P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin

Neutrophils roll on P-selectin expressed by activated platelets or endothelial cells under the shear stresses in the microcirculation. P- selectin glycoprotein ligand-1 (PSGL-1) is a high affinity ligand for P- selectin on myeloid cells. However, it has not been demonstrated that PSGL-1 contributes to the rolling of neutrophils on P-selectin. We developed two IgG mAbs, PL1 and PL2, that appear to recognize protein- dependent epitopes on human PSGL-1. The mAbs bound to PSGL-1 on all leukocytes as well as on heterologous cells transfected with PSGL-1 cDNA. PL1, but not PL2, blocked binding of 125-I-PSGL-1 to immobilized P-selectin, binding of fluid-phase P-selectin to myeloid and lymphoid leukocytes, adhesion of neutrophils to immobilized P-selectin under static conditions, and rolling of neutrophils on P-selectin-expressing CHO cells under a range of shear stresses. PSGL-1 was localized to microvilli on neutrophils, a topography that may facilitate its adhesive function. These data indicate that (a) PSGL-1 accounts for the high affinity binding sites for P-selectin on leukocytes, and (b) PSGL- 1 must interact with P-selectin in order for neutrophils to roll on P- selectin at physiological shear stresses.     

Immobilized IL-8 triggers progressive activation of neutrophils rolling in vitro on P-selectin and intercellular adhesion molecule-1

The chemokine IL-8 is found on the luminal side of vascular endothelial cells, where it is postulated to be immobilized during inflammation. In this study, we observed that immobilized IL-8 can stimulate neutrophils to firmly adhere to a substrate containing ICAM-1 in a static adhesion assay. Soluble IL-8 was then perfused over neutrophils rolling on P-selectin (P-sel) and ICAM-1, confirming that IL-8 in solution can quickly cause rolling neutrophils to arrest. To mimic a blood vessel wall with IL-8 expressed on the luminal surface of endothelial cells, IL-8 was immobilized along with P-sel and ICAM-1 at defined site densities to a surface. Neutrophils rolled an average of 200 microm on surfaces of P-sel, ICAM-1, and IL-8 before firmly adhering through ICAM-1-beta(2) integrin interactions at 2 dynes/cm(2) wall shear stress. Increasing the density of IL-8 from 60 to 350 sites/microm(2) on the surface decreased by 50% the average distance and time the neutrophils rolled before becoming firmly adherent. Temporal dynamics of ICAM-1-beta(2) integrin interactions of rolling neutrophils following IL-8 exposure suggest the existence of two classes of beta(2) integrin-ICAM-1 interactions, a low avidity interaction with a 65% increase in pause times as compared with P-sel-P-sel glycoprotein ligand-1 interactions, and a high avidity interaction with pause times 400% greater than the selectin interactions. Based on the proportionality between IL-8 site density and time to arrest, it appears that neutrophils may need to sample a critical number of IL-8 molecules presented by the vessel wall before forming a sufficient number of high avidity beta(2) integrin bonds for firm adhesion.     

P-selectin must extend a sufficient length from the plasma membrane to mediate rolling of neutrophils

Under physiological shear stress, neutrophils roll on P-selectin on activated endothelial cells or platelets through interactions with P- selectin glycoprotein ligand-1 (PSGL-1). Both P-selectin and PSGL-1 are extended molecules. Human P-selectin contains an NH2-terminal lectin domain, an EGF domain, nine consensus repeats (CRs), a transmembrane domain, and a cytoplasmic tail. To determine whether the length of P- selectin affected its interactions with PSGL-1, we examined the adhesion of neutrophils to CHO cells expressing membrane-anchored P- selectin constructs in which various numbers of CRs were deleted. Under static conditions, neutrophils attached equivalently to wild-type P- selectin and to constructs containing from 2-6 CRs. Under shear stress, neutrophils attached equivalently to wild-type and 6 CR P-selectin and nearly as well to 5 CR P-selectin. However, fewer neutrophils attached to the 4 CR construct, and those that did attach rolled faster and were more readily detached by increasing shear stress. Flowing neutrophils failed to attach to the 3 CR and 2 CR constructs. Neutrophils attached and rolled more efficiently on 4 CR P-selectin expressed on glycosylation-defective Lec8 CHO cells, which have less glycocalyx. We conclude that P-selectin must project its lectin domain well above the membrane to mediate optimal attachment of neutrophils under shear forces. The length of P-selectin may: (a) facilitate interactions with PSGL-1 on flowing neutrophils, and (b) increase the intermembrane distance where specific bonds form, minimizing contacts between the glycocalyces that result in cell-cell repulsion.     

