Correlating the kinetics of cytokine-induced E-selectin adhesion and expression on endothelial cells.

Many human diseases are mediated through the immune system. In chronic inflammatory disorders, the processes ordinarily involved in tissue healing become destructive. Endothelial cells normally recruit leukocytes to inflamed tissue using cytokine-induced adhesion receptors on the surfaces of interacting cells. Leukocyte capture depends on specialized characteristics of these receptors, particularly the binding kinetics. This study is designed to clarify the relationship between cytokine-induced changes in cell properties and binding kinetics. Here, we measure the kinetics of expression and monoclonal antibody binding for E-selectin in interleukin-1alpha-stimulated microvascular endothelium in vitro and incorporate the data into kinetic models. Quantitative flow cytometry is used to determine molecular density (expression), and micropipette assays are used to find the probability of adhesion (function). Within five hours of interleukin-1alpha stimulation, E-selectin density increases from 0 to 742 sites/microm(2), and antibody-E-selectin adhesion probability increases from a baseline of 6.3% to 64%. A kinetic model is applied to find an apparent association rate constant, k(f), of 3.7 x 10(-14) cm(2)/sec for antibody-E-selectin binding. Although the model successfully predicts experimental results, the rate constant is undervalued for a diffusion-limited process, suggesting that functional adhesion may be modified through cytokine-induced changes in microtopology and receptor localization.

Micropipette aspiration of living cells.

The mechanical behavior of living cells is studied with micropipette suction in which the surface of a cell is aspirated into a small glass tube while tracking the leading edge of its surface. Such edges can be tracked in a light microscope to an accuracy of +/-25 nm and suction pressures as small as 0.1-0.2 pN/microm2 can be imposed on the cell. Both soft cells, such as neutrophils and red cells, and more rigid cells, such as chondrocytes and endothelial cells, are studied with this technique. Interpretation of the measurements with basic continuum models leads to values for a cell's elastic and viscous properties. In particular, neutrophils are found to behave as a liquid drop with a cortical (surface) tension of about 30 pN/microm and a viscosity on the order of 100 Pa s. On the other hand, chondrocytes and endothelial cells behave as solids with an elastic modulus of the order of 500 pN/microm2 (0.5 kPa).

Myosin I contributes to the generation of resting cortical tension.

The amoeboid myosin I's are required for cellular cortical functions such as pseudopod formation and macropinocytosis, as demonstrated by the finding that Dictyostelium cells overexpressing or lacking one or more of these actin-based motors are defective in these processes. Defects in these processes are concomitant with changes in the actin-filled cortex of various Dictyostelium myosin I mutants. Given that the amoeboid myosin I's possess both actin- and membrane-binding domains, the mutant phenotypes could be due to alterations in the generation and/or regulation of cell cortical tension. This has been directly tested by analyzing mutant Dictyostelium that either lacks or overexpresses various myosin I's, using micropipette aspiration techniques. Dictyostelium cells lacking only one myosin I have normal levels of cortical tension. However, myosin I double mutants have significantly reduced (50%) cortical tension, and those that mildly overexpress an amoeboid myosin I exhibit increased cortical tension. Treatment of either type of mutant with the lectin concanavalin A (ConA) that cross-links surface receptors results in significant increases in cortical tension, suggesting that the contractile activity of these myosin I's is not controlled by this stimulus. These results demonstrate that myosin I's work cooperatively to contribute substantially to the generation of resting cortical tension that is required for efficient cell migration and macropinocytosis.

Mechanical anchoring strength of L-selectin, beta2 integrins, and CD45 to neutrophil cytoskeleton and membrane.

