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Ann Thorac Surg 2002;74:1173-1179
© 2002 The Society of Thoracic Surgeons

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Original article: cardiovascular

Increased neutrophil priming and sensitization before commencing cardiopulmonary bypass in cardiac surgical patients

^a Department of Cardiothoracic Surgery, University of Groningen, Groningen, The Netherlands
^b Department of Biomedical Engineering, University of Groningen, Groningen, The Netherlands
^c HaemoProbe BV, Groningen, The Netherlands
^d Pfizer Global Research and Development, Kent, United Kingdom

Accepted for publication May 13, 2002.

* Address reprint requests to Dr Gu, Department of Cardiothoracic Surgery, University Hospital Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands
e-mail: y.j.gu{at}med.rug.nl

	Abstract

Top Abstract Introduction Patients and methods Results Comment Acknowledgments References

 
Background. Neutrophil activation is implicated in postoperative complications in patients having cardiac surgery with cardiopulmonary bypass (CPB). This study was designed to determine the temporal fluctuations in the primability of neutrophils in the preoperative, intraoperative, and postoperative periods of CPB, and specifically whether CPB was a primary cause leading to increased neutrophil priming and elastase release.

Methods. Twenty patients undergoing multiple coronary bypass grafting, valve replacement, or both of these procedures were included in this study. Blood samples were taken 1 day before the operation and at several time points during and after the operation. For each sample, blood was divided in vitro into four subgroups: control without priming, priming alone with cytochalasin B (CytoB), priming plus stimulation with platelet-activating factor (PAF), and priming plus stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP). The elastase concentration of all these samples was determined using the enzyme immunoassay.

Results. Compared with the controls, CytoB priming increased release of elastase more than 10-fold before CPB, 1.6-fold during CPB, and 1.5-fold at the end of CPB. Further stimulation with PAF or fMLP showed greater increase of elastase than priming alone, with peak values in both found before CPB. This increased neutrophil primability prior to CPB did not differ significantly among patients who had different preoperative disease profiles.

Conclusions. Our data suggest that neutrophil priming occurs early before commencing CPB in cardiac surgical patients, and that CPB is not the primary primer. Anesthesia, surgical trauma, and other events may have been involved in neutrophil priming and sensitization before CPB, which warrants further investigation.

	Introduction

Top Abstract Introduction Patients and methods Results Comment Acknowledgments References

 
Cardiopulmonary bypass (CPB) is known to induce an over-stimulated inflammatory response in cardiac surgical patients because of exposure of blood with a large area of artificial surface in the extracorporeal circuit [1–4]. This systemic inflammatory response is largely mediated by the activation and degranulation of neutrophils and the subsequent release of various inflammatory mediators [5, 6], contributing to increased postoperative morbidity and mortality and to compromised organ function [7, 8].

Among various release products from neutrophil degranulation, elastase is considered one of the most powerful and injurious enzymes because of its strong biological effects [9]. Elastase has the capacity to degrade a wide variety of extracellular matrix proteins, including elastin, fibronectin, several types of collagen, and proteoglycans, and therefore may cause severe tissue damage and organ dysfunction, especially in the lungs [10]. Increased levels of elastase in peripheral blood has been repeatedly reported in literature, indicating the severity of leukocyte activation and inflammatory response in cardiac surgical patients [11–15].

It has been shown that, before activation, neutrophils undergo a preactivation process called "priming," which is defined as an amplification of cell response to a secondary stimulus after being exposed to a primary activating agent or event [16, 17]. Enhanced neutrophil response after priming is often the cause of increased generation of superoxide anion and release of elastase, contributing to the development of postoperative multiple organ dysfunction in cardiac surgical patients [18, 19]. Although it has been suspected that CPB is the main cause inducing neutrophil priming [17, 19], other intraoperative factors, such as anesthesia and surgical trauma, occurring before the start of CPB, may be involved in early neutrophil priming before CPB [20–25].

This study was undertaken to determine (1) the time periods during or after cardiac operation that neutrophils are most prone to priming and to releasing elastase, and (2) whether or not CPB is the primary cause leading to increased neutrophil priming.

