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J Am Coll Cardiol, 1999; 34:280-288
© 1999 by the American College of Cardiology Foundation
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EXPERIMENTAL STUDIES

P-selectin inhibition prevents early neutrophil activation but provides only modest protection against myocardial injury in dogs with ischemia and forty-eight hours reperfusion

Masazumi Arai, MDa, Yoshihiro Masui, MDa, Pascal Goldschmidt-Clermont, MDa, Anthony DiPaula, BSa, Cynthia Siu, PhDa, Takeshi Kondo, MDa and Lewis C. Becker, MD, FACCa

a Division of Cardiology, Department of Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland, USA

Manuscript received June 12, 1998; revised manuscript received February 3, 1999, accepted March 15, 1999.

Reprint requests and correspondence: Dr. Lewis C. Becker, Johns Hopkins Medical Institutions, 600 North Wolfe Street, Halsted 500, Baltimore, Maryland, 21287
lbecker{at}welchlink.welch.jhu.edu


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
OBJECTIVES

This study was designed to determine whether antibody neutralization of the adhesion protein P-selectin would prevent neutrophil activation and reduce myocardial reperfusion injury.

BACKGROUND

Although inhibition of P-selectin markedly reduces short-term myocardial injury after ischemia and reperfusion, it is unknown whether it can provide meaningful long-term protection and preserve left ventricular function.

METHODS

Closed-chest dogs underwent 90 min left anterior descending coronary artery occlusion and 48 h reperfusion, and were randomized to 1) a treatment group (n = 11) receiving 1 mg/kg of the blocking anti–P-selectin antibody PB1.3, or 2) a control group receiving 1 mg/kg PNB1.6 (nonblocking antibody against P-selectin, n = 7) or an equivalent volume of saline (n = 2) 10 min before reperfusion. Infarct size was assessed postmortem by triphenyl tetrazolium chloride staining. Contrast left ventriculography was used to measure left ventricular function. Activation of circulating polymorphonuclear neutrophils (PMNs) was assessed by an increase in surface CD18 expression.

RESULTS

Neutrophil activation was observed at 30 min after reperfusion in the control group, but was abolished in the treatment group. Infarct size was reduced about 25% in the treatment group after controlling for variations in ischemic blood flow (p = 0.003, by analysis of covariance). However, this protective effect was not associated with preservation of blood flow to the ischemic–reperfused myocardium, nor with any improvement in global or regional left ventricular function.

CONCLUSIONS

The anti–P-selectin antibody PB1.3 prevented early PMN activation, but had only a modest long-term infarct-limiting effect over 48 h reperfusion. Adhesion molecules other than P-selectin may mediate delayed PMN activation and accumulation in reperfused myocardium.

Abbreviations and Acronyms
  ANCOVA = analysis of covariance
  Ig = immunoglobulin
  LAD = left anterior descending coronary artery
  mAb = monoclonal antibody
  MPO = myeloperoxidase
  PAF = platelet-activating factor
  PMN = polymorphonuclear neutrophil

P-selectin (GMP-140, CD62) localizes in alpha-granules of platelets and in Weibel-Palade bodies of endothelial cells (1,2), and distributes to the endothelial cell plasma membrane rapidly after exposure to histamine (1), thrombin (3) or oxygen radicals (4) in vitro, or after ischemia–reperfusion of various tissues in vivo (5,6). P-selectin is believed to mediate polymorphonuclear neutrophil (PMN) rolling in vivo (7), thereby facilitating CD11/CD18-mediated attachment of PMNs to endothelial cells and PMN emigration from the vascular lumen (3,8–11). P-selectin appears to tether PMNs to promote juxtacrine activation of CD11/CD18 integrins by interaction between platelet-activating factor (PAF) and its PMN receptor (12). Although the dependence of CD11/CD18 up-regulation on P-selectin has been demonstrated in vitro, no studies have been done to show that the same dependence occurs in vivo in the setting of ischemia–reperfusion.

