Leukocyte Activation With Increased Expression of CR3 Receptors During Cardiopulmonary Bypass Y. J. Gu, MD, Willem van Oeveren, PhD, Piet W. Boonstra, MD, PhD, Jacob de Haan, MSc, and Charles R. H. Wildevuur, MD, PhD Thorax Centre, University Hospital Groningen, Groningen, the Netherlands

The effects of cardiopulmonary bypass (CPB) on the expression of leukocyte adhesive receptors, ie, complement receptor type 3 (CR3), were studied in 16 patients. The CR3 expression on leukocytes was determined by time-resolved fluoroimmunoassay on a standardized number of cells isolated from blood samples taken during various times during CPB. The results demonstrated that CR3 expression on leukocytes increased immediately after the start of CPB ( p < 0.05), concomitant with an early sharp increase of plasma concentrations of C3a (p < 0.01). After release of the aortic cross-clamp, a second peak of leukocyte CR3 expression was induced ( p < 0.05), paralleled by a significant increase of leukotriene B, ( p < 0.05) and elastase (p < 0.05) levels in the

late period of CPB. In vitro studies with leukocytes isolated from healthy donors (n = 5) showed a dosedependent increase of CR3 expression stimulated by zymosan-activated plasma, indicating that the rapid CR3 expression on leukocytes is likely mediated by complement activation. However, the mechanisms for the second peak of leukocyte CR3 expression during CPB remain to be further elucidated. In conclusion, CR3 expression on leukocytes increased immediately after the start of CPB and was followed by a second peak of expression in the late phase of CPB. Pharmacological blockage of these adhesive receptors might reduce the leukocyte-mediated deleterious effects of CPB. (Ann Thorac Surg 1992;53:83943)

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Material a n d Methods

ostoperative morbidity after cardiac operations has been related to the damaging effects of cardiopulmonary bypass (CPB) [l-31. These damaging effects are considered to be mediated mainly by the activation of leukocytes as a result of complement activation initiated by blood-material interaction [4-61. Previous studies [7] demonstrated that anaphylatoxins such as C5a generated during complement activation promote leukocyte activation through C5a receptors on the cell surface of polymorphonuclear leukocytes (PMNs). However, next to activation by C5a, PMN adhesion to the target is required to initiate their local effect and mediate endothelial cell damage [8, 91. Thus, expression of adhesive receptors, ie, complement receptor type 3 (CR3), on the PMN surface must be anticipated as a prerequisite for the local release of inflammatory mediators by leukocytes during CPB. We performed a study involving 16 patients having coronary artery bypass grafting to investigate the effect of CPB on the membrane expression of CR3 on leukocytes as well as other variables indicating complement and leukocyte activation during CPB. In addition, we performed in vitro studies on isolated leukocytes obtained from healthy donors and stimulated with zymosan-activated plasma to assess whether the increased CR3 expression on leukocytes was dependent on the increased activation of complement. Accepted for publication Oct 15, 1991 Address reprint requests to Dr Wildevuur, Cardiopulmonary Surgery Research Division, University Hospital Groningen, 59 Oostersingel, 9713 EZ Groningen, the Netherlands.

0 1992 by The Society of Thoracic Surgeons

Sixteen patients undergoing CPB for selective coronary artery bypass grafting were prospectively scheduled for this study. None of the patients had preoperative signs of infection. Informed consent was obtained from each of the patients the day before operation, and the study was approved by the ethical committee of the hospital. After premedication with diazepam (10 to 15 mg), anesthesia was induced by sufentanil citrate (3 to 5 pgkg), and muscle relaxation was achieved with pancuronium bromide (100 to 140 p e g ) . Analgesia was provided by sufentanil and midazolam hydrochloride infusion. Cefamandole nafate, 2 g, and dexamethasone, 1 mg/kg, were administered preoperatively. Anticoagulation was achieved by intravenous administration of bovine lung heparin sodium (300 IU/kg).

