The Multistep Process of Homotypic Neutrophil Aggregation: A Review of the Molecules and Effects of Hydrodynamics

Homotypic adhesion of neutrophils stimulated with chemoattractant is analogous to capture on vascular endothelium in that both processes are supported by L-selectin and β₂-integrin adhesion receptors. Under hydrodynamic shear, cell adhesion requires that receptors bind sufficient ligand over the duration of intercellular contact to withstand the hydrodynamic stresses. Using cone and plate viscometry to apply a uniform linear shear field to suspensions of neutrophils and flow cytometry to quantitate the size distribution of aggregates formed over the time course of formyl peptide stimulation, we conducted a detailed examination of the affect of shear rate and shear stress on the kinetics of cell aggregation. The efficiency of aggregate formation was fit from a mathematical model based on Smoluchowski's two-body collision theory. Over a range of venular shear rates (400–800 s^-1), β90% of the single cells are recruited into aggregates ranging from doublets to groupings larger than sextuplets. Adhesion efficiency fit to the kinetics of aggregation increased with shear rate from β20% at 100s^-1 to a maximum level of β80% at 400 s^-1. This increase to peak adhesion efficiency was dependent on L-selectin and β₂-integrin, and was resistant to shear stress up to β7 dyn/cm². When L-selectin was blocked with antibody, β₂-integrin (CD11a, b) supported adhesion at low shear rates (< 400 s^-1). Aggregates formed over the rapid phase of aggregation remain intact and resistant to shear up to 120 s. At the end of this plateau phase of stability, aggregates spontaneously dissociate back to singlets. The rate of cell disaggregation is linearly proportional to the applied shear rate. The binding kinetics of selectin and integrin appear to be optimized to function within discrete ranges of shear rate and stress, providing an intrinsic mechanism for the transition from neutrophil tethering to firm but reversible adhesion.     

Modeling the reversible kinetics of neutrophil aggregation under hydrodynamic shear.

Neutrophil emigration into inflamed tissue is mediated by beta 2-integrin and L-selectin adhesion receptors. Homotypic neutrophil aggregation is also dependent on these molecules, and it provides a model system in which to study adhesion dynamics. In the current study we formulated a mathematical model for cellular aggregation in a linear shear field based on Smoluchowski's two-body collision theory. Neutrophil suspensions activated with chemotactic stimulus and sheared in a cone-plate viscometer rapidly aggregate. Over a range of shear rates (400-800 s-1), approximately 90% of the single cells were recruited into aggregates ranging from doublets to groupings larger than sextuplets. The adhesion efficiency fit to these kinetics reached maximum levels of > 70%. Formed aggregates remained intact and resistant to shear up to 120 s, at which time they spontaneously dissociated back to singlets. The rate of cell disaggregation was linearly proportional to the applied shear rate, and it was approximately 60% lower for doublets as compared to larger aggregates. By accounting for the time-dependent changes in adhesion efficiency, disaggregation rate, and the effects of aggregate geometry, we succeeded in predicting the reversible kinetics of aggregation over a wide range of shear rates and cell concentrations. The combination of viscometry with flow cytometry and mathematical analysis as presented here represents a novel approach to differentiating between the effects of hydrodynamics and the intrinsic biological processes that control cell adhesion.     

Neutrophil string formation: hydrodynamic thresholding and cellular deformation during cell collisions