If P-selectin is an important facilitator of PMN activation, immunoneutralization of P-selectin should prevent infiltration of PMNs into ischemic–reperfused tissues and thereby block PMN-induced tissue injury. On the basis of this hypothesis, the anti–P-selectin antibody PB1.3 has been shown to markedly reduce infarct size in cats and dogs with 1.5- to 2-h coronary artery occlusion and short-term reperfusion of up to 4.5 h (13,14). Immunohistologic studies have shown that P-selectin is expressed only transiently after ischemia–reperfusion and is much less evident after 2 h of reperfusion (6). Although this finding might suggest that transient inhibition of P-selectin should result in sustained protection against PMN-mediated reperfusion injury, other adhesion molecules become expressed and may assume importance later after reperfusion, or other non–adhesion-related mechanisms may account for delayed accumulation of PMNs and tissue injury. P-selectin knockout mice have been found to have total absence of leukocyte rolling in mesenteric venules, but are able, nevertheless, to mobilize nearly normal numbers of PMNs to the peritoneal cavity during experimentally induced inflammation, albeit in a delayed fashion (15).

The current study was, therefore, designed to investigate whether P-selectin plays a role in PMN activation after myocardial ischemia–reperfusion and whether inhibition of P-selectin results in a sustained and meaningful reduction of myocardial injury after 48 h of reperfusion. A closed-chest canine infarct model was used to avoid the inflammatory response associated with surgery, and the same anti–P-selectin antibody was chosen that produced marked reductions of infarct size after short-term reperfusion in feline and canine models (13,14).


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Surgical preparation.   Adult female mongrel dogs, weighing 20 to 27 kg, were anesthetized with thiopental sodium (20 to 30 mg/kg), intubated and ventilated with 0 to 2% halothane, with the concentration adjusted to maintain a stable arterial blood pressure. Through small skin incisions, 8-F vascular sheaths were placed aseptically in a femoral vein and both femoral arteries, and 5,000 IU of heparin was administered intravenously. Under fluoroscopy, a 6- or 7-F pigtail catheter was inserted into the left ventricle for injection of radiolabeled microspheres and contrast medium. For coronary arterial occlusion, an angioplasty balloon catheter with appropriate balloon diameter (2.5 to 3.5 mm) was advanced to the proximal left anterior descending coronary artery (LAD) over a 0.014-in. (0.036 cm) guide wire through a 7-F guiding catheter positioned in the left coronary ostium. Femoral artery pressure and heart rate were monitored continuously.

Experimental protocol.   Dogs were subjected to LAD occlusion for 90 min followed by 48 h reperfusion. Complete LAD occlusion and reperfusion were confirmed angiographically after balloon inflation and deflation. The angioplasty balloon and guiding catheters were removed shortly after reperfusion. Ten minutes before reperfusion, dogs were randomized to receive intravenous administration of 1 mg/kg PB1.3 (a blocking anti–P-selectin antibody, n = 11), or PNB1.6 (a nonblocking control anti–P-selectin antibody, n = 8, 1 mg/kg) or saline (n = 2). After 3.5 h of reperfusion, all catheters were removed, the femoral arteries and veins were ligated, the incisions were closed and the dogs were allowed to recover from anesthesia. At 48 h after reperfusion, dogs were reanesthetized and intubated, and repeat cannulation of the left ventricle and left coronary ostium was performed via a femoral artery cutdown. The chest was opened after acquiring hemodynamic, hematologic, flow and angiographic data, and the heart was excised. At baseline and 70 min after occlusion, and 10 min, 1 h, 3.5 h, 24 h and 48 h after reperfusion, blood samples were withdrawn for measurement of circulating blood cell counts, and hemodynamics were recorded.

Monoclonal antibodies.   Monoclonal antibodies (mAbs) PB1.3 (Cy1747) and PNB1.6, both of which were provided pyrogen free by Cytel Corp. (San Diego, California), were murine immunoglobulin (Ig) G1 monoclonal antibodies raised against human P-selectin. PB1.3 has been shown to block the interaction between P-selectin and its receptor in various species (8,11,16–18), whereas PNB1.6 recognizes but does not block P-selectin (5,13,18,19).

