In the Elderly, Interleukin-6 Plasma Levels and the −174G>C Polymorphism Are Associated With the Development of Cardiovascular Disease
Objective— Interleukin (IL)-6–mediated inflammation is involved in cardiovascular disease (CVD). We assessed IL-6 levels and the −174G>C genotype in a case-control study of men and women (average age 73 years) within the Cardiovascular Health Study.
Methods and Results— Cases included incident angina, myocardial infarction (MI), and stroke (5-year follow-up), prevalent MI, and MRI-detectable infarcts. A control group and a group free of subclinical CVD were used for comparison. The −174C allele was associated with higher C-reactive protein (11% higher, P=0.02), fibrinogen (3% higher, P=0.02), and IL-6 (5% higher; P=0.16). IL-6 was associated with increased atherosclerosis when the control group was compared with the group free of subclinical CVD. No further association with CVD events was found when case groups were compared with the control group. Compared with its absence, presence of the −174C allele was associated with risk of MRI infarcts (odds ratio 1.5).
Conclusions— IL-6 levels differentiated those with subclinical CVD from those without. Although the −174C allele was not associated with incident events, associations of the genotype with inflammation and MRI infarcts, combined with the plasma IL-6 results, suggest that IL-6 may chronically predispose an individual to develop atherosclerosis.
Inflammation is recognized as a central component of cardiovascular disease (CVD), although the underlying regulation and molecular mechanisms remain unclear.1 Interleukin (IL)-6, a key inflammatory cytokine, is involved in a diverse range of biological activities, in particular, the synthesis of acute-phase reactants by the liver.2,3⇓ IL-6 levels are normally very low and increase during infection, trauma, or stress, and they also increase with age.4 Elevated levels of IL-6 are associated with an increased risk of death from cardiovascular and noncardiovascular causes in the elderly5–8⇓⇓⇓ and with myocardial infarction (MI) in middle-aged men.9 Elevated levels of C-reactive protein (CRP) and fibrinogen, acute-phase reactants largely regulated by IL-6,2,3⇓ are also independently associated with risk of CVD10,11⇓ over long time periods.
Our underlying hypothesis is that atherosclerosis represents a chronic inflammatory disorder and that elevated IL-6 levels may predict risk of future CVD events. In addition, polymorphisms in the 3′- and 5′-untranslated regions of the IL-6 gene may be key regulators of IL-6 and downstream protein levels and therefore may predispose an individual to CVD risk as well.
The −174G>C promoter polymorphism in the IL-6 gene may involve one such site of regulation. The −174G>C polymorphism has demonstrated functionality in vitro and in healthy middle-aged white men.12 In middle-aged men, the −174C allele was not associated with fibrinogen but was associated with higher blood pressure and conveyed a modestly higher relative risk (RR) for coronary heart disease (RR 1.54, 95% CI 1.02 to 2.23), particularly evident in smokers (RR 2.66, 95% CI 1.60 to 4.32).13 The −174G>C genotype may also contribute to susceptibility to type 1 diabetes14,15⇓ and lipid abnormalities.15,16⇓ Because a large fraction of CVD events occurs in people aged >65 years and because the IL-6 promoter polymorphism has not been studied extensively in women, we examined the effects of plasma IL-6 and the −174G>C genotype on CVD risk in a subset of the elderly men and women of the Cardiovascular Health Study (CHS).
Cardiovascular Health Study
The original CHS cohort (5201 adults aged ≥65 years) was recruited from 1989 to 1990.17 An additional cohort of 687 African American participants (minority cohort) was recruited between 1992 and 1993. Examinations included lifestyle and medical histories, including assessment of the prevalence and extent of clinical CVD,17 physical exams, blood collection, resting 12-lead echocardiography,18 ankle-arm blood pressure index (AAI),19 and carotid ultrasonography.20 Cerebral MRI was performed between 1992 and 1994.21 All subjects gave informed consent for participation in the study, and all procedures were conducted under institutionally approved protocols for use of human subjects.