Tyrosine sulfation enhances but is not required for PSGL-1 rolling adhesion on P-selectin

P-selectin glycoprotein ligand-1 (PSGL-1) is a large (240 kDa) glycoprotein found on the surface of nearly all leukocytes. The mature molecule is decorated with multiple N- and O-linked glycans and displays copies of the tetrasaccharide sialyl-Lewis(x) (sLe(X)), as well as a cluster of three tyrosine sulfate (tyr-SO(3)) groups near the N-terminus of the processed protein. Previous studies have suggested that PSGL-1 needs to be tyrosine-sulfated, in addition to glycosylated with sLe(X), to successfully interact with P-selectin. To better understand how biochemical features of the PSGL-1 ligand are related to its adhesion phenotype, we have measured the dynamics of adhesion under flow of a series of well-defined PSGL-1 variants that differ in their biochemical modification, to both P- and E-selectin-coated substrates. These variants are distinct PSGL-1 peptides: one that possesses sLe(X) in conjunction with three N-terminal tyr-SO(3) groups (SGP3), one that possesses sLe(X) without tyrosine sulfation (GP1), and one that lacks sLe(X) but has three N-terminal tyr-SO(3) groups (SP3). Although all peptides expressing sLe(X), tyr-SO(3), or both supported some form of rolling adhesion on P-selectin, only peptides expressing sLe(X) groups showed rolling adhesion on E-selectin. On P-selectin, the PSGL-1 peptides demonstrated a decreasing strength of adhesion in the following order: SGP3 > GP1 > SP3. Robust, rolling adhesion on P-selectin was mediated by the GP1 peptide, despite its lack of tyrosine sulfation. However, the addition of tyrosine sulfation to glycosylated peptides (SGP3) creates a super ligand for P-selectin that supports slower rolling adhesion at all shear rates and supports rolling adhesion at much higher shear rates. Tyrosine sulfation has no similar effect on PSGL-1 rolling on E-selectin. Such functional distinctions in rolling dynamics are uniquely realized with a cell-free system, which permits precise, unambiguous identification of the functional activity of adhesive ligands. These findings are consistent with structural and functional characterizations of the interactions between these peptides and E- and P-selectin published recently.     

Sialyl Lewis(x)-mediated, PSGL-1-independent rolling adhesion on P-selectin

Selectin-mediated cell adhesion is an essential component of the inflammatory response. In an attempt to unambiguously identify molecular features of ligands that are necessary to support rolling adhesion on P-selectin, we have used a reconstituted ("cell-free") system in which ligand-coated beads are perfused over soluble P-selectin surfaces. We find that beads coated with the saccharides sialyl Lewis(x) (sLe(x)), sialyl Lewis(a) (sLe(a)), and sulfated Lewis(x) (HSO(3)Le(x) support rolling adhesion on P-selectin surfaces. Although it has been suggested that glycosylation and sulfation of P-selectin glycoprotein ligand-1 (PSGL-1) is required for high-affinity binding and rolling on P-selectin, our findings indicate that sulfation of N-terminal tyrosine residues is not required for binding or rolling. However, beads coated with a tyrosine-sulfated, sLe(x)-modified, PSGL-1-Fc chimera support slower rolling on P-selectin than beads coated with sLe(x) alone, suggesting that sulfation improves rolling adhesion by modulating binding to P-selectin. In addition, we find it is not necessary that P-selectin carbohydrate ligands be multivalent for robust rolling to occur. Our results demonstrate that beads coated with monovalent sLe(x), exhibiting a more sparse distribution of carbohydrate than a similar amount of the multivalent form, are sufficient to yield rolling adhesion. The relative abilities of various ligands to support rolling on P-selectin are quantitatively examined among themselves and in comparison to human neutrophils. Using stop-time distributions, rolling dynamics at video frame rate resolution, and the average and variance of the rolling velocity, we find that P-selectin ligands display the following quantitative trend, in order of decreasing ability to support rolling adhesion on P-selectin: PSGL-1-Fc > sLe(a) approximately sLe(x) > HSO(3)Le(x).     

A novel glycosulfopeptide binds to P-selectin and inhibits leukocyte adhesion to P-selectin.

