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(Circulation. 2000;102:2576.)
© 2000 American Heart Association, Inc.
Clinical Investigation and Reports |
Circulating Immune Complexes in 50-Year-Old Men as a Strong and Independent Risk Factor for Myocardial Infarction
From the Immunological Research Unit (A.M., S.N., A.K.L.), Center for Molecular Medicine, Karolinska Institutet, Stockholm, and the Department of Public Health and Caring Sciences (L.B., H.L.), Geriatrics Unit, University of Uppsala, Uppsala, Sweden.
Correspondence to Prof Ann Kari Lefvert, Immunological Research Unit, CMM L8:03, Karolinska Hospital, S-171 76 Stockholm, Sweden. E-mail Ann.Kari.Lefvert{at}cmm.ki.se
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Abstract |
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Background—Circulating immune complexes (CICs) and autoantibodies against oxidatively modified LDLs (oxLDLs) and cardiolipin occur in patients with atherosclerosis and myocardial infarction (MI). The ability of such CICs and antibodies to predict myocardial infarction (MI) was investigated in a prospective nested case-control study in which healthy 50-year-old men were followed for 20 years.
Methods and Results—Two hundred fifty-seven men were included in the study, and 119 developed MI (39 died) between 50 and 70 years of age. One hundred thirty-eight randomly chosen men who did not develop MI up to 70 years of age served as controls. The prevalence of elevated levels of CICs and the concentration of CICs in men who developed MI were higher than in those who remained healthy. The concentration of CICs at age 50 was associated with a marked increased risk for MI, and this risk was independent of other conventionally recognized risk factors. There was a positive correlation between the levels of CIC and IgG antibodies to cardiolipin in men who developed MI. The level of IgG antibodies and the prevalence of elevated IgG and IgM antibodies to cardiolipin were higher in those who developed MI and had CICs than in those without CICs. Among men homozygous for C4 null alleles, those who developed MI had higher concentrations of CICs than did those who remained healthy.
Conclusions—This prospective study shows that CICs alone or in combination with autoantibodies against cardiolipin in healthy males at 50 years of age predict subsequent MI between the age of and 70 years.
Key Words: circulating immune complexes • antibodies • antigens • lipoproteins • myocardial infarction
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Introduction |
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The classic risk factors, hypercholesterolemia, smoking, hypertension, and diabetes, explain only part of the epidemiological features of atherosclerotic vascular disease. Therefore, it is likely that other, hitherto unrecognized, factors exist. In the past decade, there has been an upsurge of interest in the role of immune mechanisms in the development and regulation of atherosclerosis and its complications.1 Both humoral and cellular mechanisms have been proposed to participate in the onset and/or progression of atherosclerotic lesions, and postulates for the involvement of autoantibodies against oxidatively modified LDL (oxLDL), phospholipids, and circulating immune complexes (CICs) have met with considerable experimental support.2 3 4 5 Repeated immunization of animals with protein antigens leads to the formation of CICs and acceleration of the diet-induced atherosclerotic process in both rabbits and mice.6 7 CICs are present in
50% of cases with acute myocardial infarction
(MI),8 9 10 11
although persistence of CICs is more
rare.12 Precocious MI
occurs in individuals with chronic immune complex formation due to
inflammatory disorders, such as systemic lupus
erythematosus13 14
and rheumatoid
arthritis.15 In an earlier
study, we described persisting CICs in 20% of patients with precocious
MI 6 months to several years after the acute
event.16 In another study,
the presence of lipoprotein-containing CICs promoted the onset and
development of atherosclerotic lesions in the vessel
wall.17 Several studies
have shown that oxLDLs in combination with antibodies obtained by
immunization of animals causes an increased rate of foam cell
development in vitro via interaction of immune complexes with IgG Fc
receptors.18 19 20
That autoantibodies against cardiolipin and oxLDL can participate in
the formation of CICs has been shown by recent
studies.21 22 23 The solubilization and efficient elimination of CICs is dependent on an intact classical pathway of complement activation.24 Genetic deficiencies of complement proteins are associated with high levels of CICs and immune complex–mediated disease.16 25 Our earlier studies have shown that the presence of null alleles of complement factor C4 (C4Q0) is not related to future MI.26 However, persons with C4Q0 and past premature MI had higher prevalence and levels of CICs.16 There was also a relation between C4Q0 and CICs in persons with premature peripheral vascular disease.27 Thus, C4Q0 might predispose an individual to immune complex formation, which might play a role in vascular inflammation.
