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(Circulation. 1995;92:2276-2283.)
© 1995 American Heart Association, Inc.

Articles

Neutrophil Sequestration and Pulmonary Dysfunction in a Canine Model of Open Heart Surgery With Cardiopulmonary Bypass

Evidence for a CD18-Dependent Mechanism

William J. Dreyer, MD; Lloyd H. Michael, PhD; Ellen E. Millman, MS; Kurt L. Berens, BA; Robert S. Geske, BA

From the Lillie Frank Abercrombie Section of Cardiology (W.J.D.), the Speros P. Martel Section of Leukocyte Biology and Inflammation Research (W.J.D., E.E.M., K.L.B.), the Department of Pediatrics, Section of Cardiovascular Sciences (W.J.D., L.H.M.), the Department of Medicine and the Center for Comparative Medicine (R.S.G.), Baylor College of Medicine, Houston, Tex.

Correspondence to William J. Dreyer, MD, Pediatric Cardiology, Texas Children's Hospital, 6621 Fannin, Houston, TX 77030.

	Abstract

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Background Previous studies documented an inflammatory reaction to cardiopulmonary bypass with neutrophil (PMN) sequestration in the lungs, contributing to microvascular injury and postoperative pulmonary dysfunction. This study explored the hypothesis that the ß₂ integrin CD18, a leukocyte adhesion molecule, mediates this response.

Methods and Results Fifteen adult, mixed-breed dogs underwent 90 minutes of cardiopulmonary bypass with 3 hours of subsequent recovery. Seven additional dogs were treated before cardiopulmonary bypass with a 1-mg/kg IV bolus of R15.7 IgG, a monoclonal antibody to CD18. Both groups were compared with 5 sham bypass control dogs. Bypassed dogs demonstrated an increased number of PMNs sequestered in the lungs 3 hours after bypass compared with sham bypass control dogs (1466±75 versus 516±43 PMN/mm² alveolar surface area, mean±SEM, P<.001). Also, when PMNs from bypass dogs were compared with those from sham dogs, they produced more H₂O₂ (305±45 versus 144±48 amol H₂O₂ per phagocyte per 20 minutes, P<.05). Bypass dogs had significantly decreased arterial oxygenation 3 hours after the procedure compared with shams (457±20 versus 246±49 mm Hg, P<.05), and they had a significantly increased lung wet-to-dry weight ratio (5.38±0.14 versus 4.54±0.15, P=.003), demonstrating a significant increase in lung water. R15.7 markedly attenuated pulmonary PMN accumulation in bypass dogs (412±73 PMN/mm², P<.001) and significantly inhibited PMN production of H₂O₂ (146±18 amol H₂O₂ per phagocyte per 20 minutes, P<.05) Bypass dogs pretreated with R15.7 also had improved oxygenation (445±28 mm Hg, P<.05) and tended to have less lung water accumulation after bypass (4.99±0.20).

Conclusions Pulmonary dysfunction after cardiopulmonary bypass is caused, at least in part, by a neutrophil-mediated, CD18-dependent mechanism.

Key Words: leukocytes • lung • cardiopulmonary bypass • antibodies

	Introduction

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A growing body of literature documents an inflammatory response to cardiopulmonary bypass (CPB). Chenoweth and associates¹ first demonstrated complement activation in patients undergoing CPB in 1981. Subsequently, other studies documented neutrophil sequestration in the lungs and suggested that this localization of neutrophils in the pulmonary microvasculature may contribute to postoperative pulmonary dysfunction.^{2 3 4} Recent investigations into the molecular determinants of cellular adhesion revealed at least two classes of adhesion molecules present on the neutrophil surface that participate in the margination of neutrophils at inflammatory sites. L-Selectin, present on inactivated cells, participates in the initial rolling process of marginating neutrophils but is rapidly shed from the cell surface with chemotactic stimulation.⁵ The ß₂ integrins CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) allow firm adhesion of neutrophils and transendothelial migration.⁶ One of these molecules, CD11b/CD18, increases on the neutrophil surface in response to chemotactic activation.⁷ Thus, changes in neutrophil surface expression of L-selectin and CD11b/CD18 indicate cellular activation. Recently, neutrophil activation during CPB was suggested by demonstration of an increase in CD11b/CD18 and a decrease in L-selectin on cells circulated in isolated CPB circuits.^{8 9} Studies in animal models by our group and others documented similar changes on peripheral blood neutrophils.^{10 11} Increases in neutrophil and monocyte CD18 also were documented in adult patients after CPB.^{12 13 14}

