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Ann Thorac Surg 2003;75:534-537
© 2003 The Society of Thoracic Surgeons

Original article: cardiovascular

A tumor necrosis factor gene polymorphism influences the inflammatory response after cardiac operation

Stefan Schroeder, MDa*, Nicole Börger, MDa, Hermann Wrigge, MDa, Armin Welz, MDb, Christian Putensen, MDa, Andreas Hoeft, MD, PhDa, Frank Stüber, MDa

a Departments of Anesthesiology and Intensive Care Medicine, University of Bonn, Bonn, Germany
b Department ofCardiac Surgery, University of Bonn, Bonn, Germany

Accepted for publication August 29, 2002.

* Address reprint requests to Dr Schroeder, Department of Anesthesiology and Intensive Care Medicine, University of Bonn, Sigmund-Freud-Straße 25, D-53105 Bonn, Germany
e-mail: schroed{at}ukb.uni-bonn.de


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Comment
 Acknowledgments
 References
 
BACKGROUND: The genetic background may influence cytokine release evoked by cardiac operation. Thus we determined the allele frequency and genotype distribution of a bi-allelic tumor necrosis factor (TNF) gene polymorphism and TNF-{alpha} concentrations in patients undergoing cardiac operations with and without cardiopulmonary bypass (CPB).

METHODS: The TNF NcoI gene polymorphism was identified by polymerase chain reaction followed by restriction analysis of the polymerase chain reaction product. Reading the size of the resulting DNA bands from the agarose gel defined the genotype as homozygous or heterozygous for the two alleles TNFB1 and TNFB2. Blood samples to determine TNF-{alpha} plasma levels were drawn from the patients before induction of general anesthesia after termination of CPB or after finishing coronary revascularization on the beating heart in non-CPB patients and 12 hours postoperatively.

RESULTS: The genotype distribution and allele frequencies in 47 patients undergoing cardiac operation with CPB were comparable with those found in 36 patients undergoing cardiac operation without CPB. The TNF-{alpha} plasma levels over time were comparable in patients with and without CPB. However, patients homozygous for the TNF-B2 allele had significantly higher TNF-{alpha} plasma levels after termination of the CPB (40.2 ± 3.5 pg/mL; mean ± standard error of the mean; n = 28) compared with non-CPB patients (29.8 ± 2.5 pg/mL; mean ± standard error of the mean; n = 15) (p < 0.05).

CONCLUSIONS: Patients homozygous for the TNF-B2 allele showed significantly higher TNF-{alpha} plasma levels after termination of CPB compared with non-CPB patients. Therefore preoperative TNF genotyping may be useful as patients with genetically determined increased proinflammatory cytokine expression with multiple comorbidities may in particular benefit from avoiding the use of CPB.


   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Comment
 Acknowledgments
 References
 
Coronary artery bypass grafting with cardiopulmonary bypass (CPB) and cardioplegic arrest is known to induce a systemic inflammatory response syndrome that may effect postoperative morbidity and mortality of patients [1,2]. Thus coronary artery bypass grafting on the beating heart without CPB using a cardiac stabilizer has gained increasing interest. This technique has been postulated to cause a marked reduction of adverse effects related to cardiac operation using CPB [3, 4]. However, cardiac operation with or without CPB is associated with an inflammatory reaction. Tumor necrosis factor (TNF) has been recognized as a central mediator in patients with systemic inflammatory responses after surgical intervention, trauma, infection, and other causes that may initiate similar cascades of endogenous mediators [5]. Furthermore it was shown that a bi-allelic NcoI restriction fragment length polymorphism within the TNF locus is associated with variable TNF-{alpha} plasma concentrations and outcome of patients with severe sepsis [6]. The present study was designed to investigate whether the TNF gene polymorphism influences TNF-{alpha} expression in patients undergoing coronary artery bypass grafting with or without CPB. Determining the genetic background of the individual inflammatory response to cardiac operation could provide the clinician with information concerning the risk of developing a severe inflammatory response syndrome and possible multiple organ dysfunction. In addition this information may influence the decision whether patients with genetically determined increased pro-inflammatory cytokine expression and with multiple comorbidities may in particular benefit from avoiding CPB. Moreover, if clinically relevant, knowledge about the genetic background of the individual inflammatory response type to cardiac operation could possibly be valuable for stratifying anti-inflammatory therapies in these patients.