In the present investigation, the presence and levels of CICs and their relation to antibodies against cardiolipin and oxidatively modified LDLs and to C4Q0 were determined in samples from healthy 50-year-old men, who were followed for 20 years for the development of MI. The aim of the present study was to assess whether the presence of high concentrations of CICs was related to future MI.
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Methods |
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Study Groups
The present study was a prospective nested case-control study28 composed of 257 individuals: 119 men (mean age 49.63 years) who developed MI between 50 to 70 years of age and 138 men (mean age 49.58 years) who did not develop MI up to 70 years of age. Samples had been collected from 2322 males aged 50 years in Uppsala during 1970 to 1973. This cohort made up 82% of all 50-year-old men living in Uppsala at that time. This cohort was followed for 20 years. Of the 156 men who developed MI, samples were missing or in bad condition for 18 of them, and 19 cases were excluded because of diabetes mellitus. Of the remaining 119 men who developed MI, 39 died. A control group of 156 individuals was selected by simple random selection from those who did not develop MI up to 70 years of age. Samples were missing or in bad condition for 17 of them, and 1 individual was excluded because of diabetes. Thus, the remaining control group consisted of 138 individuals. There were no differences in the conventional risk factors for MI (serum LDL/HDL cholesterol ratio, the ratio between arachidonic acid and dihomo-
-linoleic acid from serum cholesterol, body mass
index, and smoking) between the control group selected in the present
study and the rest of the healthy individuals. The mean value of supine
blood pressure in the selected control group (130 mm Hg) was lower
than that for the rest of the healthy individuals (133 mm Hg)
(P<0.05). Diagnoses from hospital records were
obtained from the National Central Bureau of Statistics until 1983.
From 1983, these data were requested from the hospitals. Information on
causes of death was requested from the National Board of Health and
Welfare. Only MI as the main diagnosis was used for classification. At
each 10-year follow-up, hospital and death records were scrutinized,
and the accuracy of the MI diagnosis was
assessed.29 Data on the
groups regarding hypertension, dyslipoproteinemia, tobacco use, and
other conventionally recognized risk factors were presented
earlier.30
Isolation and Quantitative Determination of
Immune Complexes
Ultracentrifugation
Details were as described by
us.16 A sample of 0.5 mL
serum was centrifuged for 15.5 hours at 160 000g and
4°C on a continuous sucrose density gradient by using a Beckman L5-65
centrifuge and 12 mL tubes. The gradient was made of 5% to 20%
sucrose with 1 mL 40% sucrose as a bottom layer. Fractions of 20 drops
were collected from the bottom. Fractions containing protein complexes
with a molecular weight >175 kDa were assayed by ELISA for the
presence and concentration of
immunoglobulins.16 Pooled
purified IgG (Gammaglobulin, Pharmacia-Upjohn) was used as a standard
for the determination of IgG, and normal pooled human serum was used as
a standard for the determination of IgA and IgM. The protein content in
the fractions was measured by the method of Lowry et
al.31 Results were tested
for normality of distribution. IgG- and IgA-containing complexes with a
molecular weight >200 kDa, in a concentration of >25 mg/L, by protein
determination according to Lowry et al, were taken to be abnormally
raised. This value represents the mean+3 SD of the values of a control
population (350 healthy
individuals).16 The
density gradient was standardized by using molecular weight markers
according to established
criteria.32 The
intraexperimental coefficient of variation between duplicate samples
for the ultracentrifugation assay was 14.5%.