Neutrophils unexpectedly display flow-enhanced adhesion (hydrodynamic thresholding) to L-selectin in rolling or aggregation assays. We report that the primary collision efficiency (epsilon) of flowing neutrophils with preadhered neutrophils on intercellular adhesion molecule-1 (ICAM-1) or fibrinogen also displayed a maximum of epsilon approximately 0.4-0.45 at a wall shear rate of 100 s(-1), an example of thresholding. Primary collision lifetime with no detectable bonding decreased from 130 to 10 ms as wall shear rate increased from 30 to 300 s(-1), whereas collision lifetimes with bonding decreased from 300 to 100 ms over this shear range using preadhered neutrophils on ICAM-1, with similar results for fibrinogen. Antibodies against L-selectin, but not against CD11a, CD11b, or CD18, reduced epsilon at 100 s(-1) by >85%. High resolution imaging detected large scale deformation of the flowing neutrophil during the collision at 100 s(-1) with the apparent contact area increasing up to approximately 40 microm(2). We observed the formation of long linear string assemblies of neutrophils downstream of neutrophils preadhered to ICAM-1, but not fibrinogen, with a maximum in string formation at 100 s(-1). Secondary capture events to the ICAM-1 or fibrinogen coated surfaces after primary collisions were infrequent and short lived, typically lasting from 500 to 3500 ms. Between 5 and 20% of neutrophil interactions with ICAM-1 substrate converted to firm arrest (>3500 ms) and greatly exceeded that observed for fibrinogen, thus defining the root cause of poor string formation on fibrinogen at all shear rates. Additionally, neutrophils mobilized calcium after incorporation into strings. Static adhesion also caused calcium mobilization, as did the subsequent onset of flow. To our knowledge, this is the first report of 1). hydrodynamic thresholding in neutrophil string formation; 2). string formation on ICAM-1 but not on fibrinogen; 3). large cellular deformation due to collisions at a venous shear rate; and 4), mechanosensing through neutrophil beta(2)-integrin/adhesion. The increased contact area during deformation was likely responsible for the hydrodynamic threshold observed in the primary collision efficiency since no increase in primary collision lifetime was detected as shear forces were increased (for either surface coating).     

Molecular dynamics of the transition from L-selectin- to beta 2-integrin-dependent neutrophil adhesion under defined hydrodynamic shear.

Homotypic adhesion o2 neutrophils stimulated with chemoattractant is analogous to capture on vascular endothelium in that both processes depend on L-selectin and beta 2-integrin adhesion receptors. Under hydrodynamic shear, cell adhesion requires that receptors bind sufficient ligand over the duration of intercellular contact to withstand hydrodynamic stresses. Using cone-plate viscometry to apply a uniform linear shear field to suspensions of neutrophils, we conducted a detailed examination of the effect of shear rate and shear stress on the kinetics of cell aggregation. A collisional analysis based on Smoluchowski's flocculation theory was employed to fit the kinetics of aggregation with an adhesion efficiency. Adhesion efficiency increased with shear rate from approximately 20% at 100 s-1 to approximately 80% at 400 s-1. The increase in adhesion efficiency. Adhesion efficiency increased with shear rate from approximately 20% at 100 s-1 to approximately 80% at 400 s-1. The increase in adhesion efficiency with shear was dependent on L-selectin, and peak efficiency was maintained over a relatively narrow range of shear rates (400-800 s-1) and shear stresses (4-7 dyn/cm2). When L-selectin was blocked with antibody, beta 2-integrin (CD11a, b) supported adhesion at low shear rates (< 400 s-1). The binding kinetics of selectin and integrin appear to be optimized to function within discrete ranges of shear rate and stress, providing an intrinsic mechanism for the transition from neutrophil tethering to stable adhesion.     

Probabilistic modeling of shear-induced formation and breakage of doublets cross-linked by receptor-ligand bonds.

A model was constructed to describe previously published experiments of shear-induced formation and breakage of doublets of red cells and of latexes cross-linked by receptor-ligand bonds (. Biophys. J. 65:1318-1334; Tees and Goldsmith. 1996. Biophys. J. 71:1102-1114;. Biophys. J. 71:1115-1122). The model, based on McQuarrie's master equations (1963. J. Phys. Chem. 38:433-436), provides unifying treatments for three distinctive time periods in the experiments of particles in a Couette flow in which a doublet undergoes 1) formation upon two-body collision between singlets; 2) evolution of bonds at low shear rate; and 3) break-up at high shear rate. Neglecting the applied force at low shear rate, the probability of forming a doublet per collision as well as the evolution of probability distribution of bonds in a preformed doublet were solved analytically and found to be in quite good agreement with measurements. At high shear rate with significant force acting to accelerate bond dissociation, the predictions for break-up of doublets were obtained numerically and compared well with data in both individual and population studies. These comparisons enabled bond kinetic parameters for three types of particles cross-linked by two receptor-ligand systems to be calculated, which agreed well with those computed from Monte Carlo simulations. This work can be extended to analyze kinetics of receptor-ligand binding in cell aggregates, such as those of neutrophils and platelets in the circulation.     

