Ann Thorac Surg 2002;74:2064-2070
© 2002 The Society of Thoracic Surgeons
Original article: cardiovascular
Intracoronary E-/L-selectin blockade reduces neutrophil infiltration in heart transplantation
Yvonne M. Carter, MDa,
Robert Thomas, BSa,
Robert Bargatze, PhDc,
Veronica Poppa, MSb,
Mark Jutila, PhDd,
Charles E. Murry, MD, PhDb,
Margaret D. Allen, MDe*
a Department of Surgery, University of Washington, Seattle, Washington, USA
b Department of Pathology, University of Washington, Seattle, Washington, USA
c Ligocyte Pharmaceuticals, Inc, Bozeman, Montana, USA
d Veterinary Molecular Biology Laboratory, Montana State University, Bozeman, Montana, USA
e Hope Heart Institute, Seattle, Washington, USA
Accepted for publication July 15, 2002.
* Address reprint requests to Dr Allen, The Hope Heart Institute, 1710 E. Jefferson St, Seattle WA 98122, USA.
e-mail: mallen{at}hopeheart.org
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Abstract
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BACKGROUND: This study examined the effect of local intracoronary delivery of a unique monoclonal antibody (mAb) to both E- and L-selectin (EL-246) on neutrophil infiltration after global ischemia during cardiac transplantation.
METHODS: In 12 ovine heart transplants, allograft coronary arteries were locally perfused with EL-246 (n = 6), or isotype-matched control antibodies (n = 2) or saline (n = 4). At 24 hours posttransplant, myocardium was analyzed for neutrophil infiltration and myocardial water content.
RESULTS: The mean number of intramyocardial neutrophils per area (PMN/hpf) was greatly reduced in the allografts perfused with EL-246 (3.45 ± 0.4 PMN/hpf), compared with an average 6.5 ± 0.97 PMN/hpf in control hearts (p = 0.004). Peripheral leukocyte counts were unaffected; myocardial water content was not significantly reduced.
CONCLUSIONS: Local perfusion of cardiac allografts with blocking antibody EL-246 before reperfusion significantly reduced the neutrophilic infiltration that occurs early after transplantation. Prohibiting neutrophil–endothelial adhesion and transmigration may be useful in decreasing neutrophil-dependent post-reperfusion injury in transplantation and routine cardiac surgery.
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Introduction
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| Drs Bargatze and Jutila disclose that they have a financial relationship with Ligocyte Pharmaceuticals.
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Reperfusion injury following myocardial ischemia has been proposed to cause significant operative morbidity and mortality after both cardiac transplantation and routine cardiac surgery. The neutrophil may be the principal leukocytic mediator for the reperfusion injury that occurs in previously ischemic tissues [1]. Neutrophils are recruited to the microvasculature in areas of injury, and migrate across the endothelium in response to released cytokines and chemokine gradients. Margination and localization of neutrophils to areas of inflammation requires the selectin family of adhesion molecules, which mediate the initial slow rolling of neutrophils on the endothelial surface [2]. P-selectin, stored in the Weibel-Palade bodies of endothelial cells, is expressed rapidly in response to oxidative stress, whereas endothelial E-selectin expression requires de novo synthesis, mediated by both NF
B and AF-2 transcription factors [3]. L-selectin, constitutively expressed on neutrophils, is necessary for neutrophil–endothelial interaction in the postcapillary venule. Previous work has demonstrated that antiselectin therapies can limit myocardial injury in the first few hours of reperfusion [4–9]. However, P-selectin is downregulated soon after bypass [4] and may play a limited role afterward, whereas E-selectin expression in humans is rapidly induced during reperfusion and by cardiopulmonary bypass [10, 11] and may persist for 24 hours. Here, we examined the effect of EL-246, a unique antibody that blocks both E- and L-selectin [12–14], on myocardial neutrophil infiltration at 24 hours, using transplantation as a model of global ischemia and reperfusion.
