Effect of leukocyte depletion on endothelial cell activation and transendothelial migration of leukocytes during cardiopulmonary bypass

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Effect of Leukocyte Depletion on Endothelial Cell Activation and Transendothelial Migration of Leukocytes During Cardiopulmonary Bypass Ying-Fu Chen, MD, PhD, Wen-Chan Tsai, MD, Ching-Cheng Lin, MD, Li-Yu Tsai, PhD, Chee-Siong Lee, MD, Chiung-Hui Huang, BS, Pi-Chen Pan, PhD, and Man-Lin Chen, RN Divisions of Cardiovascular Surgery, Cardiology, and Immunology, School of Technology for Medical Sciences, and School of Public Health, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan

Background. Although leukocyte depletion from systemic circulation during cardiopulmonary bypass (CPB) has been studied, the effect of leukocyte depletion on the leukocyte-endothelial cascade remains poorly understood. So far, there has been no published work on the effects of leukocyte filters during cardiac operations from the viewpoint of endothelial activation and transendothelial neutrophil migration. Methods. Thirty-two patients undergoing elective heart operations were randomly allocated to a leukocytedepletion (LD) group or a control group. Blood samples were collected at seven time points: before sternotomy, at 30 minutes and at 60 minutes of CPB, at 5 minutes after coronary reperfusion, at the end of CPB, and at 2 hours and 24 hours after the cessation of CPB. The plasma concentrations of P-selectin, intercellular adhesion molecule-1 (ICAM-1), interleukin-8, and platelet-endothelial cell adhesion molecule-1 (PECAM-1) were measured using enzyme-linked immunosorbent assays. Plasma malondialdehyde (MDA) concentration was determined by

measurement of thiobarbituric acid-reactive substances in plasma. In addition, blood samples collected at intervals before and after operation were used for arterial blood gases. Results. Our studies show significant increases of plasma levels of P-selectin, ICAM-1, interleukin-8, PECAM-1, and MDA during and after CPB in the control group. Interestingly, a significant decrease of plasma levels of P-selectin, ICAM-1, interleukin-8, PECAM-1, and MDA, and better preservation of lung function could be found in the LD group compared with the control group. Conclusions. Our results demonstrate a rationale for using a leukocyte filter in patients undergoing cardiac surgery to attenuate the endothelial-mediated component of the CPB-induced inflammatory response by reducing endothelial activation and neutrophil transmigration.

M

gands for ␤2-integrins are intercellular adhesion molecule-1 (ICAM-1) and possibly ICAM-2. These molecules, members of the immunogloblin superfamily, are present constitutively on endothelial cells both in vitro and vivo [4]. After the firm adhesive bond between the integrins on the neutrophil and ICAM-1 on the endothelial cells, interleukin-8 is particularly important in the regulation of transendothelial neutrophil migration [5]. This migration through the endothelium seems to be in part mediated by platelet-endothelial cell adhesion molecule-1 (PECAM-1) [6]. A member of the immunoglobulin superfamily, PECAM-1 is expressed at relatively low levels on the surface of leukocyte and platelets but at higher levels on endothelium [7]. There is evidence that adherence by CD11/CD18 primes the neutrophil to degranulate [8] and to produce the respiratory burst [9]. By this binding, oxygen-derived free radicals and proteolytic enzymes are generated by the adherent neutrophils, which results in marked injury to endothelial cells and thus tissue injury. Theoretically, reduction of the leukocyte population during cardiopul-

any clinical and experimental studies have suggested that a portion of the morbidity associated with cardiopulmonary bypass (CPB) can be attributed to the generalized inflammatory response caused by extracorporeal circulation [1]. One important aspect for this inflammatory damage appears to be the interactions between activated neutrophils with the endothelium, so-called leukocyte-endothelial cell interaction [2]. As we know, adherence of neutrophils to the vascular endothelium involves the interaction of specific molecules belonging to one of the three major families: the selectin, the integrins, and the immunoglobulin gene superfamily. Of them, the selectin mediate the first stage (tethering) of the process of adhesion of neutrophils and endothelium [3]. On the other hand, strong adhesion is mediated by leukocyte ␤2-integrins (CD 11/CD18 complex) that bind to counter receptors on endothelium [4]. Important liAccepted for publication Feb 10, 2004. Address reprint requests to Dr Chen, Division of Cardiovascular Surgery, Kaohsiung Medical University Hospital, 100 Shih-Chuan 1st Rd, Kaohsiung, Taiwan; e-mail: [email protected].

© 2004 by The Society of Thoracic Surgeons Published by Elsevier Inc

(Ann Thorac Surg 2004;78:634 – 43) © 2004 by The Society of Thoracic Surgeons

0003-4975/04/$30.00 doi:10.1016/j.athoracsur.2004.02.091

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Table 1. Patient Demographic Dataa

Age (y) Sex Male Female Procedure Coronary artery bypass Valve replacement Number of grafts CPB time (min) Aortic crossclamp time (min) Systemic hypothermia (°C) a

All patients gave informed consent before participating in this study.

