G protein-coupled receptors serve as mechanosensors for fluid shear stress in neutrophils

Many cells respond to fluid shear stress but in a cell type-specific fashion. Fluid shear stress applied to leukocytes serves to control pseudopod formation, migration, and other functions. Specifically, fresh neutrophils or neutrophilic leukocytes derived from differentiated HL60 cells respond to fluid shear stress by cytoplasmic pseudopod retraction. The membrane elements that sense fluid shear and induce such a specific response are still unknown, however. We hypothesized that membrane receptors may serve as fluid shear sensors. We found that fluid shear decreased the constitutive activity of G protein-coupled receptors (GPCRs). Inhibition of GPCR constitutive activity by inverse agonists abolished fluid shear stress-induced cell area reduction. Among the GPCRs in neutrophils, the formyl peptide receptor (FPR) exhibits relatively high constitutive activity. Undifferentiated HL60 cells that lacked FPR formed few pseudopods and showed no detectable response to fluid shear stress, whereas expression of FPR in undifferentiated HL60 cells caused pseudopod projection and robust pseudopod retraction during fluid shear. FPR small interfering RNA-transfected differentiated HL60 cells exhibited no response to fluid shear stress. These results suggest that GPCRs serve as mechanosensors for fluid shear stress in neutrophils by decreasing its constitutive activity and reducing pseudopod projection. leukocyte; constitutive activity; mechanotransduction; formyl peptide receptor     

Control of neutrophil pseudopods by fluid shear: role of Rho family GTPases

Blood vessels and blood cells are under continuous fluid shear. Studies on vascular endothelium and smooth muscle cells have shown the importance of this mechanical stress in cell signal transduction, gene expression, vascular remodeling, and cell survival. However, in circulating leukocytes, shear-induced signal transduction has not been investigated. Here we examine in vivo and in vitro the control of pseudopods in leukocytes under the influence of fluid shear stress and the role of the Rho family small GTPases. We used a combination of HL-60 cells differentiated into neutrophils (1.4% dimethyl sulfoxide for 5 days) and fresh leukocytes from Rac knockout mice. The cells responded to shear stress (5 dyn/cm²) with retraction of pseudopods and reduction of their projected cell area. The Rac1 and Rac2 activities were decreased by fluid shear in a time- and magnitude-dependent manner, whereas the Cdc42 activity remained unchanged (up to 5 dyn/cm²). The Rho activity was transiently increased and recovered to static levels after 10 min of shear exposure (5 dyn/cm²). Inhibition of either Rac1 or Rac2 slightly but significantly diminished the fluid shear response. Transfection with Rac1-positive mutant enhanced the pseudopod formation during shear. Leukocytes from Rac1-null and Rac2-null mice had an ability to form pseudopods in response to platelet-activating factor but did not respond to fluid shear in vitro. Leukocytes in wild-type mice retracted pseudopods after physiological shear exposure, whereas cells in Rac1-null mice showed no retraction during equal shear. On leukocytes from Rac2-null mice, however, fluid shear exerted a biphasic effect. Leukocytes with extended pseudopods slightly decreased in length, whereas initially round cells increased in length after shear application. The disruption of Rac activity made leukocytes nonresponsive to fluid shear, induced cell adhesion and microvascular stasis, and decreased microvascular density. These results suggest that deactivation of Rac activity by fluid shear plays an important role in stable circulation of leukocytes. microcirculation; mechanotransduction; actin polymerization; transgenic mouse; leukocyte     