Gel Filtration
IgM-containing immune complexes and large IgG- and
IgA-containing CICs were isolated from serum by gel filtration with use
of a fast performance liquid chromatography system and a Sepharose-6
column (Pharmacia). The buffer used was 0.05 mol/L phosphate, pH 7.0,
with 0.15 mol/L NaCl. The fractions were assayed by ELISA for the
concentration of
immunoglobulins,16 and the
concentration of protein was measured by the method of Lowry et
al.31 Results were tested
for normality of distribution. IgM-containing complexes with a
molecular weight >900 kDa and a protein concentration >30 mg/L were
considered to be abnormally raised. This value represents the mean+3 SD
of a control population (234 healthy individuals). The
intraexperimental coefficient of variation between duplicate samples
was 16.3%.
C4 Allotyping
EDTA plasma was kept frozen at -70°C. C4
allotypes were determined by flat-bed agarose gel electrophoresis,
followed by
immunofixation.33 34
Briefly, EDTA plasma was treated with neuraminidase and
carboxypeptidase B; after dialysis, agarose gel electrophoresis was
carried out at pH 8.8, and the C4 bands were detected by immunofixation
with use of rabbit anti-human C4 antibody as described
previously.33 After a
washing, the gel was dried, and the proteins were stained with
Coomassie blue. Standard samples for the C4 allotypes frequently seen
in the white population (A2, A3, A6, B1, B2, and B3) were
included in each gel. C4 allotypes were assigned according to published
criteria.35 When bands
were intermediate between the A and B loci, C4B gene products were
distinguished by their greater hemolytic
capacity.34 The density of
the C4A and C4B bands was determined by a scanning densitometer.
Complete absence of C4A or C4B bands was taken to indicate homozygous
deficiency. Heterozygous deficiency was determined by comparison of the
densities of the C4A and C4B
bands.33 36
The effect of freezing/thawing was analyzed and showed that repeated cycles of freezing/thawing affected the intensity of the bands but not the allotyping itself.
Determination of Autoantibodies Against OxLDL
and Native LDL
LDL was isolated by ultracentrifugation and was
copper-oxidized as
described.3 23 37
Autoantibodies of IgG, IgA, and IgM isotypes against oxLDL and native
LDL were determined by ELISA as described
earlier.23 37 38
Briefly, microplates were coated with oxidized or native LDL, diluted
serum samples were added, and the plates were incubated for 3 hours at
room temperature in a humid atmosphere. After a washing, alkaline
phosphatase-labeled second antibody was added to each well, and the
plates were further incubated for 2 hours at 37°C. After another
washing, the substrate p-nitrophenyl phosphate was
added. One standard containing high concentrations of antibodies
against oxLDL, 2 samples that were earlier demonstrated to contain
antibodies against oxLDL, 2 samples lacking such antibodies, and a
blank were run on each plate. For blank wells, phosphate buffer was
added instead of serum. The plates were read at 405 nm when the
positive control reached an optical density of 1.0±0.1, which took
30 minutes. All assays were run in duplicate, and observers were
blinded to the study protocol. The cumulative interassay coefficient of
variation was <10%. The assay has been further validated with the use
of F(ab')2 fragments from purified IgG
fractions with high concentrations of specific antibodies.
The cutoff level for the presence of antibodies, as determined by the absorbance units in the ELISA, was the mean+2 SD of that of individuals who remained healthy.