Control (n ⫽ 16)

Depletion (n ⫽ 16)

p Value

61.2 ⫾ 9.1

60.3 ⫾ 9.9

NS

14 2

12 4

NS

16 0 2.8 ⫾ 0.7 130 ⫾ 28 93 ⫾ 21 27.4 ⫾ 0.8

15 1 2.6 ⫾ 0.7 133 ⫾ 37 95 ⫾ 31 27.6 ⫾ 0.7

NS NS NS NS NS

Data are shown as mean ⫾ SD.

CPB ⫽ cardiopulmonary bypass;

NS ⫽ not significant.

monary bypass would resulted in decreased neutrophilmediated tissue injury. Although leukocyte depletion from systemic circulation during CPB has been studied [10 –12], the effect of leukocyte depletion on the leukocyte-endothelial cascade remains poorly understood. So far, there has been no systemic study on the effects of leukocyte filters during cardiac operations from the viewpoint of the endothelial activation and transendothelial neutrophil migration. The present study, therefore, is designed to characterize the patterns of endothelial activation during clinical CPB and to assess the effect of a leukocyte filter on these patterns. The results are assessed by measuring circulating levels of P-selectin, ICAM-1, interleukin-8, PECAM-1, and malondialdehyde (MDA). These markers are selected because they allow to evaluate the alteration of endothelial activation and the steps sequentially involved in neutrophil transmigration. Thus, P-selectin and ICAM-1 reflect endothelial cell activation. Malondialdehyde levels reflect neutrophil degranulation associated with adhesion to the endothelial cells. Interleukin-8 and PECAM-1 are the marker of transendothelial migration of adherent neutrophils.

Material and Methods Study Population Thirty-two consecutive adult patients undergoing CPB for coronary artery bypass grafting or heart valve operation constituted the study population for this study. The patients were randomly allocated to a leukocytedepletion (LD) group (n ⫽ 16) or a control group (n ⫽ 16). Exclusion criteria were prior cardiac operation, infection, emergency operation, congestive heart failure, acute myocardial infarction within the previous 1 month, corticosteroid therapy, and severe asthma or chronic obstructive lung disease. The demographic data of patients in both groups are summarized in Table 1. There was no significant difference in baseline characteristics between the two groups. This study was approved by the Ethics Committee of Kaohsiung Medical University Hospital.

Conduction of CPB, Myocardial Protection, and Leukocyte Depletion Identical anesthetic (dormicum, fentanyl, pancuronium) and monitoring techniques (electrocardiogram, central venous and arterial catheter, pulmonary artery catheter, urinary catheter, and temperature probes) were used in both groups of patients. The heart was exposed through a median sternotomy and sodium heparin at a dose of 300 U/kg of body weight was administered intravenously before CPB to produce an activated clotted time greater than 400 seconds. A disposable membrane oxygenator (Affinity NT; Medtronic, Minneapolis, MN) was used for CPB. The management of myocardial protection was identical in both groups. Cold intermittent blood cardioplegia resulting from mixing blood with high-potassium cardioplegic solution was administered in an antegrade fashion into the aortic root. Moderate systemic hypothermia was induced with the heat exchanger of the uncoated extracorporeal circuit. Topical cold saline solution or slush was used concomitantly. The leukocyte-depleting arterial blood filter for extracorporeal circulation (LeukoGuard-6; Pall Biomedical Product Corp, East Hillis, NY) was incorporated instead of a standard arterial filter in the circuit of 16 patients of LD group.

Collection and Laboratory Measurements of Blood Samples Blood samples were collected at seven time points: after induction of anesthesia but before sternotomy, at 30 minutes and 60 minutes of CPB, 5 minutes after coronary reperfusion, at the end of CPB, and at 2 hours and 24 hours after the cessation of CPB. Blood was withdrawn from an indwelling arterial cannula into an ethylenediamine tetraacetic acid (EDTA)– containing tube. After collection, the blood was immediately centrifuged (15 minutes at 600 gm) and the plasma samples were stored at ⫺70°C. Within 4 weeks after blood sampling, the plasma concentrations of P-selectin, ICAM-1, interleukin-8, and PECAM-1 were measured using commercial enzyme-linked immunosorbent assays (P-selectin, ICAM-1, interleukin-8, and PECAM-1; British Biotechnology Products, Abingdon, UK). Plasma MDA concentration was determined by measurement of thiobarbituric acid-reactive substances in plasma by standard biochemical techniques. Total white blood cell (WBC) and differential (neutrophil) counts, and platelet counts were measured at the seven time points by means of a blood cell counter (Cell-Dyn; Abbott Laboratories, Abbott Park, IL) and expressed as the cell count ⫻ 103/mm3. Pulmonary gas exchange was measured by the partial arterial oxygen pressure from blood samples drawn from the radial arterial line. Separate blood samples for arterial blood gas analysis (PH/blood gas analyzer, model 16200-06; Instrumentation Laboratory, Milan, Italy) were carried out before the operation, and after the operation. The oxygen index (arterial oxygen tension/inspired oxy-

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gen fraction, PaO2/FiO2) was used as an indicator of lung function [13].

