Download C-Reactive Protein, Interleukin-6, and Fibrinogen as Predictors of

C-Reactive Protein, Interleukin-6, and Fibrinogen as
Predictors of Coronary Heart Disease
The PRIME Study
Gérald Luc, Jean-Marie Bard, Irène Juhan-Vague, Jean Ferrieres, Alun Evans, Philippe Amouyel,
Dominique Arveiler, Jean-Charles Fruchart, Pierre Ducimetiere on behalf of the PRIME Study Group
Downloaded from http://atvb.ahajournals.org/ by guest on October 2, 2016
Objective—This study was undertaken to examine the association of plasma inflammatory markers such as C-reactive
protein (CRP), interleukin-6, and fibrinogen with the incidence of coronary heart disease within the prospective cohort
study on myocardial infarction (PRIME study).
Methods and Results—Multiple risk factors were recorded at baseline in 9758 men aged 50 to 59 years who were free of
coronary heart disease (CHD) on entry. Nested case-control comparisons were carried out on 317 participants who
suffered myocardial infarction (MI)-coronary death (n⫽163) or angina (n⫽158) as an initial CHD event during a
follow-up for 5 years. After adjustment for traditional risk factors, incident MI-coronary death, but not angina, was
significantly associated with CRP, interleukin-6, and fibrinogen, but only interleukin-6 remained significantly associated
with MI-coronary death when the 3 inflammatory markers were included in the model. The different interleukin-6 levels
in Northern Ireland and France partly explained the difference in risk between these countries. Interleukin-6 appeared
as a risk marker of MI-coronary death, and it improved the definition of CHD risk beyond LDL cholesterol.
Conclusions—This association may reflect the underlying inflammatory reaction located in the atherosclerotic plaque or
a genetic susceptibility on the part of CHD subjects to answer a proinflammatory stimulus and subsequent increase in
hepatic CRP gene expression. (Arterioscler Thromb Vasc Biol. 2003;23:1255-1261.)
Key Words: coronary heart disease 䡲 C-reactive protein 䡲 interleukin-6 䡲 fibrinogen
E
concentrations may be subject to posttranscription regulation,
they may not reflect all of the relevant effectors of
inflammation.16
Interleukin 6 (IL-6) is the major initiator of acute phase
response by hepatocytes and a primary determinant of hepatic
CRP production,17,18 as suggested by IL-6 – deficient animals
showing impaired acute phase reaction.19 Experimental studies indicate that vascular endothelial and smooth muscle cells
produce IL-620 –26 and that IL-6 gene transcripts are expressed
in human atherosclerotic lesions.22,27,28 Given the role of IL-6
in CRP regulation and the hypothesis that atherosclerosis
fundamentally represents a chronic inflammatory disorder,1,2
the predictive value of IL-6 for cardiovascular ischemic
events was evaluated in a prospective cohort study and IL-6
was associated with increased risk of future myocardial
infarction (MI) in healthy middle-aged men.29 However,
several of the participants in this study were taking the
anti-inflammatory drug aspirin at the time of blood sampling,
vidence now indicates that inflammation contributes
considerably to the initiation and progression of atherosclerosis,1,2 and histopathological and immunochemical observations suggest that active inflammatory processes may
trigger plaque rupture and enhance the risk of coronary
thrombosis leading to a clinical ischemic event.3 Inflammation is characterized by a local reaction that may be followed
by the activation of an acute phase reaction.4 Some systemic
inflammatory markers can indicate the severity of inflammation, and their levels have actually been associated with
coronary disease. Fibrinogen, which was previously recognized as an independent coronary heart disease (CHD) risk
factor,5–7 is now considered an inflammatory marker and not
only a coagulation component.8 Another acute phase reactant
such as C-reactive protein (CRP) has also been demonstrated
to be an independent cardiovascular risk factor in prospective
population-based studies.8 –14 However, fibrinogen and CRP
are products of acute phase reaction,15 and because their
Received November 18, 2002; revision accepted May 14, 2003.
