade bodies as compared with the monomeric form. Platelet activation with thrombin receptor–activating peptide leads to the presence of P-selectin monomers and homodimers on the cell surface as well as P-selectin heterodimers, which are composed of P-selectin and an unidentified protein of approximately 81 kd molecular weight. In summary, these studies demonstrate that P-selectin is homodimeric in situ and that platelet activation leads to the formation of an additional activation-specific heterodimeric species. In addition, the homodimer has unique biochemical characteristics compared with the monomeric form, and dimerization occurs in the endoplasmic reticulum and Golgi compartments of endothelial cells.
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Park, Jinho, Kyoung Seong Choi, and J. Stephen Dumler. "Major Surface Protein 2 of Anaplasma phagocytophilum Facilitates Adherence to Granulocytes." Infection and Immunity 71, no. 7 (2003): 4018–25. http://dx.doi.org/10.1128/iai.71.7.4018-4025.2003.
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ABSTRACT Anaplasma phagocytophilum is an obligate intracellular bacterium that infects myeloid cells in the mammalian host. Msp2 (p44) is the major immunodominant outer-membrane protein of these bacteria. We hypothesized that Msp2 acts as an adhesin for A. phagocytophilum entry into granulocytes. This potential role was investigated by blocking binding with Msp2 monoclonal antibodies and by antagonizing binding and propagation with recombinant Msp2 (rMsp2) in vitro. With HL-60 cells, fresh human peripheral blood neutrophils, and a cell line devoid of the fucosylated platelet selectin glycoprotein ligand 1 (PSGL-1) receptor for A. phagocytophilum or one that was transfected to express this ligand, Msp2 monoclonal antibody and rMsp2 used as the antagonist caused concentration-dependent reductions in bacterial adhesion (P < 0.007 and P < 0.02, respectively) and propagation (P < 0.05 and P < 0.001), although inhibition of adhesion or propagation was moderate and incomplete. Likewise, rMsp2 bound to surfaces of the transfected cell at a level similar to that of extracellular A. phagocytophilum and significantly (P < 0.05) beyond that of nontransfected cells. Moreover, a dose-dependent reduction (P < 0.019) in PSGL-1 monoclonal antibody binding to HL-60 cells was elicited with rMsp2. We conclude that Msp2s of A. phagocytophilum are involved in bacterial adhesion to ligands on host myeloid cells before intracellular infection.
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Volcik, Kelly A., Diane Catellier, Aaron R. Folsom, Nena Matijevic, Bruce Wasserman, and Eric Boerwinkle. "SELP and SELPLG Genetic Variation Is Associated with Cell Surface Measures of SELP and SELPLG: The Atherosclerosis Risk in Communities Carotid MRI Study." Clinical Chemistry 55, no. 6 (2009): 1076–82. http://dx.doi.org/10.1373/clinchem.2008.119487.
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Abstract Background: P-selectin (SELP) and its ligand, P-selectin glycoprotein ligand 1 (SELPLG), play key roles in both the inflammatory response and the atherosclerotic process. Previous studies have shown genetic variation in the SELP gene [selectin P (granule membrane protein 140kDa, antigen CD62)] to be associated with plasma SELP concentrations; however, the major biological function of SELP (and SELPLG) is at the cell surface. We therefore investigated the association of SELP polymorphisms with platelet SELP measures and polymorphisms in the SELPLG gene (selectin P ligand) with lymphocyte, granulocyte, and monocyte SELPLG measures among 1870 participants in the Atherosclerosis Risk in Communities (ARIC) Carotid MRI Study. Methods: Whole-blood flow cytometry was used to analyze leukocyte and platelet markers in the ARIC Carotid MRI Study. The allele frequencies for the SELP and SELPLG polymorphisms of whites and African Americans were markedly different; therefore, all analyses were race specific. Results: SELP T715P was significantly associated with lower values for platelet SELP measures in whites (P = 0.0001), whereas SELP N562D was significantly associated with higher values for SELP measures in African Americans (P = 0.02). SELPLG M62I was significantly associated with lower granulocyte and monocyte SELPLG measures in African Americans (P = 0.003 and P = 0.0002, respectively) and with lower lymphocyte SELPLG measures in whites (P = 0.01). Conclusions: Specific SELP and SELPLG polymorphisms were associated with cell surface measures of SELP and SELPLG in both whites and African Americans in the ARIC Carotid MRI Study. To our knowledge, this study is the first to examine the association of SELP and SELPLG genetic variation with measures of cell surface SELP and SELPLG.
