Kinetics of adhesion of IgE-sensitized rat basophilic leukemia cells to surface-immobilized antigen in Couette flow.

Antigen-antibody systems provide the flexibility of varying the kinetics and affinity of molecular interaction and studying the resulting effect on adhesion. In a parallel-plate flow chamber, we measured the extent and rate of adhesion of rat basophilic leukemia cells preincubated with anti-dinitrophenyl IgE clones SPE-7 or H1 26. 82 to dinitrophenyl-coated polyacrylamide gel substrates in a linear shear field. Both of these IgEs bind dinitrophenyl, but H1 26.82 has a 10-fold greater on rate and a 30-fold greater affinity. Adhesion was found to be binary; cells either arrested irreversibly or continued at their unencumbered hydrodynamic velocity. Under identical conditions, more adhesion was seen with the higher affinity (higher on rate) IgE clone. At some shear rates, adhesion was robust with H1 26.82, but negligible with SPE-7. Reduction in receptor number or ligand density reduced the maximum level of adhesion seen at any shear rate, but did not decrease the shear rate at which adhesion was first observed. The spatial pattern of adhesion for both IgE clones is well represented by the first-order kinetic rate constant kad, and we have determined how kad depends on ligand and receptor densities and shear rate. The rate constant kad found with H1 26.82 was approximately fivefold greater than with SPE-7. The dependence of kad on site density and shear rate for SPE-7 is complex: kad increases linearly with antigen site density at low to moderate shear rates, but is insensitive to site density at high shear. kad increases with shear rate at low site density but decreases with shear at high site density. With H1 26.82, the functional dependence of kad with shear rate was similar. Although these data are consistent with the hypothesis that we have sampled both transport and reaction-limited adhesion regimes, they point out deficiencies in current theories describing cell attachment under flow.     

Simulation of cell rolling and adhesion on surfaces in shear flow: general results and analysis of selectin-mediated neutrophil adhesion.

The receptor-mediated adhesion of cells to ligand-coated surfaces in viscous shear flow is an important step in many physiological processes, such as the neutrophil-mediated inflammatory response, lymphocyte homing, and tumor cell metastasis. This paper describes a calculational method which simulates the interaction of a single cell with a ligand-coated surface under flow. The cell is idealized as a microvilli-coated hard sphere covered with adhesive springs. The distribution of microvilli on the cell surface, the distribution of receptors on microvilli tips, and the forward and reverse reaction between receptor and ligand are all simulated using random number sampling of appropriate probability functions. The velocity of the cell at each time step in the simulation results from a balance of hydrodynamic, colloidal and bonding forces; the bonding force is derived by summing the individual contributions of each receptor-ligand tether. The model can simulate the effect of many parameters on adhesion, such as the number of receptors on microvilli tips, the density of ligand, the rates of reaction between receptor and ligand, the stiffness of the resulting receptor-ligand springs, the response of springs to strain, and the magnitude of the bulk hydrodynamic stresses. The model can successfully recreate the entire range of expected and observed adhesive phenomena, from completely unencumbered motion, to rolling, to transient attachment, to firm adhesion. Also, the method can generate meaningful statistical measures of adhesion, including the mean and variance in velocity, rate constants for cell attachment and detachment, and the frequency of adhesion. We find a critical modulating parameter of adhesion is the fractional spring slippage, which relates the strain of a bond to its rate of breakage; the higher the slippage, the faster the breakage for the same strain. Our analysis of neutrophil adhesive behavior on selectin-coated (CD62-coated) surfaces in viscous shear flow reported by Lawrence and Springer (Lawrence, M.B., and T.A. Springer 1991. Cell. 65:859-874) shows the fractional spring slippage of the CD62-LECAM-1 bond is likely below 0.01. We conclude the unique ability of this selectin bond to cause neutrophil rolling under flow is a result of its unique response to strain. Furthermore, our model can successfully recreate data on neutrophil rolling as function of CD62 surface density.     

Receptor-mediated binding of IgE-sensitized rat basophilic leukemia cells to antigen-coated substrates under hydrodynamic flow.

