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Influence of Receptor and Ligand Density on the Shear Threshold Effect for Carbohydrate-Coated Particles on L-Selectin Sujata K. Bhatia and Daniel A. Hammer* Departments of Bioengineering and Chemical Engineering, and Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Received March 4, 2002. In Final Form: April 30, 2002 The selectin family of cell adhesion molecules mediates capture and rolling adhesion of white blood cells to vascular walls, an essential component of the inflammatory response. Adhesion through L-selectin requires a hydrodynamic shear stress above a threshold level, a phenomenon known as the shear threshold effect. We have reported that the shear threshold effect can be recreated in cell-free systems, in which microspheres coated with the carbohydrate ligand sialyl Lewis x (sLex) are perfused over L-selectin-coated surfaces. This paper extends the use of the cell-free system to determine the concurrent influence of receptor and ligand site density on the shear threshold effect. We find that the shear threshold effect diminishes with increasing levels of either L-selectin or sLex. At reduced site densities of either L-selectin or sLex, the shear threshold effect is present, with maximal rolling observed at a shear stress of 1.2 dyn/cm2. At higher site densities of L-selectin and sLex, the shear threshold effect disappears. These results suggest that a shear threshold is required for L-selectin-mediated rolling only when low numbers of receptorligand bonds can be formed. The quantitative data here may help elucidate the physicochemical mechanism of the shear threshold effect.
Introduction Selectins are cell adhesion molecules that initiate cell recruitment to a site of inflammation, an essential component of the inflammatory response. The selectin family of cell adhesion molecules, consisting of E-, P-, and L-selectin, mediates capture and dynamic adhesion (referred to as “rolling”) of blood borne cells onto activated vascular walls.1 E- and P-selectin expressed on the vascular wall interact with carbohydrate-presenting ligands on white blood cells; L-selectin expressed on white blood cells binds carbohydrate-presenting ligands on the vessel wall.2,3 The carbohydrate sialyl-Lewisx (sLex) has been shown to bind all selectins in static and dynamic studies,4-7 and more complex selectin ligands, such as P-selectin-glycoprotein-ligand-1 (PSGL-1), bear an sLexrelated carbohydrate that is essential for function.8 Selectin-sLex binding and force-driven unbinding generates transient adhesion and rolling of cells on the vascular wall. Adhesion through L-selectin has been studied in rolling experiments using neutrophils and the L-selectin ligand CD34. L-selectin-mediated rolling of neutrophils on CD34 was found to be unique in that a threshold level of hydrodynamic shear is required for initiation of rolling interactions.9 At wall shear stresses below 0.6 dyn/cm2, rolling of neutrophils over CD34 is decreased; rolling is maximal at wall shear stresses of approximately 1.0 dyn/ * To whom correspondence should be addressed at 120 Hayden Hall, 3320 Smith Walk, Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104. E-mail: hammer@ seas.upenn.edu. (1) (2) (3) (4) (5) (6) 2000, (7) 2000, (8) (9)
Lawrence, M. B.; Springer, T. A. Cell 1991, 65, 859. Varki, A. J. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 7390. Carlos, T. M.; Harlan, J. M. Blood 1994, 84, 2068. Foxall, C.; et al. J. Cell Biol. 1992, 117, 895. Brunk, D. K.; Hammer, D. A. Biophys. J. 1997, 72, 2820. Rodgers, S. D.; Camphausen, R. T.; Hammer, D. A. Biophys. J. 79, 694. Greenberg, A. W.; Brunk, D. K.; Hammer, D. A., Biophys. J. 79, 2391. Sako, D.; et al. Cell 1995, 83, 323. Finger, E. B.; et al. Nature 1996, 379, 266.
