Bistability of cell adhesion in shear flow

Cell adhesion plays a central role in multicellular organisms helping to maintain their integrity and homeostasis. This complex process involves many different types of adhesion proteins, and synergetic behavior of these proteins during cell adhesion is frequently observed in experiments. A well-known example is the cooperation of rolling and stationary adhesion proteins during the leukocytes extravasation. Despite the fact that such cooperation is vital for proper functioning of the immune system, its origin is not fully understood. In this study we constructed a simple analytic model of the interaction between a leukocyte and the blood vessel wall in shear flow. The model predicts existence of cell adhesion bistability, which results from a tug-of-war between two kinetic processes taking place in the cell-wall contact area—bond formation and rupture. Based on the model results, we suggest an interpretation of several cytoadhesion experiments and propose a simple explanation of the existing synergy between rolling and stationary adhesion proteins, which is vital for effective cell adherence to the blood vessel walls in living organisms.     

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.     

Influence of cell deformation on leukocyte rolling adhesion in shear flow

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

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.     

Macrophage antigens associated with adhesion: identification by a monoclonal antibody specific for Lewis lung carcinoma cells

A monoclonal antibody specific for Lewis lung carcinoma (3LL) cells (Mab 5B5) was found to recognize antigens expressed on murine macrophages and on a macrophage hybridoma line upon cell adhesion on plastic surfaces. These antigens were also present on the surface of murine macrophage tumor M5076 cells which develop solid tumors and metastases. The M5076 tumor cells freshly isolated from the primary tumor and from hepatic metastases strongly bound Mab 5B5 but lost this capacity after adhesion. Freshly isolated thioglycolate-elicited peritoneal mouse macrophages were not labeled by Mab 5B5; however, after 1 h of adhesion, 50% of the adherent macrophages were directly incubated with Mab 5B5 prior to harvesting by scraping. Permeabilization of peritoneal macrophages by saponin showed that the antigens recognized by Mab 5B5 were present inside the cells before adhesion. Similar results were obtained with the 2C11-12 macrophage hybridoma cells. P388D1 cells (a weakly adherent macrophage tumor cell line), HL60 cells (a human promyelocytic cell line), and human monocytes were poorly labeled without permeabilization but were strongly labeled by Mab 5B5 upon permeabilization. The specificity of the monoclonal antibody in relation to the adherence capacity of these cells is discussed.     

Flow-induced detachment of red blood cells adhering to surfaces by specific antigen-antibody bonds.

Fixed spherical swollen human red blood cells of blood type B adhering on a glass surface through antigen-antibody bonds to monoclonal mouse antihuman IgM, adsorbed or covalently linked on the surface, were detached by known hydrodynamic forces created in an impinging jet. The dynamic process of detachment of the specifically bound cells was recorded and analyzed. The fraction of adherent cells remaining on the surface decreased with increasing hydrodynamic force. For an IgM coverage of 0.26%, a tangential force on the order of 100 pN was able to detach almost all of the cells from the surface within 20 min. After a given time of exposure to hydrodynamic force, the fraction of adherent cells remaining increased with time, reflecting an increase in adhesion strength. The characteristic time for effective aging was approximately 4 h. Results from experiments in which the adsorbed antibody molecules were immobilized through covalent coupling and from evanescent wave light scattering of adherent cells, imply that deformation of red cells at the contact area was the principal cause for aging, rather than local clustering of the antibody through surface diffusion. Experiments with latex beads specifically bound to red blood cells suggest that, instead of breaking the antigen-antibody bonds, antigen molecules were extracted from the cell membrane during detachment.     

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.     

CRP promotes monocyte-endothelial cell adhesion via Fcgamma receptors in human aortic endothelial cells under static and shear flow conditions

Monocyte-endothelial cell adhesion is a key early event in atherogenesis. C-reactive protein (CRP), a cardiovascular risk marker, is known to stimulate ICAM and VCAM in human aortic endothelial cells (HAEC) and induces monocyte-endothelial cell adhesion. In this study, we examined the mechanisms by which native CRP promotes monocyte-endothelial cell adhesion under static conditions and tested the effect of CRP on adhesion under shear flow. Incubation of HAEC with CRP (>25 microg/ml) upregulated NF-kappaB activity, and this resulted in a significant increase in ICAM (54% increase, P<0.001), VCAM (41% increase, P<0.01), and monocyte-endothelial cell adhesion (44% increase, P<0.02) compared with those of control. Preincubation with antibodies to CD32 and CD64 but not CD16 effectively inhibited this activation. Blocking NF-kappaB activity with inhibitors or a dominant negative inhibitory kappaB significantly decreased ICAM, VCAM upregulation, and subsequent monocyte-endothelial cell adhesion. Preincubation with antibodies to CD32 and CD64 or transient transfection with small interference RNA to CD32 attenuated CRP-induced NF-kappaB activity, ICAM, VCAM, and monocyte-endothelial cell adhesion under static conditions. Also, the Syk kinase inhibitor piceatannol and MG-132, a proteasome degradation inhibitor, produced similar attenuation in NF-kappaB activity, ICAM, VCAM, and adhesion. Furthermore, CRP-activated endothelial cells supported monocyte rolling, arrest, and transmigration in shear flow (2 dyn/cm2), and this was also inhibited by preincubation with antibodies to CD32 and CD64. Thus, in HAEC, CRP upregulates monocyte-endothelial adhesion by activation of NF-kappaB through engaging the Fcgamma receptors CD32 and CD64.     

