Genotype and interleukin-10 responses after cardiopulmonary bypass{dagger}

H. F. Galley*, P. R. Lowe, R. L. Carmichael and N. R. Webster

Academic Unit of Anaesthesia and Intensive Care, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK

Corresponding author. E-mail: h.f.galley@abdn.ac.uk
{dagger}Presented in part to the Anaesthetic Research Society, Sheffield, UK, April 2002.

Accepted for publication: April 16, 2003


    Abstract
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 Abstract
 Introduction
 Methods and results
 Comment
 References
 
Background. The pro- and anti-inflammatory cytokine balance has been implicated in outcome from inflammatory conditions, and cardiopulmonary bypass is associated with a marked inflammatory response. Interleukin-10 (IL-10) is an anti-inflammatory cytokine and levels have been shown to be highest in those patients who develop sepsis after trauma or surgery. IL-10 levels vary between individuals and genotype may dictate the IL-10 response. We therefore investigated IL-10 genotype, circulating IL-10 concentrations and outcome in terms of organ dysfunction 24 h after cardiopulmonary bypass.

Methods. Blood samples were obtained from 150 patients before, and 3, and 24 h after cardiopulmonary bypass. IL-10 was measured by enzyme immunoassay. The single nucleotide polymorphism at –1082 base pairs was detected by restriction fragment length polymorphism analysis. Post-bypass organ system dysfunction was defined prospectively.

Results. IL-10 concentrations were increased 3 h after bypass (P<0.0001) and were still increased at 24 h (P<0.0001). Homozygosity for the G allele was associated with lower median (range) maximal IL-10 levels at 3 h (44 (13–136) pg ml–1) compared with the A allele (118 (39–472) pg ml–1; P=0.042). Those patients who developed at least one organ dysfunction (n=33) had higher IL-10 levels 3 h after surgery (242 (18–694) pg ml–1) compared with those without organ dysfunction (77 (7–586) pg ml–1; P=0.001, n=117).

Conclusions. The G allele of the –1082 base pair single nucleotide polymorphism in the IL-10 gene is associated with lower IL-10 release after cardiopulmonary bypass. High levels of IL-10 secretion are associated with organ dysfunction 24 h after surgery.

Br J Anaesth 2003; 91: 424–6

Keywords: complications, organ dysfunction; polypeptide, cytokines, interleukin-10; heart, cardiopulmonary bypass


    Introduction
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
The pro- and anti-inflammatory cytokine balance has been implicated in determination of outcome from inflammatory conditions.1 Perioperative inflammation, associated with postoperative complications, occurs in patients undergoing cardiopulmonary bypass (CPB). Increased levels of the anti-inflammatory cytokine, interleukin-10 (IL-10), have a negative impact on resistance to infection,24 and in septic mice, reduction of IL-10 levels improves survival.5

There is large inter-individual variation in IL-10 secretion, with a genetic component of >70%.6 There are a number of polymorphisms in the promoter and upstream regions of the IL-10 gene and a single nucleotide polymorphism (SNP) has been described. Either guanine (G) or adenine (A) can be present at position –1082 base pairs (bp) upstream of the transcription start site, associated with variable IL-10 protein release in vitro.7

We hypothesized that the genotype of the –1082 polymorphism in the IL-10 gene may dictate the IL-10 response to CPB and hence influence postoperative outcome. We determined the relationship between the polymorphism genotype and IL-10 concentrations and prospectively defined organ dysfunction as a measure of postoperative outcome, in patients undergoing cardiac surgery with CPB.


    Methods and results
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 Abstract
 Introduction
 Methods and results
 Comment
 References
 
After ethical committee approval and written informed consent was obtained, 150 consecutive eligible patients who were undergoing first time elective coronary artery bypass grafting (CABG) involving CPB were recruited. Patients with cancer, those who were receiving steroids or non-steroidal anti-inflammatory therapy, or who had an autoimmune condition, were excluded. Anaesthetic and surgical technique remained according to usual practice: patients received balanced anaesthesia with a benzodiazepine as premedication, and i.v. induction of anaesthesia was followed by maintenance with either isoflurane or enflurane, neuromuscular block and fentanyl. Outcome in terms of organ system dysfunction occurring within 24 h of bypass was recorded. Organ dysfunction was prospectively defined: respiratory system dysfunction—requirement for intermittent positive pressure ventilation beyond 24 h post-bypass; renal dysfunction—oliguria <0.5 ml h–1 despite adequate fluid resuscitation; cardiovascular dysfunction—hypotension (systolic pressure <90 mm Hg or >40 mm Hg below usual systolic pressure) requiring inotropic support; haematological dysfunction—continued bleeding or evidence of a clotting deficit requiring therapeutic intervention; and central nervous system dysfunction—Glasgow coma score of 14 or less.