The physiological function of many cells is dependent on their ability to adhere via receptors to ligand-coated surfaces under fluid flow. We have developed a model experimental system to measure cell adhesion as a function of cell and surface chemistry and fluid flow. Using a parallel-plate flow chamber, we measured the binding of rat basophilic leukemia cells preincubated with anti-dinitrophenol IgE antibody to polyacrylamide gels covalently derivatized with 2,4-dinitrophenol. The rat basophilic leukemia cells' binding behavior is binary: cells are either adherent or continue to travel at their hydrodynamic velocity, and the transition between these two states is abrupt. The spatial location of adherent cells shows cells can adhere many cell diameters down the length of the gel, suggesting that adhesion is a probabilistic process. The majority of experiments were performed in the excess ligand limit in which adhesion depends strongly on the number of receptors but weakly on ligand density. Only 5-fold changes in IgE surface density or in shear rate were necessary to change adhesion from complete to indistinguishable from negative control. Adhesion showed a hyperbolic dependence on shear rate. By performing experiments with two IgE-antigen configurations in which the kinetic rates of receptor-ligand binding are different, we demonstrate that the forward rate of reaction of the receptor-ligand pair is more important than its thermodynamic affinity in the regulation of binding under hydrodynamic flow. In fact, adhesion increases with increasing receptor-ligand reaction rate or decreasing shear rate, and scales with a single dimensionless parameter which compares the relative rates of reaction to fluid shear.     

A dynamical model for receptor-mediated cell adhesion to surfaces in viscous shear flow

We present a dynamical model for receptor-mediated cell adhesion to surfaces in viscous shear flow when the surfaces are coated with ligand molecules complementary to receptors in the cell membrane. This model considers the contact area between the cell and the surface to be a small, homogeneous region that mediates the initial attachment of the cell to the surface. Using a phase plane analysis for a system of nonlinear ordinary differential equations that govern the changes in free receptor density and bond density within the contact area with time, we can predict the conditions for which adhesion between the cell and the surface will take place. Whether adhesion occurs depends on values of dimensionless quantities that characterize the interaction of the cell and its receptors with the surface and its ligand, such as the bond formation rate, the receptor-ligand affinity, the fluid mechanical force, the receptor mobility, and the contact area. A key result is that there are two regimes in which different chemical and physical forces dominate: a rate-controlled high affinity regime and an affinity-controlled low affinity regime. Many experimental observations, including the effects of temperature and receptor mobility on adhesiveness, can be explained by understanding which of these regimes is appropriate. We also provide simple approximate analytical solutions, relating adhesiveness to cell and surface properties as well as fluid forces, which allow convenient testing of model predictions by experiment.     

Glycoprotein (GP) Ib-IX-transfected cells roll on a von Willebrand factor matrix under flow. Importance of the GPib/actin-binding protein (ABP-280) interaction in maintaining adhesion under high shear

Adhesion of platelets to sites of vascular injury is critical for hemostasis and thrombosis and is dependent on the binding of the vascular adhesive protein von Willebrand factor (vWf) to the glycoprotein (GP) Ib-V-IX complex on the platelet surface. A unique but poorly defined characteristic of this receptor/ligand interaction is its ability to support platelet adhesion under conditions of high shear stress. To examine the structural domains of the GPIb-V-IX complex involved in mediating cell adhesion under flow, we have expressed partial (GPIb-IX), complete (GPIb-V-IX), and mutant (GPIbalpha cytoplasmic tail mutants) receptor complexes on the surface of Chinese hamster ovary (CHO) cells and examined their ability to adhere to a vWf matrix in flow-based adhesion assays. Our studies demonstrate that the partial receptor complex (GPIb-IX) supports CHO cell tethering and rolling on a bovine or human vWf matrix under flow. The adhesion was specifically inhibited by an anti-GPIbalpha blocking antibody (AK2) and was not observed with CHO cells expressing GPIbbeta and GPIX alone. The velocity of rolling was dependent on the level of shear stress, receptor density, and matrix concentration and was not altered by the presence of GPV. In contrast to selectins, which mediate cell rolling under conditions of low shear (20-200 s-1), GPIb-IX was able to support cell rolling at both venous (150 s-1) and arterial (1500-10,500 s-1) shear rates. Studies with a mutant GPIbalpha receptor subunit lacking the binding domain for actin-binding protein demonstrated that the association of the receptor complex with the membrane skeleton is not essential for cell tethering or rolling under low shear conditions, but is critical for maintaining adhesion at high shear rates (3000-6000 s-1). These studies demonstrate that the GPIb-IX complex is sufficient to mediate cell rolling on a vWf matrix at both venous and arterial levels of shear independent of other platelet adhesion receptors. Furthermore, our results suggest that the association between GPIbalpha and actin-binding protein plays an important role in enabling cells to remain tethered to a vWf matrix under conditions of high shear stress.     