The EL-246 antibody has been used to reduce neutrophil infiltration in vivo in several models of lung injury [15–18] and, in plastic surgery, in skeletal muscle transfer flaps after ischemia-reperfusion [19], but has not been studied in the heart. Given the pivotal role that selectins may play in cardiac ischemia-reperfusion injury, it seemed plausible to use similar antibody blockade strategies to alter the neutrophilic infiltration that follows hypothermic, cardioplegic arrest in heart transplantation. In this pilot study, we used local intracoronary delivery of EL-246 antibody as might be applied clinically in transplantation or routine cardiac surgery.
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Material and methods
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This experimental protocol was approved by the Animal Care Committee at the University of Washington. Animals were cared for in compliance with "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health publication 85-23, revised 1996).
Heterotopic ovine cardiac transplant model
Twenty-four farm-raised sheep (25 to 40 kg) were used for 12 transplants examined at 24 hours postoperatively. An additional 12 sheep were used for six further pilot transplants in which the donor hearts were followed out to end-stage rejection. Animals were sedated with an intravenous injection of sodium thiopental (12.5 mg/kg) and underwent endotracheal intubation. Anesthesia was maintained with inhaled halothane (1.5% to 2%) in 100% oxygen.
Cardiac organ procurement
Through a median sternotomy, the donor heart great vessels were dissected out and, after systemic heparinization (300 U/kg intravenously), the aorta, innominate, and pulmonary arteries were cross-clamped. The heart was arrested with cold (4°C) Stanford cardioplegia through cannulation of the innominate artery, then excised and stored in saline slush. After pulmonary vein ligation, the heart was prepared for transplantation by closing the aortic stump and left atrium with running 5-0 Prolene (Ethicon, Somerville, NJ).
Recipient cardiac transplantation
Under sterile conditions, the left external jugular vein and carotid artery were exposed through a cervical incision. After systemic anticoagulation (heparin 150 U/kg intravenously), the donor innominate and pulmonary arteries were anastomosed to the recipient carotid artery and external jugular vein, respectively, with 7-0 Prolene. In the donor heart before reperfusion, the coronary ostia were blindly cannulated through the aortic root and each ostium perfused with 5 mL of blocking antibody to E- and L-selectin (EL-246, 0.2 mg/kg) or 5 mL of an isotype-matched negative control antibody (EL-81, 0.2 mg/kg), each diluted in normal saline, or 5 mL of the diluent (saline) alone. After flushing the coronary arteries with blood to remove air, vascular clamps were removed for reperfusion. After restoration of normal sinus rhythm, the allografts were placed in a previously constructed cervical subcutaneous pocket. In the group studied at 24 hours, the treatment group consisted of 6 donor hearts that received the treatment antibody, EL-246, and two control groups consisted of 4 donor hearts that received saline and 2 that received the isotype-matched control antibody. Of the 6 donor hearts followed to end-stage rejection, 2 were treated with a single intracoronary dose of the EL-246 antibody at the time of transplantation and 4 control donor hearts were treated with saline alone.
In the 24-hour study, a peripheral blood sample was obtained 24 hours after surgery for total white blood cell and neutrophil counts (Phoenix Central Laboratory, Everett, WA). After euthanasia (0.2 mL/kg intravenous Pentosol; Med-Pharmex, Pomona, CA) in all recipient animals, the transplanted hearts were excised for histochemical analysis, reserving the apices for assessment of myocardial water content (MWC).
Monoclonal antibodies
EL-246 is a mouse IgG1 mAb that binds to both E- and L-selectin, recognizing a conformational epitope that includes short consensus repeat domains held in common between the two selectins [12–14]. EL-81 is a nonblocking mouse anti-human E-selectin Ab that does not inhibit neutrophil binding, used here as an isotype-matched negative control antibody. Both mAbs were gifts from Dr Robert Bargatze (Ligocyte Pharmaceuticals, Inc., Bozeman, MT).
