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Ann Thorac Surg 2001;71:233-237
© 2001 The Society of Thoracic Surgeons

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Original article: cardiovascular

ß-Chemokine secretion patterns in relation to clinical course and outcome in children after cardiopulmonary bypass: continuing the search to abrogate systemic inflammatory response

^a Department of Pediatric Intensive Care, Chaim Sheba Medical Center, Tel Hashomer, Israel
^b Department of Pediatric Intensive Care, Schneider Medical Center, Petach Tikvah, Israel
^c Tel-Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

Accepted for publication May 5, 2000.

Address reprint requests to Dr Paret, Department of Pediatric Intensive Care, Chaim Sheba Medical Center, Tel Hashomer 52621, Israel
e-mail: gparet{at}post.tau.ac.il

	Abstract

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Background. Surgery involving cardiopulmonary bypass (CPB) is frequently accompanied by a systemic inflammatory response partly triggered by neutrophils and monocyte-macrophages. Certain cytokines that are powerful leukocyte-chemotactic factors have recently been characterized and shown to be important in evoking inflammatory responses: monocyte chemoattractant protein-1 (MCP-1) has monocyte-macrophage chemotactic activity, and regulated-upon-activation normal T-cell expressed and secreted (RANTES) has a potent chemoattractant activity for mononuclear phagocytes. This prospective cohort study investigated possible roles of these chemokines in the inflammatory response to CPB and relationships between the changes in chemokine levels and the clinical course and outcome.

Methods. Systemic blood of 16 children undergoing CPB was collected after induction of anesthesia (base line); at 15 minutes after bypass onset; at CPB cessation; and at 1, 2, 4, 8, 12, and 24 hours afterward to measure MCP-1 and RANTES.

Results. The significant changes of plasma ß chemokine levels following CPB were associated with patient characteristics, operative variables, and postoperative course. Cardiopulmonary bypass of more than 2 hours, longer surgical times, inotropic support, and reoperation were associated with higher MCP-1 levels and lower RANTES levels.

Conclusions. Our results suggest a relation between CPB-induced mediators and clinical effects, implying pathogenic roles for chemokines following CPB. These molecules should be considered as possible targets for therapeutic intervention.

	Introduction

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
It is well documented that cardiopulmonary bypass (CPB) is characteristically associated with a systemic inflammatory response, which is initiated by the synthesis of various cytokines, including tumor necrosis factor (TNF), interleukin-1, interleukin-6, and interleukin-8 [1, 2]. Inflammatory cytokines, endothelial activation, and endothelial–leukocyte interactions have been reported to play an important role in the induction of systemic inflammatory response syndrome, causing large fluid shifts, coagulation disturbances, and increased concentrations of catecholamines and stress hormones [3, 4]. Recently, the chemokines, a group of cytokines with chemotactic and activating effects on leukocytes, have been identified as important mediators in both acute and chronic human inflammatory disorders [5]. The chemokines, notably interleukin-8, induce neutrophil chemotaxis, whereas the ß chemokines are chemotactic for monocytes and T-cell subsets. Among the ß chemokines, monocyte chemoattractant protein (MCP-1) and regulated-upon-activation normal T-cell expressed and secreted (RANTES) attract and activate monocyte-macrophages and polymorphonuclear neutrophils.

Open heart surgery with CPB is associated with an increased inflammatory response inculpated for the high incidence of poor outcome after CPB [1, 4]. The purposes of the current study were to characterize the pattern of ß chemokine secretion in the acute postoperative period in children undergoing open heart surgery with CPB and to identify the role of chemokines in this deleterious process. We also examined whether there is a relationship between the changes in chemokine levels and the children’s clinical course and outcome.

	Material and methods

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Patients
The patients recruited for this study were 16 infants and children (10 boys and 6 girls; mean age 6.3 years, range 1.5 to 16.8 years) who were candidates for open heart surgery at the Chaim Sheba Medical Center. Entry criteria included infants and children who were to undergo open heart surgery involving the use of deep hypothermic circulatory arrest or conventional CPB techniques and in whom there were no anticipated severe residual defects after repair and no known preexisting causes of vascular injury, such as recent cardiac arrest, sepsis, history of vasculitis, or recent severe hypotension. Preoperative informed consent was obtained from the parents.

Intraoperative management
Bentley membrane oxygenators primed with blood and lactated Ringer’s solution were used for all patients. Induction and maintenance of anesthesia were carried out in a standard manner, consisting of weight-related doses of fentanyl, midazolam, and pancuronium bromide. Five minutes before CPB, 300 IU/kg bovine heparin was administered to achieve anticoagulation. A roller pump and membrane oxygenator were used for bypass. The extracorporeal perfusate was primed with dextrose 5% in addition to mannitol, aprotinin, furosemide, heparin, HCO₃, CaCl, and methylprednisolone 0.3 mg/kg. The activated clotting time was maintained between 400 to 500 seconds with a Hemochron system (International Teen 400 to 500; Techydyne Corp, Edison, NJ).