From the Department of Atherosclerosis (G.L., J.-C.F.), SERLIA-INSERM UR545, Institut Pasteur de Lille and University Lille II, France; U.F.R. of
Pharmacy (J.-M.B.), INSERM UR539, Nantes, France; Department of Hematology (I.J.-V.), Hôpital de la Timone, Marseille, France; Toulouse MONICA
Project (J.F.), INSERM U588, Department of Epidemiology, Paul Sabatier-Toulouse Purpan University, Toulouse, France; Department of Epidemiology
and Public Health (A.E.), Queen’s University Belfast, Northern Ireland; Lille Monica Project (P.A.), INSERM U508, Pasteur Institute of Lille, France;
Strasbourg MONICA Project (D.A.), Department of Epidemiology and Public Health, Faculty of Medicine, Strasbourg, France; and Coordinating Center
(P.D.), INSERM U258, Hôpital Paul Brousse, Villejuif, France.
Correspondence to Gérald Luc, Department of Atherosclerosis, SERLIA-INSERM UR545, Institut Pasteur de Lille, 1 Rue du Professeur Calmette,
59019 Lille Cedex, France. E-mail [email protected]
© 2003 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
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DOI: 10.1161/01.ATV.0000079512.66448.1D
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Arterioscler Thromb Vasc Biol.
July 2003
a drug known to affect IL-6 levels30 and so inducing a
potential bias.
The Prospective Epidemiological Study of Myocardial
Infarction (PRIME) study is a cohort study set up to prospectively investigate the association of different risk factors and
CHD simultaneously in France and Northern Ireland.31 Although several prospective cohort studies have evaluated the
role of plasma inflammatory markers such as CRP, IL-6, and
fibrinogen in predicting CHD risk, none has simultaneously
analyzed these markers to determine the most predictive ones.
Furthermore, their association has been evaluated with MI
and coronary death incidence, but none used angina pectoris
as an end point. In this work, we have studied the value of
CRP, IL-6, and fibrinogen in predicting CHD risk in the
PRIME prospective cohort according to the type of first
clinical event during follow-up: MI-coronary death on the
one hand, and angina on the other hand.
Downloaded from http://atvb.ahajournals.org/ by guest on October 2, 2016
Methods
The PRIME study has been described in great detail.32 The PRIME
study is a prospective cohort study that was set up to investigate risk
factors of ischemic heart disease. From 1991 to 1994, 10 600 men
aged 50 to 59 years living in France and Northern Ireland were
included. On entry, nurses distributed questionnaires, made physical
measurements, recorded ECGs, and analyzed them following the
Minnesota code.
In the morning between 8 and 10 AM after a 12-hour fast, blood
samples were taken and placed in tubes containing EDTA. Plasma
was separated by centrifugation at 4°C within 15 minutes at each
clinic. Aliquots of plasma were immediately frozen at ⫺80°C for
measurements of CRP, IL-6, and fibrinogen. These samples were
sent weekly by air (with the exception of those from the Lille Center)
to the Central Laboratory at the Pasteur Institute of Lille, where they
were stored in liquid nitrogen until analysis. All samples were treated
in exactly the same way (delay, temperature) whatever the center.
Other analyses such as lipid measurements were carried out by usual
methods as previously described.32,33 Additional questionnaires were
posted or phoned to participants every year over 5 years (98.5%
response). For subjects reporting a possible clinical event, clinical
information was sought directly from the hospital or general practitioners’ files. All details of ECG, hospital admissions, enzymes,
surgical operations, angioplasty, and treatment were collected and
classified according to MONICA criteria.34 Death certificates were
also used to complete information on the cause of death.
A medical committee was established to provide independent
validation and classification of coronary events. CHD categories
retained for analysis were nonfatal MI or coronary death and angina
at the first event.31 The former category included subjects who had
had at least 1 nonfatal MI or who died from CHD during follow-up.
MI was defined by at least 1 of the following sets of conditions: (1)
new diagnostic Q wave or another typical aspect of necrosis at ECG;
(2) typical or atypical pain symptoms and new (or increased)
ischemia at ECG and a myocardial enzyme level higher than twice
the upper limit; or (3) postmortem evidence of recent MI or
thrombosis. Definite coronary death was defined as death with a
documented coronary event. When a coronary death was suspected
with no other documentation or explanation, it was classified as
possible coronary death. Sudden death was defined as death occurring within 1 hour after the onset of symptoms without explanation.