Ascertainment of Events
Incident events in this case-control study occurred between baseline and June 30, 1995, and were adjudicated by a committee using standardized and published criteria.22 Case groups consisting of original cohort individuals free of clinical CVD at baseline included incident MI (n=217), stroke (n=208), and angina (n=226) and MRI-detectable infarcts (MRI infarcts, n=250). A subclinical CVD-free reference group (n=250) consisted of the original cohort of individuals free of prevalent and incident CVD who met the following criteria: maximum internal carotid intima-media thickness (IMT) ≤ the 20th percentile, AAI ≥1.00, carotid stenosis ≤25%, no major ECG abnormality, no abdominal aortic aneurysm, and no wall motion abnormalities on an echocardiogram. The control group (n=500) was composed of randomly selected original cohort members who had no prevalent or incident CVD and no MRI-detectable infarcts and who did not satisfy the criterion of being free of subclinical CVD. At the time of selection, there was insufficient follow-up of the minority cohort to include them in the case groups. A separate group of randomly selected minority cohort members free of prevalent CVD was added as a comparison (n=250). The prevalent MI case group (n=553) consisted of all subjects from both cohorts with confirmed MI at baseline.
Prevalent CVD was defined as confirmed angina, MI, stroke, or transient ischemic attack.17 Hypertension was defined as seated systolic blood pressure ≥160 mm Hg, diastolic pressure ≥95 mm Hg, or self-reported high blood pressure and use of antihypertensive medication. Borderline hypertension was seated systolic blood pressure of 140 to 159 mm Hg or diastolic pressure 90 to 94 mm Hg. Obesity was defined as body mass ≥130% of ideal body mass for age and sex, based on body mass index (BMI), which is weight (kg)/height (m2). Smoking was classified as current, former, or never. Diabetic status was classified according to the American Diabetic Association.23 Major ECG abnormalities and calculation of left ventricular mass (LVM) were previously defined.24 Subclinical CVD was defined as follows: (1) AAI ≤0.9, (2) maximum common or internal carotid artery IMT ≥80th percentile, (3) carotid artery stenosis ≥25%, (4) presence of major ECG abnormalities, or (5) positive response for angina or intermittent claudication on the Rose questionnaire.25
Procedures for blood collection and laboratory methods have been reported.26 The assays for fibrinogen (coefficient of variation [CV] 2.9%) and IL-6 (CV 6.3%) have also been described.5,26,27⇓⇓ CRP was measured in a high-sensitivity assay developed in house (CV 8.9%).28,29⇓ All assays were conducted on blood drawn at baseline.
Genotyping for IL-6 to 174G>C Promoter Polymorphism
Genotyping for the −174G>C polymorphism was carried out as previously described.12,30⇓ Genotyping was performed for 1857 white and 344 African American individuals who consented to DNA preparation and use for CVD analysis. CVD risk factor data were also available on these individuals. IL-6 levels were available on 1424 whites and 300 African Americans.
Data were analyzed by using the Statistical Package for Social Science (SPSS) for Windows (version 10.0).31 Associations of IL-6 levels with categorical variables representing CVD risk factors (sex, diabetes, hypertension, angina, and smoking) and subclinical CVD (major ECG abnormalities) were determined by ANOVA for individuals in the control group. Significance was set at P≤0.05. Bivariate associations with continuous variables representing CVD risk factors (age, BMI, fasting glucose and insulin, blood pressure, physical activity, and lipids), measures of subclinical CVD (common and internal carotid IMT, AAI, and LVM), and inflammatory factors (CRP, fibrinogen, white blood cell count, and albumin) were determined by Pearson correlation coefficients.
Logistic regression was used to estimate risk associated with IL-6 quartiles; the control group was compared with the group free of subclinical CVD, and then the cases were compared with the controls. Analyses were adjusted for age, race, sex, and clinic site.