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin on leukocytes that binds selectins. The molecular features of PSGL-1 that determine this high affinity binding are unclear. Here we demonstrate the in vitro synthesis of a novel glycosulfopeptide (GSP-6) modeled after the extreme N terminus of PSGL-1, which has been predicted to be important for P-selectin binding. GSP-6 contains three tyrosine sulfate (TyrSO(3)) residues and a monosialylated, core 2-based O-glycan with a sialyl Lewis x (C2-O-sLe(x)) motif at a specific Thr residue. GSP-6 binds tightly to immobilized P-selectin, whereas glycopeptides lacking either TyrSO(3) or C2-O-sLe(x) do not detectably bind. Remarkably, an isomeric glycosulfopeptide to GSP-6, termed GSP-6', which contains sLe(x) on an extended core 1-based O-glycan, does not bind immobilized P-selectin. Equilibrium gel filtration analysis revealed that GSP-6 binds to soluble P-selectin with a K(d) of approximately 350 nM. GSP-6 (<5 microM) substantially inhibits neutrophil adhesion to P-selectin in vitro, whereas free sLe(x) (5 mM) only slightly inhibits adhesion. In contrast to the inherent heterogeneity of post-translational modifications of recombinant proteins, glycosulfopeptides permit the placement of sulfate groups and glycans of precise structure at defined positions on a polypeptide. This approach should expedite the probing of structure-function relationships in sulfated and glycosylated proteins, and may facilitate development of novel drugs to treat inflammatory diseases involving P-selectin-mediated leukocyte adhesion.     

Membrane mobility of beta2 integrins and rolling associated adhesion molecules in resting neutrophils

The mobilities of transmembrane adhesion proteins are key underlying physical factors that contribute to neutrophil adhesion and arrest during inflammation. Here we present a novel (to our knowledge) fluorescence recovery after photobleaching system and a complementary analytical model to measure the mobility of the four key receptors involved in the adhesion cascade: L-selectin, PSGL-1, Mac-1, and LFA-1 for resting, spherical, and human neutrophils. In general, we find that β₂ integrins (Mac-1, LFA-1) have mobilities 3-7 times faster than rolling associated molecules (L-selectin; PSGL-1), but that the mobilities within each of these groups are indistinguishable. Increasing temperature (room temperature versus 37°C) results in increased mobility, in all cases, and the use of a bivalent antibody label (mAb versus Fab) decreases mobility, except in the case of rolling associated molecules at room temperature. Disrupting the actin cytoskeleton increased mobility except that the highest mobilities measured for integrins (D = 1.2 × 10⁻⁹ cm²/s; 37°C, Fab) are not affected by actin poisons and approach the expected value for free diffusion. Although evidence of cytoskeletal hindrance of integrin mobility has been found in other systems, our data suggest such hindrance does not limit bulk integrin diffusion in resting neutrophils over distances and times important for adhesive plaque formation.     

P-selectin activates integrin-mediated colon carcinoma cell adhesion to fibronectin

During hematogenous cancer metastasis, tumor cells separate from a primary mass, enter the bloodstream, disperse throughout the body, migrate across vessel walls, and generate distant colonies. The later steps of metastasis superficially resemble leukocyte extravasation, a process initiated by selectin-mediated cell tethering to the blood vessel wall followed by integrin-mediated arrest and transendothelial migration. Some cancer cells express P-selectin ligands and attach to immobilized P-selectin, suggesting that these cells can arrest in blood vessels using sequential selectin- and integrin-mediated adhesion, as do leukocytes. We hypothesize that selectin binding may regulate subsequent integrin-mediated steps in metastasis. Using a model system of cultured Colo 320 human colon adenocarcinoma cells incubated with soluble P-selectin-IgG chimeric protein, we have found that P-selectin can stimulate activation of the alpha(5)beta(1) integrin resulting in a specific increase of adhesion and spreading of these cells on fibronectin substrates. P-selectin binding also induced activation of p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol 3-kinase (PI3-K). PI3-K inhibitors blocked P-selectin-mediated integrin activation, cell attachment, and cell spreading. Inhibition of p38 MAPK activation blocked cell spreading, but not cell attachment. P-selectin binding also resulted in formation of a signaling complex containing PI3-K and p38 MAPK. These results suggest that P-selectin binding to tumor cells can activate alpha(5)beta(1) integrin via PI3-K and p38 MAPK signaling pathways leading to increased cell adhesion. We propose that P-selectin ligands are important tumor cell signaling molecules that modulate integrin-mediated cell adhesion in the metastatic process.     

