Asian Cardiovasc Thorac Ann 2007;15:214-217
© 2007 Asia Publishing EXchange Ltd
Interleukin-4 C-590T Polymorphism Has No Role in Coronary Artery Bypass Surgery
Mohamad N Bittar, FRCS C-Th,
Noman H Khasati, FRCS,
Abdul K Deiraniya, FRCS,
Nizar Yonan, FRCS C-Th
Department of Cardiothoracic Surgery, South Manchester University Hospitals NHS Trust, Wythenshawe Hospital, Manchester, United Kingdom
For reprint information contact: Mohamad N Bittar, FRCS Tel: 44 161 862 9080 Fax: 44 161 862 9080 Email: mbittar{at}doctors.org.uk, Department of Cardiac Surgery, Wythenshawe Hospital, Southmoor Road, Manchester, M23 9LT, United Kingdom.
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ABSTRACT
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Interleukin-4 exerts anti-inflammatory effects through decreased macrophage production of tumor necrosis factor-
and interleukin-1ß. We investigated genetic predisposition in the interleukin-4 response to coronary revascularization and studied the association between C-590T polymorphism, interleukin-4 levels, and outcome of surgery. DNA was obtained from 96 consecutive patients undergoing elective coronary revascularization. Patients were genotyped for interleukin-4 C-590T polymorphism using a sequence-specific primer polymerase chain reaction. Interleukin-4 levels were measured using an enzyme-linked immunosorbent assay in serum samples taken 3 hr postoperatively. The frequency of interleukin-4 C-590T genotypes CC, CT, and TT was 33.3%, 27.1%, and 39.6%, respectively. Patients with the TT genotype had significantly higher circulating levels of interleukin-4 (3.4 ± 4.6 pg·mL–1) postoperatively compared to CC (2.5 ± 0.1 pg·mL–1) and CT (2.7 ± 0.5 pg·mL–1) genotypes. Interleukin-4 C-590T polymorphism is the main determinant of postoperative interleukin-4 levels. The TT genotype is the highest producer of interleukin-4. Neither the genotype nor the serum levels seem to play any role in recovery from coronary artery bypass surgery.
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INTRODUCTION
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Systemic inflammatory response syndrome is the reaction to a variety of severe clinical insults.1 It is known to affect the outcome of cardiac surgery; in its mildest form, it has little impact, but the most severe form is often fatal.2,3 Many factors might trigger this response, including surgical trauma, contact of blood with foreign surfaces, lung injury caused by the cardiopulmonary bypass (CPB) machine, and reperfusion injury.4–6 Inflammatory cells produce cytokines that mediate various stages of inflammation and are capable of stimulating cells such as smooth muscle cells, fibroblasts, and endothelial cells.7 Although many studies have focused on the role of tumor necrosis factor- and interleukin-6 (IL-6) in the systemic inflammatory response syndrome after cardiac surgery, very little is known about IL-4 in that setting. Interleukin-4 is produced mainly by a subpopulation of activated T-cells that are biologically the most active helpers of B-cells. It promotes the proliferation and differentiation of activated B-cells.8,9 Interleukin-4 exerts its anti-inflammatory effects through decreased macrophage production of tumor necrosis factor- and IL-1ß, and its modulation appears less dramatic than that induced by IL-10.10 Interleukin-4 may have a clinically important role in inflammatory and autoimmune diseases because it inhibits the production of inflammatory cytokines. One report implied that lack of release of this anti-inflammatory cytokine may be partially to blame for complications after cardiac surgery.11 Interleukin-4 has been implicated in acute rejection following cardiac transplantation. Functional polymorphism within the IL-4 gene at position C-590T was reported to influence the production of IL-4.12 We investigated whether the magnitude of IL-4 release in response to coronary artery bypass grafting (CABG) is related to the presence of a certain allele in the functional polymorphism at position C-590T. We also examined whether C-590T polymorphism or postoperative IL-4 levels were related to clinical outcome.