The strength of anchoring of transmembrane receptors to cytoskeleton and membrane is important in cell adhesion and cell migration. With micropipette suction, we applied pulling forces to human neutrophils adhering to latex beads that were coated with antibodies to CD62L (L-selectin), CD18 (beta2 integrins), or CD45. In each case, the adhesion frequency between the neutrophil and bead was low, and our Monte Carlo simulation indicates that only a single bond was probably involved in every adhesion event. When the adhesion between the neutrophil and bead was ruptured, it was very likely that receptors were extracted from neutrophil surfaces. We found that it took 1-2 s to extract an L-selectin at a force range of 25-45 pN, 1-4 s to extract a beta2 integrin at a force range of 60-130 pN, and 1-11 s to extract a CD45 at a force range of 35-85 pN. Our results strongly support the conclusion that, during neutrophil rolling, L-selectin is unbound from its ligand when the adhesion between neutrophils and endothelium is ruptured.

Alterations in the Young's modulus and volumetric properties of chondrocytes isolated from normal and osteoarthritic human cartilage.

The mechanical environment of the chondrocyte is an important factor that influences the maintenance of the articular cartilage extracellular matrix. Previous studies have utilized theoretical models of chondrocytes within articular cartilage to predict the stress-strain and fluid flow environments around the cell, but little is currently known regarding the cellular properties which are required for implementation of these models. The objectives of this study were to characterize the mechanical behavior of primary human chondrocytes and to determine the Young's modulus of chondrocytes from non-osteoarthritic ('normal') and osteoarthritic cartilage. A second goal was to quantify changes in the volume of isolated chondrocytes in response to mechanical deformation. The micropipette aspiration technique was used to measure the deformation of a single chondrocyte into a glass micropipette in response to a prescribed pressure. The results of this study indicate that the human chondrocyte behaves as a viscoelastic solid. No differences were found between the Young's moduli of normal (0.65+/-0.63 kPa, n = 44) and osteoarthritic chondrocytes (0.67+/-0.86 kPa, n = 69, p = 0.93). A significant difference in cell volume was observed immediately and 600 s after complete aspiration of the cell into the pipette (p < 0.001), and the magnitude of this volume change between normal (11+/-11%, n = 40) and osteoarthritic (20+/-11%, n = 41) chondroctyes was significantly different at both time points (p < 0.002). This finding suggests that chondrocytes from osteoarthritic cartilage may have altered volume regulation capabilities in response to mechanical deformation. The mechanical and volumetric properties determined in this study will be of use in analytical and finite element models of chondrocyte-matrix interactions in order to better predict the mechanical environment of the cell in vivo.

Static and dynamic lengths of neutrophil microvilli.

Containing most of the L-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) on their tips, microvilli are believed to promote the initial arrest of neutrophils on endothelium. At the rolling stage following arrest, the lifetimes of the involved molecular bonds depend on the pulling force imposed by the shear stress of blood flow. With two different methods, electron microscopy and micropipette manipulation, we have obtained two comparable neutrophil microvillus lengths, both approximately 0.3 microm in average. We have found also that, under a pulling force, a microvillus can be extended (microvillus extension) or a long thin membrane cylinder (a tether) can be formed from it (tether formation). If the force is 61 pN (+/- 5 pN), a tether will be formed from the microvillus at a constant velocity, which depends linearly on the force. When the force is between 34 pN and 61 pN (transition zone), the degree of association between membrane and cytoskeleton in individual microvilli will dictate whether microvillus extension or tether formation occurs. When a microvillus is extended, it acts like a spring with a spring constant of approximately 43 pN/microm. In contrast to a rigid or nonextendible microvillus, both microvillus extension and tether formation can decrease the pulling force imposed on the adhesive bonds, and thus prolonging the persistence of the bonds at high physiological shear stresses.

The resistance to flow of individual human neutrophils in glass capillary tubes with diameters between 4.65 and 7.75 microns.