Assessment of cross-reactivity of PB1.3 to dog P-selectin.   Platelets were obtained by centrifugation of normal dog blood with prostaglandinE1 (1 µmol/liter) added to reduce in vitro platelet activation, and the pellet was resuspended in Tyrode’s buffer. Aliquots of 50 x 106 platelets were activated with 25 µmol/liter thrombin receptor–activating peptide (Ser-Phe-Leu-Leu-Arg-Asn) (20,21) (Bachem, Pennsylvania) or 200 ng/ml of PAF (22) (Sigma Chemical Co., St. Louis, Missouri) for 15 min to increase surface P-selectin expression. After fixation with 3% formaldehyde, platelets were washed, incubated with PB1.3 (0 to 100 µg/ml) for 15 min, washed again to remove free PB1.3 and incubated with fluorescein isothiocynate–labeled goat antimouse IgG (Sigma Chemical Co.) for 20 min. Cross-reactivity of PB1.3 to dog P-selectin was assessed by flow cytometry (FACScan, Becton Dickinson, Franklin Lakes, New Jersey). For each experiment and each platelet activator, one platelet sample was incubated with buffer containing secondary antibody but no PB1.3 to serve as a background for the other samples in the group and to correct for any nonspecific binding of PB1.3 to the platelet Fc receptor.

Assessment of mAb excess in plasma.   Plasma samples were obtained from 6 dogs in the PB1.3 group and 4 dogs in the PNB1.6 group before, and 30 min, 3.5 h, 24 h and 48 h after mAb administration. Plasma (250 µl) was incubated with 250 µl of human platelet suspension containing 50 million platelets activated with thrombin receptor–activating peptide. The preparation of human platelets was identical to that of dog platelets as described above. The platelets were washed and incubated with fluorescein isothiocyanate–labeled secondary antibody. The amount of mAb in the plasma was assessed by the fluorescence intensity on platelets. To obtain the fluorescence intensity resulting from a known concentration of mAb, 250 µl of 10-µg/ml PB1.3 instead of plasma was incubated with the platelets.

Regional blood flow measurement.   Radioactive microspheres (15 µm diameter) labeled with 153Gd, 113Sn, 103Ru, 95Nb or 46Sc (DuPont, North Billerica, Massachusetts) were used to measure myocardial blood flow. Two million microspheres were injected into the left ventricle through the pigtail catheter at baseline, after 75 min coronary occlusion and at 3.5 h and 48 h after reperfusion. A simultaneous reference blood sample was withdrawn from an arterial sheath. After sacrifice, tissue samples were counted with a scintillation counter, along with radionuclide standards and background and reference blood samples. Regional myocardial blood flow was calculated by standard methods (23).

Estimation of risk, infarct and no-reflow region sizes.   After 48 h of reperfusion, the left ventricle was excised and sectioned into five slices parallel to the atrioventricular ring. Each slice was weighed, incubated in a 2% solution of triphenyltetrazolium chloride for 30 min at 37°C to visualize the infarct area and photographed. Area at risk was defined by the regional blood flow during coronary occlusion. On each slice, contiguous transmural myocardial sections (2 to 3 mm deep) were dissected from a region that covered the whole area at risk. One transmural section was taken from the nonischemic region in each slice. Each transmural section was divided into five samples weighing 0.05 to 0.2 g between endocardium and epicardium. Samples with blood flow during occlusion less than 50% of transmural flow in the nonischemic area were considered ischemic (24). The no-reflow region was defined as myocardium with more than 50% reduction of blood flow at 48 h of reperfusion compared with the nonischemic area. The photographic transparencies were projected and traced, and the location of each tissue sample was marked and color coded. The masses of infarct, area at risk and no-reflow area were quantified by computerized planimetry of the appropriate areas in each slice, multiplied by the weight of the slice and summed for the entire left ventricle.

Assessment of PMN activation.   Neutrophil activation was assessed in dogs receiving PB1.3 or PNB1.6 by the surface expression of CD18 on circulating neutrophils. Blood samples were obtained at baseline, after 75 min coronary occlusion and at 10 min, 30 min, 1 h, 2 h and 3.5 h after reperfusion. Whole blood was incubated with a saturating concentration (20 µg/ml) of R15.7, a murine monoclonal antibody against CD18 (Boehringer-Ingelheim Pharmaceuticals, Ridgefield, Connecticut), for 30 min at 4°C. The cells were washed with phosphate-buffered saline with 1% bovine serum albumin and incubated with a saturating concentration of fluorescein-conjugated goat antimouse Ig (Tago, Burlingame, California) for 30 min at 4°C. Neutrophil isolation was accomplished by erythrocyte lysis with whole blood lysing reagent (Coulter Immunology, Hialeah, Florida). The cells were washed and fixed in 1% paraformaldehyde. The mean channel fluorescence was measured by flow cytometry after selective gating of viable cells, as determined by log-forward versus log-lateral scatter.

