increased concentrations of c-reactive protein and il...

Increased Concentrations of C-Reactive Protein and IL-6but not IL-18 Are Independently Associated With Incident

Coronary Events in Middle-Aged Men and WomenResults From the MONICA/KORA Augsburg Case–Cohort

Study, 1984–2002

Wolfgang Koenig, Natalie Khuseyinova, Jens Baumert, Barbara Thorand, Hannelore Loewel,Lloyd Chambless, Christa Meisinger, Andrea Schneider, Stephan Martin, Hubert Kolb, Christian Herder

Objectives—We performed a prospective case–cohort study in initially healthy, middle-aged men and women from theMONICA/KORA Augsburg studies conducted between 1984 and 2002 to assess the role of IL-18 in comparison withIL-6 and CRP in the prediction of incident coronary heart disease (CHD).

Methods and Results—Concentrations of IL-18 were measured in 382 case subjects with incident CHD and 1980 noncases.Mean follow-up was 11 years. Baseline concentrations of IL-18 were slightly higher in cases than in noncases (172.4[1.0] versus 161.3 [1.0] pg/mL, respectively; P�0.114), but were clearly elevated for C-reactive protein (CRP) and IL-6in cases compared with noncases. In multivariable analyses, accounting for classical cardiovascular risk factors andinflammatory markers, no statistically significant association was seen between increased concentrations of IL-18 andincident CHD both in men (hazard ratio [HR] and 95% confidence intervals [CIs] comparing extreme tertiles, 1.20; 95%CI, 0.85 to 1.69), and in women (HR, 1.25; 95% CI, 0.67 to 2.34). However, in this population increased concentrationsof CRP and IL-6 were found to be independent predictors of future CHD events, even after multivariable adjustment.

Conclusions—Elevated concentrations of CRP and IL-6, but not IL-18, were independently associated with risk of CHDin subjects from an area with moderate absolute risk. (Arterioscler Thromb Vasc Biol. 2006;26:2745-2751.)

Key Words: case–cohort study � coronary heart disease � IL-18 � inflammation � risk factors

Cytokine-mediated inflammation accompanies atheroscle-rosis from its initiation to the occurrence of clinical

endpoints.1 Recently IL-18, a member of the IL-1 family ofcytokines, has been suggested to play a central role in theregulation of both innate and adaptive immunity.2–3 IL-18 iswidely expressed in monocytes/macrophages, dendritic cells,Kupffer cells, adipocytes, colon carcinoma cells, keratino-cytes, and osteoblasts.3–4 It is synthesized as a 23-kDabiologically inert precursor, which is further cleaved bycaspase 1 (or IL-1�–converting enzyme) to yield the matureand active 18.3-kDa glycoprotein.3,5 IL-18 inducesinterferon-� with subsequent promotion of the Th1 immuneresponse; it also enhances the expression of matrix metallo-proteases.6–7 These 2 abilities of IL-18 characterize it as acrucial and potent mediator of atherosclerotic plaque desta-bilization and vulnerability. Indeed, some experimental stud-ies demonstrated significantly increased expression of IL-18

in human atheroma, especially in lesions prone to rupture,where it is localized mainly in plaque macrophages.6,8 Ani-mal models further support the proatherogenic role of IL-18,demonstrating that endogenous inhibition of IL-18 by IL-18binding protein reduced atherosclerotic plaque developmentand progression in apolipoprotein (apo) E-deficient mice.9

Similarly, IL-18/apoE double knockout mice exhibited re-duced lesion size.10 In contrast, direct administration of IL-18enhanced atherogenesis in an interferon-�–dependent man-ner,11 even in the absence of T cells12 and promoted a switchto a vulnerable plaque phenotype by decreasing intimalcollagen content and cap-to-core ratio.13

Although the aforementioned experimental studies seem tobe relatively consistent, the results of only few clinical studiesremain somewhat controversial. While a number of recentcross-sectional studies favored a role of IL-18 in the devel-opment of plaque instability, showing significantly increased

Original received June 26, 2006; final version accepted September 13, 2006.From Department of Internal Medicine II-Cardiology (W.K., N.K.), University of Ulm Medical Center, Ulm, Germany; GSF National Research Centre

for Environment and Health (J.B., B.T., H.L., C.M., A.S.), Institute of Epidemiology, Neuherberg, Germany; University of North Carolina at Chapel Hill(L.C.), Chapel Hill, NC; German Diabetes Clinic (S.M., H.K., C.H.), German Diabetes Centre, Leibniz Centre at Heinrich-Heine-University Dusseldorf,Dusseldorf, Germany.

W.K. and N.K. contributed equally to this work.Correspondence to Dr Barbara Thorand, MPH; GSF National Research Center for Environment and Health, Institute of Epidemiology, Postfach 1129,

85758 Neuherberg, Germany. E-mail [email protected]© 2006 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000248096.62495.73

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levels of this cytokine in patients with acute coronarysyndrome (ACS),14,15 several other investigations were notable to demonstrate any meaningful relationship with coro-nary heart disease (CHD).16–17 Furthermore, in 1 prospectivestudy IL-18 predicted cardiovascular events in patients withmanifest CHD,18 and data in initially healthy subjects arescarce and need to be confirmed.19

Therefore, we conducted a prospective case–cohort20 studywithin the MONICA/KORA Augsburg platform to assess therole of IL-18 in the prediction of incident CHD (fatal andnonfatal myocardial infarction [MI] and sudden cardiacdeath) over a mean follow-up (FU) of 11 years.

