ABSTRACTObjective: To study rolling of mouse neutrophils on E‐selectin and ICAM‐1 in an ex vivo flow chamber system.Methods: The authors developed a small autoperfused flow chamber (20 × 200‐μ m cross section) that allows direct visualization of cells with and without fluorescent labeling and does not require recirculation of blood.Results: Neutrophils rolled on E‐selectin alone, but were unable to interact with immobilized ICAM‐1. When ICAM‐1 was co‐immobilized with E‐selectin, the number of cells that rolled was doubled, but no significant firm adhesion was observed. This phenomenon was specific for E‐selectin, and no enhancement of rolling was observed when P‐selectin was immobilized with ICAM‐1. The increased neutrophil rolling seen on E‐selectin and ICAM‐1 substrates required β2 integrins. Treating mice with antibodies to the β2 integrins LFA‐1 and Mac‐1 showed that LFA‐1 was primarily responsible for mediating rolling on ICAM‐1 in this model. Increased rolling on E‐selectin and ICAM‐1 was significantly reduced following administration of a specific p38 mitogen‐activated protein kinase (MAPK) inhibitor.Conclusion: The data show that neutrophil rolling on E‐selectin leads to partial activation of LFA‐1, enabling LFA‐1‐dependent rolling on ICAM‐1. This mechanism is likely to amplify and accelerate neutrophil recruitment in inflammation.

An approach is presented that uses velocimetry data to estimate accurately the spatial distribution of viscosity in steady laminar parallel flows of incompressible linearly viscous fluids. The approach is generally applicable to Newtonian fluids with spatially varying viscosity or to particle-suspension flows where a non-uniform distribution of the particles contributes to spatial variations in the local effective viscosity of the suspension. Emphasis is placed on the application of these methods to steady axisymmetric blood flow in cylindrical glass capillary tubes and microvessels. In this context, the spatial variations in viscosity over the vessel cross-section are predicted where it is assumed that the rheological properties associated with a heterogeneous red blood cell suspension can be well approximated by a continuous generalized linearly viscous fluid having a spatially non-uniform viscosity. For such a fluid, an expression for the viscosity profile over the vessel cross-section is derived that satisfies the conservation principles of mass and momentum and depends upon the a priori determined velocity distribution, which is extracted from fluorescent micro-particle image velocimetry data obtained from microvessels in vivo. These profiles provide useful information about dynamic, kinematic and rheological properties of the flow that include expressions for the axial pressure-gradient component, the local shear stress distribution, and the relative apparent viscosity. In microvessels, the effect of the glycocalyx surface layer on the vessel wall is also accounted for in the analysis by modelling the layer as a uniformly thick porous medium. Velocimetry data are presented from in vivo measurements made in venules after the application of a light-dye treatment to degrade the glycocalyx. Results reveal that these methods are sufficiently sensitive to detect a reduction in glycocalyx thickness of similar to 0.3 mum, which represents a fractional decrease in thickness of similar to 60-70% when compared with results from a separately published data set obtained from venules having an intact glycocalyx.

The signaling events leading to the activation of integrins and firm arrest of rolling neutrophils in inflamed venules have yet to be elucidated. In vitro assays suggest that both E-selectin and chemokines can trigger arrest of rolling neutrophils, but E-selectin−/− mice have normal levels of adherent neutrophils in inflamed venules. To test whether chemokine-induced neutrophil arrest in vivo can be unmasked by blocking E-selectin, we investigated neutrophil adhesion in inflamed cremaster muscle venules in tumor necrosis factor (TNF)-α–treated CXCR2−/− or wild-type (WT) mice injected with E-selectin blocking monoclonal antibody (mAb) 9A9. To block chemokine receptor signaling, we investigated E-selectin−/− or WT mice treated with pertussis toxin (PTx) intravenously. Neutrophil adhesion was unchanged in CXCR2−/−, E-selectin−/−, PTx-treated WT, or mAb 9A9–treated WT mice. However, TNF-α–induced neutrophil adhesion was almost completely abrogated in E-selectin−/− mice treated with PTx and significantly reduced in CXCR2−/− mice treated with the E-selectin blocking mAb. In thioglycollate-induced peritonitis, PTx treatment blocked neutrophil recruitment into the peritoneum of E-selectin−/− mice, but had only a partial effect in WT animals. These data show that E-selectin– and chemokine-mediated arrest mechanisms are overlapping in this model and identify CXCR2 as an important neutrophil arrest chemokine in vivo.

To study rolling of mouse neutrophils on P‐ and E‐selectins in whole blood and without cell isolation, we constructed an autoperfused flow chamber made from rectangular microslides (0.2×2 mm) perfused from a carotid artery catheter. A differential pressure transducer served to measure wall shear stress. Green fluorescent neutrophils rolled on P‐selectin but not E‐selectin coated at 50 ng/ml, with some rolling on E‐selectin at 150 ng/ml. However, when P‐ and E‐selectins were coimmobilized, the resulting number of rolling neutrophils was sixfold and fourfold higher than on P‐ or E‐selectin alone. Velocity and flux analysis shows that P‐selectin initiates neutrophil rolling, and a small amount of E‐selectin, unable to capture many neutrophils, reduces the rolling velocity of all neutrophils by more than 90%. The unexpected synergism between E‐ and P‐selectins explains why neutrophil recruitment is enhanced when both selectins are expressed.