Differences in the −174 C allele frequency in selected white and African American participants were assessed by χ2 tests. Associations of genotype with inflammatory markers, CVD risk factors, coagulation and fibrinolytic factors, and measures of subclinical disease were determined by ANOVA for continuous variables and by χ2 statistics for categorical variables in the selected white population. Logistic regression was used to assess the association of genotype with CVD events.
Because of the association of IL-6 level and adiposity,32,33⇓ we stratified on BMI and used 2-way ANOVA to examine the association of BMI (tertiles) and IL-6 genotype with selected variables. We likewise stratified on smoking (never versus current/former) because of the smoking-related changes in IL-6 and CRP levels.34–36⇓⇓
Baseline characteristics of the CHS participants selected for the present study are shown in Table 1. Control and case groups had similar levels of subclinical CVD, with 67% of men and 62% of women in the control group and 78% of men and 67% of women in the combined case groups having some form of subclinical CVD.
IL-6 levels did not vary by race when white control subjects were compared with the African American reference group (whites 1.86±1.82 pg/mL, African Americans 1.78±1.94 pg/mL; P=NS). Levels were also similar when analyses were stratified on race and sex.
Genotype distribution and −174C allele frequency in controls (whites only) and African Americans (reference group) are shown in Table 2. Genotype distributions were in Hardy-Weinberg proportions in both populations (both P>0.3); however, the −174C allele was significantly more common in whites than in African Americans (38% versus 8%, respectively; P≤0.001). Because of the relatively small number of African American participants, genotype analyses were limited to whites.
In controls, plasma IL-6 was associated with CVD risk factors (age, BMI, glucose, and insulin), lipids, measures of subclinical CVD (common and internal carotid IMT, AAI, and LVM), and inflammatory factors (CRP, fibrinogen, albumin, and white blood cell count), with most associations remaining significant when the cohort was stratified by sex.
Overall in case-control group whites, the −174C allele was associated with higher fibrinogen (P=0.015) and CRP (P=0.014), with a trend toward higher IL-6 (P=0.163, Figure 1). Results were similar for men and women, but the associations were not significant in men. There were no associations of genotype with metabolic or CVD risk measures including the following: blood pressure, AAI, lipids, albumin, white blood cell count, obesity, carotid IMT, LVM, carotid stenosis, and ECG abnormalities.
We examined possible effect modification by adiposity (Figure 2) and smoking. For those in the lowest BMI tertile, the presence of at least 1 C allele was associated with 20% higher CRP (P=0.011), whereas only 8% higher levels (P=NS) were seen in those in the highest BMI tertile. Similar results were seen for fibrinogen, and weaker associations were seen for IL-6. In current or former smokers, the −174C allele was associated with a greater difference in levels of CRP and fibrinogen than in never smokers. The formal test for interaction approached significance for CRP (Pgenotype=0.018, Psmoking=0.001, and Pinteraction=0.068).
Prediction of Events
There were no significant differences in IL-6 levels between the controls and cases with angina, incident MI, or MRI infarcts (Table 1). IL-6 was significantly higher in the stroke group (P=0.009) and significantly lower in the subclinical CVD-free group (P≤0.001) compared with the control group. All case groups (with the exception of prevalent MI, which lacked plasma IL-6 measurements) had significantly higher IL-6 levels (P≤0.001) than the subclinical CVD-free group.
In logistic regression, after adjustment for age, sex, race, and clinic site, higher IL-6 was not associated with incident CVD events or MRI infarcts when controls were the reference group (Table 3). However, with the subclinical CVD-free group as the reference group, increasing levels of IL-6 were associated with subclinical CVD (Table 3) and with clinical CVD. Similar results were seen in men and women separately. In addition, in men, the association between the highest quartile and risk of stroke was also significant when the controls were used as the reference group (adjusted odds ratio [OR] 3.84, 95% CI 1.20 to 12.29).
