Sequential contribution of L- and P-selectin to leukocyte rolling in vivo

Leukocyte recruitment into inflammatory sites is initiated by a reversible transient adhesive contact with the endothelium called leukocyte rolling, which is thought to be mediated by the selectin family of adhesion molecules. Selectin-mediated rolling precedes inflammatory cell emigration, which is significantly impaired in both P- and L-selectin gene-deficient mice. We report here that approximately 13% of all leukocytes passing venules of the cremaster muscle of wild- type mice roll along the endothelium at < 20 min after surgical dissection. Rolling leukocyte flux fraction reaches a maximum of 28% at 40-60 min and returns to 13% at 80-120 min. In P-selectin-deficient mice, rolling is absent initially and reaches 5% at 80-120 min. Rolling flux fraction in L-selectin-deficient mice is similar to wild type initially and declines to 5% at 80-120 min. In both wild-type and L- selectin-deficient mice, initial leukocyte rolling (0-60 min) is completely blocked by the P-selectin monoclonal antibody (mAb) RB40.34, but unaffected by L-selectin mAb MEL-14. Conversely, rolling at later time points (60-120 min) is inhibited by mAb MEL-14 but not by mAb RB40.34. After treatment with tumor necrosis factor (TNF)-alpha for 2 h, approximately 24% of all passing leukocytes roll in cremaster venules of wild-type and P-selectin gene-deficient mice. Rolling in TNF- alpha-treated mice is unaffected by P-selectin mAb or E-selectin mAb 10E9.6. By contrast, rolling in TNF-alpha-treated P-selectin-deficient mice is completely blocked by L-selectin mAb. These data show that P- selectin is important during the initial induction of leukocyte rolling after tissue trauma. At later time points and in TNF-alpha-treated preparations, rolling is largely L-selectin dependent. Under the conditions tested, we are unable to find evidence for involvement of E- selectin in leukocyte rolling in mice.     

Importance of primary capture and L-selectin-dependent secondary capture in leukocyte accumulation in inflammation and atherosclerosis in vivo.

In the multistep process of leukocyte extravasation, the mechanisms by which leukocytes establish the initial contact with the endothelium are unclear. In parallel, there is a controversy regarding the role for L-selectin in leukocyte recruitment. Here, using intravital microscopy in the mouse, we investigated leukocyte capture from the free flow directly to the endothelium (primary capture), and capture mediated through interactions with rolling leukocytes (secondary capture) in venules, in cytokine-stimulated arterial vessels, and on atherosclerotic lesions in the aorta. Capture was more prominent in arterial vessels compared with venules. In venules, the incidence of capture increased with increasing vessel diameter and wall shear rate. Secondary capture required a minimum rolling leukocyte flux and contributed by approximately 20-50% of total capture in all studied vessel types. In arteries, secondary capture induced formation of clusters and strings of rolling leukocytes. Function inhibition of L-selectin blocked secondary capture and thereby decreased the flux of rolling leukocytes in arterial vessels and in large (>45 microm in diameter), but not small (<45 microm), venules. These findings demonstrate the importance of leukocyte capture from the free flow in vivo. The different impact of blockage of secondary capture in venules of distinct diameter range, rolling flux, and wall shear rate provides explanations for the controversy regarding the role of L-selectin in various situations of leukocyte recruitment. What is more, secondary capture occurs on atherosclerotic lesions, a fact that provides the first evidence for roles of L-selectin in leukocyte accumulation in atherogenesis.     

Therapeutic potential of inhibiting leukocyte rolling in ischemia/reperfusion.