Activated cells in the peripheral blood may indicate an ongoing inflammatory response within the microvasculature of the lungs and other organs. In a recent study, we documented a significant correlation between the percentage of activated peripheral blood neutrophils (as documented by CD18 expression) and postoperative arterial oxygenation.¹⁰ From this work, it follows that neutrophil sequestration within the lungs after CPB may be CD18-dependent, and a strategy to block neutrophil adhesion in the perioperative period may help to alleviate postoperative pulmonary dysfunction. This study was performed in a canine model to test this hypothesis.

	Methods

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Animal Preparation
Healthy adult mixed-breed dogs (16 to 25 kg) were anesthetized with sodium pentobarbital (30 mg/kg), intubated, and mechanically ventilated with 100% oxygen. After femoral arterial cannulation, ventilatory rate and tidal volume were adjusted to establish a normal pH and PCO₂ as determined by arterial blood gas. After a midline thoracotomy, CPB was achieved by selective venous cannulation of the inferior and superior venae cavae with arterial return directed into the distal aortic arch. The bypass circuit consisted of a cardiotomy reservoir and a variable prime membrane oxygenator (Cobe Cardiovascular, Inc), a heat exchanger, and a roller pump (Sarns/3M Healthcare). In our circuitry, an in-line arterial filter was not used. The circuit was primed with 40 to 50 mL per 1 kg body weight of lactated Ringer's solution maintained at room temperature until the CPB was begun. Before CPB, the prime solution was circulated through a Pall 0.2-µm filter for 15 to 20 minutes; subsequently, prime from the circuit was demonstrated to be free of endotoxin by limulus amoebocyte lysate assay.

Three groups of dogs were included in this study. The first group consisted of 15 dogs that underwent CPB with an aortic cross-clamp time of 60 minutes and total bypass time of 90 minutes. Before cannulation, dogs were anticoagulated with 100 U/kg porcine heparin sodium. After the aorta was cross clamped, the heart was arrested with prograde administration of cold potassium cardioplegia, given to effect (Plegisol, Abbott Laboratories), and the dogs were cooled to 24°C to 28°C. During CPB, hematocrit was maintained in the range of 23% to 25%, and flow was maintained at 60 to 70 mL · kg^-1 · min^-1 at a pressure of 40 to 60 mm Hg. Alpha-stat pH management was observed during CPB. After rewarming and weaning from CPB, dogs received protamine sulfate 1 mg/100 U heparin delivered for reversal of anticoagulation. Dogs were once again ventilated with 100% oxygen. Tidal volume and ventilator rate were returned to their original settings. Dogs were then maintained with open chests for 3 hours while blood samples were obtained. At no time in the recovery phase did dogs receive inotropic support or vasodilators. In selected dogs, a small biopsy specimen was obtained from the right upper lobe of the lung before cannulation for CPB. In all dogs, pulmonary tissue samples were obtained at the end of the experiment.

The second group consisted of 7 dogs that were treated in a similar fashion to that described, except just before CPB each dog received a single 1-mg/kg bolus injection of the anti-CD18 monoclonal antibody R15.7 (described below).

The third group consisted of 5 dogs designated sham bypass controls. These dogs served as controls in that they received general anesthesia and mechanical ventilation similar to the dogs in group 1. In addition, they underwent midline thoracotomies and cannula placement but were not placed on CPB. Also, these dogs were not subjected to cooling and rewarming, anticoagulation, or hemodilution, and there was no reduction of pulmonary blood flow at any time during the procedure. These dogs, however, were maintained with open chests for a duration similar to that for the dogs in group 1.

Monoclonal Antibody R15.7
R15.7 is an IgG1 murine monoclonal antibody that recognizes a functional epitope of the CD18 adherence glycoprotein on both human and canine cells as previously described.¹⁵ Pharmaceutical-grade R15.7 monoclonal antibody was provided by Boehringer-Ingelheim Pharmaceuticals, Inc. Endotoxin was removed by passage over a polymyxin B column, and the antibody was sterile-filtered and stored at -80°C until use.

