A stochastic model of leukocyte rolling.

Selectin-mediated leukocyte rolling under flow is an important process in leukocyte recruitment during inflammation. The rolling motion of individual cells has been observed to fluctuate randomly both in vivo and in vitro. This paper presents a stochastic model of the micromechanics of cell rolling and provides an analytical method of treating experimental data. For a homogeneous cell population, the velocity distribution is obtained in an analytical form, which is in good agreement with experimentally determined velocity histograms obtained previously. For a heterogeneous cell population, the model provides a simple, analytical method of separating the contributions of temporal fluctuations and population heterogeneity to the variance of measured rolling velocities. The model also links the mean and variance of rolling velocities to the molecular events underlying the observed cellular motion, allowing characterization of the distribution and release rate of the clusters of molecular bonds that tether the cell to substratum. Applying the model to the analysis of data obtained for neutrophils rolling on an E-selectin-coated surface at a wall shear stress of 1.2 dyn/cm2 yields estimations of the average distance between bond clusters (approximately micron) and the average time duration of a bond cluster resisting the applied fluid force (approximately 0.5 s).     

Rolling dynamics of a neutrophil with redistributed L-selectin

The most common white blood cell is the neutrophil, which slowly rolls along the walls of blood vessels due to the coordinated formation and breakage of chemical selectin-carbohydrate bonds. We show that L-selectin receptors are rapidly redistributed to form a cap at one end of the cell membrane during rolling via selectins or chemotactic stimulation. This topography significantly alters the adhesive dynamics as demonstrated by computer simulations of neutrophils rolling on a carbohydrate selectin-ligand substrate under flow. It was found that neutrophils with a redistributed L-selectin cap roll on sialyl Lewis-x with a quasi-periodic motion, as characterized by relatively low velocity intervals interspersed with regular jumps in the rolling velocity. On average, neutrophils with redistributed L-selectin rolled at a lower velocity when compared with cells having a uniform L-selectin distribution of equal average density. We speculate on the possible biological implications that these differences in adhesion dynamics will have during the inflammatory response.     

Simultaneous tether extraction contributes to neutrophil rolling stabilization: a model study

Neutrophil rolling is the initial step of neutrophil recruitment to sites of inflammation. During the rolling, membrane tethers are very likely extracted from both the neutrophil and the endothelial cell lining of vessel walls. Here, we present a two-dimensional neutrophil-rolling model to investigate whether and how membrane tethers contribute to stable neutrophil rolling. In our model, neutrophils are assumed to be rigid spheres covered with randomly distributed deformable microvilli, and endothelial cells are modeled as flat membrane surfaces decorated with evenly distributed ligands. The instantaneous rolling velocity and other unknowns of the model are calculated by coupling the hydrodynamic resistance functions, the geometric relationships, and the constitutive equations that govern microvillus extension and tether extraction. Our results show that glutaraldehyde-fixed neutrophils (without microvillus extension or tether extraction) roll unstably on a P-selectin-coated substrate with large variance in rolling velocity. In contrast, normal neutrophils roll much more stably, with small variance in rolling velocity. Compared with tether extraction from the neutrophil alone, simultaneous tether extraction from the neutrophil and endothelial cell greatly increases the lifetime of the adhesive bond that mediates the rolling, allows more transient tethers to make the transition into stable rolling, and enables rolling neutrophils to be more shear-resistant.     

Direct observation of membrane tethers formed during neutrophil attachment to platelets or P-selectin under physiological flow

Adhesion and subsequent aggregation between neutrophils and platelets is dependent upon the initial binding of P-selectin on activated platelets to P-selectin glycoprotein ligand 1 (PSGL-1) on the microvilli of neutrophils. High speed, high resolution videomicroscopy of flowing neutrophils interacting with spread platelets demonstrated that thin membrane tethers were pulled from neutrophils in 32 +/- 4% of the interactions. After capture by spread platelets, neutrophil membrane tethers (length of 5.9 +/- 4.1 microm, n = 63) were pulled at an average rate of 6-40 microm/s as the wall shear rate was increased from 100-250 s(-1). The average tether lifetime decreased significantly (P < 0.001) from 630 to 133 ms as the shear rate was increased from 100 s(-1) (F(bond) = 86 pN) to 250 s(-1) (F(bond) = 172 pN), which is consistent with P-selectin/PSGL-1 bond dynamics under stress. Tether formation was blocked by antibodies against P-selectin or PSGL-1, but not by anti-CD18 antibodies. During neutrophil rolling on P-selectin at 150 s(-1), thin membrane tethers were also pulled from the neutrophils. The characteristic jerking motion of the neutrophil coexisted with tether growth (8.9 +/- 8.8 microm long), whereas tether breakage (average lifetime of 3.79 +/- 3.32 s) caused an acute jump in the rolling velocity, proving multiple bonding in the cell surface and the tether surface contact area. Extremely long membrane tethers (>40 microm) were sometimes pulled, which detached in a flow-dependent mechanism of microparticle formation. Membrane tethers were also formed when neutrophils were perfused over platelet monolayers. These results are the first visualization of the often hypothesized tethers that shield the P-selectin/PSGL-1 bond from force loading to regulate neutrophil rolling during inflammation and thrombosis.     