MethodsStudy DesignThe design of this prospective case–cohort study conducted withinthe population-based MONICA/KORA Augsburg cohort has beenrecently described in detail.21

A combined endpoint that included incident fatal/nonfatal MI andsudden cardiac death occurring before the age of 75 years was usedas the outcome variable, and was identified through the MONICA/KORA Augsburg coronary event registry and through FU question-naires for subjects who had moved out of the study area. Because ofthe low incidence of CHD in those younger than 35 years, the presentstudy was limited to 10 718 persons (5382 men and 5336 women)between 35 to 74 years at baseline who participated in at least 1 ofthe 3 MONICA/KORA Augsburg surveys (S), conducted in 1984/1985 (S1), 1989/1990 (S2), and 1994/1995 (S3). After exclusion of1187 subjects with missing blood samples and 231 participants withself-reported prevalent CHD, the source population for the presentstudy comprised 9300 subjects (4507 men, 4793 women).

For the case–cohort study, a random sample of the sourcepopulation, called here the subcohort, containing 2163 subjects(1154 men, 1009 women) was selected stratifying by sex and survey.Participants with missing values for IL-18 or any of the covariablesused in the present analysis were excluded leading to a subcohort of2095 subjects (1101 men, 994 women). The final stratum-specificsample sizes were used together with the stratum-specific sizes of thecohort of interest to compute sampling fractions, and the inverse ofthe sampling fractions yielded the survey and sex-specific samplingweights: 3.01, 3.84, 6.25 for men, and 3.93, 4.76, 6.12 for women.

The number of cases of incident CHD until December 31, 2002was 397 (307 men, 90 women). For 295 men and 87 women,complete information on all relevant variables was available. Be-cause 92 male and 23 female cases were also part of the randomlydrawn subcohort, the present analysis comprised a total of 2362participants (295 men and 87 women with incident CHD, 1009 menand 971 women without incident CHD). For detailed information onthe study design please see the online data supplement(http://atvb.ahajournals.org).

Data Collection, Laboratory Measurements, andStatistical AnalysesMost aspects of data collection and laboratory measurements havebeen described before.21 For additional details and information onstatistical analyses, please see the online data supplement.

ResultsOverall, 2362 participants (382 subjects with incident CHDand 1980 subjects without incident CHD) of the 3 population-based MONICA/KORA Augsburg surveys were included inthis case–cohort study. The mean FU time (�SD) was 11.0(�5.0) years for the study population and ranged from 0.05 to18.2 years.

The baseline demographic, clinical, and laboratory charac-teristics of the study population are shown in Table 1.Subjects with incident CHD were older, more likely to becurrent or former smokers, were less active, and showed ahigher body mass index (BMI) and waist-to-hip ratio (WHR)compared with noncases. Furthermore, cases more frequentlyreported a history of diabetes mellitus and actual hyperten-sion, as well as a positive or unknown parental history of MI,whereas no significant differences were observed for educa-tional levels and alcohol consumption habits between the 2groups.

As expected, total cholesterol (TC) and low-density li-poprotein cholesterol (LDL-C) concentrations, and TC/high-density lipoprotein cholesterol (HDL-C) ratio were consider-ably higher and HDL-C was considerably lower in casescompared with noncases.

Although plasma concentrations of IL-18 at baselinetended to be slightly higher in CHD cases than in noncases,they did not differ statistically significantly between subjectswith incident CHD and those who remained free of an eventduring FU (weighted geometric means with antilog of stan-dard errors of log means); 172.4 (1.0) versus 161.3 (1.0)pg/mL, respectively; P�0.114) (Table 1). Geometric meansof IL-18 were 174.1 (1.1) pg/mL in cases and 162.4 (1.0)pg/mL in noncases for men (P�0.163) and 165.2 (1.1) pg/mLand 160.3 (1.0) pg/mL for women (P�0.712), respectively.Conversely, CRP and IL-6 concentrations were found to besignificantly increased in subjects with incident CHD com-pared with healthy individuals and that held true for bothgenders (CRP concentration for men: 2.5 [1.1] mg/L in casesand 1.5 [1.0] mg/L in noncases; P�0.001), for women, 2.8[1.1] versus 1.4 [1.0] mg/L (P�0.001), respectively; IL-6concentration for men: 3.0 [1.1] pg/mL in cases and 2.1 [1.0]pg/mL in noncases; P�0.001) and for women, 3.4 [1.1]versus 1.9 [1.0] pg/mL; P�0.001, respectively).

Pearson correlation coefficients (R) between log IL-18 andinflammatory markers, lipid variables or conventional riskfactors calculated in the randomly sampled subcohort of 2095individuals, revealed a weak positive, but statistically signif-icant, correlation between log IL-18 and log CRP (R�0.095,P�0.001), log IL-6 (R�0.085, P�0.001), and the TC/HDL-C ratio (R�0.061, P�0.006). Furthermore, a borderlinesignificant positive correlation between log IL-18 and WHR(R�0.071, P�0.005); diastolic blood pressure (BP)(R�0.055, P�0.012) and a negative correlation with HDL-C(R��0.063, P�0.004) were also observed. No statisticallysignificant correlations were seen with age, body mass index,TC, and LDL-C (data not shown).

