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Ann Thorac Surg 1995;59:328-335
© 1995 The Society of Thoracic Surgeons
Divisions of Cardiac Surgery, Cardiology, and Pediatric Immunology, The Johns Hopkins Medical Institutions, Baltimore, Maryland
Accepted for publication August 5, 1994.
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Abstract |
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Platelet activating factor (PAF) is a potent mediator of neutrophil integrin (CD11b/CD18) expression [12, 15]. It is a glycerol phospholipid (1-o-alkyl-2-acetyl-sn-glycerol-3-phosphorylcholine) synthesized by many cells, including platelets, granulocytes, and endothelial cells [16]. Circulating PAF is increased by 350% after CPB [17]. The role of PAF in acute inflammatory injury [18] and organ rejection has been well demonstrated. Platelet activating factor antagonists attenuated hyperacute rejection in both presensitized allograft and xenograft rat cardiac transplant models [19]. When used as a preservation adjunct, PAF antagonists also improve pulmonary function after transplantation [20].
SDZ HUL-412 (a quinolinium compound) is an established competitive inhibitor of PAF binding on the neutrophil receptor [21, 22]. We used SDZ HUL-412 in a porcine CPB model to determine the relationship between PAF and neutrophil integrin (CD11b/CD18) expression and to examine whether it protected against neutrophil-mediated lung injury after CPB.
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Material and Methods |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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The CPB circuit was primed with 1,000 mL of lactated Ringer's solution and 25 mEq of NaHCO3. A Bentley bubble oxygenator, a 40-µm millipore arterial filter, and Sarns roller pumps made up the extracorporeal circuitAu: need manufacturers' name, city & state. Cardiopulmonary bypass was initiated and a left ventricular vent was placed through the apex. By surface and core cooling temperature was lowered to 28°C. Lungs were ventilated during CPB with room air. Mean systemic arterial pressure was maintained at 50 to 55 mm Hg with extracorporeal flow rates of 70 to 80 mL/kg when the temperature was more than 32°C and 50 to 60 mL/kg when the temperature fell to less than 32°C. Phenylephrine hydrochloride was given as needed to maintain arterial blood pressure. During the last 30 minutes of CPB, the animals were rewarmed to 37°C. The total CPB time was 2 hours. All pigs were weaned from CPB on isoproterenol infusion (0.1 to 0.2 µg • kg-1 • min-1). The CPB cannulas then were removed, the animals were allowed to stabilize for 15 minutes, and isoproterenol administration was discontinued. Physiologic measurements were recorded before and during CPB and for 2 hours after CPB. The animals remained under general anesthesia and were sacrificed at the close of the experiment. All animal care and operative procedures were in accordance with the Animal Care and Use Committee of The Johns Hopkins Hospital and the ``Guide for the Care and Use of Laboratory Animals'' published by the National Institutes of Health (NIH publication 85-23, revised 1985).
Pharmacologic Intervention
Before sternotomy group I animals (n = 6) received SDZ HUL-412 (1 mg/mL in normal saline solution) intravenously through the femoral vein (3 mg/kg) for 0.5 hours, followed by a continuous intravenous infusion for 4 hours at 2 mg • kg-1 • h-1. Group II animals (n = 7) received similar volumes of normal saline solution. SDZ HUL-412 was provided as a gift from Sandoz Corporation (East Hanover, NJ).
Platelet Aggregometry
Platelet activating factor inhibition was verified by measurement of platelet aggregation ex vivo before and after infusion of SDZ HUL-412. Samples were collected before sternotomy and before CPB. Platelet-rich plasma was obtained by centrifuging whole blood at 2,000 rpm for 4 minutes at room temperature and aspirating the supernatant. The plasma (450 µL) was incubated for 3 minutes at 37°C. Aggregation was induced by the addition of 50 µL of adenosine diphosphate (ADP) (10 and 5 µmol/L concentration) and PAF (50, 5, and 0.05 µmol/L). Inhibition of PAF-induced platelet aggregation was expressed as percent of pretreatment values. ADP-induced platelet aggregation is independent of PAF and therefore served as a posttreatment control for platelet function. Aggregation curves were recorded for 5 minutes on a dual channel aggregometer (Bio/Data Corp, Philadelphia, PA) within 2 hours of blood sample collection.