cm2. A shear stress of 1.0 dyn/cm2 is physiologically relevant, as this is within the range of measured shear stresses in vivo of the venular microvasculature.10 The shear threshold effect has been confirmed in experiments with T lymphocytes rolling over the L-selectin ligands PNAd and CD34.11,12 It has been speculated that the shear threshold effect provides a hydrodynamic switch for regulating initial attachment or accumulation of cells at a site of inflammation. The shear threshold requirement may also help prevent inappropriate leukocyte aggregation and accumulation in vessels with inherently low wall shear stresses or in hypoperfusion states.13 To elucidate the interesting chemoviscous mechanism behind the shear threshold effect, we turned to cell-free systems. Cell-free systems are an ideal method for studying receptor-ligand interactions under flow, without confounding variables such as cell morphology or rheology. In cell-free experiments, microspheres coated with carbohydrate ligands, such as sLex or PSGL-1, are perfused over selectin-coated surfaces. We have shown that microspheres coated with sLex interact specifically and roll over E-, P-, and L-selectin under hydrodynamic flow.5-7 The dynamics of rolling in cell-free systems is similar to that observed with in vitro cellular assays. For example, beads coated with PSGL-1 peptides roll at the same velocity as neutrophils on P-selectin, if the beads have a similar surface density of PSGL-1 as neutrophils.6 We have reported that cell-free rolling mediated by L-selectin recreates the shear threshold effect,7 with maximal rolling observed at shear stresses of 0.7-1.0 dyn/cm2. The ability of cell-free systems to capture the shear threshold effect suggests that the origin of the effect is in the interaction between the hydrodynamic forces acting on the particle and the physical chemistry of selectin-ligand interactions rather than cellular features such as deformability or topography. (10) Malek, A. M.; Alper, S. L.; Izumo, S. JAMA 1999, 282, 2035. (11) Lawrence, M. B.; Kansas, G. S.; Kunkel, E. J.; Ley, K. J. Cell. Biol. 1997, 136, 717. (12) Puri, K. D.; Chen, S.; Springer, T. A. Nature 1998, 392, 930. (13) Alon, R.; Fuhlbrigge, R. C.; Finger, E. B.; Springer, T. A. J. Cell Biol. 1996, 135, 849.
10.1021/la0256937 CCC: $22.00 © 2002 American Chemical Society Published on Web 06/26/2002
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Figure 1. Site density of L-selectin chimera molecules on silanated glass slides as a function of bulk incubation concentration. Slides were incubated with up to 15.0 µg/mL human L-selectin-IgG chimera. Points represent the average of two fluorescence measurements. Error bars are standard deviations.
In this paper, we extend the use of the cell-free system to determine whether site densities of L-selectin and sLex may modulate the shear threshold effect. In these experiments, 10.9 mm diameter polystyrene latex microspheres are coated with sLex, while L-selectin-IgG chimera is adsorbed to silanated glass slides. The microspheres are perfused over the L-selectin-coated surfaces in a parallel plate flow chamber at varying wall shear stresses, and rolling flux and average rolling velocities are determined. We find that the appearance of the shear threshold effect may be controlled via either receptor or ligand density. At low site densities of either L-selectin or sLex, the effect is observed, and microsphere rolling flux over L-selectin substrates goes through a clear maximum at 1.2 dyn/cm2. The effect diminishes with increasing site densities of L-selectin and sLex. The results suggest that the shear threshold effect relies on the formation of low numbers of receptor-ligand bonds, as well as the unique features of L-selectin recognition. Materials and Methods L-Selectin Surfaces. Human L-selectin IgG chimera was obtained from R&D Systems (Minneapolis, MN). The chimera consists of the lectin, epidermal growth factor, and multiple short consensus repeat domains for human L-selectin linked to the Fc region of human IgG. L-selectin chimera was adsorbed to silanated glass microscope slides. Briefly, the desired concentration of L-selectin chimera (5.0-15.0 µg/mL) in PBS, pH 7.4, was incubated on silanated glass microscope slides (Sigma, St. Louis, MO) using modified flexiPERM wells (Sigma) for at least 2 h at room temperature with gentle mixing. Slides were then washed with PBS and incubated with blocking buffer for at least 1 