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Table 2. Perioperative Clinical Data Study Group

Other Clinical Variables CARDIOVASCULAR

Duration of postoperative intubation was recorded during each patient’s stay in the surgical intensive care unit. The postoperative mediastinal chest tube drainage was measured at hourly interval. Cumulative mediastinal drainage was calculated 24 hours after the patient first reached the surgical intensive care unit.

Statistical Analysis All values were expressed as the mean ⫾ SD. Comparisons between groups were made using two-way analysis of variance for repeated measurements over time of the study. Comparisons within groups were performed using one-way analysis of variance followed by Tukey’s test for multiple comparisons among the sampling points. To evaluate the lung function of a certain group, preoperative and postoperative data within group were compared using paired Student’s t test. The data from the two groups were compared preoperatively or postoperatively with unpaired Student’s t test. A probability value of less than 0.05 was considered statistically significant.

Results All patients recovered uneventfully after operation except a 75-year-old woman in the control group who died on postoperative day 13 as a result of sepsis due to peritonitis.

Blood Cell Counts Before CPB, the total WBC counts were similar in both groups (5.3 ⫾ 1.6 ⫻ 103 cells/mm3 in the control group versus 5.5 ⫾ 2.2 ⫻ 103 cells/mm3 in the LD group, p ⫽ 0.3667). In the control group, no significant change in the total WBC count during CPB was followed by significant leukocytosis after reperfusion (5.3 ⫾ 1.6 ⫻ 103 cells/mm3 before CPB versus 10.1 ⫾ 3.2 ⫻ 103 cells/mm3 2 hours after CPB, p ⬍ 0.0001). Conversely, a significant decrease in the total WBC count at 30 minutes of CPB was noted in the LD group (5.5 ⫾ 2.2 ⫻ 103 cells/mm3 before CPB versus 3.8 ⫾ 1.5 ⫻ 103 cells/mm3 at 30 minutes of CPB, p ⫽ 0.0146), although significant leukocytosis was also noted after termination of CPB (5.5 ⫾ 2.2 ⫻ 103 cells/mm3 before CPB versus 8.2 ⫾ 2.6 ⫻ 103 cells/mm3 2 hours after CPB, p ⫽ 0.0144). Analysis of the neutrophil count revealed a pattern similar to those described by the total WBC counts. After CPB, the neutrophil counts increased significantly compared with counts before CPB in both groups (4.0 ⫾ 1.5 ⫻ 103 cells/mm3 before CPB versus 9.0 ⫾ 2.9 ⫻ 103 cells/ mm3 2 hours after CPB for the control group, p ⬍ 0.0001; and 4.2 ⫾ 1.8 ⫻ 103 cells/mm3 before CPB versus 7.2 ⫾ 2.3 ⫻ 103 cells/mm3 2 hours after CPB for the LD group, p ⫽ 0.0037). The efficacy of neutrophil depletion during CPB was significant in the LD group (4.2 ⫾ 1.8 ⫻ 103 cells/mm3 before CPB versus 3.2 ⫾ 1.3 ⫻ 103 cells/mm3 at 5 minutes after coronary reperfusion, p ⫽ 0.0493). However, there

Control (n ⫽ 16) Oxygen index before CPB (mm Hg) Oxygen index after CPB 10 hours (mm Hg) Intubation time (hours) Mean ⫾ SD Median Mediastinal drainage (mL/24 hours)

LD (n ⫽ 16)

p Value

446.3 ⫾ 102.8 453.9 ⫾ 86.2

0.8294

253.6 ⫾ 75.5a 337.5 ⫾ 95.7b

0.0123

21.4 ⫾ 14.0 15 577 ⫾ 182

17.8 ⫾ 9.0 14 568 ⫾ 195

0.1937 0.9043

p ⫽ 0.0031, compared with respective group oxygen index before b CPB. p ⫽ 0.0302, compared with respective group oxygen index before CPB. a

CPB ⫽ cardiopulmonary bypass;

LD ⫽ leukocyte depletion.