	Patients and methods

Top Abstract Introduction Patients and methods Results Comment Acknowledgments References

 
Patients
Twenty patients undergoing a coronary artery bypass grafting, a heart valve replacement, or a combined procedure were included in this study. Patients’ demographic data, including their age, gender, as well as body surface area are listed in Table 1. The study protocol was approved by the Medical Ethics Committee of the University Hospital in Groningen, and informed consent was obtained from all patients.

Cardiopulmonary bypass
The extracorporeal circuit consisted of roller pumps (Stöckert Instrumentation, Munich, Germany) and a membrane oxygenator, and was primed with 1,500 ml ringers lactate solution plus 500 ml 10% HES solution (Fresenius, Bad Homburg, Germany). Myocardial preservation during aortic cross-clamping was provided by 1 L of St. Thomas cardioplegia solution (4°C) infused into the aortic root. A standard hemodilution technique was used including dilution of the circulating blood volume to a hematocrit of approximately 20% to 25%. During bypass, the pump flow was set at 2.4 L/m2/min under moderate hypothermia. Mean arterial pressure was maintained at 50 to 60 mm Hg during bypass. Anticoagulation during bypass was monitored by the activated clotting time (ACT, Hemochron 800, International Technidyne Corp, Edison, NJ). Additional heparin was administered if the ACT was shorter than 400 seconds. Heparin was neutralized by means of protamine chloride infusion (3 mg/kg) after the completion of cardiopulmonary bypass.

Blood sampling
Blood samples were taken at the following time points: 1 day prior to operation as a baseline (T1), 3 minutes after heparization prior to the start of cardiopulmonary bypass (T2), 30 minutes after commencement of bypass (T3), at the end of bypass (T4), 2 hours after arriving in the intensive care unit (T5), and postoperatively on the first (T6), second (T7), and seventh (T8) day. Blood was drawn either from the indwelling central venous catheter or by venous puncture, except for samples T3 and T4, which were taken directly from the venous side of the bypass circuit. After sampling, blood was immediately mixed and anticoagulated with 3.06% sodium citrate solution in a blood to citrate ratio of 9:1. Cell count was measured by an electronic cell counter (Cell-Dyn 610, Sequoia Turner, Mountain View, CA).

In vitro study protocol
Reagents
Cytochalasin B (CytoB) from Helminthosporium dematioideum was obtained from Sigma (St. Louis, MO). A stock solution of 10 mg/mL was prepared in dimethylsulfoxide (DMSO, Sigma). A final concentration of CytoB in blood was 10 µg/mL. Octadecyl-platelet activating factor (PAF), obtained from Bachem AG (Bubendorf, Switzerland), was dissolved to a concentration of 20 mM in phosphate buffer solution (PBS, Sigma) containing 0.35% (w/v) of bovine serum albumin (BSA, fraction V, Sigma). N-formyl-methionyl-leucyl-phenylalanine (fMLP), obtained from Sigma, was dissolved at a concentration of 10 mM in DMSO. Aliquots of these solutions were stored at -20°C for further use. Prior to blood processing, cytochalasin B was diluted to a concentration of 0.5 mg/mL with PBS (final concentration in blood 10 µg/mL); PAF was diluted to a concentration of 0.5 mM with PBS containing 0.35% (w/v) BSA (final concentration 10 µM); and fMLP was diluted to a concentration of 5 µM with PBS (final concentration 100 nM).

Neutrophil priming and stimulation
Each blood sample taken from patients was divided into four subgroups: group 1, control without priming and stimulation; group 2, priming alone with cytochalasin B; group 3, priming plus stimulation with PAF; and group 4, priming plus stimulation with fMLP. Within 30 minutes of sampling, samples of whole blood were deposited in eight tubes (480 µL per tube), resulting in 4 subgroups in duplicate. In group 1, blood from the first two tubes was centrifuged immediately at 13,000 g for 1 minute in a microcentrifuge and the supernatant was stored at -80°C for further determination of elastase. The concentration measured in group 1 was also regarded as the circulating elastase level at the time of sampling. The remaining six tubes for groups 2 (CytoB), group 3 (CytoB + PAF), and group 4 (CytoB + fMLP) were mixed with 10 µL of cytochalasin B at 37°C for 5 minutes (neutrophil priming). Thereafter, 10 µL of PBS, PAF or fMLP solution, respectively, was added to each of the two tubes and incubated at 37°C for another 5 minutes. After these incubation episodes, the samples were cooled down to 0°C on melting ice for 5 minutes. Finally, these six tubes were centrifuged at 13,000 g for 1 minute and the supernatant was stored at -80°C for further elastase determination.