Leukocyte rolling has been postulated to be mandatory for subsequent leukocyte adhesion and tissue injury observed during ischemia/reperfusion. The objective of this study was to systematically assess this hypothesis at the microvascular level by examining the effects of various concentrations of a selectin-binding carbohydrate (fucoidin) on the increased rolling and adhesion of leukocytes in postischemic venules. The contribution of L-selectin and/or P-selectin to leukocyte rolling were also assessed in this model. Using intravital microscopy we observed that 60 min of ischemia followed by reperfusion caused a profound increase in leukocyte rolling and adhesion. A high dose of fucoidin (25 mg/kg) reduced leukocyte rolling by > 90% and significantly reduced leukocyte adhesion, whereas a lower dose of fucoidin still reduced leukocyte rolling by 60% but had no effect on leukocyte adhesion. Moreover, despite the profound reduction in leukocyte rolling with fucoidin, the remaining rolling cells were able to firmly adhere via a CD18-dependent mechanism, particularly in those postcapillary venules with reduced (30-50%) shear rates. The increased rolling was also reduced 60% by either an anti-P-selectin antibody, an anti-L-selectin antibody, or a combination of the two antibodies, but this reduction in rolling cells did not translate into significantly reduced leukocyte adhesion. Our data suggest that L-selectin, P-selectin, and a fucoidin-sensitive pathway contribute to the significant increase in reperfusion-induced leukocyte rolling. However, targeting leukocyte rolling as a form of therapy requires very significant efficacy (> 90%) to achieve reasonable (approximately 50%) attenuation in leukocyte adhesion in postischemic venules.     

P-selectin glycoprotein ligand-1-deficient mice have impaired leukocyte tethering to E-selectin under flow

P-selectin glycoprotein ligand-1 (PSGL-1) mediates rolling of leukocytes on P-selectin under flow. The glycoproteins that enable leukocyte tethering to or rolling on E-selectin are not known. We used gene targeting to prepare PSGL-1-deficient (PSGL-1-/-) mice, which were healthy but had moderately elevated total blood leukocytes. Fluid-phase E-selectin bound to approximately 70% fewer sites on PSGL-1-/- than PSGL-1+/+ neutrophils. Compared with PSGL-1+/+ leukocytes, significantly fewer PSGL-1-/- leukocytes rolled on E-selectin in vitro, because their initial tethering to E-selectin was impaired. The residual cells that tethered rolled with the same shear resistance and velocities as PSGL-1+/+ leukocytes. Compared with PSGL-1+/+ mice, significantly fewer PSGL-1-/- leukocytes rolled on E-selectin in TNF-alpha-treated venules of cremaster muscle in which P-selectin function was blocked by an mAb. The residual PSGL-1-/- leukocytes that tethered rolled with slow velocities equivalent to those of PSGL-1+/+ leukocytes. These results reveal a novel function for PSGL-1 in tethering leukocytes to E-selectin under flow.     

Soluble L-selectin at levels present in septic patients diminishes leukocyte-endothelial cell interactions in mice in vivo: a mechanism for decreased leukocyte delivery to remote sites in sepsis

OBJECTIVE: Recent in vivo studies of both septic humans and animals demonstrate that leukocyte delivery is attenuated to sites remote from the primary infection. The mechanisms for this are not entirely clear. L-selectin is integral to rolling, the first step in leukocyte recruitment to an inflammatory site. L-selectin is shed from leukocytes in sepsis, resulting in increased levels of soluble L-selectin in plasma (2.33 microg/mL). This study investigates the effects of soluble L-selectin at levels found in sepsis on leukocyte trafficking in vivo. DESIGN: Prospective, controlled trial. SETTING: Surgical research laboratory in a university hospital. SUBJECTS: Swiss white male mice of 25-35 g. INTERVENTIONS: Mice were randomized to one of three study groups: soluble L-selectin 2.33, soluble L-selectin 8.0, or albumin. Intravital microscopy was performed on postcapillary venules of 20-40 microm in diameter in the cremaster muscle of mice. Leukocyte-endothelial cell interactions (rolling, adherence, and rolling velocity) were measured pre- and post- (1, 15, 30, and 45 mins) intravenous infusion of human recombinant soluble L-selectin (2.33 and 8.0 microg/mL) or human albumin (8.0 microg/mL). MEASUREMENTS AND MAIN RESULTS: The intravenous administration of soluble L-selectin to a systemic concentration of 2.33 microg/mL diminished rolling significantly. Soluble L-selectin at 8.0 microg/mL decreased rolling and increased rolling velocity to a greater degree. Injection of albumin did not alter leukocyte-endothelial cell interactions at any time point. No difference between groups in blood pressure, shear rate, or leukocyte counts was detected. CONCLUSIONS: Soluble L-selectin diminishes leukocyte rolling at levels present in sepsis (2.33 microg/mL). This effect is dose dependent, and could not be explained by differences in blood pressure, shear rate, or leukocyte counts. These findings identify increased soluble L-selectin levels as one of the mechanisms for decreased leukocyte delivery and exudation to remote sites in septic patients.     