Left ventriculography.   Left ventriculograms were performed at baseline, during occlusion and 10 min and 48 h after reperfusion. Nonionic contrast medium was injected into the left ventricle through the pigtail catheter at a speed of 7 ml/s for 4 s, and images were recorded in the 20° right anterior oblique projection at 30 frames per second. For analysis, the end-diastolic and end-systolic ventricular contours were digitized using an ImageComm System (Sunnyvale, California). Ejection fraction was calculated by the area–length method, assuming a prolate spheroid shape of the left ventricle (25). The left ventricle was divided into five regions of 20 chords each (anterobasal, midanterior, apical, midinferior and inferobasal). The motion of each region was represented by the shortening of the center chord expressed as a percent of the perimeter length (26). The motion of the wall corresponding to the LAD region was expressed as the average of the center chord shortenings of the midanterior and apical regions (anterior wall motion); wall motion in the circumflex perfused region was expressed as the average of the inferobasal and midinferior regions (inferior wall motion).

Myeloperoxidase assay.   Transmural myocardial samples weighing 0.5 to 1.0 g were obtained from both the center of the area at risk and the normal area on a midventricular slice. The samples were frozen and maintained at –70°C immediately after counting microsphere radioactivity. The activity of myeloperoxidase (MPO) was measured as previously described (27). Results were expressed as units of MPO per 100 mg wet weight.

Statistical analysis.   All data are presented as the mean ± SEM. Hematologic, hemodynamic, flow and ejection fraction variables during ischemia and reperfusion in control and PB1.3 groups were compared by repeated measures analysis of variance. Two-way analysis of variance, combined with Dunnett’s post hoc test for multiple comparisons, was used to test for an overall difference between groups for each variable, and for differences at each time point within and between groups. Comparisons of infarct size and tissue MPO were made by the Student t test. The relation between infarct size and collateral blood flow was determined by linear regression analysis, and an analysis of covariance (ANCOVA) was performed to determine whether treatment effect was significant after controlling for the influence of collateral blood flow. To test the hypothesis that PB1.3 prevented early PMN activation, repeated measures analysis of variance with Dunnett’s post hoc test was applied to determine the time points at which there were significant differences in CD18 expression between groups and in comparison with baseline within each group. Profile analysis was also used to compare the two groups by performing analysis of variance for the transformed variable representing the difference in CD18 expression between adjacent time points.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Of the 22 dogs undergoing LAD occlusion, one died before randomization (at 20 min after occlusion) and one injected with PNB1.6 died at 5 min after reperfusion because of ventricular fibrillation. Accordingly, 20 dogs were used for the data analyses. They were divided into two groups: the PB1.3 group (n = 11) and the control group (seven dogs treated with PNB1.6 and two dogs given saline).

Antibody binding to P-selectin on activated platelets.   The affinity of PB1.3 to dog P-selectin was assessed by flow cytometry of activated dog platelets. Mean channel fluorescence, representing the amount of bound PB1.3, increased in a concentration-dependent manner (Fig. 1). Similar results were observed when adenosine diphosphate or hydrogen peroxide was used as a platelet activator. A dose of 1 mg/kg PB1.3 was chosen for the in vivo studies, since this dose resulted in a concentration of about 10 µg/ml in blood, which was sufficient for near maximal binding (Fig. 1).



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  		Figure 1 In vitro PB1.3 binding to dog platelets stimulated by thrombin receptor–activating peptide (TRAP) or platelet-activating factor (PAF). Mean channel fluorescence increased in a concentration-dependent manner, indicating cross-reactivity of PB1.3 to dog P-selectin.

 
Assessment of mAb excess in plasma.   Mean channel fluorescence of human platelets incubated with plasma samples acquired before and at 30 min, 3.5 h, 24 h and 48 h after PB1.3 injection was 3 ± 1, 358 ± 13, 349 ± 21, 333 ± 20 and 343 ± 17, respectively. Results at the same time points before and after PNB1.6 injection were 22 ± 4, 200 ± 18, 231 ± 17, 206 ± 9 and 207 ± 12, respectively. Incubation of platelets with 10 µg/ml PB1.3 instead of plasma resulted in a mean channel fluorescence of 386. These data suggest that after injection, both PB1.3 and PNB1.6 stayed in the circulation at a high concentration throughout the 48-h experiment and that the dose of PB1.3 was sufficient to achieve near maximal P-selectin blockade.