Determination of Autoantibodies Against
Cardiolipin
Anticardiolipin antibodies of IgG, IgA, and IgM
isotypes were analyzed by ELISA according to the method of Harris et
al,39 with minor
modifications.37 38
Briefly, polyvinylchloride microplates were coated with bovine heart
cardiolipin and blocked by 1% BSA. Diluted serum samples were added,
and the plates were incubated for 3 hours at room temperature in a
humid atmosphere. After a washing, alkaline phosphatase–labeled
secondary antibody was added to each well, and the plates were further
incubated for 2 hours at 37°C. After another washing, the substrate
p-nitrophenyl phosphate was added. One standard
containing high concentrations of antibodies against cardiolipin, 2
samples that were earlier demonstrated to contain antibodies against
cardiolipin, 2 samples lacking such antibodies, and a blank were run on
each plate. For blank wells, phosphate buffer with BSA was added
instead of serum. The plates were read at 405 nm when the positive
control reached an optical density of 1.0±0.1, which was 25 minutes
for the IgG assay and 30 to 40 minutes for the IgM and IgA assays. The
cumulative interassay coefficient of variation was <10%. The cutoff
level for the presence of antibodies, as determined by the absorbance
units in the ELISA, was the mean+2 SD of that of individuals who
remained healthy.
Statistical Evaluation
The nonparametric Mann-Whitney U
test and Fisher exact test were performed for group comparisons. A
2-tailed value of P<0.05 was regarded as significant.
Simple regression was used to analyze the correlation between the
concentrations of CICs and levels of antibodies against oxLDL and
cardiolipin.
The association between the concentration of CICs and the
health status of the men (1 indicates healthy; 2, survival of MI; and
3, death by MI) was tested by Spearman rank correlation analysis. The
importance of the concentration of CICs when adjusted for the
conventionally recognized risk factors for MI (supine blood pressure,
serum LDL/HDL cholesterol ratio, the ratio between arachidonic acid and
dihomo--linoleic acid from serum cholesterol, body mass index, and
smoking)30 was examined by
use of partial Spearman rank correlation analysis. A logistic
regression analysis was used to test the predictive importance of the
concentration of CICs alone and after adjustment for the
above-mentioned risk factors. The statistical package was SAS for
Windows, version 6.12.
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Results |
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Conventionally Recognized Risk Factors
Men who developed MI had higher systolic and diastolic blood pressure, body mass index, serum triglycerides, serum cholesterol, LDL/HDL ratio, and ratio between arachidonic acid and dihomo-
-linoleic acid from serum
cholesterol.37 Persons
with diabetes mellitus were excluded from both populations because of
the high prevalence of increased levels of CICs and the increased
incidence of cardiovascular
diseases.40
Prevalence of High CICs and Concentration
of CICs
The prevalence of elevated levels of CICs and the
concentration of CICs in men who developed MI compared with those who
remained healthy are shown in Table 1
.
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The prevalence of elevated levels of CICs was 36% (43 of 119 individuals) in those who developed MI and 13% (18 of 138 individuals) in those who remained healthy (P<0.0001). The concentration of CICs was higher in those who developed MI (P<0.0001).
Association of CICs With Other Risk Factors
for MI
The association of CICs with other risk factors for MI
is shown in Table 2.
The concentration of CICs at age 50 years was associated with an
increased risk for future MI, with an uncorrected odds ratio of 3.17
(95% CI 1.68 to 5.95, P<0.0001). The odds ratio
corrected for the conventional risk factors (supine blood pressure,
serum LDL/HDL cholesterol ratio, ratio between arachidonic acid and
dihomo--linoleic acid from serum cholesterol, body mass index, and
smoking) was 2.95 (95% CI 1.52 to 5.75,
P<0.0001).
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The results from the Spearman rank correlation analysis
(Table 3)
show that the concentration of CICs at age 50 years was related not
only to future MI but also to death by MI.
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Levels of CIC and Antibodies to OxLDL and
Cardiolipin in Men Who Developed MI
There was a positive correlation between the levels of
CICs and IgG antibodies to cardiolipin (r=0.4,
P=0.0001) in men who developed MI. There was no
correlation between levels of CICs and IgA or IgM antibodies to
cardiolipin or to levels of antibodies against oxLDL. The levels of IgG
antibodies against cardiolipin was higher in men who developed MI and
had CICs compared with men without CICs (P<0.0001)
(Figure).
The prevalence of elevated levels of IgG and IgM antibodies against
cardiolipin was higher in patients who developed MI and had CICs
compared with those without CICs (P<0.001 and
P<0.02, respectively)
(Table 4).
