(Stroke. 2000;31:2661.)
© 2000 American Heart Association, Inc.
Original Contributions |
Polymorphism in the Promoter of Lipopolysaccharide Receptor CD14 and Ischemic Cerebrovascular Disease
From the Departments of Neurology (D.I., N.T., H.S., Y.F.), Laboratory Medicine (M.M., A.S., K.W.), Hematology (M.M.), and Health Center (I.S.), School of Medicine, Keio University, Tokyo, Japan.
Correspondence to Daisuke Ito, MD, Department of Neurology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
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Abstract |
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Background and Purpose—A growing amount of evidence suggests that infectious and inflammatory processes may be involved in the initiation of arteriosclerosis, but the mechanisms are conceivably multifactorial and complex. Two European groups have recently demonstrated that a C(-260)
T polymorphism in the
promoter of the CD14 lipopolysaccharide receptor may be a risk
factor for coronary artery disease (CAD). The T allele of
this polymorphism reportedly increases the expression of CD14 and
may be involved in atherogenesis. In the present study we
investigated a possible association between the C(-260)T
polymorphism in the CD14 promoter and the occurrence of
symptomatic ischemic cerebrovascular disease
(CVD).
Methods—Genotype frequencies of the C(-260)T
polymorphism in the CD14 promoter were determined in 235 patients
with CVD, as confirmed by brain CT and/or MRI, and 309 age- and
sex-matched control subjects.
Results—The distribution of genotypes was as follows: CVD
patients, T/T 24.3%,
C/T 53.2%, and
C/C 22.6%; controls,
T/T 26.9%,
C/T 50.2%, and
C/C 23.0%. There was no significant
difference between the CD14 promoter genotypes of the CVD
patients and the controls (
2=0.601,
P=0.741). We also measured the concentration of serum
soluble CD14 and the density of membranous CD14 on monocytes in the CVD
patients, but the polymorphism was not associated with either the
concentration of soluble CD14 or the density of membranous CD14
(P=0.358, P=0.238, respectively).
Conclusions—Our results indicate that the C(-260)T
polymorphism in the CD14 promoter is not associated with an
increased risk for CVD.
Key Words: lipopolysaccharides • polymorphism (genetics) • risk factors • stroke
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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There are accumulating data indicating that infection may be linked to atherosclerotic disease.1 2 3 Although the true role of infection as a risk factor for atherosclerosis is unclear, several known mechanisms may play at least a partial role in this process. One of the most likely mechanisms involves lipopolysaccharide (LPS) and its receptor, CD14, both of which have been implicated in atherogenesis.3 LPS, a structural component of gram-negative bacteria, is bound in plasma by LPS binding protein.4 The LPS–LPS binding protein complex then binds to a glycosylphosphatidylinositol-anchored membrane protein, membranous CD14 (mCD14), on monocytes and macrophages and activates these cells. The activated phagocytes in turn secrete inflammatory cytokines through which LPS indirectly activates endothelial cells. Soluble CD14 (sCD14), which lacks a glycosylphosphatidylinositol anchor, can also be found in plasma. Endothelial cells and smooth muscle cells, lacking their own mCD14, are directly activated by LPS-sCD14 complex.5 6 Directly and indirectly activated endothelial cells express cell adhesion molecules and increased procoagulant activity, and they release free radicals, thereby mediating the initiation and development of atherosclerosis.
Two European groups have recently reported that genetic variation in
the CD14 promoter may be a risk factor for coronary artery
disease (CAD).7 8 Both groups, one in Germany and the
other in the Czech Republic, identified a C(-260)T
nucleotide change in the promoter region of the CD14
gene.9 The German study7 of 2228 patients
reported no significant association between this polymorphism and
CAD within the total study group, but T homozygosity was associated
with an increased risk of CAD in a subgroup with low coronary
risk. The Czech study8 demonstrated that T allele
frequency was significantly higher in myocardial infarction survivors
and that the density of monocyte mCD14 was higher in T/T homozygotes
than in other genotypes. Thus, the CD14 promoter
genotype may affect inflammatory processes and be involved in
atherogenesis, and it is therefore possible that this genotype
might also be associated with other major forms of thrombotic disease,
such as ischemic cerebrovascular disease.
The primary aim of this study was to determine whether the C(-260)T
polymorphism in the promoter of the CD14 gene is associated with
symptomatic ischemic cerebrovascular disease
(CVD).
