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Circulation. 1997;96:2302-2310

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(Circulation. 1997;96:2302-2310.)
© 1997 American Heart Association, Inc.

Articles

Antithrombin III Prevents and Rapidly Reverses Leukocyte Recruitment in Ischemia/Reperfusion

Lena Ostrovsky; Richard C. Woodman, MD; Derrice Payne; Diane Teoh; ; Paul Kubes, PhD

From the Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada.

Correspondence to Dr Paul Kubes, Immunology Research Group, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1.


*    Abstract
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 *Abstract
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 down arrowResults
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Background P-selectin has recently been shown to be essential for leukocyte rolling after the reperfusion of ischemic mesentery. However, the mediators responsible for neutrophil rolling in ischemic microvessels remain entirely unclear.

Methods and Results Intravital microscopy was used to examine leukocyte kinetics in a feline mesentery ischemia/reperfusion model. Sixty minutes of ischemia followed by reperfusion caused a profound increase in leukocyte rolling and adhesion. Pretreatment with the endogenous antithrombotic agent antithrombin III (ATIII) infused as a bolus (250 U/kg) reduced neutrophil rolling and adhesion to preischemic levels during reperfusion. No effect was seen with heat-inactive ATIII. Importantly, ATIII posttreatment also significantly reduced neutrophil rolling and adhesion during reperfusion, suggesting that ATIII can reverse the leukocyte recruitment response induced by ischemia/reperfusion. Vascular permeability was also reduced by 50% after ATIII administration. To determine whether ATIII could reverse thrombin-induced rolling directly, neutrophil rolling was performed on human endothelium in flow chambers. Indeed, thrombin-induced rolling, but not histamine-induced rolling, could be rapidly reversed with ATIII on endothelium, suggesting that ATIII affects thrombin rather than directly affecting neutrophils or the endothelium.

Conclusions This study demonstrates for the first time that thrombin plays an important role in ischemia-induced leukocyte rolling and adhesion and that ATIII can be used therapeutically postreperfusion to attenuate the leukocyte recruitment response in inflammation without the nonspecific effects associated with anti–adhesion molecule therapy.


Key Words: antithrombin III • reperfusion • ischemia • leukocytes


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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Infiltrating neutrophils have been implicated as key mediators of I/R injury associated with the heart,1 2 brain,3 intestine,4 skeletal muscle,5 as well as various other tissues.6 7 8 There are three pieces of evidence to support this view. First, neutrophils infiltrate postischemic tissue.1 2 4 Second, depleting circulating neutrophils reduces reperfusion-induced tissue injury.2 9 Third, antiadhesion therapy that successfully inhibits neutrophil influx into postischemic vessels concomitantly provides protection against reperfusion injury.6 10 11 12 Neutrophil infiltration from the mainstream of blood into postischemic tissue is a multistep process that includes an initial, transient adhesive interaction manifested as neutrophil rolling, which is thought to be primarily P-selectin mediated,13 14 although L-selectin may also contribute.12 14 Identification of the mediators that induce neutrophil rolling after ischemia may be of tremendous importance therapeutically, at least in part because it is thought that successful inhibition of rolling will impair subsequent adhesion and emigration and may therefore serve as a target for drug intervention. In addition, chemotactic agents, including PAF, coexpressed on the surface of the endothelium (with P-selectin), induce firm neutrophil adhesion to the vessel wall by activating the CD18/glycoprotein complex, which ultimately leads to emigration of neutrophils out of the vasculature.15 16 Because many of the same mediators that cause P-selectin expression also induce PAF synthesis, inhibition of the molecule that prevents leukocyte rolling may also inhibit PAF-induced leukocyte recruitment.17 18

To date, the mediator involved in the early induction of P-selectin and rapid recruitment of rolling neutrophils in reperfusion remains elusive. It has been demonstrated that histamine, oxidants, cysteinyl leukotrienes, and thrombin are all capable of rapidly activating endothelium to express P-selectin on the cell surface within minutes and thereby induce early leukocyte rolling.19 20 Interestingly, inhibition of histamine has been unremarkable in reversing reperfusion-induced vascular and/or tissue injury, suggesting only a minor role for this mediator in P-selectin–mediated leukocyte recruitment.21 Cysteinyl leukotrienes have been shown to have no role in I/R-induced leukocyte recruitment,22 and numerous other reports failed to document any apparent inhibition of leukocyte rolling using antioxidants.23 24 Thrombin, like oxidants, histamine, and cysteinyl leukotrienes, can induce P-selectin expression and PAF production.18 19 25 However, whether it contributes to reperfusion-induced neutrophil rolling and/or adhesion has not been examined. In addition to the evidence that thrombin enhances endothelial P-selectin expression19 26 and endothelial PAF production,27 it has been shown to induce rapid ICAM-1 expression28 causing firm leukocyte adhesion. Because thrombin appears to increase in I/R,29 this protease may be a prime candidate as a contributor to the multistep recruitment pathway of neutrophils into postischemic tissues. Therefore, it is conceivable that antithrombin therapy reduces neutrophil rolling and adhesion and thereby decreases reperfusion-induced tissue and vascular dysfunction.