Recoil and stiffening by adherent leukocytes in response to fluid shear

Prolonged exposure to fluid shear stress alters leukocyte functions associated with the immune response. We examined the initial response of freshly isolated human leukocytes to fluid shear stress under high magnification. Adherent leukocytes exhibit a rapid biomechanical response to physiological levels of fluid shear stress. After passive displacement in the direction of a constant fluid shear stress, adherent leukocytes actively recoil back in the opposite direction of the fluid flow. Recoil is observed within seconds of the applied fluid shear stress. Simultaneously, fluid shear stress induces a stiffening of the cell. The immediate cell displacement in response to a step increase in fluid shear stress is greatly attenuated in subsequent steps compared to the initial fluid shear stress step. Recoil is not mediated by actin polymerization-dependent mechanisms, as cytochalasin D had no effect on this early response. However, stiffening was determined in part by an intact actin cytoskeleton. Inhibiting myosin force generation with ML-7 abolished the recoil and stiffening responses, implicating force generation by myosin as an important contributor to the early leukocyte response to fluid shear stress. This initial shear stress response may be particularly important in facilitating leukocyte attachment under sustained fluid shear stress by the flowing blood in the microcirculation.     

Fluid shear-induced activation and cleavage of CD18 during pseudopod retraction by human neutrophils

Surface membrane expression and conformational activation of CD18 integrins into an open molecular configuration play critical roles in neutrophil ligand binding, membrane attachment, spreading on the endothelium, and cell migration to sites of inflammation. Previously, we observed pseudopod retraction and concomitant cleavage of CD18 by human neutrophils upon exposure to fluid shear stress. But the underlying cellular mechanism(s) linking these phenomena remains unknown. We hypothesize here that activation of CD18 under the influence of fluid shear stress leads to its increased susceptibility to proteolytic cleavage by lysosomal proteases such as cathepsin B and is a requirement for CD18 cleavage and subsequent pseudopod retraction. Specifically, we report conformational changes in the CD18 extracellular domain on neutrophils exposed to physiological fluid shear stresses. Western blot analysis using a CD18 antibody targeted against the intracellular domain revealed reduced levels of full-length CD18 after stimulation of neutrophils with either fluid shear stress or with the Ca2+ ionophore phorbol 12-myristate 13-acetate (PMA; 100 nM) in the presence of exogenous cathepsin B (0.5 U/ml). Moreover, we identified cathepsin B as one protease that may be released by neutrophils under flow and required for shear-induced pseudopod retraction. These results suggest that a putative mechanotransduction mechanism involving shear-induced changes in the conformation of CD18 and its subsequent cleavage from the cell surface serves to regulate pseudopod activity of neutrophils under physiologic shear stress.     

Leukocyte biophysics. An invited review.

The biophysical properties of leukocytes in the passive and active state are discussed. In the passive unstressed state, leukocytes are spherical with numerous membrane folds. Passive leukocytes exhibit viscoelastic properties, and the stress is carried largely by the cell cytoplasm and the nucleus. The membrane is highly deformable in shearing and bending, but resists area expansion. Membrane tension can usually be neglected but plays a role in cases of large deformation when the membrane becomes unfolded. The constant membrane area constraint is a determinant of phagocytic capacity, spreading of cells, and passage through narrow pores. In the active state, leukocytes undergo large internal cytoplasmic deformation, pseudopod projection, and granule redistribution. Several different measurements for assessment of biophysical properties and the internal cytoplasmic deformation in form of strain and strain rate tensors are presented. The current theoretical models for active cytoplasmic motion in leukocytes are discussed in terms of specific macromolecular reactions.     

Leukocyte biophysics

The biophysical properties of leukocytes in the passive and active state are discussed. In the passive unstressed state, leukocytes are spherical with numerous membrane folds. Passive leukocytes exhibit viscoelastic properties, and the stress is carried largely by the cell cytoplasm and the nucleus. The membrane is highly deformable in shearing and bending, but resists area expansion. Membrane tension can usually be neglected but plays a role in cases of large deformation when the membrane becomes unfolded. The constant membrane area constraint is a determinant of phagocytic capacity, spreading of cells, and passage through narrow pores. In the active state, leukocytes undergo large internal cytoplasmic deformation, pseudopod projection, and granule redistribution. Several different measurements for assessment of biophysical properties and the internal cytoplasmic deformation in form of strain and strain rate tensors are presented. The current theoretical models for active cytoplasmic motion in leukocytes are discussed in terms of specific macromolecular reactions.     