However, when significant coronary atheroma was present at autopsy, death was considered as definite coronary death. The 3 death
categories were grouped together as coronary deaths. Angina pectoris was defined by the presence of chest pain at rest or on exertion
and 1 of the following criteria: (1) angiographic stenosis greater than
50%; (2) a positive scintigraphy (if no angiographic data); (3)
positive exercise stress test (if no angiographic or scintigraphic data);
or (4) ECG changes at rest (if no angiographic, scintigraphic, or
exercise stress test data) but without any set of conditions for MI and
no evidence of a noncoronary cause in the clinical history. Unstable
angina was defined as a crescendo pain (change in frequency or
severity of chest pain on exertion or appearance of chest pain at rest
following preexisting pain on exertion) or chest pain at rest with
either enzyme changes or ischemic ECG changes. In the absence of
enzyme or ECG data, the diagnosis was rejected.
The number of subjects lost to follow-up, ie, those who could not
be contacted in the fifth year of surveillance or who refused to
participate any longer in the study at any time during the follow-up,
was 228. As LDL cholesterol used in statistical analysis was
calculated according to the Friedewald formula,35 subjects with
triglycerides up to 400 mg/dL (n⫽217) were excluded. Furthermore,
only subjects who were without any history of CHD on entry were
included in this study. Therefore, the number of subjects free of CHD
on entry and not lost to follow-up was 9758, 7359 living in France
and 2399 in Northern Ireland.
To evaluate CRP, IL-6, and fibrinogen as markers of coronary risk
in the PRIME study, assays were made on baseline plasma samples
of the 320 study participants who subsequently developed a coronary
ischemic event during follow-up and from 2 controls per case.
Matched controls were study participants recruited in the same center
and on the same day (⫾2 days) as the corresponding case and were
free of CHD at the date of the ischemic event of the case.
CRP was measured by immunonephelometry (Dade Behring),
IL-6 by ELISA (R&D Systems) according to the instructions
available from the supplier, and fibrinogen according to the method
of Clauss.36 CRP, IL-6, and fibrinogen were measured in different
central laboratories, at the University Hospital of Nantes, Pasteur
Institute of Lille, and Laboratory of Hemostasis of La Timone
Hospital in Marseille, France, respectively. Plasma samples were
sent from the central plasma bank (Lille) to each laboratory in dry
ice. For all 3 parameters, measurements were carried out as batch
analyses. Accuracy and precision were assured by a strict internal
quality control program using quality control from the supplier
(CRP) or a single batch of normal plasma pooled from 50 healthy
subjects. The coefficients of variation were 4.4%, 7.8%, and 4.3%
for CRP, IL-6, and fibrinogen, respectively. Laboratory personnel
was unaware of case or control status.
Statistical Analysis
All statistical analyses were carried out using the statistical SAS
package (SAS Institute). Values of continuous variables are expressed as mean⫾SD, but the median value of triglycerides, CRP,
IL-6, and fibrinogen are given because of their rightward skewed
distribution. A conditional logistic regression analysis suitable for a
nested case-control design was performed to identify discriminating
predictive parameters. The same type of analysis was used to
determine the relative risks of future CHD event after controlling for
the presence of diabetes, hypertension, or smoking and possibly for
LDL cholesterol, HDL cholesterol, and triglyceride levels. Relative
risks related to IL-6 and LDL cholesterol distribution among controls
were assessed using conditional logistic regression analysis after
controlling for nonlipid risk markers, HDL cholesterol, and triglycerides. Correlations between continuous variables were calculated
using Spearman’s rank correlation coefficients. All tests were considered significant at the 0.05 level.
Results
The characteristics and biological values of 317 cases and 609
controls included in the nested case-control study are presented
in Table 1. Compared with their matched controls without CHD
events, the subjects with incident CHD during the 5-year
follow-up were of similar age. As expected, body-mass index
(BMI), cholesterol, LDL cholesterol, and triglycerides were
significantly higher in the case group, whereas HDL cholesterol
was lower. Furthermore, the prevalence of smoking, hypertension, and diabetes was also higher in cases (Table 1). In these
Luc et al
TABLE 1.
