Dependence of adhesive behavior of neutrophils on local ... - IOS Press

Biorheology 38 (2001) 213–227 IOS Press

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Dependence of adhesive behavior of neutrophils on local fluid dynamics in a region with recirculating flow Christopher Skilbeck a , Susan M. Westwood b , Peter G. Walker b , Tim David b and Gerard B. Nash a,∗ a Department b Department

of Physiology, The University of Birmingham, Birmingham, UK of Mechanical Engineering, University of Leeds, Leeds, UK

Received 5 February 2001 Accepted in revised form 12 March 2001 Abstract. 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 µm, downstream depth 600 µm, maximum wall shear stress ∼0.1 Pa) and shallower (upstream depth 260 µm, downstream depth 450 µm, maximum wall shear stress ∼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 depeletion 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.

1. Introduction – Rheological constraints on leukocyte adhesion from flow Adhesion of flowing leukocytes to vascular endothelium is constrained by rheological factors. Initial contact with the vessel wall is dependent on margination of leukocytes in the blood stream. The more numerous red blood cells tend to migrate towards to the centreline, especially when aggregated at low shear rate, displacing leukocytes outward [1–3]. Collisions with red cells increase diffusion of leukocytes (see [5] for review), helping repopulate blood near the wall after leukocytes have attached. * Address for correspondence: Dr. G.B. Nash, Division of Medical Sciences, The Medical School, The University of Birmingham, Birmingham B15 2TT, UK. Tel: +44 121 414 3670; Fax: +44 121 414 6919; E-mail: [email protected].

0006-355X/01/$8.00  2001 – IOS Press. All rights reserved

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C. Skilbeck et al. / Neutrophil adhesion in recirculating flow

Flow from smaller into larger vessels (e.g., capillary into venule) also favours margination, as leukocytes are retained in the laminae next to the wall [4]. Conversion of contact into initial adhesive bond formation (capture) is only likely if receptors are available which have rapid forward rate constants (e.g., selectins on endothelial cells which bind carbohydrate ligands on leukocytes) [6,7]. The rapid rate is necessary to make bond formation probable within a period limited by the velocity of the leukocyte. Selectin bonds also have rapid reverse rate constant (>1/s) [8]. This rate constant, and hence bond breakage, are accelerated by increasing fluid shear stress which effectively applies force on the formed bonds [6,8]. Depending on receptor density, the result is that leukocytes may quickly detach from the surface if no further bonds can be made immediately, or they may roll erratically over the surface as bonds are lost at the rear of the cells and formed at the front. The velocity of rolling depends on the receptor/ligand density, the local shear stress, and deformation of the cell on the surface [9–11]. Deformation tends to increase contact area (so that more bonds may form) and decrease the projection into flow (so that torque on the cell is decreased). After rolling adhesion, immobilisation of leukocytes and active migration into tissue require signal-mediated activation of integrin adhesion receptors with slower forward and reverse kinetics. The integrins are not generally rapid enough to enable capture directly, except in regions where flow is slow [12]. The above concepts are based mainly on observations in post-capillary venules of animals and in vitro experiments with purified leukocyte suspensions and laminar flow. Selectin-mediated adhesion is observed in post capillary venules where cell velocities are typically ∼mm/s, wall shear rates of ∼hundreds/s and calculated shear stresses (based on viscosity of blood) ∼1 Pa [13–16]. In vitro, capture occurs at comparable wall shear rates upto ∼400/s, but stresses only ∼0.3 Pa (based on viscosity of simple buffers) [17–19]. However, we have shown that rolling leukocytes can be observed in whole blood in vitro at shear stress up to ∼1.2 Pa [20]. Adhesion is not generally detected in arterioles, partly because of the high shear rates and stresses, but also because of differences in endothelial presentation of receptors [14,21]. Adhesion has not been quantified in large arteries because of technical limitations. We were interested in whether physical constraints on adhesion in arteries might be cirumvented in regions where flow is disturbed, such as arterial bifurcations, anastomoses of surgical reconstructions, or after angioplasty or other traumatic injury. In these conditions, adhesion molecules may be available in arteries. For instance, endothelium overlying atheroma does express P-selectin, while activated platelets deposited in damaged areas may also capture leukocytes via this receptor [22–24]. We recently reported that purified immobilised P-selectin supported adhesion of freshly-isolated neutrophils, lymphocytes and monocytes in the region downstream of a backward facing step in a flow chamber [25]. Importantly, disturbance of flow allowed capture when the wall shear stress in a straight tube would have been too high to allow adhesion. Moreover, rolling adhesion allowed leukocytes to spread to regions of high shear stress where they could not initially attach. Others have also shown that recirculating flow can promote adhesion of monocytic cells and platelets [26,27]. Here we present calculated patterns of flow velocity and shear stress downstream of steps, and compare these to measurements of free-flow velocities of cells. These, in turn, are linked to the previously reported patterns of neutrophil adhesion [25], to evaluate the critical rheological factors controlling capture. We also give details of rolling velocities of neutrophils on P-selectin, and relate these to local shear stresses and components of flow velocity parallel and perpendicular to the wall. We aimed to gain insight into the rheological parameters that influence adhesive behavior of leukocyte in different regions of the circulation.