Receptor-mediated cell attachment and detachment kinetics. II. Experimental model studies with the radial-flow detachment assay.

Quantitative information regarding the kinetics of receptor-mediated cell adhesion to a ligand-coated surface are crucial for understanding the role of certain key parameters in many physiological and biotechnology-related processes. Here, we use the probabilistic attachment and detachment models developed in the preceding paper to interpret transient data from well-defined experiments. These data are obtained with a simple model cell system that consists of receptor-coated latex beads (prototype cells) and a Radial-Flow Detachment Assay (RFDA) using a ligand-coated glass disc. The receptors and ligands used in this work are complementary antibodies. The beads enable us to examine transient behavior with particles that possess fairly uniform properties that can be varied systematically, and the RFDA is designed for direct observation of adhesion to the ligand-coated glass surface over a range of shear stresses. Our experiments focus on the effects of surface shear stress, receptor density, and ligand density. These data provide a crucial test of the probabilistic framework. We show that these data can be explained with the probabilistic analyses, whereas they cannot be readily interpreted on the basis of a deterministic analysis. In addition, we examine transient data on cell adhesion reported from other assays, demonstrating the consistency of these data with the predictions of the probabilistic models.     

A dynamical model for receptor-mediated cell adhesion to surfaces.

We present a dynamical model for receptor-mediated adhesion of cells in a shear field of viscous fluid to surfaces coated with ligand molecules complementary to receptors in the cell membrane. We refer to this model as the "point attachment model" because it considers the contact area between the cell and the surface to be a small, homogeneous region that mediates the initial attachment of the cell to the surface. Using a phase plane analysis of a system of nonlinear ordinary differential equations which govern the changes in free receptor density and bond density within the contact area with time, we can predict the conditions for which adhesion between the cell and the surface will take place. Whether adhesion occurs depends on values of dimensionless quantities that characterize the interaction of the cell and its receptors with the surface and its ligand, such as the bond formation rate, the receptor-ligand affinity, the fluid mechanical force, the receptor mobility, and the contact area. A key result is that there are two regimes in which different chemical and physical forces dominate: a rate-controlled high affinity regime and an affinity-controlled low-affinity regime. Many experimental observations can be explained by understanding which of these regimes is appropriate. We also provide simple approximate analytical solutions, relating adhesiveness to cell and surface properties as well as fluid forces, which allow convenient testing of model predictions by experiment.     

L-selectin-mediated lymphocyte-cancer cell interactions under low fluid shear conditions

Cell migration in blood flow is mediated by engagement of specialized adhesion molecules that function under hemodynamic shear conditions, and many of the effectors of these adhesive interactions, such as the selectins and their ligands, are well defined. However, in contrast, our knowledge of the adhesion molecules operant under lymphatic flow conditions is incomplete. Among human malignancies, head and neck squamous cell cancer displays a marked predilection for locoregional lymph node metastasis. Based on this distinct tropism, we hypothesized that these cells express adhesion molecules that promote their binding to lymphoid tissue under lymphatic fluid shear stress. Accordingly, we investigated adhesive interactions between these and other cancer cells and the principal resident cells of lymphoid organs, lymphocytes. Parallel plate flow chamber studies under defined shear conditions, together with biochemical analyses, showed that human head and neck squamous cell cancer cells express heretofore unrecognized L-selectin ligand(s) that mediate binding to lymphocyte L-selectin at conspicuously low shear stress levels of 0.07-0.08 dynes/cm(2), consistent with lymphatic flow. The binding of head and neck squamous cancer cells to L-selectin displays canonical biochemical features, such as requirements for sialylation, sulfation, and N-glycosylation, but displays a novel operational shear threshold differing from all other L-selectin ligands, including those expressed on colon cancer and leukemic cells (e.g. HCELL). These data define a novel class of L-selectin ligands and expand the scope of function for L-selectin within circulatory systems to now include a novel activity within shear stresses characteristic of lymphatic flow.     

