Leukocyte Rolling In Postcapillary Venules Research Articles

Alteration of the sublingual microvascular glycocalyx in critically ill patients

Glycocalyx degradation may contribute to microvascular dysfunction and tissue hypoperfusion during systemic inflammation and sepsis. In this observational study we evaluated the alteration of the sublingual microvascular glycocalyx in 16 healthy volunteers and 50 critically ill patients. Sidestream Dark Field images of the sublingual microcirculation were automatically analyzed by dedicated software. The Perfused Boundary Region (PBR) was calculated as the dimensions of the permeable part of the glycocalyx allowing the penetration of circulating red blood cells, providing an index of glycocalyx damage. The PBR was increased in ICU patients compared to healthy controls (2.7 [2.59–2.88] vs. 2.46 [2.37–2.59]μm, p<0.0001) and tended to be higher in the 32 septic patients compared to non-septics (2.77 [2.62–2.93] vs. 2.67 [2.55–2.75]μm, p=0.05), suggesting more severe glycocalyx alterations. A PBR of 2.76 showed the best discriminative ability towards the presence of sepsis (sensitivity: 50%, specificity: 83%; area under the receiver operating characteristic curve: 0.67, 95% CI 0.52–0.82, p=0.05). A weak positive correlation was found between PBR and heart rate (r=0.3, p=0.03). In 17 septic patients, a correlation was found between PBR and number of rolling leukocytes in post-capillary venules (RL/venule) (r=0.55, p=0.02), confirming that glycocalyx shedding enhances leukocyte–endothelium interaction.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery. Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion. Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor. While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM). Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968. Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality. We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method. In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Tyrosine sulfation of native mouse Psgl-1 is required for optimal leukocyte rolling on P-selectin in vivo.

BackgroundWe recently demonstrated that tyrosine sulfation is an important contributor to monocyte recruitment and retention in a mouse model of atherosclerosis. P-selectin glycoprotein ligand-1 (Psgl-1) is tyrosine-sulfated in mouse monocyte/macrophages and its interaction with P-selectin is important in monocyte recruitment in atherosclerosis. However, whether tyrosine sulfation is required for the P-selectin binding function of mouse Psgl-1 is unknown. Here we test the function of native Psgl-1 expressed in leukocytes lacking endogenous tyrosylprotein sulfotransferase (TPST) activity.Methodology/Principal FindingsPsgl-1 function was assessed by examining P-selectin dependent leukocyte rolling in post-capillary venules of C57BL6 mice transplanted with hematopoietic progenitors from wild type (WT→B6) or Tpst1;Tpst2 double knockout mice (Tpst DKO→B6) which lack TPST activity. We observed that rolling flux fractions were lower and leukocyte rolling velocities were higher in Tpst DKO→B6 venules compared to WT→B6 venules. Similar results were observed on immobilized P-selectin in vitro. Finally, Tpst DKO leukocytes bound less P-selectin than wild type leukocytes despite equivalent surface expression of Psgl-1.Conclusions/SignificanceThese findings provide direct and convincing evidence that tyrosine sulfation is required for optimal function of mouse Psgl-1 in vivo and suggests that tyrosine sulfation of Psgl-1 contributes to the development of atherosclerosis.