Kinetics of beta2-integrin and L-selectin bonding during neutrophil aggregation in shear flow.

Activated neutrophils aggregate in a shear field via bonding of L-selectin to P-selectin glycoprotein ligand-1 (PSGL-1) followed by a more stable bonding of LFA-1 (CD11a/CD18) to intercellular adhesion molecule 3 (ICAM-3) and Mac-1 (CD11b/CD18) to an unknown counter receptor. Assuming that the Mac-1 counter receptor is ICAM-3-like in strength and number, rate processes were deconvoluted from neutrophil homoaggregation data for shear rates (G) of 100-3000 s-1 with a two-body hydrodynamic collision model (. Biophys. J. 73:2819-2835). For integrin-mediated aggregation (characteristic bond strength of 5 microdynes) in the absence of L-selectin contributions, an average forward rate of kf = 1.57 x 10(-12) cm2/s predicted the measured efficiencies for G = 100-800 s-1. For a selectin bond formation rate constant equal to the integrin bond formation rate constant, the colloidal stability of unactivated neutrophils was satisfied for a reverse rate of the L-selectin-PGSL bond corresponding to an average bond half-life of 10 ms at a characteristic bond strength of 1 microdyne. Colliding neutrophils initially bridged by at least one L-selectin-PSGL-1 bond were calculated to rotate from 8 to 50 times at G = 400 to 3000 s-1, respectively, before obtaining mechanical stability in sheared fluid of either 0.75 or 1.75 cP viscosity. Thus for G > 400 s-1, the interaction time needed for the rotating aggregates to become stable was relatively constant at 52.5 +/- 8.5 ms, largely independent of shear rate or shear stress. Aggregation data and the colloidal stability criterion can provide a consistent set of forward and reverse rate constants and characteristic bond strengths for a known time-dependent stoichiometry of receptors on cells interacting in a shear flow field.     

Shear-dependent capping of L-selectin and P-selectin glycoprotein ligand 1 by E-selectin signals activation of high-avidity beta2-integrin on neutrophils

Two adhesive events critical to efficient recruitment of neutrophils at vascular sites of inflammation are up-regulation of endothelial selectins that bind sialyl Lewis(x) ligands and activation of beta(2)-integrins that support neutrophil arrest by binding ICAM-1. We have previously reported that neutrophils rolling on E-selectin are sufficient for signaling cell arrest through beta(2)-integrin binding of ICAM-1 in a process dependent upon ligation of L-selectin and P-selectin glycoprotein ligand 1 (PSGL-1). Unresolved are the spatial and temporal events that occur as E-selectin binds to human neutrophils and dynamically signals the transition from neutrophil rolling to arrest. Here we show that binding of E-selectin to sialyl Lewis(x) on L-selectin and PSGL-1 drives their colocalization into membrane caps at the trailing edge of neutrophils rolling on HUVECs and on an L-cell monolayer coexpressing E-selectin and ICAM-1. Likewise, binding of recombinant E-selectin to PMNs in suspension also elicited coclustering of L-selectin and PSGL-1 that was signaled via mitogen-activated protein kinase. Binding of recombinant E-selectin signaled activation of beta(2)-integrin to high-avidity clusters and elicited efficient neutrophil capture of beta(2)-integrin ligands in shear flow. Inhibition of p38 and p42/44 mitogen-activated protein kinase blocked the cocapping of L-selectin and PSGL-1 and the subsequent clustering of high-affinity beta(2)-integrin. Taken together, the data suggest that E-selectin is unique among selectins in its capacity for clustering sialylated ligands and transducing signals leading to neutrophil arrest in shear flow.     

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.     

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.     