Baseline Patient Characteristics of Participants
Inflammatory Markers as CHD Risk Factors
TABLE 2. Spearman’s Rank Correlation Coefficient Between
CRP, IL-6 and Fibrinogen With Age, BMI and Lipid Parameters
Cases (n⫽317)
Controls (n⫽609)
P
Age, y
55.3⫾2.9
55.2⫾2.7
NS
BMI, kg/m2
27.3⫾3.6
26.7⫾3.5
0.008
Age
0.09†
0.08*
0.11‡
0.24‡
0.13‡
0.05
0.04
0.00
0.09†
CRP
IL-6
Fibrinogen
Cholesterol, mg/dL
234⫾39
223⫾41
0.0003
BMI
LDL cholesterol, mg/dL
154⫾35
144⫾37
0.0001
Cholesterol
HDL cholesterol, mg/dL
44⫾13
47⫾13
0.0002
LDL cholesterol
0.00
⫺0.03
0.0001
HDL cholesterol
⫺0.22‡
⫺0.20‡
⫺0.15‡
0.01
ApoA1
⫺0.14‡
⫺0.14‡
⫺0.14‡
0.13‡
0.17‡
1
...
0.53‡
0.52‡
1
0.39‡
ApoA1, mg/dL
Triglycerides, mg/dL
141⫾23
147⫾24
137 (99 to 1.96)
125 (02 to 179)
Current smokers, %
56
41
0.01
Triglycerides
Hypertension, %
27
15
0.0001
CRP
IL-6
Diabetes mellitus, %
10
6
0.004
CRP, mg/L
2.00 (0.77 to 3.59)
1.33 (0.64 to 2.70)
0.0001
IL-6, pg/mL
1.58 (1.04 to 2.62)
1.25 (0.84 to 1.98)
0.0001
340 (290 to 415)
314 (275 to 372)
0.0003
Fibrinogen, mg/dL
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Mean⫾SD is presented for age, BMI, cholesterol, and LDL and HDL
cholesterol; median (25th to 75th percentile) for triglycerides, CRP, IL-6, and
fibrinogen. The comparison between cases and controls was performed by
using conditional logistic regression analysis. NS indicates not significant
(P⬎0.05).
univariate analyses, CRP, IL-6, and fibrinogen were significantly higher in cases than in controls.
Correlations between inflammatory markers and anthropometric or lipid parameters shown in Table 2 were calculated
over the whole group of subjects because they were similar to
those obtained separately in cases and controls. CRP and IL-6
were positively correlated with BMI and triglycerides and
inversely with HDL cholesterol. No correlation was noted
between CRP or IL-6 and total cholesterol or LDL cholesterol. Fibrinogen was positively and moderately correlated
with age and LDL cholesterol and inversely with HDL
cholesterol but not with triglycerides. Thus, less than 5% of
the variance in the levels of these inflammatory marker levels
was determined by lipid factors. There was a strong mutual
correlation between inflammatory markers, as for instance
CRP with IL-6 (r⫽0.53).
The Prime Medical Committee enabled us to divide cases
into the 2 categories of MI and coronary death (n⫽163) and
angina pectoris (n⫽158) according to the first occurrence of
the disease. The sum of the numbers of the 2 clinical
categories is higher than the total number of cases, because 4
cases had angina before MI and were included in both
analyses, MI-coronary death, and angina. Medians of plasma
levels for MI-coronary death and angina, respectively, were
CRP, mg/L
⫹0.050 (0.019)
6.63
IL-6, pg/mL
⫹0.200 (0.059)
11.58
Fibrinogen, mg/dL
⫹0.00291 (0.00107)
4.51
0.04
2.00 and 1.92 mg/L for CRP, 1.65 and 1.29 pg/mL for IL-6,
and 353 and 329 mg/dL for fibrinogen. These levels were
separately compared with control subjects using conditional
logistic regression after adjustment for nonlipid (diabetes,
hypertension, smoking) and lipid (LDL-cholesterol, HDLcholesterol, triglycerides) risk factors in each of the 2 clinical
categories. CRP, IL-6, and fibrinogen were significantly
associated with the appearance of future MI-coronary death
events (Table 3). On the contrary, none of these parameters
was significantly associated with angina pectoris. However,
the comparison of ␤ regression coefficients computed in the
2 clinical categories for each parameter showed that only IL-6
was differentially associated with MI-coronary death and
angina events (Z score⫽2.21, P⬍0.05).