Myocardial water content
Apical sections from both donor and native hearts were weighed immediately after harvest (wet weight) at 24 hours posttransplant and again after 48 hours of desiccation at 85°C (dry weight). Myocardial water content was then calculated as:
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Immunohistochemistry
Donor hearts were sectioned transversely for immunocytochemistry. Myocardial specimens representative of both ventricles and the interventricular septum were taken from five standardized sites in each donor heart (Fig 1)
and fixed in formalin. Tissue sections were embedded in paraffin, and 4-µm thick sections were cut and transferred to Vectabond-coated slides (Vector Laboratories, Inc, Burlingame, CA). Neutrophils were identified on immunocytochemistry with a rabbit antibody to human myeloperoxidase (MPO) (DAKO Corp, Carpinteria, CA), using the avidin-biotin immunoperoxidase technique. Briefly, slides were deparaffinized in xylene for 10 minutes, followed by tissue dehydration in 100% ethanol for 10 minutes. Slides were immersed in 30% hydrogen peroxide diluted in 100% methanol for 30 minutes. Nonspecific binding was blocked with 1.5% normal goat serum (Vector Laboratories) in 0.5 M Tris-HCl (pH 7.4) for 30 minutes at room temperature. Tissue sections were incubated in a 1:500 dilution of the MPO mAb in the same buffer for 1 hour at 37°C, followed by sequential incubations with biotinylated anti-rabbit IgG and avidin-biotin-peroxidase complex (Vector Laboratories) for 1 hour and 30 minutes, respectively. The reaction product was visualized with 3,3'-diaminobenzidine for 2 minutes, and the slides counterstained with methyl green. Positive staining was defined as a cell displaying the brown immunoperoxidase reaction product.
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Fig 1. Locations of myocardial specimen sampling. (A) Five wedge sections were taken from each transverse section. (B) Each wedge section was divided into two epicardial and two endocardial fields. Twenty fields were examined per heart.
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Morphometric analysis with a computerized image analysis program (Optimus, Bothell, WA) was used to determine the absolute neutrophil counts on four randomly chosen high-powered microscopic fields (hpf) at 200x magnification, examining two epicardial and two endocardial fields from each tissue section (Fig 1). Epicardial and endocardial fields were evaluated separately because of the potential that surgical manipulation could affect the neutrophilic infiltration in the epicardial specimens. Five sections, consisting of 20 high power fields, from each heart were analyzed. Neutrophils located within the vascular spaces were not included in the analyses. The quantified neutrophilic infiltrate determined by this method is expressed as the mean number of neutrophils per high power field (PMN/hpf).
Statistical analysis
All data are expressed as means ± the standard error of the mean. Means were compared by unpaired two-tailed Student’s t tests.
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Results
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Data from control hearts treated with saline and the isotype-matched control antibody, EL-81, were compared and found to be similar (p = 0.35 to 0.57 for all comparisons), so the two control groups were combined for the following analyses.
Ischemic time
Ischemic time was defined as the time from aortic cross-clamp application in the donor animal to the removal of the vascular clamps in the recipient. No significant difference was noted between the hearts in the treatment (87.8 ± 3.9 minutes) and control (combined saline and EL-81-treated) groups (82.2 ± 1.8 minutes, p = 0.19) in the 24-hour study. Ischemic times in the long-term follow-up group were similar, but the sample was too small for statistical comparison.
Myocardial neutrophil infiltration
Histologic sections of myocardium confirmed that, in this model, this period of ischemia did produce neutrophilic infiltration in the donor myocardium at the 24-hour time point after transplantation (Fig 2).