Physiologic Measurements
Arterial blood gases analyses were performed on a Radiometer ABL-30 analyzer (Radiometer, Copenhagen, Denmark). Blood pressure and dynamic peak airway pressure were recorded on an eight-channel monitoring system (Hewlett-Packard 7758B; Hewlett-Packard, Andover, MA). Cardiac output was determined by thermodilution technique using an Edwards Laboratories 9520 cardiac output computer (Edwards Laboratories, Santa Ana, CA). Tidal volume was measured by microspirometry (Micro Medical Ltd, Rochester, Kent, England). Physiologic measurements were recorded before sternotomy, immediately before cannulation, at 15, 30, 60, 90, and 120 minutes of CPB, and 15, 30, 60, and 120 minutes after CPB. Dynamic lung compliance was calculated as tidal volume (mL) divided by peak airway pressure (mm Hg). Pulmonary vascular resistance (PVR) was determined according to the equation PVR = 79.92 x (Mean pulmonary artery pressure - Mean left atrial pressure)/Cardiac output (L/min). Arterial pressure, pump flow rate, and temperature were monitored continuously throughout the experiment.
CD18 Expression
Heparinized blood samples (100 µL) were placed in polypropylene tubes and washed twice with phosphate-buffered saline without Ca2+ and Mg2+ (Dulbecco-PBS [D-PBS]; 500 µL). Blood was resuspended in PBS (100 µL). The primary antibody was a murine monoclonal immunoglobin G raised from a hybridoma cell line specifically against porcine CD18 (gift from Dr James E. K. Hildreth) [23]. Primary antibody (100 µL) was added and cells were incubated in the dark at room temperature for 15 minutes. The cells were washed twice with D-PBS (500 µL). The secondary antibody was a fluorescein-labeled goat antimouse FC receptor-specific immunoglobulin G (Sigma, St. Louis, MO). A secondary antibody (10 µL) was added and the incubation step repeated. Erythrocytes were lysed using the Coulter Whole Blood Lysing Reagent Kit (Coulter Immunology, Coulter Corp, Hialeah, FL) and vortexed, then incubated at room temperature in the dark for 12 minutes. Cells were centrifuged at 1,800 rpm for 6 minutes. The supernatant was removed and cells washed with D-PBS (500 µL), vortexed, and centrifuged at 1,800 rpm for 6 minutes. The white blood cell pellet was fixed with 0.5% paraformaldehyde (500 µL). Suspended cells were stored at 4°C in the dark. CD18 receptor quantification was determined using an EPICS-Profile II flow cytometer (Coulter Corp, Miami, FL). The increase in receptor expression was determined by comparison of mean channel fluorescence. Results were expressed as percent increase over presternotomy samples, with each animal serving as its control. Ten thousand neutrophils were counted at each time point.
Cell Counts
Neutrophil counts were determined by Coulter Counter, but differentials were performed manually. Absolute granulocyte numbers were adjusted for hemodilution. Blood was collected simultaneously during physiologic measurements. Pulmonary leukosequestration after CPB was determined by analysis of blood samples drawn simultaneously from left and right atria 30, 60, and 120 minutes after CPB.
Tissue Analysis
Biopsy was performed, removing a small portion of the right upper lobe of the lung, before CPB and 2 hours after CPB. Biopsy specimens were flash frozen in liquid nitrogen, and myeloperoxidase assay was performed later in the following manner. Tissue was disrupted by homogenization at 4°C and placed into 0.5% hexyldecyltrimethylammonium bromide in 50 mmol/L of potassium phosphate solution, pH 6.0 (1 mL/100 mg lung tissue). Tissue was disrupted further by sonication and then underwent three freeze (liquid nitrogen bath)–thaw (37°C water bath) cycles. The solution was centrifuged at 18,500 g for 20 minutes at 4°C. Aliquots (0.040 mL) of supernatant were added to 0.960 mL of assay buffer (0.17 mg/mL o-dianisidine, 0.05% H2O2, 50 µmol/L sodium phosphate, pH 6.0). Absorbance at 460 nm was measured after 5 minutes of incubation by spectrophotometry (Beckman, Silver Springs, MD). Lung tissue myeloperoxidase activity was expressed as percent increase over baseline. All activity was normalized to normal lung dry weight of 16%.