Flow Cytometry
In dogs from all three groups, CD18 expression was determined on peripheral blood neutrophils by indirect immunofluorescence with whole-blood samples from the femoral artery. Blood samples were collected in citrate-phosphate-dextrose buffer (1.4 mL/10 mL blood) and were immediately fixed in 0.25% paraformaldehyde until all samples were collected. Duplicate 100-µL aliquots were then incubated for 15 minutes at room temperature with 10 µg/mL R15.7. The samples were washed twice in 1 mL Dulbecco's PBS and were then incubated with a 1:30 dilution of fluorescein-conjugated goat anti-mouse F(ab')₂ (Zymed Laboratories) for 15 minutes at room temperature and washed once in 1 mL Dulbecco's PBS. Red blood cells were lysed in 1.5 mL FACS lysing solution (Becton Dickinson) for 10 minutes at room temperature. The samples were then washed once in Dulbecco's PBS, resuspended in 1% paraformaldehyde, and stored overnight at 4°C. Samples were subsequently analyzed in duplicate on a Becton Dickinson FACScan. Neutrophils in suspension were gated on from light-scatter characteristics, and fluorescence intensity was measured and interpreted as the surface expression of the CD18 molecule.

In selected dogs from the group systemically treated with R15.7, we wished to determine the percent saturation of binding sites by the systemically administered R15.7 antibody. For this purpose, the protocol was altered as previously described.¹⁶ In this case, a duplicate set of blood samples was obtained at the indicated time points and processed immediately with no paraformaldehyde fixation. Blood samples received no primary antibody but were initially washed twice in 1 mL Dulbecco's PBS, resuspended, and then incubated with fluorescein-conjugated goat anti-mouse F(ab')₂ for 15 minutes at room temperature. Samples were then handled as described above. By comparing the mean fluorescence values from the initial set of blood samples that received exogenous R15.7 with the second set of samples described above, we could determine the total number of R15.7 binding sites present on the neutrophils, the number of binding sites occupied by systemically administered R15.7, and the percent saturation of available binding sites by exogenously administered R15.7 throughout the protocol.

Also, to document the presence of R15.7 in plasma in sufficient quantity to produce antibody excess, additional blood samples from the test dogs were centrifuged at 800g for 5 minutes at room temperature, and the plasma was removed. The cell button from 100 µL whole blood drawn from a separate donor dog was suspended and incubated with either a saturating concentration of R15.7 or plasma from the test dog for 15 minutes at room temperature. The cells were washed, incubated with fluorescein-conjugated goat anti-mouse F(ab')₂, and processed for flow cytometry as described above. Comparison between the samples allowed us to determine whether a saturating concentration of R15.7 remained in the plasma throughout the protocol.

White Blood Cell Counts
White blood cell counts were determined from femoral arterial whole-blood samples by use of an automated counter (Coulter Electronics, Ltd). Counts obtained during and after CPB were corrected for hemodilution with the formula WBC_corr=WBC_obsxHct_bl/Hct_obs, where WBC_corr is the corrected white blood cell count, WBC_obs is the observed white blood cell count, Hct_bl is the beginning or "baseline" hematocrit, and Hct_obs is the observed hematocrit at the time of the white blood cell count determination.

Morphometric Analysis
Canine lung tissue was immersion-fixed in 10% buffered zinc-formalin and embedded in paraffin, and 4-µm sections were cut with a Leica microtome. Tissue sections were incubated with SG8H6, an IgG1 murine monoclonal antibody to a canine neutrophil–specific antigen,¹⁷ with standard immunohistochemical techniques applicable to an alkaline phosphatase reporter system. The chromagen used was nitroblue tetrazolium, and the sections were counterstained with eosin.

Stained tissue sections were analyzed with an image analysis software program (OPTIMAS, Bioscan). Images of lung tissue were captured with a black and white camera (CCD72, Dage MTI) connected to a Leitz Diaplan microscope with frame grabber support through an ALR 486/66-MHz personal computer. The number of neutrophils in a given x40 field were distinguished by their differential gray-scale intensity compared with the eosin-counterstained lung tissue. Neutrophils were counted and expressed per cross-sectional area of alveolar air space.

