Interaction between soluble P-selectin and soluble P-selectin glycoprotein ligand 1: equilibrium binding analysis

Leukocyte rolling in the vasculature is mediated by the interaction of endothelial P-selectin and leukocyte P-selectin glycoprotein ligand 1 (PSGL-1). Since cell-cell interaction mediated by P-selectin and PSGL-1 is cooperative and complex, we have developed a model system to examine the binding of P-selectin to PSGL-1 in a soluble system. Equilibrium binding analyses were performed with truncated forms of soluble human P-selectin and dimeric PSGL-1, both lacking the transmembrane domain and both produced in Chinese hamster ovary (CHO) cells. Soluble PSGL-1 (sPSGL-1), which contains no tryptophan residues and exhibits no intrinsic fluorescence, was harvested from CHO cells cotransfected with either fucosyltransferase III (sPSGL-1/Fuc-TIII) or fucosyltransferase VII (sPSGL-1/Fuc-TVII). Both fucosylation isoforms of sPSGL-1 bound to sP-selectin. The interaction of sP-selectin and sPSGL-1 was studied by monitoring changes in the intrinsic fluorescence of sP-selectin upon binding to sPSGL-1. Binding of sPSGL-1 to sP-selectin in the presence of calcium caused an increase in tryptophan fluorescence that could be reversed by the addition of ethylenediaminetetraacetic acid (EDTA). The fluorescence enhancement of sP-selectin by sPSGL-1 was used to generate binding isotherms, and these data were fitted to a bimolecular binding model. The binding constant, Kd, for the binding of sPSGL-1/Fuc-TIII and sPSGL-1/Fuc-TVII to sP-selectin was 3 +/- 2 nM and 80 +/- 44 nM, respectively. Monomeric sP-selectin bound to dimeric sPSGL-1 with a 2:1 stoichiometry. In a system in which both protein species are soluble and lack transmembrane domains, these results indicate high-affinity interaction between P-selectin and PSGL-1. Furthermore, the fucosylation pattern of PSGL-1 can affect its affinity for P-selectin. These binding constants can be used to explore models of cell adhesion in flow systems.     

P-selectin mediates adhesion of the human melanoma cell line NKI-4: identification of glycoprotein ligands

Activated endothelial cells and stimulated platelets express the cell adhesion molecule P-selectin (CD62P), which mediates adhesion to various leukocytes and certain types of cancer cells. In this study, we show Ca2+-dependent binding of P-selectin to NKI-4 cells, a cell line derived from a human melanoma. The binding is inhibited by P7 (a leukocyte adhesion blocking mAb against P-selectin), but not by PL5 (a leukocyte adhesion blocking mAb against P-selectin glycoprotein ligand-1; PSGL-1). Further, expression of PSGL-1 could not be detected on NKI-4 cells by either PL5 mAb or an Ab against a synthetic peptide corresponding to a portion of the PSGL-1 sequence. P-selectin affinity chromatography of lysates from in vivo [3H]-glucosamine-labeled NKI-4 cells resulted in the isolation of three glycoproteins, with apparent molecular masses of approximately 250, approximately 110, and approximately 100 kDa under reducing conditions and approximately 230, approximately 105, and approximately 85 kDa under nonreducing conditions. These molecules could be precipitated by P-selectin, but not by E-selectin. EDTA and the P7 mAb, but not the PL5 mAb, inhibited the binding of P-selectin to the purified ligands. Surprisingly, we found that sodium chlorate, a sulfation inhibitor, did not inhibit the binding of P-selectin to NKI-4 cells and that [35S]-sulfate did not label the NKI-4 cell ligands. We conclude that P-selectin-dependent adhesion of the human melanoma cell line NKI-4 is mediated by a novel class of glycoprotein ligands.     

Regulation of spontaneous rhythmic contractions in rat pregnant myometrium by corticotrophin-releasing factor