A Model for a Correlated Random Walk Based on the Ordered Extension of Pseudopodia

Cell migration in the absence of external cues is well described by a correlated random walk. Most single cells move by extending protrusions called pseudopodia. To deduce how cells walk, we have analyzed the formation of pseudopodia by Dictyostelium cells. We have observed that the formation of pseudopodia is highly ordered with two types of pseudopodia: First, de novo formation of pseudopodia at random positions on the cell body, and therefore in random directions. Second, pseudopod splitting near the tip of the current pseudopod in alternating right/left directions, leading to a persistent zig-zag trajectory. Here we analyzed the probability frequency distributions of the angles between pseudopodia and used this information to design a stochastic model for cell movement. Monte Carlo simulations show that the critical elements are the ratio of persistent splitting pseudopodia relative to random de novo pseudopodia, the Left/Right alternation, the angle between pseudopodia and the variance of this angle. Experiments confirm predictions of the model, showing reduced persistence in mutants that are defective in pseudopod splitting and in mutants with an irregular cell surface.     

Macrorheology and adaptive microrheology of endothelial cells subjected to fluid shear stress

Vascular endothelial cells (ECs) respond to temporal and spatial characteristics of hemodynamic forces by alterations in their adhesiveness to leukocytes, secretion of vasodilators, and permeability to blood-borne constituents. These physiological and pathophysiological changes are tied to adaptation of cell mechanics and mechanotransduction, the process by which cells convert forces to intracellular biochemical signals. The exact time scales of these mechanical adaptations, however, remain unknown. We used particle-tracking microrheology to study adaptive changes in intracellular mechanics in response to a step change in fluid shear stress, which simulates both rapid temporal and steady features of hemodynamic forces. Results indicate that ECs become significantly more compliant as early as 30 s after a step change in shear stress from 0 to 10 dyn/cm² followed by recovery of viscoelastic parameters within 4 min of shearing, even though shear stress was maintained. After ECs were sheared for 5 min, return of shear stress to 0 dyn/cm² in a stepwise manner did not result in any further rheological adaptation. Average vesicle displacements were used to determine time-dependent cell deformation and macrorheological parameters by fitting creep function to a linear viscoelastic liquid model. Characteristic time and magnitude for shear-induced deformation were 3 s and 50 nm, respectively. We conclude that ECs rapidly adapt their mechanical properties in response to shear stress, and we provide the first macrorheological parameters for time-dependent deformations of ECs to a physiological forcing function. Such studies provide insight into pathologies such as atherosclerosis, which may find their origins in EC mechanics. viscoelasticity; atherosclerosis; cell mechanics; particle tracking; mechanotransduction     

Mechanotransduction in leukocyte activation: a review

We review recent evidence which suggests that leukocytes in the circulation and in the tissue may readily respond to physiological levels of fluid shear stress in the range between about 1 and 10 dyn/cm 2, a range that is below the level to achieve a significant passive, viscoelastic response. The response of activated neutrophilic leukocytes to fluid shear consists of a rapid retraction of lamellipodia with membrane detachment from integrin binding sites. In contrast, a subgroup of non-activated neutrophils may project pseudopods after exposure to fluid shear stress. The evidence suggests that G-protein coupled receptor downregulation by fluid shear with concomitant downregulation of Rac-related small GTPases and depolymerization of F-actin serves to retract the lamellipodia in conjunction with proteolytic cleavage of beta 2 integrin to facilitate membrane detachment. Furthermore, there exists a mechanism to up- and down-regulate the fluid shear-response, which involves nitric oxide and the second messenger cyclic guanosine monophosphate (cGMP). Many physiological activities of circulating leukocytes are under the influence of fluid shear stress, including transendothelial migration of lymphocytes. We describe a disease model with chronic hypertension that suffers from an attenuated fluid shear-response with far reaching implications for microvascular blood flow.     