Luminometry
A single photon-counting luminometer (Autolumat LB953, Berthold) was used to measure phagocytic hydrogen peroxide production. Blood samples were collected in citrate-phosphate-dextrose buffer and run within 4 hours of collection. A 100-µL aliquot of blood was mixed with 9.9 mL blood-diluting medium (ExOxEmis), a buffered balanced salt solution containing 5.0 mmol 2-[N-morpholino]ethane sulfonate, porcine gelatin (0.05% wt/vol), and the following electrolyte composition: Na⁺ 142 mmol/L, K⁺ 5 mmol/L, Cl^- 147 mmol/L, HnPO42^- 0.8 mmol/L, and D-glucose 5.5 mmol/L, with pH 7.30 and 295-mosm/kg osmolality. The chemilumingenic medium was a similar buffered balanced salt solution with added calcium (1.3 mmol/L Ca²⁺), magnesium (0.9 mmol/L Mg²⁺), and 0.15 mmol/L luminol (5-amino-2,3,-dihydro-1,4-phtalazinedione) as the chemiluminigenic substrate.

Human complement opsonized zymosan (100 µL) was added to optical-quality polystyrene tubes (ExOxEmis) that had been coated with platelet-activating factor (10^-9 mol/L). Platelet-activating factor was used as a priming stimulus and zymosan as a phagocytic substrate to stimulate the respiratory burst activity of neutrophils. Automatic injectors dispensed first 100 µL of blood-diluting medium containing test blood and then 600 µL chemiluminigenic substrate into these tubes. Photon counts were measured for 30 minutes and were plotted against time. The integral from 0 to 20 minutes was used to calculate the luminescence activity (counts per phagocyte) and hydrogen peroxide production (attomoles H₂O₂ per phagocyte), a method based on the method of Stevens et al.¹⁸

Systemic Oxygenation
The change in arterial oxygen tension over the course of the experiment was measured as a marker of pulmonary dysfunction. Throughout the study, all dogs were ventilated with 100% oxygen. Tidal volume and ventilator rate were set to optimize pH and PCO₂ and were not changed subsequently. When dogs were being weaned from CPB, their lungs were reexpanded and hyperinflated just briefly to reestablish lung volume, but the ventilator was then adjusted to its original settings and not altered. Despite subsequent changes in arterial PO₂, pH and PCO₂ remained relatively constant. Given an FIO₂ of 1.0 and a relatively constant PCO₂, alveolar PO₂ was assumed to be constant, and changes in arterial PO₂ were interpreted to represent changes in intrapulmonary shunt. Femoral arterial blood gases and pH were determined in standard fashion by use of a pH and blood gas analyzer (model 170, Ciba Corning Diagnostics Corp).

Wet-to-Dry Weight Ratios
The wet weight of an excised lobe of the lung was measured immediately after termination of the experiment. The lung tissue was then dried in an Equatherm vacuum oven (Labline Instruments) at 130°C. After 6 hours, the lung tissue was weighed hourly until there was no variation in weight. The wet-to-dry ratio of the lung tissue was then calculated, and this was used as a marker of lung water.

Statistical Analysis
For descriptive analysis, values for individual dogs were pooled within their designated group at the time point at which they were measured and were expressed as mean±SEM. For the data presented in Tables 1 and 2 and Figs 1, 4, and 5, values were measured on a repeated basis over time for each individual dog. Group comparisons were made by use of a two-way ANOVA for repeated measures. When ANOVA indicated a significant group-by-time interaction, specific within-group and between-group comparisons of different time points were made with a multiple comparison procedure (Student-Newman-Keuls). For the data presented in Figs 3 and 6, a simple one-way ANOVA was performed. A value of P.05 was considered significant.

View this table: [in this window] [in a new window]   Table 1. Percent Baseline Expression of Peripheral Blood Neutrophil CD18

View this table: [in this window] [in a new window]   Table 2. Hemodynamic Data

View larger version (16K): [in this window] [in a new window]   Figure 1. Plot showing peripheral white blood cell counts throughout the course of the experiment in sham bypass controls (SHAM), cardiopulmonary bypass dogs (CPB), and CPB dogs treated with R15.7 (CPB+R15.7). Samples were obtained after anesthesia but before thoracotomy (PRE-THORAC); after thoracotomy but before bypass (PRE-CPB); at 20 and 40 minutes of aortic cross clamping (Ao X CLAMP); and at 10 and 20 minutes and 1, 2, and 3 hours after cessation of bypass (POST-CPB). White blood cell counts are expressed as a percentage of the baseline values.