   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Comment
 Acknowledgments
 References
 
After approval by the local ethics committee and obtaining written informed consent from each individual, 83 consecutive patients with single or double vessel coronary disease were enrolled in the study. The decision whether safe and complete revascularization either with CPB or without CPB seemed feasible was made by preoperative study of coronary angiography. Forty-seven patients were selected for conventional myocardial revascularization with CPB and 36 patients received coronary artery bypass grafts on the beating heart without extracorporal circulation using a cardiac stabilizer. Median sternotomy was performed in both groups. The 91 control individuals were healthy, white, blood donors. They were judged healthy on the basis of medical history, physical examination, and laboratory analysis. The control individuals were used to investigate genotype distribution and allele frequencies in comparison with patients.

Blood samples for plasma tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-8 (IL-8) concentrations were drawn from the patients before induction of general anesthesia (T1, time point 1), after termination of CPB or after finishing coronary revascularization on the beating heart (T2) and 12 hours postoperatively (T3). TNF-{alpha} and IL-8 plasma concentrations were measured by immunoenzymometric assays (Biosource, Belgium). The assay procedures were performed according to the supplier’s recommendations.

DNA extraction and typing of individuals for the bi-allelic gene polymorphism within the TNF locus was done as reported previously [6]. A 782 basepairs fragment of genomic DNA, including the polymorphic site of the restriction enzyme NcoI within the TNF locus, was amplified by means of polymerase chain reaction. The genotype of each individual was determined after NcoI digestion of the amplified product and subsequent agarose gel electrophoresis. Reading the size of the resulting DNA bands from the agarose gel demonstrated the genotype as defined by the two alleles TNF-B1 and TNF-B2.

Statistical analysis was performed by using the Wilcoxon–Mann–Whitney U test for group differences in patients undergoing coronary artery bypass grafting with and without CPB in terms of clinical characteristics unless otherwise noted. Statistical analysis of genotype distribution and allele frequency was done by {chi}2 test. Three-way analysis of variance, including post hoc comparisons (Scheffé’s test), was used to determine group differences and group trends over time for the plasma TNF-{alpha} and IL-8 concentrations. We regarded p less than 0.05 as statistically significant.


   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Comment
 Acknowledgments
 References
 
The general characteristics of the study population are shown in Table 1. There was no statistical difference with regard to age, gender, and Simplified Acute Physiology Score II [7] comparing both groups of patients (p >= 0.05). The Simplified Acute Physiology Score II is an indicator of severity of illness with higher values reflecting a more critical condition. However, postoperative maximal plasma levels of creatine kinase, MB-CK isoenzyme, and cardiac troponin-I were significantly higher in patients after myocardial revascularization with CPB compared with patients after myocardial revascularization without CPB (p < 0.01).


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  		Table 1. General Characteristics of Study Population

 
NcoI digestion of the amplified polymerase chain reaction product revealed homozygosity for the allele TNF-B1 in 8% of the patients (7 of 83) versus 52% homozygous patients (43 of 83) for the allele TNF-B2. A total of 40% of the patients (33 of 83) were heterozygous (TNF-B1/TNF-B2). Allelic frequencies for both alleles TNF-B1 (0.28) and TNF-B2 (0.72), as well as the genotype distribution, did not differ from findings in healthy whites (0.33 and 0.67; p > 0.05; 11%, 45%, and 44%; p > 0.05) (Table 2). In addition, no differences in genotype and allelic frequencies between patients with and without CPB were detected (Table 2).


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  		Table 2. Tumor Necrosis Factor Genotypes and Allelic Frequencies in Patients and Controls

 
The TNF-{alpha} plasma levels before and after coronary bypass graft operation were comparable with patients treated with or without CPB (Fig 1). However, in assigning patients to different TNF gene polymorphisms we found significantly higher TNF-{alpha} concentrations in patients homozygous for the TNF-B2 allele after finishing the use of CPB compared with non-CPB patients undergoing myocardial revascularization on the beating heart (40.2 ± 3.5 pg/mL, [n = 28] vs 29.8 ± 2.5 pg/mL, [n = 15]; mean ± standard error of the mean; p < 0.05) (Fig 2). In addition, patients homozygous for the TNF-B2 allele showed significantly higher IL-8 plasma levels after CPB when compared with patients undergoing cardiac operation on the beating heart (20.0 ± 4.1 pg/mL, [n = 28] vs 1.2 ± 1.0 pg/mL, [n = 15]; mean ± standard error of the mean; p < 0.05) (Fig 3). Avoiding the use of cardiopulmonary bypass resulted in a blunted increase in TNF-{alpha} and IL-8 levels, which otherwise occurs in patients homozygous for the TNF-B2 allele.



