Enhancement of L-selectin, but not P-selectin, bond formation frequency by convective flow

Abstract

L-selectin-mediated leukocyte rolling has been proposed to require a high rate of bond formation compared to that of P-selectin to compensate for its much higher off-rate. To test this hypothesis, a microbead system was utilized to measure relative L-selectin and P-selectin bond formation rates on their common ligand P-selectin glycoprotein ligand-1 (PSGL-1) under shear flow. Using video microscopy, we tracked selectin-coated microbeads to detect the formation frequency of adhesive tether bonds. From velocity distributions of noninteracting and interacting microbeads, we observed that tether bond formation rates for P-selectin on PSGL-1 decreased with increasing wall shear stress, from 0.14 +/- 0.04 bonds/microm at 0.2 dyn/cm(2) to 0.014 +/- 0.003 bonds/microm at 1.0 dyn/cm(2). In contrast, L-selectin tether bond formation increased from 0.017 +/- 0.005 bonds/microm at 0.2 dyn/cm(2) to 0.031 +/- 0.005 bonds/microm at 1.0 dyn/cm(2). L-selectin tether bond formation rates appeared to be enhanced by convective transport, whereas P-selectin rates were inhibited. The transition force for the L-selectin catch-slip transition of 44 pN/bond agreed well with theoretical models (Pereverzev et al. 2005. Biophys. J. 89:1446-1454). Despite catch bond behavior, hydrodymanic shear thresholding was not detected with L-selectin beads rolling on PSGL-1. We speculate that shear flow generated compressive forces may enhance L-selectin bond formation relative to that of P-selectin and that L-selectin bonds with PSGL-1 may be tuned for the compressive forces characteristic of leukocyte-leukocyte collisions during secondary capture on the blood vessel wall. This is the first report, to our knowledge, comparing L-selectin and P-selectin bond formation frequencies in shear flow.

Figure 1

Schematic diagram of L-selectin- or P-selectin-coated bead flowing over a PSGL-1 substrate. Recombinant selectin IgG molecules were adsorbed to 6 μm polystyrene microbeads at 500 ng/ml. PSGL-1 was randomly adsorbed to the floor of the flow chamber at bulk concentrations of 1, 10, or 100 ng/ml. Geometrically, microbeads translate convectively in the x-direction, parallel to the floor of the flow chamber. γ = wall shear rate (s⁻¹). Experimentally, the viscosity of the fluid is 1 cP, so that 1 dyn/cm² wall shear stress = 100 s⁻¹ wall shear rate.

Figure 2

Experimentally measured velocities of noninteracting microbeads on a 1% Tween-20-coated surface. Microbeads coated with either P-selectin or L-selectin IgG were flowed over a 1% Tween-20-coated surface to obtain a population of noninteracting velocities. The average velocity is plotted as a function of wall shear stress. Data points represent the average of at least 20 beads in each of three independent experiments (mean ± SE). Average velocities follow a linear relationship with wall shear stress (R² > 0.99). Theoretical predictions for near-wall velocities using the “method of reflections” (solid line) and asymptotic, lubrication theory (dashed line) outlined by Goldman, Cox, and Brenner (23) are shown.

Figure 3

Measurement of diffusion of microbeads. (A) Distribution of y-displacement (Fig. 1) of noninteracting microbeads in shear flow. The population of displacements is normally distributed with mean 0 μm and standard deviation 0.025 μm (p < 0.05). Displacements of exactly zero are included in the 0⁺ bin. (B) The diffusion coefficient of the microbead was calculated from the RMS displacement in the y-direction using the equation 〈y²〉 = 2Dt. Because of interpolation limits in our tracking program, 4 ms time intervals were too small to measure Brownian motion, inflating the diffusion coefficient. By expanding the time window, we were able to compute a diffusion coefficient of 0.038 μm²/s. Data shown are the result of at least 15 microbeads (mean ± SE).

Figure 4

Representative individual L-selectin bead histories interacting with PSGL-1 (A–F). Microbeads of 6 μm, coated with 500 ng/ml L-selectin IgG, were flowed over a plate coated with 10 ng/ml PSGL-1. Beads drop to velocities near zero when a bond is engaged and return to hydrodynamic velocity between bond events. Velocities were recorded at 250 fps with a 40× objective (1 pixel = 0.185 μm). Similar measurements were taken with L-selectin at 1 and 100 ng/ml and P-selectin (not shown).

Figure 5

Velocity distributions of interacting and noninteracting microbeads. Velocities of 6 μm noninteracting microbeads are plotted in a histogram (shaded). A normal (A and B) or lognormal (C–F) distribution was fit to each population. Goodness of fit was assessed using a χ² test with p < 0.05 for each wall shear stress. As an example, velocity populations from 6 μm beads coated with 500 ng/ml L-selectin interacting with 10 ng/ml PSGL-1 are shown (open). A bimodal distribution is obtained with the interacting beads. One part of the population closely resembles the noninteracting beads, and the other population lies to the left of the best-fit line (black). This subpopulation of interacting velocities falls outside of the 99% confidence interval and, thus, is a population of bond events.

Figure 6

Bond formation rates and apparent off-rate of L-selectin or P-selectin microbeads interacting with PSGL-1. L-selectin- (A and C) or P-selectin- (B and D) coated microbeads were flowed over 1 ng/ml (circles), 10 ng/ml (squares), or 100 ng/ml (diamonds) PSGL-1 over a range of wall shear stresses. Using statistically determined definitions, bond formation rates were measured on a per time (A and B) or per distance (C and D) scale. Note that the y axes are different for C and D. The bond formation rates increased for L-selectin and decreased for P-selectin over the range of wall shear stresses tested. The inset in D shows bond formation rates for P-selectin on 1 ng/ml PSGL-1 using the same scale as C. At 1 and 10 ng/ml, P-selectin had the higher bond formation rate up to 0.7 dyn/cm² wall shear stress. Above 0.7 dyn/cm², L-selectin had the higher rate. Apparent off-rates were measured (E and F) and, at each PSGL-1 site density, the apparent off-rate for L-selectin was 2–10-fold higher than P-selectin. Catch bond behavior was recorded for L-selectin at all PSGL-1 site densities, but not for P-selectin.

Figure 7

L-selectin and P-selectin bead flux measurement on PSGL-1. Microbeads (500 ng/ml L-selectin or P-selectin coated) were flowed over a 250 ng/ml PSGL-1-coated surface. The number of beads rolling in 10 fields-of-view within 1 min was counted using two different methods. A bead was considered rolling if at least five bond events took place in five cell diameters of movement. In the wash method (dashed), beads were completely clear from the floor chamber before increasing the wall shear stress. In the ramp method (solid), beads were not cleared from the flow chamber in between shear stress increases.