View larger version (19K): [in this window] [in a new window]   Figure 4. Bar graph comparing in vitro neutrophil H2O2 production in sham bypass controls, cardiopulmonary bypass (CPB) dogs, and CPB dogs treated with R15.7. Blood samples were obtained at the time points indicated, and H2O2 production was measured by luminometry as described in the text.

View larger version (17K): [in this window] [in a new window]   Figure 5. Plot showing changes in systemic arterial oxygenation throughout the experiment in untreated cardiopulmonary bypass (CPB) dogs and CPB dogs treated with R15.7. Patterns of arterial oxygenation were similar between groups before and during CPB; after CPB, however, untreated dogs demonstrated a progressive deterioration in oxygenation, whereas dogs treated with R15.7 demonstrated recovery of oxygenation after the first hour after CPB. Legend as described for Fig 1.

View larger version (13K): [in this window] [in a new window]   Figure 3. Bar graph comparing neutrophil sequestration within the lung before cardiopulmonary bypass (CPB) and 3 hours after CPB in the three experimental groups. Neutrophils per millimeter squared of alveolar surface area were determined by computerized image analysis as described in the text.

View larger version (13K): [in this window] [in a new window]   Figure 6. Bar graph depicting lung wet-to-dry weight ratios at 3 hours after cardiopulmonary bypass (CPB) in sham, untreated, and R15.7-treated dogs. Wet-to-dry ratio was used as an index of lung water.

Approval
The animal studies reported here were reviewed and approved by the Baylor College of Medicine Animal Care and Use Committee. These studies conformed with the "Guide for the Care and Use of Laboratory Animals" published by the NIH (NIH publication No. 85-23, revised 1985).

	Results

Top Abstract Introduction Methods Results Discussion References

 
Adhesion Molecule Expression
As we previously demonstrated in this model,¹⁰ compared with baseline prethoracotomy levels, CD18 surface expression on circulating neutrophils increased during CPB and remained elevated until the study ended 3 hours after CPB (see Table 1). In contrast, sham-operated dogs demonstrated no increase in CD18 surface expression. Dogs that received R15.7 before CPB demonstrated an initial rise in CD18 surface expression during CPB but subsequently demonstrated a downregulation of CD18 expression on the neutrophil surface as the study progressed. This phenomenon was documented previously in healthy, awake dogs receiving a comparable dose of R15.7.¹⁶ Also, the systemically administered antibody appeared capable of saturating a very high percentage of available CD18 binding sites throughout the protocol (91±4% at 10 minutes after administration, 86±5% at the end of the experiment), and a saturating concentration of R15.7 antibody remained in the plasma throughout the course of the study interval (data not shown).

Peripheral White Blood Cell Counts
Mean peripheral white blood cell counts were comparable between groups before thoracotomy (6495±1188, 5995±809, and 7941±662 cells/mm³ for sham, CPB, and CPB+R15.7 dogs, respectively; P=NS). After thoracotomy, peripheral white blood cell counts in all groups dropped 25% (Fig 1). In the sham controls, there was a subsequent rise in white blood cell count throughout the duration of the protocol. In dogs undergoing CPB, there was a significant drop in white blood cell count during extracorporeal circulation to 35% of baseline levels. This decrease in white blood cell count during CPB was similar in both untreated and R15.7-treated dogs. In both groups, white blood cell counts began to recover immediately after CPB. In the untreated group, white blood cell count rose more slowly throughout the protocol than in the R15.7-treated group, perhaps indicative of greater leukocyte sequestration in peripheral vascular beds. The difference between untreated and R15.7-treated dogs, however, did not reach statistical significance.

Pulmonary Neutrophil Sequestration
As Fig 2 demonstrates, neutrophils present within alveolar capillaries were readily identified by immunohistochemistry by use of the SG8H6 antibody. Compared with baseline biopsy samples obtained before CPB, sham bypass controls had no significant increase in pulmonary neutrophils at the end of the experiment; in untreated dogs undergoing CPB, however, there was approximately a threefold increase in sequestered neutrophils within the lungs 3 hours after CPB (1466±75 versus 516±43 neutrophils per 1 mm² alveolar surface area, P<.001). In general, very few neutrophils were noted within the alveolar space, although after CPB single neutrophils and neutrophil aggregates (Fig 2B, arrows) were noted within the alveolar capillaries. CPB dogs treated with R15.7 demonstrated a marked reduction in pulmonary neutrophil sequestration 3 hours after CPB compared with the untreated CPB group, and post-CPB samples had virtually the same number of neutrophils as pre-CPB samples (Fig 3).