Neutrophilic infiltration for all sites was 3.45 ± 0.40 PMN/hpf for the EL-246-treated group, almost a two-fold decrease from the control allografts, which averaged 6.5 ± 0.97 PMN/hpf (Fig 3,
p = 0.004). Presuming the epicardial infiltrate could be due to surgical manipulation alone, pooled endocardial and pooled epicardial halves of the myocardial specimens were also evaluated separately. The neutrophilic infiltration was less in both the endocardial and epicardial specimens treated with the EL-246 antibody (Fig 4),
although the difference was greater in the endocardial than in the epicardial specimens (p values of 0.02 and 0.11, respectively). The extent of neutrophil sequestration in the epicardium and endocardium was also evaluated separately for right and left ventricular specimens and for specimens taken from the interventricular septum (Fig 5).
Regardless of location, the neutrophilic infiltrate was attenuated in the EL-246-treated allograft, but, because of sample size, this difference did not reach statistical significance.
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Fig 2. Neutrophil infiltration demonstrated on histology. Histologic sections from (A) saline-treated control allograft and (B) an allograft treated with the antiselectin antibody EL-246. Immunocytochemistry with rabbit antihuman myeloperoxidase stain. Neutrophils are denoted by positive (dark brown) staining (arrows). (The sections are magnified x200 with the high-magnification inserts at x400, before 30% reduction.)
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Fig 3. Mean number of neutrophils per high-powered field (PMNs/hpf) from all myocardial specimens in transplanted hearts at 24 hours posttransplant. Error bars represent the standard error of the mean. The EL-246 treatment group contains six hearts. The control group includes those hearts perfused with saline (n = 4) and the isotype-matched negative control antibody (n = 2). *p< 0.005.
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Fig 4. Neutrophils in epicardial and endocardial specimens from the control (black squares) and EL-246 (open squares) groups. Values are mean neutrophils per high-powered field (PMNs/hpf) ± the standard error. Neutrophil infiltration was less severe in EL-246-treated myocardium compared with control allografts in endocardial specimens (*p= 0.02). A similar effect was also seen in the epicardium but this trend was not statistically significant at this sample size (p= 0.11).
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Fig 5. Mean number of neutrophils per high-powered field (PMNs/hpf) for each of the five sampled regions of the heart at 24 hours posttransplant. Neutrophil infiltration was reduced in the antibody group at all myocardial sites, although the effect was more evident in specimens from the right ventricular endocardium and septum. • = group treated with the EL-246 antibody blocking E- and L-selectin;  = controls. (IVS= interventricular septum,LV= left ventricle,RV= right ventricle.)
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A similar picture was seen in the donor hearts followed to end-stage rejection. Although the mean time to terminal rejection did not differ between 4 saline-treated control donor hearts (7.25 ± 1.5 days) and 2 EL-246-treated donor hearts (8.5 ± 0.5 days, p = 0.39), reduced numbers of neutrophils were again seen in the antibody-treated hearts in all locations (Fig 6).
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Fig 6. Mean number of neutrophils per high-powered field (PMNs/hpf) for each of the five sampled regions in specimens from EL-246-treated (•) and control () donor hearts followed out to end-stage rejection (cardiac asystole). Although the sample size is small, the trends seen at 24 hours are still evident. (IVS= interventricular septum;LV= left ventricle;RV= right ventricle.)
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Myocardial water content
At 24 hours after transplantation, MWC was lower in the treated donor hearts (77% ± 1.1%) than in the control hearts (82% ± 4.6%), but this difference did not reach statistical significance at this sample size (p = 0.35). The MWC in control donors hearts was higher than in the pooled native hearts (78% ± 2.3%, p = 0.39) whereas, in the EL-246-treated donor hearts, MWC was essentially the same as in the native hearts (p = 0.91); again, these differences were not significant. As expected, at end-stage rejection, the MWC was not different between control and treatment groups (Fisher exact p = 0.75).
White blood cell count
At 24 hours, the peripheral white blood cell counts were similar for the control (7.1 ± 0.9 x 103/µL) and EL-246-treated animals (6.6 ± 0.4 x 103/µL; p = 0.59). The peripheral neutrophil counts were also indistinguishable between the two animal groups (4059 ± 977 PMN/µL in controls versus 4193 ± 358 PMN/µL in EL-246 treated; p = 0.90).