Table 2 shows the results of Cox proportional hazardsanalysis, in which the association of baseline IL-18 concen-trations with incident CHD was assessed. In age and surveyadjusted analyses (Model 1), there was a statistically signif-icant association between increased concentrations of IL-18and incident CHD in men (HR and 95% CI comparing tertileextremes were 1.43; 95% CI, 1.05 to 1.96; P for trend 0.019,respectively), whereas the association of similar size inwomen (HR, 1.47; 95% CI, 0.87 to 2.48; P for trend 0.108,respectively) was not statistically significant. In multivariableanalyses, accounting for traditional cardiovascular risk fac-

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tors and TC/HDL-C ratio, this association was attenuated andno longer statistically significant in men (HR, 1.21; 95% CI,0.86 to 1.69; P for trend 0.241) and remained nonsignificantin women (HR, 1.23; 95% CI, 0.67 to 2.28; P for trend 0.461)(Model 2). Additional adjustment for the inflammatory mark-ers CRP and IL-6 (Model 3) did not have a further impact onthe relation between elevated IL-18 concentrations and risk ofsubsequent coronary events. Finally, when the prognosticvalue of IL-18 was assessed in the whole population(n�2362), the magnitude of such association was consistentwith that observed in men. A statistically significant associ-ation between increased IL-18 concentration (T3 versus T1)and incident CHD was found only in age and survey adjustedanalyses, whereas further adjustment for various traditionalcardiovascular risk factors, TC/HDL-C ratio and inflamma-tory biomarkers led to a loss of the statistical significance ofthe association (HR for T3 versus T1 in the fully adjustedmodel including CRP and IL-6: 1.22, 95% CI, 0.91 to 1.64, Pfor trend 0.164) (Table 2). Moreover, almost identical resultshave been obtained if risk estimates for the top tertile of theIL-18 distribution were compared with the combined first andsecond tertiles with a HR of 1.22 (95% CI, 0.95 to 1.57;P�0.11) in the fully adjusted model.

Taking into account that increased IL-18 concentrationsmight be related to more severe disease, we also assessed theassociation between this biomarker and fatal MI among 202cases (157 male and 45 female) who died from MI during 11years of FU and 1980 noncases. Although plasma concentra-tions of IL-18 tended to be slightly higher in fatal MI casescompared with nonfatal cases (175.8 versus 168.7 pg/mL);this difference was not statistically significant (P�0.59).Similarly, we found no statistically significant associationbetween increased IL-18 concentrations and incident fatal MIin multivariable analysis, although the HRs were slightlyhigher compared with those obtained in the whole studypopulation (HR, 1.39; 95% CI, 0.93 to 2.06 for T3 versus T1in the fully adjusted model including CRP and IL-6; P fortrend 0.11). Finally, no statistically significant associationswere seen between IL-18 concentrations and future coronaryevents, if IL-18 was used as a continuous (untransformed andlog-transformed) variable instead of tertiles (data not shown).

However, when we investigated the prognostic values ofCRP and IL-6 in this population (Figure), we found an�2-fold increase in coronary risk in men associated withCRP levels in the top tertile compared with the lowest tertileafter adjustment for a range of traditional cardiovascular riskfactors and the TC/HDL-C ratio (HR, 1.89; 95% CI, 1.28 to2.77; P�0.014, Model 2). This held true in further analysestaking into account a potent inflammatory marker such asIL-6 (HR, 1.87; 95% CI, 1.27 to 2.76; P�0.018, Model 3)(Figure, upper left panel). No statistically significant associ-ation was found in women with a multivariable adjusted HRof 1.35 (95% CI, 0.64 to 2.84; P�0.245) in Model 2 and a HRof 1.32 (95% CI, 0.62 to 2.81; P�0.283) in a modeladditionally adjusted for IL-6 (Figure, upper right panel).Conversely, increased levels of IL-6 revealed a strong andindependent association with first-ever coronary events inwomen after multivariable adjustment (HR, 2.99; 95% CI,1.38 to 6.49; P�0.009; Model 2) (Figure, lower right panel).

TABLE 1. Baseline Demographic, Clinical, and LaboratoryCharacteristics of the Participants With and Without IncidentCHD During Follow-Up

Characteristic CHD Cases Noncases P*

No. (women/men) 87/295 971/1009 —

Age (years) 57.3 (0.4) 52.3 (0.3) �0.001

Education (�12 years) 78.8 76.7 0.361

Smoking status �0.001

Current smoker 40.1 23.7

Former smoker 31.4 27.7

Never smoker 28.5 48.6

Frequency of exercise �0.001

Active 27.5 38.5

Inactive 72.5 61.5

Alcohol consumption† 0.195

0 g/d 28.0 31.7

�0 to 39.9/19.9 g/d 41.6 42.1

�40/20 g/d 30.4 26.3

Body mass index, kg/m2 28.5 (0.2) 27.1 (0.1) �0.001

Waist to hip ratio¶ 0.93 (0.005) 0.87 (0.002) �0.001

History of diabetes mellitus 18.1 4.8 �0.001

Parental history of MI 0.030

Positive 22.8 19.8

Unknown 25.4 20.9

Negative 51.8 59.3

History of actual hypertension 65.5 41.5 �0.001

Systolic blood pressure, mm Hg 142.6 (1.1) 133.5 (0.5) �0.001

Diastolic blood pressure mm Hg 83.5 (0.6) 81.5 (0.3) 0.004

Current HRT‡ 5.4 10.3 0.104

Current use of OC§ 0.0 14.3 –

Total cholesterol, mg/dL 258.0 (2.5) 237.1 (1.0) �0.001

LDL cholesterol, mg/dL� 170.0 (3.0) 146.8 (1.2) �0.001

HDL cholesterol, mg/dL 49.3 (0.8) 57.2 (0.4) �0.001

Ratio TC/HDL cholesterol 5.7 (0.1) 4.5 (0.04) �0.001

C-reactive protein, mg/L** 2.6 (1.1) 1.4 (1.0) �0.001

Interleukin-6, pg/mL** 3.1 (1.1) 2.0 (1.0) �0.001

Interleukin-18, pg/mL** 172.4 (1.0) 161.3 (1.0) 0.114

Survey �0.001

S1 38.5 28.0

S2 42.1 36.4

S3 19.4 35.5

Data are weighted percentages for categorical variables, weighted means(standard errors) for normally distributed continuous variables and **weightedgeometric means with (antilog of standard errors of log means) for skewedcontinuous variables.