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Pierce, Elizabeth, Sung Choi, Krystyna Olkiewicz, et al. "Critical role for selectin receptor:ligand interactions in the development of IPS following allogeneic BMT (145.32)." Journal of Immunology 184, no. 1_Supplement (2010): 145.32. http://dx.doi.org/10.4049/jimmunol.184.supp.145.32.
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Abstract Idiopathic pneumonia syndrome (IPS) is a frequently fatal complication of allogeneic BMT and limits successful outcomes. The pathophysiology of IPS involves the release of soluble inflammatory proteins and the recruitment of donor lymphoid and myeloid effectors into the lung. The entry of leukocytes into inflamed tissue is mediated by several molecules that are orchestrated in a sequential manner. We used well-established mouse BMT models to investigate the role of selectin (sel) receptor:ligand interactions in the development of IPS. Firstly, E and P sel ligands are up-regulated on donor T cells when activated by host APCs in vitro. Next, we found that sel receptor (mRNA) and ligand (protein) expression is significantly increased in the lungs of allo-BMT recipients during the development of IPS. BMT using donor cells deficient in E and P sel ligands resulted in a significant reduction in lung inflammation (cellularity, pathology, cytokines), and mixing studies uncovered a contribution to cells of both lymphoid and myeloid lineage. Experiments using isolated MHC class I or class II disparate strain combinations revealed a more significant effect on donor CD4+ vs. CD8+ T cells. Finally absence of E and P sel in BMT recipient mice conferred near complete protection from IPS especially during CD4+ mediated disease. These data demonstrate a critical role for selectin receptor:ligand interactions in the pathogenesis of IPS and uncover novel strategies to regulate disease severity.
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Underhill, Gregory H., Heather A. Minges-Wols, Jamie L. Fornek, Pamela L. Witte, and Geoffrey S. Kansas. "IgG plasma cells display a unique spectrum of leukocyte adhesion and homing molecules." Blood 99, no. 8 (2002): 2905–12. http://dx.doi.org/10.1182/blood.v99.8.2905.
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Abstract Long-lived antibody-secreting plasma cells are formed in the secondary lymphoid organs and subsequently home to the bone marrow, although the mechanisms that control this migration remain primarily unknown. In this study, we show that IgG plasma cells constitute a significant fraction of cervical lymph node cells from older mice deficient in both E- and P-selectin (E/P−/−), and that these cells can be prospectively isolated by phenotype. These IgG plasma cells were polyclonal, cytoplasmic Ig+, spontaneously secreted antibody, were in the G0/G1 phase of the cell cycle, and failed to express multiple B-cell surface markers. The plasma cells exhibited up-regulated cell surface expression of multiple adhesion molecules, including α4 and leukocyte function-associated antigen 1 (LFA-1) integrins, CD44, and P-selectin glycoprotein ligand 1 (PSGL-1). IgG plasma cells bound to vascular cell adhesion molecule 1 (VCAM-1) significantly better than IgM+B cells, indicating that the α4 integrins were constitutively active. A subset of IgG plasma cells also bound hyaluronic acid, the ligand for CD44. In addition, the IgG plasma cells interacted strongly with E-selectin, but poorly with P-selectin, despite elevated levels of PSGL-1 protein. The preferential interaction of plasma cells with E-selectin, but not P-selectin, correlated with elevated α1,3-fucosyltransferase-VII messenger RNA levels, but selective down-regulation of core 2 β1-6-N-glucosaminyltransferase levels, compared to B cells. These results demonstrate a unique adhesion profile for murine IgG plasma cells. Furthermore, the E/P−/− mice represent a novel system to isolate and purify significant numbers of primary IgG plasma cells.