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Comment
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A pivotal role for neutrophils in the development of ischemia-reperfusion injury has been demonstrated in multiple animal models and in different organs [1]. Neutrophils have been cited to alter endothelial permeability and vasomotor function, to account for microvasular "no-reflow" phenomena in prolonged ischemia, and, with degranulation, to contribute to the generation of reactive oxygen intermediates that may jeopardize cell viability. The release of proteolytic enzymes and oxygen-free radicals is ascribed, almost exclusively, to adherent neutrophils. Thus, blocking neutrophil adherence to cells or extracellular matrix proteins within an organ might not only inhibit the transendothelial migration of neutrophils but also potentially reduce local neutrophil toxicity.
Therapies directed at neutrophils—including organ reperfusion with leukocyte-depleted blood [20], and antibodies against the integrin adhesion molecules [21, 22]—have all been shown to attenuate reperfusion injury and improve myocardial function after ischemic injury. Because neutrophil influx, trapping, and egression occur during the first 4 hours of reperfusion [1], antineutrophil therapies must be delivered at the earliest stages of reperfusion. Selectins and integrins are known to function sequentially. In these experiments, we targeted the selectin adhesion molecules, because they mediate what is presumed to be the initial step in neutrophil binding, pre-dating integrin engagement [2]. In animal models of cardiac ischemia-reperfusion, E-selectin expression can be upregulated within the first 30 minutes of reperfusion and remain elevated for 24 hours [9]. EL-246 is an attractive choice as an immunomodulating agent for ischemia-reperfusion injury, because it has the potential to block both E- and L-selectin [12–14], and to "home" to sites of E-selectin upregulation [14].
In human hearts during cardiac surgery, E-selectin mRNA rises 17-fold during the first 40 minutes of reperfusion [10]. During cold ischemia, E-selectin mRNA transcription occurs unabated [23], and translation may occur during prolonged ischemia [19] or be delayed until rewarming [23], suggesting that an upsurge of E-selectin expression would be expected upon reperfusion. In the clinical setting in both organ donors with histories of prior injury or shock, and in patients undergoing nontransplant cardiac surgery with preceding unstable angina or heart failure, E-selectin may also already be upregulated at the onset of surgery [10]. Thus, this antibody treatment could potentially be even more effective in clinical than in experimental surgery. This intracoronary delivery approach may allow first-pass binding to already upregulated E-selectin ligands, but administered antibody will also bind to L-selectin on neutrophils. Importantly for cardiac surgery, EL-246 binds L-selectin in cold temperatures and so could be effective even under hypothermic conditions.
Recently, El-246-coated leukocytes have been shown to transfer antibody unidirectionally from neutrophil binding sites to E-selectin ligands, for which the antibody has a higher binding affinity [14]. Thus, besides directly targeting E-selectin ligands through intragraft delivery, excess circulating antibody that binds to neutrophils should "home" back to the graft as a site of selective E-selectin upregulation. This may explain how small amounts of antibody might have an effect even at less than saturating levels [14]. Also, because this antibody does not attach to shed L-selectin, unlike other L-selectin-directed antibodies, inhibition of neutrophil rolling has been observed with this antibody at lower doses than with comparable L-selectin- (or E-selectin)-binding antibodies [14].
In sheep, saturating levels of EL-246 antibody have been sustained out to at least 6 hours after a 1 mg/kg dose, given systemically [15]. Here, we saw an organ-specific effect using local delivery of a lower dose. In plastic surgery, local delivery of a similarly low dose of EL-246 antibody has also been shown to be effective in inhibiting neutrophil infiltration in latissimus dorsi flaps subjected to ischemia-reperfusion injury [19]. Future studies might focus on determining the smallest effective dose that would achieve a response and whether local delivery provides an important advantage.