Relevance of L-selectin shedding for leukocyte rolling in vivo

The velocity of rolling leukocytes is thought to be determined by the expression of adhesion molecules and the prevailing wall shear stress. Here, we investigate whether rapid cleavage of L-selectin may be an additional physiologic regulatory parameter of leukocyte rolling. A unique protease in the membrane of leukocytes cleaves L-selectin after activation, resulting in L-selectin shedding. The hydroxamic acid-based metalloprotease inhibitor KD-IX-73-4 completely prevented L-selectin shedding in vitro and significantly decreased the rolling velocity of leukocytes in untreated wild-type C57BL/6 mice from 55 to 35 micrometer/seconds in vivo. When E-selectin was expressed on the endothelium (tumor necrosis factor [TNF]-alpha treatment 2.5-3 h before the experiment), rolling velocity was 4 micrometer/seconds and did not change after the application of KD-IX-73-4. However, KD-IX-73-4 decreased mean rolling velocity by 29% from 23 to 16 micrometer/seconds in E-selectin-deficient mice treated with TNF-alpha. The reduction of velocity caused by KD-IX-73-4 was immediate (<5 s) after injection of KD-IX-73-4 as shown by a novel method using a local catheter. These results establish a role for L-selectin shedding in regulating leukocyte rolling velocity in vivo.     

Subpleural microvascular flow velocities and shear rates in normal and septic mechanically ventilated rats

Changes in pulmonary microhemodynamics are important variables in a large variety of pathological processes. We used in vivo fluorescent videomicroscopy of the subpleural microvasculature in mechanically ventilated rats to directly monitor microvascular flow velocity (FV) and shear rate in pulmonary arterioles, capillaries, and venules in healthy rats and in septic rats 20 h after cecal ligation and puncture (CLP). Observations were made through a small thoracotomy after injection of fluorescent microspheres (D = 1 microm) into the systemic circulation. The FVs were calculated off-line by frame-by-frame measurements of the distance covered by individual microspheres per unit of time. In healthy rats, inspiratory FV were 1322 +/- 142 microm/s in subpleural arterioles and 599 +/- 25 microm/s in capillaries. The highest FV was found in venules (1552 +/- 132 microm/s). The calculated shear rates were 547 +/- 62/s in arterioles and 619 +/- 19/s in capillaries. The highest shear rates were detected in venules (677 +/- 59/s). No significant changes in FV and shear rates were observed throughout the 1-h observation period in any of the microvascular compartments. Pulmonary microvascular FV and shear rates found in sham-operated rats in the CLP experiments were not significantly different from values of healthy rats. The CLP caused a significant increase in leukocyte sequestration in the lungs and a mean of 27% to 34% decrease in FV in all sections of the pulmonary microvasculature (P < 0.001 in capillaries and P < 0.05 in venules). Also, CLP caused a 23% decrease in capillary shear rate that reached only borderline statistical significance (P < 0.06) and a significant 35% decrease in mean shear rate in venules (P < 0.05). Fluorescent videomicroscopy is offered as a stable and reproducible method for in vivo determinations of pulmonary microhemodynamics in clinically relevant models of sepsis.     

Reduction in intestinal leukocyte adherence in rat experimental endotoxemia by treatment with the 21-aminosteroid U-74389G

OBJECTIVES: To investigate leukocyte adherence in intestinal venules in experimental endotoxemia after treatment with the 21-aminosteroid U-74389G. DESIGN AND SETTING: Prospective, randomized, controlled animal study in an experimental laboratory. SUBJECTS: Twenty-one male Wistar rats weighing 190 +/- 40 g. INTERVENTIONS: The rats were divided equally into three groups: (a) control group, (b) endotoxemia (5 mg/kg lipopolysacharide from Escherichia coli O55:B5), and (c) endotoxemia and U-74389G administration 30 min before (3 mg/kg) and 60 min after endotoxin challenge (1.5 mg/ kg). MEASUREMENTS AND MAIN RESULTS: The distal small intestine of the animals was examined using intravital fluorescence videomicroscopy 2 h after endotoxin challenge. Leukocytes were stained in vivo by means of rhodamine 6G. In the endotoxemic animals we observed a fourfold increase in the count of firmly adherent leukocytes in submucosal post-capillary and collecting venules. Treatment with the 21-aminosteroid U-74389G significantly attenuated the count of sticking leukocytes in the collecting venules (control, 61 +/- 10 cells/mm2; lipopolysaccharide, 237 +/- 42 cells/mm2; U-74389G 125 +/- 9 cells/mm2; p < 0.05). In these venules leukocyte rolling behavior was comparable to that in the control group without endotoxin challenge. CONCLUSIONS: Administration of U-74389G, which has radical scavenging properties, attenuates leukocyte adherence in selected populations of intestinal venules which is found increased during endotoxemia. Thus, 21-aminosteroids may have an impact in the treatment of endotoxin-induced intestinal injury.     