Cardiopulmonary Bypass The extracorporeal circuit consisted of roller pumps and either a hollow-fiber membrane oxygenator (BOS-CM50; Bentley/Baxter Inc, Irvine, CA) or a microporous polypropylene membrane oxygenator (CML EXCEL; COBE Laboratories, Inc, Lakewood, CO). The circuit was primed with a gelatin containing crystalloid solution (Gelifundol; Biotest Pharma GmbH, Dreieich, Germany) and 1,500 IU of heparin. After institution of CPB, the aorta was cross-clamped, and 1 L of St. Thomas’ cardioplegic solution (4°C) was infused into the aortic root to provide myocardial preservation. At the same time, moderate hypothermia was induced to maintain the nasopharyngeal temperature 0003-4975/92/$5.00

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GUETAL LEUKOCYTE CR3 A N D CPB

between 26" and 28°C. The mean arterial pressure was kept between 50 and 60 mm Hg during bypass. After completion of all the distal anastomoses, the aortic crossclamp was removed, and the proximal anastomoses were performed while the patient was being rewarmed to 37°C. After CPB, heparin was neutralized by slow infusion of protamine hydrochloride (3 mg/kg) through the right atrium. The mean duration of CPB was 109.6 2 8.3 minutes.

Meas u rements Blood samples were taken after heparinization but before CPB, at 5 minutes and 30 minutes after the initiation of CPB, 5 minutes before and after release of the aortic cross-clamp, and at the end of CPB from the indwelling radial artery catheter. Circulating leukocyte count and sample hematocrit were measured by a cell counter (CellDyn 610; Sequoia-Turner, Mountain View, CA). Samples for determination of leukocyte CR3 expression and measurement of leukocyte release products were mixed with 3.06% sodium citrate, whereas samples for measurement of complement were anticoagulated with EDTA (ethylenediaminetetraacetic acid). Plasma was obtained by centrifugation of whole blood at 1,000 g and stored at -20°C for further studies. LEUKOCYTE ISOLATION. Leukocytes were isolated by combined sedimentation and hypotonic lysis technique according to the method described previously [lo]. The harvested leukocytes were resuspended in phosphatebuffered saline solution containing a leukocyte concentration of 4.0 x 109/Lincluding approximately 70% PMNs.

Expression Of CR3 on the isolated leukocytes was determined by time-resolved fluoroimmunoassay [ll]. Monoclonal antibodies against human CR3 (Dakopatts a/s, Glostrup, Denmark) were labeled with europium 3+, and this labeled antibody solution (containing 4.7 nmol/mL immunoglobulin G) was incubated with 4 x 106/mLleukocytes for 30 minutes at room temperature. After incubation, the mixture was layered onto 1 mL of sucrose (20%) and centrifuged at 2,800 g for 5 minutes. The leukocyte pellet was then mixed with 200 pL of enhancement solution, and the amount of europium label released into the solution was measured in a time-resolved fluorometer (Delfia; Pharmacia Wallac, Turku, Finland). Nonspecific binding of the label was controlled by preincubation of isolated leukocytes with excess unlabeled monoclonal antibodies against CR3. The final results were expressed as counts per second/103 cells. LEUKOCYTE CR3 RECEPTORS.

COMPLEMENT ~ 3 a Concentrations . of C3a in plasma were determined by radioimmunoassay according to the manufacturer's instructions (The Upjohn Company, Kalamazoo, MI).

To measure leukotriene B, (LTB,), plasma was first acidified with acetic acid to pH 3 to 4. The LTB, was then extracted by ethyl acetate and the concenLEUKOTRIENE

B,.

tration determined by enzyme immunoassay (Cayman, Ann Arbor, MI). LEUKOCYTE ELASTASE. Plasma elastase concentrations were quantitated in complex with a,-proteinase inhibitor by enzyme-linked immunosorbent assay (Merck, Darmstadt, Germany).