Flow-conditioned HUVECs support clustered leukocyte adhesion by coexpressing ICAM-1 and E-selectin

Endothelial sequestration of circulating monocytes is a key event in early atherosclerosis. Hemodynamics is proposed to regulate monocyte-endothelial cell interactions by direct cell activation and establishment of flow environments that are conducive or prohibitive to cell-cell interaction. We investigated fluid shear regulation of monocyte-endothelial cell adhesion in vitro using a disturbed laminar shear system that models in vivo hemodynamics characteristic of lesion-prone vascular regions. Human endothelial cell monolayers were flow conditioned for 6 h before evaluation of monocyte adhesion under static and dynamic flow conditions. Results revealed a distinctive clustered cell pattern of monocyte adhesion that strongly resembles in vivo leukocyte adhesion in early- and late-stage atherosclerosis. Clustered monocyte cell adhesion correlated with endothelial cells coexpressing intercellular adhesion molecule-1 (ICAM-1) and E-selectin as result of a flow-induced, selective upregulation of E-selectin expression in a subset of ICAM-1-expressing cells. Clustered monocyte cell adhesion assayed under static conditions exhibited a spatial variation in size and frequency of occurrence, which demonstrates differential regulation of endothelial cell adhesiveness by the local flow environment. Dynamic adhesion studies conducted with circulating monocytes resulted in clustered cell adhesion only within the disturbed flow region, where the monocyte rate of motion is sufficiently low for cell-cell interaction. These studies provide evidence and reveal mechanisms of local hemodynamic regulation of endothelial adhesiveness and endothelial monocyte interaction that lead to localized monocyte adhesion and potentially contribute to the focal origin of arterial diseases such as atherosclerosis.     

Simulation of cell rolling and adhesion on surfaces in shear flow

The adhesion of cells to ligand-coated surfaces in viscous shear flow is an important step in many physiological processes, such as the neutrophil-mediated inflammatory response, lymphocyte homing, and tumor cell metastasis. This article describes a calculational method that allows simulation of the interaction of a single cell with a ligandcoated surface. The cell is idealized as a microvilli-coated hard sphere covered with adhesive springs. The distribution of microvilli on the cell surface, the distribution of receptors on microvilli tips, and the forward and reverse reaction between receptor and ligand are all simulated using random number sampling of appropriate probability functions. The velocity of the cell at each time step in the simulation results from a balance of hydrodynamic, colloidal, and bonding forces; the bonding force is derived by summing the individual contributions of each receptor-ligand tether. The model can simulate the effect of many parameters on adhesion, such as the number of receptors on microvilli tips, the density of ligand, the rates of reaction between receptor and ligand, the stiffness of the springs, the response of springs to extension, and the magnitude of hydrodynamic stresses. By varying these parameters, the model can successfully recreate the entire range of expected and observed adhesive phenomena, from completely unencumbered motion, to rolling, to transient attachment, to firm adhesion. Also, the model can provide meaningful statistical measures of adhesion, including the mean and variance in velocity, rate constants for ceil attachment and detachment, and the frequency of adhesion. We find a critical modulating parameter of adhesion is the fractional spring slippage, which relates the extension of a bond to its rate of breakage; the higher the slippage, the faster the breakage for the same extension. Changes in the fractional spring slippage can radically change the adhesive behavior of a cell. We show that stiffer springs will only serve to increase adhesion if the fractional slippage remains small. In addition, our simulations emphasize the importance of reaction rates between receptor and ligand, rather than affinity, as being the key determinant of adhesion under flow. These results suggest reaction rates and response to stress of adhesion molecules must be independently measured to understand how adhesion is controlled at the molecular level.     