Because thrombin has recently been recognized to have important proinflammatory functions, we propose that antithrombin therapy may interfere at multiple steps of the adhesion cascade, thereby providing a potent anti-inflammatory function during reperfusion of postischemic vessels. In this study, we examined whether in addition to its anticoagulant function, an endogenous inhibitor of thrombin, ATIII, could reduce reperfusion-induced vascular dysfunction, in part by reducing neutrophil rolling and/or adhesion. We used both an in vivo, intravital microscopy approach that permitted visualization of the multistep recruitment of leukocytes and alterations in vascular integrity and an in vitro, flow-chamber approach that directly permitted assessment of mechanisms of action of ATIII. Our data show that ATIII not only prevents the reperfusion-induced neutrophil recruitment but also, in a rapid and extremely efficient manner, reverses neutrophil trafficking both in vivo, in reperfused microvessels, and in vitro, on thrombin-stimulated endothelium.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Intravital Microscopic Studies
The experimental preparation used in this study is the same as previously described.14 Briefly, cats (1.2 to 2.4 kg) were fasted for 24 hours and initially anesthetized with ketamine hydrochloride (75 mg IM). The jugular vein was cannulated, and anesthesia was maintained by the administration of pentobarbital sodium. A tracheotomy was performed to support breathing by artificial ventilation. Systemic arterial pressure was monitored by a pressure transducer (Statham P23A; Gould) connected to a catheter in the left carotid artery. A midline abdominal incision was made, and a segment of small intestine was isolated from the ligament of Treitz to the ileocecal valve. The remainder of the small and large intestines was extirpated. Body temperature was maintained at 37°C using an infrared heat lamp. All exposed tissues were moistened with saline-soaked gauze to prevent evaporation. Heparin sodium (10 000 U; Elkins-Sinn) was administered; then, an arterial circuit was established between the SMA and left femoral artery. SMA blood flow was continuously monitored using an electromagnetic flowmeter (Carolina Medical Electronics). Blood pressures were continuously recorded with a physiological recorder (Grass Instruments).

Cats were placed in a supine position on an adjustable Plexiglas microscope stage, and a segment of midjejunum was exteriorized through the abdominal incision. The mesentery was prepared for in vivo microscopic observation as previously described.14 The mesentery was draped over an optically clear viewing pedestal that allowed for transillumination of a 3-cm segment of tissue. The temperature of the pedestal was maintained at 37°C with a constant temperature circulator (model 80; Fisher Scientific). The exposed bowel was draped with saline-soaked gauze while the remainder of the mesentery was covered with Saran Wrap (Dow Corning). The exposed mesentery was suffused with warmed bicarbonate-buffered saline (pH 7.4) that was bubbled with a mixture of 5% CO2 and 95% N2. The mesenteric preparation was observed through an intravital microscope (Optiphot-2; Nikon) with a x25 objective lens (Wetzlar L25/0.35; E. Leitz) and a x10 eyepiece. The image of the microcirculatory bed (x1400 magnification) was recorded using a video camera (Digital 5100; Panasonic) and a video recorder (NV8950; Panasonic).

Single unbranched mesenteric venules (25- to 40-µm diameter, 250-µm length) were selected for each study. Venular diameter was measured either on- or off-line using a video caliper (Microcirculation Research Institute, Texas A&M University). The number of rolling and adherent leukocytes was determined off-line during playback analysis. Rolling leukocytes were defined as white blood cells that moved at a velocity less than that of erythrocytes in a given vessel. The number of rolling leukocytes (flux) was counted using frame-by-frame analysis. To obtain a complete leukocyte rolling velocity profile, the rolling velocity of all leukocytes entering the vessel was measured. A leukocyte was defined as adherent to venular endothelium if it remained stationary for >30 seconds. Adherent cells were measured at 10-minute intervals, as described in "Experimental Protocol," and expressed as the number per 100-µm length of venule. VRBC was measured using an optical Doppler velocitometer (Microcirculation Research Institute), and mean Vmean was determined as VRBC/1.6.30 Wall shear rate was calculated based on the newtonian definition: shear rate=(Vmean/Dv)x(8/time [seconds]), where Dv is the venular diameter.