h at room temperature to prevent nonspecific adhesion. Blocking buffer was PBS containing 2% BSA that was heated at 56 °C for 30 min before blocking to denature the BSA. L-selectin site density estimates were obtained using a FITClabeled monoclonal antibody to human L-selectin, DREG56 (mouse IgG1, Caltag, Burlingame, CA), and measuring the fluorescence in counts/s with a Nikon Diaphot inverted microscope (Melville, NY) equipped with a fluorescein cube and connected to a photomultiplier tube (Photoscan, Nikon). The construction of a calibration curve to convert fluorescence into site densities has been described previously.5 A graph of estimated L-selectin site density as a function of bulk human L-selectin concentration during incubation is given in Figure 1. These estimates are based on the assumption that there is 1:1 binding between the antibodies and L-selectin. Microspheres. Superavidin microspheres (10.9 mm diameter polystyrene latex) were obtained from Bangs Laboratories (Fishers, IN). Synthetic carbohydrate probes were obtained from
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Figure 2. Site density of sLex on polysterene microspheres as a function of sLex bulk percentage during incubation. Microspheres were incubated with carbohydrate solutions containing 70% biotin-sLex/30% biotin-Lex, 90% biotin-sLex/10% biotinLex, or 100% biotin-sLex at a total carbohydrate concentration of 10.0 µg/mL. Points are calculated on the basis of flow cytometry peak fluorescence; error bars are standard deviations of fluorescence measurements. GlycoTech (Rockville, MD). The carbohydrates are incorporated into a polyacrylamide matrix substituted with biotin. This creates a multivalent polymer of ∼30 kDa, with a ratio of biotin molecules to carbohydrate molecules of 1:4. The biotin-conjugated carbohydrates used in these experiments were biotin-Lewis x (biotinLex), a carbohydrate that does not interact with selectins,5 and biotin sialyl Lewis x (biotin-sLex), a carbohydrate that is recognized by all three selectins under flow. Biotin-Lex and biotinsLex have similar molecular weights. The binding properties of biotin and avidin were utilized to attach biotinylated carbohydrate to Superavidin microspheres. A 100 µL solution of 10 µg/mL carbohydrate in PBS+ was added to 106 Superavidin beads and incubated for 1 h with continuous inversion at room temperature. To vary the site densities of sLex on the particle surface, microspheres were incubated with a mixture of 70% biotin-sLex/30% biotin-Lex, 90% biotin-sLex/10% biotin-Lex, or 100% biotin-sLex, with a total carbohydrate concentration of 10 µg/mL. The site density of sLex on the microspheres was measured by flow cytometry, using a method similar to that described previously.6 Briefly, a primary monoclonal antibody to sLex (20 µg/mL, HECA-452, Pharmingen, San Diego, CA) was added to 105 carbohydrate-coated microspheres and allowed to incubate at room temperature for 45 min. After removal of the primary antibody solution, a fluorescent monoclonal secondary antibody (20 µg/mL, FITC-labeled anti-mouse IgG1, Pharmingen, San Diego, CA) was added and allowed to incubate at room temperature for 45 min before flow cytometry analysis. The sLex site density on the microspheres as a function of sLex bulk percentage during incubation is given in Figure 2. This relationship is nonlinear; microsphere coverage with sLex drops off rapidly as sLex bulk percentage is decreased. Adhesion Experiments. All experiments were conducted in a parallel plate flow chamber with a tapered channel design that allows for a linear variation of shear stress down the length of the flow channel at a single flow rate and channel height. This design is ideal for these experiments, as it allowed us to measure adhesion at many different shear stresses in a single experiment. The design is based on Hele-Shaw flow theory between parallel plates and has been previously described.14 The plates were separated by 250 µm Duralastic sheeting (Allied Biomedical, Paso Robles, CA), which compressed to 180 µm when the flow chamber was fully assembled and tightened. The selectin-coated microscope slides serve as the bottom plate of the flow chamber. During experiments, the chamber was secured to the stage of a Nikon Diaphot inverted phase contrast microscope connected to a monochrome CCD video camera (Cohu, Inc., San Diego, CA) (14) Usami, S.; Chen, H. H.; Zhao, Y.; Chien, S.; Skalak, R. Ann. Biomed. Eng. 1993, 21, 77.