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We analyzed 235 unrelated Japanese patients with CVD and 309 age- and sex-matched control subjects. All CVD patients had visited the outpatient clinic of Keio University Hospital in Tokyo for regular follow-up examinations. We selected CVD patients aged
70 years at the
onset of CVD. On the basis of the Classification of Cerebrovascular
Diseases III report from the committee established by the National
Institute of Neurological Disorders and Stroke,10 CVD
patients who were diagnosed with atherothrombotic infarction, lacunar
infarction, or transient ischemic attack were enrolled in this
study. CVD patients with cardioembolic cerebral infarction and cerebral
hemorrhage were not included in this group. We initially
recruited 246 CVD patients who fulfilled the aforementioned criteria,
but 11 subjects were excluded because of unwillingness to participate
after an explanation of this study. Two hundred thirty-five CVD
patients were finally enrolled in the present study. The mean
interval between the onset of CVD and genotyping was 5.1±4.4 years.
The control subjects consisted of employees of Keio University. The
control subjects were recruited by first registering 603 subjects who
came for a routine screening examination and gave their informed
consent. We excluded 2 subjects with a history of CVD and 1 subject
with a history of CAD from this study and ultimately selected 309 age-
and sex-matched control subjects. There were 4 control subjects with
hyperuricemia, 3 with postoperative gastric cancer, and 1 each with
chronic nephritis, chronic hepatitis, idiopathic thrombocytopenic
purpura, and postoperative bladder carcinoma. Written informed consent
was obtained from all subjects after a full explanation of the study
and a guarantee of total privacy. Brain CT and/or MRI was performed on
all CVD patients within 2 days after the onset of CVD. MR angiography
and/or extracranial duplex ultrasonography studies were available in
>80% of the cases. Hypertension was defined as systolic blood pressure >140 mm Hg and/or diastolic pressure >90 mm Hg or current treatment with antihypertensive drugs. Smokers were defined as current smokers. Hypercholesterolemia was defined as a cholesterol level >220 mg/dL or current treatment with a cholesterol-lowering drug.
Polymorphism Analysis
Whole blood was collected into sodium citrate tubes. A direct
DNA amplification kit (Shimadzu Co), which enabled us to amplify
DNA from whole blood without DNA extraction steps, was used as
described previously.11 12 13 Amplification of the 418-bp
fragment of the CD14 promoter was performed with the 5' primer
5'-CTAAGGCACTGAGGATCATCC-3' and 3' primer 5'-CATGGTCGATAAGTCTTCCG-3'. A
0.5-µL volume of whole blood, 12.5 pmol of each primer, 200
µmol/L of each deoxynucleotide triphosphate, 5 µL of
5x Ampdirect-A, 5 µL of 5x Amp Addition-1, and 1.25 U
Taq polymerase (TOYOBO Co) and water were added to the
reaction to achieve a total volume of 25 µL. The polymerase chain
reaction (PCR) consisted of 1 cycle of 15 minutes at 85°C and 4.5
minutes at 94°C; 42 cycles of 30 seconds at 94°C, 1 minute at
55°C, and 1 minute at 72°C; and 7 minutes at 72°C in a Gene Amp
PCR system 2400 (Perkin Elmer). The PCR product (4 µL) was
cleaved in appropriate buffer with 8 U of HaeIII restriction
enzyme (New England Biolabs). The DNA fragments were separated by
electrophoresis through a 2% agarose gel containing 0.5 µg/mL of
ethidium bromide and visualized under UV light. Digestion of the PCR
products yielded bands of 418 bp in TT homozygotes, 263 and 155 bp
in CC homozygotes, and all 3 bands in the heterozygotes.
Biochemical Analysis
The serum sCD14 levels were determined with a commercially
available enzyme-linked immunosorbent assay kit supplied by IBL. A
total of 16 T/T homozygotes, 51 C/T heterozygotes, and 22 C/C
homozygotes (mean±SD age, 65.5±8.4 years; 66 men) were randomly
recruited from all CVD patients for this assay. The mean interval
between the onset of CVD and blood sampling was 7.0±3.4 years.
The density of mCD14 on the monocyte surface was measured by flow cytometry. A total of 10 T/T homozygotes, 10 C/T heterozygotes, and 10 C/C homozygotes (mean±SD age, 66.5±6.4; 19 men) were randomly selected from all CVD patients after genotyping. The mean interval between the onset of CVD and blood sampling was 5.6±2.8 years. Anti-CD14 (Leu-M3) monoclonal antibody, purchased from Becton Dickinson, directly conjugated with fluorescein isothiocyanate, was used in the analysis. Whole blood was analyzed with a FACScan (Becton Dickinson) flow cytometer. Distinct monocyte clusters were identified with a combination of CD14 monoclonal antibody intensity and forward light scatter. This display was used for gating the monocytes, and a minimum of 5000 cells per sample was analyzed.
Statistical Analysis
The differences in the frequencies of the CD14 genotypes
and alleles and other risk factors were analyzed by the
2 test. Mean age in the 2 groups was compared
by Student’s t test. Multiple logistic regression methods
were used to control for possible confounding factors. The
relationships between mCD14 densities, serum sCD14 levels, and CD14
genotypes were tested by 1-way ANOVA. Associations and
differences with probability value <0.05 were considered significant.
