© 1995 American Heart Association, Inc.
Articles |
Association Between Circulating Immune Complexes, Complement C4 Null Alleles, and Myocardial Infarction Before Age 45 Years
From the Immunological Research Laboratory (A.K.L., G.H.), and the Atherosclerosis Research Unit (A.H.), King Gustaf V Research Institute, and the Department of Medicine (A.K.L., A.H., G.H.), Division of Haematology and Medical Immunology, Karolinska Hospital, Stockholm, Sweden.
Correspondence to Dr Ann Kari Lefvert, Department of Medicine, Karolinska Hospital, S-171 76 Stockholm, Sweden.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterialsResultsDiscussionReferences |
|---|
Abstract One hundred patients who had survived a myocardial infarction before the age of 45 years and 90 age- and sex-matched healthy individuals were investigated for circulating immune complexes (CICs) and the presence of complement C4 null alleles (C4Q0). CICs were found in increased concentrations in 20% of patients and 6.7% of control subjects. Patients and control subjects had the same prevalence of C4Q0. CICs were present in all patients and in 36% of the control subjects homozygous for C4Q0. Patients and control subjects heterozygous for C4Q0 had CICs in 71% and 0%, respectively. The high prevalence and a high concentration of CICs were particularly associated with C4A*Q0. Patients homozygous for C4A*Q0 had concentrations of LDL that were lower than found in other patients. The increased concentration of CICs associated with genetic deficiencies of the complement factor C4 might thus be an additional etiological factor for the development of chronic vascular damage and premature myocardial infarction.
Key Words: myocardial infarction • immune complex disease • complement C4
|
Introduction |
|---|
|
TopAbstractIntroductionMaterialsResultsDiscussionReferences |
|---|
The development of atherosclerosis is connected with disturbances of the lipoprotein metabolism mainly attributed to genetic and/or environmental factors; such factors could account for about 60% of cases of premature myocardial infarction (MI).1 2 However, in the remaining 40% of patients, no apparent such risk factor can be identified.3 The chain of events that leads to premature MI should thus include other as yet largely unidentified components. One such element might be circulating immune complexes (CICs). CICs are associated with premature vascular disease in both humans and experimental animals. Repeated immunization of animals with protein antigens leads to the formation of circulating antigen-antibody complexes and acceleration of the spontaneous and diet-induced atherosclerotic process in both rabbits and mice.4 5 Premature atherosclerosis is observed in patients with chronic immune complex formation due to inflammatory disorders,6 7 and there is an increased incidence of premature MIs in patients with systemic lupus erythematosus and CICs.8 9 10 Thus, the abnormal presence of CICs contributes to an inflammatory vascular lesion that ultimately might progress to atherosclerosis.
The solubilization and efficient elimination of CICs is dependent on an intact classic pathway of complement activation.11 Genetic deficiencies of complement proteins are clearly associated with high levels of CICs and immune complex–mediated disease. Total deficiencies of C1q, C1r, C1s, C4, C2, and C3 are usually accompanied by systemic lupus erythematosus or lupus-like diseases.12 13 14 15 Unlike other complement factors, C4 is encoded at two polymorphic gene loci, C4A and C4B, and null or unexpressed alleles are common.12 An increased prevalence of null alleles has been reported in a variety of diseases as diverse as insulin-dependent diabetes mellitus, rheumatoid arthritis, IgA nephropathy, and systemic lupus erythematosus.12 16 Individuals who are heterozygous for C4A*Q0 or C4B*Q0 also have an increased risk of developing such autoimmune diseases as well as immune complex–mediated diseases.12 The aim of this study was to investigate the prevalence of CICs and the association between CICs and C4 null alleles (C4Q0) in young patients with MI.
|
Materials |
|---|
|
TopAbstractIntroductionMaterialsResultsDiscussionReferences |
|---|
Patients
One hundred consecutive patients who had survived a first MI before the age of 45 years in the 11 coronary care units in the greater Stockholm area were included in the study. Patients with insulin-dependent diabetes mellitus and other concomitant severe diseases such as uremia or collagen vascular diseases were not considered for the study. Patients with familial hypercholesterolemia were also excluded.
Blood samples from the patients were taken 3 to 6 months after the MI at a time when the acute-phase reaction due to the myocardial damage had subsided. Samples were taken between 8 and 10 AM after 12 hours of fasting, during which time smokers were asked to refrain from smoking. Venous blood was drawn into evacuated tubes and allowed to coagulate. Serum was recovered by low-speed centrifugation (1400g for 20 minutes) and stored at -70°C.