Leukocyte-independent plasma extravasation during endotoxemia

OBJECTIVES: To determine the meaning of leukocyte-endothelial interactions for the development of endotoxin-induced vascular leakage. DESIGN: Randomized, blinded, controlled trial. SETTING: Experimental laboratory. SUBJECTS: Twenty-four male Wistar rats. INTERVENTIONS: After application of fucoidin to prevent leukocyte rolling and adherence (25 mg/kg; n = 8; fucoidin/LPS group) or saline 0.9% (n = 8; LPS group), animals were given an intravenous infusion of endotoxin (Escherichia coli lipopolysaccharide 026:B6; 2 mg/kg/hr) over 120 mins. Animals in the control group (n = 8) received an equivalent volume of saline 0.9%. MEASUREMENTS AND MAIN RESULTS: Leukocyte rolling and leukocyte adherence, red cell velocity, vessel diameters, venular wall shear rate, volumetric blood flow, and macromolecular leakage were determined in mesenteric postcapillary venules using in vivo videomicroscopy at baseline, 60 mins, and 120 mins after start of a continuous endotoxin infusion. Fucoidin prevented leukocyte rolling (baseline, 3+/-2 rollers; 120 mins, 3+/-1 rollers; not significant vs. baseline; p < .01 vs. LPS group) and reduced the adherence of leukocytes at baseline and during endotoxemia and showed only a slight increase in adherent leukocytes (baseline, 100+/-38 cells/mm2; 120 mins, 244+/-68 cells/mm2; p < .05 vs. baseline; p < .01 vs. LPS group). In the LPS group, endotoxin exposure induced a marked increase in adherent leukocytes (baseline, 248+/-24 cells/mm2; 120 mins, 560+/-57 cells/mm2; p < .01). Leukocyte adherence in control animals (control group) did not increase significantly. Macromolecular leakage, expressed as the ratio of perivenular to intravenular fluorescence intensity after injection of fluorescence-labeled albumin, increased from 0.16+/-0.03 to 0.49+/-0.04 (p < .01 vs. baseline; p < .05 vs. control) during the infusion of endotoxin in the LPS group. Fucoidin application did not diminish the extravasation of albumin (baseline, 0.09+/-0.03; 120 mins, 0.61+/-0.10; p < .01 vs. baseline; p < .01 vs. control). CONCLUSIONS: These results demonstrate that despite a significant reduction of adherent leukocytes to the endothelium by fucoidin, there is no reduction in macromolecular leakage, indicating that leukocyte-endothelial interactions only play a minor role for the development of macromolecular leakage and microvascular damage in the early phase of endotoxemia.     

The alpha 4-integrin supports leukocyte rolling and adhesion in chronically inflamed postcapillary venules in vivo

A role for the alpha 4-integrin (alpha 4 beta 1 or alpha 4 beta 7), has been implicated in the recruitment of peripheral blood mononuclear cells (PBMCs) to sites of inflammation. However, the adhesive interactions (i.e., tethering, rolling, and adhesion) mediated by the alpha 4-integrin have not been characterized in vivo. The objective of this study was to establish a model wherein postcapillary venules were chronically inflamed, and then use intravital microscopy to identify the adhesive interactions mediated by the alpha 4-integrin in vivo. Between 4 and 20 d after immunization with Mycobacterium butyricum, animals developed a systemic vasculitis characterized by large increases in the numbers of rolling and adhering leukocytes within mesenteric venules. The selectins could only account for approximately 50% of the leukocyte rolling whereas the remaining cells rolled exclusively via the alpha 4-integrin. Anti-alpha 4 therapy also eliminated the increase in leukocyte adhesion observed in this model, whereas selectin therapies and an anti-CD18 (beta 2-integrin) monoclonal antibody (mAb) did not reduce adhesion. A serum against polymorphonuclear leukocytes (PMNs) was used to confirm that a significant proportion of rolling cells, and most of the adhering cells were PBMCs. Sequential treatment with anti-PMN serum and the anti-alpha 4 mAb demonstrated that alpha 4-dependent rolling was distinct from PMN rolling populations. Initial leukocyte tethering via the alpha 4- integrin could not be demonstrated in this model, whereas L-selectin did support leukocyte tethering. These data suggest that the alpha 4- integrin can mediate both rolling and adhesion in the multistep recruitment of PMBCs in vivo, and these interactions occur independently of the selectins and beta 2-integrins.     