Lattice-Boltzmann simulation of blood flow in digitized vessel networks

Efficient flow of red blood cells (RBCs) and white blood cells (WBCs) through the microcirculation is necessary for oxygen and nutrient delivery as well as immune cell function. Because blood is a dense particulate suspension, consisting of 40% RBCs by volume, it is difficult to analyze the physical mechanisms by which individual blood cells contribute to the bulk flow properties of blood. Both experimental and computational approaches are hindered by these non-Newtonian properties, and predicting macroscopic blood flow characteristics such as viscosity has historically been an empirical process. In order to examine the effect of the individual cells on macroscopic blood rheology, we developed a lattice-Boltzmann model that considers the blood as a suspension of particles in plasma, accounting explicitly for cell–cell and cell–wall interactions. Previous studies have concluded that the abundance of leukocyte rolling in postcapillary venules is due to the interactions between red blood cells and leukocytes as they enter postcapillary expansions. Similar fluid dynamics may be involved in the initiation of rolling at branch points, a phenomenon linked to atherosclerosis. The lattice-Boltzmann approach is used to analyze the interactions of red and white blood cells as they flow through vascular networks digitized from normal and tumor tissue. A major advantage of the lattice-Boltzmann method is the ability to simulate particulate flow dynamically and in any geometry. Using this approach, we can accurately determine RBC–WBC forces, particle trajectories, the pressure changes in each segment that accompany cellular traffic in the network, and the forces felt by the vessel wall at any location. In this technique, intravital imaging using vascular contrast agents produces the network outline that is fed to the lattice-Boltzmann model. This powerful and flexible model can be used to predict blood flow properties in any vessel geometry and with any blood composition.

Cerebral Endothelial Nitric Oxide Synthase Expression is Reduced After Very Low Flow Bypass

In previous studies we have shown that delayed capillary reperfusion after low flow bypass predicts neurologic injury. In this acute study, we hypothesized that low flow reduces endothelial nitric oxide synthase (eNOS) expression, which may lead to more profound inflammatory response and delayed capillary perfusion. Twelve piglets (13.2 +/- 0.7 kg) had a cranial window placed over the parietal cerebral cortex for direct examination of the microcirculation using intravital fluorescence microscopy. Animals were cooled to 15 degrees C or 34 degrees C on cardiopulmonary bypass (pH stat, hematocrit 30%, pump flow 100 mL/kg/minute) followed by 2 hours of low flow (50 mL/kg/minute) or very low flow (10 mL/kg/minute). Rhodamine staining was used to observe adherent and rolling leukocytes in postcapillary venules. The eNOS protein expression was determined by Western immunoblotting. High temperature and low flow rate correlated with significantly reduced eNOS expression (p < 0.01). Univariate comparisons based on Student t tests indicated that eNOS protein levels were lower at 34 degrees C than at 15 degrees C (0.7 +/- 0.6 vs 1.7 +/- 0.5, p < 0.01) and at 10 mL/kg per minute compared with 50 mL/kg per minute (0.8 +/- 0.7 vs 1.6 +/- 0.5, p = 0.03). Moreover, two-way analysis of variance revealed that temperature (F = 21.6, p < 0.001) and flow rate (F = 13.8, p = 0.005) were independent multivariate predictors of eNOS expression. During low flow bypass, eNOS was inversely correlated with numbers of adherent (p = 0.002) and rolling (p = 0.006) leukocytes, following an exponential decay curve closely. eNOS expression is reduced after very low flow bypass, particularly at a higher bypass temperature. This is associated with delayed capillary reperfusion. Reduced eNOS is also associated with increased white cell activation which may lead to greater neurologic injury.

Influence of erythrocyte aggregation on leukocyte margination in postcapillary expansions: A lattice Boltzmann analysis

Leukocyte rolling on the vascular endothelium requires initial contact between the circulating leukocytes in the blood and the vessel wall. Although specific adhesion mechanisms are involved in leukocyte–endothelium interactions, adhesion patterns in vivo suggest other rheological mechanisms are involved as well. Previous studies have proposed that the abundance of leukocyte rolling in postcapillary venules is due to interactions between red blood cells and leukocytes as they enter capillary expansions as well as red blood cell (RBC) aggregation. We have established a lattice Boltzmann approach to analyze the interactions of RBC aggregates and leukocytes as they flow through a postcapillary expansion. The lattice Boltzmann technique provides the complete solution of the flow field and quantification of the particle–particle forces. Our results show that RBC aggregation strongly influences leukocyte–endothelium interactions.

Role of shear forces and adhesion molecule distribution on P-selectin-mediated leukocyte rolling in postcapillary venules.