Frequency of the −174C allele in selected CHS participants is shown in Table 1. The C allele was more common in the MRI infarct group than in the control group (adjusted OR 1.50, 95% CI 1.05 to 2.14). There were no other significant associations with CVD events. Similar results were seen for men and women. In secondary analyses, stratifying on smoking revealed that the association of the C allele with MRI infarcts was primarily in nonsmokers (adjusted OR 1.70, 95% CI 1.01 to 2.88) and in the lowest BMI tertile (adjusted OR 2.37, 95% CI 1.25 to 4.49). When the subclinical CVD-free reference group was used, male nonsmokers with at least 1 C allele had a significantly increased OR for incident MI (adjusted OR 8.93, 95% CI 1.70 to 46.83), prevalent MI (adjusted OR 4.78, 95% CI 1.23 to 18.46), and MRI-detectable infarcts (adjusted OR 3.80, 95% CI 1.14 to 12.71). This association was not evident in women.
The major findings of this population-based study of elderly individuals were as follows: (1) a strong correlation of plasma IL-6 with markers of subclinical CVD; (2) significantly higher ORs for subclinical and clinical CVD, with increased levels of IL-6 when those with subclinical and clinical CVD were compared with the subclinical CVD-free reference group, but no discrimination between those with CVD events and the controls with subclinical disease; (3) a general lack of association of IL-6 promoter genotype with metabolic measures and CVD risk factors; (4) a stronger association of genotype with inflammation and blood pressure in subgroups based on obesity and smoking, known correlates of inflammation; and (5) evidence from subset analyses suggesting that in never-smoking men the presence of a C allele was associated with increased CVD.
In the selected CHS sample, the level of subclinical CVD in the control group was similar to that in the case groups (Table 1), and IL-6 levels were not predictive of CVD events when the controls were used as the reference group. However, when the subclinical CVD-free group was used for comparison, IL-6 levels were significantly associated with incident, prevalent, and subclinical CVD. Similarly, the −174C allele was associated with CVD events in nonsmoking men compared with the subclinical CVD-free group, supporting the position that the C allele may play an important role in the development of atherosclerosis, even in the absence of smoking. The lack of association in male current or former smokers may be due to survivor effects, inasmuch as there were fewer C allele carriers in this group compared with never smokers. However, the number of men in these subgroups is small, and our results need to be confirmed in future studies.
Our results are consistent with the position that adipose tissue is a major regulator of total circulating IL-632,33⇓ and may, in more obese individuals, overwhelm the contribution of the −174G>C genotype to plasma levels. Cigarette smoking has likewise been reported to influence IL-6 expression34,35⇓ and has been previously demonstrated to affect the association of CRP with other variables.36 This suggests that the polymorphism may exert an influence in the presence of a constant inflammatory stimulus, such as that provided by smoking, but not in the presence of obesity.
These findings are in apparent contrast to a previous report12 suggesting that the C allele was associated with lower IL-6 levels in a small group of younger healthy men and women. The conflicting results may be due to chance, although it is also possible that the −174G>C polymorphism exhibits different effects in older individuals compared with younger healthier people. In support of this, a larger study of patients with aneurysmal disease found a strong association between the −174C allele and higher levels of IL-6. In addition, subjects with the C allele had a higher mortality during the 5-year follow-up of the trial.37 The C allele has also been reported to be associated with risk of coronary heart disease in a case-control study.38 Likewise, in a large cohort of healthy middle-aged men from the Second Northwick Park Heart Study (NPHS II), the C allele was associated with significantly higher systolic blood pressure, CRP levels, and risk of coronary artery disease.13 In that study, smoking did not modify the effects of the C allele, and the effects of the C allele were more marked in men in the upper tertiles of BMI compared with the lowest tertile. These differences from our results may reflect survivor effects, inasmuch as the men and women of the CHS were considerably older than the men of NPHS II.
In summary, we report that in the CHS, plasma IL-6 appears to function as a marker for subclinical CVD and is associated with CVD events when individuals essentially free of subclinical CVD are used as a reference group. The −174G>C promoter polymorphism appears to function in the regulation of IL-6 and acute-phase reactants influenced by IL-6 expression. However, the effects of the genotype are modified by environmental factors, such as adiposity, which may contribute more significantly to IL-6 production than genotype. Although the −174G>C genotype does not appear to directly influence CVD risk in the elderly, the C allele appears likely to contribute to the development and progression of atherosclerotic disease.