Soluble L-selectin attenuates tumor necrosis factor-alpha-mediated leukocyte adherence and vascular permeability: a protective role for elevated soluble L-selectin in sepsis

OBJECTIVE: We have previously demonstrated that leukocyte delivery to remote sites is decreased in sepsis and that increased concentrations of soluble L-selectin are, in part, responsible for this finding. Given that leukocytes have been implicated in the pathogenesis of vascular leakage, we hypothesized that the elevated soluble L-selectin concentrations in sepsis may translate into decreased inflammation-mediated leukocyte-endothelial cell interactions and vascular leakage at these sites. DESIGN: Prospective, controlled animal study. SETTING: Surgical research laboratory in a university hospital. SUBJECTS: Swiss white male mice weighing 25-35 g. INTERVENTIONS: Mice were randomized to one of three study groups: intracremaster tumor necrosis factor-alpha with subsequent intravenous bicarbonate buffered solution; intracremaster tumor necrosis factor-alpha with intravenous soluble L-selectin (10 microg/mL); and intracremaster bicarbonate buffered solution with intravenous bicarbonate buffered solution. The cremaster muscle was prepared for both light and fluorescence intravital microscopy 2 hrs after intracremaster injection, and fluorescein isothiocyanate-labeled albumin was injected intravenously. Leukocyte-endothelial interactions (rolling flux, rolling velocity, and adherence) were counted off-line. Postcapillary venule leakage was determined by the permeability index (perivenular/intravenular fluorescence) after intravenous injection of fluorescent albumin. MEASUREMENTS AND MAIN RESULTS: Soluble L-selectin significantly attenuated tumor necrosis factor-alpha-mediated increases in leukocyte adherence and vascular leakage. Leukocyte rolling velocity was restored to baseline with soluble L-selectin; however, rolling flux was not altered. Blood pressure, shear rate, and leukocyte counts did not differ between groups. CONCLUSIONS: Soluble L-selectin decreases local inflammation-mediated leukocyte adherence and vascular leakage in vivo. The increased concentrations of soluble L-selectin in sepsis may represent a protective mechanism by which the host attempts to diminish the deleterious systemic effects of activated leukocytes during sepsis.     

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.     

Transit time of leukocytes rolling through venules controls cytokine-induced inflammatory cell recruitment in vivo.

Leukocyte recruitment requires leukocyte rolling, activation, firm adhesion, and transmigration. Injection of the proinflammatory cytokine TNF-alpha induces expression of E-selectin, interleukin-8, and other adhesion molecules and chemoattractants on the endothelial surface. TNF-alpha- treated CD18 null mouse cremaster muscle venules show increased leukocyte rolling velocity and reduced leukocyte recruitment efficiency. Leukocyte recruitment in CD18 null but not wild-type mice is significantly blocked by an mAb to E-selectin. To understand this overlap between adhesion events previously considered separate, we introduce a quantitative analysis of the efficiency of induction of rolling, conversion of rolling to adhesion, and of adhesion to transmigration. We find that CD18 and E-selectin cooperate to control the time a leukocyte needs to roll through an inflamed area and to convert rolling to firm adhesion. Leukocyte rolling time, defined as the time it takes for a rolling leukocyte to pass through a defined length of a vessel segment, emerges as a unifying parameter determining the efficiency of inducing firm adhesion, which is a rate-limiting step controlling leukocyte recruitment in inflammation. We conclude that leukocytes integrate chemoattractant signals while rolling along the endothelial surface until they reach a critical level of activation and become firmly adherent.     