Blood samples were obtained immediately before, and 3 and 24 h after bypass. Samples were centrifuged at 700 g for 10 min and stored at –80°C until analysis. IL-10 was determined in plasma using the IL-10 optimized reagents enzyme immunoassay system (Becton Dickinson, Oxford, UK) according to the manufacturer’s instructions. DNA was extracted from EDTA blood samples with the Nucleon BACC DNA extraction system (Tepnel Life Sciences Plc, Manchester, UK), and stored at 4°C until required. Polymerase chain reaction (PCR) and restriction fragment polymorphism analysis was carried out as described previously.7 8 Genotype was assigned as homozygous G/G, homozygous A/A or heterozygous G/A. Data were not normally distributed and are presented as median (range). Data were analysed using Analyse-It statistical add-in for Excel (Microsoft, UK) by Kruskal–Wallis (change in IL-10 levels with genotype) or Friedman (change in IL-10 levels with time) analysis of variance, with Mann–Whitney U-test or Wilcoxon signed ranks post hoc testing respectively. Differences in IL-10 between patients with and without organ dysfunction were analysed using Mann–Whitney U-test. The {chi}2-test was used to analyse allele frequency.

Patient characteristics, bypass time, and cross clamp time are shown in Table 1. IL-10 levels were detectable in all patients before bypass, and were higher at 3 h after bypass (P<0.0001, Fig. 1), but were not related to bypass or cross clamp time. At 24 h, concentrations were lower than at 3 h, but were still higher than before bypass (P<0.0001). The patients with evidence of organ dysfunction (n=33) had higher IL-10 levels 3 h after bypass than those who did not (242 (18–694) pg ml–1 compared with 77 (8–586) pg ml–1; P=0.001).


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Table 1 Physical characteristics of patients and CPB details
 


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Fig 1 (A) IL-10 concentrations before, 3, and 24 h after cardiopulmonary bypass in patients undergoing cardiac surgery. *P<0.0001 compared with before bypass (Wilcoxon signed ranks post hoc test). (B) Relationship with genotype of the polymorphism at position –1082 bp in the IL-10 gene and circulating IL-10 concentrations 3 h after cardiopulmonary bypass in patients undergoing cardiac surgery. **P=0.042 compared with the A/A genotype (Mann–Whitney post hoc test). Box and whisker plots show median, 25th and 75th percentile and range. P value refers to analysis of variance.

 
Genotype frequency was 24.0% (n=36) homozygous for the G allele, 32.7% (n=49) homozygous for the A allele and 43.3% (n=65) were heterozygotes. This frequency is similar to that in 130 healthy volunteers from the same geographical area (24.2% G/G, 32.0% A/A and 43.8% G/A, P=0.38).8 Thirty-three (22.0%) patients fulfilled the criteria for at least one dysfunctional organ and 18 (12%) patients had two or more dysfunctional organ systems. Of those patients with at least one organ dysfunction (n=33), 14 (42.4%) were genotype A/A; 5 (15.2%) were genotype G/G; and 14 (42.4%) were heterozygotes. In contrast, the genotype frequency in those patients without organ dysfunction was 29.1% A/A, 24.8% G/G, and 46.1% A/G.

IL-10 concentrations 3 h after bypass were significantly lower in those patients who were homozygous for the G allele compared with those who were homozygous for the A allele (P=0.042, Fig. 1).


    Comment
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
We have shown that IL-10 concentrations increase early after cardiopulmonary bypass. Peak levels 3 h after bypass were higher in patients who had a poorer outcome in terms of prospectively defined organ systems failures within 24 h of bypass. We also showed that the G allele of the single nucleotide polymorphism at position –1082 bp was associated with lower circulating IL-10 levels after bypass. This is the first report of IL-10 polymorphisms in patients undergoing cardiac surgery.