Carbohydrate-Coated Particles on L-Selectin and an S-VHS videocassette recorder (model SVO-9500MD; Sony Electronics, Park Ridge, NJ). The chamber was perfused with PBS+, and carbohydrate-coated microspheres at a concentration of 5 × 105/mL in PBS+ were drawn through the flow chamber by an infusion/withdrawal syringe pump (Harvard Apparatus, South Natick, MA). Microsphere interactions with the surface were recorded for future analysis at a total magnification of 300×. All experiments were done at room temperature. Data Analysis. Velocity measurements were obtained from recorded data using National Instruments (Austin, TX) image acquisition (IMAQ) PCI-140 frame grabber board, IMAQ software, and LabVIEW 4.0. Virtual Instruments (VIs) used in LabVIEW were developed to determine rolling velocities and have been described previously.15 Briefly, VIs automatically advanced the VCR a specified number of frames, grabbed a designated number of frames, and converted each captured frame into a binary image. Particle positions were recorded as coordinates in a two-dimensional array. Another VI was then used to plot particle trajectories; instantaneous and average rolling velocities were computed on the basis of the particle trajectories. Each rolling microsphere was observed for a minimum of 3 s to obtain a trajectory. To determine the rolling flux (number of microspheres rolling/ (min/mm2)), the number of rolling microspheres in each field of view, consisting of a 0.32 mm2 area, were counted for 1 min at each shear stress. This is consistent with the definition of rolling flux used by the Springer laboratory, which first identified the shear threshold effect.9 Yet, the flux of particles is not through the area but rather over the area. However, the rolling flux is still an adequate metric of the magnitude of the extent of rolling and is related to the rolling flux through an area by a geometric factor. For a microsphere to be objectively counted as rolling, it had to move at least 50% slower than the calculated free stream velocity of a noninteracting microsphere. Free stream velocities were calculated using the theory of Goldman, Cox, and Brenner16 for a 10.9 µm diameter sphere at a particle to surface separation of 50 nm; calculated free stream velocities range from 105 µm/s at a wall shear stress of 0.4 dyn/cm2 to 664 µm/s at a wall shear stress of 2.5 dyn/cm2. At this particle-to-surface separation, it is assumed that a particle would be able to interact with and roll on the selectin-coated surface. Firm attachment was measured by counting the number of nonmoving microspheres. Nonmoving microspheres were defined as spheres remaining stationary on the surface for at least 10 s. Firm adhesion was insignificant in experiments done with human L-selectin.
Experimental Results Sialyl Lewis x-coated microspheres interact and roll over L-selectin-coated surfaces; we have shown previously that this interaction is specific.7 Data are presented as rolling velocities and rolling fluxes of microspheres. Rolling velocities are fast, between 50 and 150 µm/s, typical of the rapid binding kinetics observed for L-selectin.7 Rolling fluxes are measured as the number of particles interacting with the L-selectin surface per time per area (no./(min/ mm2)) and range from 0 to 1500 rolling particles/(min/ mm2). The rolling fluxes are determined by counting the number of rolling microspheres in a 0.32 mm2 field of view and dividing by elapsed time. The concentration of particles introduced over each surface is constant at 5 × 105 microspheres/mL (see Materials and Methods), so rolling fluxes may be reliably compared from experiment to experiment. Effect of Wall Shear Stress on Rolling Velocity. Average rolling velocities were determined at 0.7, 1.0, 1.2, 1.4, 1.7, and 2.0 dyn/cm2 for sLex-coated microspheres over surfaces to which L-selectin-IgG was adsorbed. Although we tested wall shear stresses up to 2.5 dyn/cm2, (15) Greenberg, A. W.; Kerr, W. G.; Hammer, D. A. Blood 2000, 95, 478. (16) Goldman, A. J.; Cox, R. G.; Brenner, H. Chem. Eng. Sci. 1967, 22, 637.
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Figure 3. Average rolling velocity of sLex-coated microspheres over human L-selectin as a function of wall shear stress. Rolling velocity increases with increasing wall shear stress. In these experiments, the site density of L-selectin on surfaces is 5200 molecule/µm2. The site density of sLex on microspheres is 95 molecules/µm2. Rolling velocties are the result of averaging measurements from 5 to 20 microspheres. Error bars are standard deviations.