Inhaled nitric oxide modulates leukocyte kinetics in the mesenteric venules of endotoxemic rats

OBJECTIVE: to determine whether inhaled nitric oxide (NO) would alter leukocyte kinetics in the septic microvasculature. DESIGN: Randomized, controlled trial. SETTING: Experimental laboratory. SUBJECTS: Male Sprague Dawley rats. INTERVENTIONS: Rats were treated with either saline or endotoxin (10 mg/kg, iv) and then allowed to breathe either air or air plus NO (10 ppm). MEASUREMENTS AND MAIN RESULTS: After a 4-hr period, rolling, firm adhesion, and emigration of leukocytes and endothelial dysfunction were monitored in mesenteric venules by using intravital videomicroscopy. Compared with controls, endotoxemic rats exhibited a profound influx in mesenteric venule rolling leukocytes (55+/-17 vs. 70+/-19 leukocytes/min; p < .05), associated with a reduction of leukocyte rolling velocity (83+/-14 vs. 34+/-3 microm/sec; p < .05). In endotoxemic rats, venular endothelium leukocyte firm adhesion (1.15+/-0.32 vs. 4.08+/-0.96 leukocytes/ 100 microm; p < .05) and emigration (0.84+/-0.47 vs. 4.23+/-1.2 leukocytes/100 microm; p < .05) increased compared with controls. Inhaled NO had no effect on leukocyte kinetics in control rats. Inhaled NO significantly attenuated endotoxin-induced venular endothelium leukocyte adhesion (4.08+/-0.96 vs. 1.86+/-0.76 leukocytes/100 microm; p < .05) and emigration (4.23+/-1.2 vs. 1.68+/-0.72 leukocytes/100 microm; p < .05). Compared with control rats, macromolecular (FITC-dextran) vascular leakage, expressed as the perivenular/intravenular fluorescence intensity ratio, increased in endotoxemic rats (0.56+/-0.02 vs. 0.81+/-0.05; p < .01). Endotoxin-induced macromolecular vascular leakage increases were partially prevented by inhaled NO (0.66+/-0.01 vs. 0.56+/-0.02; p < .05). CONCLUSION: These observations suggest that inhaled NO reduces leukocyte adhesion and the degree of vascular permeability dysfunction in mesenteric venule of endotoxemic rats.     

Increase in capillary perfusion following low-intensity ultrasound and microbubbles during postischemic reperfusion

OBJECTIVES: We postulated that the increase in shear stress caused by microbubbles in the presence of low-intensity ultrasound increases vasodilation in ischemia/reperfusion. DESIGN: Prospective, randomized, and blinded experimental study. SETTING: Research laboratory. SUBJECTS: Forty hamsters were subjected to ischemia/reperfusion and observed by intravital microscopy. INTERVENTIONS: Ultrasound (2.5 MHz, 1.3 mechanical index, 2.0 peak pressure) was applied to the hamster cheek pouch in ischemia/reperfusion with and without microbubbles (Levovist or Sono Vue) at baseline (15 mins) and at the beginning (15 mins) of reperfusion after ischemia (30 mins). MEASUREMENTS AND MAIN RESULTS: Arterial diameter (A2-A3, 38.5 +/- 5.3 microm; A4,15.0 +/- 7.0 microm), red blood cell velocity, wall shear stress, permeability, perfused capillary length, and adherent leukocytes in venules were evaluated. Lipid peroxides were also determined in the systemic blood. Ultrasound and microbubbles in reperfusion significantly increased the diameter (A2-A3 Sono Vue, 33%; Levovist, 53% vs. ischemia/reperfusion, p < .05; A4, Sono Vue, 93%; Levovist, 104% vs. ischemia/reperfusion, p < .05), red blood cell velocity, flow, and shear stress in both A4 and A2-A3 arterioles. Shear stress was significantly higher with Levovist (A2-A3, 105%; A4, 185%) and Sono Vue (A2, 108%; A4, 140% vs. ischemia/reperfusion, p < .05) than ultrasound alone in arterioles. With ischemia/reperfusion, perfused capillary length was reduced significantly, whereas it increased with Levovist and Sono Vue (43%, 41% vs. ischemia/reperfusion p < .05). Lipid peroxides increased early during reperfusion and remained at increased levels throughout reperfusion. Lipid peroxides were unchanged after ultrasound alone or ultrasound with Sono Vue or Levovist during ischemia/reperfusion. With ultrasound there was a significant increase in vascular permeability vs. ischemia/reperfusion. Treatment with Sono Vue (-36%) and Levovist (-57%) decreased permeability vs. ischemia/reperfusion in reperfusion (p < .001). Ischemia/reperfusion had significantly increased leukocyte adhesion. Ultrasound alone (-39%) or with Sono Vue (-64%) and Levovist (-57%) caused smaller increases in leukocyte adhesion than ischemia/reperfusion (p < .05). CONCLUSIONS: Ultrasound and microbubbles equilibrate microvascular shear stress, thus avoiding the failure of capillary perfusion in postischemic reperfusion.     