Experimental Protocol
In Vivo Experiments
Baseline measurements of blood pressure, SMA blood flow, VRBC, and vessel diameter were obtained. Experiments were carried out in three groups of animals: (1) untreated, (2) ATIII treatment before ischemia, and (3) ATIII treatment once reperfusion-induced leukocyte recruitment was evident (10 to 20 minutes postischemia). In the first group of animals, the preparation was videotaped for 10 minutes, and then SMA blood flow was mechanically reduced (Gaskell clamp) to 20% of control for 1 hour. The final 10 minutes of the ischemic period were videotaped, and the clamp was removed to restore intestinal blood flow. Video recordings were made at 10 minutes, 1 hour, and the final 10 minutes of the 2-hour reperfusion period. In the second series of animals, an identical protocol was completed, but the animals received a bolus of ATIII (250 U/kg; Bayer Inc of Canada), infused into the jugular vein immediately before ischemia. The third group of animals were given ATIII at 10 to 20 minutes after reperfusion. This concentration of ATIII is a standard dose for humans. To test the specificity of ATIII activity, we performed additional experiments (1) using inactivated ATIII in our I/R model and (2) examining the effect of ATIII on histamine-induced rolling in the cat. ATIII was inactivated by boiling (100°C) for 10 minutes and was measured for activity using a chromatographic assay. In our histamine-induced rolling model, the cat mesentery was exteriorized, as described above, and superfused with 100 µmol/L histamine (Sigma Chemical) for 3 hours. ATIII was administered exactly the same in all models.

Cell Culture
HUVECs were harvested from freshly obtained umbilical cords as previously described.31 Briefly, umbilical cord veins were rinsed of formed blood products with warm PBS, after which the vein was filled with collagenase (320 U/mL in PBS). After a 20-minute incubation period at 37°C, the cords were gently massaged to ensure detachment of endothelial cells from the vessel wall. The digest was collected into centrifuge tubes, and the collagenase inactivated with heat-inactivated FBS, after which the tube was centrifuged (400g for 10 minutes). The pellet was resuspended in Medium 199 supplemented with 20% FBS and antibiotics but no endothelial cell mitogen. The cells were then seeded into fibronectin-coated T25 culture flasks and grown to confluence (2 to 5 days). At confluence, the HUVECs were rapidly detached from the flasks with Trypsin-EDTA and seeded heavily onto fibronectin-coated glass coverslips. The heavy seeding minimizes cell growth and thereby makes cells most responsive to P-selectin upregula-tion. Only first-passaged HUVECs were used for all in vitro experiments because previous work in our laboratory has shown that cultured endothelium loses its ability to express P-selectin.31

In additional experiments, CHO cells transfected with P-selectin cDNA and cultured in Dulbecco's modified Eagle's medium with 10% FBS were seeded at confluence onto glass coverslips as described for HUVECs.

Neutrophil Isolation
Human neutrophils were harvested from acetate-citrate-dextrose–anticoagulated venous blood collected from healthy donors. All isolation steps were performed at room temperature. Neutrophils were purified by dextran sedimentation followed by centrifugation through a Ficoll-Hypaque density gradient. Isolated neutrophils were resuspended in Hanks' balanced salt solution buffer and used at a density of 106 cells/mL. The neutrophil suspensions were warmed in a 37°C water bath for 5 minutes before all flow chamber experiments.

Flow Chamber Assay
To study neutrophil/endothelial cell interactions under shear conditions, a flow chamber assay was used as previously described.32 Briefly, glass coverslips with confluent monolayers of HUVECs or P-selectin transfectants were mounted into a polycarbonate chamber with parallel plate geometry. The flow chamber was placed onto an inverted microscope stage, and monolayers were visualized (x10 objective, x10 eyepiece) using phase contrast imagery. The stage was enclosed in a warm air cabinet, and the temperature was maintained at 37°C. A syringe pump (Harvard Apparatus) was used to draw the freshly isolated neutrophils over monolayers at a shear of 2 dynes/cm2.