Measurement of elastase
The concentration of polymorphonuclear (PMN) elastase was determined in its complex with alpha-1-proteinase inhibitor by a commercially available 96-well enzyme immunoassay (Milenia PMN Elastase Kit, Diagnostic Products Corporation, Breda, The Netherlands). These assay kits contained all reagents required to perform elastase measurements. All kits used in this study were from the same lot and had an identical expiration date. The assay was performed according to the manufacturer’s instruction. The first antibody, coated on the surface of the wells, is a specific polyclonal chicken-egg-yolk anti-PMN elastase antibody which captures neutrophil elastase/alpha-1-proteinase inhibitor complex by binding neutrophil elastase. In the second stage, a polyclonal rabbit antibody, which is conjugated with horseradish peroxidase, binds to the captured alpha-1-proteinase inhibitor. Unreacted material was removed by washing, and the amount of conjugate in the well was then measured with a chromogenic substrate (3,3'',5,5''-tetramethyl-benzidine). Color development is terminated after 20 minutes. Absorbance was read at 450 nm, with 550 nm as reference, with a Bio-Tek PowerWave 200 reader (Bio-Tek Instruments, Winooski, VT) operated with KC4 software (Version 2.5, Bio-Tek Instruments).

Statistics
Patient demographic data and postoperative observations are expressed as mean ± standard deviation, whereas data for cell counts and elastase are expressed as mean and the standard error of the mean, except when otherwise noted. Before performing statistics, all data were analyzed by the Komogorov-Smirnov goodness-of-fit test to check data distribution. Analysis of variance with repeated measures was performed to examine the difference between the different time points, whereas the two-way analysis of variance was performed to examine the difference of neutrophil priming between different patient subgroups. For all these tests, a p-value <0.05 was considered statistically significant.

	Results

Top Abstract Introduction Patients and methods Results Comment Acknowledgments References

 
Patient clinical course
All operations went without incident with an average CPB duration of 108 ± 40 minutes. One patient died during the night of the operative day because of circulatory collapse. There was 1 coronary artery bypass graft (CABG) patient who stayed in the intensive care unit (ICU) for 12 days due to postoperative renal failure and another aortic valve replacement (AVR) patient had a hospital stay of 69 days due to postoperative wound infection. Postoperative fluid balance, urine output, chest drainage, ventilation support, and duration of ICU stay in hospital are summarized in Table 2.

View larger version (13K): [in this window] [in a new window]   Fig 1. Circulating levels of leukocytes, granulocytes, and elastase in venous samples taken from 20 cardiac surgical patients with cardiopulmonary bypass (CPB). Data are expressed as mean ± standard error. *p < 0.05 and **p < 0.01 compared with T1. (T1 = before operation; T2 = prior to CPB; T3 = 30 min on CPB; T4 = end of CPB; T5 = 2 h arriving intensive unit; T6 = postoperative day 1; T7 = day 2; T8 = day 7.)

View larger version (11K): [in this window] [in a new window]   Fig 2. The primability of neutrophils in vitro stimulated by cytochalasin B (CytoB) before, during, and after cardiac operations with cardiopulmonary bypass (CPB) (n = 20). Data are comparisons (ratio) of elastase concentrations between the CytoB-stimulated and unstimulated samples and are expressed as mean ± standard error based on comparison of each individual patient. (T1 = before operation; T2 = prior to CPB; T3 = 30 min on CPB; T4 = end of CPB; T5 = 2 h arriving intensive unit; T6 = postoperative day 1; T7 = day 2; T8 = day 7.)