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

CD24 mediates rolling of breast carcinoma cells on P-selectin

P-selectin mediates rolling of neutrophils and other leukocytes on activated endothelial cells and platelets through binding to P-selectin glycoprotein ligand-1 (PSGL-1). Certain PSGL-1 negative tumor cell lines can bind P-selectin under static conditions through the GPI-linked surface mucin, CD24, but the physiological significance of this interaction and whether it can occur under flow conditions is not known. Here, we show that CD24+ PSGL-1- KS breast carcinoma cells attach to and roll on recombinant P-selectin under a continuous wall shear stress, although at a lower density and higher velocity than CD24+ PSGL-1+ cells, such as HL-60. Adding excess soluble CD24 or removing CD24 from the cell surface with phosphatidylinositol-phospholipase C (PI-PLC) significantly reduced KS cell rolling on P-selectin. The ability of KS cells to roll on P-selectin was positively correlated with the CD24 expression level. Comparison with three other CD24+ cell lines established that expression of sialyl-Lewis(x) antigen was also necessary for CD24-mediated rolling on P-selectin. CD24 purified from KS cells supported rolling of P-selectin transfectants, but not L-selectin transfectants. Finally, KS cells rolled on vascular endothelium in vivo in a P-selectin-dependent manner. Together our data show that CD24 serves as a ligand for P-selectin under physiological flow conditions. Interaction of tumor cells with P-selectin via CD24 may be an important adhesion pathway in cancer metastasis.     

Affinity and kinetic analysis of P-selectin binding to P-selectin glycoprotein ligand-1

Leukocytes use the cell-surface mucin P-selectin glycoprotein ligand-1 (PSGL-1) to tether to and roll on P-selectin on activated endothelial cells and platelets. By using surface plasmon resonance, we measured the affinity and kinetics of binding of soluble monomeric human P-selectin to immobilized PSGL-1 from human neutrophils. Binding was specific, as documented by its Ca2+-dependence, its inhibition by specific monoclonal antibodies to P-selectin and PSGL-1, and its abrogation by treating PSGL-1 with sialidase. Similar binding was observed for soluble P-selectin that contained the lectin and epidermal growth factor domains plus all nine consensus repeats, and for a soluble construct that contained only the lectin and epidermal growth factor domains. Soluble P-selectin bound saturably to a single class of sites on PSGL-1 with a dissociation constant (Kd) of 320 +/- 20 nM. The measured koff was 1.4 +/- 0.1 s-1, and the calculated kon was 4.4 x 10(6) M-1 s-1. We conclude that monomeric P-selectin binds to PSGL-1 with fast association and dissociation rates and relatively high affinity. These features may be important for efficient tethering and rolling of leukocytes at physiologic densities of PSGL-1 and P-selectin.     

L-selectin binds to P-selectin glycoprotein ligand-1 on leukocytes: interactions between the lectin, epidermal growth factor, and consensus repeat domains of the selectins determine ligand binding specificity

The selectins mediate cellular interactions by binding carbohydrate determinants present on a limited number of glycoprotein ligands. L-selectin binds multiple ligands expressed on endothelial cells, while P-selectin interacts exclusively with P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes. In this study, L-selectin was shown to bind leukocytes through the P-selectin ligand, PSGL-1, although at lower levels than P-selectin. L-selectin binding to PSGL-1 is specific since it was blocked by Abs to L-selectin or PSGL-1, required appropriate glycosylation of PSGL-1, and was Ca2+ dependent. The contributions of the extracellular domains of the selectins to ligand binding was assessed using a panel of chimeric selectins created by exchange of domains between L-selectin and P- or E-selectin. The lectin and epidermal growth factor domains of L- and P-selectin contributed significantly to binding through similar, if not identical, regions of PSGL-1. The different chimeric selectins revealed that the lectin domain was the dominant determinant for ligand binding, while cooperative interactions between the lectin, epidermal growth factor, and short consensus repeat domains of the selectins also modified ligand binding specificity. L-selectin binding to PSGL-1 expressed by leukocytes may mediate neutrophil rolling on stationary leukocytes bound to cytokine-induced endothelial cells, which was previously reported to be a L-selectin-dependent process.     

Platelet/polymorphonuclear leukocyte interaction: P-selectin triggers protein-tyrosine phosphorylation-dependent CD11b/CD18 adhesion: role of PSGL-1 as a signaling molecule

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 beta2-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 approximately 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 approximately 110. The inhibition of P approximately 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 approximately 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 beta2-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 beta2-integrin to engage with its ligands on the neighboring cells is necessary for the tyrosine phosphorylation of P approximately 110.     

The importance of L-arginine metabolism in melanoma: an hypothesis for the role of nitric oxide and polyamines in tumor angiogenesis

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.     