OBJECTIVE: The resistance to flow of individual human neutrophils flowing through smooth-walled glass capillary tubes at velocities ranging between 0 and 30 microns/s is measured for six different capillary tube diameters between 4.65 and 7.75 microns. METHODS: The experiments were performed with a micropipet manipulation system. The velocity of individual human neutrophils was measured under different constant pressures and compared to the velocity of the cell-free fluid in the same tube. With the aid of a theory that describes the motion of a concentric smooth-walled, sausage-shaped body in a tube, the experimental measurements are used to calculate an apparent gap width between the cell and the capillary wall. RESULTS: The maximum calculated gap width in the larger capillary tubes was on the order of 0.1 micron, whereas the minimum gap width in the smaller capillaries was about 0.015 micron. These small gap widths mean that even one neutrophil can cause a significant increase in the apparent viscosity when a cell flows through 100- to 200-micron-long capillary tubes. The neutrophils often exhibited a small static friction that tended to increase as the capillary diameter decreased. Maximum values for the adhesive force caused by the static friction were on the order of 80 pN. CONCLUSIONS: Even a single white cell entirely within a capillary can cause a significant increase in the resistance of flow.

Micropipette suction for measuring piconewton forces of adhesion and tether formation from neutrophil membranes.

A new method for measuring piconewton-scale forces that employs micropipette suction is presented here. Spherical cells or beads are used directly as force transducers, and forces as small as 10-20 pN can be imposed. When the transducer is stationary in the pipette, the force is simply the product of the suction pressure and the cross-sectional area of the pipette minus a small correction for the narrow gap that exists between the transducer and the pipette wall. When the transducer is moving along the pipette, the force on it is corrected by a factor that is proportional to the ratio of its velocity relative to its drag-free velocity. With this technique, the minimum force required to form a membrane tether from neutrophils is determined (45 pN), and the length of the microvilli on the surface of neutrophils is inferred. The strength of this technique is in its simplicity and its ability to measure forces between cells without requiring a separate theory or a calibration against an external standard and without requiring the use of a solid surface.

F-actin network formation in tethers and in pseudopods stimulated by chemoattractant.

Micropipets are used either to deliver a given concentration of the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) to a local region of a human neutrophil or to create a membrane tether. Pseudopods, which have a cylindrical shape and grow at a constant rate, are formed in either case. After reaching a maximum extension, they retract, even in the presence of chemoattractant. As a pseudopod grows, cell granules begin to penetrate the pseudopod region to a "boundary" that defines a distance to the pseudopod's leading edge that is almost constant. The exclusion of granules from this domain indicates that it is filled with a dense network. The formation of this network involves the plasma membrane because pseudopod growth ceases when a membrane tether is pulled away from the leading edge. The rate of pseudopod growth depends on fMLP concentration just as the number of occupied N-formyl peptide receptors depends on this concentration. The experimental data are explained by assuming that F-actin network is formed next to the plasma membrane. The newly formed network displaces the membrane and the dominant process in the network region then becomes F-actin depolymerization. The rate of pseudopod growth is determined by the rate of the process leading to network formation. This process is apparently an enzymatic type of reaction. It has a positive enthalpy change and, therefore, is endothermic.

Comparison of different drying procedures for scanning electron microscopy using human leukocytes.

Using human leukocytes as test specimens, three different drying procedures for scanning electron microscopy: critical-point drying (CPD), Peldri II, and tetramethylsilane (TMS), were compared. All three procedures produced identical surface morphology preservation. An equal amount of volume shrinkage was observed regardless of the dehydrants and drying techniques employed. Considering the simplicity, convenience, and time saved, air-drying with TMS is by far the best choice for preparing animal cells for scanning electron microscopy.

Effect of cytochalasin D on the mechanical properties and morphology of passive human neutrophils.

The actin-rich cortex plays a major role in neutrophil chemotaxis and phagocytosis. In passive neutrophils, 30-50% of the actin molecules are in the F (filamentous) form, and it is the shifting of equilibrium with its monomeric G (globular) form that controls cell motility and phagocytosis. Cytochalasins have been shown to inhibit cell phagocytosis and ruffling. In purified actin, cytochalasins have been shown to decrease the amount of F-actin by capping the fast-growth end of actin filaments. Recent studies with intact cells, however, reveal that the most potent cytochalasin, cytochalasin D (CD), actually increases F-actin content suggesting that CD disrupts the actin network so as to increase the number of actin-filament ends for further actin polymerization. In this paper, we report the effects of CD on the passive mechanical behavior and morphology of human neutrophils with 1, 2, 10, and 20 microM CD. At 1 and 2 microM CD, the cells remain spherical. However, in the presence of 10 and 20 microM CD, cells are severely deformed and "blebby" as shown by light and scanning electron microscopy. After 1 and 2 microM CD treatment, the cells show a decrease of 43 and 66%, respectively, in cortical tension when measured by static micropipet aspiration experiments. Similarly, the cytoplasmic viscosities of 1 and 2 microM CD-treated cells are decreased, but only by 17 and 24%, respectively. A proportionally greater effect on the cortical tension suggests that CD acts mainly on the actin-rich cortex by disrupting the filament network.