Experimental Protocol
In Vitro Experiments
Based on the in vivo experiments, we sought to determine whether ATIII could reverse the thrombin-induced neutrophil rolling in vitro. First, ATIII pretreatment experiments were performed to establish an appropriate dose of thrombin and ATIII. In pretreatment studies, ATIII was added to the neutrophil suspension after a brief 1-minute control period, and then thrombin was added at 6 minutes and perfused over the HUVECs. In ATIII posttreatment experiments, after an initial control period of 6 minutes, thrombin (1 U/mL; Sigma Chemical) was added to the neutrophil suspension and coperfused over the HUVEC monolayers. After the induction of neutrophil rolling, ATIII (5 U/mL) was added to the neutrophil suspension. ATIII was excluded from control experiments. To determine the specificity of ATIII in inhibition of thrombin-induced neutrophil rolling and to ensure ATIII was not having any adverse effects on the neutrophils, ATIII posttreatment experiments were carried out on HUVECs stimulated with histamine (25 µmol/L; Sigma Chemical) and on P-selectin–transfected CHO cells (generously donated by Dr R.P. McEver).

Statistical Analysis
The data were analyzed using standard statistical analysis (ANOVA and Student's t test with Bonferroni's correction for multiple comparisons where appropriate). All values are mean±SEM. Statistical significance was set at P<.05.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Table 1Down summarizes the effects of ischemia followed by reperfusion on hemodynamic parameters in the feline mesenteric microvasculature. Mean arterial blood pressure consistently increased during the ischemic episode and then returned to or dropped below the control value at the time of reperfusion. SMA blood flow was reduced for 60 minutes to 20% of control value, and when the clamp was removed, a hyperemic response was always noted. Blood flow had returned to control levels by 10 minutes and then decreased to {approx}60% of control by 1 to 2 hours. The VRBC and shear rates of vessels chosen for study followed a similar pattern; both VRBC and shear forces dropped below control values at 1 and 2 hours of reperfusion. The diameter of the venules did not change throughout the experimental protocol. Table 2Down demonstrates that mean arterial blood pressure, VRBC, shear rates, and venular diameter were not different with ATIII pretreatment under preischemic conditions. Although hemodynamic responses were similar between the two experimental groups (Table 1Down versus Table 2Down), the reduction in blood flow and shear rate reached significance only in the untreated group.


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  		Table 1. Control Hemodynamic Parameters After I/R in the Feline Mesenteric Microvasculature


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  		Table 2. Hemodynamic Parameters After I/R in the ATIII-Pretreated Feline Mesenteric Microvasculature

Fig 1Down demonstrates that neutrophil rolling velocity is {approx}80 to 90 µm/s in control postcapillary microvessels. The rolling velocity is dramatically decreased during ischemia, primarily due to the dramatically reduced shear forces: the ratio of neutrophil rolling velocity to VRBC does not change during ischemia (data not shown). In addition, the neutrophils retained their rounded shape and skipped along the length of the vessel. However, at the time of reperfusion (10 minutes after ischemia), when VRBC and shear forces returned to control levels, the neutrophils continued to roll very slowly (16.5±3.8 µm/s). Qualitatively, the rolling is noticeably different during this high shear from that of both the control and ischemic periods. During reperfusion, most of the rolling cells adopt a teardrop shape and make greater surface area contact with the endothelium. At 1 and 2 hours of reperfusion, the cells increase their velocity to 50% of preischemic values. Pretreatment of animals with ATIII did not affect neutrophil rolling velocity before or during ischemia. Immediately on reperfusion in the ATIII-treated vessels, the neutrophils rolled slower (48.2±12.3 µm/s) than before ischemia (P<.05) but had increased their velocity relative to untreated cells (P<.05). By 60 minutes of reperfusion, neutrophil rolling velocity reached preischemic values in animals receiving ATIII, a value higher than the untreated group.



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  		Figure 1. Effect of ATIII on leukocyte rolling velocity in untreated (n=3), ATIII-pretreated (n=4), and ATIII-posttreated (n=6) cats. ATIII-pretreated animals received a bolus of ATIII (250 U/kg) immediately before ischemia. ATIII-posttreated cats received ATIII 10 or 20 minutes after reperfusion. *P<.05 relative to control value. {dagger}P<.05 relative to untreated value.