Human ZP3 restores fertility in Zp3 null mice without affecting order-specific sperm binding

The mammalian zona pellucida surrounding ovulated eggs mediates sperm binding at fertilization, provides a postfertilization block to polyspermy, and facilitates passage of pre-implantation embryos down the oviduct. Although the three zona proteins (ZP1, ZP2, ZP3) are well conserved, mammalian fertilization is relatively specific and human sperm do not bind to the mouse zona pellucida. There are considerable in vitro data that ZP3 acts as a primary sperm adhesion molecule in mice and, by analogy, a similar role has been postulated for human ZP3. Genetically altered mice lacking ZP3 (Zp3(tm/tm)) do not form a zona pellucida and are infertile. To rescue this phenotype, transgenic mice expressing human ZP3 (67% identical to mouse ZP3) were produced and bred with Zp3(tm/tm) null mice. The resultant human ZP3 rescue females had chimeric zonae pellucidae composed of mouse ZP1, mouse ZP2 and human ZP3. Human ZP3 expressed in mouse oocytes had an apparent mass (64 kDa) indistinguishable from native human ZP3 and distinct from mouse ZP3 (83 kDa). Despite the presence of human ZP3, human sperm did not bind to the chimeric zona pellucida, and notwithstanding the absence of mouse ZP3, mouse sperm bound to ovulated eggs in vitro and fertility was restored in vivo. These data have implications regarding the molecular basis of mouse and human sperm binding to their respective zonae pellucidae.     

Factors influencing sperm-zona pellucida binding in vitro using the intact zona model

The zona pellucida binding assay assesses the ability of spermatozoa to bind to the zona pellucida. The present study investigated the influence of: (i) prior oocyte exposure to spermatozoa on subsequent sperm-zona pellucida binding in vitro; and (ii) cryopreservation of oocytes. Only oocytes obtained from fertile donors were used and the binding capacity of non-inseminated, cryopreserved oocytes was compared with both inseminated/unfertilized, cryopreserved oocytes and inseminated/unfertilized, non-cryopreserved oocytes recovered from in-vitro fertilization cultures on sperm-zona pellucida binding using an intact zona model. There was no statistically significant difference in sperm-zona binding between non-inseminated, cryopreserved oocytes (range 9.6-23.2), inseminated/unfertilized, cryopreserved oocytes (range 15.0-16.0) and inseminated/ unfertilized, non-cryopreserved oocytes (range 3.3-23.0). The coefficient of variation for sperm binding to all oocyte groups was very large (range 37-121%). We conclude that neither prior exposure of human oocytes to human spermatozoa nor cryopreservation of human oocytes influences the subsequent binding of a different population of spermatozoa to the zona pellucida. However, large oocyte-to-oocyte variation of sperm-zona binding may diminish the usefulness of this assay in clinical practice and as a research tool.     

Tyrosylprotein sulfotransferase: purification and molecular cloning of an enzyme that catalyzes tyrosine O-sulfation, a common posttranslational modification of eukaryotic proteins

Tyrosine O-sulfation is a common posttranslational modification of proteins in all multicellular organisms. This reaction is mediated by a Golgi enzyme activity called tyrosylprotein sulfotransferase (TPST) that catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to tyrosine residues within acidic motifs of polypeptides. Tyrosine O-sulfation has been shown to be important in protein-protein interactions in several systems. For example, sulfation of tyrosine residues in the leukocyte adhesion molecule P-selectin glycoprotein ligand 1 (PSGL-1) is required for binding to P-selectin on activated endothelium. In this report we describe the purification of TPST from rat liver microsomes based on its affinity for the N-terminal 15 amino acids of PSGL-1. We have isolated human and mouse TPST cDNAs that predict type II transmembrane proteins of 370 amino acid residues with almost identical primary structure. The human cDNA encodes a fully functional N-glycosylated enzyme with an apparent molecular mass of approximately 54 kDa when expressed in mammalian cells. This enzyme defines a new class of Golgi sulfotransferases that may catalyze tyrosine O-sulfation of PSGL-1 and other protein substrates involved in diverse physiologic functions including inflammation and hemostasis.     