*The t test for continuous variables and � 2 test for categorical variables.†Men: 0, �0 to 39.9 g/d, �40 g/d; Women: 0, �0 to 19.9 g/d, �20 g/d.‡Only for women aged �50 years (n�628) with no current use of oral

contraceptives.§Only for women aged �50 years (n�413) with no current hormone

replacement therapy.¶Only measured in participants of survey 2 and 3 (cases: n�234; noncases:

n�1259).�Only measured in participants of survey 2 and 3 (cases: n�235; noncases:

n�1263).Weights: cases�1; noncases�1/sampling fraction with sampling

fraction�subcohort/full cohort without cases for each gender and survey.OC indicates oral contraceptive; HRT, hormone replacement therapy.

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Further inclusion of CRP in the model did not affect itspredictive ability in women (HR, 2.94; 95% CI, 1.31 to 6.62;P�0.005; Model 3), whereas no statistically significantassociation was found in men, comparing the top tertile of theIL-6 distribution to the bottom tertile (HR, 1.42; 95% CI, 0.97to 2.07; P�0.266; Model 3) (Figure, lower left panel).Additional adjustment for IL-18, which was attempted in afinal model for CRP and IL-6, as expected, had no impact onthe observed HRs (data not shown).

Taking into account that IL-18 was previously consideredas a short-term or intermediate-term risk marker, we exam-ined the prognostic value of increased IL-18 concentrations inaccordance with the occurrence of a first coronary events overdifferent FU periods (1, 2, 3, 4, 5, and 6 years) and found thatmultivariable adjusted HRs estimated for various time pointswere similar to the HRs estimated for the full FU period (datanot shown).

DiscussionData from this large prospective case–cohort study did notshow a statistically significant independent association be-tween increased IL-18 concentrations and subsequent coro-nary events in apparently healthy, middle-aged subjects. Bycontrast, increased concentrations of CRP in men and IL-6 inwomen remained strong and independent predictors of CHDrisk in this population even after adjustment for traditionalcardiovascular risk factors, and for IL-6 in case of CRP andvice versa and thus confirmed results from various cohorts.

Our data on IL-18 are in contrast to 2 prospective cohortsreported in the literature.18,19 Results from 1 large prospectivestudy, conducted in 1229 patients with angiographicallyconfirmed CAD showed that increased concentrations ofIL-18 at baseline were associated with future cardiovascular(CV) death during a 3.9-year FU, even after adjustment forpotential confounders (HR, 3.3; 95% CI, 1.3 to 8.4; P�0.01).

TABLE 2. Hazard Ratios (95% CI) for the Risk of Incident CHD According to Baseline Concentration of IL-18

Tertiles of IL-18

T1 T2 T3 P for Trend

Men

Median (IQR), pg/mL 90.3 (63.6) 181.1 (35.8) 302.5 (127.6)

No. cases/noncases 89/331 85/333 121/345

HR (95% CI) adjusted for age and survey 1.00 (Ref.) 1.03 (0.74–1.43) 1.43 (1.05–1.96) 0.019

P — 0.859 0.024

HR (95% CI) adjusted for age, survey, and traditional CV risk factors* 1.00 (Ref.) 1.00 (0.71–1.42) 1.21 (0.86–1.69) 0.241

P — 0.999 0.271

HR (95% CI) additionally adjusted for CRP and IL-6 1.00 (Ref.) 1.00 (0.70–1.41) 1.20 (0.85–1.69) 0.266

P — 0.991 0.298

Women

Median (IQR), pg/mL 98.8 (47.4) 167.2 (34.1) 278.9 (104.6)


HR (95% CI) adjusted for age and survey 1.00 (Ref.) 0.93 (0.52–1.66) 1.47 (0.87–2.48) 0.108

P — 0.795 0.154

HR (95% CI) adjusted for age, survey, and traditional CV risk factors* 1.00 (Ref.) 0.99 (0.52–1.87) 1.23 (0.67–2.28) 0.461

P value — 0.967 0.501


P — 0.987 0.481

Total

Median (IQR), pg/mL 94.7 (56.0) 174.1 (36.7) 292.7 (116.5)


HR (95% CI) adjusted for age, gender, and survey 1.00 (Ref.) 1.01 (0.76–1. 34) 1.46 (1.11–1.91) 0.004

P value — 0.975 0.006

HR (95% CI) adjusted for age, gender, survey, and traditional CV risk factors* 1.00 (Ref.) 1.00 (0.73–1.35) 1.23 (0.92–1.65) 0.149

P — 0.978 0.169


P — 0.967 0.185

Hazard ratios were estimated by Cox proportional hazard model. Correction for standard errors was made by SAS macro ROBPHREG using themethod by Barlow.