Tertiles of CRP, IL-6, and fibrinogen were derived from
the distribution of control subjects and used to model the risk
of MI-coronary death using stratified conditional logistic
regression after adjustment for nonlipid and lipid parameters
(LDL cholesterol, HDL cholesterol, and triglycerides). Increases in CRP, IL-6, and fibrinogen levels were significantly
associated with an increase in coronary event risk (Table 4).
The linear trend test for all 3 parameter levels was highly
significant in all models. Moreover, Table 4 shows that the
risk of MI-coronary death was considerably higher in the
second and third tertiles than for those of CRP and fibrinogen.
To evaluate whether CRP, IL-6, and fibrinogen were
independent markers of MI-coronary death, their levels were
introduced into a set of conditional logistic regression analyses with age, presence of diabetes, smoking, and high blood
pressure in model 1 and the same parameters plus LDL
cholesterol, HDL cholesterol, and triglycerides in model 2. In
MI-Coronary Death
Wald ␹2
0.08*
*P⬍0.05; †P⬍0.01; ‡P⬍0.0001; otherwise not significant.
TABLE 3. Univariate Conditional Logistic Regression of CHD Risk on
Inflammatory Parameters
Logistic Regression
Coefficient (SD)
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Angina Pectoris
P
Logistic Regression
Coefficient (SD)
Wald ␹2
P
0.01
0.047 (0.025)
3.60
0.06
0.0007
0.038 (0.044)
0.77
NS
0.04
0.00240 (0.00145)
2.72
NS
Subjects with MI or coronary death on the one side, angina pectoris on the other side were separately compared
with controls.
1258
Arterioscler Thromb Vasc Biol.
July 2003
TABLE 4. Relative Risk (RR) of Future MI-Coronary Death Among Apparently
Healthy Men Included in the Prime Study According to Tertiles of Baseline
Plasma Concentrations of CRP, IL-6, and Fibrinogen
Tertile
1
CRP, mg/L
⬍0.75
RR (95% CI)
P value
1.0
IL-6, pg/mL
⬍0.93
Fibrinogen, mg/dL
ⱖ0.75-⬍1.97
ⱖ1.97
0.81 (0.47 to 1.40)
2.16 (1.26 to 3.72)
NS
0.005
ⱖ0.93 to ⬍1.50
ⱖ1.50
3.10 (1.77 to 5.44)
1.0
0.02
0.0001
⬍290
ⱖ290 to ⬍350
ⱖ350
1.09 (0.63 to 1.89)
2.02 (1.19 to 3.42)
NS
0.009
RR (95% CI)
P value
3
1.97 (1.11 to 3.50)
RR (95% CI)
P value
2
1.0
P for Linear Trend
0.002
⬍0.0001
0.008
The analysis was performed after adjustment for age, diabetes, smoking, hypertension, LDL
cholesterol, HDL cholesterol, and triglycerides.
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both models, IL-6 level seemed to be an independent risk
factor for MI-coronary death, unlike CRP and fibrinogen
(Table 5).
Because LDL cholesterol was a strong lipid risk marker for
CHD and IL-6 seemed to be the most discriminating risk
marker among the 3 inflammatory parameters that were
analyzed in this study, we assessed their bivariate relationship
to the risk of MI-coronary death. To do so, we divided the
sample into 9 subgroups defined by the tertiles of the
distribution level of controls, the limit values being 127 and
159 mg/dL and 0.93 and 1.58 pg/mL for LDL cholesterol and
IL-6, respectively. As expected, the risk increased in each
IL-6 tertile along with the increase in LDL cholesterol
(Figure). Relative risk also increased with IL-6 in each LDL
cholesterol tertile with the exception of the subgroup of
subjects with the highest levels of IL-6 and LDL cholesterol,
even if the calculated relative risk of this subgroup remained
clearly higher than that of subjects with low LDL cholesterol
and IL-6. The risk was particularly high (⬎6- to 10-fold) in
subjects with both high LDL cholesterol and IL-6 compared
with subjects with low values for both.
The analysis of MI-coronary death risk according to IL-6
level after adjustment for nonlipid and lipid variables was
performed separately on French and Northern Irish subjects.