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  		Fig 1. Tumor necrosis factor-{alpha} (TNF{alpha}) plasma levels were comparable between the different time points (1 to 3) in patients undergoing cardiac operation with cardiopulmonary bypass (CPB) (n = 47) (closed circles) and without CPB (n = 36) (open circles). 1 = before induction of general anesthesia; 2 = after the use of CPB or after finishing coronary revascularization on the beating heart; 3 = 12 hours postoperatively.

 


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  		Fig 2. Tumor necrosis factor-{alpha} (TNF{alpha}) plasma levels were significantly higher in patients homozygous for the TNF-B2 allele after finishing cardiopulmonary bypass (CPB) (n = 28) (closed circles) compared with patients without CPB (n = 15) (open circles). 1 = before induction of general anesthesia; 2 = after the use of CPB or after finishing coronary revascularization on the beating heart; 3 = 12 hours postoperatively. *p less than 0.05.

 


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  		Fig 3. Patients homozygous for the tumor necrosis factor-B2 allele (TNF-B2/TNF-B2) showed significantly higher interleukin-8 (IL-8) plasma levels after cardiopulmonary bypass (CPB) (n = 28) (closed circles) when compared with patients undergoing cardiac operation on the beating heart (n = 15) (open circles). 1 = before induction of general anesthesia; 2 = after the use of CPB or after finishing coronary revascularization on the beating heart; 3 = 12 hours postoperatively. *p less than 0.05.

 

   Comment
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Comment
Acknowledgments
 References
 
Coronary artery bypass grafting with cardiopulmonary bypass (CPB) is associated with an inflammatory response mainly caused by surgical trauma, contact of blood with the artificial surface of the circuit, and reperfusion injury. The inflammatory reaction includes activation of the humoral and cellular immune system with enhanced release of cytokines. This may result in increased capillary permeability, respiratory distress, low cardiac output, and multiple organ failure [1, 4]. Therefore avoiding the use of CPB is believed to be beneficial. Recent clinical trials showed that coronary artery bypass grafting without CPB was associated with reduction of the inflammatory response and with decreased postoperative morbidity and mortality [2, 3, 8]. However we demonstrated that TNF-{alpha} plasma levels revealed no significant changes during the period of investigation comparing patients undergoing cardiac operation with and without CPB. In contrast, a recent published study found a marked difference in inflammatory response comparing coronary artery bypass grafting with and without CPB [9]. In this study coronary artery bypass grafting with CPB was performed by median sternotomy, whereas in patients without CPB, the lateral thoracotomy was used for surgical access. It was shown that median sternotomy results in a more extended inflammatory response than anterolateral thoracotomy [10]. In the present study all patients were operated by median sternotomy. Thus direct tissue injury is comparable between groups and may explain the similar pattern of cytokine release in our patients undergoing coronary artery bypass grafting without CPB compared with the patients with CPB.

Furthermore, a genetic background may play a role in influencing cytokine plasma levels induced by cardiac operations. Therefore we evaluated the genotype distribution and allele frequency of the TNF NcoI gene polymorphism with regard to plasma TNF-{alpha} concentrations in patients undergoing cardiac operation. TNF-{alpha} is believed to be a pivotal proinflammatory mediator in the pathogenesis of the systemic inflammatory response syndrome. In addition, excessive production of TNF-{alpha} may lead to organ dysfunction and death [5]. Recently it has been shown that a genomic restriction fragment length polymorphism within the TNF locus is correlated with increased TNF-{alpha} plasma concentrations and poor prognosis in sepsis [6]. In the present study the overall allele frequency and genotype distribution was comparable in patients undergoing coronary artery bypass grafting with and without CPB. However, in assigning patients to different TNF gene polymorphisms we found significantly higher TNF-{alpha} concentrations in patients homozygous for the TNF-B2 allele after termination of CPB compared with non-CPB patients who received myocardial revascularization on the beating heart. We demonstrated that 60% of the CPB patients were carriers of the TNF-B2/B2 allele, the one which was shown to be associated with the higher postoperative TNF-{alpha} levels. Only 42% of the non-CPB patients carried this allele. Although the overall genotype distribution was comparable between the groups it may be possible that this somewhat biased the results.