were no significant changes in neutrophil count during CPB in the control group (4.0 ⫾ 1.5 ⫻ 103 cells/mm3 before CPB versus 3.7 ⫾ 1.4 ⫻ 103 cells/mm3 at 5 minutes after coronary reperfusion, p ⫽ 0.4544). Platelet counts were significantly decreased at the time points of cross clamp in both groups, but then increased at the end of CPB. It remained below baseline at 24 hours after CPB, and showed no significant change compared with respective baseline levels in both groups (177.6 ⫾ 45.1 ⫻ 103 cells/mm3 before CPB versus 148.6 ⫾ 33.0 ⫻ 103 cells/mm3 24 hours after CPB for the control group, p ⫽ 0.0903; 181.9 ⫾ 42.7 ⫻ 103 cells/mm3 before CPB versus 155.7 ⫾ 60.0 ⫻ 103 cells/mm3 24 hours after CPB for the LD group, p ⫽ 0.2078). Furthermore, the analysis of the time course of platelet count did not reveal any significant difference between the groups (p ⫽ 0.3533).

Clinical Observations Pulmonary gas exchange, measured by oxygen index, was significantly deteriorated at 10 hours after termination of CPB in both groups (control group: 446.3 ⫾ 102.8 mm Hg before CPB versus 253.6 ⫾ 75.5 mm Hg 10 hours after termination of CPB, p ⫽ 0.0031; LD group: 453.9 ⫾ 86.2 mm Hg before CPB versus 337.5 ⫾ 95.7 mm Hg 10 hours after termination of CPB, p ⫽ 0.0302; Table 2). However, there was a significantly higher oxygen index in the LD group compared with the control group at 10 hours after termination of CPB (p ⫽ 0.0123; Table 2), although pulmonary gas exchange was similar before CPB in both groups (p ⫽ 0.8294). Duration of intubation time after operation was slightly shorter in the LD group than in the control group, but this difference was not statistically significant. In addition, there was no statistical difference between two groups with regard to mediastinal drainage (Table 2).

P-Selectin The time course of plasma concentrations of P-selectin during and after CPB in both groups is illustrated in

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Fig 1. Time course of plasma concentrations of P-selectin during and after cardiopulmonary bypass (CPB) in the control group (open circles, n ⫽ 16) and in the LD group (solid squares, n ⫽ 16). There is a significant difference between control and LD groups (p ⫽ 0.0003 by ANOVA). *Significant difference compared with the LD group, with p values in parentheses. Values are expressed as mean ⫾ SD. (ANOVA ⫽ analysis of variance; CPB 30 and CPB 60 ⫽ 30 and 60 minutes after start of cardiopulmonary bypass; LD ⫽ leukocyte depletion.)

Figure 1 and Table 3. The plasma concentration of Pselectin increased significantly after the initiation of CPB in either group. Although both groups showed a rise in P-selectin over time, the rise was considerably blunted in the LD group (p ⬍ 0.0001, Table 3; p ⫽ 0.0003, Fig 1).

Intercellular Adhesion Molecule-1 Cardiopulmonary bypass was associated with a rapid and statistically significant increase of plasma ICAM-1 in the control group (p ⫽ 0.0006, Fig 2; p ⬍ 0.0001, Table 3). Similarly, there was also a significant increase of circulating ICAM-1 in the LD group (p ⫽ 0.0352, Fig 2; p ⬍ 0.0001, Table 3). Nonetheless, patients in the control group displayed significantly higher plasma concentrations of ICAM-1 than in LD group during and after bypass (p ⫽ 0.0013, Fig 2; p ⬍ 0.0001, Table 3).

Interleukin-8 In the control group, interleukin-8 plasma levels did increase significantly during CPB and remained elevated throughout the observation period (p ⬍ 0.0001, Fig 3; p ⬍ 0.0001, Table 3). The changes of interleukin-8 level of LD group was also significant during CPB, and reaching a peak at the end of CPB, and the level fell steadily thereafter toward the preoperative level (p ⬍ 0.001, Fig 3; p ⬍ 0.0001, Table 3). However, two-way analysis of variance indicated a significant interaction effect in plasma concentrations of interleukin-8 between the control and the LD group (p ⬍ 0.0001, Fig 3; p ⬍ 0.0001, Table 3).

Malondialdehyde Figure 4 and Table 3 show the time course of plasma MDA during and after CPB in both groups. There was a significant increase in plasma MDA level during and after CPB in both groups. Similarly, CPB resulted in greater production of MDA in control patients than in the patients of LD group (p ⫽ 0.0098, Fig 4; p ⬍ 0.0001, Table 3).

Platelet-Endothelial Cell Adhesion Molecule-1 In the control group, circulating concentrations of PECAM-1 showed a rapid increase during CPB and remained increase throughout the observation period (p

⫽ 0.0005, Fig 5; p ⬍ 0.0001, Table 3). In the LD group, the plasma PECAM-1 level revealed an initial mild increase in PECAM-1 level from the baseline; however, this increase was not statistically significant. On the contrary, a significant decrease of circulating level after bypass was observed (p ⫽ 0.004, Fig 5; p ⫽ 0.0004, Table 3). Thus, there was a significantly higher circulating level of PECAM-1 in the control group compared with the LD group (p ⫽ 0.0017, Fig 5; p ⬍ 0.0001, Table 3).