In Vitro Study Whole blood (9 mL) drawn from healthy donors (n = 5) was mixed immediately with 1 mL of 3.8% sodium citrate for anticoagulation. Leukocytes were isolated by combined sedimentation and hypotonic lysis according to the technique previously presented [lo]. Zymosan-activated plasma was prepared by incubation of autologous plasma with 1mg/mL of zymosan (Sigma Chemical Co, St. Louis, MO) for 30 minutes at 37°C. After zymosan particles were separated from plasma by centrifugation at 1,000 g for 10 minutes, serial log dilutions of zymosan-activated plasma were made with phosphate-buffered saline solution. Each of the 100-pL phosphate-buffered saline solutions containing different concentrations of zymosan-activated plasma was then incubated with 100 pL of isolated leukocyte suspension for 60 minutes. Finally, CR3 expression on these cells was determined by time-resolved fluoroimmunoassay.

Statistical Analysis Values for cell counts obtained and biochemical tests done during CPB were corrected for hemodilution using the hematocrit levels measured before bypass. Statistical analysis was performed using Student's t test, and the results were expressed as the mean k the standard error of the mean.

Results Leukocyte CR3 Receptors Expression of CR3 on leukocytes increased significantly 5 minutes after the start of CPB compared with the prebypass values ( p < 0.05). This early peak of leukocyte CR3 expression gradually returned to the prebypass value until release of the aortic cross-clamp. After release of the clamp, a second peak of leukocyte CR3 expression was induced ( p < 0.05), and this high CR3 expression was maintained to the end of CPB ( p < 0.05) (Fig 1).

Leukocyte Count In association with the increased CR3 expression, there was an initial drop in the number of circulating leukocytes 5 minutes after the start of CPB (from 4.9 f 0.3 X 109/Lto 4.4 f 0.3 x 109/L;p > 0.05). Leukocytosis to about three times the prebypass count developed late during CPB (Fig 2).

Complement C3a Plasma C3a concentrations increased significantly after the start of CPB to a peak level of 2,210 k 440 ng/mL

GUETAL LEUKOCYTE CR3 AND CPB

Ann Thorac Surg 1992;53:83943

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time (min) Fig 1 . Expression of complement receptors type 3 (CR3) on leukocytes from patients undergoing cardiopulmonary bypass (CPB). The CR3 expression increased immediately after the start of CPB and was followed by a second increase after aortic cross-clamp release (triangle) during the late period of CPB. (* = p < 0.05 versus baseline.)

during CPB ( p < 0.01). The concentrations remained high until the end of bypass ( p < 0.01) (Fig 3).

Leukotriene B, The level of LTB, increased immediately after the start of CPB and remained high thereafter, with an increase 5 minutes after release of the aortic cross-clamp ( p < 0.05) (Fig 4).

Leukocyte Elastase There was only a slight increase of elastase in plasma early during CPB. However, the plasma elastase concentration increased significantly 5 minutes after release of the aortic cross-clamp ( p < 0.05) and remained high until the end of CPB ( p < 0.05) (Fig 5).

In Vitro Studies In vitro studies with leukocytes isolated from healthy donors showed that the increased CR3 expression corre-

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lated with an increased concentration of zymosanactivated plasma. The CR3 expression on leukocytes increased at a concentration of zymosan-activated plasma as low as lo-' in phosphate-buffered saline solution (voVvo1) with the maximal increase at a lo-' concentration (Fig 6 ) .

Comment The present study demonstrates that CR3 expression on leukocytes increased immediately after the start of CPB, concomitant with an early sharp increase of C3a. Moreover, a second peak of CR3 expression on leukocytes was induced after release of the aortic cross-clamp and was paralleled by a significant increase of LTB, and release of leukocyte elastase. The rapid leukocyte CR3 expression early during CPB is likely mediated by complement activation, as a dosedependent increase of CR3 expression was similarly stimulated by zymosan-activated plasma in vitro (see Fig 6).