After animal sacrifice, a portion of right lung (20 to 30 g) was removed for determination of wet lung weight. The sample was incubated at 100°C for 48 hours and reweighed. Percent wet weight was determined as follows: % wet weight = (wet weight - dry weight)/wet weight.
The left lung was perfusion-fixed using 10% formalin, 0.5% cacodylate solution. In each animal, five representative samples were taken from several pulmonary areas and stained with hematoxylin and eosin for histologic analysis.
A blinded quantitative analysis of the percentage of high-power fields (x200) affected with alveolar edema was performed by examination of the five lung specimens from each animal using light microscopy. Quantification of percent of alveolar spaces affected by edema was also performed by examination of contiguous highpower fields (x200) and the use of a 10 x 10 reticle grid.
Statistical Analysis
All measurements are reported as mean ± standard error of the mean. Comparisons between groups were made using analysis of variance for repeated measures. Myeloperoxidase activities, differential right and left atrial neutrophil counts, and percent wet weight were compared with a Student's t test. Significance was assumed at a p value less than 0.05.
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Results |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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). Platelet counts were similar between samples in each animal. After treatment with SDZ HUL-412, addition of exogenous 50 µmol/L PAF induced 54% ± 25% of baseline aggregation and 5 µmol/L PAF caused 21% ± 13% of baseline aggregation, whereas 0.5 µmol/L PAF solution failed to induce aggregation. Control animals exhibited 104% ± 28%, 109% ± 27%, and 25% ± 16% of baseline aggregation with addition of PAF in concentrations of 50, 5, and 0.5 µmol/L, respectively. Thus, SDZ HUL-412 inhibited PAF-induced platelet aggregation significantly at 50, 5, and 0.5 µmol/L concentrations (p < 0.05). ADP-induced aggregation was measured as a control for platelet function. At 10- and 5-µmol/L concentrations of ADP, inducible aggregation was 150% ± 37% and 160% ± 42% of baseline, respectively, in group I. In group II controls, ADP induced 68% ± 10% and 88% ± 21% of baseline aggregation at concentration of 10 and 5 µmol/L, respectively (p = not significant).
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). Fifteen minutes after conclusion of CPB, PaO2 in group I was 401 ± 47 mm Hg and remained stable to the end of the experiment: at 120 minutes PaO2 was 425 ± 61 mm Hg. In group II (15 minutes after CPB) PaO2 was 276 ± 62 mm Hg and decreased further to 234 ± 76 mm Hg by 120 minutes (p < 0.05). Baseline PVR was 255 ± 50 dynes • s • cm-5 in group I and 269 ± 29 dynes • s • cm-5 for group II. Pulmonary vascular resistance rose dramatically in group II when compared with group I after CPB (Fig 3). Fifteen minutes after CPB, PVR rose to 531 ± 177 dynes • s • cm-5 in group II versus 323 ± 55 dynes • s • cm-5 in group I. Group II PVR rose further to 884 ± 419 dynes • s • cm-5 by the end of the post-CPB observation period, whereas in group I PVR increased to 553 ± 106 dynes • s • cm-5 (p < 0.05).
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). Fifteen minutes after separation from CPB, group I compliance decreased to 91% ± 12% of baseline, compared with 49% ± 5.2% of baseline in group II. At 120 minutes after CPB, compliance decreased to 69% ± 3.6% of baseline in group I and to 56% ± 6.4% in group II.
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). In both groups, the greatest increase occurred during cooling (the first 30 minutes of CPB) and during the last 30 minutes of rewarming, before weaning from CPB.