Tertiles of the weighted distributions of IL-18 in the subcohort, stratified for gender, were used: men 139.2 and 222.4; women 133.8 and 208.9.*Adjustment for traditional cardiovascular (CV) risk factors includes adjustment for body mass index, smoking status, alcohol consumption, physical

activity, systolic blood pressure, TC/HDL-C, parental history of MI, and history of diabetes



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Such association held further true if the prognostic value ofincreased IL-18 on subsequent CV events was assessedseparately in patients with stable and unstable patients onadmission.18 However, when the FU was extended from 3.9to 5.9 years, IL-18 concentrations were no longer predictiveof outcome, thus questioning the value of IL-18 as anindependent risk marker for future CV events.22

These results, however, cannot be directly compared withour findings, because they were based on data from subjectswith pre-existing CHD. Our data, by contrast, is based on alarge prospective case– cohort study including initiallyhealthy subjects from a population based sample.

To date, the prognostic value of elevated IL-18 for futurecoronary events in apparently healthy subjects has beeninvestigated only in the PRIME study,19 representing a cohortof 10 600 middle-aged men without preexisting CHD, re-cruited from 2 European populations (French and Irishpopulations). Three hundred thirty-five subjects who experi-enced at least one CHD event (nonfatal MI, coronary death,and angina pectoris) during a 5-year FU were compared with670 age-matched controls. Baseline concentrations of IL-18were significantly higher among cases compared with thosewho remained free of disease (225.1 versus 203.9 pg/mL,P�0.005). Furthermore, elevated IL-18 concentrations wereassociated with a 2-fold increased risk for subsequent coro-nary events after multivariable adjustment, if the upper tertileof the IL-18 distribution was compared with the bottomtertile. However, such an association was statistically signif-icant only when data from both populations were pooled foranalysis. When the prognostic value of IL-18 was investi-gated separately in the French and the Belfast cohort, thestatistical significance of the association between increasingIL-18 tertiles and future coronary events was completely lostin France (for both, combined endpoints and for coronarydeath/MI), whereas in the Irish populations it even increased.One possible explanation for such discrepancy could bebecause of the fact that the French and the Belfast PRIME

cohorts represent heterogeneous populations. For instance,differences between these 2 cohorts with regard to theincidence of CHD are marked and have been reportedpreviously.23–24 Additionally, the distribution of several car-diovascular risk factors in the Irish PRIME population wasalso different from the French cohort and thus might account,at least in part, for the excess risk of CHD seen in Belfast.Indeed, several studies demonstrated that the Belfast partici-pants, as compared with the French, had much higher levelsof triglycerides and LDL-C and significantly lower concen-trations of HDL-C.23,25 In addition, increased concentrationsof several inflammatory biomarkers such as CRP, fibrinogen,plasminogen activator inhibitor-1, IL-6, and IL-18 were alsoobserved in Belfast as opposed to France.19,26–27 Therefore,the Irish PRIME population might represent a populationwith a high risk for CHD, whereas the French and the Germancohorts represent populations with moderate or even lowabsolute CHD risk. Thus, such existing differences couldexplain the observed discrepancies between our cohort andthe PRIME study.

Our study has several strengths that need to be mentioned.First, it represents a population-based prospective studyconducted in initially healthy subjects. Second, we minimizedthe likelihood of survival bias because fatal and nonfatalcoronary events were included in our study. Third, wecarefully adjusted for conventional risk factors by multivari-able methods.

The present study has also several limitations that shouldbe considered. The mean FU period of 11 years is relativelylong and might be responsible for the weakening of theassociation between the risk marker and the disease outcome.This, however, cannot completely explain the lack of asignificant association between IL-18 and coronary risk,because increased concentrations of CRP and IL-6 werestrongly and independently associated with future CHD, asseen in other cohorts. Moreover, in the same cohort increasedconcentrations of IL-18 strongly and independently predicted

Hazard ratios (95% confidence interval) forthe risk of incident CHD according to tertilesof CRP (upper panel) and IL-6 (lower panel)for men and women separately. Model 1,Adjusted for age and survey. Model 2,Adjusted for age, survey, body mass index,smoking status, alcohol consumption, physi-cal activity, systolic blood pressure,TC/HDL-C, parental history of myocardialinfarction and history of diabetes. Model 3,Additional adjustment for IL-6 (in case ofCRP) and for CRP (in case of IL-6). Tertile 1served as a referent tertile. Hazard ratioswere estimated by Cox proportional hazardmodel. Correction for standard errors wasmade by SAS macro ROBPHREG using themethod by Barlow. Tertiles of the weighteddistributions of CRP in the subcohort strati-fied for gender were used: men 0.88 and2.34; women 0.84 and 2.44 mg/L. Tertiles ofthe weighted distributions of IL-6 in the sub-cohort stratified for gender were used: men1.75 and 3.24; women 1.62 and 2.91 pg/mL.



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incident type 2 diabetes mellitus over 10.8 years.21 Further,the lack of association between increasing concentrations ofIL-18 and incident CHD in women should be interpreted withcaution because of the relatively low number of CHD eventsin this group (n�87). However, if multivariable analysis wasperformed in the total study population that had a power of97% to detect a HR of 1.5, almost identical results werefound, with similar hazard ratios as can be seen in Table 2.Moreover, testing for interaction of gender and IL-18 re-vealed P�0.7 in all 3 models, indicating no modification ofthe association between IL-18 and CHD by gender. Anotherpoint that may be mentioned here is that IL-18 might be moresuitable to predict short-term risk19 versus long-term risk.However, we did not find any significant association betweenincreased concentrations of IL-18 and occurrence of a firstcoronary event during short or intermediate FU periods aswell. Regarding the method of IL-18 detection, we used aLuminex assay with a somewhat higher interassay CV thanthe enzyme-linked immunosorbent assays used in previousstudies,18,19 which might have attenuated the associationbetween IL-18 concentrations and incident CHD slightly.Although higher quality assays might have provided similarHRs, CIs most probably would be narrower, however. An-other limitation of our study may result from the fact that onlymiddle-aged men and women of German nationality wereincluded. Thus, these data may not be extrapolated to otherpopulations, ethnic/racial minorities and other age-groups.