Fig 2ADown illustrates that the flux of rolling neutrophils was {approx}30 to 50 cells/min and either did not change or decreased during ischemia. However, at the time of reperfusion, the flux of rolling cells increased dramatically, surpassing 200 cells/min, and remained at this level throughout the 2 hours of reperfusion in untreated animals. ATIII pretreatment did not affect neutrophil rolling during preischemic or ischemic values. Moreover, at 10 minutes of reperfusion, a significant influx of rolling leukocytes was still noted (128.3±46.1 cells/min). However, as the reperfusion continued, the flux of rolling cells progressively decreased by >=70% of the untreated value (P<.05). Therefore, at 1 and 2 hours, there were fewer cells rolling, and these cells were rolling much faster in the ATIII-treated group.



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  		Figure 2. Flux of rolling leukocytes (A) and number of cells rolling within a 100-µm segment of venule (B) in untreated (n=3) and ATIII-pretreated (n=4) cats. ATIII-pretreated animals received a bolus of ATIII (250 U/kg) immediately before ischemia. *P<.05 relative to control value. {dagger}P<.05 relative to untreated value.

The rolling velocity is an important parameter because as cells roll slower, more of them accumulate along the vessel wall. However, because they are rolling slower, the flux of cells (cells passing a certain point) underestimates the actual number of white cells rolling within a vessel at any given time. When the data are presented as the number of rolling cells within a given length of venule (dependent on flux and velocity), the differences between the two groups become very evident (Fig 2BUp). Under normal conditions, {approx}1 cell can be seen rolling within the venule at any given time. This increased to 22.9±4.3 cells/100-µm-length venule immediately on reperfusion and then decreased to a new plateau (8 to 10 cells/100-µm-length venule) for the remainder of the reperfusion phase in untreated animals. There was still an increase in the number of rolling cells within the venule in ATIII-pretreated animals, but this was attenuated by >=70% at 10 minutes and by >90% at 1 and 2 hours of reperfusion.

Fig 3Down demonstrates that the number of adherent cells increases from {approx}1 cell/100-µm-length venule in control to 10 cells/100-µm-length venule at 10 minutes of reperfusion and then to 15 cells/100-µm-length venule at both 1 and 2 hours of reperfusion. ATIII pretreatment still increased neutrophil adhesion at 10 minutes (P<.05) but returned the number of adherent cells to near-control values by 1 and 2 hours of reperfusion. A significant number of neutrophils emigrated out of the vasculature at 10 minutes of reperfusion in control animals, and emigration persisted at a rate of {approx}1 cell every 4 minutes. In the ATIII-treated group, neutrophils emigrated out of the vasculature and reached peak values at 10 minutes. Emigration was not observed after the 10 minutes of reperfusion (data not shown).



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  		Figure 3. Number of adherent leukocytes in untreated (n=3) and ATIII-pretreated (n=4) cats. ATIII-pretreated animals received a bolus of ATIII (250 U/kg) immediately before ischemia. *P<.05 relative to control value. {dagger}P<.05 relative to untreated value.

A viable form of therapy for reperfusion injury would require that the ATIII be effective when administered at the reperfusion phase. Fig 4Down demonstrates that ATIII given after 1 hr of ischemia and 10 minutes of reperfusion caused a significant reduction in the flux of rolling cells (Fig 4ADown), the number of rolling cells (Fig 4BDown), and the number of adherent cells (Fig 4CDown). Moreover, the velocity with which neutrophils rolled was dramatically increased after ATIII administration (see Fig 1Up). Although recordings were only taken at 1-hour intervals during reperfusion, in each animal there was a remarkable reduction in the number of adherent cells within 10 to 20 minutes of ATIII administration. Posttreatment with ATIII did not alter shear but greatly reduced leukocyte recruitment, suggesting that the effects of ATIII are independent of the hemodynamic factors.



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  		Figure 4. Flux of rolling leukocytes (A), number of cells rolling per 100-µm venule (B), and leukocyte adhesion (C) in untreated (n=3) and ATIII-posttreated (n=6) cats. ATIII was added as a bolus (250 U/kg) 10 or 20 minutes after reperfusion. *P<.05 relative to control value. {dagger}P<.05 relative to untreated value.

In addition, microvascular permeability was measured as an indicator of vascular damage. FITC-albumin was given intravenously, and the leakage of protein from the mesenteric microvasculature was determined before and at reperfusion. Animals received ATIII after the 10-minute recording. FITC-albumin began to leak from venules within minutes of reperfusion, reaching near-peak values by 10 minutes, which was maintained for the remainder of the reperfusion phase. Administration of ATIII to the experimental group significantly reversed the microvascular dysfunction at 2 hours (Fig 5Down). Clearly, reversal of leukocyte rolling and adhesion at 60 minutes does not translate into immediate repair of the endothelium, but beneficial responses can be seen within 2 hours.