Isolated P-selectin glycoprotein ligand-1 dynamic adhesion to P- and E-selectin

Leukocyte adhesion to vascular endothelium under flow involves an adhesion cascade consisting of multiple receptor pairs that may function in an overlapping fashion. P-selectin glycoprotein ligand-1 (PSGL-1) and L-selectin have been implicated in neutrophil adhesion to P- and E-selectin under flow conditions. To study, in isolation, the interaction of PSGL-1 with P- and E-selectin under flow, we developed an in vitro model in which various recombinant regions of extracellular PSGL-1 were coupled to 10-microm-diameter microspheres. In a parallel plate chamber with well defined flow conditions, live time video microscopy analyses revealed that microspheres coated with PSGL-1 attached and rolled on 4-h tumor necrosis factor-alpha-activated endothelial cell monolayers, which express high levels of E-selectin, and CHO monolayers stably expressing E- or P-selectin. Further studies using CHO-E and -P monolayers demonstrate that the first 19 amino acids of PSGL-1 are sufficient for attachment and rolling on both E- and P-selectin and suggest that a sialyl Lewis x-containing glycan at Threonine-16 is critical for this sequence of amino acids to mediate attachment to E- and P-selectin. The data also demonstrate that a sulfated, anionic polypeptide segment within the amino terminus of PSGL-1 is necessary for PSGL-1-mediated attachment to P- but not to E-selectin. In addition, the results suggest that PSGL-1 has more than one binding site for E-selectin: one site located within the first 19 amino acids of PSGL-1 and one or more sites located between amino acids 19 through 148.     

The P-selectin glycoprotein ligand-1 is important for recruitment of neutrophils into inflamed mouse peritoneum

The P-selectin glycoprotein ligand-1 (PSGL-1) is a high-affinity ligand of P-selectin on myeloid cells and certain subsets of lymphoid cells. We generated the rat monoclonal antibody (MoAb) 2PH1 that recognizes an epitope within the first 19 amino acids at the N-terminus of the processed form of mouse PSGL-1. This antibody blocks attachment of mouse myeloid cells to P-selectin under both static and flow conditions. Intravenous administration of saturating amounts of 2PH1 reduced the number of rolling leukocytes in venules of the acutely exposed mouse cremaster muscle by 79% (+/-5.7%), whereas an anti-P-selectin MoAb reduced it completely. Examining the effect of the MoAb 2PH1 on the recruitment of neutrophils into chemically inflamed mouse peritoneum showed that blocking PSGL-1 inhibited neutrophil accumulation in the peritoneum by 82% (+/-7%) at 2 hours and by 59% (+/-7.9%) at 4 hours after stimulation. A similar effect was seen with the MoAb against P-selectin. Simultaneous administration of both antibodies at the 4-hour time point blocked neutrophil accumulation by 86% (+/-4.2%), arguing for an additional partner molecule for PSGL-1 besides P-selectin. This is the first demonstration of the importance of PSGL-1 in the recruitment of mouse neutrophils into inflamed tissue.     

Gamete recognition in mammals: sperm and zona pellucida interactions

Association of sperm with the acellular protective envelope of the oocyte, the zona pellucida, and their penetration is a determinant step in fertilization process. It is at this stage that species barriers take place to prevent cross fertilizations. This association is dependent upon binding of ZP3 oligosaccharides to specific sperm receptors. Their activation triggers the acrosome reaction via transduction pathways and release of proteolytic enzymes that dissociate the zona pellucida network. Then, secondary binding to zona pellucida components allows sperm to penetrate and cross the zona pellucida barrier. Several sperm surface proteins are putative receptors for zona pellucida partners. Repercussion of these studies on human fertility are discussed.     

Phosphorylation of the cytoplasmic domain of E-selectin is regulated during leukocyte-endothelial adhesion

E-selectin, a selectin expressed on activated vascular endothelium, supports rolling and stable adhesion of leukocytes at sites of inflammation. Previously, we have reported that leukocyte adhesion to cultured endothelial cells induces association of the cytoplasmic domain of E-selectin with cytoskeletal elements, suggesting that outside-in signaling may occur during E-selectin-mediated adhesion. To investigate this potential signaling function of E-selectin, HUVEC activated with recombinant human IL-1beta (10 U/ml, 4 h) were labeled with [32P]orthophosphate, and E-selectin was immunoprecipitated using mAb H18/7. Autoradiography revealed constitutive phosphorylation of E-selectin in these cells and time-dependent dephosphorylation following adhesion of HL-60 cells. Cross-linking of cell surface E-selectin using H18/7 and a polyclonal secondary Ab induced E-selectin dephosphorylation, as did adhesion of beads coated with recombinant P-selectin glycoprotein ligand-1 (PSGL-1), an E-selectin ligand. Using adenoviral vector-mediated transfection in HUVEC of a tail-less E-selectin and phosphoamino acid analysis, we documented phosphorylation occurring exclusively within the cytoplasmic domain and involving serine residues. Additional experiments using a series of cytoplasmic domain mutants of E-selectin expressed in COS-7 cells localized the regions that were constitutively phosphorylated. Preincubation with okadaic acid and sodium vanadate abrogated adhesion-induced dephosphorylation of E-selectin. Thus, E-selectin, which is constitutively phosphorylated in cytokine-activated human endothelial cells, undergoes an enzymatically regulated dephosphorylation following leukocyte adhesion. This process appears to be triggered by multivalent ligand binding and/or cross-linking of cell surface E-selectin. Ligand-dependent regulation of the phosphorylation of E-selectin's cytoplasmic domain provides additional evidence for a transmembrane signaling function of this molecule during leukocyte-endothelial interactions.     