Cell-free rolling mediated by L-selectin and sialyl Lewis(x) reveals the shear threshold effect

The selectin family of adhesion molecules mediates attachment and rolling of neutrophils to stimulated endothelial cells. This step of the inflammatory response is a prerequisite to firm attachment and extravasation. We have reported that microspheres coated with sialyl Lewis(x) (sLe(x)) interact specifically and roll over E-selectin and P-selectin substrates (Brunk et al., 1996; Rodgers et al 2000). This paper extends the use of the cell-free system to the study of the interactions between L-selectin and sLe(x) under flow. We find that sLe(x) microspheres specifically interact with and roll on L-selectin substrates. Rolling velocity increases with wall shear stress and decreases with increasing L-selectin density. Rolling velocities are fast, between 25 and 225 microm/s, typical of L-selectin interactions. The variability of rolling velocity, quantified by the variance in rolling velocity, scales linearly with rolling velocity. Rolling flux varies with both wall shear stress and L-selectin site density. At a density of L-selectin of 800 sites/microm(2), the rolling flux of sLe(x) coated microspheres goes through a clear maximum with respect to shear stress at 0.7 dyne/cm(2). This behavior, in which the maintenance and promotion of rolling interactions on selectins requires shear stress above a threshold value, is known as the shear threshold effect. We found that the magnitude of the effect is greatest at an L-selectin density of 800 sites/microm(2) and gradually diminishes with increasing L-selectin site density. Our study is the first to reveal the shear threshold effect with a cell free system and the first to show the dependence of the shear threshold effect on L-selectin site density in a reconstituted system. Our ability to recreate the shear threshold effect in a cell-free system strongly suggests the origin of the effect is in the physical chemistry of L-selectin interaction with its ligand, and largely eliminates cellular features such as deformability or topography as its cause.     

Ethanol enhances neutrophil membrane tether growth and slows rolling on P-selectin but reduces capture from flow and firm arrest on IL-1-treated endothelium

The effects of ethanol at physiological concentrations on neutrophil membrane tether pulling, adhesion lifetime, rolling, and firm arrest behavior were studied in parallel-plate flow chamber assays with adherent 1-microm-diameter P-selectin-coated beads, P-selectin-coated surfaces, or IL-1-stimulated human endothelium. Ethanol (0.3% by volume) had no effect on P-selectin glycoprotein ligand-1 (PSGL-1), L-selectin, or CD11b levels but caused PSGL-1 redistribution. Also, ethanol prevented fMLP-induced CD11b up-regulation. During neutrophil collisions with P-selectin-coated beads at venous wall shear rates of 25-100 s(-1), ethanol increased membrane tether length and membrane growth rate by 2- to 3-fold but reduced the adhesion efficiency (detectable bonding per total collisions) by 2- to 3-fold, compared with untreated neutrophils. Without ethanol treatment, adhesion efficiency and adhesion lifetime declined as wall shear rate was increased, whereas ethanol caused the adhesion lifetime over all events to increase from 0.1 s to 0.5 s as wall shear rate was increased, an example of pharmacologically induced hydrodynamic thresholding. Consistent with this increased membrane fluidity and reduced capture, ethanol reduced rolling velocity by 37% and rolling flux by 55% on P-selectin surfaces at 100 s(-1), compared with untreated neutrophils. On IL-1-stimulated endothelium, rolling velocity was unchanged by ethanol treatment, but the fraction of cells converting to firm arrest was reduced from 35% to 24% with ethanol. Overall, ethanol caused competing biophysical and biochemical effects that: 1) reduced capture due to PSGL-1 redistribution, 2) reduced rolling velocity due to increased membrane tether growth, and 3) reduced conversion to firm arrest.     