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  		Figure 5. Vascular permeability (percent FITC-albumin leakage) in untreated (n=3) and ATIII-posttreated (n=6) cats. ATIII was added as a bolus (250 U/kg) 10 or 20 minutes after reperfusion. *P<.05 relative to control value. {dagger}P<.05 relative to untreated value.

In a final series of in vivo experiments, we tested the specificity of ATIII in our I/R model. Pretreatment with heat-inactivated ATIII had absolutely no effect on attenuation of leukocyte recruitment in the postischemic mesentery. Fig 6Down shows that adhesion and flux of rolling leukocytes increased significantly during reperfusion. Heat inactivation of ATIII attenuated the beneficial effects of ATIII. The vascular permeability, in response to reperfusion, was still evident after inactive ATIII administration (data not shown). The partial effect of inactive ATIII may be directly related to residual (<25%) ATIII activity remaining after boiling. Finally, histamine induced a significant increase in leukocyte rolling, adhesion, and vascular permeability in our feline model; however, ATIII was not able to reverse leukocyte recruitment in this model (data not shown).



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  		Figure 6. Effect of inactivated ATIII on flux of rolling leukocytes in untreated (n=3) and ATIII-pretreated (n=4) cats. ATIII-pretreated animals received a bolus of ATIII (250 U/kg) that has been inactivated by boiling for 10 minutes. The inactivated ATIII was determined to be deficient in ATIII activity before use (<25% activity), and it was administered immediately before ischemia. *P<.05 relative to control value.

In Vitro Neutrophil Rolling Kinetics
Because activation of the thrombin receptor on endothelium occurs irreversibly, the turnover of thrombin receptor and P-selectin must be very rapid. To test whether ATIII can directly reverse thrombin-induced, P-selectin–dependent neutrophil rolling, experiments were performed in vitro.

ATIII Pretreatment Inhibits Thrombin-Induced Neutrophil Rolling on HUVECs
Fig 7Down demonstrates that exposure of thrombin (1 U/mL) to confluent HUVEC monolayers resulted in a rapid (<4 minutes) and sustained increase in neutrophil rolling. Pretreatment of the monolayers for 5 minutes with ATIII (5 U/mL), followed by thrombin addition, almost completely inhibited the rolling response to thrombin, suggesting that ATIII can prevent thrombin-induced rolling prophylactically.



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  		Figure 7. Effect of ATIII pretreatment on thrombin-induced neutrophil rolling on HUVECs. Thrombin (1 U/mL) addition induced a rapid and sustained increase in neutrophil rolling (n=4). ATIII pretreatment (5 U/mL) attenuated this response (n=4). *P<.05 relative to thrombin-only value.

ATIII Posttreatment Reverses the Thrombin-Induced Neutrophil Rolling on HUVECs
To more closely mimic the posttreatment experiments in vivo, in some experiments, the HUVECs were exposed to thrombin until neutrophil rolling was evident ({approx}4 minutes); ATIII (5 U/mL) was then added to the perfusion buffer. As shown in Fig 8Down, thrombin induced a progressive increase in the number of rolling neutrophils. In contrast, the addition of ATIII caused the number of rolling neutrophils to reach a plateau after a 6-minute exposure; then, the number of rolling cells diminished within the next 10 minutes. This suggests that the thrombin-induced neutrophil rolling can be rapidly reversed, which is consistent with the time frame observed in the in vivo feline system, and that the reversibility of rolling is not particular to the feline system but also occurs on human endothelium. The thrombin-induced increase in neutrophil rolling was entirely P-selectin dependent in that the anti–P-selectin–blocking antibody (G1; 2 µg/mL) completely reversed the rolling response (data not shown).



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  		Figure 8. Effect of ATIII posttreatment on thrombin-induced neutrophil rolling on HUVECs. Thrombin (1 U/mL), by itself, rapidly induced neutrophil rolling, which increased with time (n=4). ATIII posttreatment (5 U/mL) was able to reverse this response within minutes (n=4). *P<.05 relative to thrombin-only value.