we did not observe any microsphere interaction with the surface at shear stresses greater than 2.0 dyn/cm2. Figure 3 illustrates the dependence of average rolling velocity of sLex-coated microspheres over L-selectin-IgG substrates on wall shear stress. In these experiments, the site density of L-selectin-IgG molecule on surfaces is 5200 molecules/ µm2, and the site density of sLex on microspheres is 95 molecules/µm2. Rolling velocity increases with increasing shear stress. As wall shear stress is increased from 0.7 to 2.0 dyn/cm2, the mean rolling velocity increases 3-fold. Effect of Receptor and Ligand Site Density on Rolling Velocity. To determine the effect of L-selectin site density on rolling velocity, we examined rolling on surfaces with various L-selectin site densities (5200, 4700, and 3300 molecules/µm2) while keeping the sLex site density constant at 95 molecules/µm2. The dependence of average rolling velocity on L-selectin site density is illustrated in Figure 4A. Particle rolling velocity increases with decreasing L-selectin site density. This effect is most dramatic at higher wall shear stresses. At a wall shear stress of 1.4 dyn/cm2, the mean rolling velocity increases by 75% as the L-selectin site density is decreased from 5200 to 3300 molecules/µm2. To determine the effect of sLex site density on rolling velocity, we examined rolling of microspheres with various sLex site densities (95, 70, and 30 molecules/µm2) while keeping the L-selectin site density constant at 5200 molecules/µm2. The dependence of average rolling velocity on sLex site density is illustrated in Figure 4B. Particle rolling velocity increases with decreasing sLex site density, a similar trend to that observed for L-selectin site density. At a wall shear stress of 1.4 dyn/cm2, the mean rolling velocity increases by 50% as the sLex site density is decreased from 95 to 30 molecules/µm2. Effect of Wall Shear Stress on Rolling Flux: Shear Threshold Effect. To ascertain the presence of the shear threshold effect in the sLex/human L-selectin cell-free system, the rolling flux of sLex microspheres was determined at each wall shear stress. Reduced site densities of L-selectin (3300 molecules/µm2) and sLex (30 molecules/ µm2) were used. Figure 5 illustrates the dependence of microsphere rolling flux on wall shear stress. Rolling flux increases with increasing wall shear stress up to 1.2 dyn/ cm2 and then decreases for wall shear stresses greater
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Figure 4. (A) Average rolling velocity of sLex-coated microspheres over human L-selectin as a function of wall shear stress and L-selectin site density. Rolling velocity increases with increasing wall shear stress and decreasing L-selectin site density. In these experiments, the site density of sLex on microspheres is 95 molecules/µm2. The site density of L-selectin on surfaces varies from 3300 to 5200 molecules/µm2. (B) Average rolling velocity of sLex-coated microspheres over human Lselectin as a function of wall shear stress and sLex site density. In these experiments, the site density of L-selectin on surfaces is 5200 molecules/µm2. The site density of sLex on microspheres varies from 30 to 95 molecules/µm2. Rolling velocities are the result of averaging measurements from 5 to 20 microspheres.
than 1.2 dyn/cm2. A threshold level of shear is thus required for optimal rolling interactions in this system. Effect of Receptor and Ligand Site Density on the Shear Threshold Effect. Both receptor and ligand site densities were systematically varied to determine the influence of receptor and ligand site density on the shear threshold effect. The results of these experiments are given in Figure 6. Overall rolling flux decreases with decreasing L-selectin site density; overall rolling flux also decreases with decreasing sLex site density. The appearance of the shear threshold effect is dependent on ligand density and receptor site density. At reduced L-selectin site density (3300 molecules/µm2), the shear threshold effect is present at all sLex site densities tested (95, 70, and 30 molecules/ µm2). At an L-selectin site density of 4700 molecules/µm2, the shear threshold effect is not present when sLex site density is 95 molecules/µm2. When the sLex site density is lowered to 70 molecules/µm2 under these conditions,
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Figure 5. (A) Rolling flux of sLex-coated microspheres over human L-selectin as a function of wall shear stress. Rolling flux is maximal at a shear stress of 1.2 dyn/cm2, demostrating the shear threshold effect. (B) Image consisting of 10 superimposed video frames (0.07 s apart) to show fast-moving nonadherent microspheres at a shear stress of 2.0 dyn/cm2. (C) When shear stress is lowered to 1.2 dyn/cm2, rolling is observed, with six rolling microspheres in the image shown. (D) When shear stress is lowered further to 1.0 dyn/cm2, microspheres begin to detach from the surface, with zero rolling microspheres in the image shown. In these experiments, the site density of L-selectin on surfaces is 5200 molecules/µm2. The site density of sLex on microspheres is 30 molecules/µm2. Rolling fluxes are the result of averaging three measurements. Error bars are standard deviations.