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time (min) Fig 4. Leukotriene B4 (LTB4), presented as percentage of baseline value, increased immediately after the start of cardiopulmonary bypass (CPB) with the highest level 5 minutes after aortic cross-clamp release (triangle) in the late period of CPB. (* = p < 0.05 versus baseline.)

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time (min) Fig 5. Elastase concentration increased significantly 5 minutes after

release of the aortic cross-clamp (triangle) and remained high until the end of cardiopulmonary bypass (CPB).(* = p < 0.05 versus baseline.)

During CPB, the complement system is indeed activated by blood-material interaction, as reflected by the sharp increase of plasma C3a (see Fig 3). Although leukocyte CR3 expression is known to be induced by C5a, this peptide is difficult to detect because of its very short half-life and rapid binding on PMNs in vivo [5, 71. In previous studies [7],receptors for C5a have been demonstrated on the surface of PMNs that are involved directly in mediating the chemotactic response of these cells including the changes in intracellular oxygen metabolism and cellular aggregation. Besides these changes, however, the binding of C5a on PMNs also leads to the immediate increase of adhesive receptor expression, such as CR3, and increases leukocyte adhesion [12, 131. The consequences of the increased leukocyte adherence will be, in the early period of CPB, increased leukocyte adhe-

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Log Concentrations of ZAP in PBS (voVvol) Fig 6 . Expression of complement receptors type 3 (CR3) on leukocytes isolated from donor blood stimulated by zymosan-activated plasma (ZAP). The CR3 expression increased with increased concentrations of ZAP. (PBS = phosphate-buffered saline solution.)

sion to the surfaces of foreign material. On these surfaces, C3b is chemically bound to the surface hydroxyl groups through ester bonding when the complement system is activated [14, 151. The expressed CR3 on leukocytes recognizes the inactivated form of C3b (C3bi) on the material surfaces and in turn mediates leukocyte adhesion to the foreign material [16], resulting in a commonly observed pattern of initial leukopenia in patients undergoing both hemodialysis and CPB [4, 5, 171. The second peak of leukocyte CR3 expression in the later period of CPB is less likely due to the materialdependent complement activation but rather to stimuli that are considered material-independent [18, 191. After release of the aortic cross-clamp late during CPB, a unique pattern of blood activation is induced presumably by organ reperfusion [19]. The generation of chemotactic mediators such as LTB, is enhanced after reperfusion in the late period of CPB, as observed in the present study as well as in others [19,20]. Actually, LTB, is mainly released by the activated leukocytes through the lipooxygenase pathway. However, this lipid peptide is also a strong and fast mediator stimulating leukocyte CR3 expression and mediating leukocyte adhesion to the endothelial cells [21]. On the other hand, release of endogenous endotoxin was found to occur at this specific moment during CPB followed by the generation of tumor necrosis factors [22], both of which are known to be potential stimuli for the expression of adhesive molecules on endothelial cells promoting leukocyte adhesion [23]. As a result, an enhanced interaction between leukocytes and host endothelial cells is induced, leading to intensified leukocyte sequestration and leukocyte-mediated tissue injury [24, 251, the consequence of which tends to be more deleterious than leukocyte adhesion to the material surfaces. It has already been demonstrated in both animal experiments and clinical reports that leukocyte sequestration together with the release of oxygen free radicals occurs massively in the lungs after pulmonary reperfusion [26-281. This deleterious effect was also demonstrated in the present study by the release of leukocyte elastase, a specific protease mediating endothelial cell injury [29], which increased dramatically after cross-clamp release in the late period of CPB. In conclusion, the CR3 expression on leukocytes increased immediately after blood contacted the extracorporeal circuit in the early period of CPB. This was followed by a second peak of expression that occurred after reperfusion and was associated with a strong release of elastase known to cause tissue injury. It can therefore be anticipated that if these adhesive receptors on leukocytes could be transiently blocked by monoclonal antibodies or pharmacologically suppressed by other substances [30,31], the sequential damaging effects of leukocyte-mediated endothelial injury induced by CPB could possibly be diminished or even eliminated. The technical assistance of J. Haan in the performance of the assays and D. Njoo in the collection and processing of the blood samples is greatly appreciated.