ATIII Specifically Inhibits Thrombin
To ensure that ATIII was not affecting (1) the ability of endothelium to directly express P-selectin or (2) the ability of neutrophils to roll on P-selectin, additional experiments were performed on histamine-stimulated HUVECs and P-selectin–transfected CHO cells. Fig 9ADown demonstrates that histamine also rapidly induced neutrophil rolling on HUVECs (also P-selectin dependent). However, the administration of ATIII had absolutely no effect on the rolling response, suggesting that ATIII specifically blocks thrombin activity and does not impair the endothelium in a nonspecific manner. Finally, neutrophils rolled as effectively on P-selectin–transfected CHO cells in the presence or absence of ATIII, suggesting the lack of a direct effect of ATIII on the ability of neutrophils to interact with P-selectin (Fig 9BDown).



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  		Figure 9. A, Neutrophil rolling on histamine-stimulated HUVECs. Histamine (25 µmol/L) induced neutrophil rolling on HUVECs, which was not affected by ATIII treatment (n=4). B, Neutrophil rolling on P-selectin–transfected CHO cells. ATIII (5 U/mL) exposure to P-selectin transfectants had no effect on the number of neutrophils rolling (n=6). *P<.05 relative to control value.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The inadvertent activation of the inflammatory cascade via the production of one or more mediators may result in an abnormal recognition of host cells as "foreign," leading to a variety of acute inflammatory conditions, including that observed during reperfusion. Central to the onset of reperfusion-induced pathology is the accumulation of neutrophils, which by virtue of their ability to synthesize and release large quantities of reactive oxygen metabolites and proteases, are thought to injure tissue. For neutrophils to gain access to a potential site, they must leave the mainstream of blood, make initial contact with or tether to the endothelium, and then roll along the length of postcapillary venules before adhering and emigrating out of the vasculature. It is clear that interruption of the early rolling event in I/R prevents subsequent leukocyte adhesion, emigration, and associated tissue injury.15 33 34 35 Therefore, mechanisms that regulate leukocyte rolling as well as adhesion have become the focus of extensive investigation because they may be potential targets for therapeutic intervention in inflammatory diseases.

Thrombin, the terminal serine protease of the coagulation cascade, is best known for its ability to cleave fibrinogen and activate platelets. Thrombin, however, has also been implicated in several other important cellular functions, including inflammation, wound healing, angiogenesis, and atherosclerosis. Although the involvement of thrombin represents an important link between these cellular events, its precise role in inflammation has not been elucidated and requires further investigation. In this study, we visualized single inflamed vessels to assess whether inhibition of thrombin activity would have an impact on the inflammatory response associated with reperfusion. We used an anti-thrombin strategy to demonstrate that (1) thrombin plays a critical role in reperfusion-induced leukocyte recruitment (rolling and adhesion) as well as increased microvascular permeability alterations and (2) administration of ATIII (but not inactive ATIII) to directly inactivate thrombin activity is an extremely effective approach to prevent the sequelae of reperfusion. ATIII inhibits thrombin directly through the formation of a stable stoichiometric covalent complex in which ATIII actually acts as a substrate for thrombin. This approach is likely the only approach to inhibit the activity of thrombin in vivo because there is no known agent that specifically interrupts thrombin receptor activation.36 Furthermore, the possibility that multiple thrombin receptor subtypes exist37 38 39 40 41 may further complicate thrombin inhibition; blockade of the platelet-type thrombin receptor may not be entirely effective for cellular targets that have multiple receptors for thrombin-mediated activation.

Surprisingly, our data also revealed that posttreatment with ATIII reversed the leukocyte responses to thrombin within {approx}20 minutes. This would not have been predictable when one considers the irreversible manner in which thrombin activates the thrombin receptor.37 42 The thrombin receptor contains a thrombin-cleavage site within its extracellular amino terminus. Thrombin cleavage at this site unmasks a new amino terminus, which then acts as its own tethered ligand, binding and activating the receptor. Because ATIII does not reverse this interaction but can inactivate thrombin that has not yet interacted with its receptor, our data suggest that P-selectin expression on human umbilical vein endothelium and feline postcapillary venules is dependent on continued activation of new thrombin receptors, and prevention of further thrombin/thrombin ligand interactions interrupts the inflammatory cascade. Indeed, removal of thrombin from the perfusate was also effective in attenuating the rolling response on human umbilical vein endothelium (L.O. and P.K., unpublished observation), which is consistent with the view that in the absence of thrombin, endothelium rapidly turns off the thrombin response. Results from other biological systems, such as smooth muscle vasoconstriction, support the view that a single dose of exogenous thrombin induces a brief biological response (constriction) that dissipates within 10 minutes. Moreover, Ishii et al43 demonstrated that shut-off of signal in fibroblasts occurred despite the continued presence of cleaved and activated thrombin receptors on the cell surface.