The dynamics and mechanics of endothelial cell spreading

Cell adhesion to extracellular matrix is mediated by receptor-ligand interactions. When a cell first contacts a surface, it spreads, exerting traction forces against the surface and forming new bonds as its contact area expands. Here, we examined the changes in shape, actin polymerization, focal adhesion formation, and traction stress generation that accompany spreading of endothelial cells over a period of several hours. Bovine aortic endothelial cells were plated on polyacrylamide gels derivatized with a peptide containing the integrin binding sequence RGD, and changes in shape and traction force generation were measured. Notably, both the rate and extent of spreading increase with the density of substrate ligand. There are two prominent modes of spreading: at higher surface ligand densities cells tend to spread isotropically, whereas at lower densities of ligand the cells tend to spread anisotropically, by extending pseudopodia randomly distributed along the cell membrane. The extension of pseudopodia is followed by periods of growth in the cell body to interconnect these extensions. These cycles occur at very regular intervals and, furthermore, the extent of pseudopodial extension can be diminished by increasing the ligand density. Measurement of the traction forces exerted by the cell reveals that a cell is capable of exerting significant forces before either notable focal adhesion or stress fiber formation. Moreover, the total magnitude of force exerted by the cell is linearly related to the area of the cell during spreading. This study is the first to monitor the dynamic changes in the cell shape, spreading rate, and forces exerted during the early stages (first several hours) of endothelial cell adhesion.     

Leukocytes of Biomphalaria glabrata: Morphology and behavior of granulocytic cells in vitro

Blood cells from Biomphalaria glabrata hemolymph were studied by phase microscopy in vitro. The most common cell is a granulocytic leukocyte, present in the hemolymph at a concentration of 334 ± 105 cells/mm³. This cell ranges in length from 14.1 ± 2.8 μm (including pseudopodia), when suspended in hemolymph, to 77.2 ± 15.1 μm, when allowed to spread on a glass substrate. Cytoplasmic inclusions and other organelles are described. Observations on the behavior of these granulocytic leukocytes in vitro confirm that a strong phagocytic response develops toward a variety of particles in the virtual absence of snail hemolymph.     

Quantitation of cell area on glass and fibronectin-coated surfaces by digital image analysis.

By using digital image processing and analysis, two procedures were developed to rapidly measure the projected area of a field of adherent 3T3 fibroblasts without staining of cell borders. The cell area of newly attached and rounded cells with well-resolved borders was obtained by a gray value thresholding procedure. For cells that had undergone an appreciable degree of spreading, cell boundaries were less distinct and a nonlinear spatial Sobel filter was used, followed by thresholding. For both procedures, linear relations were observed between cell areas obtained from image analysis and cell areas obtained by tracing. The areas of a population of traced cells were not statistically different from the area distribution obtained by using the standard curves for the processed images. Uncertainty in the estimated mean area depended only upon the number of cells examined. Approximate numbers of cells required to obtain estimates of the mean are calculated. As an application of these procedures, cell areas were measured for 3T3 cells attached to glass and fibronectin-coated surfaces and were found to be significantly larger for cells spreading on fibronectin-coated glass than on glass alone. Increased cell area during spreading on fibronectin-coated surfaces was proportional to increased cell adhesivity after exposure to a shear stress of 58 dyn/cm2.     

Shear-induced resistance to neutrophil activation via the formyl peptide receptor

The application of fluid shear stress on leukocytes is critical for physiological functions including initial adhesion to the endothelium, the formation of pseudopods, and migration into tissues. The formyl peptide receptor (FPR) on neutrophils, which binds to formyl-methionyl-leucyl-phenylalanine (fMLP) and plays a role in neutrophil chemotaxis, has been implicated as a fluid shear stress sensor that controls pseudopod formation. The role of shear forces on earlier indicators of neutrophil activation, such as L-selectin shedding and α(M)β(2) integrin activation, remains unclear. Here, human neutrophils exposed to uniform shear stress (0.1-4.0 dyn/cm(2)) in a cone-and-plate viscometer for 1-120 min showed a significant reduction in both α(M)β(2) integrin activation and L-selectin shedding after stimulation with 0.5 nM of fMLP. Neutrophil resistance to activation was directly linked to fluid shear stress, as the response increased in a shear stress force- and time-dependent manner. Significant shear-induced loss of FPR surface expression on neutrophils was observed, and high-resolution confocal microscopy revealed FPR internalized within neutrophils. These results suggest that physiological shear forces alter neutrophil activation via FPR by reducing L-selectin shedding and α(M)β(2) integrin activation in the presence of soluble ligand.     