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Xie, Huijuan, Yaw-Chyn Lim, Francis W. Luscinskas, and Andrew H. Lichtman. "Acquisition of Selectin Binding and Peripheral Homing Properties by CD4+ and CD8+ T Cells." Journal of Experimental Medicine 189, no. 11 (1999): 1765–76. http://dx.doi.org/10.1084/jem.189.11.1765.
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Different T cell subsets exhibit distinct capacities to migrate into peripheral sites of inflammation, and this may in part reflect differential expression of homing receptors and chemokine receptors. Using an adoptive transfer approach, we examined the ability of functionally distinct subsets of T cells to home to a peripheral inflammatory site. The data directly demonstrate the inability of naive T cells and the ability of effector cells to home to inflamed peritoneum. Furthermore, interleukin (IL)-12 directs the differentiation of either CD4+ or CD8+ T cells into effector populations that expresses functional E- and P-selectin ligand and that are preferentially recruited into the inflamed peritoneum compared with T cells differentiated in the presence of IL-4. Recruitment can be blocked by anti–E- and –P-selectin antibodies. The presence of antigen in the peritoneum promotes local proliferation of recruited T cells, and significantly amplifies the Th1 polarization of the lymphocytic infiltrate. Preferential recruitment of Th1 cells into the peritoneum is also seen when cytokine response gene 2 (CRG-2)/interferon γ–inducible protein 10 (IP-10) is used as the sole inflammatory stimulus. We have also found that P-selectin binds only to antigen-specific T cells in draining lymph nodes after immunization, implying that both antigen- and cytokine-mediated signals are required for expression of functional selectin-ligand.
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Scalia, Rosario, Valerie E. Armstead, Alexander G. Minchenko, and Allan M. Lefer. "Essential Role of P-Selectin in the Initiation of the Inflammatory Response Induced by Hemorrhage and Reinfusion." Journal of Experimental Medicine 189, no. 6 (1999): 931–38. http://dx.doi.org/10.1084/jem.189.6.931.
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Resuscitation from hemorrhage induces profound pathophysiologic alterations and activates inflammatory cascades able to initiate neutrophil accumulation in a variety of tissues. This process is accompanied by acute organ damage (e.g., lungs and liver). We have previously demonstrated that significant leukocyte–endothelium interactions occur very early in other forms of ischemia/reperfusion (i.e., splanchnic ischemia/reperfusion and traumatic shock) which are largely mediated by increased expression of the adhesion molecule, P-selectin, on the vascular endothelium. Here we postulated that increased endothelial expression of P-selectin in the microvasculature would play an essential role in initiating the inflammatory signaling of hemorrhagic shock. Using intravital microscopy, we found that hemorrhagic shock significantly increased the number of rolling and adherent leukocytes in the mouse splanchnic microcirculation. In contrast, mice genetically deficient in P-selectin, or wild-type mice given either an anti–P-selectin monoclonal antibody or a recombinant soluble P-selectin glycoprotein ligand (PSGL)-1 immunoglobulin, exhibited markedly attenuated leukocyte–endothelium interaction after hemorrhagic shock. Thus, activation of P-selectin protein on the microvascular endothelium is essential for the initial upregulation of the inflammatory response occurring in hemorrhagic shock. Moreover, endogenous levels of PSGL-1 mRNA were significantly increased in the lung, liver, and small intestine of wild-type mice subjected to hemorrhagic shock. Since PSGL-1 promotes adhesive interactions largely through P-selectin expressed on the vascular endothelium, this result further supports the crucial role played by P-selectin in the recruitment of leukocytes during hemorrhagic shock.
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Lichtenthaler, Stefan F., Diana-ines Dominguez, Gil G. Westmeyer, et al. "The Cell Adhesion Protein P-selectin Glycoprotein Ligand-1 Is a Substrate for the Aspartyl Protease BACE1." Journal of Biological Chemistry 278, no. 49 (2003): 48713–19. http://dx.doi.org/10.1074/jbc.m303861200.