Rolling on the venular endothelium is a critical step in the recruitment of leukocytes during the inflammatory response. P-selectin is a key mediator of leukocyte rolling, which is an early event in the inflammatory cascade; this rolling is likely to be directly regulated by both local fluid shear forces and P-selectin site densities in the microvasculature. However, neither the spatial pattern of P-selectin expression in postcapillary venules nor the effect of local expression patterns on rolling behavior in intact functional venules is known. We investigated the influence of local shear forces and the spatial distribution of endothelial P-selectin in intact blood perfused post capillary venules in anesthetized mice using intravital confocal microscopy, high temporal resolution particle tracking, and immunofluorescent labeling. We demonstrated a shear-dependent increase in average leukocyte rolling velocity that was attributable to a shear-dependent increase in the occurrence of transient leukocyte detachments from the endothelial surface: translational velocity during leukocyte contact with the vessel wall remained constant. P-selectin expression was not different in venules with characteristically different shear rates or diameters but varied significantly within individual venules. In postcapillary venules, regions of high P-selectin expression correlated with regions of slow leukocyte rolling. Thus the characteristically variable leukocyte rolling in vivo is a function of the spatial heterogeneity in P-selectin expression. The study shows how the local hydrodynamic forces and the nonuniform pattern of P-selectin expression affect the behavior of interacting leukocytes, providing direct evidence for the local variation of adhesion molecule expression as a mechanism for the regulation of leukocyte recruitment.

Higher bypass temperature correlates with increased white cell activation in the cerebral microcirculation

Objective Cardiopulmonary bypass induces a systemic inflammatory response, which in turn promotes a cascade of leukocyte and endothelial cell activity. We investigated whether differences in bypass temperature and flow rate affect endothelial cell and leukocyte adhesion in the cerebral microcirculation. Methods Thirty-six piglets (13.0 ± 1.1 kg) had a cranial window placed over the parietal cortex to evaluate the microcirculation by means of intravital microscopy. Animals were cooled to a temperature of 15°C, 25°C, or 34°C on cardiopulmonary bypass with hematocrit levels of 20% or 30% by using pH-stat management, followed by 60 minutes of reduced flow (10, 25, or 50 mL · kg −1 · min −1). Rhodamine staining was used to observe adherent and rolling leukocytes in postcapillary venules. Results Higher bypass temperature correlated with significantly more adherent and rolling leukocytes during the full 60 minutes of low-flow bypass ( P < .05). Poisson regression revealed more adherent leukocytes at 34°C than at 15°C and at a flow rate of 10 mL · kg −1 · min −1 compared with a flow rate of 50 mL · kg −1 · min −1. There was an inverse correlation between flow rate and the number of adherent and rolling leukocytes at 30, 45, and 60 minutes of low-flow bypass ( P < .05). Temperature was a multivariable predictor of histologic score, with greater neurologic damage found after bypass at 34°C ( P < .01). Conclusions Leukocyte activation in cerebral microcirculation is increased with higher temperature and lower flow rate, suggesting that these variables influence the inflammatory response during cardiopulmonary bypass.

Red Blood Cells Initiate Leukocyte Rolling in Postcapillary Expansions: A Lattice Boltzmann Analysis

Leukocyte rolling on the vascular endothelium requires initial contact between leukocytes circulating in the blood and the vessel wall. Although specific adhesion mechanisms are involved in leukocyte-endothelium interactions, adhesion patterns in vivo suggest other rheological mechanisms also play a role. Previous studies have proposed that the abundance of leukocyte rolling in postcapillary venules is due to interactions between red blood cells (RBCs) and leukocytes as they enter postcapillary expansions, but the details of the fluid dynamics have not been elucidated. We have analyzed the interactions of red and white blood cells as they flow from a capillary into a postcapillary venule using a lattice Boltzmann approach. This technique provides the complete solution of the flow field and quantification of the particle-particle forces in a relevant geometry. Our results show that capillary-postcapillary venule diameter ratio, RBC configuration, and RBC shape are critical determinants of the initiation of cell rolling in postcapillary venules. The model predicts that an optimal configuration of the trailing red blood cells is required to drive the white blood cell to the wall.