A finite element model predicts the mechanotransduction response of tendon cells to cyclic tensile loading

The importance of fluid-flow-induced shear stress and matrix-induced cell deformation in transmitting the global tendon load into a cellular mechanotransduction response is yet to be determined. A multiscale computational tendon model composed of both matrix and fluid phases was created to examine how global tendon loading may affect fluid-flow-induced shear stresses and membrane strains at the cellular level. The model was then used to develop a quantitative experiment to help understand the roles of membrane strains and fluid-induced shear stresses on the biological response of individual cells. The model was able to predict the global response of tendon to applied strain (stress, fluid exudation), as well as the associated cellular response of increased fluid-flow-induced shear stress with strain rate and matrix-induced cell deformation with strain amplitude. The model analysis, combined with the experimental results, demonstrated that both strain rate and strain amplitude are able to independently alter rat interstitial collagenase gene expression through increases in fluid-flow-induced shear stress and matrix-induced cell deformation, respectively.     

Evidence for contractile protein translocation in macrophage spreading, phagocytosis, and phagolysosome formation

Macrophage pseudopodia that surround objects during phagocytosis contain a meshwork of actin filaments and exclude organelles. Between these pseudopodia at the base of developing phagosomes, the organelle exclusion ceases, and lysosomes enter the cell periphery to fuse with the phagosomes. Macrophages also extend hyaline pseudopodia on the surface of nylon wool fibers and secrete lysosomal enzymes into the extracellular medium instead of into phagosomes. To analyze biochemically these concurrent alterations in cytoplasmic architecture, we allowed rabbit lung macrophages to spread on nylon wool fibers and then subjected the adherent cells to shear. This procedure caused the selective release of β-glucoronidase into the extracellular medium and yielded two fractions, cell bodies and isolated pseudopod blebs resembling podosomes, which are plasma-lemma-bounded sacs of cortical cytoplasm. Cytoplasmic extracts of the cell bodies eluted from nylon fibers contained two-thirds less actin-binding protein and myosin, and approximately 20 percent less actin and two-thirds of the other two proteins were accounted for in podosomes. The alterations in protein composition correlated with assays of myosin-associated EDTA-activated adenosine triphosphatase activity, and with a diminution in the capacity of extracts of nylon wool fiber-treated cell bodies to gel, a property dependent on the interaction between actin-binding protein and F-actin. However, the capacity of the remaining actin in cell bodies to polymerize did not change. We propose that actin-binding protein and myosin are concentrated in the cell cortex and particularly in pseudopodia where prominent gelation and syneresis of actin occur. Actin in the regions from which actin-binding protein and myosin are displaced disaggregates without depolymerizing, permitting lysosomes to gain access to the plasmalemma. Translocation of contractile proteins could therefore account for the concomitant differences in organelle exclusion that characterize phagocytosis.     

Myosin I phosphorylation is increased by chemotactic stimulation

Directed cell migration occurs in response to extracellular cues. Following stimulation of a cell with chemoattractant, a significant rearrangement of the actin cytoskeleton is mediated by intracellular signaling pathways and results in polarization of the cell and movement via pseudopod extension. Amoeboid myosin Is play a critical role in regulating pseudopod formation in Dictyostelium, and their activity is activated by heavy chain phosphorylation. The effect of chemotactic stimulation on the in vivo phosphorylation level of a Dictyostelium myosin I, myoB, was tested. The myoB heavy chain is phosphorylated in vivo on serine 322 (the myosin TEDS rule phosphorylation site) in chemotactically competent cells. The level of myoB phosphorylation increases following stimulation of starving cells with the chemoattractant cAMP. A 3-fold peak increase in the level of phosphorylation is observed at 60 s following stimulation, a time at which the Dictyostelium cell actively extends pseudopodia. These findings suggest that chemotactic stimulation results in increased myoB activity via heavy chain phosphorylation and contributes to the global extension of pseudopodia that occurs prior to polarization and directed motility.     