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Guan, Jun, Sundee Dees, Tony Chadderton, and Alejandro Amador Arjona. "Abstract 6373: P-selectin glycoprotein ligand-1 modulates the functions of human T cells and macrophages in vitro." Cancer Research 83, no. 7_Supplement (2023): 6373. http://dx.doi.org/10.1158/1538-7445.am2023-6373.
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Abstract P-selectin glycoprotein ligand-1 (PSGL-1) is a type I transmembrane protein expressed on the surface of most hematopoietic cells. PSGL-1 can engage multiple ligands (e.g., selectins, VISTA, Siglec-5, versican, CCL19, and CCL21). Apart from being a key adhesion molecule involved in immune cell trafficking, PSGL-1 has been shown to function as a negative immune checkpoint receptor in both T cells and macrophages. PSGL-1 is highly expressed in tumor-infiltrating T cells (TILs) and in tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). PSGL-1 signaling in TILs induces development of T-cell exhaustion and, in TAMs, it promotes immunosuppressive activity of macrophages. In the TME, PSGL-1 is also highly expressed on the surface of myeloid-derived suppressor cells, regulatory T cells, and some types of cancer cells. We hypothesized that PSGL-1 may also act as a ligand to modulate the effector functions of human T cells and macrophages in the TME. In the present study, flow cytometry analysis showed that monocytes and granulocytes had a higher PSGL-1 expression than CD4+ and CD8+ T cells isolated from normal human blood cells. To mimic the cell-expressing PSGL-1, a recombinant human PSGL-1-Fc protein (rhPSGL-1-Fc, R&D Systems) was coated onto 96-well plates overnight. Human peripheral blood mononuclear cells (PBMCs), purified human CD4+ T cells, or CD8+ T cells were cultured in the PSGL-1-coated plates for 3 days in the presence of anti-CD3 antibody. In some experiments, a commercial tool PSGL-1 monoclonal antibody (mAb) with the ability to block PSGL-1/P-selectin interaction was added into the culture system. Cytokine secretion in the culture supernatants was measured using Meso Scale Discovery assays. Similar experiments were also conducted with commercially supplied human M1 and M2 macrophages. The results demonstrated that plate-bound rhPSGL-1-Fc dose-dependently inhibited IFN-γ, IL-2, and TNF-α cytokine production in human PBMCs, CD4+ T cells, and in CD8+ T cells following T-cell receptor stimulation. The tool PSGL-1 mAb was able to partially rescue the suppression of cytokine production. Plate-bound rhPSGL-1-Fc also inhibited TNF-α secretion from human M1 macrophage, and the PSGL-1 mAb significantly enhanced TNF-α production in both human M1 and M2 macrophages treated with plate-bound PSGL-1-Fc. Together, these data indicate that PSGL-1 expressed on immune cells in TME may act as both an inhibitory ligand and receptor to impact on T-cell and macrophage function. Acknowledgements: Thanks to Patrick Mayes and Ricardo Macarron for scientific input and thanks to Qian Wang Yao and Lynn Leffet for technical assistance. Trial Registration: N/A Ethics Approval: N/A Citation Format: Jun Guan, Sundee Dees, Tony Chadderton, Alejandro Amador Arjona. P-selectin glycoprotein ligand-1 modulates the functions of human T cells and macrophages in vitro. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6373.
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Mueller, Helena, Mari Tenno, Hugo Van Aken, Jamey D. Marth, Klaus Ley, and Alexander Zarbock. "Severe impairment of leukocyte recruitment into inflamed tissue of ppGalNAcT1-deficient mice (94.1)." Journal of Immunology 182, no. 1_Supplement (2009): 94.1. http://dx.doi.org/10.4049/jimmunol.182.supp.94.1.