Comment In the present study, total WBC counts did not changes significant during CPB in the control group. However, the number of circulating total WBCs decreased markedly during the CPB period in the LD group. In addition, after reperfusion stage, an earlier significant leukocytosis could be found in the control group compared with the LD group. Animal [12] and clinical [10, 11] studies also supported these findings. As is known, CPB activates complement factors, inducing neutrophil and endothelial cell activation, which is believed to be primarily responsible for CPB-related inflammatory response [1]. Our recent study [11] demonstrated the clinical use of a leukocyte-depleting filter could down-regulate the expression of neutrophil CD11b and L-selectin compared with the control group. This down-regulation was probably due to the retention of most of the activated leukocytes by the leukocyte filter [12]. The present study also demonstrated that the LD group had favorably affected postoperative lung function, as was exemplified by better preserved Pao2/FiO2 ratios during the postbypass period. This view was consistent with that of recent several investigators [10, 12], in which the authors demonstrated that leukocyte depletion during and after CPB resulted in better preservation of pulmonary function [10] and to ameliorate free radical–mediated lung injury [12]. One of the concerns regarding leukocyte depletion during cardiac operations is that the simultaneous removal of platelets might affect postoperative hemostasis. In this study, little influence on circulating platelet counts and postoperative hemostasis was observed in patients receiving leukocyte-depleted blood. Our findings appear to be in agreement with those of other investigators [10].

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Time Measurement P-selectin (ng/mL) Control LD ICAM-1 (ng/mL) Control LD IL-8 (pg/mL) Control LD MDA (umol/L) Control LD PECAM-1 (ng/mL) Control LD

ANOVA p Value

Baseline

CPB 30

CPB 60

Reperfusion

CPB Off

2 Hours After CPB

59.6 ⫾ 16.0 69.6 ⫾ 23.1

79.9 ⫾ 27.7 77.5 ⫾ 24.3

94.1 ⫾ 36.4 89.3 ⫾ 33.3

111.9 ⫾ 35.3 98.6 ⫾ 43.5

138.5 ⫾ 41.4 103.9 ⫾ 47.3

182.9 ⫾ 82.2 106.2 ⫾ 47.0

202.2 ⫾ 90.9 75.0 ⫾ 32.5

⬍0.0001 ⬍0.0001

0.0097

⬍0.0001

151.8 ⫾ 27.2 156.8 ⫾ 25.9

164.9 ⫾ 35.4 160.0 ⫾ 24.2

190.6 ⫾ 38.1 159.5 ⫾ 28.4

218.3 ⫾ 47.7 169.1 ⫾ 28.1

234.4 ⫾ 36.7 171.9 ⫾ 30.1

237.8 ⫾ 42.4 162.8 ⫾ 32.8

209.6 ⫾ 39.5 138.8 ⫾ 25.6

⬍0.0001 ⬍0.0001

0.0002

⬍0.0001

5.2 ⫾ 1.5 7.4 ⫾ 1.9

5.9 ⫾ 1.6 8.0 ⫾ 1.2

7.5 ⫾ 2.2 9.9 ⫾ 2.7

10.4 ⫾ 4.3 10.5 ⫾ 3.1

12.5 ⫾ 5.3 10.6 ⫾ 3.1

12.3 ⫾ 5.2 9.3 ⫾ 2.7

11.5 ⫾ 5.2 7.3 ⫾ 2.0

⬍0.0001 ⬍0.0001

0.7385

⬍0.0001

2.26 ⫾ 0.6 2.54 ⫾ 0.8

2.47 ⫾ 0.6 2.57 ⫾ 0.7

2.63 ⫾ 0.7 2.62 ⫾ 0.7

2.69 ⫾ 0.9 2.80 ⫾ 0.8

3.13 ⫾ 1.3 2.89 ⫾ 0.7

3.39 ⫾ 1.4 2.97 ⫾ 0.7

4.04 ⫾ 1.3 2.68 ⫾ 0.6

⬍0.0001 0.0155

0.4369

⬍0.0001

16.2 ⫾ 4.8 20.4 ⫾ 4.0

18.6 ⫾ 2.9 20.3 ⫾ 3.0

20.7 ⫾ 4.0 21.3 ⫾ 3.7

22.6 ⫾ 4.9 20.6 ⫾ 5.2

24.9 ⫾ 4.9 19.0 ⫾ 3.7

26.2 ⫾ 5.1 17.3 ⫾ 3.8

27.0 ⫾ 8.8 16.8 ⫾ 4.0

⬍0.0001 0.0004

0.0158

⬍0.0001

a P Values were tabulated for time effect (within group) by one-way ANOVA. interaction (group/time) effect by two-way ANOVA.

b

24 Hours After CPB

Time

Groupb

Interactionc

a

P Values were tabulated for group effect (between groups) by two-way ANOVA.