the shear threshold effect is observed. At an L-selectin site density of 5200 molecules/µm2, the shear threshold effect is not evident at sLex site densities of 95 or 70 molecules/µm2; when the sLex site density is lowered to 30 molecules/µm2, the shear threshold effect appears. Taken together, these results suggest that the shear threshold effect diminishes with increasing receptor or ligand site density. Discussion Using the cell-free system, we have shown that the rolling behavior of sLex-coated microspheres over Lselectin surfaces may be tuned via receptor and ligand site densities. This study goes significantly beyond the previous demonstration of the shear threshold effect
Carbohydrate-Coated Particles on L-Selectin
Figure 6. Rolling flux of sLex-coated microspheres over human L-selectin as a function of wall shear stress, receptor site density, and ligand site density. (A) At an L-selectin site density of 3300 molecules/µm2, the shear threshold effect is evident at all sLex site densities. Maximal rolling flux is observed at 1.2 dyn/cm2. Overall rolling flux decreases with decreasing sLex site density. (B) At an L-selectin site density of 4700 molecules/µm2, the shear threshold effect is evident at sLex site densities e 70 molecules/µm2. (C) At an L-selectin site density of 5200 molecules/µm2, the shear threshold effect is evident only at an sLex site density of 30 molecules/µm2.
published by our laboratory, as it explored the role of sLex density in the dynamics of rolling.7 Rolling velocity of microspheres increases with decreasing receptor site density and increases with decreasing ligand site density. Rolling exhibits a shear threshold effect, with maximum rolling interactions at a wall shear stress of 1.2 dyn/cm2. When the shear threshold is observed, the location of the threshold does not change with receptor or ligand site
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density; maximal rolling is always observed at a shear stress of 1.2 dyn/cm2, despite changes in molecular site densities. The shear threshold effect is only evident when either receptor or ligand site density is low. At a reduced L-selectin site density of 3300 molecules/µm2, the shear threshold effect is present regardless of sLex site density. At a reduced sLex site density of 30 molecules/µm2, the shear threshold effect is present regardless of L-selectin site density. However, when both receptor and ligand site densities are increased, the shear threshold effect disappears. The ability to reproduce the shear threshold effect in a cell-free system proves that the effect is due to the physical chemistry of receptor-ligand binding, perhaps modulated by viscous interactions. A shear threshold is observed for L-selectin-mediated rolling only when low numbers of receptor-ligand bonds can be formed. We propose a mechanism to explain the shear threshold effect and its dependence on molecular binding; the mechanism originates in a recent study from our laboratory,17 which models the overall reaction rate of species bound to surfaces under relative motion. The study shows that the rate of collision between receptor and ligand increases with shear rate, and the encounter duration decreases. Depending on the rate of bimolecular reaction, increases in shear rate can increase adhesion (by increasing the encounter frequency) or decrease adhesion (by decreasing the encounter time). The shear threshold may occur at shear rates at which these effects are counterbalanced. This suggested mechanism also explains why the shear threshold effect disappears when both L-selectin and sLex site densities are high. At very high site densities of receptor and ligand, the collision rate is very high, even at low shear stresses, resulting in an increased probability of bond formation. We plan to further investigate the mechanism of the shear threshold effect through adhesive dynamics simulations.18 The shear threshold effect is a potential physiologic mechanism for the regulation of leukocyte attachment and the prevention of inappropriate leukocyte accumulation.12 The dependence of the shear threshold effect on receptor and ligand density may be of physiologic significance, as it suggests that the effect can be modulated in vivo to regulate cell attachment and accumulation at a site of inflammation. Neutrophils rapidly shed L-selectin from the cell surface upon stimulation with chemokines;19 chemical inhibition of L-selectin shedding results in increased neutrophil accumulation and arrest on endothelium.20 L-selectin shedding may thus represent a natural mechanism for controlling the appearance of the shear threshold effect and, consequently, the recruitment of cells to inflammatory sites. This paper further supports the use of cell-free systems to mimic and elucidate cell phenomena systematically. Our future plans involve exploration of chemical alterations or mutations in selectins and sLex, to determine the chemical and structural dependence of the shear threshold effect. Acknowledgment. The authors are grateful to NIH Grants GM59100 and HL18208, and the Whitaker Foundation for support. LA0256937 (17) Chang, K.-C.; Hammer, D. A. Biophys. J. 1999, 76, 1280. (18) Hammer, D. A.; Apte, S. M. Biophys. J. 1992, 63, 35. (19) Kishimoto, T. K.; Jutila, M. A.; Berg, E. L.; Butcher, E. C. Science 1989, 245, 1238. (20) Hafezi-Moghadam, A.; Thomas, K. L.; Prorock, A. J.; Huo, Y.; Ley, K. J. Exp. Med. 2001, 193, 863.