P-selectin glycoprotein ligand-1 mediates L-selectin-dependent leukocyte rolling in venules.

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.

Desmopressin Induces Endothelial P-Selectin Expression and Leukocyte Rolling in Postcapillary Venules

Desmopressin, (DDAVP; 1-desamino-8-D-arginine vasopressin) increases the release and activity of von Willebrand factor (vWF); however, its effects on the other major constituent of endothelial Weibel-Palade bodies, P-selectin, has not been investigated. DDAVP-induced P-selectin expression may explain DDAVP's efficacy in bleeding disorders in which vWF levels are normal. Therefore, the objective of this study is to assess the effect of DDAVP on P-selectin expression on endothelial cells of postcapillary venules in vivo and on human umbilical vein endothelium in vitro, and to determine whether DDAVP has direct effects on leukocyte behavior in postcapillary venules. DDAVP (0.1 and 1.0 microgram/mL) induced a significant but transient increase in P-selectin expression on human umbilical vein endothelial cells as well as on rat and human platelets. Immunohistochemical analysis of rat postcapillary venules showed that in contrast to saline, DDAVP injection (1 microgram/kg, intravenous) induced significant endothelial P-selectin expression. DDAVP administration also induced a rapid and significant increase in leukocyte rolling in rat mesenteric venules in vivo. This response was entirely dependent on P-selectin, as an anti-P-selectin antibody rapidly reversed the DDAVP-induced increase in leukocyte rolling. DDAVP induced leukocyte rolling in medium (20 to 40 microns) and large (> 40 microns), but not small (< 20 microns), postcapillary venules. In animals that were treated with DDAVP, there was a steady and significant increase in leukocyte adhesion. This study shows that DDAVP can directly induce P-selectin expression on endothelium in vitro and in vivo and that the latter response is capable of supporting prolonged leukocyte rolling in rat postcapillary venules.

Desmopressin induces endothelial P-selectin expression and leukocyte rolling in postcapillary venules

Desmopressin, (DDAVP; 1-desamino-8-D-arginine vasopressin) increases the release and activity of von Willebrand factor (vWF); however, its effects on the other major constituent of endothelial Weibel-Palade bodies, P-selectin, has not been investigated. DDAVP-induced P-selectin expression may explain DDAVP's efficacy in bleeding disorders in which vWF levels are normal. Therefore, the objective of this study is to assess the effect of DDAVP on P-selectin expression on endothelial cells of postcapillary venules in vivo and on human umbilical vein endothelium in vitro, and to determine whether DDAVP has direct effects on leukocyte behavior in postcapillary venules. DDAVP (0.1 and 1.0 microgram/mL) induced a significant but transient increase in P-selectin expression on human umbilical vein endothelial cells as well as on rat and human platelets. Immunohistochemical analysis of rat postcapillary venules showed that in contrast to saline, DDAVP injection (1 microgram/kg, intravenous) induced significant endothelial P-selectin expression. DDAVP administration also induced a rapid and significant increase in leukocyte rolling in rat mesenteric venules in vivo. This response was entirely dependent on P-selectin, as an anti-P-selectin antibody rapidly reversed the DDAVP-induced increase in leukocyte rolling. DDAVP induced leukocyte rolling in medium (20 to 40 microns) and large (&gt; 40 microns), but not small (&lt; 20 microns), postcapillary venules. In animals that were treated with DDAVP, there was a steady and significant increase in leukocyte adhesion. This study shows that DDAVP can directly induce P-selectin expression on endothelium in vitro and in vivo and that the latter response is capable of supporting prolonged leukocyte rolling in rat postcapillary venules.

Leukocyte rolling in venules of striated muscle and skin is mediated by P-selectin, not by L-selectin.