CPB ⫽ cardiopulmonary bypass; CPB 30 and CPB 60 ⫽ 30 and 60 minutes after the start of cardiopulmonary bypass; leukocyte-depletion; MDA ⫽ malondialdehyde; PECAM-1 ⫽ platelet-endothelial cell adhesion molecule-1.

ICAM-1 ⫽ intercellular adhesive molecule-1;

c

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Table 3. Time Course of Plasma Concentrations of P-Selectin, ICAM-1, IL-8, MDA, and PECAM-1 During and After CPB in Control Group and LD Group

P Values were tabulated for

IL-8 ⫽ interleukin-8;

LD ⫽

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Fig 2. Time course of plasma concentrations of ICAM-1 during and after cardiopulmonary bypass (CPB) in the control group (open circles, n ⫽ 16) and in the LD group (solid squares, n ⫽ 16). There is a significant difference between control and LD groups (p ⫽ 0.0013 by ANOVA).*Significant difference compared with the LD group, with p values in parentheses. Values are expressed as mean ⫾ SD. (ANOVA ⫽ analysis of variance; CPB 30 and CPB 60 ⫽ 30 and 60 minutes after start of cardiopulmonary bypass; ICAM-1 ⫽ intercellular adhesion molecule-1; LD ⫽ leukocyte depletion.)

Effect of CPB With or Without Leukocyte Filtration on Circulating Adhesion Molecules, Interleukin-8, and Malondialdehyde In this study, we demonstrated that significant increases of plasma levels of P-selectin, ICAM-1, interleukin-8, PECAM-1, and MDA during and after CPB in the control group. Interestingly, a significant decrease of plasma levels of P-selectin, ICAM-1, interleukin-8, PECAM-1, and MDA could be found in the LD group compared with the control group.

onstrates that the plasma level of soluble P-selectin was significant lower in the LD group compared with the control group. The importance of circulating adhesion molecules is still a much debated issue [3, 18]. Nevertheless, Sakamaki and associates [19] demonstrated elevated plasma levels of P-selectin in patients with neutrophilmediated lung injury, especially in those who subsequently died. Thus, increased concentrations of soluble adhesion molecules may considered to be markers of inflammation, endothelial activation or damage [18].

P-Selectin

Intercellular Adhesion Molecule-1

P-selectin (PADGEM,GMP-140,CD62P) is constitutively found in the membranes of Weibel-Palade bodies within endothelial cells and in the ␣-granules of platelets [14]. Ley and coworkers [15] demonstrated that “early” leukocyte rolling is entirely dependent on P-selectin function, that L- and P-selectin synergize to produce rolling in an intermediate time period, and that rolling is largely L-selectin dependent at later time points. Leukocyte rolling was reported to be almost absent in venules of P-selectin gene-deficient mice [16]. Several investigators have shown the reduction of ischemia and reperfusion injury by anti–P-selectin monoclonal antibody in vivo [17]. Our study has demonstrated that plasma concentration of soluble P-selectin showed a continuing increase up to 24 hours after the cessation of CPB in the control group. Similar increases in soluble P-selectin during CPB or after the termination at CPB have been reported in the majority of studies [13, 18]. Interestingly, our study dem-

Intercellular adhesion molecule-1 is constitutively expressed at low levels in unstimulated endothelial cells in the venular system but can be markedly upregulated by cytokine (eg, interleukin-1 or tumor necrosis factor) stimulation [20]. The importance of ICAM-1 in neutrophilmediated tissue injury has been shown by blocking the interaction between CD11/CD18 complex and ICAM-1 or by creating ICAM-1 deficiency, which reduced tissue injury and organ dysfunction after ischemia/reperfusion, and endotoxin challenge in animal models [21, 22]. Our findings indicated that plasma levels of ICAM-1 continued to increase during CPB, reaching a peak 2 hours after the end of CPB in the control group. Similar increases of plasma ICAM-1 levels during and after CPB had been reported in other studies [18, 23]. Some investigators, however, did not find an increase in the plasma levels of ICAM-1 during and after CPB [13]. Heparin coating [24], hypothermia [25], high-dose aprotinin [26], and the use of Fig 3. Time course of plasma concentrations of interleukin-8 during and after CPB in the control group (open circles, n ⫽ 16) and in the LD group (solid squares, n ⫽ 16). There is a significant difference between control and LD groups (p ⬍ 0.0001 by ANOVA). *Significant difference compared with the LD group, with p values in parentheses. Values are expressed as mean ⫾ SD. (ANOVA ⫽ analysis of variance; CPB 30 and CPB 60 ⫽ 30 and 60 minutes after start of cardiopulmonary bypass; LD ⫽ leukocyte depletion.)