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Association of CICs With C4 Null Alleles in
MI
The prevalence of elevated levels of CICs was higher in
men who developed MI and were homozygous for C4Q0 (C4A*Q0 or C4B*Q0)
than in men who developed MI and were heterozygous for C4Q0 (C4A*Q0 or
C4 B*Q0) (P<0.03,
Table 5).
Among individuals homozygous for C4Q0, men who developed MI had higher
concentrations of CICs (P<0.01) than did those who
remained healthy.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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This prospective study shows CICs to be a strong independent risk factor for the development of future MI. Moreover, because there was a positive correlation between elevated levels of CICs and IgG antibodies to cardiolipin, the combination of CICs with autoantibodies to cardiolipin might further increase the pathogenic potential of these humoral factors.
A major obstacle in understanding the pathogenetic significance of CICs in MI has been that most earlier studies have been retrospective, and the formation of CICs could be related to the event. We have previously described a high prevalence of persisting CICs after premature MI.16 However, our present prospective study has shown that CICs are present before the MI and thus are likely to be more directly related to the event. What induces the formation of CICs in these cases is mostly unknown.
Several studies have shown that autoantibodies to epitopes of oxLDL exist in human serum,41 are present as part of immune complexes with oxLDL, and can recognize material in atherosclerotic lesions of rabbits and humans.42 43 However, we did not find a correlation between elevated levels of antibodies against oxLDL and CICs in the present study.
Several independent groups reported that LDL-containing immune complexes prepared in vitro or isolated from patients’ sera could induce intracellular accumulation of cholesterol esters.18 19 20 44 45 46 47 48 High levels of oxLDL-containing immune complexes have been suggested to be a risk factor for the development of coronary arterial disease in patients with insulin-dependent diabetes mellitus, a fact that could not be deduced from the measurement of free oxLDL antibody concentrations and the interference of circulating oxLDL-containing immune complexes with the assay of free oxLDL antibodies.22 Thus, one pathogenic effect of autoantibodies to oxLDL might be participation in CIC formation. However, we did not analyze the constituent of CICs found in the present study.
In one previous report, CICs present in postinfarction patients were found to contain lipopolysaccharides from Chlamydia species.49 We have observed high persistent concentrations of CICs containing alimentary proteins in patients with precocious MI before 45 years of age (authors’ unpublished data, 2000).
The positive correlation between levels of antibodies to cardiolipin and levels of CICs would indicate that these autoantibodies are involved in the formation of CICs. Our earlier data indicate that these antibodies might be involved in the formation of CICs.21 We have previously shown a high prevalence of cardiolipin antibodies and CICs in patients with type 1 diabetes mellitus with vascular complications. CICs from 6 patients were further investigated and contained antibodies to cardiolipin.40 Thus, one major pathogenic effect of these autoantibodies might be participation in the formation of CICs.
The concentration and chronicity of CICs are important for the induction of vascular damage.50 CICs can lead to endothelial perturbation by complement activation, leading to low-grade chronic inflammation of the vascular wall that might enhance the atherosclerotic process. The molecular size of the immune complexes observed by us was in the range that is pathogenic and likely to get deposited in vessel walls.16 50 A slightly decreased capacity to clear the complexes is present in carriers of C4Q0,24 25 and in the present study, such a defect was indeed accompanied by higher prevalence of CICs. Thus, as found in our earlier studies,16 27 this investigation also identified C4Q0 as one factor that predisposes to CICs.
In conclusion, increased prevalence and concentrations of CICs alone or in combination with raised levels of autoantibodies against cardiolipin in a cohort of healthy 50-year-old Swedish men predicted subsequent MI. The predictive power was strong and independent of that of other risk factors.
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Acknowledgments |
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This study was supported by grants from the Swedish Heart-Lung Foundation and the King Gustaf V:s 80-Years Foundation.
Received May 4, 2000; revision received July 6, 2000; accepted July 7, 2000.
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