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Abstract P-selectin glycoprotein ligand-1 (PSGL-1) plays an important role in leukocyte recruitment. Its binding affinity to selectins is modulated by posttranslational modifications. The polypeptide N-acetylgalactosamine transferase-1 (ppGalNAcT1) initiates core-type protein O-glycosylation. To address whether the glycosylation of PSGL-1 by ppGalNAcT1 is important for leukocyte recruitment, we investigated leukocyte rolling and velocity in untreated and TNF-α treated cremaster muscles and autoperfused flow chambers comparing ppGalNAcT1-/-(Galnt1-/-) with WT mice. In untreated and TNF-α treated Galnt1-/- mice, leukocyte rolling was significantly reduced with markedly increased rolling velocity compared to control mice. Flow chamber experiments showed that Galnt1-/-- and WT-neutrophils had the same rolling velocity on E-selectin, but the rolling velocity of Galnt1-/--neutrophils on E-selectin/ICAM-1 was significantly elevated, suggesting that E-selectin-dependent neutrophil activation may be defective. Thioglycollate-induced peritonitis experiments with chimeric mice revealed that hematopoietic ppGalNAcT1 is important for leukocyte recruitment. These data show that the loss of ppGalNAcT1 led to reduced leukocyte rolling and recruitment and increased rolling velocity, suggesting a predominant role of ppGalNAcT1 in attaching functionally relevant O-linked glycans to PSGL-1.
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Kyriakides, Constantinos, William Austen, Yong Wang, et al. "Skeletal muscle reperfusion injury is mediated by neutrophils and the complement membrane attack complex." American Journal of Physiology-Cell Physiology 277, no. 6 (1999): C1263—C1268. http://dx.doi.org/10.1152/ajpcell.1999.277.6.c1263.
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The relative inflammatory roles of neutrophils, selectins, and terminal complement components are investigated in this study of skeletal muscle reperfusion injury. Mice underwent 2 h of hindlimb ischemia followed by 3 h of reperfusion. The role of neutrophils was defined by immunodepletion, which reduced injury by 38%, as did anti-selectin therapy with recombinant soluble P-selectin glycoprotein ligand-immunoglobulin (Ig) fusion protein. Injury in C5-deficient and soluble complement receptor type 1-treated wild-type mice was 48% less than that of untreated wild-type animals. Injury was restored in C5-deficient mice reconstituted with wild-type serum, indicating the effector role of C5–9. Neutropenic C5-deficient animals showed additive reduction in injuries (71%), which was lower than C5-deficient neutrophil-replete mice, indicating neutrophil activity without C5a. Hindlimb histological injury was worse in ischemic wild-type and C5-deficient animals reconstituted with wild-type serum. In conclusion, the membrane attack complex and neutrophils act additively to mediate skeletal muscle reperfusion injury. Neutrophil activity is independent of C5a but is dependent on selectin-mediated adhesion.
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Bestebroer, Jovanka, Kok P. M. van Kessel, Hafida Azouagh, et al. "Staphylococcal SSL5 inhibits leukocyte activation by chemokines and anaphylatoxins." Blood 113, no. 2 (2009): 328–37. http://dx.doi.org/10.1182/blood-2008-04-153882.
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Abstract Staphylococcus aureus secretes several virulence factors modulating immune responses. Staphylococcal superantigen-like (SSL) proteins are a family of 14 exotoxins with homology to superantigens, but with generally unknown function. Recently, we showed that SSL5 binds to P-selectin glycoprotein ligand 1 dependently of sialyl Lewis X and inhibits P-selectin–dependent neutrophil rolling. Here, we show that SSL5 potently and specifically inhibits leukocyte activation by anaphylatoxins and all classes of chemokines. SSL5 inhibited calcium mobilization, actin polymerization, and chemotaxis induced by chemokines and anaphylatoxins but not by other chemoattractants. Antibody competition experiments showed that SSL5 targets several chemokine and anaphylatoxin receptors. In addition, transfection studies showed that SSL5 binds glycosylated N-termini of all G protein–coupled receptors (GPCRs) but only inhibits stimuli of protein nature that require the receptor N-terminus for activation. Furthermore, SSL5 increased binding of chemokines to cells independent of chemokine receptors through their common glycosaminoglycan-binding site. Importance of glycans was shown for both GPCR and chemokine binding. Thus, SSL5 is an important immunomodulatory protein of S aureus that targets several crucial, initial stages of leukocyte extravasation. It is therefore a potential new antiinflammatory compound for diseases associated with chemoattractants and their receptors and disorders characterized by excessive recruitment of leukocytes.