Ninety age- and sex-matched healthy individuals randomly selected from the general population of Stockholm County were used as control subjects. All control subjects were without symptoms and signs of coronary heart disease as assessed by a symptom-limited exercise stress test and a structured interview aimed at detecting individuals with myocarditis, angina pectoris, or any other severe illness. Patients with known diseases of the immune system were excluded. Recruitment, representativeness, and clinical and metabolic characteristics of the patient and control groups have been described.3
C4 Allotyping
C4 allotypes were determined by agarose gel electrophoresis of
neuraminidase- and carboxypeptidase-treated plasma followed by
immunofixation.17 When bands were positioned
intermediately between the A and B loci, B locus products were
distinguished by their greater hemolytic capacity.17 The
gels were overlaid with a 0.6% agarose gel containing
5x108/mL sheep erythrocytes that had been
sensitized with rabbit antiserum and 2% C4-deficient guinea pig serum
and incubated for 1 hour at 37°C. The numbers of C4 null alleles were
determined as described from the C4 phenotype17 and by
comparing the relative densities of C4A and C4B bands using a scanning
laser photometer. This method cannot detect samples with one null
allele at both the A and the B locus, and the number of null alleles
may thus be underestimated. There is also a quantitative variation of
C4 variant proteins associated with many MHC haplotypes that may lead
to an underestimation or an overestimation of the C4A*Q0 and C4B*Q0
heterozygotes.18 Two null alleles were assigned in the
absence of bands for one C4 locus and the presence of one or two
separate alleles at the other. Zero null alleles were assigned in the
presence of three separate alleles, with the fourth allele deduced
because of a densitometric ratio of approximately one.
Numbers for the prevalence of null alleles in a Swedish population and in other Caucasian populations were taken from the literature.12 16
Quantitative Determination and Separation of Immune Complexes
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 by 5% to 20% sucrose with 1 mL 40% sucrose as a bottom
layer.19 20 Fractions of 20 drops were collected from the
bottom. Peaks containing immunoglobulins with higher molecular weights
than 175 kD were assayed by enzyme-linked immunosorbent assay for the
presence of immunoglobulins19 and for binding to
C1q.21 IgG-containing complexes with molecular weights
higher than 175 kD in a concentration of >25 mg/L were considered to
be abnormally raised. This value represents the mean+3 SD of
the mean of the control population.20 All these fractions
bound to C1q.21
The density gradient was standardized by using molecular weight markers according to established methods.22 23
Gradient centrifugation of sera analyzed before freezing and after storage in -70°C showed that there was no difference in the concentration of immune complexes in sera with four C4 allele products and those homozygous for C4A*0 and C4B*0.
The intraexperimental coefficient of variation between duplicate samples for the ultracentrifugation assay was 14.5%.
Statistical Evaluation
Distributions of categorical data were compared by using a
2 test with Yates' correction. Group differences
in continuous variables were determined by the Mann-Whitney
U test.
|
Results |
|---|
|
TopAbstractIntroductionMaterialsResultsDiscussionReferences |
|---|
The prevalence of C4A*Q0 and C4B*Q0 in healthy control subjects and in patients is shown in Table 1
. The prevalence of
null alleles in patients with MI was not different from that in healthy
control subjects. The prevalence of both C4A*Q0 and C4B*Q0 was higher
than reported for both a Swedish population16 and other
Caucasian populations.12 The distribution of different C4
allele products was the same in the patient and control
populations.
|
A typical density-gradient centrifugation showing immune complexes in
two patient sera compared with the pattern of serum from a healthy
individual is shown in Fig 1.
|
) and one serum is from a
healthy individual (
). Fractions 1-10 have an estimated molecular
weight >175 kD and are considered to contain circulating immune
complexes.
The prevalence of raised concentrations of CICs was higher in patients
with previous MI than in the control subjects (P<.001)
(Table 2). All patients homozygous for C4A*Q0 and C4B*Q0
had increased levels of CICs. This was found in only 4/11 (36%) of the
control subjects with the same C4 allotypes. Of the 16 patients with
C4A*Q0, 11 (69%) had raised levels of CICs compared with 6/26 (23%)
in the control population. Patients with C4B*Q0 had elevated levels of
CICs (8/29; 28%) compared with 1 of the 30 (3%) control subjects. One
of the patients and none of the healthy control subjects with four C4
allele products had increased levels of CICs.
|
The concentrations of CICs were higher in patients than in control
subjects (Fig 2). This difference was significant when
comparing all patients and control subjects (P<.001), patients and
control subjects homozygous for C4A*Q0 (P<.0002), and
patients and control subjects heterozygous for C4A*Q0
(P<.001). The high prevalence and the higher concentration
of immune complexes were thus particularly associated with C4A*Q0
(Table 2
and Fig 2).
|
The estimated size of the immune complexes was 175 to 300 kD in 3 patients and in 1 control subject, between 300 and 900 kD in 15 patients and in 5 control subjects, and equal to or greater than 900 kD in 1 patient and in no control subjects. Eleven patients and 3 control subjects had a mixed pattern with complexes of different sizes.