Membrane tether extraction from human umbilical vein endothelial cells and its implication in leukocyte rolling

During the rolling of human neutrophils on the endothelium, tethers (cylindrical membrane tubes) are likely extracted from the neutrophil. Tether extraction reduces the force imposed on the adhesive bond between the neutrophil and endothelium, thereby facilitating the rolling. However, whether tethers can be extracted from the endothelium is still unknown. Here, with the micropipette-aspiration technique, we show that tethers can be extracted from either suspended or attached human umbilical vein endothelial cells. We also show that a linear relationship between the pulling force and tether growth velocity exists and this relationship does not depend on the receptor type (used to impose point forces), tumor necrosis factor-alpha stimulation, or cell attachment state. With linear regression, we determined that the threshold force was 50 pN and the effective viscosity was 0.50 pN.s/microm. Therefore, tethers might be simultaneously extracted from the neutrophil and endothelial cell during the rolling and, more importantly, the endothelial cell might contribute much more to the total composite tether length than the neutrophil. Compared with tether extraction from the neutrophil alone, simultaneous tether extraction results in a larger increase in the lifetime of the adhesive bond, and thus further stabilizes the rolling of neutrophils under high physiological shear stresses.     

P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin

Neutrophils roll on P-selectin expressed by activated platelets or endothelial cells under the shear stresses in the microcirculation. P- selectin glycoprotein ligand-1 (PSGL-1) is a high affinity ligand for P- selectin on myeloid cells. However, it has not been demonstrated that PSGL-1 contributes to the rolling of neutrophils on P-selectin. We developed two IgG mAbs, PL1 and PL2, that appear to recognize protein- dependent epitopes on human PSGL-1. The mAbs bound to PSGL-1 on all leukocytes as well as on heterologous cells transfected with PSGL-1 cDNA. PL1, but not PL2, blocked binding of 125-I-PSGL-1 to immobilized P-selectin, binding of fluid-phase P-selectin to myeloid and lymphoid leukocytes, adhesion of neutrophils to immobilized P-selectin under static conditions, and rolling of neutrophils on P-selectin-expressing CHO cells under a range of shear stresses. PSGL-1 was localized to microvilli on neutrophils, a topography that may facilitate its adhesive function. These data indicate that (a) PSGL-1 accounts for the high affinity binding sites for P-selectin on leukocytes, and (b) PSGL- 1 must interact with P-selectin in order for neutrophils to roll on P- selectin at physiological shear stresses.     

Sialyl Lewis(x)/E-selectin-mediated rolling in a cell-free system.

Selections mediate transient adhesion of neutrophils to stimulated endothelial cells at sites of inflammation by binding counter-receptors that present carbohydrates such as sialyl Lewis(x). We have developed a cell-free adhesion assay using sialyl Lewis(x)-coated microspheres and E-selection-IgG chimera-coated substrates to investigate the premise that rolling primarily results from functional properties of selection-carbohydrate bonds, whereas cellular morphology and signaling act as secondary effects. Sialyl Lewis(x)-coated microspheres attach to and roll over E-selectin-IgG chimera-coated substrates between the physiological wall shear stresses of 0.7 and 2 dynes/cm2. Rolling velocities vary with time and depend on E-selectin-IgG chimera site density and wall shear stress. Our results show that sialyl Lewis(x) is a minimal functional recognition element required for rolling on E-selectin under flow.     

A 3-D computational model predicts that cell deformation affects selectin-mediated leukocyte rolling

Leukocyte recruitment to sites of inflammation is initiated by their tethering and rolling on the activated endothelium under flow. Even though the fast kinetics and high tensile strength of selectin-ligand bonds are primarily responsible for leukocyte rolling, experimental evidence suggests that cellular properties such as cell deformability and microvillus elasticity actively modulate leukocyte rolling behavior. Previous theoretical models either assumed cells as rigid spheres or were limited to two-dimensional representations of deformable cells with deterministic receptor-ligand kinetics, thereby failing to accurately predict leukocyte rolling. We therefore developed a three-dimensional computational model based on the immersed boundary method to predict receptor-mediated rolling of deformable cells in shear flow coupled to a Monte Carlo method simulating the stochastic receptor-ligand interactions. Our model predicts for the first time that the rolling of more compliant cells is relatively smoother and slower compared to cells with stiffer membranes, due to increased cell-substrate contact area. At the molecular level, we show that the average number of bonds per cell as well as per single microvillus decreases with increasing membrane stiffness. Moreover, the average bond lifetime decreases with increasing shear rate and with increasing membrane stiffness, due to higher hydrodynamic force experienced by the cell. Taken together, our model captures the effect of cellular properties on the coupling between hydrodynamic and receptor-ligand bond forces, and successfully explains the stable leukocyte rolling at a wide range of shear rates over that of rigid microspheres.     