View larger version (15K): [in this window] [in a new window]   Fig 3. (A) Platelet-activating factor-induced release of elastase in whole blood primed with cytochalasin B. (B) N-formyl-methionyl-leucyl-phenylalanine-induced release of elastase in whole blood primed with cytochalasin B. Elastase concentrations are corrected for the sample granulocyte count (ng/mL per million granulocytes). Blood samples were taken from venous samples taken before, during, and after cardiac operations with cardiopulmonary bypass (CPB). Data are expressed as mean ± standard error. *p < 0.05 and **p < 0.01 compared with T1. (T1 = before operation; T2 = prior to CPB; T3 = 30 min on CPB; T4 = end of CPB; T5 = 2 h arriving intensive unit; T6 = postoperative day 1; T7 = day 2; T8 = day 7.)

Neutrophil primability in different subgroup patients
In the baseline samples taken 1 day before operation, the primability of neutrophils did not differ significantly from patients who had different underlying diseases and different preoperative profiles, such as preexisting myocardial infarction and chronic obstructive pulmonary diseases. Prior to CPB, however, all samples from these patient subgroups had a significant increase of elastase release when they were primed in vitro by cytochalasin B. The average of elastase concentration increased from 46 ± 12 ng/mL to 546 ± 120 ng/mL in patients with myocardial infarction (n = 5), from 49 ± 11 ng/mL to 433 ± 83 ng/mL in patients with chronic obstructive pulmonary diseases (n = 6), from 62 ± 13 ng/mL to 367 ± 83 ng/mL in valve replacement patients (n = 9), and from 53 ± 10 ng/mL to 469 ± 70 ng/mL in CABG patients (n = 11). There was no significant difference among these subgroup patients with regard to their neutrophil primability.

	Comment

Top Abstract Introduction Patients and methods Results Comment Acknowledgments References

 
Neutrophil activation with increased release of elastase has been extensively investigated in cardiac surgical patients during the past decades [15–19], but the discussion on etiology was largely focused on the role of CPB because of the contact of blood with the artificial surfaces in the extracorporeal circuit. In the current study, we demonstrated that the primability of neutrophils and its subsequent release of elastase increased and peaked before the commencement of CPB, when neutrophils were stimulated in vitro by cytochalasin B, a known primer to neutrophils for the release of azurophil enzymes [26]. Contrary to the statement that has informed the literature [17], the primability of neutrophil was relatively low during and after CPB as compared with the time period before CPB, suggesting that neutrophils are "preactivated" before blood comes into contact with the CPB circuit.

Several factors and events may have been involved in the early neutrophil priming and activation before CPB, including anesthesia, surgical trauma, cannulation, and the insertion of indwelling catheters. In general, anesthetic drugs are known to inhibit neutrophil function instead of activating neutrophils. Sufentanil and midazolam, the two main agents applied in our clinic for anesthetic induction, are known to have little effect on neutrophil function [21, 22]. However, one recent report has stated that although midazolam alone inhibited neutrophil activation, it may promote activation in the presence of other chemoattractant agents such as lipopolysaccharide [23]. Furthermore, surgical trauma with extensive tissue injury may contribute to neutrophil priming and activation [24]. We have reported recently that the complement system was activated by the surgical incision in patients undergoing coronary bypass surgery without CPB [25]. It is thus possible that released anaphylatoxins during complement activation may subsequently result in neutrophil activation [1, 2, 5, 8].

In this study, dexamethason was administered after anesthetic induction in every patient as part of the routine patient care protocol in our clinic. As is known from the literature, corticosteroids may inhibit elastase release from neutrophils that have been exposed to stimulating agents [27]. This is a possible explanation for the inhibition of our in vivo elastase release, especially in the time period before commencing CPB, as seen from the relatively low elastase concentration in comparison with the baseline concentration. The inhibitory effect of corticosteroids on neutrophils may also be reflected in the relatively low elastase level observed in our patients in comparison with other reported patients who had received no corticosteroids [14, 15]. Whatever the inhibitory effect of corticosteroids on neutrophils, our in vitro data nonetheless revealed a high sensitivity of neutrophils before CPB, suggesting that dexamethason administered in a concentration of 1 mg/kg is not sufficient to stabilize neutrophils.