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Fig 4. Time course of plasma concentrations of MDA during and after CPB in the control group (open circles, n ⫽ 16) and in the LD group (solid squares, n ⫽ 16). There is a significant difference between control and LD groups (p ⫽ 0.0098 by ANOVA). *Significant difference compared with the LD group, with p values in parentheses. Values are expressed as mean ⫾ SD. (ANOVA ⫽ analysis of variance; CPB 30 and CPB 60 ⫽ 30 and 60 minutes after start of cardiopulmonary bypass; LD ⫽ leukocyte depletion; MDA ⫽ malondialdehyde.)

membrane or bubble oxygenators [27] failed to influence plasma concentration of ICAM-1 during CPB. Interestingly, our studies demostrated that the use of a leukocyte filter significantly decreased the plasma levels of ICAM-1 during and after CPB in the LD group compared with the control group. Recent evidence suggests that the measurement of circulating ICAM-1 levels could be a marker in inflammation or tissue damage [28]. Plasma levels of ICAM-1 have been also reported to be of prognostic value after heart transplantation [29] and in patients with the systemic inflammatory response syndrome [30].

Interleukin-8 Interleukin-8 appears to be the most important endogenous neutrophil chemoattractant. In addition to attracting neutrophils along a chemotactic gradient, endogenous endothelial interleukin-8 also activates these cells, triggering degranulation, increased expression of surface adhesion molecules, and stimulation of the respiratory burst [31], and it is an important regulator of transendothelial neutrophil migration [5]. Treatment of experimental animals with antibodies against interleukin-8 has been shown to improve survival or prevent pulmonary injury in models of sepsis or ischemia/reperfusion injury [32]. A significant systemic increase of interleukin-8 due to extracorporeal circulation has already reported by Kalfin and coworkers [33]. In our study, we have found similar systemic increases of interleukin-8 in the blood of the control group. Interestingly, a consistently lower level of plasma inteleukin-8 was observed during and after CPB in the LD group compared with the control group. Fig 5. Time course of plasma concentrations of PECAM-1 during and after CPB in the control group (open circles, n ⫽ 16) and in the LD group (solid squares, n ⫽ 16). There is a significant difference between control and LD groups (p ⫽ 0.0017 by ANOVA). *Significant difference compared with the LD group, with p values in parentheses. Values are expressed as mean ⫾ SD. (ANOVA ⫽ analysis of variance; CPB 30 and CPB 60 ⫽ 30 and 60 minutes after start of cardiopulmonary bypass; LD ⫽ leukocyte depletion; PECAM-1 ⫽ platelet-endothelial cell adhesion molecule-1.)

Malondialdehyde Oxygen free radicals or reactive oxygen species are generated at sites of inflammation and injury. Malondialdehyde, a by-product of lipid peroxidation, was measured to determine the antioxidant reserve capacity. The more MDA produced, the greater was depletion of tissue antioxidants secondary to oxygen free radical formation during oxygenation [34]. Our findings indicated that MDA production continued to increase over the observation period in the control group. Similar observations of increased MDA production during and after CPB have been reported in the majority of studies [35]. Interestingly, the level of MDA production was significantly lower in the LD group than in the control group during and after CPB. That the use of a leukocyte filter significantly reduced MDA production during and after CPB has been also reported in other studies [12, 34].

Platelet-Endothelial Cell Adhesion Molecule-1 Platelet-endothelial cell adhesion molecule-1, also known as CD31 or endoCAM, is expressed in large amounts on endothelial cells at intercellular junctions and to a lesser extent on platelets and most leukocytes [7]. There is good evidence to suggest that PECAM-1 is a key participant in the adhesion cascade leading to extravasation of leukocytes during the inflammatory process. Our finding of markedly increased plasma PECAM-1 concentration during and after CPB was seen in the control group. However, PECAM-1 was not significantly altered during CPB, and it underwent a progressive decline thereafter in the LD group. In vitro experimental

preparations [36], activated neutrophils exhibited a rapid shedding of PECAM-1. Although the pathophysiologic significance of this rapid shedding remains unclear, it has been suggested that the circulating adhesion molecules may serve as markers of endothelial activation and vascular inflammation [37]. Thus, strategies to inhibit the cleavage of PECAM-1 may have therapeutic relevance to ameliorate the inflammatory response prompted by CPB.

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6. 7. 8.