Intracellular heterogeneity in adhesiveness of endothelium affects early steps in leukocyte adhesion

Endothelial cell junctions are thought to be preferential sites for transmigration. However, the factors that determine the site of transmigration are not well defined. Our data show that the preferential role of endothelial cell junctions is not limited to transmigration but extends to earlier steps of leukocyte recruitment, such as rolling and arrest. We used primary mouse neutrophils and mouse aortic endothelium in a flow chamber system to compare adhesive interactions near endothelial cell junctions to interactions over endothelial cell centers. We found differences in both rolling velocity and arrest frequency for neutrophils at endothelial cell junctions vs. more central areas of endothelial cells. Differences were governed by adhesion molecule interactions, not local topography. Interestingly, the role of particular adhesion molecules depended on their location on the endothelial cell surface. Although ICAM-1 stabilized and slowed rolling over central areas of the cell, it did not influence rolling velocity over endothelial cell junctions. P-selectin and VCAM-1 were more important for rolling near endothelial cell junctions than E-selectin. This demonstrates that adhesive properties of endothelial cell junctions influence early events in the adhesion cascade, which may help explain how leukocytes are localized to sites of eventual transmigration. endothelial cells; rolling; selectins; integrins     

Leukocyte rolling on P-selectin: a three-dimensional numerical study of the effect of cytoplasmic viscosity

Rolling leukocytes deform and show a large area of contact with endothelium under physiological flow conditions. We studied the effect of cytoplasmic viscosity on leukocyte rolling using our three-dimensional numerical algorithm that treats leukocyte as a compound droplet in which the core phase (nucleus) and the shell phase (cytoplasm) are viscoelastic fluids. The algorithm includes the mechanical properties of the cell cortex by cortical tension and considers leukocyte microvilli that deform viscoelastically and form viscous tethers at supercritical force. Stochastic binding kinetics describes binding of adhesion molecules. The leukocyte cytoplasmic viscosity plays a critical role in leukocyte rolling on an adhesive substrate. High-viscosity cells are characterized by high mean rolling velocities, increased temporal fluctuations in the instantaneous velocity, and a high probability for detachment from the substrate. A decrease in the rolling velocity, drag, and torque with the formation of a large, flat contact area in low-viscosity cells leads to a dramatic decrease in the bond force and stable rolling. Using values of viscosity consistent with step aspiration studies of human neutrophils (5-30 Pa·s), our computational model predicts the velocities and shape changes of rolling leukocytes as observed in vitro and in vivo.     

Influence of cell deformation on leukocyte rolling adhesion in shear flow

Blood cell interaction with vascular endothelium is important in microcirculation, where rolling adhesion of circulating leukocytes along the surface of endothelial cells is a prerequisite for leukocyte emigration under flow conditions. HL-60 cell rolling adhesion to surface-immobilized P-selectin in shear flow was investigated using a side-view flow chamber, which permitted measurements of cell deformation and cell-substrate contact length as well as cell rolling velocity. A two-dimensional model was developed based on the assumption that fluid energy input to a rolling cell was essentially distributed into two parts: cytoplasmic viscous dissipation, and energy needed to break adhesion bonds between the rolling cell and its substrate. The flow fields of extracellular fluid and intracellular cytoplasm were solved using finite element methods with a deformable cell membrane represented by an elastic ring. The adhesion energy loss was calculated based on receptor-ligand kinetics equations. It was found that, as a result of shear-flow-induced cell deformation, cell-substrate contact area under high wall shear stresses (20 dyn/cm2) could be as much as twice of that under low stresses (0.5 dyn/cm2). An increase in contact area may cause more energy dissipation to both adhesion bonds and viscous cytoplasm, whereas the fluid energy input may decrease due to the flattened cell shape. Our model predicts that leukocyte rolling velocity will reach a plateau as shear stress increases, which agrees with both in vivo and in vitro experimental observations.     