The concentration of plasma IgG, IgA, and IgM did not differ between patients and control subjects, nor was there any correlation between the amount of immunoglobulins and the amount of CICs.
Patients with different C4 allotypes were also compared with regard to the lipoprotein composition of plasma. The one difference was that the four patients homozygous for C4A*Q0 had concentrations of LDL that were significantly lower (P<.05) than found in other patients.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterialsResultsDiscussionReferences |
|---|
This article reports on increased concentrations of CICs in 20% of patients who had an MI before the age of 45 years compared with only 6.7% of healthy control subjects. The frequent presence of CICs in the acute stage and during the 2 to 4 weeks after MI has been documented in several studies using indirect techniques such as C1q binding and binding to conglutinin.24 25 26 27 CICs were present for up to 2 to 4 weeks in 40% to 76% of patients after MI and usually disappeared within 4 to 6 weeks. Persisting CICs as found by using the C1q binding assay were found in only 8% of patients.27
The cause of immune complex formation in MI and the role of CICs in the pathogenesis of MI are incompletely known. Immune reactions against both exogenous and endogenous antigens have been implicated. Recent respiratory tract infection was more prevalent in MI patients with CICs than in those without a history of such infections.27 Antibodies against microbial antigens and alimentary antigens such as bovine proteins have been suggested as being involved in the etiology of the CICs that accompany MI.28 29 Moreover, antibodies against endogenous antigens such as lipoproteins and DNA are more prevalent in immune complex form in patients with MI.10 30
With the exception of one individual, CICs were found only in patients carrying C4*Q0. Not only the prevalence but also the concentration of CICs was higher in patients than in control subjects having the same C4 allotype. In systemic lupus erythematosus, high concentrations of CICs contribute to the vascular damage and are clearly correlated with the presence of C4A*Q0.11 13 15 16 An association between C4B*Q0 and the incidence of severe MI with high mortality has been reported in Hungarian patients.31 This increased MI mortality was suggested as an explanation for the marked decreased in prevalence of the C4B*Q0 allele in healthy elderly individuals compared with young persons in Hungary.32 In these studies, the presence of CICs as a cause of the vascular disease was not investigated. If individuals with the C4B*Q0 allele have a higher MI mortality, then this could explain that our surviving patients have the same prevalence of C4Q0 alleles as healthy individuals. A recent prospective study by us of 410 Swedish men in whom C4 allotypes were analyzed at the ages of 50 and 70 years failed to show any correlation between MI between ages 50 to 70 and C4A*Q0 or C4B*Q0. In this study, the prevalence of the different C4 allotypes was the same at age 50 as at age 70 and the same as found in the present study (S. Nityanand, et al, unpublished data, 1995). Thus, in a Swedish population, there is so far no evidence that people bearing the C4B*Q0 allele have a higher incidence of MI or higher MI mortality or that the prevalence of the C4B*Q0 allele is lower in elderly individuals.
The mechanisms for CIC-induced vascular damage are not known in detail. Generally, the concentrations of CICs and time of exposure to CICs seem to be important for the induction of vascular damage.4 30 Immune complex formation is a normal event and has important biological functions in the elimination of antigens. CICs are temporarily present in all individuals, especially during infectious diseases.33 A slightly lower capacity to clear the complexes, such as in individuals with C4A*Q0, results in a prolonged exposure to CICs. The immune complex fractions in our patients did bind to complement C1q and were thus capable of activating complements, a feature that is important for the pathological effect of complexes.11 Moreover, in most persons the measured molecular size of the immune complexes was between 300 and 900 kD, and thus of a size that in experimental models does lead to deposits of the complexes in vessel walls.34
The higher prevalence and higher concentrations of CICs in patients than in healthy control subjects having the same C4A*Q0 and C4B*Q0 alleles suggest that other as yet unidentified factors contribute to the formation of CICs in the patients. Such factors might be increased immunological reactivity to endogenous antigens or abnormal exposition for or reactivity to exogenous antigens such as alimentary or microbial proteins. The persistent CICs associated with genetic deficiencies of the complement factor C4 might thus be an additional etiological factor for the development of chronic vascular damage and premature MI.
|
Acknowledgments |
|---|
This study was supported by grants from the Wallenberg Foundation, the Nanna Svartz Foundation, and Hedlund's Foundation. The technical assistance of Ragnhild Östman is gratefully acknowledged.