Clinical Implications Numerous clinical and experimental studies have demonstrated a complex systemic inflammatory response in operations involving CPB. The main reason for this generalized inflammatory reaction is CPB circuitinduced contact activation and ischemia/reperfusion injury. Experimental observations have demonstrated that these events have profound effects on activating endothelial cells to recruit neutrophils from the circulation. Therefore, understanding the signals that result in endothelial activation and transendothelial neutrophil transmigration is important in assessing potential therapy to block this response. Several principal endothelial cell activation proteins (eg, P-selectin, ICAM-1, interleukin-8, PECAM-1) are expressed after CPB. The protein Pselectin mediates the initial rolling of the leukocyte through low-affinity binding, ICAM-1 forms the firm bond, and interleukin-8 and PECAM-1 activate neutrophils and facilitate transendothelial cell migration to the underlying tissue, where the neutrophil does its damage through oxygen-derived free radicals and proteolytic enzymes [35]. Our studies show a significant deterioration of postoperative lung gas exchange function and significant increases of plasma levels of P-selectin, ICAM-1, interleukin-8, PECAM-1, and MDA during and after CPB in the control group. Conversely, a significant decrease of plasma levels of P-selectin, ICAM-1, interleukin-8, PECAM-1, and MDA and better preservation of pulmonary function could be found in the LD group compared with the control group. Thus, our results demonstrate the rationale of therapeutic strategies using a leukocyte filter in patients undergoing cardiac surgery, targeted at attenuating the endothelial-mediated component of CPB-induced inflammatory response through the mechanism of reducing endothelial activation and subsequent neutrophil transmigration.

9.

10.

11.

12. 13.

14.

15. 16. 17.

18. 19. 20.

This study was supported by grants from the National Science Council of the Republic of China (NSC 89 –2314-B037– 095).

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INVITED COMMENTARY This is a very interesting piece of clinical research, focusing on the continued investigation of the clinical efficacy and efficiency of leukocyte filtration technology, applied during routine cardiopulmonary bypass (CPB). The authors focus on molecules thought to be involved in neutrphil-endothelial interaction, in particular the study focused on molecules known to be involved in tethering (P-Selectin), firm adherence (ICAM-1), transmigration of neutrophils (IL-8, PECAM-1), and degranualtion (MDA). The authors found that there was a general upregulation in all of these factors associated with CPB, but there was a substantially less effect in those patients exposed to leukocyte depletion. The authors conclude that the application of leukocyte depletion filtration technology attenuates the very important neutrophil-endothelial interaction, known to be central to the inflammatory response to CPB. The authors are to be congratulated for a performing a very good clinical study, albeit on a small group of patients. Despite the high quality of this research, this study does little to calm the leukocyte depletion controversy. The popular understanding of the major benefit of leukocyte depletion is that this technique removes activated neutrophils from the systemic circulation during CPB, and that this is a desirable characteristic is not in doubt. However, the use of leukocyte filtration remains controversial when viewed from the standpoint of the molecular basis of neutrophil-endothelial interaction, but less controversial in the context of clinical outcome studies. To illustrate this, using MDA as a marker of lipid peroxidation in a much larger study of 80 patients, Scholz and coworkers [1] found that there was no difference between filter and nonfilter groups, indeed they found that there were greater levels of PMNE and MPO in peripheral venous blood of patients undergoing CPB with the leukocyte filters, but surmised that this was due © 2004 by The Society of Thoracic Surgeons Published by Elsevier Inc

to degranulation of neutrophils captured by the filters. In an earlier study, Baksaas and colleagues [2] also found no difference between leukocyte-filtered groups when compared with controls in terms of markers of inflammation (MPO, IL-6, complement), and that the filters produced no significant drop in white cell or neutrophil count. Clinical outcome studies tend to tell a somewhat different story. Many clinical outcome studies have shown that the application of leukocyte depleting technology, used in the arterial line of the perfusion circuit, is associated with considerable clinical advantages. Olivencia-Yurvati and associates [3], for example, found that leukocyte depletion was associated with a reduction in pulmonary vascular pressure and an improvement in postoperative pulmonary function. These findings were further enhanced by reduction in intensive care units (ICU) and hospital stay in patients in which leukocyte depletion was used. Our own experience with leukocyte depletion reflects the experiences of others investigating this field. In one brief clinical study (see Stefanou and coworkers [4]), we found absolutely no difference between filtered and unfiltered groups in terms of molecular markers of inflammation (CD11b, MPO, lactoferrin), but we found a significant difference in terms of blood and crystalloid transfusion requirements, and ICU stay, in favor of leukocyte filtration. Indeed our experience with leukocyte depletion indicates that the most significant advantage is to be gained by positioning the leukocyte filter not in the arterial line, but, in the cardioplegia line of the bypass circuit (Samankatiwat and associates [5]). In summary, I salute the elegant systematic approach to clinical study demonstrated by the authors, but I believe that this study does little to dispel the controversy surrounding the use of leukocyte filters in CPB. However, it may add to the weight of evidence in favor of leukocyte 0003-4975/04/$30.00 doi:10.1016/j.athoracsur.2004.04.025