Surface cooling was instituted with a low ambient room temperature, a cooling mattress, and ice packs to the head. Cardiopulmonary bypass and core cooling were instituted until core temperature was reduced to 25.7 ± 2.9°C. The aorta was cross-clamped, and cold (4°C) crystalloid antegrade cardioplegia was infused through the aortic root. At the end of the procedure, the cross-clamp was removed, and the patient was rewarmed while remaining under conditions of bypass. No modified ultrafiltration or leukocyte depletion techniques were employed for any of the patients throughout the study period. The heparin administered at the conclusion of surgery was neutralized with protamine sulfate in a 1:1 ratio.

Collection of samples
Serial blood samples were collected in ethylenediaminetetraacetic acid–containing tubes from the arterial line of the patient after induction of anesthesia (base line); at 15 minutes after the onset of CPB; at the cessation of CPB; and at 1, 2, 4, 8, 12, and 24 hours after the cessation of bypass while the child was still in the intensive care unit. Plasma was recovered immediately from these samples and was aliquoted and frozen at -70°C until use. Circulating chemokine levels were measured with a sandwich enzyme-linked immunosorbent assay (ELISA) technique (Endogen, Woburn, MA). The assay was performed according to the manufacturer’s instructions, and all samples were analyzed at a dilution resulting in concentrations within the range of the standard curve. The minimum detectable concentration of MCP-1 and RANTES was 10 pg/mL and 2 pg/mL, respectively. All results from the ELISA measurements represent the means from duplicate measurements.

Selected patient data
Demographic data, medical history, and medical variables were recorded preoperatively. Postoperative data, including ventilator status, neutrophil count, and blood gas variables, were collected daily while the patients were in the intensive care unit and at the time of discharge from the ward. Each patient’s severity of illness was assessed postoperatively using the Pediatric Risk of Mortality Scoring system (PRISM). This score is based on the observation that physiologic dysfunction in the context of intensive care is the main predictor of mortality risk. The score has been shown to be an excellent predictor of mortality in pediatric intensive care units. To identify the possible association between chemokine concentration and the patient’s clinical status and morbidity, we correlated surgical time, duration of CPB, duration of aortic cross-clamping (cardiac ischemia), PRISM score, inotropic support, use of antiinflammatory drugs, and confirmed sepsis with chemokine concentrations.

Statistical analysis
All measurements are expressed as mean values ± standard error of the mean. Analysis of variance for repeated measures was applied for the comparison of values at the various times with the basal levels. Comparison of measured data between groups was made using the two-sampled Student’s t test. A p value of less than 0.05 was considered significant.

	Results

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
The variety of the patients’ diagnoses and surgical procedures reflected the heterogeneity of congenital heart disease routinely seen at our center (Table 1). Patient characteristics, types of surgical repair, and intraoperative data from their CPB procedures are summarized in Table 1.

View larger version (25K): [in this window] [in a new window]   Fig 1. Changes in the median circulating concentration of monocyte chemoattractant protein-1 (MCP-1; upper panel) and regulated-upon-activation normal T-cell expressed and secreted (RANTES; lower panel) at various times before, during, and after cardiopulmonary bypass. Results are expressed as mean ± standard error of mean. • Significantly different from the preceding time period (p < 0.03). Significantly different versus induction (p < 0.040). * Significantly different versus induction (p < 0.002).

View larger version (37K): [in this window] [in a new window]   Fig 2. Changes in the median circulating concentration of monocyte chemoattractant protein-1 (MCP-1; upper panel) and regulated-upon-activation normal T-cell expressed and secreted (RANTES; lower panel) versus time in children undergoing cardiopulmonary bypass: children requiring surgical time more than 200 minutes (n = 7; black bars) versus children requiring surgical time less than 200 minutes (n = 9; open bars). Results are mean ± standard error of mean. * p less than 0.02.

View larger version (17K): [in this window] [in a new window]   Fig 3. Changes in the median circulating concentration of monocyte chemoattractant protein-1 (MCP-1) with time in children undergoing cardiopulmonary bypass: children requiring inotropic support (n = 7; black bars) versus children without inotropic support (n = 9; open bars). Results are mean ± standard error of mean. * p less than 0.04.

View larger version (32K): [in this window] [in a new window]   Fig 4. Changes in the median circulating concentration of monocyte chemoattractant protein-1 (MCP-1; upper panel) and regulated-upon-activation normal T-cell expressed and secreted (RANTES; lower panel) with time in children undergoing cardiopulmonary bypass: children reoperated (n = 4; black bars) versus children not reoperated (n = 12; open bars). Results are mean ± standard error of mean. * p less than 0.035.