The respective ␤ regression coefficients, 0.251⫾0.089
(P⫽0.005) and 0.170⫾0.081 (P⫽0.03), were not significantly different (Z score⫽0.36; P⬎0.05). The hazard ratio for
MI or coronary death between Northern Irish and French men
in their fifties was estimated at 1.79.31 Because the mean
value of IL-6 is higher in Northern Irish controls than in
French ones (2.06⫾0.18 [SEM] pg/mL versus 1.58⫾0.08
pg/mL), we might speculate that an IL-6 increase, if causal,
might partly explain the between-country difference in coronary risk. Using ␤ regression coefficient for IL-6 in the
multivariate analysis of risk, we estimated that the adjusted
hazard ratio attributable to higher IL-6 in Northern Ireland
compared with France was 1.10 (95% CI, 1.04 to 1.38),
which represents approximately 13% of the marginal excess
relative risk between the 2 countries. LDL cholesterol levels
were also different in controls in the 2 countries, 149 and 141
mg/dL in Northern Ireland and France, respectively. This
difference explains approximately 10% of the marginal excess relative risk between the 2 countries.
TABLE 5. Multivariate Conditional Logistic Regression of
MI-Coronary Risk on Inflammatory Parameters
Logistic Regression
Coefficient (SD)
Wald ␹2
CRP
⫹0.018(0.021)
0.8
NS
IL-6
⫹0.165(0.066)
6.3
0.01
Fibrinogen
⫹0.043(0.130)
0.11
NS
CRP
⫹0.011(0.022)
0.25
NS
IL-6
⫹0.152(0.063)
5.87
0.02
Fibrinogen
⫹0.090(0.122)
0.55
NS
P
Model 1
Model 2
In model 1, CRP, IL-6, and fibrinogen were included after adjustment for
nonlipid parameters (age, diabetes, smoking, high blood pressure). In model 2,
the same nonlipid parameters as in model 1 were included plus LDL
cholesterol, HDL cholesterol, and triglyceride levels.
Relative risk of MI or coronary death according to IL-6 and LDL
cholesterol. The PRIME study. The relative risk was arbitrary
fixed at 1 (reference) for subjects with LDL cholesterol and IL-6
lower than 127 mg/dL and 0.93 pg/mL, respectively. *P⬍0.05;
**P⬍0.01; ***P⬍0.001.
Luc et al
Discussion
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Prospective data from the PRIME population– based study
reported in the present paper shows that in apparently healthy
men, baseline plasma concentrations of CRP, IL-6, and
fibrinogen are predictive of the risk of a first coronary heart
ischemic event. However, these 3 risk markers are strongly
correlated with each other, and IL-6 appears as the most
discriminating marker. Moreover, IL-6 is associated with
MI-coronary death but not with angina end points. Finally,
IL-6 improves the prediction of CHD risk when this parameter is added to models already including CRP or LDL
cholesterol.
Prospective data on IL-6 and CHD risk are limited. Ridker
et al29 observed the same predictive value of IL-6 in the
Physician’s Health Study as in the Prime Study, where IL-6
remained significantly associated with CHD risk after adjustment for CRP. These results also concord with the finding
that IL-6 is a marker of mortality in the elderly.37 The present
study is the first study devoted to examining the predictive
value of such parameters in nontreated patients, whereas
participants in the Physician’s Health Study were treated with
aspirin, which decreases IL-6 level.30
IL-6 is secreted by macrophages and smooth muscle cells
present in the atherosclerotic lesion, and so the IL-6 plasma
level could reflect the extent of inflammatory reactions in the
atherosclerotic vessels.8 This could explain why IL-6 is a
predictive factor of MI-coronary death but not of angina.