Monoclonal antibody to murine PECAM-1 (CD31) blocks acute inflammation in vivo

A murine model of peritonitis was used to test the role of platelet/endothelial cell adhesion molecule 1 (PECAM-1/CD31) in acute inflammation. A monoclonal antibody (mAb) specific for murine PECAM-1 injected intravenously 4 h before the intraperitoneal injection of thioglycollate broth blocked leukocyte emigration into the peritoneal cavity for up to 48 h. This block was particularly evident for neutrophils. Control mAb, including one that bound to murine CD18 without blocking its function, failed to block emigration when used at the same or higher concentrations. The decreased emigration seen with the anti-PECAM-1 antibody was not due to neutropenia or neutrophil sequestration in the lung, spleen, or other organs; peripheral blood leukocyte counts were not diminished in these mice. In the mesenteric venules of the mice treated with anti-PECAM-1 mAb, leukocytes were frequently seen in association with the luminal surface of the vessel, but did not appear to emigrate. Thus, the requirement for PECAM-1 in the transendothelial migration of leukocytes previously seen in an in vitro model holds true in this in vivo model of acute inflammation.     

Platelets adhered to endothelial cell-bound ultra-large von Willebrand factor strings support leukocyte tethering and rolling under high shear stress.

Leukocyte rolling on vascular endothelium is mediated by an interaction between P-selectin expressed on endothelial cells and P-selectin glycoprotein ligand-1 on leukocytes. This interaction reduces the velocity of leukocyte movements to allow subsequent firm adhesion and transmigration. However, the interaction has so far been observed only under low venous shear stress and cannot explain the accumulation of monocytes in atherosclerotic plaques found in arteries, where shear stress is much higher. We have previously shown that newly released ultra-large von Willebrand factor (ULVWF) forms extremely long string-like structures to which platelets tether. Here, we investigated whether platelets adhered to ULVWF strings are activated and form aggregates. We also determined whether activated platelets on ULVWF strings can support leukocyte tethering and rolling under high shear stresses. We found that platelets adhered to ULVWF expressed P-selectin and bound PAC-1, suggesting their rapid activation. We also found that leukocytes tethered to and rolled on these platelet-decorated ULVWF strings, but not directly on endothelial cells, under high shear stresses of 20 and 40 dyn/cm(2) in a P-selectin dependent manner. These results suggest that the endothelial cell-bound ULVWF provide an ideal matrix to aggregate platelets and recruit leukocytes to endothelial cells under high shear stress. The observed phenomenon delineates a mechanism for leukocytes to be tethered to arterial endothelial cells under high shear, providing a potential link between inflammation and thrombosis.     

Neutrophil-neutrophil interactions under hydrodynamic shear stress involve L-selectin and PSGL-1. A mechanism that amplifies initial leukocyte accumulation of P-selectin in vitro.