In addition, patients homozygous for the TNF-B2 allele showed significantly higher IL-8 plasma levels after CPB when compared with non-CPB patients. TNF-{alpha} is known as a potent inducer of the synthesis of secondary proinflammatory mediators such as IL-8 [11]. IL-8 is a crucial cytokine known to attract and activate neutrophils and is supposed to play a role in the pathophysiology of capillary leak [12–14]. As TNF-{alpha} is a major mediator in the pathogenesis of systemic inflammation and sepsis, individuals with a genetic determination for high TNF-{alpha} responses with the consequence of increased expression of secondary proinflammatory mediators may be at high risk for development of organ failure and death when challenged with stimuli that can evoke a generalized systemic inflammatory response like the CPB. Preopera-tive TNF genotyping may be useful to decide whether patients with genetically determined increased pro-inflammatory cytokine expression and multiple comorbidities in particular may benefit from avoiding CPB. Furthermore, genotyping may be used to select patients at high risk for intensive monitoring or prophylactic treatment.


   Acknowledgments
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Comment
 Acknowledgments
References
 
This study was supported by departmental funding.


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Comment
 Acknowledgments
 References
 

  1. Cremer J., Martin M., Redl H., et al. Systemic inflammatory response syndrome after cardiac operations. Ann Thorac Surg 1996;61:1714-1720.[Abstract/Free Full Text]
  2. Cleveland J.C., Jr, Shroyer A.L., Chen A.Y., Peterson E., Grover F.L. Off-pump coronary artery bypass grafting decreases risk-adjusted mortality and morbidity. Ann Thorac Surg 2001;72:1282-1289.[Abstract/Free Full Text]
  3. Matata B.M., Sosnowski A.W., Galinanes M. Off-pump bypass graft operation significantly reduces oxidative stress and inflammation. Ann Thorac Surg 2000;69:785-791.[Abstract/Free Full Text]
  4. Czerny M., Baumer H., Kilo J., et al. Inflammatory response and myocardial injury following coronary artery bypass grafting with or without cardiopulmonary bypass. Eur J Cardiothorac Surg 2000;17:737-742.[Abstract/Free Full Text]
  5. Strieter R.M., Kunkel S.L., Bone R.C. Role of tumor necrosis factor-alpha in disease states and inflammation. Crit Care Med 1993;21:S447-463.[Medline]
  6. Stüber F., Petersen M., Bokelmann F., Schade U. A genomic polymorphism within the tumor necrosis factor locus influences plasma tumor necrosis factor-alpha concentrations and outcome of patients with severe sepsis. Crit Care Med 1996;24:381-384.[Medline]
  7. Le Gall J.R., Lemeshow S., Saulnier F. A new Simplified Acute Physiology Score (SAPS II) based on a European/North American multicenter study. JAMA 1993;270:2957-2963.[Abstract/Free Full Text]
  8. Gu Y.J., Mariani M.A., van Oeveren W., Grandjean J.G., Boonstra P.W. Reduction of the inflammatory response in patients undergoing minimally invasive coronary artery bypass grafting. Ann Thorac Surg 1998;65:420-424.[Abstract/Free Full Text]
  9. Struber M., Cremer J.T., Gohrbandt B., et al. Human cytokine responses to coronary artery bypass grafting with and without cardiopulmonary bypass. Ann Thorac Surg 1999;68:1330-1335.[Abstract/Free Full Text]
  10. Gu Y.J., Mariani M.A., Boonstra P.W., Grandjean J.G., van Oeveren W. Complement activation in coronary artery bypass grafting patients without cardiopulmonary bypass: the role of tissue injury by surgical incision. Chest 1999;116:892-898.[Abstract/Free Full Text]
  11. Schade F.U., Stuber F., Borgermann J., Majetschak M. Relation of the bi-allelic NcoI restriction fragment length polymorphism within the tumour necrosis factor B gene to the development of mediastinitis. Eur J Surg 1999;584:S73-78.
  12. Elliott M.J., Finn A.H. Interaction between neutrophils and endothelium. Ann Thorac Surg 1993;56:1503-1508.[Abstract]
  13. Moat N.E., Rebuck N., Shore D.F., Evans T.W., Finn A.H. Humoral and cellular activation in a simulated extracorporeal circuit. Ann Thorac Surg 1993;56:1509-1514.[Abstract]
  14. Finn A., Naik S., Klein N., Levinsky R.J., Strobel S., Elliott M. Interleukin-8 release and neutrophil degranulation after pediatric cardiopulmonary bypass. J Thorac Cardiovasc Surg 1993;105:234-241.[Abstract]



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