Energy of hydrogen bonds probed by the adhesion of functionalized lipid layers

It is now well admitted that hydrophobic interactions and hydrogen bonds are the main forces driving protein folding and stability. However, because of the complex structure of a protein, it is still difficult to separate the different energetic contributions and have a reliable estimate of the hydrogen bond part. This energy can be quantified on simpler systems such as surfaces bearing hydrogen-bonding groups. Using the surface force apparatus, we have directly measured the interaction energy between monolayers of lipids whose headgroups can establish hydrogen bonds in water: nitrilotriacetate, adenosine, thymidine, and methylated thymidine lipids. From the adhesion energy between the surfaces, we have deduced the energy of a single hydrogen bond in water. We found in each case an energy of 0.5 kcal/mol. This result is in good agreement with recent experimental and theoretical studies made on protein systems showing that intramolecular hydrogen bonds make a positive contribution to protein stabilization.     

A flow system for the study of shear forces upon cultured endothelial cells

A parallel plate chamber in a flow system has been designed to study the effects of fluid shear stresses on cells. The system was applied to the study of cultured endothelial cells grown on cover slips which were accommodated in recessed wells in the base plate. Dye injection studies in the chamber indicated laminar flow over the cells. Shear rates measured over the cover slips by an electrochemical technique were found to be linear with flow rate. Laser doppler anemometry showed parabolic profiles between the plates. Endothelial cells subjected to flow showed a correlation between the time required for orientation and the magnitude of the shear stress.     

Suggestions for the operation of radial flow cells in cell adhesion and biofouling studies

An analysis of the radial flow cell is presented to show that the assumption of creeping laminar flow should be used cautiously. Simple models which account for the influence of fluid inertial forces over most of the width of the plate are reviewed. A modified Reynolds number is introduced which may be used to test the validity of the creeping flow solution.     

Granulocyte-endothelium initial adhesion. Analysis of transient binding events mediated by E-selectin in a laminar shear flow.

The adhesion of moving cells to receptor-bearing surfaces is a key step to many important biological processes. Attachment was subjected to extensive modeling. However, the numerical values of kinetic bonding parameters relevant to realistic models of cell adhesion remain poorly known. In this report, we describe the motion of human granulocytes to interleukin-1-activated endothelial cells in presence of a low hydrodynamic drag (a few piconewtons) estimated to be much weaker than a standard ligand-receptor bond. It was thus expected to visualize the formation and rupture of individual bonds. We observed multiple short-time cell arrests with a median duration of 2.43 s. Stop frequency, not duration, was significantly inhibited by anti-E-selectin antibodies. Binding efficiency exhibited an almost linear relationship with the inverse of cell velocity. The distribution of arrest duration was determined: results were consistent with the view that these arrests reflected the formation/dissociation of single ligand-receptor bonds with a spontaneous dissociation rate of 0.5 s-1. The rate of bond formation was on the order of 0.04 s-1 when cells were freely rolling (mean velocity: 19 microns/s) and it exhibited an approximately 10-fold increase after the formation of a first adhesion.     

Investigation of human platelet adhesion under low shear conditions in a rotational flow chamber

Even though blood pumps have come into clinical usage, thrombo-embolic complications still pose a major problem, and they have not yet been clarified and quantified. However, it is known that the basis of thrombus formation is platelet adhesion, which is thought to be closely associated with the shear rate. Therefore, our current interest focuses on the effect of shear conditions on platelet adhesion. We have designed and carried out an experimental setup allowing fluorescent microscopy of whole blood within a rotational viscometer under controllable shear conditions. A small area of the bottom plate was coated with type I collagen, which provided a model of the injured vessel as a target for platelet adhesion. Using this setup, the time course of platelet adhesion under several different shear rates, ranging from 127 to 723 s^?1, was studied. Platelet adhesion increased along with shear rates up to 283 s^?1, followed by a gradual decrease when the shear rate exceeded 346 s^?1. The adhesion amounts were statistically significant between 283 and 173 s^?1 (p = 0.02), 173 and 127 s^?1 (p = 0.035), and 283 and 503 s^?1 (p = 0.03), respectively. This result suggests that there is an optimal shear condition around 300 s^?1 for platelet adhesion to type I collagen.     