Outcome after major surgery is determined in part by the intensity of the inflammatory response, which is variable between individuals, and can be regulated at the genetic level.1 3 6 8 The response to surgery, trauma and infection is orchestrated by cytokines including IL-10. IL-10 has been said to represent a likely candidate gene for predisposition to sepsis.1 9 The pro- and anti-inflammatory cytokine balance seems to be important for outcome,1 4 and high IL-10 release is related to poor outcome in infection.24 In septic mice, anti-IL-10 treatment improved survival.5 We found that patients with poorer outcome in terms of simple pre-defined measures of organ system dysfunction had higher peak IL-10 levels compared with those who did not have any organ dysfunction.

IL-10 synthesis is tightly regulated. In the promoter region of the IL-10 gene are several motifs known to modulate transcription. These include a nuclear factor {kappa}B-like recognition site, an interferon inducibility sequence, a cAMP responsive element, and a glucocorticoid responsive element. Suspected regulatory elements in the 3' untranslated portion of the gene include sequences that have been implicated in the regulation of stability and nucleo-cytoplasmic transport of other short-lived mRNA species. Common polymorphisms have been described within the IL-10 gene promoter and upstream region that may influence production of the protein in response to inflammatory stimuli, and may therefore affect the outcome after major surgery. We found that the G allele of the SNP located at –1082 bp was associated with lower circulating IL-10 levels after CPB.

Studying cytokine gene polymorphisms in disease has a number of possible positive end-points, including: enhanced understanding of disease processes; identification of potential markers of susceptibility, severity and outcome; identification of targets for therapeutic intervention and potential responders or non-responders to such therapy; and perhaps identification of strategies for disease prevention. High levels of IL-10 secretion may be associated with poorer outcome after cardiac surgery, and identification of those patients likely to fare worse may enable novel targeted therapy in the future.


    Acknowledgement
 
Financial support for this work was obtained from the Scottish Hospitals Endowment Research Trust and the British Journal of Anaesthesia.


    References
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
1 Oberholzer A, Oberholzer C, Moldawer LL. Interleukin-10: a complex role in the pathogenesis of sepsis syndromes and its potential as an anti-inflammatory drug. Crit Care Med 2002; 30: S58–63[CrossRef][ISI]

2 van Dissel JT, van Langevelde P, Westendorp RGJ, Kwappenberg K, Frolich M. Anti-inflammatory cytokine profile and mortality in febrile patients. Lancet 1998; 351: 950–3[CrossRef][ISI][Medline]

3 Westendorp RG, Langermans JA, Huizinga TW, et al. Genetic influence on cytokine production and fatal meningococcal disease. Lancet 1997; 349: 170–3[CrossRef][ISI][Medline]

4 Lyons A, Kelly JL, Rodrick ML, Mannick JA, Lederer JA. Major injury induces increased production of interleukin-10 by cells of the immune system with a negative impact on resistance to infection. Ann Surg 1997; 226: 450–8[CrossRef][ISI][Medline]

5 Lyons A, Goebel A, Mannick JA, Lederer JA. Protective effects of early interleukin-10 antagonism on injury-induced immune dysfunction. Arch Surg 1999; 134: 1317–23[Abstract/Free Full Text]

6 van der Linden MW, Huizinga TW, Stoeken DJ, Sturk A, Westendorp RG. Determination of tumour necrosis factor-alpha and interleukin-10 production in a whole blood stimulation system: assessment of laboratory error and individual variation. J Immunol Methods 1998; 218: 63–71[CrossRef][ISI][Medline]

7 Lowe PR, Galley HF, Webster NR. A novel PCR-rFLP assay for the detection of the single nucleotide polymorphism at position –1082 in the human IL-10 gene promoter. Eur J Immunogenetics 2001; 28: 563[Medline]

8 Lowe PR, Galley HF, Webster NR. Influence of interleukin-10 polymorphisms on interleukin-10 expression and survival in critically ill patients. Crit Care Med 2003; 31: 34–8[CrossRef][ISI][Medline]

9 Stuber F. Another definite candidate gene for genetic predisposition to sepsis: Interleukin-10. Crit Care Med 2003; 31: 314–15[CrossRef][ISI][Medline]