Preferential binding of platelets to monocytes over neutrophils under flow

This study was undertaken to systematically investigate the binding kinetics of platelet recruitment by monocytes relative to neutrophils in bulk suspensions subjected to shear as well as the molecular requirements of leukocyte-platelet binding. Hydrodynamic shear-induced collisions augment the proportion of monocytes with adherent platelets more drastically than that of neutrophils with bound platelets. These heterotypic interactions are further potentiated by platelet activation with thrombin or to a lesser extent by monocyte stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP). Monocyte-platelet heteroaggregation increases with increasing shear rate and shear exposure time. Platelet P-selectin binding to monocyte P-selectin-glycoprotein-ligand-1 is solely responsible for maximal platelet adhesion to unstimulated monocytes in shear flow. However, the enhanced platelet binding to fMLP-treated monocytes involves a sequential two-step process, wherein P-selectin-PSGL-1 interactions are stabilized by CD18-integrin involvement. Blocking platelet alpha(IIb)beta(3) or monocyte beta(1)-integrin function had no effect. This study underscores the preferential recruitment of platelets by monocytes relative to neutrophils in shear flow, and demonstrates that the shear environment of the vasculature coupled to the state of cell activation modulates the dynamics and molecular constituents mediating monocyte-platelet adhesion.     

Hypotonicity induces L-selectin shedding in human neutrophils

Expression levels of adhesion molecules on neutrophils are affectedunder various conditions, including ischemia, possibly becauseof associated increases in cell volume. We examined the effects of cellswelling in hypotonic media on the level of L-selectin (CD62L) and₂-integrin (CD18) on human neutrophils. In hypotonic media, neutrophils shed L-selectin. The shedding was greatly reduced by30 µM RO31-9790, the metalloprotease (sheddase) inhibitor. Hypotonicity-induced L-selectin shedding was also time and tonicity dependent. Decreasing tonicity caused increased shedding. In 0.6× medium (0.6× the normal tonicity of 300 mosmol/kgH₂O),shedding increased over a 2-h period, after which >70% of theneutrophils had lost L-selectin. In contrast to L-selectin, the levelof ₂-integrin on the neutrophil surface was notsignificantly affected. Thus L-selectin shedding, which occurs onneutrophil activation and is usually accompanied by₂-integrin upregulation, was selectively induced byhypotonicity without a corresponding effect on₂-integrin.

     

Cytoskeletal interactions regulate inducible L-selectin clustering

L-selectin (CD62L) amplifies neutrophil capture within the microvasculature at sites of inflammation. Activation by G protein-coupled stimuli or through ligation of L-selectin promotes clustering of L-selectin and serves to increase its adhesiveness, signaling, and colocalization with ₂-integrins. Currently, little is known about the molecular process regulating the lateral mobility of L-selectin. On neutrophil stimulation, a progressive change takes place in the organization of its plasma membrane, resulting in membrane domains that are characteristically enriched in glycosyl phosphatidylinositol (GPI)-anchored proteins and exclude the transmembrane protein CD45. Clustering of L-selectin, facilitated by E-selectin engagement or antibody cross-linking, resulted in its colocalization with GPI-anchored CD55, but not with CD45 or CD11c. Disrupting microfilaments in neutrophils or removing a conserved cationic motif in the cytoplasmic domain of L-selectin increased its mobility and membrane domain localization in the plasma membrane. In addition, the conserved element was critical for L-selectin-dependent tethering under shear flow. Our data indicate that L-selectin’s lateral mobility is regulated by interactions with the actin cytoskeleton that in turn fortifies leukocyte tethering. We hypothesize that both membrane mobility and stabilization augment L-selectin’s effector functions and are regulated by dynamic associations with membrane domains and the actin cytoskeleton. membrane domains; adhesion; leukocyte; inflammation     

L-selectin signaling of neutrophil adhesion and degranulation involves p38 mitogen-activated protein kinase