Mechanically stimulated cytoskeleton rearrangement and cortical contraction in human neutrophils.

A mechanical test with micropipets is used to characterize cytoskeleton rearrangement and contraction induced by mechanical stresses in human neutrophils. The yield shear resultant of the cell cortex is on the order of 0.06 to 0.09 mN.m-1. The measured yield shear resultant suggests that the neutrophil cortex is a weakly cross-linked structure. When a tether is pulled out from the cell surface, a polymer structure starts to fill it and spreads out from the cell body. The rate of advancement of the polymerization front is almost constant and, therefore, is not diffusion limited. The measured rate is much smaller than the one of spontaneous actin polymerization, suggesting that the limiting process is either the dissociation of actin monomers from their dimers with the capping proteins or the rate of formation of new nucleation sites or both. Polymerization is also observed after applying sufficient mechanical stresses on a small portion of the cell surface. The polymerization is followed by mass transfer from the cell into the prestressed region and later on by contraction of the main cell body. The pressure generating the flow is located in the prestressed region and most probably is a result of its "swelling" and contraction. The contraction of the main cell body is very similar (in its time dependence and magnitude) to the contraction during phagocytosis. The measured maximum cortical tension is on the order of 0.5 mN.m-1, which for a 3.5-microns diameter pipet corresponds to a maximum contraction force of 11 nN.

Role of the membrane cortex in neutrophil deformation in small pipets.

The simplest model for a neutrophil in its "passive" state views the cell as consisting of a liquid-like cytoplasmic region surrounded by a membrane. The cell surface is in a state of isotropic contraction, which causes the cell to assume a spherical shape. This contraction is characterized by the cortical tension. The cortical tension shows a weak area dilation dependence, and it determines the elastic properties of the cell for small curvature deformations. At high curvature deformations in small pipets (with internal radii less than 1 micron), the measured critical suction pressure for cell flow into the pipet is larger than its estimate from the law of Laplace. A model is proposed where the region consisting of the cytoplasm membrane and the underlying cortex (having a finite thickness) is introduced at the cell surface. The mechanical properties of this region are characterized by the apparent cortical tension (defined as a free contraction energy per unit area) and the apparent bending modulus (introduced as a bending free energy per unit area) of its middle plane. The model predicts that for small curvature deformations (in pipets having radii larger than 1.2 microns) the role of the cortical thickness and the resistance for bending of the membrane-cortex complex is negligible. For high curvature deformations, they lead to elevated suction pressures above the values predicted from the law of Laplace. The existence of elevated suction pressures for pipets with radii from 1 micron down to 0.24 micron is found experimentally. The measured excess suction pressures cannot be explained only by the modified law of Laplace (for a cortex with finite thickness and negligible bending resistance), because it predicts unacceptable high cortical thicknesses (from 0.3 to 0.7 micron). It is concluded that the membrane-cortex complex has an apparent bending modulus from 1 x 10(-18) to 2 x 10(-18) J for a cortex with a thickness from 0.1 micron down to values much smaller than the radius of the smallest pipet (0.24 micron) used in this study.

A novel micropipet method for measuring the bending modulus of vesicle membranes.