Heparin has been described as a "primer" in inducing subsequent granulocyte activation, and it was especially so in the clinical dose between 3 to 5 IU/mL that is usually administered before the start of CPB [28]. These early data may largely explain our observation of increased neutrophil primability before CPB, although methodologically there are considerable differences between this study and the present one. In the former study, heparin was added in vitro to isolated granulocytes, and the release of myeloperoxidase was used as a marker of neutrophil activation. In our study, however, heparin was administered in vivo in the systemic circulation and elastase was selected as a marker. With regard to the direct effect on elastase, heparin is traditionally understood as an inhibitor, rather than a stimulator [29]. However, heparin was also found to strongly decrease the rate of inhibition of neutrophil elastase by alpha 1-proteinase inhibitor [30]. Therefore, the overall effect of heparin on in vivo elastase release remains unclear.

Recently, CPB has been described as a "primer" acting synergistically with the inflammatory stimulus in cardiac surgical patients [17]. In a study reported by Schwartz and coworkers [17], neutrophils, when primed in vitro with PAF and activated with fMLP, were found to generate significantly more superoxide products 6 hours after CPB than the baseline production before CPB. This finding, however, was based on the comparison of two samples lasting for more than 6 hours and may have included a mixture of coexisting factors, such as anesthesia, surgical trauma, heparin effect, blood-material interaction, and temperature changes during CPB, all of which could contribute to neutrophil priming and activation. In our study, however, by comparing directly the stimulated (in vitro) and unstimulated (in vivo) elastase release from the same blood sample, we found that the increased neutrophil sensitivity peaked before the start of CPB, which suggests that the massive contact of blood with the CPB circuit is, at the very least, not the primary primer or stimulus for neutrophil activation.

One limitation of this study was that we have used citrate as the anticoagulant for blood sampling. Compared with heparin, the in vitro neutrophil priming effect of different agonists was somewhat reduced as shown by our additional data. Therefore, the overall stimulating effect of cytochalasin B, PAF, and fMLP on neutrophils may have been underestimated by our experiments. However, the activation pattern of these stimulating agents on neutrophils remained similar by the two anticoagulants. Another limitation of this study was that our in vitro study protocol did not include subgroups of neutrophil stimulation without cytochalasin B priming, such as direct stimulation of neutrophils by PAF. This was mainly due to the fear that the direct stimulating effect of PAF on neutrophil was weak if the elastase was used as a marker of neutrophil degranulation [31]. On the contrary, cytochalasin B is an ideal stimulus for elastase release because it disrupts the neutrophil skeleton and thus stimulates cell degranulation [26]. In the presence of cytochalasin B, PAF and fMLP can greatly increase elastase release from neutrophils [32]. Given that our study was performed under these circumstances, our results may specifically indicate that neutrophils are most susceptible to cytochalasin B priming prior to CPB when the priming is measured by elastase release stimulated by PAF and fMLP.

In conclusion, in this study, we have demonstrated that neutrophil priming and sensitization increased and peaked before commencing CPB, rather than during and after CPB. It is thus speculated that anesthesia, surgical trauma, and other events may have been involved in neutrophil priming and sensitization before CPB, which warrant further investigation.

	Acknowledgments

Top Abstract Introduction Patients and methods Results Comment Acknowledgments References

 
We acknowledge the funding of this study by Pfizer Global Research and Development, Kent, United Kingdom; the performance of elastase assays by HaemoProbe BV, Groningen, The Netherlands; as well as the help of Dr Donald Miller with the language of the manuscript.

	References

Top Abstract Introduction Patients and methods Results Comment Acknowledgments References

 

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Y. John Gu Tjark Ebels Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gu, Y. J. Articles by van Oeveren, W. Search for Related Content PubMed PubMed Citation Articles by Gu, Y. J. Articles by van Oeveren, W. Related Collections Extracorporeal circulation