The adhesion molecules known as selectins mediate the capture of neutrophils from the bloodstream. We have previously reported that ligation and cross-linking of L-selectin on the neutrophil surface enhances the adhesive function of beta(2)-integrins in a synergistic manner with chemotactic agonists. In this work, we examined degranulation and adhesion of neutrophils in response to cross-linking of L-selectin and addition of interleukin-8. Cross-linking of L-selectin induced priming of degranulation that was similar to that observed with the alkaloid cytochalasin B. Activation mediated by L-selectin of neutrophil shape change and adhesion through CD11b/CD18 were strongly blocked by Merck C, an imidazole-based inhibitor of p38 mitogen-activated protein kinase (MAPK), but not by a structurally similar non-binding regioisomer. Priming by L-selectin of the release of secondary, tertiary, and secretory, but not primary, granules was blocked by inhibition of p38 MAPK. Peak phosphorylation of p38 MAPK was observed within 1 min of cross-linking L-selectin, whereas phosphorylation of ERK1/2 was highest at 10 min. Phosphorylation of p38 MAPK, but not ERK1/2, was inhibited by Merck C. These data suggest that signal transduction as a result of clustering L-selectin utilizes p38 MAPK to effect neutrophil shape change, integrin activation, and the release of secondary, tertiary, and secretory granules.     

A direct comparison of selectin-mediated transient, adhesive events using high temporal resolution

Leukocyte capture and rolling on the vascular endothelium is mediated principally by the selectin family of cell adhesion receptors. In a parallel plate flow chamber, neutrophil rolling on purified selectins or a selectin-ligand substrate was resolved by high speed videomicroscopy as a series of ratchet-like steps with a characteristic time constant (Kaplanski, G., C. Farnarier, O. Tissot, A. Pierres, A.-M. Benoliel, M. C. Alessi, S. Kaplanski, and P. Bongrand. 1993. Biophys. J. 64:1922-1933; Alon, R., D. A. Hammer, and T. A. Springer. 1995. Nature (Lond.). 374:539-542). Under shear, neutrophil arrests due to bond formation events were as brief as 4 ms. Pause time distributions for neutrophils tethering on P-, E-, L-selectin, or peripheral node addressin (PNAd) were compared at estimated single bond forces ranging from 37 to 250 pN. Distributions of selectin mediated pause times were fit to a first order exponential, resulting in a molecular dissociation constant (k(off)) for the respective selectin as a function of force. At estimated single bond forces of 125 pN and below, all three selectin dissociation constants fit the Bell and Hookean spring models of force-driven bond breakage equivalently. Unstressed k(off) values based on the Bell model were 2.4, 2.6, 2.8, 3.8 s(-1) for P-selectin, E-selectin, L-selectin, and PNAd, respectively. Bond separation distances (reactive compliance) were 0.39, 0.18, 1.11, 0.59 A for P-selectin, E-selectin, L-selectin, and PNAd, respectively. Dissociation constants for L-selectin and P-selectin at single bond forces above 125 pN were considerably lower than either Bell or Hookean spring model predictions, suggesting the existence of two regimes of reactive compliance. Additionally, interactions between L-selectin and its leukocyte ligand(s) were more labile in the presence of flow than the L-selectin endothelial ligand, PNAd, suggesting that L-selectin ligands may have different molecular and mechanical properties. Both types of L-selectin bonds had a higher reactive compliance than P-selectin or E-selectin bonds.     

Physical and chemical effects of red cells in the shear-induced aggregation of human platelets.