Leukocyte rolling in post-capillary venules is mediated by adhesion molecules of the selectin family expressed on both leukocytes (L-selectin) and endothelial cells (E- and P-selectin). With the use of intravital fluorescence microscopy, the effects of antibodies against these selectins were analyzed in the skinfold chamber model of BALB/c mice and the ear model of homozygous hairless mice (hr/hr) that permit chronic observation of striated muscle and skin microcirculation in awake animals, respectively. Mice were injected intravenously with monoclonal antibodies (MAb) to murine L-selectin and E-selectin and affinity-purified polyclonal antibodies to P-selectin. The antibodies, which are known to block cell adhesion, were tested by immunoprecipitation to selectively bind to L-, E-, or P-selectin. Leukocyte rolling was a constant finding in both microcirculation models in the absence of inflammatory stimuli. In both models, injection of anti-P-selectin antibodies completely prevented baseline leukocyte rolling over an observation period of 2 h (P < 0.01 vs. baseline), while no effects were seen after administration of either anti-L-selectin or anti-E-selectin MAb. Treatment with the isotype-matched control antibodies did not affect leukocyte rolling in either model. We conclude that leukocyte rolling in postcapillary venules of murine striated muscle and skin is a physiological process mediated via P-selectin, whereas L- and E-selectin appear not to play a significant role under these circumstances.

Thrombin receptor peptide-mediated leukocyte rolling in rat mesenteric venules: roles of P-selectin and sialyl Lewis X.

Neutrophil adhesion to monolayers of cultured endothelial cells is enhanced, via a P-selectin-mediated mechanism, by a 14-amino acid peptide fragment (TRP-14) of the thrombin receptor. The objective of this study was to determine whether TRP-14 promotes P-selectin-mediated sialyl Lewis X-dependent leukocyte rolling in postcapillary venules. Superfusion of the rat mesentery with TRP-14 for 30 min resulted in the recruitment of rolling leukocytes and a concomitant reduction in leukocyte rolling velocity. Analogues of TRP-14 were largely ineffective in promoting leukocyte-endothelial cell adhesion. Treatment with either a monoclonal antibody directed against rat P-selectin or soluble sialyl Lewis X oligosaccharide (the carbohydrate ligand to P-selectin found on leukocytes) significantly attenuated the TRP-14-induced recruitment of rolling leukocytes. However, no effect was observed with a nonbinding antibody or a control fucose-deficient oligosaccharide. These results indicate that TRP-14 elicits the recruitment of rolling leukocytes in postcapillary venules via a P-selectin-dependent mechanism. The results also support the view that sialyl Lewis X participates in P-selectin-mediated leukocyte-endothelial cell adhesion.

Histamine induces leukocyte rolling in post-capillary venules. A P-selectin-mediated event.

The objective of this study was to systematically assess the molecular mechanisms and kinetics of histamine-induced leukocyte rolling in rat mesenteric venules using intravital microscopy. A complicating factor in these studies is surgical preparation-induced leukocyte rolling (spontaneous rolling), which leads to a lack of effect of histamine on this parameter. Therefore, we identified the source of the surgery-induced leukocyte rolling (partial mast cell degranulation) and established that pretreatment of animals with sodium cromoglycate (connective tissue mast cell stabilizer) inhibited spontaneous leukocyte rolling. Superfusion of the mast cell-stabilized rat mesentery with histamine caused a profound increase in leukocyte rolling which persisted for the entire hour of experimentation. Diphenhydramine (H1-receptor antagonist) but not cimetidine (H2-receptor antagonist) prevented the rise in histamine-induced leukocyte rolling. An anti-P-selectin Ab but not an anti-CD18 Ab reversed the histamine-induced leukocyte rolling in a dose-dependent fashion. In this model of low base line rolling, exposure of the mesentery to the chemotactic agent platelet-activating factor did not induce leukocyte rolling or adhesion. However, co-administration of histamine with platelet-activating factor did indeed promote leukocyte adhesion suggesting that the presence of at least one effector of P-selectin is a minimal requirement for chemotactically-stimulated leukocytes to adhere to postcapillary venules. This study demonstrates for the first time that histamine induces leukocyte rolling via a P-selectin-dependent mechanism in vivo. This is a prolonged, H1 receptor-mediated event that may contribute significantly to the early phase of inflammation.