These experiments demonstrate that local intracoronary administration of antibody to E- and L-selectin immediately before reperfusion can reduce migration of neutrophils into cardiac allografts in a large animal model. The effectiveness of antibody treatment in the endocardium and the interventricular septum was not unexpected, as these areas are most susceptible to ischemic injury. Although epicardial neutrophils were similarly reduced in the EL-246-treated hearts, this difference did not reach significance at this sample size. We presume that some epicardial neutrophilic infiltration was a response to surgical manipulation and to local perigraft inflammatory responses that would occur independently of the intravascular extravasation of neutrophils that we expected to inhibit with intravascular antibody administration. Dreyer and coworkers [24] found that subendocardial, but not epicardial, neutrophil localization was the hallmark of ischemic injury in a large animal model, and that the subendocardium was similarly the site most improved by antibody therapy directed against integrin adhesion molecules. Additionally, other studies have found relative differences between right and left ventricular tissue oxidative stress after ischemia-reperfusion that are similar to the trends we saw in these experiments [25]. The apparent effectiveness of EL-246 treatment in the right ventricle at 24 hours may be of importance in cardiac transplantation, because early right ventricular dysfunction is a particular problem in the immediate posttransplant period.
Although MWC tended to be somewhat lower in the EL-246-treated hearts, this difference did not reach statistical significance. Potentially, the lack of significance is related to the sample size, the relatively short ischemic times overall, and the 24-hour assessment time point. The mannitol in the Stanford cardioplegia that we used to simulate clinical practice may have had a protective effect that mitigated tissue edema at the 24-hour end point. This might explain why the MWC of the control hearts was also not significantly different from the native hearts at 24 hours.
An alternative and also plausible explanation is that E-selectin blockade may be insufficient in itself to alter vascular leak phenomena. Certainly, the relative roles that oxidant-induced injury alone [26], neutrophils [27], elaborated cytokines and extracellular matrix, and, most recently, apoptosis [28] play as the final effectors of ischemia-reperfusion injury are continually being reevaluated. Paralleling our findings, in one lung ischemia-reperfusion study in sheep using this same antibody [15], neutrophil infiltration was significantly reduced and survival rates increased three-fold in EL-246-treated animals, even though lung water content was unaffected. Similarly, Miura and colleagues [6] found improvement in left ventricular dynamics, but no change in MWC when an oligosaccharide was used to block E-selectin in an isolated working heart model of myocardial infarction, also in sheep. Certainly, one limitation of the heterotopic heart transplant model used in this transplant study is that myocardial hemodynamics cannot be accurately assessed because the workload on the left ventricle is subnormal.
In summary, this pilot study demonstrated that a single dose of antibody to E- and L-selectin can limit the transendothelial migration of neutrophils into myocardium after transplant ischemia-reperfusion injury. Additionally, we have shown that intraoperative local delivery of this antibody into the heart is an effective mode of therapy. In transplantation, local modulation of selectin-based adhesion in the donor heart by antibodies or other means may be an important adjunct to current cardioprotective strategies. Equally important is the fact that the aortic cross-clamp times and selective cardiac perfusion techniques in routine cardiac surgery and interventional cardiology procedures could serve as standard routes for such local intracoronary antibody or small molecule delivery. In both human organ transplantation, in which donor injury has preceded organ procurement, and in routine heart surgery for patients with unstable angina or heart failure, E-selectin may be upregulated in the heart at the time of surgery, thus further enhancing the effectiveness of a local delivery approach.
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Acknowledgments
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We thank Dr Akiko Iwata for producing the photographic images, Robert Bergelin for the statistical review, and Dr Bargatze of Ligocyte Pharmaceuticals for supplying the antibodies for these experiments.
This work was supported by a Roche Surgical Scientist Award from the American Society of Transplant Surgeons and a National Institutes of Health National Research Scientist Award (F32 HL10151-02).
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Abstract
Introduction