A theoretical model and an experiment are presented for determining the bending modulus of a bilayer vesicle membrane. The vesicle is held with a pipet having a radius between 1 and 2 microns, and the tension in the membrane is changed by changing the suction pressure. Then the vesicle membrane is deformed by aspirating it into a smaller pipet having a radius on the order of 0.5 microns. The relationship between the suction pressures in the two pipets is found to be linear, as predicted by the theoretical model. The curvature of the vesicle membrane at the pipet orifice and the bending modulus are found with the help of the model from the slope and the intercept of the linear experimental relationship between the suction pressures in the two pipets. The bending modulus for the two SOPC membranes studied in these experiments was found to be either 0.6 or 1.15 x 10(-19) J, which is similar to the values measured previously.

Numerical simulation of the flow of highly viscous drops down a tapered tube.

The flow of a highly viscous drop surrounded by an inviscid fluid inside a tapered tube is analyzed according to a Newtonian, liquid-drop model in which a variational method is used to simultaneously solve the hydrodynamic equations for low Reynolds-number flow and the equations for membrane equilibrium with a constant membrane tension. It is found that the flow in the end caps is plug and radial in the conical section of the drop. The results are compared to a simplified analytical theory that makes these assumptions. Very good agreement is found between the two approaches. Both approaches are used to analyze existing experimental results of passive neutrophils flowing down a tapered tube. The theoretical models give a good fit to published experimental data by Bagge et al. (1977) at driving pressures of 20 and 40 mm H2O for a membrane cortical tension of 0.024 dyn/cm and an apparent cytoplasmic viscosity of about 2400 and 1400 poise, respectively.

Measuring the mechanical properties of individual human blood cells.

The largest human blood cells--the red cells (erythrocytes) and white cells (leukocytes)--must undergo a significant amount of deformation as they squeeze through the smallest vessels of the circulation and the small openings between bone, vessel and tissue. This ability to deform in response to external forces shows that cells exhibit material behavior and behave as either elastic solids or viscous liquids. The question then is "how can we measure the deformation and flow of something as small as a blood cell and what kinds of constitutive equations describe cellular deformation"? In this paper the use of the micropipet to measure red cell and white cell, especially neutrophil, deformation will be described and the viscoelastic models used to describe the deformation behavior of red cell membrane and neutrophil cytoplasm will be discussed. Values for the elasticities of a red cell membrane subjected to shear, area expansion and bending will be given. The viscosity of red cell membrane in shear will also be discussed. Finally, the cortical tension of the neutrophil and the Newtonian and Maxwell models used to characterize its apparent viscosity will be discussed even though neither is wholly successful in describing the viscous behavior of the neutrophil. Thus, alternate models will be suggested.

Volume and osmotic properties of human neutrophils.

Quantitative models describing the dynamics of human neutrophils in the microcirculation require accurate morphometric parameters such as volume and surface membrane area. Using both a micropipette technique and video light microscopy (LM) to measure the diameters of the spherical cells, we have accurately determined the volume of the human neutrophil. Our value, 299 +/- 32 microns 3, is in good agreement with our earlier results, but 55% larger than that reported by Schmid-Schönbein et al (Blood 56:866, 1980). However, the measurements of Schmid-Schönbein et al were based on the actual mass of the cells derived from transmission electron microscopic (TEM) images. The membrane surface area, at lysis, was calculated to be 2.6 times its initial projected area. After lysis, the cells do not reduce their size, indicative of the possibility of a F-actin network formation that would stiffen the structure. Further, we show that neutrophils behave as ideal osmometers when exposed to anisotonic solutions at 21 degrees C, as predicted by the Boyle-Van't Hoff relationship. The calculated Ponder's value, R, is 0.77, which corresponds to 77% of the cell volume being osmotically active under isotonic conditions. However, at 37 degrees C, the cells are able to regulate their volumes toward the original volumes after an osmotic stress.

Viscosity of passive human neutrophils undergoing small deformations.