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Remmler, Jill, Dorothea Zucker-Franklin, and Claire Philipp. "The Effects of MPL-Ligand, Interleukin-6 and Interleukin-11 on Megakaryocyte and Platelet Alpha-granule Proteins." Thrombosis and Haemostasis 80, no. 12 (1998): 968–75. http://dx.doi.org/10.1055/s-0037-1615397.
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SummaryThrombopoietin (Mpl ligand), interleukin-6 (IL-6), and interleukin-11 (IL-11) differ in their effects on megakaryocyte maturation and development. In the present study, the impact of these thrombocytopoietic cytokines on biochemical and structural granule and membrane components was examined. Western blotting was performed on equivalent amounts of isolated megakaryocyte or platelet protein and the relative intensities of the enhanced chemiluminescent-visualized bands were quantitated by densitometry. Megakaryocyte growth and development factor (MGDF), a recombinant thrombopoietin-related molecule, significantly increased megakaryocyte fibronectin (72%), thrombospondin (55%), von Willebrand factor (28%) and p-selectin (CD62p) (37%) when compared to equivalent amounts of protein from saline-treated controls (p < 0.01). Megakaryocyte fibrinogen was not increased. Ultrastructurally, there was a marked increase in ribosomes and rough endoplasmic reticulum even in mature-appearing megakaryocytes. Platelets from MGDF-treated mice showed small increases in fibronectin (8%), and CD62p (18%), but did not show increases in other measured α-granule proteins. Neither IL-6 nor IL-11 increased megakaryocyte or platelet α-granule proteins over levels observed in saline controls. IL-11 treated megakaryocytes, while also exhibiting an increase in ribosomes, were characterized by prominent cytoplasmic fragmentation. The study demonstrates the impact of Mpl ligand in increasing synthesized megakaryocyte α-granule proteins and of IL-11 in promoting megakaryocyte fragmentation.
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Piccardoni, Paola, Rita Sideri, Stefano Manarini, et al. "Platelet/polymorphonuclear leukocyte adhesion: a new role for SRC kinases in Mac-1 adhesive function triggered by P-selectin." Blood 98, no. 1 (2001): 108–16. http://dx.doi.org/10.1182/blood.v98.1.108.
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Abstract Adhesion of polymorphonuclear leukocytes (PMNLs) to activated platelets requires a P-selectin–triggered, tyrosine kinase–dependent adhesiveness of Mac-1 and is accompanied by tyrosine phosphorylation of a 110-kd protein (P-110) in PMNLs. Inhibitors of SRC tyrosine kinases were found to inhibit PMNL adhesion to activated platelets or to P-selectin expressing Chinese hamster ovary (CHO-P) cells and the tyrosine phosphorylation of P-110. Adhesion of PMNLs to activated platelets or to CHO-P cells stimulated activity of LYN and HCK. Monoclonal antibody blockade of P-selectin or β2-integrins reduced the activation of both kinases. In PMNLs either adherent to platelets or aggregated by P-selectin–IgG chimera, Mac-1 was rapidly redistributed to the Triton X-100–insoluble cytoskeletal fraction, and large clusters of Mac-1 colocalized with patches of F-actin at the sites of cell-cell contact. In PMNLs stimulated by P-selectin–IgG chimera, SRC kinase inhibition impaired Mac-1 clustering, F-actin accumulation, and CD18 redistribution to the cytoskeleton. Disruption of the actin filament network by cytochalasin D prevented PMNL-platelet adhesion and P-selectin–induced PMNL aggregation and impaired the clustering of Mac-1. In agreement with the requirement for the β2-integrin in the functional up-regulation of LYN and HCK, integrin blockade by monoclonal antibodies resulted in a complete inhibition of P-selectin–induced Mac-1 clustering and F-actin accumulation. Taken together, the results indicate that, after an initial P-selectin–triggered β2-integrin interaction with the ligand, SRC kinases are activated and allow the remodeling of cytoskeleton-integrin linkages and integrin clustering that finally strengthen cell-cell adhesion. This model highlights a new role for SRC kinases in a regulatory loop by which the Mac-1 promotes its own adhesive function.