All statistical analyses were performed with the use of
Statview software (version 5.0 for Windows, SAS Institute).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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A total of 235 CVD patients and 309 control subjects were recruited for this study. Table 1
summarizes the clinical features of the CVD patients and the control
subjects studied. There were no significant differences in age or sex
between the 2 groups. The risk factors hypertension, diabetes mellitus,
and smoking were significantly more common in the CVD patients.
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The distributions of genotypes and the allelic frequencies of
the polymorphism in the CD14 promoter in the control and CVD groups
are shown in Table 2. Among the CVD
patients, 24.3% were T/T, 53.2% were
C/T, and 22.6% were C/C.
This genotype distribution was not significantly different from
the distribution in the control group (T/T,
26.9%; C/T, 50.2%; C/C,
23.0%,) (2=0.601, P=0.741). T
allele frequencies were also comparable between CVD patients and
control subjects (50.9% and 51.9%, respectively). This was confirmed
by the results of multiple logistic regression analysis with
the established risk factors hypertension,
hypercholesterolemia, diabetes mellitus, and
smoking (2=0.692, P=0.405). In
further calculations, subgroups were formed as low-risk groups
(excluding subjects with hypertension,
hypercholesterolemia, diabetes mellitus, or
smoking). The CD14 promoter genotype was not related to CVD in
any of the subgroups (data not shown). As shown in Table 2,
analysis by CVD subtype (atherothrombotic infarction, lacunar
infarction, transient ischemic attack) also failed to yield any
difference in genotype distribution compared with the control
group.
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We also measured the serum concentration of sCD14 and the density of
mCD14 on monocytes in the CVD patients (Table 3). Although the sCD14 concentration
tended to be slightly higher in the T/T
genotype patients, there were no significant differences in
either sCD14 concentrations or the density of mCD14 between any of the
genotypes.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The present study is the first to examine the relationship between CVD and the CD14 promoter polymorphism. The Japanese population has a slightly higher T allele frequency than European populations.7 8 Our study demonstrated that the CD14 promoter polymorphism is not associated with CVD, even in low-risk subjects. Although the number of subjects in the CVD subtypes was relatively small, no differences in genotype distribution were found in the analysis by CVD subtypes either. In addition, there were no significant correlations between the genotypes of the CVD patients and the sCD14 concentration in their serum or the density of mCD14 on their monocytes.
A Czech group8 demonstrated that the T allele was
associated with a higher density of mCD14 on monocytes in healthy
volunteers. They suggested that the C(-260) T change in the
promoter region affects the level of CD14 gene expression. Their
findings are in clear contrast to the results of our own studies.
Several reasons may account for these conflicting results. First, the
genetic background of Japanese and European populations is different.
The T allele in the CD14 promoter may be in linkage disequilibrium,
with other mutations involved in the expression or function of CD14 in
Europeans but not in Japanese. Second, the choice of subjects for
measurement of mCD14 was different in the 2 studies. The Czech group
recruited healthy volunteers, whereas our subjects were selected from
CVD patients. Although we excluded CVD patients with acute CVD and
other acute illnesses from the analysis of mCD14 and sCD14,
chronic thrombotic disease in itself may affect CD14 gene expression.
Third, the age of the subjects was another important difference between
the 2 studies. The Czech group selected young subjects (aged 20 to 30
years), whereas the mean ages for measurement of sCD14 and mCD14 in our
study were 65.5±8.4 and 66.5±6.4 years, respectively. It is possible
that the level of expression of CD14 changes with age.
The most important finding in our study was that a polymorphism in
the CD14 promoter is not associated with the occurrence of CVD.
However, several points should be borne in mind when one interprets the
results of the present study. All CVD patients in this study were
selected from patients who had visited the outpatient clinic for a
regular checkup, and they were therefore cerebrovascular attack
survivors. Therefore, both selection and survival bias cannot be
avoided in this disease-association study, and it is likely that early
mortality of CVD in patients could lead to underestimation of the
incidence of this polymorphism. Moreover, patients with
cardioembolic cerebral infarction were not included in the present
study because the primary aim of our study was to examine atherogenesis
of the cerebrovascular system. Therefore, we cannot exclude the
possibility that this polymorphism could be related to pathogenesis
of cardioembolic cerebral infarction. Finally, our study refers to the
association between this polymorphism and CVD only in the Japanese
population. The relevance of this polymorphism should be
investigated in other populations and by prospective and family
studies. Nevertheless, our findings suggested that the C(-260)T
polymorphism in the CD14 promoter may have less effect on the
occurrence of CVD than on that of CAD.
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Acknowledgments |
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This study was supported by grant-in-aid for scientific research No. 11557206 (to Dr Watanabe) from the Ministry of Education, Science, and Culture of Japan.
Received June 12, 2000; revision received August 2, 2000; accepted August 2, 2000.
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