Role of cytoskeleton and deformability in laminin-mediated cell rolling

Recent mathematical models show that molecular events that mediate rolling interactions also have an impact on the stochastic features of rolling. In spherical cells, statistical fluctuations in cell displacement were shown to be an indication that only a few adhesion bonds are involved in rolling interactions. In this study, we investigated whether cell shape and cell deformability could also modulate the stochastic features of rolling. As an experimental model we considered the flow-initiated rolling of MCF-10 breast epithelial cells on laminin. The dynamic adhesion of MCF-10A cells to laminin, which involves integrin alpha 6 beta 4, occurs slow enough to allow for an accurate determination of the trajectories of rolling cells. The data from high-magnification videomicroscopy showed that cell shape, cell deformability, and the level of fluid shear stress were all strong determinants of the rolling velocity and the extent of fluctuations in the trajectory of rolling cells. MCF-10A cells with large surface projections rolled faster and wobbled more extensively than spherical cells under the same flow conditions. The extent of wobbling decreased and the variation of rolling velocity increased with increasing fluid shear stress. MCF-10A cells treated with cytochalasin B, which increased cell deformability and caused extensive blebbing without significantly altering surface expression of laminin integrins, reduced mean rolling velocity and increased its variance. Because leukocytes change shape as they roll in postcapillary blood venules at high shear rates, results indicate the need for further expanding the present biophysical models of rolling to the case of deformable cells.     

Molecular dynamics of the transition from L-selectin- to beta 2-integrin-dependent neutrophil adhesion under defined hydrodynamic shear.

Homotypic adhesion o2 neutrophils stimulated with chemoattractant is analogous to capture on vascular endothelium in that both processes depend on L-selectin and beta 2-integrin adhesion receptors. Under hydrodynamic shear, cell adhesion requires that receptors bind sufficient ligand over the duration of intercellular contact to withstand hydrodynamic stresses. Using cone-plate viscometry to apply a uniform linear shear field to suspensions of neutrophils, we conducted a detailed examination of the effect of shear rate and shear stress on the kinetics of cell aggregation. A collisional analysis based on Smoluchowski's flocculation theory was employed to fit the kinetics of aggregation with an adhesion efficiency. Adhesion efficiency increased with shear rate from approximately 20% at 100 s-1 to approximately 80% at 400 s-1. The increase in adhesion efficiency. Adhesion efficiency increased with shear rate from approximately 20% at 100 s-1 to approximately 80% at 400 s-1. The increase in adhesion efficiency with shear was dependent on L-selectin, and peak efficiency was maintained over a relatively narrow range of shear rates (400-800 s-1) and shear stresses (4-7 dyn/cm2). When L-selectin was blocked with antibody, beta 2-integrin (CD11a, b) supported adhesion at low shear rates (< 400 s-1). The binding kinetics of selectin and integrin appear to be optimized to function within discrete ranges of shear rate and stress, providing an intrinsic mechanism for the transition from neutrophil tethering to stable adhesion.     

Expression of P-selectin at low site density promotes selective attachment of eosinophils over neutrophils

The selective interaction of neutrophils with E-selectin and eosinophils with P-selectin has been previously reported, but the relevance of selectin site density and fluid shear has not been studied in detail. We have developed a new approach to examine these interactions in cell suspensions that integrates an on-line cone-plate viscometer with a flow cytometer. We find that eosinophils and neutrophils both use P-selectin glycoprotein ligand-1 to form stable conjugates with P-selectin Chinese hamster ovary cell transfectants, with a preferential adhesion of eosinophils. Further, the difference in cell adhesion between neutrophils and eosinophils is magnified at P-selectin expression levels below approximately 20 sites/microm2, a range likely to be relevant to endothelial cell expression levels in conditions associated with eosinophilia. The unique behavior is retained over shear rates ranging from 100 to 1500/s but is magnified at low shear. Results from parallel-plate flow chamber assays suggest that preferential eosinophil adhesion reflects an enhanced efficiency of initial PSGL-1 bond formation with P-selectin rather than a unique ability of eosinophils to mediate rolling interactions of longer duration on low-density P-selectin substrates. These differences may account in part for the increase in eosinophil accumulation in allergic diseases.     