Participating Institutions and Principal Investigators, CHS
Wake Forest University School of Medicine: Gregory L. Burke, MD; Wake Forest University, ECG Reading Center: Pentti M. Rautaharju, MD, PhD; University of California, Davis: John Robbins, MD, MHS; The Johns Hopkins University: Linda P. Fried, MD, MPH; The Johns Hopkins University, MRI Reading Center: Nick Bryan, MD, PhD, and Norman J. Beauchamp, MD; University of Pittsburgh: Lewis H. Kuller, MD, DrPH; University of California, Irvine, Echocardiography Reading Center (baseline): Julius M. Gardin, MD; Georgetown Medical Center, Echocardiography Reading Center (follow-up): John S. Gottdiener, MD; New England Medical Center, Boston, Ultrasound Reading Center: Daniel H. O’Leary, MD; University of Vermont, Central Blood Analysis Laboratory: Russell P. Tracy, PhD; University of Arizona, Tucson, Pulmonary Reading Center: Paul Enright, MD; University of Wisconsin, Retinal Reading Center: Ronald Klein, MD; University of Washington, Coordinating Center: Richard A. Kronmal, PhD; and National Heart, Lung, and Blood Institute Project Office: Jean Olson, MD, MPH.
This research was supported by contracts N01-HC-85079 through N01-HC-85086, N01-HC-35129, and N01-HC-15103 from the National Heart, Lung, and Blood Institute and RO1 HL-46696 (R.P.T., N.S.J.) from the National Institutes of Health. S.E.H. and M.S.O. were supported by the British Heart Foundation (RG95007, SH/CH/02025, and PG99153), and L.A.L. was supported by the Commission of the European Communities (HIFMECH study, contract BMH4-CT96-0272).
Received July 24, 2002; revision accepted September 18, 2002.
- ↵Ridker PM, Cushman M, Stampfer MJ, Tracy RP, Hennekens CH. Plasma concentration of C-reactive protein and risk of developing peripheral vascular disease. Circulation. 1998; 97: 425–428.
- ↵Volpato S, Guralnik JM, Ferrucci L, Balfour J, Chaves P, Fried LP, Harris TB. Cardiovascular disease, interleukin-6, and risk of mortality in older women: the women’s health and aging study. Circulation. 2001; 103: 947–953.
- ↵Ridker PM, Rifai N, Stampfer MJ, Hennekens CH. Plasma concentration of interleukin-6 and the risk of future myocardial infarction among apparently healthy men. Circulation. 2000; 101: 1767–1772.
- ↵Tracy RP, Bovill EG, Yanez D, Psaty BM, Fried LP, Heiss G, Lee M, Polak JF, Savage PJ. Fibrinogen and factor VIII, but not factor VII, are associated with measures of subclinical cardiovascular disease in the elderly: results from The Cardiovascular Health Study. Arterioscler Thromb Vasc Biol. 1995; 15: 1269–1279.
- ↵Tracy RP, Lemaitre RN, Psaty BM, Ives DG, Evans RW, Cushman M, Meilahn EN, Kuller LH. Relationship of C-reactive protein to risk of cardiovascular disease in the elderly: results from the Cardiovascular Health Study and the Rural Health Promotion Project. Arterioscler Thromb Vasc Biol. 1997; 17: 1121–1127.
- ↵Fishman D, Faulds G, Jeffery R, Mohamed-Ali V, Yudkin JS, Humphries S, Woo P. The effect of novel polymorphisms in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic-onset juvenile chronic arthritis. J Clin Invest. 1998; 102: 1369–1376.
- ↵Humphries SE, Luong LA, Ogg MS, Hawe E, Miller GJ. The interleukin-6 −174 G/C promoter polymorphism is associated with risk of coronary heart disease and systolic blood pressure in healthy men. Eur Heart J. 2001; 22: 2243–2252.