Received September 9, 1994; accepted February 8, 1995.
|
References |
|---|
|
TopAbstractIntroductionMaterialsResultsDiscussionReferences |
|---|
1. Solberg LA, Strong JP. Risk factors and atherosclerotic lesions: a review of autopsy studies. Atherosclerosis. 1983;3:187-198.
2. Pooling Project Research Group. Relationship of blood pressure, serum cholesterol, smoking habits, relative weight and ECG abnormalities to incidence of major coronary events: final report of the Pooling Project. J Chronic Dis. 1978;31:201-306. [Medline] [Order article via Infotrieve]
3. Hamsten A. Myocardial infarction at a young age: mechanisms and management. Vasc Med Rev. 1991;2:45-60.
4. Mannik M, Arend WP, Hall AP, Gilliand BC. Studies on antigen-antibody complexes, I: elimination of soluble complexes from rabbit circulation. J Exp Med. 1971;133:713-739. [Abstract]
5.
Qiao JH, Castellani LW, Fishbein MC, Lusis AJ.
Immune complex–mediated vasculitis increases coronary artery
lipid accumulation in autoimmune-prone MRL mice.
Arterioscler Thromb. 1993;13:932-943.
6. Beaumont JL. Autoimmune hyperlipidemia: an atherogenic metabolic disease of immune origin. Rev Eur Etud Clin Biol. 1970; 15:1037-1041.
7. Poston RN, Davies DF. Immunity and inflammation in the pathogenesis of atherosclerosis: a review. Atherosclerosis. 1974; 19:353-367.
8. Urowitz MB, Bookman AA, Koehler BE, Gordon DA, Smythe HA, Ogryzlo MA. The bimodal mortality pattern of systemic lupus erythematosus. Am J Med. 1976;60:221-225. [Medline] [Order article via Infotrieve]
9. Spiera H, Rothenberg RR. Myocardial infarction in four young patients with SLE. J Rheumatol. 1983;10:464-466. [Medline] [Order article via Infotrieve]
10. Gladman DD, Urowitz MB. Morbidity in systemic lupus erythematosus. J Rheumatol. 1987;13:223-226.
11. Schifferli JA, Yin CNG, Peters DK. The role of complement and its receptor in the elimination of immune complexes. N Engl J Med. 1986;315:488-495. [Medline] [Order article via Infotrieve]
12. Dawkins RL, Christiansen FT, Kay PH, Garlepp M, McCluskey JM, Hollingsworth PN, Zilko PJ. Disease association with complotypes, supratypes and haplotypes. Immunol Rev. 1983;70:5-22.
13. Fielder AHL, Walport MJ, Batchelor JR, Rynes RI, Black C, Dodi IA, Hughes GR. Family study of the major histocompatibility complex in patients with systemic lupus erythematosus: importance of null alleles of C4A and C4B in determining susceptibility. BMJ. 1983;286: 425-428.
14. Hauptman G, Goetz J, Uring-Lambert B, Grosshans E. C4 deficiency. Prog Allergy. 1986;39:232-249. [Medline] [Order article via Infotrieve]
15. Agnello V. Lupus diseases associated with hereditary and acquired deficiencies of complement Springer Semin Immunopathol. 1986;9:161-178. [Medline] [Order article via Infotrieve]
16. Sturfelt G, Truedsson L, Johansen P, Johnsson H, Nived O, Sjöholm AG. Homozygous C4A deficiency in systemic lupus erythematosus: analysis of patients from a defined population. Clin Genet. 1990;38:427-433. [Medline] [Order article via Infotrieve]
17. Zhang WJ, Kay PH, Cobain TJ, Dawkins RL. C4 allotyping on plasma or serum: application to routine laboratories. Hum Immunol. 1988;21:165-171. [Medline] [Order article via Infotrieve]
18. Truedsson L, Awdeh Z, Yunis EJ, Mrose S, Moore B, Alper CA. Quantitative variation of C4 variant proteins associated with many MHC haplotypes. Immunogenetics. 1989;30:414-421. [Medline] [Order article via Infotrieve]
19. Sundewall AC, Lefvert AK. Acetylcholine receptor antibodies in primary biliary cirrhosis: characterization of antigen and idiotypic specificity. Scand J Immunol. 1990;31:477-484. [Medline] [Order article via Infotrieve]
20. Lefvert AK. Circulating immune complexes containing anti-bovine serum albumin antibodies as a cause of severe angioedema. Int Arch Allergy Immunol. 1993;102:112-116. [Medline] [Order article via Infotrieve]
21. Wener MH, Mannik M, Schwartz MM, Lewis EJ. Relationship between renal pathology and the size of circulating immune complexes in patients with systemic lupus erythematosus. Medicine. 1987;66:85-97. [Medline] [Order article via Infotrieve]
22. Hinton R, Dobrota M. Density gradient centrifugation. In: Work TS, Work E, eds. Laboratory Techniques in Biochemistry and Molecular Biology, VI. Part 1. Amsterdam, Netherlands: Elsevier/North-Holland Publishing Co; 1976.