P-selectin mediates neutrophil rolling on histamine-stimulated endothelial cells.

In postcapillary venules, marginating neutrophils (PMNs) are often seen rolling along the vessel wall prior to stopping and emigrating. There is substantial evidence in vitro and in vivo that the adhesion receptors E- and L-selectin participate in this phenomenon on cytokine-stimulated endothelium, and recent evidence has shown that a closely related adhesion receptor, P-selectin, is capable of mediating neutrophil rolling on an artificial membrane. Here we demonstrate and characterize PMN rolling on monolayers of human umbilical vein endothelial cells (HUVECs) stimulated with histamine to induce surface expression of P-selectin. Peak association of PMNs with the HUVECs occurs 10 min after histamine stimulation, and at a postcapillary venular wall shear stress of 2.0 dyn/cm2 the rolling velocity is 14 microns/s. Approximately 95% of the PMNs roll on the endothelial cells, 5% adhere firmly, and none migrate beneath the endothelial monolayer. Monoclonal antibody (MAb) G1, which binds P-selectin and blocks its adhesive function, completely prevents association of the PMNs with histamine-stimulated HUVEC, whereas the nonblocking anti-P-selectin MAb S12 does not. Treatment of PMNs with the anti-L-selectin MAb DREG56 reduces PMN adherence by approximately 50%. Anti-CD54 MAb R6.5 and anti-CD18 MAb R15.7 have little effect on the number of PMNs rolling on the HUVECs but completely prevent PMNs from stopping and significantly increase rolling velocity. Nonblocking control MAbs for R6.5 (CL203) and R15.7 (CL18/1D1) lack these effects. Rolling adhesion of PMNs on histamine-stimulated HUVECs therefore appears to be completely dependent on endothelial cell P-selectin, with a minor adhesion-stabilizing contribution from intercellular adhesion molecule 1 and beta 2 integrins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Limited adhesion of biodegradable microspheres to E- and P-selectin under flow

In a variety of disease settings the expression of the endothelial selectins E- and P-selectin appears to be increased. This feature makes these molecules attractive targets around which to design directed drug-delivery schemes. One possible approach for achieving such delivery is to use polymeric biodegradable microspheres bearing a humanized monoclonal antibody (MAb) for E- and P-selectin, MAb HuEP5C7.g2. Perhaps the simplest technique for "coupling" HuEP5C7.g2 to the microspheres is via nonspecific adsorption. Previous studies suggest, however, that the adsorption of proteins onto microspheres fabricated in the presence of a stabilizer such as poly(vinyl alcohol) (PVA) is limited. It is unclear to what extent this limited level of adsorbed HuEP5C7.g2 would be able to support adhesion to E- and P-selectin under flow conditions. To explore this issue, we prepared microspheres from the biodegradable polymer, poly(epsilon-caprolactone) (PCL), using a single emulsion process and PVA as a stabilizer. We then incubated the PCL microspheres with HuEP5C7.g2 and studied the adhesion of the resulting HuEP5C7.g2 microspheres to E- and P-selectin under in vitro flow conditions. We found that the HuEP5C7.g2 PCL microspheres exhibit specific adhesion to Chinese hamster ovary cells stably expressing P-selectin (CHO-P) and 4-h IL-1beta-activated human umbilical vein endothelial cells (HUVEC). In contrast, HuEP5C7.g2 PCL microspheres exhibit little adhesion to parental CHO cells or unactivated HUVEC. The attachment efficiency to the selectin substrates was quite low, with appreciable attachment occurring only at low shear (0.3 dyn/cm(2)). Other supporting data strongly suggest that the limited attachment efficiency is due to a low level of HuEP5C7.g2 adsorbed to the PCL microspheres. Although the attachment was limited, a significant percentage of the HuEP5C7.g2 PCL microspheres were able to remain adherent at relatively high shear (8 dyn/cm(2)). Combined, our data suggest that HuEP5C7.g2 PCL microspheres exhibit selective limited adhesion to cellular substrate expressing E- and P-selectin.     