Despite these limitations, this study does not suggest anindependent, clinically relevant association between in-creased concentrations of IL-18 and risk of future CHD inapparently healthy subjects from an area with moderateabsolute risk of CHD. However, the lack of prognostic impactof increased IL-18 concentrations on the occurrence of futurecoronary events among apparently healthy subjects does notcompletely exclude the potential significance of IL-18–regulated mechanisms in the pathophysiology of athero-thrombotic disease, especially taking into account potentplaque destabilizing properties of this molecule. Moreover,IL-18 might serve as a marker of future cardiovascular eventsin men with manifest CHD and/or in areas of high absoluterisk of CHD. Thus, further studies are warranted to establishthe role of IL-18 in the prediction of CHD risk.

AcknowledgmentsWe thank all members of the GSF-Institute of Epidemiology whowere involved in the planning and conduct of the surveys and theMONICA/KORA myocardial infarction registry and survey teams(Augsburg). We also thank Gerlinde Trischler (University of Ulm),Gabi Gornitzka, Ulrike Poschen and Karin Rohrig (all from theGerman Diabetes Center) for excellent technical assistance andWilco de Jager (University Medical Center Utrecht/Immune Toler-ance Network (ITN) Luminex Core Facility, Netherlands) for hissupport with establishing the Luminex assay for IL-18 measure-ments. Finally, we express our appreciation to all study participants.

Sources of FundingThe KORA research platform and the MONICA Augsburg studieswere initiated and funded by the GSF-National Research Center forEnvironment and Health (GSF), Neuherberg, Germany and sup-ported by grants from the Federal Ministry of Education andResearch (FMER), Berlin, Germany. The present study was primar-

ily supported by a research grant from the German ResearchFoundation (TH-784/2-1). Additional funds were provided by theEuropean Foundation for the Study of Diabetes, the German Diabe-tes Center, the GSF and the FMER (NGFN-2).

DisclosuresNone.

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6. Gerdes N, Sukhova GK, Libby P, Reynolds RS, Young JL, Schonbeck U.Expression of Interleukin (IL)-18 and functional IL-18 receptor on humanvascular endothelial cells, smooth muscle cells, and macrophages: impli-cations for atherogenesis. J Exp Med. 2002;195:245–257.

7. Ishida Y, Migita K, Izumi Y, Nakao K, Ida H, Kawakami A, Abiru S,Ishibashi H, Eguchi K, Ishii N. The role of IL-18 in the modulation ofmatrix metalloproteinases and migration of human natural killer (NK)cells. FEBS Letters. 2004;569:156–160.

8. Mallat Z, Corbaz A, Scoazec A, Besnard S, Leseche G, Chvatchko Y,Tedgui A. Expression of Interleukin-18 in human atherosclerotic plaquesand relation to plaque instability. Circulation. 2001;104:1598–1603.

9. Mallat Z, Corbaz A, Scoazec A, Graber P, Alouani S, Esposito B,Humbert Y, Chvatchko Y, Tedgui A. Interleukin-18/Interleukin-18binding protein signaling modulates atherosclerotic lesion developmentand stability. Circ Res. 2001;89:e41–e45.

10. Elhage R, Jawien J, Rudling M, Ljunggren HG, Takeda K, Akira S,Bayard F, Hansson GK. Reduced atherosclerosis in interleukin-18deficient apolipoprotein E-knockout mice. Cardiovasc Res. 2003;59:234–240.

11. Whitman SC, Ravisankar P, Daugherty A. Interleukin-18 enhances ath-erosclerosis in Apolipoprotein E-/- mice through release of Interferon-�.Circ Res. 2002;90:e34–e38.

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13. de Nooijer R, von der Thusen JH, Verkleij CJ, Kuiper J, Jukema JW, vander Wall EE, van Berkel JC, Biessen EA. Overexpression of IL-18decreases intimal collagen content and promotes a vulnerable plaquephenotype in apolipoprotein-E-deficient mice. Arterioscler Thromb VascBiol. 2004;24:2313–2319.

14. Rosso R, Roth A, Herz I, Miller H, Keren G, George J. Serum levels ofinterleukin-18 in patients with stable and unstable angina pectoris. IntJ Cardiol. 2005;98:45–48.

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16. Narins CR, Lin DA, Burton PB, Jin Z-G, Berk BC. Interleukin-18 andinterleukin-18 binding protein levels before and after percutaneouscoronary intervention in patients with and without recent myocardialinfarction. Am J Cardiol. 2004;94:1285–1287.

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Christian HerderLloyd Chambless, Christa Meisinger, Andrea Schneider, Stephan Martin, Hubert Kolb and

Wolfgang Koenig, Natalie Khuseyinova, Jens Baumert, Barbara Thorand, Hannelore Loewel,2002−Cohort Study, 1984−rom the MONICA/KORA Augsburg Case

Associated With Incident Coronary Events in Middle-Aged Men and Women: Results FIncreased Concentrations of C-Reactive Protein and IL-6 but not IL-18 Are Independently

Print ISSN: 1079-5642. Online ISSN: 1524-4636 Copyright © 2006 American Heart Association, Inc. All rights reserved.