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Fig. 1. Experimental system for examining adhesion of neutrophils perfused over a backward facing step and predicted patterns of flow. A: Photograph of vertical step flow (VSF) chamber components, based on that described by Chiu et al. [28]. Two silicone rubber gaskets (each either ∼200 or 300 µm nominal thickness) with channels cut in them, were placed one above the other on a coverslip (75 × 26 mm) coated with desired protein(s), and clamped between a lower holding plate and an upper plate with inlet and outlet ports drilled in it. Inflow was via a push-fit seal with stiff polythene tubing, to minimise bubble formation at junctions at high flow. B: Schematic of system for perfusion of neutrophils or cell-free buffer through the flow chamber, and for observation of the coverslip surface. C and D: Patterns of flow predicted for “deep” and “shallow” channels made with the different gaskets. Measured depths are shown. Experimental flow rates were 3.5 and 8.4 ml/min respectively, so that Reynolds numbers where 9 and 21, and wall shear stress downstream in re-established flow should be 0.07 and 0.3 Pa. Reattachment points exist at the wall, at the right-hand end of the vortices, where forward and backward flow seperate.

2. Recirculating flow downstream of a backward-facing step: Predicted and observed patterns of flow and particle paths 2.1. Apparatus We built a vertical-step flow chamber based upon the design of Chiu et al. [28], and linked it to a perfusion system previously described and used for adhesion studies with laminar flow [18,29]. The apparatus is illustrated in Fig. 1. Neutrophils were isolated from venous blood from healthy volunteers by density fractionation as described [18]. They were washed and resuspended at 0.5 × 106 /ml in phosphate

