Dimerization of a selectin and its ligand stabilizes cell rolling and enhances tether strength in shear flow

Abstract

Selectins mediate rolling of leukocytes by rapid formation and dissociation of selectin-ligand bonds, which are assumed to require high mechanical strength to prevent premature dissociation by the forces applied in shear flow. This assumption is based largely on the observation that increasing wall shear stress increases only modestly the dissociation of transient leukocyte tethers on very low selectin densities. P-selectin binds to the N-terminal region of P-selectin glycoprotein ligand-1 (PSGL-1), a mucin on leukocytes. Both PSGL-1 and P-selectin are extended homodimers. We perfused transfected cells expressing wild-type dimeric PSGL-1 or a chimeric monomeric form of PSGL-1 on immobilized dimeric or monomeric forms of P-selectin. Cells expressing dimeric or monomeric PSGL-1 tethered to P-selectin at equivalent rates. However, cells expressing dimeric PSGL-1 established more stable rolling adhesions, which were more shear resistant and exhibited less fluctuation in rolling velocities. On low densities of dimeric P-selectin, increasing wall shear stress more rapidly increased transient tether dissociation of cells expressing monomeric PSGL-1 than dimeric PSGL-1. Tether dissociation on low densities of monomeric P-selectin was even more shear sensitive. We conclude that dimerization of both PSGL-1 and P-selectin stabilizes tethering and rolling, probably by increasing rebinding within a bond cluster. Because transient tethers may have more than one bond, the mechanical strength of selectin-ligand bonds is likely to be lower than initially estimated. Tether strength may rely more on bond clusters to distribute applied force.

Figure 1

Expression of PSGL-1 and sLe^x on transfected CHO and K562 cells. Cells were incubated with the anti-PSGL-1 mAb PL1, the anti-sLe^x mAb HECA-452, or an isotype-matched control mAb. Bound antibody was detected with FITC-conjugated goat-antimouse IgG/IgM.

Figure 2

Rolling of cells expressing wild-type or CD43TMD PSGL-1 on mP-selectin. (A and B) CHO cells or K562 cells expressing wild-type or CD43TMD PSGL-1 were perfused at the indicated wall shear stress over mP-selectin at the indicated density. After 4 min, the number of rolling cells was quantified. (C and D) CHO cells or K562 cells expressing wild-type or CD43TMD PSGL-1 were allowed to accumulate on mP-selectin at 0.5 dyne/cm². Wall shear stress was increased every 30 s, and the percentage of remaining adherent cells was determined. The data represent the mean ± SD of three to six experiments.

Figure 3

Rate of tethering of cells expressing wild-type or CD43TMD PSGL-1 on mP-selectin. CHO cells expressing wild-type or CD43TMD PSGL-1 were perfused over mP-selectin at 66 sites/μm² (A) or 31 sites/μm² (B). The number of cells that tethered to mP-selectin during the first 60 s was quantified and normalized by dividing by the number of cells delivered across the field of view in the focal plane of the substrate. The percentage of tethers that were transient or that were converted to rolling adhesion is also indicated. The data represent the mean ± SD of three experiments. At each wall shear stress, the difference in percentage of cells that converted to rolling adhesion between wild-type and CD43TMD PSGL-1 was significant at P < 0.05.

Figure 4

Rolling velocities of cells expressing wild-type or CD43TMD PSGL-1 on mP-selectin. (A and B) Frame-by-frame velocities of representative cells rolling at 1 dyn/cm². (C–F) Mean velocities and variances of velocities for cell populations rolling on mP-selectin at the indicated densities. The data were derived from three to four experiments. At each shear stress, the difference in variance of velocity between wild-type and CD43TMD PSGL-1 was significant at P < 0.05.

Figure 5

Kinetics of dissociation and mechanical strength of transient tethers of cells expressing wild-type or CD43TMD PSGL-1 on very low densities of mP-selectin or sP-selectin. (A and B) Representative pseudofirst-order dissociation kinetics for transient tethers of CHO cells expressing wild-type or CD43TMD PSGL-1 on mP-selectin at the indicated wall shear stress. (C and D) Effect of increasing wall shear stress on tether dissociation rates for transfected CHO cells or neutrophils on mP-selectin or sP-selectin. Each group of points represents a single wall shear stress from each of five independent experiments. The points were displaced horizontally so that all points could be seen. The data were fit to the Bell equation (26).