We show that many salient hemodynamic flow properties, which have been difficult or impossible to assess in microvessels in vivo , can be estimated by using microviscometry and fluorescent microparticle image velocimetry in microvessels >20 μm in diameter. Radial distributions in blood viscosity, shear stress, and shear rate are obtained and used to predict axial pressure gradient, apparent viscosity, and endothelial-cell surface-layer thickness in vivo . Based solely on microparticle image velocimetry data, which are readily obtainable during the course of most intravital microscopy protocols from systemically injected particle tracers, we show that the microviscometric method consistently predicted a reduction in local and apparent blood viscosity after isovolemic hemodilution. Among its clinical applications, hemodilution is a procedure that is used to treat various pathologies that require reduction in peripheral vascular-flow resistance. Our results are directly relevant in this context because they suggest that the fractional decrease in systemic hematocrit is ≈25–35% greater than the accompanying fractional decrease in microvascular-flow resistance in vivo . In terms of its fundamental usefulness, the microviscometric method provides a comprehensive quantitative analysis of microvascular hemodynamics that has applications in broad areas of medicine and physiology and is particularly relevant to quantitative studies of angiogenesis, tumor growth, leukocyte adhesion, vascular-flow resistance, tissue perfusion, and endothelial-cell mechanotransduction.

To investigate the role of adenosine formed extracellularly in vascular homeostasis, mice with a targeted deletion of the cd73/ecto-5'-nucleotidase were generated. Southern blot, RT-PCR, and Western blot analysis confirmed the constitutive knockout. In vivo analysis of hemodynamic parameters revealed no significant differences in systolic blood pressure, ejection fraction, or cardiac output between strains. However, basal coronary flow measured in the isolated perfused heart was significantly lower (-14%; P<0.05) in the mutant. Immunohistochemistry revealed strong CD73 expression on the endothelium of conduit vessels in wild-type (WT) mice. Time to carotid artery occlusion after ferric chloride (FeCl3) was significantly reduced by 20% in cd73-/- mice (P<0.05). Bleeding time after tail tip resection tended to be shorter in cd73-/- mice (-35%). In vivo platelet cAMP levels were 0.96+/-0.46 in WT versus 0.68+/-0.27 pmol/106 cells in cd73-/- mice (P<0.05). Under in vitro conditions, platelet aggregation in response to ADP (0.05 to 10 micromol/L) was undistinguishable between the two strains. In the cremaster model of ischemia-reperfusion, the increase in leukocyte attachment to endothelium was significantly higher in cd73-/- compared with WT littermates (WT 98% versus cd73-/- 245%; P<0.005). The constitutive adhesion of monocytes in ex vivo-perfused carotid arteries of WT mice was negligible but significantly increased in arteries of cd73-/- mice (P<0.05). Thus, our data provide the first evidence that adenosine, extracellularly formed by CD73, can modulate coronary vascular tone, inhibit platelet activation, and play an important role in leukocyte adhesion to the vascular endothelium in vivo.

Leukocyte rolling in postcapillary venules of inflamed tissues is reduced in L-selectin-deficient mice and mice treated with L-selectin blocking antibodies, but the glycoprotein ligand for L-selectin in inflamed venules is unknown. Here, we show that L-selectin-dependent rolling after P-selectin blockade is completely absent in P-selectin glycoprotein ligand-1 (PSGL-1)(-/-) mice or wild-type mice treated with a PSGL-1 blocking monoclonal antibody. Immunohistochemistry and flow cytometry failed to show PSGL-1 expression on resting or inflamed endothelium or on platelets. To investigate whether leukocyte-expressed PSGL-1 is mediating L-selectin-dependent rolling, we reconstituted lethally irradiated wild-type mice with PSGL-1(-/-) bone marrow cells. These chimeric mice showed no L-selectin-dependent rolling, suggesting that leukocyte-expressed PSGL-1 mediates L-selectin-dependent rolling. Frame-to-frame video analysis of L-selectin-dependent rolling in wild-type mice showed that the majority of observed L-selectin-dependent leukocyte rolling was between free flowing leukocytes and already adherent leukocytes or possibly leukocyte fragments, followed by E-selectin-dependent leukocyte rolling along the endothelium. Leukocyte rolling was significantly slower for leukocyte-endothelial than leukocyte-leukocyte interactions. We conclude that leukocyte-expressed PSGL-1 serves as the main L-selectin ligand in inflamed postcapillary venules. L-selectin binding to PSGL-1 initiates tethering events that enable L-selectin-independent leukocyte-endothelial interactions. These findings provide a molecular mechanism for the inflammatory defects seen in L-selectin-deficient mice.

ABSTRACTObjective: To determine whether selectin‐mediated leukocyte‐rolling velocity in inflamed venules in vivo is determined by wall shear rate (WSR) or by wall shear stress (WSS).Methods: WSS was manipulated independently of WSR by altering the viscosity of blood plasma in mice with an isovolemic exchange of blood for low‐ or high‐viscosity dextran solutions. Rolling of neutrophils or beads coated with P‐selectin glycoprotein ligand‐1 (PSGL‐1) was reconstituted on P‐selectin immobilized on the wall of a parallel plate flow chamber at two different viscosities of the perfusion medium.Results: Leukocytes in vivo showed no increase in rolling velocity when shear stress was doubled by doubling viscosity. Neutrophils in the parallel‐plate flow chamber in vitro showed the same dependence on WSR as leukocytes in vivo, but bead‐rolling velocities correlated best with WSS. Rolling leukocytes, but not beads, deformed significantly in shear flow, and deformation correlated better with WSS.Conclusion: These data suggest leukocyte deformation during rolling offsets increased bond breakage at higher shear stress. The stable rolling velocity allows sufficient surveillance of the endothelial surface, even in venules with high WSS.