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buffered saline containing 1 mM Ca2+ and 0.5 mM Mg2+ and 0.1% bovine serum albumin (PBSA). Neutrophils or cell-free PBSA were perfused over the backward-facing step in the chamber using a withdrawal syringe pump (PHD 2000; Harvard Apparatus, Kent, UK) equipped with a 140 ml syringe. The chamber was maintained at 37◦ C in a perspex box, mounted on a microscope stage, so that flowing cells could be observed and video recorded. Two channels with different depths were made using different sets of gaskets (Fig. 1). Channel depths were measured using a microscope fine focus control with micrometer. The deeper channel had a depth upstream of the step of 300 µm and a downstream depth of 600 µm (a 300 µm step height). The shallower channel had an upstream depth of 260 µm, and a downstream depth of 450 µm (190 µm step height). Channel width was always 10 mm. The shallower channel was constructed to provide a higher wall shear stress for a given volume flow rate, since the volume of cell suspension available was always limited. 2.2. Predicted patterns of flow c Fluent Incorporated, We used a Computational Fluid Dynamics (CFD) analysis package (Fidap 8, USA) to model flow over the step. Fidap is a finite element based simulation package which solves the Navier–Stokes equations for motion for a fluid within complex geometries. The inputs to the CFD were the depths of the different channels upstream and downstream of the steps, and the Reynold’s numbers, with the assumption that flow was fully-developed upstream of the step. CFD was used to predict two-dimensional streamlines (i.e., ignoring effects of side walls). Velocities of flow along streamlines (components parallel, V x, and normal, V z, to the channel floor) were computed, as well as velocties at fixed distances above the floor. The wall shear stress (τw ) was also computed. In addition, paths of particles (with diameter of 8 µm and density equal to water) were predicted from chosen starting points, taking into account Stoke’s drag and buoyancy forces. As expected, CFD predicted existence of vortices in the regions where flow expanded as it passed over the backward facing steps (Fig. 1C,D). Reattachment points were defined on the wall at characteristic distances from the steps, i.e., where separation between forward and backward flowing fluid occurred. The wall shear stress was zero at these points, which were predicted to occur 250 µm from the step in the deeper (low shear) channel, and 265 µm from the step in the shallower (high shear) channel. For the chosen flow rates, wall shear stress rose to 0.07 or 0.3 Pa as simple laminar flow was re-established downstream of the reattachment points in the deeper and shallower chambers respectively (see, e.g., Figs 5 and 6 where patterns of wall shear stress and adhesion are compared). Wall shear stress was predicted to be constant by about 1500 µm from the step in either channel. Inside the recirculation zones, wall shear stresses rose (peaking at only 0.012 and 0.11Pa, respectively) and then decreased again to zero at a stagnation point up against the step. Figures 2A–D shows variation in fluid flow components V x and V z with distance downstream of the steps at a discrete set of distances above the floor. For V x, distances were chosen to cover the depth of focus when focussing on the channel floor, so that predictions should cover the range seen when actually measuring velocity of flowing neutrophils (see below). Values of V x = 0 occur at each height, at increasing but small distance upstream of the reattachment point. Inside the recirculation zone, V x is least near the reattachment point and approaching the step, where values for V z are maximal. V z is negative (towards the wall) in the former region, and positive in the latter. At a distance 5 µm from the wall (the approximate nearest approach of a non-adherent cell, based on a cell diameter ∼10 µm) V x peaks at ∼70 µm/s in the deeper chamber or ∼800 µm/s in the shallower chamber at positions approximately midway between the step and the reattachment point. V z is zero near the midpoint of the


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Fig. 2. Variation in fluid velocity components with distance downstream from the step, predicted by CFD. A, C and E refer to the deeper (low shear) channel and B, D and F refer to the shallower (high shear) channel. In A–D, families of curves are shown for the velocity of fluid at specific distances above the floor (i.e., not along streamlines). A, B: velocity parallel with the flow channel axis (V x) with flow towards the step defined as positive. Distances from the floor are 5, 10, 15, 20, 30, 40 µm. B, C: velocity normal to the channel flow (V z) with upward flow defined as positive. Distances from the floor are 5, 10, 15 µm. In E and F, V x is shown for particles with diameter of 8 µm and density equal to water, which started at different distances (5, 10, 20, 60 µm) from the floor at a point 200 µm from the step. The particles approach the wall as they travel backward toward the step, and predicted distances of closest approach to the floor are 3.8, 7.5, 14 and 24 µm in E, and 4.1, 7.5, 10 and 25 µm in F.

recirculation zone, and also far downstream of the step (i.e., re-established flow, not shown). Values for V x far downstream reach steady values of 500 or 2100 µm/s at a height 5 µm above the floor. The velocities shown in Figs 2A–D are not the same as those for particles on particular streamlines, but are useful because they indicate the velocities of cells in close proximity to or fixed heights above the wall. Figures 2E,F show velocities of particles following streamlines that approach the wall in the recirculation zone. Different streamlines will deliver cells at 5 µm from the wall at different points in the downstream half of the zone. While the streamlines can go closer to the wall as they progress, cells cannot, so that they must collide and adhere or be displaced across streamlines as they travel parallel to the wall, remaining 5 µm from the wall until the V z becomes positive and they are lifted from the wall. Practically, the result of these patterns is that cells can be seen to move into focus as they recirculate near