Simulation of cell rolling and adhesion on surfaces in shear flow. Microvilli-coated hard spheres with adhesive springs.

The adhesion of cells to ligand-coated surfaces in viscous shear flow is an important step in many physiological processes, such as the neutrophil-mediated inflammatory response, lymphocyte homing, and tumor cell metastasis. This article describes a calculational method that allows simulation of the interaction of a single cell with a ligand-coated surface. The cell is idealized as a microvilli-coated hard sphere covered with adhesive springs. The distribution of microvilli on the cell surface, the distribution of receptors on microvilli tips, and the forward and reverse reaction between receptor and ligand are all simulated using random number sampling of appropriate probability functions. The velocity of the cell at each time step in the simulation results from a balance of hydrodynamic, colloidal, and bonding forces; the bonding force is derived by summing the individual contributions of each receptor-ligand tether. The model can simulate the effect of many parameters on adhesion, such as the number of receptors on microvilli tips, the density of ligand, the rates of reaction between receptor and ligand, the stiffness of the springs, the response of springs to extension, and the magnitude of hydrodynamic stresses. By varying these parameters, the model can successfully recreate the entire range of expected and observed adhesive phenomena, from completely unencumbered motion, to rolling, to transient attachment, to firm adhesion. Also, the model can provide meaningful statistical measures of adhesion, including the mean and variance in velocity, rate constants for cell attachment and detachment, and the frequency of adhesion. We find a critical modulating parameter of adhesion is the fractional spring slippage, which relates the extension of a bond to its rate of breakage; the higher the slippage, the faster the breakage for the same extension. Changes in the fractional spring slippage can radically change the adhesive behavior of a cell. We show that stiffer springs will only serve to increase adhesion if the fractional slippage remains small. In addition, our simulations emphasize the importance of reaction rates between receptor and ligand, rather than affinity, as being the key determinant of adhesion under flow. These results suggest reaction rates and response to stress of adhesion molecules must be independently measured to understand how adhesion is controlled at the molecular level.     

The forward rate of binding of surface-tethered reactants: effect of relative motion between two surfaces

The reaction of molecules confined to two dimensions is of interest in cell adhesion, specifically for the reaction between cell surface receptors and substrate-bound ligand. We have developed a model to describe the overall rate of reaction of species that are bound to surfaces under relative motion, such that the Peclet number is order one or greater. The encounter rate between reactive species is calculated from solution of the two-dimensional convection-diffusion equation. The probability that each encounter will lead to binding depends on the intrinsic rate of reaction and the encounter duration. The encounter duration is obtained from the theory of first passage times. We find that the binding rate increases with relative velocity between the two surfaces, then reaches a plateau. This plateau indicates that the increase in the encounter rate is counterbalanced by the decrease in the encounter duration as the relative velocity increases. The binding rate is fully described by two dimensionless parameters, the Peclet number and the Damk?hler number. We use this model to explain data from the cell adhesion literature by incorporating these rate laws into "adhesive dynamics" simulations to model the binding of a cell to a surface under flow. Leukocytes are known to display a "shear threshold effect" when binding selectin-coated surfaces under shear flow, defined as an increase in bind rate with shear; this effect, as calculated here, is due to an increase in collisions between receptor and ligand with increasing shear. The model can be used to explain other published data on the effect of wall shear rate on the binding of cells to surfaces, specifically the mild decrease in binding within a fixed area with increasing shear rate.     

Simulation of detachment of specifically bound particles from surfaces by shear flow.