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PATIENTS AND METHODS
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A prospective study of 96 patients undergoing first-time elective CABG was conducted at the Wythenshawe Hospital, Manchester, UK. Patients with unstable angina, recent myocardial infarction (< 30 days), pre-existing autoimmune diseases, or renal failure were excluded from the study. Patients on immunosuppressive therapy or anti-inflammatory agents were also excluded. Anti-platelet therapy was routinely stopped 7 days prior to surgery. Patients gave informed consent for the collection and storage of blood, isolation of DNA, and determination of cytokine gene polymorphism. Ethical approval was obtained from the South Manchester Medical Research Ethics Committee.
All operations were performed by consultant surgeons through a midline sternotomy approach. Cardiopulmomary bypass was instituted in 82 patients by cannulation of the right atrium and ascending aorta. Myocardial protection was provided by intermittent antegrade (with or without retrograde) blood cardioplegia. Aprotinin was not used. In 14 patients, CABG was performed without CPB, using an intracoronary shunt (Medtronic, Minneapolis, MN, USA) and an Octopus stabilizer (Medtronic, Minneapolis, MN, USA). Perioperative anticoagulation with heparin was reversed with protamine sulphate. Venous blood samples were collected preoperatively in the outpatient clinic and at 3 hr postoperatively in the intensive care unit. They were placed in ethylenediamine tetraacetic acid (EDTA) vacutainer tubes (Becton Dickinson, Oxford, UK) and centrifuged within 30 min at 3,000 rpm for 10 min. Plasma and blood cells were separated and stored separately at -80°C.
DNA was obtained from EDTA-anticoagulated blood using the double-lysis method. Briefly, 4 mL of blood was centrifuged, blood cells were transferred to 13-mL polypropylene tubes containing 9 mL of lysis buffer 1 (155 mM ammonium chloride, 10 mM potassium hydrogen carbonate, 1 mM EDTA) and mixed for 15 min. Samples were centrifuged, and the supernatant was removed. The cell pellets were re-suspended and lysed again. A nuclear membrane lysis step was performed using 25 mM EDTA and 2% sodium dodecyl sulfate in a 3-mL volume with 1 mL of 10 M ammonium acetate. Samples were centrifuged and the supernatant was added to propan-2-ol to wash out DNA. After centrifuging again, they were re-suspended in 350 µL double-distilled water.
DNA was amplified using specific oligonucleotide primers based on the published sequence (GenBank accession number AF 5224 41-43 Genosys, UK). The primer sequences used for IL-4 genotyping are shown in Table 1
. Each reaction mixture comprised 1.25 U Thermoprime Plus DNA polymerase (ABgene, Epsom, UK), 75 mM Tris-HCl, 20 mM (NH4)2SO4, 2.0 mM MgCl2, 0.01% Tween 20, and 0.2 mM each of 2'-deoxycytidine-5'-triphosphate, 2'-deoxyguanosine-5'-triphosphate, 2'-deoxyadenosine-5'-triphosphate, 2'-deoxythymidine-5'-triphosphate, and 2'-deoxyuridine-5'-triphosphate. Precipitant and red dye was also present for electrophoresis. This reaction mixture was added to human growth hormone primers (control) in a ratio of 10:1, and 10 µL of this was mixed with 1.5 µL DNA; 5 µL of this master mix was added to 5 µL of specific C or T primers (Table 1) in separate tubes. Cycling conditions included an initial denaturation at 95°C for 1 min followed by 95°C for 15 sec, 65°C for 50 sec and 72°C for 40 sec (10 cycles), followed by 95°C for 50 sec, 59°C for 50 sec and 72°C for 50 sec (20 cycles). The final holding temperature was 4°C. The polymerase chain reaction products were electrophoresed on a 2% agarose gel (Life Technologies, St. Paul, MN, USA) and visualized using ethidium bromide (Life Technologies, St.Paul, MN, USA) under ultraviolet illumination.13,14
Serum levels of IL-4 were measured using a solid-phase sandwich enzyme-linked immunosorbent assay (Diaclone, France). Monoclonal IL-4 antibody was coated onto the wells of a 96-well plate. Standards of known IL-4 concentration were added along with control samples and patient serum. After washing, a biotinylated polyclonal IL-4 antibody was added and incubated. After washing, streptavidin-coupled peroxidase was added. Following incubation, a wash step was performed to remove chromogenic peroxidase. Finally, tetramethylbenzidine substrate was added, and the absorbance was measured using a MRX plate reader (Dynex Technologies Ltd, West Sussex, UK) with a primary wavelength of 450 nm and a reference wavelength of 620 nm.