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); the most rapid reduction occurred during the first 60 minutes of CPB: neutrophil counts fell from 5,304 ± 478 to 1,253 ± 271 cells/µL in group I and from 6,964 ± 1,984 to 1,335 ± 247 cells/µL in group II (p = not significant). Neutropenia persisted throughout the remainder of the experiment. Two hours after CPB, neutrophil counts were 1,037 ± 176 cells/µL in group I and 1,805 ± 376 cells/µL in group II. Pulmonary leukosequestration (assessed by difference in granulocyte counts in samples drawn simultaneously from left and right atria was greater in group I (Fig 7). Thirty minutes after CPB, RA-LA neutrophil differentials were 477 ± 173 cells/µL in group I and 1,286 ± 324 cells/µL in group II (p < 0.05). This difference between groups ceased by 1 hour after CPB.
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Wet lung weight was similar between groups. Group I lungs were 87.9% ± 1.0% water, whereas group II lungs contained 88.9% ± 1.7% water (p = not significant).
Histologic evaluation of group II lungs revealed marked intraalveolar hemorrhage, edema, and abundant intraalveolar neutrophils. Extensive neutrophil extravasation caused disruption of normal pulmonary parenchyma (Fig 8). Capillary plugging by neutrophils gave the interstitium a hypercellular appearance. In group I animals, there was preservation of normal pulmonary parenchyma (Fig 9), with only occasional small foci of fluid without cells into alveolae. There were abundant intracapillary neutrophils but no interstitial migration. Quantitative microscopy at x200 revealed that 5.05% ± 1.54% of group I fields had edema, compared with 41.0% ± 11.7% of group II (p < 0.02). By grid analysis, percentage of alveolar spaces affected by edema was significantly different between groups I and II at all percentile comparisons (Table 1)tab 1.
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Early experimental efforts to reduce neutrophil-mediated CPB injury included mechanical filtration to remove circulating neutrophils; this resulted in improved pulmonary function after CPB [24]. More recently, neutrophil activation and neutrophil-mediated tissue injury have been shown to be dependent on neutrophil adhesion to activated endothelium [25–27]. This raised the possibility that inhibition of neutrophil adhesion might decrease pulmonary injury after CPB.
Neutrophil activation and subsequent diapedesis into tissue is dependent on adhesion to activated endothelium [13, 15]. The adhesion molecules primarily responsible for this have been termed ``integrins.'' The B2 integrin family consists of three glycoprotein heterodimers (CD11a/CD18, CD11b/CD18, and CD11c/CD18), each containing different units but sharing the same B2 subunit. Although all three integrins are present on neutrophils, the receptor most frequently implicated in neutrophil activation, chemotaxis, and diapedesis is CD11b/CD18 (also known as MAC-1, CR3, or Mo-1 receptor) [26]. This receptor is present within intracytoplasmic vesicles, but appears on the neutrophil surface within minutes of activation. Binding of neutrophils to endothelium alters actin cytoskeletal properties and intracellular activity, resulting in conformational change and ultimately in diapedesis, an oxidative respiratory burst, and neutrophil degranulation [25, 26]. Recently, we demonstrated that CD18 is up-regulated by 150% during CPB [28]. Pharmacologic inhibition of up-regulation of CD18 in vivo in a porcine CPB model decreased pulmonary leukocyte sequestration, myeloperoxidase activity, and free radical generation during CPB [8]. Postoperative pulmonary function was also improved.
The precise mechanism of neutrophil activation and adhesion receptor up-regulation during CPB remains unclear. There is evidence to suggest several potential mediators: PAF, exposure to foreign surfaces, C5a, leukotriene B4, and kallikrein [2, 6, 9, 17]. Platelet activating factor seems an important mediator of neutrophil activation because of its known physiologic effects. It is known to increase PVR and decrease vascular permeability [18], but whether these effects are direct or mediated through neutrophils is unknown.
A variety of PAF competitive inhibitors have been used to delineate the role of PAF in inflammation. In canine lung transplantation, PAF antagonists given before organ reperfusion improve postoperative pulmonary function and decrease edema [29]. Platelet activating factor inhibition can reverse the inflammation of the guinea pig arthus reaction [30] and reduce hyperacute rejection in presensitized cardiac rat recipients and rat recipients of guinea pig hearts [19]. Platelet activating factor inhibition also has reduced ischemia/reperfusion injury in the myocardium [31] and gut [32, 33].