Breakage by hydrodynamic shear of the bonds between cohered ends of lambda-DNA molecules

The rate of breakage by hydrodynamic shear of the cohered ends of λ-DNA molecules has been observed for the circular monomers, joined half molecules, and joined quarter molecules, in a capillary apparatus with known flow parameters. The rate constant for breakage has been measured as a function of shear stress, temperature, ionic strength, and molecular length. There is a large temperature coefficient, with an activation energy of 120 ± 20 kcal./mole. The values of d ln k/dG, where k is the rate constant for breaking and G is shear gradient, in aqueous solution at 25°C. are about 3.8 ± 0.3 × 10^?4 see. The shear stresses needed for breakage of joined quarter molecules and of circular monomers, respectively, are about equal, and about half that needed for breakage of joined half molecules. The rate of breakage at a given shear stress increases with decreasing ionic strength, approximately as [Na⁺]^?1.6. Self-protection effects are not observed for opening of circular monomers at a DNA concentration of 5 μg./ml. but are observed for breakage of joined half molecules at concentrations down to 0.5 μg./ml. The large temperature coefficient which is approximately equal to that of the thermal dissociation of the cohered ends is interpreted to mean that shear breakage is a mechanically assisted thermal reaction in which the thermal fluctuations provide most of the free energy of activation for breakage. A detailed model for this interpretation is presented. The self-protection effect implies that those molecules which break are not average molecules but exceptional ones which, due to some fluctuation, are more fully extended in the flow field.     

Inflammatory responses of endothelial cells experiencing reduction in flow after conditioning by shear stress

Exposure of endothelial cells (EC) to shear stress reduces their response to tumour necrosis factor-alpha (TNF). We tested how shear-conditioned EC responded to reduction in flow, either by spontaneously binding leukocytes, or by increasing sensitivity to TNF. Human umbilical vein EC were exposed to shear stress of 2.0 Pa (20 dyn/cm(2)) for 24 h. Shear was then reduced to stasis (30 sec perfusion each hour to exchange medium) or 0.003 Pa (creeping flow). At chosen times, neutrophils were perfused over the EC at 0.1 Pa (effective reperfusion). EC developed an ability to capture flowing neutrophils that lasted from 1 to 3 h after flow reduction, which was reduced by antibody against P-selectin or pre-treatment of EC with an inhibitor of NADPH-oxidase. Adhesion of neutrophils to TNF-treated EC was greatly suppressed by shear-conditioning, remained suppressed immediately after cessation of flow and then took 48 h to approach the level in static cultures. Interestingly, the response to TNF remained suppressed in cultures switched to creeping flow. Gene array analysis confirmed that differently recovered cells had separate phenotypes. Thus, an acute response of EC to reduction in shear may contribute to leukocyte recruitment, along with hypoxia, in ischaemia and reperfusion. Prolonged cessation of flow may increase the sensitivity of EC to inflammatory stimuli, but this effect may be suppressed by residual flow.     

Transient strong reduction of PTEN expression by specific RNAi induces loss of adhesion of the cells

The tumor suppressor gene pten encodes a lipid phosphatase that dephosphorylates D3 of phosphatidylinositol(3,4,5)trisphosphate, producing phosphatidylinositol(4,5)bisphosphate. Although PTEN has been implicated in cell adhesion and migration, the underlying molecular mechanism is unknown. To investigate the role of PTEN in cell adhesion, we designed three different siRNAs (siRNA PTEN-a, siRNA PTEN-b, and siRNA PTEN-c) and transfected into 293T cells. Two days later, only the cells transfected with siRNA PTEN-b became round and detached from the culture dishes, whereas cells transfected with a control siRNA against GFP or the two other siRNAs against PTEN did not. Evaluation of the RNAi effect revealed that siRNA PTEN-b inhibited >95% of PTEN expression, the most effective among the three siRNAs. To check for non-specific effects such as interferon response and inhibition of off-target genes, we then used quantitative PCR analysis and DNA microarray analysis. None was detected, indicating that the RNAi system was highly specific. Immunofluorescence studies using PTEN-knockdown HeLa cells revealed that the loss of adhesion was accompanied by a reduction in the number of focal adhesion plaques and disorganization of the actin cytoskeleton. Transient and near-complete loss of PTEN expression induces loss of adhesion of the cells.     

流动剪切力对鼠脑微血管内皮细胞ICAM—1表达的影响

利用内皮细胞流动小室方法,对大鼠脑微血管内皮细胞的剪切力作用下细胞内粘附分子－1（ICAM－1,intercellular adhesion molecule-1）的表达进行了研究。图像分析结果提示,脑微血管内皮细胞在剪切力作用下ICAM－1的表达呈特异上调,且存在着时间依赖性,与一定范围内的剪切力强度无关,用对细胞施加剪切力作用后提取上清液孵育内皮细胞的方法证明：剪切力对鼠脑微血管内皮细胞ICAM－1表达的影响,是直接作用于内皮细胞引起的细胞内的直接反应,而不是剪切力导致细胞先释放细胞因子,释放的细胞因子再引起ICAM－1变化的间接反应。该工作为进一步开展剪切力对微血管内皮细胞信号转导机制的影响提供了实验数据。     

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.     

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.