The receptor-mediated adhesion of cells to ligand-coated surfaces is important in many physiological and biotechnological processes. Previously, we measured the detachment of antibody-coated spheres from counter-antibody- and protein A-coated substrates using a radial-flow detachment assay and were able to relate mechanical adhesion strength to chemical binding affinity (Kuo and Lauffenburger, Biophys. J. 65:2191-2200 (1993)). In this paper, we use "adhesive dynamics" to simulate the detachment of antibody-coated hard spheres from a ligand-coated substrate. We modeled the antibody-ligand (either counter-antibody or protein A) bonds as adhesive springs. In the simulation as in the experiments, beads attach to the substrate under static conditions. Flow is then initiated, and detachment is measured by the significant displacement of previously bound particles. The model can simulate the effects of many parameters on cell detachment, including hydrodynamic stresses, receptor number, ligand density, reaction rates between receptor and ligand, and stiffness and reactive compliance of the adhesive springs. The simulations are compared with experimental detachment data, thus relating measured bead adhesion strength to molecular properties of the adhesion molecules. The simulations accurately recreated the logarithmic dependence of adhesion strength on affinity of receptor-ligand recognition, which was seen in experiments and predicted by analytic theory. In addition, we find the value of the reactive compliance, the parameter which relates the strain of a bond to its rate of breakage, that gives the best match between theory and experiment to be 0.01. Finally, we analyzed the effect of varying either the forward or reverse rate constants as different ways to achieve the same affinity, and showed that adhesion strength depends uniquely on the equilibrium affinity, not on the kinetics of binding. Given that attachment is independent of affinity, detachment and attachment are distinct adhesive phenomena.     

Shear and time-dependent changes in Mac-1, LFA-1, and ICAM-3 binding regulate neutrophil homotypic adhesion

We examined the relative contributions of LFA-1, Mac-1, and ICAM-3 to homotypic neutrophil adhesion over the time course of formyl peptide stimulation at shear rates ranging from 100 to 800 s-1. Isolated human neutrophils were sheared in a cone-plate viscometer and the kinetics of aggregate formation was measured by flow cytometry. The efficiency of cell adhesion was computed by fitting the aggregate formation rates with a model based on two-body collision theory. Neutrophil homotypic adhesion kinetics varied with shear rate and was most efficient at 800 s-1, where approximately 40% of the collisions resulted in adhesion. A panel of blocking Abs to LFA-1, Mac-1, and ICAM-3 was added to assess the relative contributions of these molecules. We report that 1) LFA-1 binds ICAM-3 as its primary ligand supporting homotypic adhesion, although the possibility of other ligands was also detected. 2) Mac-1 binding to an unidentified ligand supports homotypic adhesion with an efficiency comparable to LFA-1 at low shear rates of approximately 100 s-1. Above 300 s-1, however, Mac-1 and not LFA-1 were the predominant molecules supporting cell adhesion. This is in contrast to neutrophil adhesion to ICAM-1-transfected cells, where LFA-1 binds with a higher avidity than Mac-1 to ICAM-1. 3) Following stimulation, the capacity of LFA-1 to support aggregate formation decreases with time at a rate approximately 3-fold faster than that of Mac-1. The results suggest that the relative contributions of beta2 integrins and ICAM-3 to neutrophil adhesion is regulated by the magnitude of fluid shear and time of stimulus over a range of blood flow conditions typical of the venular microcirculation.     

The LFA-1 integrin supports rolling adhesions on ICAM-1 under physiological shear flow in a permissive cellular environment

The LFA-1 integrin is crucial for the firm adhesion of circulating leukocytes to ICAM-1-expressing endothelial cells. In the present study, we demonstrate that LFA-1 can arrest unstimulated PBL subsets and lymphoblastoid Jurkat cells on immobilized ICAM-1 under subphysiological shear flow and mediate firm adhesion to ICAM-1 after short static contact. However, LFA-1 expressed in K562 cells failed to support firm adhesion to ICAM-1 but instead mediated K562 cell rolling on the endothelial ligand under physiological shear stress. LFA-1-mediated rolling required an intact LFA-1 I-domain, was enhanced by Mg2+, and was sharply dependent on ICAM-1 density. This is the first indication that LFA-1 can engage in rolling adhesions with ICAM-1 under physiological shear flow. The ability of LFA-1 to support rolling correlates with decreased avidity and impaired time-dependent adhesion strengthening. A beta2 cytoplasmic domain-deletion mutant of LFA-1, with high avidity to immobilized ICAM-1, mediated firm arrests of K562 cells interacting with ICAM-1 under shear flow. Our results suggest that restrictions in LFA-1 clustering mediated by cytoskeletal attachments may lock the integrin into low-avidity states in particular cellular environments. Although low-avidity LFA-1 states fail to undergo adhesion strengthening upon contact with ICAM-1 at stasis, these states are permissive for leukocyte rolling on ICAM-1 under physiological shear flow. Rolling mediated by low-avidity LFA-1 interactions with ICAM-1 may stabilize rolling initiated by specialized vascular rolling receptors and allow the leukocyte to arrest on vascular endothelium upon exposure to stimulatory endothelial signals.     