Platelet activating factor is a potent up-regulator of CD11b/CD18 expression on neutrophils in vitro [12, 15]. Neutrophils possess a specific PAF receptor, as do pulmonary capillary membranes in guinea pig and human [34]. Experimental intravenous injection of PAF results in pulmonary leukosequestration [35] and the neutrophil oxidative burst [36–40].
Our porcine model of CPB produces a reproducible pulmonary injury similar to the pathophysiologic effects of PAF: increased PVR, pulmonary edema, and pulmonary leukosequestration, along with complement activation, generation of oxygen free radicals, and profound neutropenia.
In this study, SDZ HUL-412 significantly reduced pulmonary injury after CPB. Pulmonary vascular resistance and PaO2 were better in treated animals than in controls. Although there was no significant difference in wet lung weight, there was a trend toward more pulmonary edema in the control lungs. A large section of lung was used to determine wet lung weight. This contained subsegmental atelectatic areas in both groups. These areas would tend to imply greater wet lung weight because of edema associated with atelectasis even in animals with no alveolar membrane disruption and thus eliminate a significant difference between the groups by this parameter. Smaller nonatelectatic lung sections were examined by quantitative light microscopy, and intraalveolar edema fluid was less in treated animals and normal pulmonary parenchymal architecture was preserved. In both groups, intracapillary neutrophils were seen in all microscopic fields, and similar degrees of circulating neutropenia were seen during and after CPB. In treated animals, there were no intraalveolar and few interstitial neutrophils and there was significantly less pulmonary leukosequestration at 30 minutes after CPB. The latter finding was supported by qualitative histology showing a marked increase in intraalveolar and interstitial neutrophils in the untreated group. More important, there was less lung tissue myeloperoxidase in treated animals after CPB than in controls.
Because the increase in CD18 expression on circulating neutrophils was similar in control and experimental groups, PAF antagonism probably did not inhibit neutrophil adhesion significantly, although it may have inhibited diapedesis. This is consistent with previous findings that PAF is an important neutrophil chemotactic agent [35, 41, 42]. However, other studies have shown PAF to be a primary inducer of CD11b/CD18 expression [12, 15]. Several possibilities may account for this discrepancy. The complement component C5a [43] and arachodonic pathway product leukotriene B4 [44, 45] have been shown to result in increased neutrophil adhesion to endothelium. We did not inhibit these cascades in our experiment. The neutrophils measured are circulating and may not reflect the surface expression of CD11b/CD18 on the sequestered pool of neutrophils in the pulmonary and other vascular endothelium. This is a problem that vexes all current studies of neutrophil adhesion. The antibody we used to measure the adhesion molecules binds to the CD18 ß-subunit. This subunit is also a component of CD11a/CD18 and CD11c/CD18 and the antibody has been shown to bind to CD11a/CD18 [23]. CD11a/CD18 is known to be constitutively expressed on activated neutrophils, although it is not thought to contribute directly to the diapedesis process. The measurement of this adhesion molecule may contribute to the equal increase in expression of CD18 in groups I and II. It has been shown that the CD11b/CD18 molecule can mediate neutrophil adhesion and modulate degranulation independent of its quantitative cell surface expression thought to be attributable to a conformational change in the glycoprotein during neutrophil activation [46]. Finally, neutrophil adherence may result in part in both groups from a process involving P-selectin. This may account for binding without diapedesis in group I.
In summary, a competitive PAF inhibitor (SDZ HUL-412) ameliorated CPB-induced pulmonary injury in a porcine model. It decreased extravascular diapedesis of neutrophils without affecting their CD11b/CD18 integrin expression. These data suggest that PAF is an important neutrophil chemotactic agent in CPB-mediated lung injury, and PAF inhibition may have some usefulness in clinical strategies to reduce organ injury in cardiac operations.
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Acknowledgments |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Footnotes |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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