Leukocytes attach to and roll on inflamed endothelium and on leukocyte monolayers that form on the endothelial cells. Leukocyte-leukocyte interactions occurring under hydrodynamic shear stress are mediated by binding of L-selectin to unknown sialomucin-like glycoproteins. We show that purified neutrophil PSGL-1, a sialomucin glycoprotein that serves as a ligand for both P- and E-selectin, can also support the attachment and rolling of free flowing neutrophils in vitro. Neutrophil rolling on PSGL-1 was abolished by the anti-L-selectin mAb DREG200 and by the anti-PSGL-1 mAb PL1, indicating that L-selectin can interact directly with PSGL-1. Neutrophil rolling on neutrophil monolayers was also blocked by PL1 (60 +/- 9% SEM inhibition); however, DREG200 blocked more efficiently (93 +/- 7% SEM inhibition), suggesting that other L-selectin ligands may exist on the neutrophil surface. These studies demonstrate that PSGL-1 on the neutrophil surface is a major functional ligand for L-selectin. The avidity of this L-selectin-dependent adhesion event was sufficient to allow individual neutrophils rolling on P-selectin to capture free flowing neutrophils, which progressed to form linear strings and discrete foci of rolling neutrophils. Neutrophil accumulation on P-selectin accelerated with time as a result of neutrophil-assisted capture of free flowing neutrophils. When neutrophil-neutrophil interactions were blocked by DREG200, neutrophils accumulated on P-selectin in a random pattern and at a uniform rate. Thus, leukocyte-assisted capture of flowing leukocytes may play an important role in amplifying the rate of initial leukocyte recruitment at sites of inflammation.     

Platelet activation In mice and human Helicobacter pylori infection.

Extracts of Helicobacter pylori (HP) have been shown to induce leukocyte adhesion in mesenteric venules, but the effects of HP infection on gastric microvessels are unknown. Inflammatory cell interactions in the gastric microcirculation were studied by intravital videomicroscopy in mice inoculated with either saline or fresh isolates of HP. Platelet aggregates were detected and quantified in murine portal blood, while endothelial P-selectin expression was determined using the dual radiolabeled mAb technique. Platelet activation and aggregation were studied in HP-infected patients and controls by measuring the platelet-aggregate ratio and platelet P-selectin expression. HP infection induced a marked increase in the flux of rolling leukocytes and the appearance of platelet and leukocyte- platelet aggregates in murine gastric venules. The HP-induced rolling and platelet aggregate formation was abrogated by mAbs against L- or P-, but not E- selectin. Endothelial cell expression of P-selectin was not altered, but platelet P-selectin expression was enhanced in HP-infected mice. Circulating platelet aggregates and activated platelets were also detected in HP-infected patients. These findings indicate that platelet activation and aggregation contribute to the microvascular dysfunction and inflammatory cell recruitment associated with HP infections.     

Absence of trauma-induced leukocyte rolling in mice deficient in both P- selectin and intercellular adhesion molecule 1

Leukocyte recruitment during inflammation is achieved through a multistep paradigm that includes margination, selectin-mediated rolling, beta 2 integrin-mediated firm adhesion, emigration, and migration into the site of inflammation. We have used the mouse cremaster muscle as a model of trauma- and cytokine-induced inflammation to study the possible role of intercellular adhesion molecule (ICAM) 1 in leukocyte rolling using gene-targeted mice deficient in ICAM-1, P-selectin, and a combination of P-selectin and ICAM-1. Rolling flux and average leukocyte rolling velocity in ICAM-1- deficient mice was not different from wild-type mice, but P- selectin/ICAM-1-deficient mice showed a total absence of rolling for at least 2 h after surgical trauma. Rolling in both wild-type and ICAM-1- deficient mice 60-120 min after trauma was significantly inhibited by a P-selectin monoclonal antibody (mAb) (RB40.34). In contrast, an mAb (KAT-1) blocking ICAM-1 binding to leukocyte function-associated antigen 1 did not block residual rolling in P-selectin-deficient mice. TNF-alpha induced leukocyte rolling in P-selectin/ICAM-1-deficient mice, but the rolling flux fraction was significantly lower than in TNF- alpha-treated ICAM-1-deficient mice. Leukocyte rolling in P- selectin/ICAM-1-deficient mice treated with TNF-alpha for 3 h was completely blocked by an E-selectin mAb (9A9E3), and partially by an L- selectin mAb (MEL-14). This clearly demonstrates E-selectin-dependent rolling in vivo. Leukocyte rolling velocities were significantly reduced after TNF-alpha treatment and were similar in wild-type and gene-targeted strains. We conclude that the residual trauma-induced leukocyte rolling seen in P-selectin-deficient mice is completely abolished by concomitant ICAM-1 deficiency. This severe defect in leukocyte rolling may explain the absence of leukocyte recruitment into the inflamed peritoneal cavity of P-selectin/ICAM-1-deficient mice at early time points (< or = 4 h).