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the reattachment point, can be tracked and their velocity measured if they become within about 40 µm/s of the floor, and then lift off again and go out of focus (see below). Only those coming within about 5 µm of the wall can be captured, regardless of their velocity. 2.3. Experimental patterns of flow Video recordings of free-flowing neutrophils were made using a high-resolution variable shutter-speed CCD camera (PULNiX TM-765E, PULNiX, Basingstoke, UK), typically operated at a shutter speed of 4 ms to improve resolution for fast moving cells. The base of the chamber was coated with albumin so that there was negligible adhesion (80% of adherent cells rolled downstream of the reattachment point, and ∼60% rolled in the recirculation zone. Cells adhering beyond the reattachment points rolled steadily downstream. In the regions of recirculation, neutrophils rolled towards the step. Average velocities of rolling are illustrated in Fig. 7. It was notable that in the higher shear channel, cells tended to roll steadily and then lift off the surface as they approached the step. Some cells could be seen to roll right up to the step before lifting off (see Fig. 8). However, in the lower shear channel, cells tended to become stationary before reached the step, and then to slowly lift off the surface after a period. They were not seen to roll right up to the step (see Fig. 8). In Fig. 7, the average rolling velocities in the regions upstream and downstream of the reattachment points in the two chambers are plotted against the predicted wall shear stresses in those regions. Comparing the two channels, it can be seen that lower shear stress in the deeper channel is associated with lower rolling velocity in either the region of recirculation or in the laminar flow far downstream. However, the rolling velocity in the recirculation zone is faster than predicted from the values in re-established flow downstream. To examine this further, cells rolling in the recirculation regions were followed as they moved through the regions and wall shear stress varied. Velocity is shown as a function of distance from the step in Fig. 8. Velocities of rolling in either of the two chambers were much slower than axial velocities of free-flowing non-adherent cells measured in the same regions (Fig. 4), showing that they were indeed rolling adherent. On the same graphs, the local wall shear stress is shown. It seems that not only was velocity faster than expected for a given wall shear stress, but it was also insensitive to stress in the recirculation zone. One contributing factor to the may be the vertical component of fluid velocity and its lifting action. In the region near the step where wall shear stress is low, V z increases (Figs 2C,D) and

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Fig. 7. Rolling velocity (V r) of neutrophils measured in the recirculation zone (, ) or in re-established flow (∆, ) in the deeper chamber (open symbols) or in the shallower chamber (closed symbols) plotted against the local wall shear stress. Data are mean ± SEM of mean rolling velocities from 3 experiments. Lines join velocities measured in the same zones of different channels. The floors of the channels were coated with 1 µg/ml P-selectin.

Fig. 8. Variation in rolling velocities of cells (V r) on P-selectin, with distance downstream from the step in A. the deeper (low shear channel) or B. the shallower (high shear) channel. A total of 19 cells were measured at 150 different points in A, and 15 cells at 220 points in B, in 3 experiments with each channel. The variations in predicted wall shear stress across the same regions are also shown, as continuous lines. Regression analysis showed no significant variation in V r with distance from the step in either chamber, even if the analysis was done separately on either side of the mid-point where shear stress peaked.