View larger version (103K): [in this window] [in a new window]   Figure 2. Photomicrographs (x100) of lung biopsy specimens obtained at the time points indicated. Neutrophils were identified by immunohistochemical staining as described in the text. A, Pre–cardiopulmonary bypass (CPB) specimen. Relatively few, isolated single neutrophils (dark-staining cells) were present within alveolar capillaries. B, Dogs 3 hours after CPB. There was a substantial increase in the number of neutrophils present in the alveolar wall. Many neutrophil aggregates were present (arrows), although the majority of cells appeared to be retained within the capillary lumen. Significant numbers of neutrophils were not present within the alveolar space. C, Sham control time-matched to "3 hours after CPB." Compared with pre-CPB dogs, there was no significant increase in neutrophils retained in the lung. D, Dogs 3 hours after CPB treated with R15.7. Neutrophil sequestration was markedly attenuated. There were no neutrophil aggregates present.

Luminometry
Our luminometry experiments were designed to examine the functional ability of peripheral neutrophils to produce H₂O₂ in response to CPB when presented in vitro with a substrate for phagocytosis (human complement–opsonized zymosan) and presented with a chemotactic stimulus (platelet-activating factor). As Fig 4 shows, neutrophils from all three study groups demonstrated a similar degree of H₂O₂ production before CPB. In the sham group, this H₂O₂ production tended to increase over time, although the increase was not significant. Neutrophils from dogs undergoing CPB, however, showed a marked increase in H₂O₂ production in response to CPB. The presence of R15.7 in the serum of CPB dogs significantly attenuated this response so that, 3 hours after CPB, H₂O₂ production in the R15.7-treated dogs was comparable to that seen in shams.

Arterial Oxygenation
Baseline arterial oxygenation before thoracotomy was similar in sham, CPB, and R15.7-treated dogs (479±25, 546±14, 494±51 mm Hg, respectively). Arterial oxygenation in the sham controls did not change significantly throughout the protocol, and their data are not shown. Similarly, pH, PCO₂, and hematocrit remained comparable between CPB groups during the study and therefore are not reported. Significant differences did occur, however, between the arterial oxygenation patterns of the untreated and R15.7-treated dogs (Fig 5). Oxygenation was similar for both groups before and during CPB, with very efficient oxygenation occurring during the CPB procedure. Also, with the first post-CPB sample, both groups were close to baseline. Within 20 minutes after CPB, however, the groups began to separate. Untreated CPB dogs had a continued deterioration in oxygenation that appeared to plateau between 2 and 3 hours after CPB. R15.7-treated dogs, however, showed a slower rate of decline and a significant recovery of oxygenation after the first post-CPB hour.

Lung Water
Wet-to-dry weight ratios were determined in lung specimens obtained at the end of the experiment as an estimate of lung water. As Fig 6 demonstrates, CPB dogs had a significant increase in lung water compared with shams. This increase appeared to be attenuated in CPB dogs treated with R15.7, but the difference between R15.7-treated and untreated CPB dogs did not reach statistical significance.

Hemodynamics
Finally, hemodynamic parameters, including heart rate, mean systemic arterial pressure, mean pulmonary arterial pressure, and mean left atrial pressure, were measured in each of the study groups (Table 2). Mean left atrial pressure and mean pulmonary artery pressure were low and remained low in all groups throughout the study protocol. After CPB, heart rate and mean systemic arterial pressure were significantly decreased compared with baseline values in both R15.7-treated and untreated dogs. Post-CPB heart rates, however, were not different between the two groups. The R15.7-treated dogs tended to have a higher systemic arterial pressure after CPB than the untreated group, although this difference was not statistically significant. Hemodynamic changes did not correlate with changes in the other parameters noted above.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
As stated earlier, recent studies documented an activation of both humoral and cellular components of the inflammatory system in response to CPB. Sequestration of activated neutrophils in the microcirculation of the lungs and other organs, with release of proteolytic enzymes and reactive oxygen, has been implicated as a pathophysiological mechanism causing end-organ injury and contributing to postoperative morbidity.^{19 20 21 22} Previous studies using neutrophil depletion or nonspecific inhibition of neutrophil function support this concept.^{23 24 25}