Along the same line of reasoning, our data suggest that P-selectin is also rapidly removed from the cell surface once the inflammatory stimulus is inhibited. Indeed, P-selectin–dependent rolling was rapidly reversed after inhibition of thrombin with ATIII. This suggests that intracellular P-selectin is rapidly mobilized to the surface of endothelium after stimulation and that surface P-selectin, in turn, is either shed or reinternalized. Clearly, the endothelium has the capacity to rapidly turn off the inflammatory response once the activating agent dissipates or is removed by endogenous inhibitors. In support of this view, Hattori et al44 demonstrated that P-selectin is rapidly reinternalized from the cell surface of stimulated endothelial cells. The internalized P-selectin is then sorted into secretory granules or lysosomes for degradation. The latter prevents further cycling of reinternalized P-selectin to the cell surface and, as such, is a mechanism limiting leukocyte recruitment.45 Clearly, these data and our data suggest P-selectin is tightly regulated and rapidly turned off and therefore can be therapeutically targeted.

Many reports to date have postulated that numerous mediators, including PAF, leukotriene B4, anaphylatoxins, and oxidants, contribute to reperfusion-induced neutrophil adhesion.46 47 48 49 However, none of these studies have reported that these agents affect the flux of rolling neutrophils, despite the fact that a number of these mediators (oxidants and anaphylotoxins) induce the rapid expression of P-selectin.20 50 We report for the first time that thrombin is an important mediator of neutrophil rolling in reperfused vessels. To our knowledge, this is the first documentation of a proinflammatory mediator that can induce neutrophil rolling in reperfused vessels in vivo. However, the fact that ATIII did not have an impact on the profound rolling response within the first 10 minutes of reperfusion suggests that there may be another mediator or mediators involved in the recruitment of neutrophils, presumably formed during ischemia or early in reperfusion.

Because rolling is the first step in the recruitment of leukocytes, one can conclude that thrombin induces neutrophil rolling. However, because adhesion is dependent on rolling, it becomes more difficult to establish whether thrombin directly contributes to the reduced adhesive response or if the decrease in adhesion is the result solely of reduced rolling. Based on our previous report that >60%, and probably >=90%, of rolling cells had to be inhibited to impact on adhesion and the fact that posttreatment with ATIII reduced both rolling and adhesion by 50% to 80%, it is likely that thrombin is also contributing to the adhesive response. Although thrombin does not activate the neutrophils or directly affect their adhesiveness (R.C.W. and P.K., unpublished observation),51 it activates the endothelium to synthesize PAF, a proadhesive molecule previously reported by us48 to induce adhesion in this model of reperfusion. In addition, thrombin may contribute to the cascade of events by stimulating the production of MCP-1,27 increasing expression of ICAM-1,26 28 52 or inducing platelet/neutrophil interactions,53 which permits transcellular biosynthesis of certain mediators (leukotriene B4) that would not be produced in significant quantities by either cell type alone.54 This leukotriene has been demonstrated to play a very important role in the proadhesive response to reperfusion.46

It is well known that ATIII binds to endothelial proteoglycans and inhibits the proteolytic activity of thrombin. In view of the abundance of glycosaminoglycans expressed by the endothelium, there is considerable binding potential for ATIII (and antithrombin activity) directly at the site of reperfusion injury. Indeed, in this study, we demonstrate that in addition to antithrombotic activity, ATIII has important anti-inflammatory functions at the blood vessel/wall interface and that ATIII can be used not just to prophylactically inhibit neutrophil rolling and adhesion but also to therapeutically reverse neutrophil/endothelial interactions, a response that leads to the reversal of microvascular dysfunction.


*    Selected Abbreviations and Acronyms
 
ATIII = antithrombin III
CHO = Chinese hamster ovary
FITC = fluorescein isothiocyanate
HUVEC = human umbilical vein endothelial cell
I/R = ischemia/reperfusion
PAF = platelet-activating factor
SMA = superior mesenteric artery
Vmean = mean red blood cell velocity
VRBC = red blood cell velocity


*    Acknowledgments
 
This work was supported by a grant from the Canadian Medical Research Council and the Bayer Inc of Canada/Canadian Red Cross Society Research and Development Fund. Drs Kubes and Woodman are scholars of the Alberta Heritage Foundation for Medical Research, and Dr Kubes is an MRC scientist. The authors would like to thank Nursing Unit 51 at the Foothills Hospital for generously providing the umbilical cords for this study.

Received April 21, 1997; accepted May 6, 1997.


*    References
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