Hemodynamics.   Blood pressure decreased in both control and PB1.3 groups after coronary occlusion. No significant differences in blood pressure or heart rate were seen between the two groups at any time point. Antibody injection produced no hemodynamic effect.

Hematologic parameters.   Leukocyte and neutrophil counts were increased at 24 h reperfusion but returned toward baseline levels at 48 h in both groups. Compared with baseline, hematocrit and platelet count were decreased at 48 h in both groups. Except for a lower hematocrit at baseline in the PB1.3 group (39% vs. 45%, p < 0.05), there were no significant differences between the two groups at any time point in any hematologic parameter. Antibody injection had no significant effect on any hematologic variable.

Regional myocardial blood flow.   Myocardial blood flow to endo-, mid- and epicardium in the ischemic-reperfused region and transmural myocardium in the nonischemic region is shown in Table 1. There were no significant differences in blood flow between the two groups in any region at any time point.


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  		Table 1 Myocardial Blood Flows in Control and PB1.3 Groups

 
Infarct size.   Infarct size expressed as a percent of the area at risk was inversely related to transmural collateral flow in both groups, and the regression line for the PB1.3 group (y = 54.3 – 5.1x, r = 0.87) was shifted downwards compared with that of the control group (y = 66.4 – 4.4x, r = 0.91) (p = 0.003 by ANCOVA) (Fig. 2), indicating that both severity of ischemia and PB1.3 treatment contributed significantly to infarct size. The downward shift of the regression line in the PB1.3 group was also observed when infarct size was plotted against endo- or epicardial collateral flow (p = 0.005 for endocardial flow and p = 0.012 for epicardial collateral flow by ANCOVA). The average infarct size as a percent of area at risk was 47.7 ± 7.3% in the control group and 35.5 ± 5.4% in the PB1.3 group (p = 0.19), respectively, without adjustment for the severity of ischemia. The transmural collateral flow and area at risk as a percent of left ventricle in control and PB1.3 groups were 4.3 ± 1.5 versus 3.7 ± 0.9 ml/min/100 g and 25.4 ± 2.4 versus 27.6 ± 1.6%, respectively. The size of the no-reflow area as a percent of the area at risk did not differ between the two groups (26.6 ± 4.1% in the control and 20.4 ± 4.3% in the PB1.3 group), consistent with the observation that PB1.3 treatment did not attenuate the blood flow reduction at 48 h reperfusion.



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  		Figure 2 Relation between infarct size and the severity of ischemia in control (open circles) and PB1.3 antibody (closed circles) groups. The regression line for PB1.3 was shifted significantly downward (p = 0.003 by analysis of covariance).

 
Effect of treatment with PB1.3 on PMN activation.   In the control group, mean channel fluorescence, representing CD18 expression on circulating PMNs, significantly increased at 30 min after reperfusion compared with the PB1.3 group (113.7 ± 5.6% vs. 96.1 ± 4.0% of baseline fluorescence, p < 0.01 by analysis of variance and Dunnett’s post hoc test) and returned to the baseline level by 2 h (Fig. 3). CD18 expression on PMNs at 30 min after reperfusion was correlated with the amount of ischemic myocardium, represented by the area at risk as a percent of left ventricle, in the control group (r = 0.83) but not in the group receiving PB1.3 (r = 0.18) (Fig. 4). The time course of CD18 expression was significantly different between the two groups after 1 h reperfusion. In contrast to down-regulation of CD18 in the control group, CD18 expression on PMNs from dogs in the PB1.3 group showed significant up-regulation during this period (p = 0.0026 vs. control group by profile analysis), suggesting that delayed PMN activation occurred in PB1.3-treated dogs.



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  		Figure 3 Time course of CD18 expression on circulating polymorphonuclear neutrophils (PMNs). In the control group, CD18 expression increased significantly at 30 min after reperfusion, and returned to the baseline level by 2 h. However, treatment with PB1.3 prevented up-regulation of CD18 at 30 min after reperfusion. The curves were also significantly different after 1 h reperfusion by profile analysis.

 


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  		Figure 4 CD18 expression on circulating neutrophils at 30 min after reperfusion was correlated with the size of the area at risk in the control group (open circles, y = 58.8 + 1.6x, r = 0.83) but not in the group receiving PB1.3 (closed circles, y = 86.9 + 0.3x, r = 0.18). This suggests that the amount of neutrophil activation is dependent on the amount of ischemic–reperfused myocardium and that this activation can be prevented by PB1.3. LV = left ventricle.