Indeed, most coronary thromboses responsible for fatal and
nonfatal MI result from a thrombus overlying the protective
fibrous caps of the fissured plaque,38 with a now-recognized
inflammatory phenomenon playing a decisive role. In subjects with MI-coronary death, there is a more intense process
of lesions in transition from clinically stable to unstable
atherosclerotic plaques,39 whereas the absence of elevated
IL-6 in patients with angina corresponds to an anatomical
aspect of severely stenotic plaques, which tend to be fibrotic
and stable with low inflammatory components.40
In both the present study and the Physician’s Health
Study,29 a high IL-6 level was present in individuals several
years before the occurrence of the ischemic event. Because
plaque rupture is an acute phenomenon, it suggests that the
inflammatory process at the origin of fissuration or rupture
could appear a relatively short time before the acute clinical
event. However, Ojio et al41 have recently shown that
considerable time elapses between the onset of plaque rupture
and the onset of MI. Indeed, most fissures reseal and
incorporate thrombus at the same time but do not produce
clinical symptoms.42 Therefore, these data suggest that subjects suffering MI-coronary death are likely to have an
intensive and perhaps prolonged inflammatory reaction in the
artery wall.
Besides its expression and secretion by arterial macrophages present in the atherosclerotic plaque, IL-6 is also
known to be produced by adipose tissue.43 This observation
explains the relationship between plasma IL-6 levels and
anthropometric measurements such as BMI and markers
associated with the insulin resistance syndrome.44 The higher
adipose tissue mass in cases rather than in controls (as noted
by their respective BMI [Table 1]) can partly explain the IL-6
Inflammatory Markers as CHD Risk Factors
1259
increase in cases compared with controls, but the moderate
difference in BMI disappears in multivariate analysis and
cannot entirely explain IL-6 difference. Furthermore, BMI
was similar in subjects with MI-coronary death and in those
with angina, whereas IL-6 was higher in MI-coronary death
than in angina cases. It can be hypothesized that IL-6 is
expressed to a greater extent by cells in subjects with
MI-coronary death than in subjects with angina, possibly
because of a different gene-environment interaction45 or
greater genetic susceptibility in CHD subjects to have a
strong immunological activation in response to a proinflammatory stimulus. A retrospective case-control study on MI
(ECTIM study)46 established an association between an IL-6
genetic polymorphism and MI, which concords with our
hypothesis.
CRP has been measured in several prospective studies of
fatal and nonfatal MI. A meta-analysis of 14 available
prospective studies of CRP has given a combined risk ratio of
1.9 (95% CI, 1.5 to 2.3) in individuals in the top third
compared with those in the bottom third of baseline measurements,47 a relative risk similar to that observed in the present
study (1.92; 95% CI, 1.14 to 3.22; data not shown). As in
several prospective studies, fibrinogen was a CHD factor
independently of lipid and nonlipid risk factors.48 The difference between MI-coronary death and stable angina had
already been observed for CRP and fibrinogen in one casecontrol study48 but not in another.49 This suggests that the
chronic inflammatory component of atherosclerotic lesions
might be less pronounced in subjects with angina and much
more intense in subjects with plaques prone to instability and
consequently likely to induce MI-coronary death.
There are potential limitations to our study. First, we
cannot exclude the possibility that protein degradation appeared during storage and affected the results, even if plasma
were stored at very low temperature (⫺196°C). However,
inflammatory marker levels measured in the present study are
similar to those reported in previous ones that used fresh
plasma samples, and the analysis of longitudinal stability of
several risk factors including CRP and fibrinogen in plasma
kept at ⫺70°C for 5 years has shown no sample degradation
over time.50 Furthermore, even if protein degradation appeared in our study, this effect could not have led to any
systematic bias, because samples from case and control
subjects were handled identically throughout the procedure
from blood drawing to analytical analysis. Also, first clinical
events were as precisely documented as possible, but it was
not possible to distinguish various case subgroups in the
analysis because of low numbers. Subjects with stable and
possible unstable angina were put together in the group
“angina,” although the atherosclerotic process at the origin of
each pathology could be different. Most subjects with angina
suffered a first episode of stable angina (n⫽114), and no
association of IL-6 with unstable angina (n⫽44) was statistically significant, although its mean value was intermediate
between that of stable angina and MI-coronary death cases. A
longer follow-up of the cohort would enable us to analyze
more precisely the association of IL-6 level with the different
clinical forms of CHD events.
