Dynamic alterations of membrane tethers stabilize leukocyte rolling on P-selectin.

Leukocytes rolling on selectins extrude thin membrane tethers that might stabilize rolling velocities despite marked alterations in wall shear stress. To test this hypothesis, we used differential interference contrast videomicroscopy to visualize formation and breakage of membrane tethers as neutrophils rolled on P-selectin under flow. Neutrophils rapidly increased tether number as wall shear stress rose and decreased tether number as wall shear stress declined. Membrane tethers invariably accompanied slower, more uniform rolling steps that translated into lower mean rolling velocities and variances in velocity. Unexpectedly, neutrophils, but not fixed cells or microspheres bearing selectin ligands, rolled progressively more slowly and uniformly over time. Scanning electron microscopy revealed that neutrophils developed larger, more complex tether structures as they rolled for longer periods. These data provide evidence that neutrophils stabilize selectin-mediated rolling by rapidly adjusting tether number in response to changes in wall shear stress. Gradual remodeling of tether architecture may further reduce rolling velocities, facilitating integrin-dependent deceleration and arrest on inflamed vascular surfaces.

N-terminal residues in murine P-selectin glycoprotein ligand-1 required for binding to murine P-selectin.

P-selectin binds to the N-terminal region of human P-selectin glycoprotein ligand-1 (PSGL-1). For optimal binding, this region requires sulfation on 3 tyrosines and specific core-2 O-glycosylation on a threonine. P-selectin is also thought to bind to the N terminus of murine PSGL-1, although it has a very different amino acid sequence than human PSGL-1. Murine PSGL-1 has potential sites for sulfation at Tyr13 and Tyr15 and for O-glycosylation at Thr14 and Thr17. We expressed murine PSGL-1 or constructs with substitutions of these residues in transfected Chinese hamster ovary cells that coexpressed the glycosyltransferases required for binding to P-selectin. The cells were assayed for binding to fluid-phase P-selectin and for tethering and rolling on P-selectin under flow. In both assays, substitution of Tyr13 or Thr17 markedly diminished, but did not eliminate, binding to P-selectin. In contrast, substitution of Tyr15 or Thr14 did not affect binding. Substitution of all 4 residues eliminated binding. Treatment of cells with chlorate, an inhibitor of sulfation, markedly reduced binding of wild-type PSGL-1 to P-selectin but did not further decrease binding of PSGL-1 with substitutions of both tyrosines. These data suggest that sulfation of Tyr13 and O-glycosylation of Thr17 are necessary for murine PSGL-1 to bind optimally to P-selectin. Because it uses only one tyrosine, murine PSGL-1 may rely more on other peptide components and O-glycosylation to bind to P-selectin than does human PSGL-1.

Discordant expression of selectin ligands and sialyl Lewis x-related epitopes on murine myeloid cells.

Murine leukocytes are thought to express alpha2-3-sialylated and alpha1-3-fucosylated selectin ligands such as sialyl Lewis x (sLe(x)), although monoclonal antibodies (mAbs) to sLe(x) or Le(x) reportedly do not bind to murine leukocytes. We observed that P- and E-selectin bound to pronase-sensitive ligands on murine monocytic WEHI-3 cells and murine neutrophils, indicating that the ligands for both selectins are glycoproteins. CSLEX-1, HECA-452, and other widely used mAbs to sLe(x) and Le(x) did not bind to WEHI-3 cells and bound at very low levels to murine neutrophils. Only the anti-sLe(x) mAbs 2H5 and KM93, which also recognize nonfucosylated glycans, bound to WEHI-3 cells. 2H5 and KM93 bound to pronase-resistant structures, indicating that the mAbs did not identify selectin ligands. Treatment of WEHI-3 cells with glycosidases or chlorate demonstrated that sialic acid modifications, alpha1-3-galactosylation, or sulfation did not mask epitopes for mAbs to sLe(x) or Le(x). Compared to human promyelocytic HL-60 cells, WEHI-3 cells and murine neutrophils expressed low alpha1-3-fucosyltransferase activities. Consistent with very low endogenous fucosylation, forced fucosylation of intact WEHI-3 cells or murine neutrophils by exogenous alpha1-3-fucosyltransferase FTVI and GDP-fucose created many new epitopes for anti-sLe(x) mAbs such as HECA-452 and CSLEX-1. Nevertheless, forced fucosylation of intact cells did not significantly augment their ability to bind to fluid-phase P- or E-selectin or to roll on immobilized P- or E-selectin under flow. These data suggest that murine myeloid leukocytes fucosylate only a few specific glycans, which interact preferentially with P- and E-selectin.