 
Left ventricular function.   Left ventricular ejection fraction decreased markedly during coronary occlusion and recovered slightly during reperfusion in both groups (Table 2). Similar changes were seen in left ventricular regional chord shortening (Fig. 5). There were no significant differences between the two groups in either global or regional left ventricular function at any time point.


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  		Table 2 Left Ventricular Ejection Fraction

 


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  		Figure 5 Regional myocardial function assessed by left ventriculography. Chord shortening was averaged in the anterior (infarct) and posterior (control) regions. Wall motion was markedly reduced in the infarct region during occlusion and recovered slightly after reperfusion (**p < 0.01 vs. baseline). However, there was no significant difference in regional wall motion between the two groups at any time point.

 
Myocardial myeloperoxidase activity.   The transmural MPO activity in the ischemic–reperfused region was significantly increased compared with that in the nonischemic region in both groups (0.71 ± 0.20 vs. 0.03 ± 0.01 U/100 mg in the control group, p < 0.01, and 0.59 ± 0.14 vs. 0.04 ± 0.01 U/100 mg in the PB1.3 group, p < 0.01). There was no significant difference between the two groups.


   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
Previous studies in animal models with myocardial ischemia and up to 4.5 h reperfusion have demonstrated that administration of PB1.3 produces marked myocardial protection with a reduction of infarct size of 50% or more (13,14,17). Our study, which used 48 h reperfusion, demonstrated that PB1.3 prevents the early activation of PMNs after reperfusion, but reduces infarct size by only 25%, and does not preserve global or regional left ventricular function or prevent no-reflow in the infarcted myocardium. These results suggest that inhibition of P-selectin may temporarily prevent PMN-mediated reperfusion injury, but that full myocardial protection is not sustained and ultimately the benefit afforded by specific P-selectin blockade is rather modest.

P-selectin and PMN activation.   P-selectin facilitates PMN activation on stimulated endothelium (12,28,29). By binding to its PMN carbohydrate ligand, P-selectin tethers the PMN for efficient juxtacrine activation by PAF, thereby up-regulating CD11/CD18. In addition, purified P-selectin can increase PMN CD11/CD18 adhesiveness, associated with the appearance of an "activation" epitope (30). Selectin-mediated PMN rolling is a prerequisite for firm adhesion dependent on CD11/CD18 and intercellular adhesion molecule 1 (9,10,31). Soluble P-selectin or P-selectin bound to platelets may also play a role (32). Ours is the first study to demonstrate the dependence of CD18 up-regulation on P-selectin in vivo in the setting of ischemia-reperfusion.

P-selectin and myocardial reperfusion injury.   Several studies have reported very significant myocardial protection by PB1.3 in large animal models with 1- to 2-h coronary artery occlusion and 1- to 4.5-h reperfusion (13,14,17), with infarct size reductions of 50% to 60% (13,14). Although we found a statistically significant reduction in infarct size at 48 h in PB1.3-treated animals, the magnitude of myocardial protection was modest and there was no concomitant preservation of left ventricular function or perfusion of the infarct region. Ours is the first study to examine the effects of PB1.3 on myocardial reperfusion injury and left ventricular function over such a long time period.

The dose of antibody we used was most likely sufficient, since the same dose was used by others who reported more favorable short-term results, and our own data demonstrated that excess antibody was present in the blood for the entire 48 h of reperfusion. Despite being developed against human protein, PB1.3 demonstrated cross-reactivity with P-selectin on dog platelets in our study, similar to previously reported results (14,33). PB1.3 has been shown to effectively inhibit P-selectin in a number of canine models of ischemia–reperfusion: PB1.3 prevented skeletal muscle no-reflow (16), loss of coronary flow reserve and contractile function (17), development of microvascular thrombi during coronary hypoperfusion (33) and myocardial neutrophil accumulation (14,17) and myocyte injury (14) in various studies. In vitro, PB1.3 has been shown to reduce by >50% the binding of canine neutrophils to thrombin-activated dog coronary artery segments, whereas PNB1.6 was without significant effect (14). Most recently, PB1.3, in a concentration similar to the circulating blood concentration in our study (10 µg/ml), was shown to completely inhibit the thrombin-stimulated binding between canine neutrophils and platelets mediated by P-selectin (33). In theory, being a holoantibody, PB1.3 could have bound to platelet Fc receptors rather than P-selectin. However, background measurements included an Fc-containing IgG secondary antibody without any PB1.3; binding of PB1.3 to nonactivated platelets, which should have had Fc receptors but minimal surface P-selectin, was insignificant.