The CD11/CD18 complex has been shown to be fundamentally important to the margination and emigration of neutrophils at inflammatory sites and to the adhesion-dependent release of H₂O₂ and elastase as mediators of target-cell injury. In models of sepsis,^{26 27} hemorrhagic shock,^{28 29} and ischemia-reperfusion,³⁰ anti-CD18 therapy has proved to be an effective means of preventing tissue injury. It follows then that neutrophil sequestration in the lungs after CPB and the potential injury caused by endothelial cell disruption and microvascular permeability changes may, in fact, be CD18-dependent. Although increased surface expression of CD18 on circulating neutrophils has been documented in response to CPB, increased surface expression of CD18 on circulating cells alone does not translate to the participation of this molecule in sequestration of neutrophils within the microcirculation.

Verrier and Shen³¹ briefly reported in a limited number of primate experiments that treatment with the anti-CD18 monoclonal antibody 60.3 reduces postoperative weight gain and improves oxygenation after CPB. Gillinov et al³² reported similar results in pigs using a leumedin compound that includes among its anti-neutrophil properties the inhibition of neutrophil adhesion. The study reported here, however, provides the first detailed documentation of the use of an anti-CD18 monoclonal antibody in an animal model of CPB and clearly confirms a role for CD18 in pulmonary neutrophil sequestration after CPB. Treatment with a 1-mg/kg bolus of R15.7 before CPB resulted in a saturating concentration of antibody in the serum throughout the protocol. Antibody treatment virtually eliminated the increase in post-CPB neutrophil sequestration in the lungs and reduced neutrophil numbers back to baseline levels. In addition, anti-CD18 therapy attenuated postoperative pulmonary dysfunction. Both oxygenation and lung water accumulation were improved by treatment. This study also demonstrated that CPB can act as a priming stimulus to potentiate the release of H₂O₂ in circulating neutrophils. This H₂O₂ release also was significantly inhibited by the systemically administered antibody.

R15.7 was chosen for these experiments for two reasons. First, although R15.7 was developed from canine peritoneal macrophages, it cross-reacts with and can block the function of CD18 in a number of species, including cats,³⁰ rabbits,³³ cows,³⁴ nonhuman primates,³⁵ and humans.³⁶ This cross-reactivity suggests that the antibody recognizes a fundamentally important and functional epitope of the molecule that has been well conserved across species. Second, in this study, we wanted to inhibit the broadest range of integrin-mediated functions possible. Because R15.7 recognizes CD18, or the ß subunit common to all leukocyte integrins, it can block the function of both LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18). Both molecules have been demonstrated to participate in firm attachment and transendothelial migration of neutrophils. In addition, the Mac-1 molecule appears to play a fundamental role in adhesion-dependent release of H₂O₂ in chemotactically stimulated neutrophils.³⁶

The applicability of these studies to therapeutic intervention in patients to reduce postoperative pulmonary dysfunction and its associated morbidity remains to be seen. Our study was limited by a short postoperative follow-up interval. Whether changes seen between treated and untreated groups within this interval would be apparent after a longer recovery time is unknown. Also, whether changes in pulmonary dysfunction seen in the model would translate to a reduced time of mechanical ventilation, a reduced recovery room stay, or reduced mortality, particularly in higher-risk patients, remains a matter of conjecture. The potential side effects of treatment also have not been fully evaluated. Nevertheless, this study suggests that anti-adhesion monoclonal antibodies may have a role in treating the postoperative CPB patient and indicates the need for further investigation of a potential new therapy.

	Acknowledgments

 
This work was supported in part by NIH grants HL-47163 and HL-42550. Dr Dreyer's work was done during the tenure of a Clinician-Scientist Award from the American Heart Association and Boehringer Ingelheim Pharmaceuticals, Inc. We gratefully acknowledge the expert technical assistance of Gary Liedtke and Peggy Jackson with the surgical preparation of the dogs used in this study and the assistance of Susan Greenwood with development of the SG8H6 antibody. We would also like to thank C. Wayne Smith, MD, and Mark L. Entman, MD, for their critical review of the manuscript.

	Footnotes

 
Guest Editor Lewis C. Becker, MD, Johns Hopkins Hospital, Baltimore, Md.

Received February 16, 1995; revision received May 1, 1995; accepted May 13, 1995.

	References

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