A high affinity human antibody antagonist of P-selectin mediated rolling

We have characterized the IgG form of a previously isolated and engineered single-chain Fv (scFv), named RR2r3s4-1, that binds to human PSGL-1. This fully human IgG was determined to have a Kd of 1.8+/-0.7 nM by fluorescence quenching titration. It better inhibits P-selectin-PSGL-1 interactions than a commercially available murine monoclonal antibody KPL1 and better inhibits neutrophil rolling than KPL1. Thus, RR2r3s4-1 is the most effective antibody at inhibiting P-selectin-PSGL-1 interactions known. Specificity analysis reveals that RR2r3s4-1 does not cross react with murine PSGL-1 and thus requires more than tyrosine sulfate for binding to human PSGL-1. This evidence demonstrates the therapeutic potential of this antibody as a potent anti-inflammatory therapeutic.     

The distribution of rolling neutrophils in venular convergences

The aim of this study was to characterize the distribution of adherent leukocytes in branched venular convergences in vivo. Intravital microscopy was used to obtain video images of leukocyte adhesion in multiple branched sites in mouse cremaster muscle, during the mild inflammatory response induced by surgical preparation. The average number of cells/vessel length was obtained over several minutes for seven venular convergences with varying geometrical configurations. Results from this study demonstrate a strong tendency of leukocytes to adhere at junctional points between converging vessels. Different vessel configurations were studied and results were shown to be insensitive to precise vessel geometry. Thus, in post-capillary venules, leukocytes are most likely to adhere at points between converging vessels, regardless of the precise geometrical properties or configuration of the vessels. Hydrodynamic mechanisms due to flow behavior through convergences likely play a significant role in determining locations of cellular adhesion. Future work should concentrate on quantifying the relative contributions of hydrodynamic and biochemical mechanisms to aid in understanding disease processes and development of treatments or therapeutics.     

Granulocyte colony-stimulating factor promotes adhesion of neutrophils

Granulocyte colony stimulating factor(G-CSF) is well known for its ability to drive the maturation andmobilization of neutrophils. G-CSF also appears to have the potentialto activate functions of mature neutrophils, influencing recruitment atsites of inflammation and tissue injury. We investigated the ability ofG-CSF to stimulate adhesion of isolated blood neutrophils. G-CSFinduced significant adherence to intercellular adhesion molecule(ICAM)-1 that was both macrophage antigen-1 (Mac-1) and leukocytefunction-associated antigen-1 dependent. The kinetics ofG-CSF-stimulated adhesion to ICAM-1 peaked at 11 min without detectablesurface upregulation of Mac-1. This was in marked contrast tochemokines, in which peak activation of adhesion is seen within 1 minof stimulation. In contrast to chemokine-induced adhesion, G-CSFstimulation was not inhibited by pertussis toxin. G-CSF also augmentedthe attachment of neutrophils to activated human umbilical veinendothelial cells (HUVEC) through specific effects on neutrophils,because HUVEC appear to lack functional G-CSF receptors.

     

Superoxide converts indigo carmine to isatin sulfonic acid: implications for the hypothesis that neutrophils produce ozone

Recently, it was proposed that neutrophils generate ozone (Wentworth, P. J., McDunn, J. E., Wentworth, A. D., Takeuchi, C., Nieva, J., Jones, T., Bautista, C., Ruedi, J. M., Gutierrez, A., Janda, K. D., Babior, B. M., Eschenmoser, A., and Lerner, R. A. (2002) Science 298, 2195-2199; Babior, B. M., Takeuchi, C., Ruedi, J., Gutierrez, A., and Wentworth, P. J. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 3031-3034). Evidence for the proposal was based largely on the chemistry of ozone reacting with indigo carmine to produce isatin sulfonic acid. In this investigation, we have examined the specificity of this reaction and whether it can be used as unequivocal evidence of ozone production by neutrophils. Stimulated neutrophils promoted the loss of indigo carmine and formation of isatin sulfonic acid in a reaction that was completely inhibited by superoxide dismutase. Methionine, which scavenges ozone, singlet oxygen, and hypochlorous acid, had no effect on the reaction. Neither did catalase or azide, which scavenge hydrogen peroxide and inhibit myeloperoxidase, respectively. From these results, it is apparent that superoxide was responsible for bleaching indigo carmine. Superoxide generated using xanthine oxidase and acetaldehyde also converted indigo carmine to isatin sulfonic acid in a reaction that was completely inhibited by superoxide dismutase and unaffected by catalase. When the xanthine oxidase reaction was carried out in H(2)(18)O, the proportion of (18)O incorporated into the isatin sulfonic acid was the same as that found for ozone. Thus, reactions of ozone and superoxide with indigo carmine are indistinguishable with respect to isatin sulfonic acid formation. We conclude that bleaching of indigo carmine cannot be used to invoke ozone production by neutrophils. Studies using indigo carmine to implicate ozone in other biological processes should also be interpreted with caution.     