The chi-squared test was used to analyze the relationship between categorical data. The nonparametric Mann-Whitney U test and Kruskal-Wallis test were used as appropriate to compare IL-4 levels and different outcomes between subgroups. Nonparametric testing was chosen because (apart from age which was analyzed using one-way analysis of variance) the data did not follow a normal distribution. Association between two continuous variables was determined using the Spearman rank correlation. Interleukin-4 levels are given as mean ± standard deviation, or median and range when applicable. The chi-squared test was used to compare the observed numbers of each genotype with those expected for a population, to establish if they were in Hardy-Weinberg equilibrium. Regression analyses were used to adjust for confounding factors. All statistical analyses were performed with SPSS software (SPSS, Inc., Chicago, IL, USA). All tests were two-sided. Significance was established at a value of p < 0.05.
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RESULTS
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Ninety-six patients undergoing elective CABG were recruited; their baseline characteristics are shown in Table 2. All planned procedures were completed and patients left the operating room in sinus rhythm. The median number of grafts per patient was 3 (range, 1–5), the mean aortic cross clamp time was 52 ± 31 min, and CPB time was 80 ± 50 min. There were 2 hospital deaths. Interleukin-4 was not detected in preoperative serum samples, whereas all patients had detectable levels 3 hr postoperatively. The median serum IL-4 level was 2.6 pg·mL–1 (range, 2.5–31 pg·mL–1). Interleukin-4 levels were comparable between patients operated on with or without CPB: 2.6 pg·mL–1 (range, 2.5–5.1 pg·mL–1) in CPB patients vs 2.6 pg·mL–1 (range, 2.5–31 pg·mL–1) in off-pump patients ( p = 0.7). The prevalence of CC, CT, and TT genotypes in the study population was 33.3%, 27.1%, and 39.6% respectively. Patients with the TT genotype had higher circulating levels of IL-4 (3.4 ± 4.6 pg·mL–1) compared to CC (2.5 ± 0.1 pg·mL–1) and CT genotypes (2.7 ± 0.5 pg·mL–1; p = 0.039). These results remained statistically significant following risk adjustment for age, sex, blood loss, ventilation time, duration of CPB, and aortic cross clamp time (Figure 1). We found no significant difference between the IL-4 genotypes or serum IL-4 levels in relation to recovery from, and outcome of, bypass surgery (Tables 3 and 4).
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DISCUSSION
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Serum IL-4 levels have shown minor individual variations that suggested they may provide reliable information regarding the immune response following CABG.5,15 Interleukin-4 has been the subject of numerous studies on heart failure, and more recently on allograft rejection. Serum IL-4 levels were found to be significantly increased during acute heart failure in the absence of infection or coronary events, and returned to normal after treatment.16 In transplantation, IL-4 was noted to be associated with graft survival. An experimental study showed longer graft survival after intra-graft infusion of IL-4.17 Donor heart rejection has been related to the pre-transplant levels of IL-4; high levels of IL-4 are associated with no or fewer episodes of graft rejection.12
In this study, the changes in the serum levels of IL-4 in response to CABG were minimal. This finding is consistent with previously published data.5 We found no significant relationship between the IL-4 genotypes or IL-4 serum levels and the outcome of CABG. Previous studies have focused on the much more potent anti-inflammatory cytokines such as IL-10 or interferon gamma, whereas IL-4 has not been the focus of attention in CABG patients, and thus its role, if any, was unclear.
It was concluded from this study that IL-4 C-590T polymorphism is the main determinant of IL-4 postoperative levels. The TT genotype patients have higher IL-4 levels. However, neither the genotype nor the serum levels seem to have any role in the recovery from coronary artery bypass surgery.
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ABSTRACT
INTRODUCTION