Heterogeneity of the zona pellucida carbohydrate distribution in human oocytes failing to fertilize in vitro

The mammalian zona pellucida contains several glycoproteins whose oligosaccharide moieties are known to play a key role in the interaction with spermatozoa. Since zona pellucida defects may represent one of the most likely causes of failed fertilization in human in-vitro reproduction, we have studied the carbohydrate composition and distribution over the human zona pellucida by means of lectins. Donated, not inseminated cumulus-oocyte complexes, from cohorts with high fertilization rates, and fertilization-failed oocytes from cohorts inseminated with proven fertile donor semen, were analysed using 11 fluorescein-labelled lectins, on deplasticized semi-thin epoxy sections. Results showed that wheat germ agglutinin (WGA), Maclura pomifera (MPA) and Pisum sativum (PSA) bound to the extracellular matrix bordering the zona pellucida-corona radiata interface of cumulus-oocytes complexes, while the zona pellucida was labelled by WGA, Concanavalin A (ConA) and PSA. WGA labelling and correlative electron microscopy on the cumulus-oocyte complexes demonstrated that this lectin is a useful tool to trace the cortical granule distribution in the human oocyte. Surprisingly, in the failed-fertilized oocytes the zona pellucida was also labelled by MPA and showed three different patterns: (i) labelling of the zona pellucida outer surface; (ii) uniform labelling; (iii) labelling of an outer zona pellucida layer with variable thickness. Comparative analysis of WGA and MPA labelling on single failed-fertilized oocytes demonstrated that MPA zona pellucida patterns are not related to the cortical reaction. The nature and meaning of the MPA pattern of failed-fertilized oocytes were discussed in the light of zona pellucida defects impairing sperm receptivity.     

A novel P-selectin glycoprotein ligand-1 monoclonal antibody recognizes an epitope within the tyrosine sulfate motif of human PSGL-1 and blocks recognition of both P- and L-selectin

Interactions between P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) mediate the earliest "rolling" of leukocytes on the lumenal surface of endothelial cells at sites of inflammation. Previously, PSGL-1 has been shown to be the primary mediator of interactions between neutrophils and P-selectin, but studies on the ability of PSGL-1 to mediate interactions between P-selectin and other subsets of leukocytes have yielded variable and conflicting results. A novel IgG monoclonal antibody (MoAb) to human PSGL-1 was generated, and the specificity of this MoAb was confirmed by both flow cytometric analysis and Western blotting of cells transfected with human PSGL-1. This newly developed MoAb, KPL1, inhibited interactions between P-selectin expressing COS cells and either HL60 cells, neutrophils, or lymphocytes. Furthermore, KPL1 completely inhibited interactions between P-selectin and either purified CD4 T cells or neutrophils in a flow assay under physiological conditions, but had no effect on interactions of T cells or neutrophils with E-selectin. In addition, KPL1 blocked interactions between lymphoid cells transfected with L-selectin and COS cells expressing PSGL-1. The KPL1 epitope was mapped to a site within a consensus tyrosine sulfation motif of PSGL-1, previously shown to be essential for interaction with P-selectin and now shown to be essential for interaction with L-selectin, and to be distinct from the epitope identified by the PL1 function blocking anti-PSGL-1 MoAb. Two-color flow cytometry of normal leukocytes showed that while natural killer (NK) cells (CD16(+)), monocytes, CD4 and CD8 T cells, and alpha/beta and gamma/delta T cells were uniformly positive for PSGL-1, B cells expressed low levels of the KPL1 epitope. This low level of KPL1 staining was also observed immunohistologically in germinal centers, which had no detectable KPL1 staining, whereas T-cell areas (interfollicular region) were positive for KPL1. Interestingly, plasma cells in situ and interleukin-6-dependent myeloma cell lines were KPL1(+). Thus, PSGL-1 is expressed on essentially all blood neutrophils, NK cells, B cells, T cells, and monocytes. Variation in tyrosine sulfation during B-cell differentiation may affect the ability of B cells to interact with P- and L-selectin.     