P-selectin binding promotes the adhesion of monocytes to VCAM-1 under flow conditions.

This study examined the adhesive interaction of peripheral blood monocytes with VCAM-1 and analyzed the effect of P-selectin binding to monocytes on the adhesive interaction with VCAM-1 under flow conditions. P-selectin glycoprotein ligand-1 is expressed on most monocytes. Furthermore, most monocytes bind soluble P-selectin derived from platelets. P-selectin binding to monocytes did not alter the amount of expression of alpha4 integrin on monocytes. However, the mean channel fluorescence value for binding Cy2-conjugated soluble VCAM-1 to P-selectin-bound monocytes was slightly more than that for binding Cy2-conjugated soluble VCAM-1 to untreated monocytes. Under flow conditions, the number of P-selectin-bound monocytes bound to VCAM-1 was much higher than that of untreated monocytes bound to VCAM-1. These bindings were abolished by pretreatment of untreated monocytes and P-selectin-bound monocytes with anti-VCAM-1 mAb or anti-alpha4 integrin mAb. Furthermore, P-selectin binding to monocytes increased shear resistance and thus increased the adhesive strength of monocytes to VCAM-1. These findings indicate that P-selectin binding to monocytes enhances the adhesive interaction of monocytes with VCAM-1. It is suggested that P-selectin glycoprotein ligand-1/P-selectin interaction and alpha4 integrin/VCAM-1 interaction can act sequentially in the adhesion cascade that regulates monocyte trafficking to inflammatory and atherosclerotic lesion.     

Binding of ADAM28 to P-selectin glycoprotein ligand-1 enhances P-selectin-mediated leukocyte adhesion to endothelial cells

ADAMs (a disintegrin and metalloproteinases) are a recently discovered gene family of multifunctional proteins with the disintegrin-like and metalloproteinase domains. To analyze the biological functions of ADAM28, we screened binding molecules to secreted-type ADAM28 (ADAM28s) by the yeast two-hybrid system and identified P-selectin glycoprotein ligand-1 (PSGL-1). Binding between the disintegrin-like domain of ADAM28s and the extracellular portion of PSGL-1 was determined by yeast two-hybrid assays, binding assays of the domain-specific recombinant ADAM28s species using PSGL-1 stable transfectants and leukocyte cell lines expressing native PSGL-1 (HL-60 cells and Jurkat cells), and co-immunolocalization and co-immunoprecipitation of the molecules in these cells. Incubation of HL-60 cells with recombinant ADAM28s enhanced the binding to P-selectin-coated wells and P-selectin-expressing endothelial cells. In addition, intravenous injection of ADAM28s-treated HL-60 cells increased their accumulation in the pulmonary microcirculation and alveolar spaces in a mouse model of endotoxin-induced inflammation. These data suggest a novel function that ADAM28s promotes PSGL-1/P-selectin-mediated leukocyte rolling adhesion to endothelial cells and subsequent infiltration into tissue spaces through interaction with PSGL-1 on leukocytes under inflammatory conditions.     

Leukocyte rolling on P-selectin: a three-dimensional numerical study of the effect of cytoplasmic viscosity

Rolling leukocytes deform and show a large area of contact with endothelium under physiological flow conditions. We studied the effect of cytoplasmic viscosity on leukocyte rolling using our three-dimensional numerical algorithm that treats leukocyte as a compound droplet in which the core phase (nucleus) and the shell phase (cytoplasm) are viscoelastic fluids. The algorithm includes the mechanical properties of the cell cortex by cortical tension and considers leukocyte microvilli that deform viscoelastically and form viscous tethers at supercritical force. Stochastic binding kinetics describes binding of adhesion molecules. The leukocyte cytoplasmic viscosity plays a critical role in leukocyte rolling on an adhesive substrate. High-viscosity cells are characterized by high mean rolling velocities, increased temporal fluctuations in the instantaneous velocity, and a high probability for detachment from the substrate. A decrease in the rolling velocity, drag, and torque with the formation of a large, flat contact area in low-viscosity cells leads to a dramatic decrease in the bond force and stable rolling. Using values of viscosity consistent with step aspiration studies of human neutrophils (5-30 Pa·s), our computational model predicts the velocities and shape changes of rolling leukocytes as observed in vitro and in vivo.