Immobilized IL-8 triggers progressive activation of neutrophils rolling in vitro on P-selectin and intercellular adhesion molecule-1

The chemokine IL-8 is found on the luminal side of vascular endothelial cells, where it is postulated to be immobilized during inflammation. In this study, we observed that immobilized IL-8 can stimulate neutrophils to firmly adhere to a substrate containing ICAM-1 in a static adhesion assay. Soluble IL-8 was then perfused over neutrophils rolling on P-selectin (P-sel) and ICAM-1, confirming that IL-8 in solution can quickly cause rolling neutrophils to arrest. To mimic a blood vessel wall with IL-8 expressed on the luminal surface of endothelial cells, IL-8 was immobilized along with P-sel and ICAM-1 at defined site densities to a surface. Neutrophils rolled an average of 200 microm on surfaces of P-sel, ICAM-1, and IL-8 before firmly adhering through ICAM-1-beta(2) integrin interactions at 2 dynes/cm(2) wall shear stress. Increasing the density of IL-8 from 60 to 350 sites/microm(2) on the surface decreased by 50% the average distance and time the neutrophils rolled before becoming firmly adherent. Temporal dynamics of ICAM-1-beta(2) integrin interactions of rolling neutrophils following IL-8 exposure suggest the existence of two classes of beta(2) integrin-ICAM-1 interactions, a low avidity interaction with a 65% increase in pause times as compared with P-sel-P-sel glycoprotein ligand-1 interactions, and a high avidity interaction with pause times 400% greater than the selectin interactions. Based on the proportionality between IL-8 site density and time to arrest, it appears that neutrophils may need to sample a critical number of IL-8 molecules presented by the vessel wall before forming a sufficient number of high avidity beta(2) integrin bonds for firm adhesion.     

Neutrophil tethering on E-selectin activates beta 2 integrin binding to ICAM-1 through a mitogen-activated protein kinase signal transduction pathway

On inflamed endothelium selectins support neutrophil capture and rolling that leads to firm adhesion through the activation and binding of beta 2 integrin. The primary mechanism of cell activation involves ligation of chemotactic agonists presented on the endothelium. We have pursued a second mechanism involving signal transduction through binding of selectins while neutrophils tether in shear flow. We assessed whether neutrophil rolling on E-selectin led to cell activation and arrest via beta 2integrins. Neutrophils were introduced into a parallel plate flow chamber having as a substrate an L cell monolayer coexpressing E-selectin and ICAM-1 (E/I). At shears >/=0.1 dyne/cm2, neutrophils rolled on the E/I. A step increase to 4.0 dynes/cm2 revealed that approximately 60% of the interacting cells remained firmly adherent, as compared with approximately 10% on L cells expressing E-selectin or ICAM-1 alone. Cell arrest was dependent on application of shear and activation of Mac-1 and LFA-1 to bind ICAM-1. Firm adhesion was inhibited by blocking E-selectin, L-selectin, or PSGL-1 with Abs and by inhibitors to the mitogen-activated protein kinases. A chimeric soluble E-selectin-IgG molecule specifically bound sialylated ligands on neutrophils and activated adhesion that was also inhibited by blocking the mitogen-activated protein kinases. We conclude that neutrophils rolling on E-selectin undergo signal transduction leading to activation of cell arrest through beta 2 integrins binding to ICAM-1.     

Dependence of adhesive behavior of neutrophils on local fluid dynamics in a region with recirculating flow

We have recently described patterns of adhesion of different types of leukocytes downstream of a backward facing step. Here the predicted fluid dynamics in channels incorporating backward facing steps are described, and related to the measured velocities of flowing cells, patterns of attachment and characteristics of rolling adhesion for neutrophils perfused over P-selectin. Deeper (upstream depth 300 microm, downstream depth 600 microm, maximum wall shear stress approximately 0.1 Pa) and shallower (upstream depth 260 microm, downstream depth 450 microm, maximum wall shear stress approximately 0.3 Pa) channels were compared. Computational fluid dynamics (CFD) predicted the presence of vortices downstream of the steps, distances to reattachment of flow, local wall shear stresses and components of velocity parallel and perpendicular to the wall. Measurements of velocities of perfused neutrophils agreed well with predictions, and suggested that adhesion to P-selectin should be possible in the regions of recirculating flow, but not downstream in re-established flow in the high shear channel. When channels were coated with a P-selectin-Fc chimaera, neutrophils were captured from flow and immobilised. Capture showed local maxima around the reattachment points, but was absent elsewhere in the high shear chamber. In the low shear chamber there was depression of adhesion just beyond the reattachment point because of expansion of flow and depletion of neutrophils near the wall. Inside the recirculation zones, adhesion decreased approaching the step because of an increasing, vertically upward velocity component. When channels were coated with P-selectin, neutrophils rolled in all regions, but lifted off the surface as they rolled backwards into low shear regions near the step. Rolling velocity in the recirculation zone was independent of shear stress, possibly because of the effects of vertical lift. We conclude that while local wall shear stress influences adhesive behavior, delivery of cells to the wall and their behavior after capture also depend on components of flow perpendicular to the wall.