Investigating the effects of membrane deformability on artificial capsule adhesion to the functionalized surface

Understanding, manipulating and controlling cellular adhesion processes can be critical in developing biomedical technologies. Adhesive mechanisms can be used to the target, pattern and separate cells such as leukocytes from whole blood for biomedical applications. The deformability response of the cell directly affects the rolling and adhesion behavior under viscous linear shear flow conditions. To that end, the primary objective of the present study was to investigate numerically the influence of capsule membrane’s nonlinear material behavior (i.e. elastic-plastic to strain hardening) on the rolling and adhesion behavior of representative artificial capsules. Specifically, spherical capsules with radius of \(3.75\, \upmu \hbox {m}\) were represented using an elastic membrane governed by a Mooney–Rivlin strain energy functions. The surfaces of the capsules were coated with P-selectin glycoprotein-ligand-1 to initiate binding interaction with P-selectin-coated planar surface with density of \(150\,\upmu \hbox {m}^{-2}\) under linear shear flow varying from 100 to \(400\,\hbox {s}^{-1}\). The numerical model is based on the Immersed Boundary Method for rolling of deformable capsule in shear flow coupled with Monte Carlo simulation for receptor/ligand interaction modeled using Bell model. The results reveal that the mechanical properties of the capsule play an important role in the rolling behavior and the binding kinetics between the capsule contact surface and the substrate. The rolling behavior of the strain hardening capsules is relatively smoother and slower compared to the elastic-plastic capsules. The strain hardening capsules exhibits higher contact area at any given shear rate compared to elastic-plastic capsules. The increase in contact area leads to decrease in rolling velocity. The capsule contact surface is not in complete contact with the substrate because of thin lubrication film that is trapped between the capsule and substrate. This creates a concave shape on the bottom surface of the capsule that is referred to as a dimple. In addition, the present study demonstrates that the average total bond force from the capsules lifetime increases by 37 % for the strain hardening capsules compared to elastic-plastic capsules at shear rate of \(400\,\hbox {s}^{-1}\). Finally, the model demonstrates the effect of finite membrane deformation on the coupling between hydrodynamic and receptor/ligand interaction.     

Tissue Factor-Expressing Tumor Cells Can Bind to Immobilized Recombinant Tissue Factor Pathway Inhibitor under Static and Shear Conditions In Vitro

Mammary tumors and malignant breast cancer cell lines over-express the coagulation factor, tissue factor (TF). High expression of TF is associated with a poor prognosis in breast cancer. Tissue factor pathway inhibitor (TFPI), the endogenous inhibitor of TF, is constitutively expressed on the endothelium. We hypothesized that TF-expressing tumor cells can bind to immobilized recombinant TFPI, leading to arrest of the tumor cells under shear in vitro. We evaluated the adhesion of breast cancer cells to immobilized TFPI under static and shear conditions (0.35 – 1.3 dyn/cm²). We found that high-TF-expressing breast cancer cells, MDA-MB-231 (with a TF density of 460,000/cell), but not low TF-expressing MCF-7 (with a TF density of 1,400/cell), adhered to recombinant TFPI, under static and shear conditions. Adhesion of MDA-MB-231 cells to TFPI required activated factor VII (FVIIa), but not FX, and was inhibited by a factor VIIa-blocking anti-TF antibody. Under shear, adhesion to TFPI was dependent on the TFPI-coating concentration, FVIIa concentration and shear stress, with no observed adhesion at shear stresses greater than 1.0 dyn/cm². This is the first study showing that TF-expressing tumor cells can be captured by immobilized TFPI, a ligand constitutively expressed on the endothelium, under low shear in vitro. Based on our results, we hypothesize that TFPI could be a novel ligand mediating the arrest of TF-expressing tumor cells in high TFPI-expressing vessels under conditions of low shear during metastasis.     