Both chemical and physical effects of red cells have been implicated in the spontaneous aggregation of platelets in sheared whole blood (WB). To determine whether the chemical effect is due to ADP leaking from the red cells, a previously described technique for measuring the concentration and size of single platelets and aggregates was used to study the shear-induced aggregation of platelets in WB flowing through 1.19-mm-diameter polyethylene tubing in the presence and absence of the ADP scavenger enzyme system phosphocreatine-creatine phosphokinase (CP-CPK). Significant spontaneous aggregation was observed at mean tube shear rates, (G) = 41.9 and 335 s-1 (42% and 13% decrease in single platelets after a mean transit time (t) = 43 s, compared to 89 and 95% decrease with 0.2 microM ADP). The addition of CP-CPK, either at the time of, or 30 min before each run, completely abolished aggregation. In the presence of 0.2 microM ADP, CP-CPK caused a reversal of aggregation at (t) = 17 s after 30% of single cells had aggregated. To determine whether red cells exert a physical effect by increasing the time of interaction of two colliding platelets (thereby increasing the proportion of collisions resulting in the formation of aggregates), an optically transparent suspension of 40% reconstituted red cell ghosts in serum containing 2.5-micron-diameter latex spheres (3 x 10(5)/microliters) flowing through 100-microns-diameter tubes was used as a model of platelets in blood, and the results were compared with those obtained in a control suspension of latex spheres in serum alone. Two-body collisions between microspheres in the interior of the flowing ghost cell or serum suspensions at shear rates from 5 to 90 s-1 were recorded on cine film. The films were subsequently analyzed, and the measured doublet lifetime, tau meas, was compared with that predicted by theory in the absence of interactions with other particles, tau theor. The mean (tau meas/tau theor) for doublets in ghost cell suspensions was 1.614 +/- 1.795 (SD; n = 320), compared to a value of 1.001 +/- 0.312 (n = 90) for doublets in serum. Whereas 11% of doublets in ghost cell suspensions had lifetimes from 2.5 to 5 times greater than predicted, in serum, no doublets had lifetimes greater than 1.91 times that predicted. There was no statistically significant correlation between tau meas/tau theor and shear rate, but the values of tau meas/tau theor for low-angle collisions in ghost cell suspensions were significantly greater than for high-angle collisions.     

Neutrophil tethering on E-selectin activates beta 2 integrin binding to ICAM-1 through a mitogen-activated protein kinase signal transduction pathway

On inflamed endothelium selectins support neutrophil capture and rolling that leads to firm adhesion through the activation and binding of beta 2 integrin. The primary mechanism of cell activation involves ligation of chemotactic agonists presented on the endothelium. We have pursued a second mechanism involving signal transduction through binding of selectins while neutrophils tether in shear flow. We assessed whether neutrophil rolling on E-selectin led to cell activation and arrest via beta 2integrins. Neutrophils were introduced into a parallel plate flow chamber having as a substrate an L cell monolayer coexpressing E-selectin and ICAM-1 (E/I). At shears >/=0.1 dyne/cm2, neutrophils rolled on the E/I. A step increase to 4.0 dynes/cm2 revealed that approximately 60% of the interacting cells remained firmly adherent, as compared with approximately 10% on L cells expressing E-selectin or ICAM-1 alone. Cell arrest was dependent on application of shear and activation of Mac-1 and LFA-1 to bind ICAM-1. Firm adhesion was inhibited by blocking E-selectin, L-selectin, or PSGL-1 with Abs and by inhibitors to the mitogen-activated protein kinases. A chimeric soluble E-selectin-IgG molecule specifically bound sialylated ligands on neutrophils and activated adhesion that was also inhibited by blocking the mitogen-activated protein kinases. We conclude that neutrophils rolling on E-selectin undergo signal transduction leading to activation of cell arrest through beta 2 integrins binding to ICAM-1.     

Interplay between membrane cholesterol and ethanol differentially regulates neutrophil tether mechanics and rolling dynamics

Using microfluidic assays at a 100 s?1 wall shear rate, we examined the effects of ethanol on cholesterol-loaded neutrophils with respect to: (1) collision efficiency and membrane tethering to P-selectin-coated microbeads, (2) rolling on P-selectin-coated surfaces, and (3) primary and secondary interactions with neutrophils pre-adhered to intercellular adhesion molecule-1 (ICAM-1). Using methyl-β-cyclodextrin:cholesterol complexes, membrane cholesterol was increased over control by 4.6-fold (no ethanol), 3.6-fold (0.3% ethanol pre-loading), and 1.6-fold (0.3% ethanol post-loading). These treatments did not alter CD11b expression; however, PSGL-1 and L-selectin were lowered by cholesterol enrichment (±ethanol). Cholesterol enrichment enhanced microbead collision efficiency, which was abrogated by ethanol. Ethanol had no effect on elevation of tethering fraction by cholesterol enrichment. Incubation of cholesterol-loaded neutrophils with ethanol resulted in significantly longer membrane tethers, due to tether lifetime enhancement. On P-selectin-coated surfaces, cholesterol-enriched neutrophils exposed to ethanol rolled faster and with more variability than cholesterol-enriched neutrophils. Ethanol reduced homotypic collision efficiency of cholesterol-loaded neutrophils without effect on tethering fraction or secondary collision efficiency. Tether length during cholesterol-loaded neutrophil homotypic collisions was enhanced by ethanol, in part due to increased L-selectin/PSGL-1 bond tether lifetime. Overall, ethanol attenuated cholesterol-induced adhesion increases while increasing membrane fluidity as indicated by tether length.     