- ↵Fernandez-Real JM, Broch M, Vendrell J, Gutierrez C, Casamitjana R, Pugeat M, Richart C, Ricart W. Interleukin-6 gene polymorphism and insulin sensitivity. Diabetes. 2000; 49: 517–520.
- ↵Gardin JM, Wong ND, Bommer W, Klopfenstein HS, Smith VE, Tabatznik B, Siscovick D, Lobodzinski S, Anton-Culver H, Manolio TA. Echocardiographic design of a multicenter investigation of free-living elderly subjects: the Cardiovascular Health Study. J Am Soc Echocardiogr. 1992; 5: 63–72.
- ↵Newman AB, Siscovick DS, Manolio TA, Polak J, Fried LP, Borhani NO, Wolfson SK. Ankle-arm index as a marker of atherosclerosis in the Cardiovascular Health Study: Cardiovascular Heart Study (CHS) Collaborative Research Group. Circulation. 1993; 88: 837–845.
- ↵O’Leary DH, Polak JF, Wolfson SK, Bond MG, Bommer W, Sheth S, Psaty BM, Sharrett AR, Manolio TA. Use of sonography to evaluate carotid atherosclerosis in the elderly: the Cardiovascular Health Study CHS Collaborative Research Group. Stroke. 1991; 22: 1155–1163.
- ↵Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. Diabetes Care. 1997; 20: 1183–1197.
- ↵Furberg CD, Manolio TA, Psaty BM, Bild DE, Borhani NO, Newman A, Tabatznik B, Rautaharju PM. Major electrocardiographic abnormalities in persons aged 65 years and older (the Cardiovascular Health Study): Cardiovascular Health Study Collaborative Research Group. Am J Cardiol. 1992; 69: 1329–1335.
- ↵Kuller LH, Shemanski L, Psaty BM, Borhani NO, Gardin J, Haan MN, O’Leary DH, Savage PJ, Tell GS, Tracy R. Subclinical disease as an independent risk factor for cardiovascular disease. Circulation. 1995; 92: 720–726.
- ↵Cushman M, Cornell ES, Howard PR, Bovill EG, Tracy RP. Laboratory methods and quality assurance in the Cardiovascular Health Study. Clin Chem. 1995; 41: 264–270.
- ↵Kuller LH, Tracy RP, Shaten J, Meilahn EN. Relation of C-reactive protein and coronary heart disease in the MRFIT nested case-control study: Multiple Risk Factor Intervention Trial. Am J Epidemiol. 1996; 144: 537–547.
- ↵Rifai N, Ridker PM. High-sensitivity C-reactive protein: a novel and promising marker of coronary heart disease. Clin Chem. 2001; 47: 403–411.
- ↵Macy EM, Hayes TE, Tracy RP. Variability in the measurement of C-reactive protein in healthy subjects: implications for reference intervals and epidemiological applications. Clin Chem. 1997; 43: 52–58.
- ↵SPSS for Windows. Chicago, Ill: SPSS, Inc; 1999.
- ↵Mendall MA, Patel P, Asante M, Ballam L, Morris J, Strachan DP, Camm AJ, Northfield TC. Relation of serum cytokine concentrations to cardiovascular risk factors and coronary heart disease. Heart. 1997; 78: 273–277.
- ↵Tracy RP, Psaty BM, Macy E, Bovill EG, Cushman M, Cornell ES, Kuller LH. Lifetime smoking exposure affects the association of C-reactive protein with cardiovascular disease risk factors and subclinical disease in healthy elderly subjects. Arterioscler Thromb Vasc Biol. 1997; 17: 2167–2176.
- ↵Jones KG, Brull DJ, Brown LC, Sian M, Greenhalgh RM, Humphries SE, Powell JT. Interleukin-6 (IL-6) and the prognosis of abdominal aortic aneurysms. Circulation. 2001; 103: 2260–2265.