Ann Thorac Surg 1992;53:83943

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T. Molecular understanding of cellular adhesion on artificial surfaces. Trans Am SOCArtif Intern Organs 1989;35:354-6. 17. Arnaout MA, Hakim RM, Todd RF 111, Dana N, Colten HR. Increased expression of an adhesion-promoting surface glycoprotein in the granulocytopenia of hemodialysis. N Engl J Med 1985;312:457-62. 18. Craddock PR, Hammerschmidt DE. Complement-mediated granulocyte activation and down-regulation during hemodialysis. ASAIO J 1984;7:5&6. 19. Jansen NJG, van Oeveren W, van der Broek L, et al. Inhibition by dexamethasone of the reperfusion phenomena in cardiopulmonary bypass. J Thorac Cardiovasc Surg 1991; 102:51525. 20. Semb AG, Forsdahl K, Vaage J. Granulocyte and eicosanoid gradients across the coronary circulation during myocardial reperfusion in cardiac surgery. Eur J Cardiothorac Surg 1990;4543-8. 21. Gimbrone MA, Brock AF, Schafer AI. Leukotriene B, stimulates polymorphonuclear leukocytes adhesion to endothelial cells. J Clin Invest 1984;74:1552-5. 22. Jansen NJG, van Oeveren W, Gu YJ, van Vliet M, Eysman L, Wildevuur CRH. Endotoxin release and tumor necrosis factor formation during cardiopulmonary bypass. Ann Thorac Surg (in press). 23. Pohlman TH, Stanness KA, Beatty PG, Ochs HD, Harlan JM. An endothelial cell surface factor@) induced in vitro by lipopolysaccharide, interleukin-1, and tumor necrosis factor increases neutrophil adherence by a CDwl8-dependent mechanism. J Immunol 1986;136:4548-53. 24. Harlan JM. Leukocyte-endothelial interactions. Blood 1985; 65:513-25. 25. Ward PA, Varani J. Mechanisms of neutrophil-mediated killing of endothelial cells. J Leukocyte Biol 1990;48:97-102. 26. Gu YJ, Wang YS, Chiang BY, Gao XD, Ye CX, Wildevuur CRH. Membrane oxygenator prevents lung reperfusion injury in canine cardiopulmonary bypass. Ann Thorac Surg 1991;51:573-8. 27. Royston D, Fleming JS, Desai JB, Westaby S, Taylor KM. Increased production of peroxidation products associated with cardiac operations. Evidence for free radical generation. J Thorac Cardiovasc Surg 1986;91:759-66. 28. Howard RJ, Crain C, Franzini DA, Hood CI, Hugli TE. Effects of cardiopulmonary bypass on pulmonary leukostasis and complement activation. Arch Surg 1988;123:1496-501. 29. LeRoy EC, Ager A, Gordon JL. Effects of neutrophil elastase and other proteases on porcine aortic endothelial prostaglandin I, production, adenine nucleotide release, and response to vasoactive agents. J Clin Invest 1984;74:1003-11. 30. Bochsler PN, Slauson DO, Neilsen NR. Modulation of an adhesion-related surface antigen on equine neutrophils by bacterial lipopolysaccharide and antiinflammatory drugs. J Leukocyte Biol 1990;48:306-15. 31. Kirklin JK. Prospects for understanding and eliminating the deleterious effects of cardiopulmonary bypass [Editorial]. Ann Thorac Surg 1991;51:529-31.

Leukocyte activation with increased expression of CR3 receptors during cardiopulmonary bypass.

The effects of cardiopulmonary bypass (CPB) on the expression of leukocyte adhesive receptors, ie, complement receptor type 3 (CR3), were studied in 1...
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