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July 2003
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The incidence of CHD was higher in Northern Ireland than
in France.31,32 Predicted risk of CHD as estimated from
logistic regression equations using classical risk factors could
not explain the much higher level of CHD incidence experienced in Northern Ireland as opposed to France.32 We found
that different levels of apolipoprotein (apo) AI and LDL
cholesterol between the 2 countries explain approximately
7%33 and 10% (present study) of relative coronary risk. Now
the difference in IL-6 between the 2 populations seems to
explain a higher proportion of relative risk (13%) than apoAI
and LDL cholesterol. However, if apoA1 and LDL interact
with arterial cells and probably have a direct role in the
atherosclerotic process, the causal role of IL-6 appears more
hypothetical. Either it is only a marker of inflammation
within the atherosclerotic lesion or it has a direct role in the
pathogenesis of atherosclerosis through autocrine, paracrine,
and endocrine mechanisms.51 Observational cohort studies
cannot test these hypotheses, and more mechanistic experimental studies are needed to answer these questions.
In conclusion, levels of IL-6, CRP, and fibrinogen are
associated with incident acute coronary events, but not angina
among healthy men, independently of traditional risk factors
for CHD. From a clinical perspective, it is important to
recognize that the simultaneous measurement of lipids, particularly LDL cholesterol, and IL-6 improves the prediction
of risk of future MI-coronary death compared with that
associated with lipids or IL-6 alone. Because treatment such
as statin decreases CRP,52 its anti-inflammatory properties
could be additionally assessed by testing levels of IL-6 rather
than of CRP. Finally, IL-6 could be used clinically as a CHD
risk marker, especially as fully automated measurements of
IL-6 are now available.
Appendix
The PRIME Study Group
The PRIME Study is organized under an agreement between
INSERM and the Merck, Sharpe, and Dohme-Chibret Laboratory,
with the following participating laboratories: Strasbourg MONICA
Project, Department of Epidemiology and Public Health, Faculty of
Medecine, Strasbourg, France (D. Arveiler, B. Haas); Toulouse
MONICA Project, INSERM U558, Department of Epidemiology,
Paul Sabatier-Toulouse Purpan University, Toulouse, France (J.
Ferrières, J.B. Ruidavets); Lille MONICA Project, INSERM U508,
Pasteur Institute, Lille, France (P. Amouyel, M. Montaye); Department of Epidemiology and Public Health, Queen’s University of
Belfast, Northern Ireland (A. Evans, J. Yarnell); Department of
Atherosclerosis, SERLIA-INSERM U325, Lille, France (G. Luc,
J.M. Bard, L. Elkhalil, J.-C. Fruchart); Laboratory of Hematology,
La Timone Hospital, Marseilles, France (I. Juhan-Vague); Laboratory of Endocrinology, INSERM U326, Toulouse, France (B. Perret); Vitamin Research Unit, University of Bern, Switzerland (F.
Gey); Trace Element Laboratory, Department of Medicine, Queen’s
University, Belfast, Northern Ireland (D. McMaster); DNA Bank,
INSERM U525/SC7, Paris, France (F. Cambien); and Coordinating
Center, INSERM U258, Paris-Villejuif, France (P. Ducimetière, P.Y.
Scarabin, A. Bingham).
Acknowledgments
We are indebted to Ms Emmanuelle Lee for her technical assistance
with this project. We thank the following organizations which
authorized the recruitment of the PRIME subjects: the Health
screening centers organized by the Social Security of Lille (Institut
Pasteur), Strasbourg, Toulouse and Tourcoing; Occupational Medi-
cal Services of Haute-Garonne, of the Urban Community of Strasbourg; the Association Inter-entreprises des Services Médicaux du
Travail de Lille et environs; the Comité pour le Développement de la
Médecine du Travail; the Mutuelle Générale des PTT du Bas-Rhin;
the Laboratoire d’Analyses de l’Institut de Chimie Biologique de la
Faculté de Médecine de Strasbourg; the Department of Health (NI)
and the Northern Ireland Chest Heart and Stroke Association.
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C-Reactive Protein, Interleukin-6, and Fibrinogen as Predictors of Coronary Heart
Disease: The PRIME Study
Gérald Luc, Jean-Marie Bard, Irène Juhan-Vague, Jean Ferrieres, Alun Evans, Philippe
Amouyel, Dominique Arveiler, Jean-Charles Fruchart and Pierre Ducimetiere
on behalf of the PRIME Study Group
Arterioscler Thromb Vasc Biol. 2003;23:1255-1261; originally published online May 29, 2003;
doi: 10.1161/01.ATV.0000079512.66448.1D
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