The CD81 tetraspanin facilitates instantaneous leukocyte VLA-4 adhesion strengthening to vascular cell adhesion molecule 1 (VCAM-1) under shear flow

Leukocyte integrins must rapidly strengthen their binding to target endothelial sites to arrest rolling adhesions under physiological shear flow. We demonstrate that the integrin-associated tetraspanin, CD81, regulates VLA-4 and VLA-5 adhesion strengthening in monocytes and primary murine B cells. CD81 strengthens multivalent VLA-4 contacts within subsecond integrin occupancy without altering intrinsic adhesive properties to low density ligand. CD81 facilitates both VLA-4-mediated leukocyte rolling and arrest on VCAM-1 under shear flow as well as VLA-5-dependent adhesion to fibronectin during short stationary contacts. CD81 also augments VLA-4 avidity enhancement induced by either chemokine-stimulated Gi proteins or by protein kinase C activation, although it is not required for Gi protein or protein kinase C signaling activities. In contrast to other proadhesive integrin-associated proteins, CD81-promoted integrin adhesiveness does not require its own ligand occupancy or ligation. These results provide the first demonstration of an integrin-associated transmembranal protein that facilitates instantaneous multivalent integrin occupancy events that promote leukocyte adhesion to an endothelial ligand under shear flow.     

Specific adhesion of glycophorin liposomes to a lectin surface in shear flow.

The adhesion of cells to other cells or to surfaces by receptor-ligand binding in a shear field is an important aspect of many different biological processes and various cell separation techniques. The purpose of this study was to observe the adhesion of model cells with receptor molecules embedded in their surfaces to a ligand-coated surface under well-defined flow conditions in a parallel plate flow chamber. Liposomes containing glycophorin were used as the model cells to permit a variation in the adhesion parameters and then to observe the effect on adhesion. A mathematical model for cell sedimentation was created to predict the deposition time and the velocity preceding adhesion for the selection of experimental operating conditions and the methods useful for data analysis. The likelihood of cell attachment was represented by a quantity called the sticking probability which was defined as the inverse of the number of times a liposome made contact with the surface before attachment occurred. The sticking probability decreased as the cell receptor concentration was lowered from approximately 10(4) to 10(2) receptors per 4-microns diam liposome and as the shear rate increased from 5 to 22 s-1. The effect of the wall shear rate and particle diameter on detachment of liposomes from a surface was also observed.     

Inhibition of L-selectin binding by polyacrylamide-based conjugates under defined flow conditions

Selectins mediate tethering and rolling of leukocytes along the endothelium in a shear force-dependent manner. This key step in the cellular immune response is a target for experimental anti-inflammatory therapies. In the present paper we have examined the inhibitory activity of the minimal selectin ligand sialyl Lewis x (SiaLe(x)), its isomer sialyl Lewis a (SiaLe(a)) and sulfated tyrosine (sTyr) residues under dynamic flow reflecting the rheological conditions in the blood stream. The monomeric ligands were compared to multivalent polyacrylamide (PAA)-based conjugates under defined flow conditions on the molecular level, using surface plasmon resonance (SPR) technology, and on the cellular level, using a parallel-plate flow chamber. SPR measurements showed that a spatial arrangement of binding epitopes mimicking the selectin binding motif of the natural ligand PSGL-1 inhibits L-selectin binding successfully with IC(50) values in the nanomolar range. Using a flow chamber adhesion assay it could be shown that the multivalent inhibitors efficiently blocked rolling and tethering of NALM-6 pre-B cells transfected with human L-selectin to activated endothelium and that the inhibitory activity increased with rising shear stress. While PAA-conjugates were almost not inhibitory at low shear stress, NALM-6 cell rolling was nearly completely inhibited at high shear stress. The results indicate that multimeric conjugates of SiaLe(x), SiaLe(a) and sTyr are highly effective inhibitors of L-selectin-mediated cell adhesion particularly under flow conditions. Consequently, SiaLe(x), SiaLe(a) and/or sTyr on macromolecular carriers may be promising candidates for anti-inflammatory therapy.