	Comment

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

Several reports have shown that dramatic changes of serum cytokine levels occur during and after CPB in both children and adults [2–4, 6]. An increased inflammatory response due to elevated sequestration of inflammatory mediators is believed to be responsible for the high incidence of poor outcome following bypass [4]. In particular, interleukin-8, a chemokine that induces neutrophil activation and migration into the peripheral tissue, has been considered responsible for some of the negative consequences of CPB in children [7–11].

Two categories of molecules direct leukocyte migration into inflammatory sites—adhesion molecules and chemoattractants. Among the chemoattractants, chemokines have recently attracted particular interest because of their potential role in pathogenic inflammation [5]. Chemokines selectively attract leukocyte subsets: some chemokines act specifically on neutrophils or eosinophils, and yet others on monocytes or T-cells. Chemokines appear to act in at least two ways: first, through direct chemoattraction, and second, by activating leukocyte integrins to bind to their adhesion receptors on endothelial cells. MCP-1 activates monocytes and T lymphocytes and is a potent activator of exocytosis in basophils and mast cells. MCP-1 production has been demonstrated in vascular endothelium, tissue monocytes, and macrophages. RANTES attracts and activates monocyte-macrophages and polymorphonuclear neutrophils.

Because of their number and diversity, chemokines present a picture of bewildering complexity. Only recently have specific functions of chemokines in physiology and disease begun to be elucidated. A number of studies have examined the expression of various cytokines following CPB [1, 2, 4]; however, there is little information on chemokine regulation after CPB [7, 8, 12] and none on how this regulation might help abrogate systemic post-CPB inflammation.

Our data show that MCP-1 appeared in the systemic circulation in the early post-CPB phase and reached a peak 1 hour after bypass. The MCP-1 levels correlated with the children’s clinical outcomes. Elevated MCP-1 levels have been described in adults after CPB [12–14]. Kawahito and colleagues [12] and Ernofsson and colleagues [13] reported that MCP-1 appeared in the systemic circulation in the early post-CPB period and that it reached a peak level at 3 hours after bypass. Kumar and colleagues [15] showed that MCP-1 reaches its highest levels by the 3rd hour following reperfusion injury of the ischemic myocardium. MCP-1 may contribute to deleterious sequelae in a number of ways. It is a chemoattractant for mononuclear phagocytes, natural killer cells, T-cells, mast cells, and basophils, and it has been implicated in transendothelial monocyte recruitment to sites of inflammation [5]. Furthermore, it has been shown to induce respiratory burst activity and to stimulate lysosomal enzyme release from monocytes [5]. Whether it is these effects that occur following CPB needs to be studied by using agents that block the activity of this cytokine in vivo.

The data on MCP-1 after CPB are scarce: to the best of our knowledge, no comparable data on RANTES whatsoever are available. Published data concerning plasma RANTES levels have been obtained primarily by studying both acute and chronic inflammatory disorders [5, 16]. The reduced RANTES levels we found to be associated with a complicated clinical course could provide new insight into the immunoregulatory characteristics of the postpump syndrome. We have shown that RANTES, a potent stimulator of macrophages and monocytes, was decreased in serum following CPB and that these lower levels reflected patient outcome. These results can explain previous reports describing changes in lymphocyte subsets following CPB [17–19]. According to Ide and colleagues [18], the total number of T lymphocytes was markedly reduced during CPB. Thus, we speculate that reduced RANTES concentrations correspond to the reduced lymphocyte counts, acting as a mediator of lymphocyte depletion following CPB, as recently reported by Hisatomi and colleagues [19].

Several points must be taken into consideration when interpreting our results: all of our patients were given methylprednisolone, based on publications that documented improved postoperative hemodynamics after corticosteroids administration during CPB [20]. Other cytokines, such as interleukin-6, have been shown to decrease in number after the use of corticosteroids [21], and we cannot rule out potential blunting of cytokine release by corticosteroid treatment. However, our results are relevant to the clinical setting, because corticosteroid administration to these patients is now commonly used during deep hypothermia and circulatory arrest and is gaining popularity in other types of pediatric open heart surgery. Another limitation of our study is that we have shown that CPB of 200 minutes is associated with worse outcomes. Because the duration of CPB is a continuous variable, our results do not address the question of whether there is an ordered relationship between CPB duration and chemokine levels: we suggest that further large-scale studies incorporating various durations of CPB time and large numbers of patients are warranted.

In conclusion, the results of this study show a significant correlation between chemokines and the intra- and postoperative courses. These data support the need for further in vivo and in vitro clinical trials aimed at determining the pathophysiology caused by these chemokines and assessing whether their inhibition plays a part in reducing the inflammatory response induced by CPB.

	Acknowledgments

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
The authors thank Esther Eshkol for editorial assistance.

	References

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 

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