Guanylyl cyclase protein and cGMP product independently control front and back of chemotaxing Dictyostelium cells

Chemotaxis of amoeboid cells is driven by actin filaments in leading pseudopodia and actin-myosin filaments in the back and at the side of the cell to suppress pseudopodia. In Dictyostelium, cGMP plays an important role during chemotaxis and is produced predominantly by a soluble guanylyl cyclase (sGC). The sGC protein is enriched in extending pseudopodia at the leading edge of the cell during chemotaxis. We show here that the sGC protein and the cGMP product have different functions during chemotaxis, using two mutants that lose either catalytic activity (sGCDelta cat) or localization to the leading edge (sGCDeltaN). Cells expressing sGCDeltaN exhibit excellent cGMP formation and myosin localization in the back of the cell, but they exhibit poor orientation at the leading edge. Cells expressing the catalytically dead sGCDelta cat mutant show poor myosin localization at the back, but excellent localization of the sGC protein at the leading edge, where it enhances the probability that a new pseudopod is made in proximity to previous pseudopodia, resulting in a decrease of the degree of turning. Thus cGMP suppresses pseudopod formation in the back of the cell, whereas the sGC protein refines pseudopod formation at the leading edge.     

Orientation of endothelial cells in shear fields in vitro

Vascular endothelial cells subjected to fluid shear stress change their shape from polygonal to ellipsoidal and become uniformly oriented with the flow. In order to study the mechanisms of this response, we have measured the relaxation of bovine aortic endothelial cells that were grown on glass coverslips and exposed to fluid shear stress for 72 hours. An image analysis system was developed to quantify the cell shape relaxation that occurs following the cessation of shear stress. This method provides two different quantitative measures of relaxation: the loss of elongated shape by the cells and the change in cell direction with time. After equilibration to a fluid shear stress level of 8 dynes/cm2, cells immersed in static medium relax their shape in about 20 hours. After 72 hours in this static condition, the cell elongation is comparable to that of unstressed control cells but vestiges remain of the original orientation in the flow direction. This relaxation process contributes to our understanding of the response of vascular endothelium to fluid shear stress.     

A continuum model of protrusion of pseudopod in leukocytes.

The morphology of human leukocytes, the biochemistry of actin polymerization, and the theory of continuum mechanics are used to model the pseudopod protrusion process of leukocytes. In the proposed model, the pseudopod is considered as a porous solid of F-actin network, the pores of which are full of aqueous solution. G-actin is considered as a "solute" transported by convection and diffusion in the fluid phase. The pseudopod grows as actin filaments elongate at their barbed ends at the tip of the pseudopod. The driving force of extension is hypothesized as being provided by the actin polymerization. It is assumed that elongation of actin filaments, powered by chemical energy liberated from the polymerization reaction, does mechanical work against opposing pressure on the membrane. This also gives rise to a pressure drop in the fluid phase at the tip of the pseudopod, which is formulated by an equation relating the work done by actin polymerization to the local state of pressure. The pressure gradient along the pseudopod drives the fluid filtration through the porous pseudopod according to Darcy's Law, which in turn brings more actin monomers to the growing tip. The main cell body serves as a reservoir of G-actin. A modified first-order equation is used to describe the kinetics of polymerization. The rate of pseudopod growth is modulated by regulatory proteins. A one-dimensional moving boundary problem based on the proposed mechanism has been constructed and approximate solutions have been obtained. Comparison of the solutions with experimental data shows that the model is compatible with available observations. The model is also applicable to growth of other cellular systems such as elongation of acrosomal process in sperm cells.