Regulation of L-selectin-dependent hydrodynamic shear thresholding by leukocyte deformability and shear dependent bond number.

BACKGROUND: During inflammation leukocyte attachment to the blood vessel wall is augmented by capture of near-wall flowing leukocytes by previously adherent leukocytes. Adhesive interactions between flowing and adherent leukocytes are mediated by L-selectin and P-selectin Glycoprotein Ligand-1 (PSGL-1) co-expressed on the leukocyte surface and ultimately regulated by hydrodynamic shear thresholding. OBJECTIVE: We hypothesized that leukocyte deformability is a significant contributory factor in shear thresholding and secondary capture. METHODS: Cytochalasin D (CD) was used to increase neutrophil deformability and fixation was used to reduce deformability. Neutrophil rolling on PSGL-1 coated planar surfaces and collisions with PSGL-1 coated microbeads were analyzed using high-speed videomicroscopy (250 fps). RESULTS: Increased deformability led to an increase in neutrophil rolling flux on PSGL-1 surfaces while fixation led to a decrease in rolling flux. Abrupt drops in flow below the shear threshold resulted in extended release times from the substrate for CD-treated neutrophils, suggesting increased bond number. In a cell-microbead collision assay lower flow rates were correlated with briefer adhesion lifetimes and smaller adhesive contact patches. CONCLUSIONS: Leukocyte deformation may control selectin bond number at the flow rates associated with hydrodynamic shear thresholding. Model analysis supported a requirement for both L-selectin catch-slip bond properties and multiple bond formation for shear thresholding.

L-selectin shear thresholding modulates leukocyte secondary capture.

Transient homotypic adhesions between flowing leukocytes and those previously adherent on the vessel wall has been proposed to amplify the accumulation of leukocytes at sites of inflammation. While adhesion of leukocytes to the vessel wall (primary capture) is mediated primarily by P-selectin on the endothelium and P-selectin Glycoprotein Ligand-1 (PSGL-1) on the leukocyte, the homotypic interactions leading to downstream leukocyte adhesion (secondary capture) are mediated primarily by reciprocal interactions between PSGL-1 and L-selectin on apposing leukocytes. One consequence of leukocyte secondary capture events are the formation of strings of adherent leukocytes as each recently captured leukocyte in turn captures another one flowing over its surface. Interestingly, PSGL-1-L-selectin interactions also mediate leukocyte hydrodynamic shear thresholding, whereby leukocyte rolling on purified L-selectin ligands such as PSGL-1 is maximized at a wall shear stress of approximately 1 dyne/cm(2) and minimized at both higher and lower flow rates. Using a novel quantitative method, we analyzed leukocyte string formation in vitro and found that hydrodynamic shear thresholding precluded secondary capture at low shear stresses yet amplified it at high shear stresses. Addition of the L-selectin mAb DREG-56 strongly inhibited leukocyte string formation, suggesting adhesion contributed significantly to hydrodynamic interactions in secondary capture processes. Taken together, the data suggest that secondary capture is modulated by the shear thresholding property of L-selectin. L-selectin mediated shear thresholding may therefore play a significant role in the regulation of leukocyte secondary capture in addition to recently described hydrodynamic recruitment mechanisms.

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

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.