23. Rickwood D, ed. Centrifugation. 2nd ed. Oxford, England: IRL Press; 1984.
24. Farrel C, Bloth B, Nielsen H, Daugharty H, Lundman T, Svehag S. A survey for circulating immune complexes in patients with acute myocardial infarction: use of a C1q-binding assay with soluble protein A as indicator. Scand J Immunol. 1977:6:1233-1240.
25. Füst G, Szondy E, Szekely J, Nänai I, Gerö S. Studies on the occurrence of circulating immune complexes in vascular diseases. Atherosclerosis. 1978;29:181-190. [Medline] [Order article via Infotrieve]
26.
Szondy E, Mezey Z, Füst G, Szekeley J,
Gerö S. Serial measurement of circulating immune complexes
in myocardial infarction. Br Heart J. 1981;46:93-98.
27. Steffen C, Böhm H, Menzel J. Immune complexes in myocardial infarction. Wien Klin Wochenschr. 1986;98:161-165. [Medline] [Order article via Infotrieve]
28. Leinonen M, Linnanmäki E, Mattila K, Nieminen MS, Valtonen V, Leirisalo RM, Saikku P. Circulating immune complexes containing clamydial lipopolysaccharide in acute myocardial infarction. Microb Pathog. 1990;9:67-73. [Medline] [Order article via Infotrieve]
29. Davies DF, Elwood PC. Milk antibodies and myocardial infarction. Lancet. 1974;874:219-220. Letter.
30. Szondy E, Horvath M, Mezey Z, Szekeley J, Lengyel E, Füst G, Gerö S. Free and complexed anti-lipoprotein antibodies in vascular disease. Atherosclerosis. 1983;49:69-77. [Medline] [Order article via Infotrieve]
31.
Kramer J, Rajczy K, Hegyi L, Fülöp T,
Mohacsi A, Mezei Z, Keltai M, Blasko G, Ferenczy E, Anh Tuan N,
Füst G. Markedly increased incidence of severe myocardial
infarction and higher mortality in patients with C4B*Q0
allotype. BMJ. 1994;309:313-314.
32. Kramer J, Fülöp T, Rajczy K, Nguyen AT, Füst G. A marked drop in the incidence of the null allele of the B gene of the fourth component of complement (C4B*Q0) in elderly subjects: C4B*Q0 as a probable negative selection factor for survival. Hum Genet. 1991;86:595-598. [Medline] [Order article via Infotrieve]
33. Lambert PH, Morel PA, Renversez JC, Goldman M. Idiotypic interactions and immune complexes in infectious diseases. Prog Immunol. 1983;5:1343-1346.
34. Haakenstad AO, Striker GE, Mannik M. The disappearance kinetics and glomerular deposition of small-lattice soluble immune complexes. Immunology. 1982;47:407-414.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  A. Mustafa, S. Nityanand, L. Berglund, H. Lithell, and A. K. Lefvert Circulating Immune Complexes in 50-Year-Old Men as a Strong and Independent Risk Factor for Myocardial Infarction Circulation, November 21, 2000; 102(21): 2576 - 2581. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Wu, S. Nityanand, L. Berglund, H. Lithell, G. Holm, and A. K. Lefvert Antibodies Against Cardiolipin and Oxidatively Modified LDL in 50-Year-Old Men Predict Myocardial Infarction Arterioscler. Thromb. Vasc. Biol., November 1, 1997; 17(11): 3159 - 3163. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||