there will be a lifting force as well as the wall shear stress. This may effectively maintain velocity by accelerating rate of bond breakage which would otherwise decrease because of reducing wall shear stress. Eventually, when the wall shear stress becomes low enough, the vertical force dominates and cells lift off rather than rolling forward. However, suprisingly, velocity is also maintained near the reattachment point where wall shear stress is also low, but V z is negative and cells are pushed onto wall. Thus, in general, rolling velocity may depend on both wall shear stress and components of flow normal to the wall which may act to lift or possibly flatten cells. In a region of recirculation, cells may continue to roll until they enter a stagnant zone where they may be held stationary by selectin bonds, even without integrin activation. Delay and build up of cells in such regions would, nevertheless, increase the likelihood of activation by locally released agents. It might also potentiate signalling through selectin receptors, which has been described when the receptors are cross-linked or occupied for prolonged periods (e.g., [38,39]). We have previously pointed out that rolling downstream of a reattachment point may cause spreading of adhesion to new areas where it cannot be initiated [25]. Figure 9 illustrates this phenomenon, showing that adhesion gradually spread during washout with cell-free medium, so that cells were found further downstream. The leading front moved ∼4 mm in 3 minutes. This is rather further than predicted from rolling velocities in Fig. 7, but the leading edge is presumably influenced by some unusually fast-rolling cells. Thus, rolling from just beyond the reattachment point allows spreading and population of regions where adhesion cannot occur directly because the shear stress is too high.


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Fig. 9. Spreading of adhesion in the shallower (high shear) chamber due to rolling. The pattern of adhesion is compared at the end of a bolus of neutrophils (closed symbols) and after 3 minutes further washout (open symbols), in a single experiment on P-selectin.

5. Conclusions We have used CFD predictions and direct visual observations of flowing cells to explain patterns of neutrophil capture described in a previous report [25], and previously unreported rolling and detachment behavior. Cells rotating in a vortex migrated gradually outward and either adhered to the wall or were released into a streamline that passed close to the wall downstream of the reattachment point where they might also attach. Thus, in low or high shear chambers there were local maxima in adhesion around the reattachment points. Within the recirculation zones, wall shear stress was never so high as to disallow adhesion. However, adhesion fell away upstream towards the step because the pattern of streamlines did not deliver cells to the wall in the half of the recirculation zone nearest the step, and also tended to lift cells which were already close to the wall. Downstream of the reattachment point, adhesion quickly dropped off as wall shear stress increased towards 0.3 Pa in the high shear channel. In the low shear chamber, there was an unexpected depression of adhesion downstream of the reattachment point, where wall shear stress was low. This can be explained by the expansion of streamlines as they pass from the narrower to the wider part of the channel, so that cells initially move away from the wall. Thus, perturbation of flow, while allowing adhesion in high shear regions where it may not otherwise occur, can also inhibit adhesion in other regions where shear stress is low. Rolling velocity in the recirculation zone was rather more rapid than expected for the local shear stresses (judged from values on the same coated surfaces further downstream) and apparently independent of shear stress in that zone. Many cells continued to roll at very low shear stresses, even though we previously showed that most rolling neutrophils became stationary when wall shear stress was ∼0.025 Pa and below [40]. A vertically upward component of flow could increase bond off rate and increase rolling velocity as cells approach the step. Rolling cells often lifted off, showing that selectins cannot necessarily hold a cell to the surface in the presence of a normal force. Bonds will break under these conditions and not be able to reform as they do when a cell rolls forward when shear is tangential to a surface. Thus, rolling behavior is sensitive to components of flow perpendicular as well as parallel to the vessel wall. Overall, patterns of adhesion are critically influenced by delivery of cells to the wall. Subsequent adhesive behavior then depends on local components of velocity parallel and perpendicular to the wall, as well as the local wall shear stress. Disturbance of flow can enable adhesion in conduits that otherwise have too high a wall shear stress, and once adhesion has been established, adhesion can spread by rolling to other regions where initial attachment is not possible (either downstream or possibly in “shadows” of steps where streamlines fail to deliver cells initally). Thus adhesion may be promoted by rheological mechanisms in regions of recirculation in large arteries where atheromatous plaque are prevalent [41]

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and capture receptors such as P-selection and VCAM-1 are presented [22,42]. Quite small irregularites in the wall of vessels at anastomoses or sites of injury may also promote adhesion just downstream.

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