Signaling functions of L-selectin in neutrophils: alterations in the cytoskeleton and colocalization with CD18.

Ligation and clustering of L-selectin by Ab ("cross-linking") or physiologic ligands results in activation of diverse responses that favor enhanced microvascular sequestration and emigration of neutrophils. The earliest responses include a rise in intracellular calcium, enhanced tyrosine phosphorylation, and activation of extracellular signal-regulated kinases. Additionally, cross-linking of L-selectin induces sustained shape change and activation of beta2 integrins, leading to neutrophil arrest under conditions of shear flow. In this report, we examined several possible mechanisms whereby transmembrane signals from L-selectin might contribute to an increase in the microvascular retention of neutrophils and enhanced efficiency of emigration. In human peripheral blood neutrophils, cross-linking of L-selectin induced alterations in cellular biophysical properties, including a decrease in cell deformability associated with F-actin assembly and redistribution, as well as enhanced adhesion of microspheres bound to beta2 integrins. L-selectin and the beta2 integrin became spatially colocalized as determined by confocal immunofluorescence microscopy and fluorescence resonance energy transfer. We conclude that intracellular signals from L-selectin may enhance the microvascular sequestration of neutrophils at sites of inflammation through a combination of cytoskeletal alterations leading to cell stiffening and an increase in adhesiveness mediated through alterations in beta2 integrins.     

Ethanol enhances neutrophil membrane tether growth and slows rolling on P-selectin but reduces capture from flow and firm arrest on IL-1-treated endothelium

The effects of ethanol at physiological concentrations on neutrophil membrane tether pulling, adhesion lifetime, rolling, and firm arrest behavior were studied in parallel-plate flow chamber assays with adherent 1-microm-diameter P-selectin-coated beads, P-selectin-coated surfaces, or IL-1-stimulated human endothelium. Ethanol (0.3% by volume) had no effect on P-selectin glycoprotein ligand-1 (PSGL-1), L-selectin, or CD11b levels but caused PSGL-1 redistribution. Also, ethanol prevented fMLP-induced CD11b up-regulation. During neutrophil collisions with P-selectin-coated beads at venous wall shear rates of 25-100 s(-1), ethanol increased membrane tether length and membrane growth rate by 2- to 3-fold but reduced the adhesion efficiency (detectable bonding per total collisions) by 2- to 3-fold, compared with untreated neutrophils. Without ethanol treatment, adhesion efficiency and adhesion lifetime declined as wall shear rate was increased, whereas ethanol caused the adhesion lifetime over all events to increase from 0.1 s to 0.5 s as wall shear rate was increased, an example of pharmacologically induced hydrodynamic thresholding. Consistent with this increased membrane fluidity and reduced capture, ethanol reduced rolling velocity by 37% and rolling flux by 55% on P-selectin surfaces at 100 s(-1), compared with untreated neutrophils. On IL-1-stimulated endothelium, rolling velocity was unchanged by ethanol treatment, but the fraction of cells converting to firm arrest was reduced from 35% to 24% with ethanol. Overall, ethanol caused competing biophysical and biochemical effects that: 1) reduced capture due to PSGL-1 redistribution, 2) reduced rolling velocity due to increased membrane tether growth, and 3) reduced conversion to firm arrest.