Greenville Avenue, Dallas, TX 75231is published by the American Heart Association, 7272Arteriosclerosis, Thrombosis, and Vascular Biology

doi: 10.1161/01.ATV.0000248096.62495.732006;

2006;26:2745-2751; originally published online September 28,Arterioscler Thromb Vasc Biol.

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Increased Concentrations of C-Reactive Protein and Interleukin-6 but not Interleukin-18 are

Independently Associated With Incident Coronary Events in Middle-aged Men and Women.

Results From the MONICA/KORA Augsburg Case-Cohort Study, 1984-2002

Wolfgang Koenig, Natalie Khuseyinova, Jens Baumert, Barbara Thorand, Hannelore Loewel, Lloyd

Chambless, Christa Meisinger, Andrea Schneider, Stephan Martin, Hubert Kolb, Christian Herder

ON-LINE SUPPLEMENT

Methods

Study Design

Based on data from the MONICA/KORA Augsburg studies we conducted a prospective case-cohort

study1 in initially healthy, middle-aged men and women. The MONICA Augsburg project (Monitoring of

Trends and Determinants in Cardiovascular Disease) was part of the multinational WHO MONICA

project2-3. Briefly, three independent population-based MONICA/KORA Augsburg surveys (S), with a

total number of 13,427 participants (6,725 men, 6,702 women) aged 25-64 (S1) or 25-74 years (S2-S3),

were conducted in 1984/85 (S1), 1989/90 (S2) and 1994/95 (S3). All subjects were prospectively

followed within the framework of KORA (Kooperative Gesundheitsforschung in der Region Augsburg).

The case-cohort design used in the present study has been recently described in detail4.

A combined endpoint that included incident fatal/non-fatal MI and SCD occurring before the age of 75

years was used as the outcome variable, and was identified through the MONICA/KORA Augsburg

coronary event registry and through FU questionnaires for subjects who had moved out of the study

area. Until December 2000, the diagnosis of a major non-fatal MI event was based on the MONICA

algorithm taking into account symptoms, cardiac enzymes and ECG changes. Since January 1, 2001 all

patients with MI diagnosed according to ESC (European Society of Cardiology) and ACC (American

College of Cardiology) criteria were included5-6. Deaths from MI were validated by autopsy reports,

death certificates, chart review, and information from the last treating physician. Due to the low

incidence of CHD under 35 years, the present study was limited to 10,718 persons (5,382 men and

5,336 women) between 35-74 years at baseline who participated in at least one of the three surveys.

After exclusion of 1,187 subjects with missing blood samples and 231 participants with self-reported

prevalent CHD, the source population for the present study comprised 9,300 subjects (4,507 men, 4,793

women).

For the case-cohort study, a random sample of the source population, called here the subcohort,

containing 2,163 subjects (1,154 men, 1,009 women) was selected stratifying by sex and survey.

Participants with missing values for IL-18 or any of the covariables used in the present analysis were

excluded leading to a subcohort of 2,095 subjects (1,101 men, 994 women). The final stratum-specific

sample sizes were used together with the stratum-specific sizes of the cohort of interest to compute

sampling fractions, and the inverse of the sampling fractions yielded the survey and sex specific

sampling weights: 3.01, 3.84, 6.25 for men, and 3.93, 4.76, 6.12 for women.

The number of cases of incident CHD until December 31st, 2002 was 397 (307 men, 90 women). For

295 men and 87 women complete information on all relevant variables was available. Since 92 male

and 23 female cases were also part of the randomly drawn subcohort, the present analysis comprised a

total of 2,362 participants (295 men and 87 women with incident CHD, 1,009 men and 971 women

without incident CHD).

Data collection

All participants completed a standardized questionnaire, including medical history, lifestyle, and drug

history. In addition, all subjects underwent a medical examination. Systolic and diastolic blood pressures

(BP), body height (m), body weight (kg), body mass index (BMI, kg/m2), smoking behavior, history of

diabetes, history of MI, alcohol consumption (g/d) were determined as described previously7. Obesity

was defined as a BMI ≥30 kg/m2. Hypertension was defined as BP values ≥140/90 mmHg and/or use of

antihypertensive medication given that the subjects were aware of being hypertensive. The number of

education years was calculated on the basis of the highest level of formal education completed. Leisure-

time physical activity was assessed by a four-level graded scale assessing sports activities during

summer and winter time (0, <1, 1-2, >2 hours/week)8. The study was approved by the local authorities.

Written informed consent was obtained from all subjects.

Laboratory methods

A non-fasting venous blood sample was obtained from all study participants at baseline. All samples

were stored at –80°C until further analysis.

Total (TC) and HDL cholesterol (HDL-C) were measured by routine enzymatic methods (CHOD-PAP;

Boehringer Mannheim, Mannheim, Germany). Serum concentrations of IL-18 were determined in single

measurements by Luminex technology using an antibody pair and recombinant IL-18 protein from MBL

(Nagoya, Japan). The assay was based on a protocol of de Jager et al9. The lower detection limit of IL-

18 in this assay was approximately 9.8 pg/mL. In general, the intra- and inter-assay coefficients of

variation (CV) of this assay are <10.0 and <25.0%, respectively. In this study, the median inter-assay

CV was 14.4% based on duplicate measurements of 61 random serum samples of cases and non-

cases.