Computer network for a diagnostic virology laboratory

We previously reported that fucoidin (a polymer of predominantly sulfated L-fucose) significantly inhibits: (1) tight binding of human sperm to human zona pellucida in vitro and (2) stimulation of the acrosome reaction by acid solubilized human zona pellucida. Here, we determined fucoidin binding activity on human spermatozoa and its localization on both live and permeabilized human sperm populations. A typical binding curve was demonstrated with biotinylated fucoidin. In competitive inhibition assays with unlabelled fucoidin or human sperm membrane extracts, IC50's were 4.0 micrograms/ml and 31.4 micrograms/ml, respectively. Fucoidin binding was localized over the acrosomal region of methanol-fixed human sperm and this pattern of binding significantly decreased from 92 +/- 3% to 74 +/- 6% with calcium ionophore A23187 treatment (p < 0.01). Binding of fucoidin-coated beads to live (non-permeabilized) human sperm was less than 1%. Addition of the detergent, Triton-X, to permeabilize sperm membranes resulted in a significant increase in binding (p = 0.001). These results provide evidence for the presence of a fucoidin binding compound in human spermatozoa that is localized to the membranes of the acrosomal region and can be extracted by a mild detergent extraction. Absence of binding by fucoidin to intact but not permeabilized spermatozoa suggests that the heteropolysaccharide binds to a receptor within the acrosomal matrix. However, further investigation is warranted to determine whether a fucoidin binding site is present both at the sperm's surface for the initial contact with the zona pellucida, and also for secondary binding after exposure of the acrosomal membranes.     

Comparison of tethering and rolling of eosinophils and neutrophils through selectins and P-selectin glycoprotein ligand-1

We compared the abilities of selectins and the selectin ligand, P-selectin glycoprotein ligand-1 (PSGL-1), to support tethering and rolling of eosinophils and neutrophils under physiologic flow conditions. Eosinophils and neutrophils accumulated on P-selectin at similar site densities and rolled at similar velocities, but fewer eosinophils than neutrophils accumulated at any P-selectin density. Compared with neutrophils, few eosinophils accumulated on E-selectin except at high densities, and those cells that did accumulate rolled faster than neutrophils. Examination of the mechanisms for accumulation revealed that eosinophils and neutrophils formed similar numbers of primary tethers to P-selectin, whereas eosinophils formed fewer primary tethers to E-selectin than did neutrophils. Compared with neutrophils, adherent eosinophils also supported fewer leukocyte-leukocyte interactions, resulting in diminished secondary tethers to either P- or E-selectin. Studies with mAbs to L-selectin and PSGL-1 demonstrated that neither cell type used L-selectin to form primary tethers to P- or E-selectin. Both eosinophils and neutrophils used the NH2 terminus of PSGL-1 to form primary tethers to P-selectin, but not to E-selectin. Both cell types used L-selectin and PSGL-1 to promote leukocyte-leukocyte interactions and secondary tethers to P- or E-selectin. However, eosinophils developed significantly fewer secondary interactions, probably because they express less L-selectin than do neutrophils.     

Induction of the human sperm acrosome reaction with mannose-containing neoglycoprotein ligands

In the interest of classifying cases of male factor infertility, we have paid particular attention to the sugar ligand binding properties of the human sperm surface and the functional capacity of the acrosome for exocytosis--key parameters for assessing sperm fertilizing ability. Zona recognition and binding involve the interactions of sperm surface mannose receptors (lectins) with mannose ligands on the zona pellucida. Sperm surface mannose lectins can be visualized by their ability to bind a synthetic model zona ligand, fluorescein isothiocyanate (FITC)-conjugated mannosylated bovine serum albumin (BSA) (Man-FITC-BSA). We now report that Man-FITC-BSA biologically also mimics the effects of solubilized authentic human zonae, in that binding of Man-FITC-BSA results in a time-dependent receptor aggregation and the induction of acrosome exocytosis in capacitated sperm populations from fertile donors. In our assay, the addition of mM amounts of mannose monosaccharide to Man-FITC-BSA increases the number of polyvalent mannose ligands bound by individual spermatozoa and increases the rate of the acrosome reactions induced by Man-FITC-BSA, thereby increasing specimen processing efficiency. We conclude that exposure of human spermatozoa to polyvalent mannose ligands + D-mannose monosaccharide offers a new, convenient and readily available system to study sperm capacity for induced acrosome loss.