At issue is the type of constitutive equation that can be used to describe all possible types of deformation of the neutrophil. Here a neutrophil undergoing small deformations is studied by aspirating it into a glass pipet with a diameter that is only slightly smaller than the diameter of the spherically shaped cell. After being held in the pipet for at least seven seconds, the cell is rapidly expelled and allowed to recover its undeformed, spherical shape. The recovery takes approximately 15 s. An analysis of the recovery process that treats the cell as a simple Newtonian liquid drop with a constant cortical (surface) tension gives a value of 3.3 x 10(-5) cm/s for the ratio of the cortical tension to cytoplasmic viscosity. This value is about twice as large as a previously published value obtained with the same model from studies of large deformations of neutrophils. This discrepancy indicates that the cytoplasmic viscosity decreases as the amount of deformation decreases. An extrapolated value for the cytoplasmic viscosity at zero deformation is approximately 600 poise when a value for the cortical tension of 0.024 dyn/cm is assumed. Clearly the neutrophil does not behave like a simple Newtonian liquid drop in that small deformations are inherently different from large deformations. More complex models consisting either of two or more fluids or multiple shells must be developed. The complex structure inside the neutrophil is shown in scanning electron micrographs of osmotically burst cells and cells whose membrane has been dissolved away.

The effect of flunarizine on erythrocyte suspension viscosity under conditions of extreme hypoxia, low pH, and lactate treatment.

Flunarizine is a class IV calcium channel blocker which increases oxygen delivery to hypoxic regions in solid tumours, exerting a radiosensitising effect in vivo in animal tumour models. Precisely how the drug improves oxygenation is not well understood. We hypothesised that metabolic conditions present within solid tumours reduce red blood cell (RBC) deformability and that flunarizine exerts its in vivo effect by preventing this loss of RBC deformability. A microrheometer was used to compare the viscosity of rat and human RBC suspensions in conditions of hypoxia (pO2 < 10 mmHg), acidic environment (pH 6.8), and elevated lactate concentration (lactate 5 mMol l-1), without or with flunarizine at concentrations of 5, 10, and 50 mg l-1. The effects of flunarizine on RBC density and morphology were also recorded. Hypoxia, low pH, and lactate exposure together increased both human and rat RBC suspension viscosity. Flunarizine at concentrations of 5 and 10 mg l-1 prevented the increases in viscosity. The drug caused dose-dependent shifts toward lower cell density while inducing a characteristic cupped shape (stomatcytic morphology), suggesting a mechanism involving calmodulin inhibition. The results support the hypothesis that flunarizine improves tumour blood flow and oxygenation by enhancing flow properties of RBC's in solid tumours.

A sensitive measure of surface stress in the resting neutrophil.

The simplest parameterized model of the "passive" or "resting receptive" neutrophil views the cell as being composed of an outer cortex surrounding an essentially liquid-like highly viscous cytoplasm. This cortex has been measured to maintain a small persistent tension of approximately 0.035 dyn/cm (Evans and Yeung. 1989. Biophys. J. 56:151-160) and is responsible for recovering the spherical shape of the cell after large deformation. The origin of the cortical tension is at present unknown, but speculations are that it may be an active process related to the sensitivity of a given cell to external stimulation and the "passive-active" transition. In order to characterize further this feature of the neutrophil we have used a new micropipet manipulation method to give a sensitive measure of the surface stress as a function of the surface area dilation of the highly ruffled cellular membrane. In the experiment, a single cell is driven down a tapered pipet in a series equilibrium deformation positions. Each equilibrium position represents a balance between the stress in the membrane and the pressure drop across the cell. For most cells that seemed to be "passive," as judged by their spherical appearance and lack of pseudopod activity, area dilations of approximately 30% were accompanied by only a small increase in the membrane tension, indicative of a very small apparent elastic area expansion modulus (approximately 0.04 dyn/cm). Extrapolations back to zero area dilation gave a value for the tension in the resting membrane of 0.024 +/- 0.003 dyn/cm, in close agreement with earlier measures. A few cells showed virtually no change in cortical tension and fit the persistent cortical tension model of Evans and Yeung (1989. Biophys. J. 56:151-160). However, other cells that also appeared "passive," as judged by their spherical appearance, had membrane tensions that increased as the apparent surface area was increased. Thus, the postulated,persistent "cortical tension" does not appear to be a unique and constant parameter for all cells as the membrane area is dilated.This measurement of membrane tension could represent a sensitive indication of the first stages of cell activation and the"passive-active" transition.