The most likely reason for our findings is that we assessed the results over 48 h reperfusion rather than the 1- to 4-h period commonly used in acute studies. Analogous to our results, Simpson et al. (34) showed that infusion of a prostacyclin analogue for the first 2 h of reperfusion resulted in a smaller infarct size at 6 h, but not at 72 h after reperfusion. The beneficial effect of P-selectin immunoneutralization may therefore be temporary rather than permanent in myocardial ischemia–reperfusion injury.

P-selectin and other models of injury.   PB1.3 has been reported to attenuate complement-induced lung injury (18) and systemic injury from hemorrhagic shock (35), both acute models. However, Winn et al. (5) found that PB1.3 markedly reduced necrosis in the rabbit ear seven days after a prolonged 6-h period of hypothermic ischemia. Immunohistologic studies showed positive staining for vascular endothelial P-selectin for at least 4 h after reperfusion in this model, considerably longer than the duration of expression found after 90 min of normothermic myocardial ischemia (6).

Inflammation in P-selectin gene–deficient mice.   P-selectin deficient mice, despite an absence of detectable P-selectin on platelets or vascular endothelium and virtual absence of PMN rolling in mesenteric venules (15), nevertheless exhibit PMN rolling 4 h after injection of an inflammatory irritant (36). After the intraperitoneal injection of thioglycollate to produce inflammation, the influx of PMNs was markedly delayed in the P-selectin deficient mice compared with that in the wild type. After 75 min, the mutant mice had 25-fold fewer PMNs in the peritoneal cavity, but by 4 h, there was only a two-fold difference, as the P-selectin–deficient animals began to mobilize PMNs (15).

In mice with P-selectin or L-selectin gene deficiencies, PMN rolling in the cremaster muscle was dependent on P-selectin for the first 60 min after surgical preparation, but from 60 to 120 min rolling was L-selectin dependent (37). Furthermore, rolling induced by tumor necrosis factor alpha was largely dependent on L-selectin and was unaffected by an anti–P-selectin antibody. These data suggest that P-selectin mediates early targeting of PMNs to inflammatory sites but that other mechanisms come into play to mediate later PMN rolling and trapping as the expression of P-selectin wanes.

P-selectin–independent mechanisms for PMN rolling.   Neutrophils may roll under conditions of reduced blood flow independent of selectin molecules. Gaboury et al. (38) showed that after a 50% reduction in shear rate in postcapillary venules, PAF induced a 10-fold increase in PMN rolling and a five-fold increase in adhesion, both dependent on CD18 and not selectins. Edema and vasoconstriction, combined with release of chemotactic factors, could therefore reduce blood flow in the reperfused microcirculation, and result in PMN trapping, adhesion and activation, all mediated through interactions between CD11/CD18 and intercellular adhesion molecule-1.

Prevention of reperfusion injury by blocking adhesion.   Neutralization of P-selectin represents an intrinsically attractive approach for preventing PMN-mediated reperfusion injury, because the tissues with up-regulated adhesion molecules would be targeted. However, Kubes et al. (39) have argued that this approach is inefficient, because >90% inhibition of PMN rolling is required to produce a modest 50% attenuation in PMN adhesion in postischemic venules.

We recently found that an anti-CD18 monoclonal antibody, R15.7, provided significantly better myocardial protection at 48 h reperfusion in the dog than did PB1.3 in the current study (40). With R15.7 there was a 44% reduction in infarct size, combined with significant preservation of global and regional left ventricular function. However, because this antibody inhibits all CD18-dependent cellular functions throughout the body for up to 48 h, there is a concern about possible increased susceptibility to infection (41). Further studies will be needed to define the relative risks and benefits of therapies given to prevent PMN-mediated reperfusion injury in the heart and other organs.


   Footnotes
 
Supported by USPHS Grant # P50 HL52315 (Specialized Center of Research in Ischemic Heart Disease) from the National Heart, Lung, and Blood Institute, Bethesda, Maryland. Tables of hemodynamic and hematologic data are available on request.


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

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Involvement of neutrophils in the pathogenesis of lethal myocardial reperfusion injury
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