Serum levels of IL-6 were determined using a previously described sandwich ELISA 10. In addition, CRP

serum concentrations were determined by a high-sensitive immunoradiometric assay (IRMA) using a 5-

point calibration with the WHO international reference standard 85/50611 (range, 0.05-10 mg/L) (S1:

men aged 45-64; S3) or a high sensitivity immunonephelometric assay (Dade Behring, Marburg,

Germany) on a BN II analyzer (S1: men aged 35-44 and all women; S2); both methods gave similar

results when the same samples were analyzed12. The intra- and inter-assay CV for CRP and IL-6 were:

CRP-IRMA: 4.0 and 12.0%, CRP immunonephelometry: 2.5 and 5.1%, IL-6: <10.0 and <10.0%. All

analyses were run in a blinded fashion.

Statistical analysis

Means or proportions for baseline demographic and clinical characteristics were computed using the

SAS procedures SURVEYREG or SURVEYFREQ which estimated standard errors appropriate to the

sampling scheme. In case of non-normality, tests were carried out with log-transformed variables and

results are presented as geometric means with antilogs of standard errors of the adjusted log means.

Associations between continuous variables were assessed by weighted Pearson Correlations with p

values obtained by SURVEYREG.

Cox proportional hazards analysis was used to assess the association between IL-18, CRP and IL-6

and incident CHD. Due to the case-cohort design, standard errors were corrected using a SAS macro

with a “sampling weight” approach developed by Barlow et al1 The weighted tertiles of IL-18, CRP and

IL-6 in the subcohort were used to classify subjects in different risk groups for each inflammation

parameter. In multivariable analyses, we adjusted for the continuous variables age, BMI, BP, TC/HDL-C

ratio, CRP, IL-6 and the categorical indicator variables sex, survey, smoking status (never smoker,

former smoker, current smoker), alcohol consumption (men 0, 0.1-39.9, ≥40 g/d; women 0, 0.1-19.9,

≥20 g/d), physical activity (inactive vs. active, i.e. regular physical activity of ≥1 hour/week in both

summer and winter), prevalent diabetes and parental history of CHD (negative, positive, unknown).

Results are presented for each inflammation parameter tertile (coded as dummy variables with the first

tertile as the reference category) as hazard ratios (HRs) and their 95% confidence intervals (CIs). P

values are based on robust variance estimates using the Barlow method. For test for trends, tertiles

were coded by their median values. Interactions between tertiles (coded as dummy variables) and other

risk factors were examined by additionally including respective interaction terms in the model. The

power estimation, computed by the method of Cai and Zheng13, revealed a power of 84% to detect an

HR of 1.35 and of 97% to detect an HR of 1.5 for subjects above the upper tertile of the IL-18

distribution compared to subjects below the upper tertile. For all statistical analyses p values were two-

tailed and values lower than 0.05 were considered to be statistically significant. All statistical analyses

were performed using the Statistical Analysis System (SAS) software package (Version 8.02 for Unix

and Version 9.1 for Windows, SAS-Institute Inc., Cary, NC, USA).

References

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MONICA Project Principal Investigators. J Clin Epidemiol. 1988;41:105-114.

3. WHO MONICA Project. WHO MONICA project: objectives and design. Int J Epidemiol.

1989;18 (suppl 1):29-37.

4. Thorand B, Kolb H, Baumert J, Koenig W, Chambless L, Meisinger C, Illig T, Martin S,

Herder C. Elevated levels of interleukin-18 predict the development of type 2 diabetes:

results from the MONICA/KORA Augsburg Study, 1984-2002. Diabetes. 2005;54:2932-

2938.

5. Luepker RV, Apple FS, Christenson RH, Crow RS, Fortmann SP, Goff D, Goldberg RJ,

Hand MM, Jaffe AS, Julian DG, Levy D, Manolio T, Mendis S, Mensah G, Pajak A, Prineas

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H; AHA Council on Epidemiology and Prevention; AHA Statistics Committee; World Heart

Federation Council on Epidemiology and Prevention; European Society of Cardiology

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7. Hense HW FB, Doering A, Stieber J, Liese A, Keil U. Ten-year trends of cardiovascular risk

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1989/1990, and 1994/1995 surveys. CVD Prevention.1998;1:318-327.

8. Koenig W, Sund M, Döring A, Ernst E. Leisure-time physical activity but not work-related

physical activity is associated with decreased plasma viscosity. Results from a large

population sample. Circulation. 1997;95:335-341.

9. De Jager W, te Velthuis H, Prakken BJ, Kuis W, Rijkers GT. Simultaneous detection of 15

human cytokines in a single sample of stimulated peripheral blood mononuclear cells. Clin

Diagn Lab Immunol. 2003;10:133-139.

10. Müller S, Martin S, Koenig W, Hanifi-Moghaddam P, Rathmann W, Haastert B, Giani G, Illig

T, Thorand B, Kolb H. Impaired glucose tolerance is associated with increased serum

concentrations of interleukin 6 and co-regulated acute-phase proteins but not TNF-alpha or

its receptors. Diabetologia. 2002;45:805-812.

11. Hutchinson WL, Koenig W, Frohlich M, Sund M, Lowe GD, Pepys MB. Immunoradiometric

assay of circulating C-reactive protein: age-related values in the adult general population.

Clin Chem. 2000;46:934-938.

12. Khuseyinova N, Imhof A, Trischler G, Rothenbacher D, Hutchinson WL, Pepys MB, Koenig

W. Determination of C-reactive protein: comparison of three high-sensitivity immunoassays.

Clin Chem. 2003;49:1691-1695.

13. Cai J, Zeng D. Sample size/power calculation for case-cohort studies. Biometrics.

2004;60:1015-1024.

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