© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Oxidized LDL–Induced NF-
B Activation and Subsequent Expression of Proinflammatory Genes Are Defective in Monocyte-Derived Macrophages From CD36-Deficient Patients
From the Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, Osaka, Japan.
Correspondence to Shizuya Yamashita, MD, PhD, Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, 2–2 Yamadaoka, Suita, Osaka 565-0871 Japan. E-mail shizu{at}imed2.med.osaka-u.ac.jp
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Abstract |
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Abstract—CD36 is 1 of the class B scavenger receptor expressed on monocytes, monocyte-derived macrophages (M
), platelets, and adipocytes. In our
previous studies, we reported that the uptake of oxidized low density
lipoproteins (OxLDLs) is reduced by 
50% in M from CD36-deficient
patients compared with that in control subjects. Recently, we have
shown that CD36 is highly expressed in human atherosclerotic aorta.
Possibilities have been raised that besides the wide distribution and
multifunctional characteristics of CD36, this molecule may also be
involved in the mediation of intracellular signaling. The aim of the
present study was to elucidate the role of CD36 in cytokine
secretion and to investigate the CD36-mediated intracellular signaling
stimulated by OxLDL. On addition of OxLDL or thrombospondin-1,
the M from CD36-deficient patients secreted significantly less
amounts of tumor necrosis factor-
(TNF-) and interleukin-1ß
(IL-1ß) compared with those from controls. RNase protection assay
with multiprobe template sets demonstrated that after incubation with
OxLDL, the mRNAs of a variety of cytokines, including genes
encoding IL-1Ra, IL-1ß, IL-6, TNF- and -ß, and interferon
(IFN)-
and -ß, were significantly lower in the M of patients.
The addition of antibody against CD36 attenuated this OxLDL-induced
response in controls. We also observed a reduced response in nuclear
factor-
B (NF-B) activity in OxLDL-stimulated M from
CD36-deficient patients. Unlike OxLDL, stimulation by
lipopolysaccharide induced an increase in NF-B activity in
M from CD36-deficient patients, suggesting that
lipopolysaccharide-mediated signaling was conserved. These
results demonstrate that in addition to the reduced OxLDL uptake that
we reported previously, CD36-deficient patients may also have an
impaired response of OxLDL-induced NF-B activation and subsequent
cytokine expression.
Key Words: atherosclerosis • CD36 • monocyte-derived macrophages • oxidized LDL • cytokines
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Hypercholesterolemia is 1 of the major and most extensively studied risk factors of atherosclerosis.1 Recent studies have contributed to our understanding of the role of oxidatively modified LDLs (OxLDLs) in the process of atherosclerosis. Furthermore, administration of antioxidants such as probucol inhibits the development of atherosclerosis in some animal models, which also supports the role of OxLDL in the pathogenesis of atherosclerosis.2 3 CD36 is 1 of the family members of class B scavenger receptors that are widely expressed in human tissues and that bind and take up OxLDL. A variety of effects of this lipoprotein on different cell functions has been demonstrated, including the expression of tumor necrosis factor-
(TNF-) and
interleukin-1ß (IL-1ß).4 Previous data have shown that
scavenger receptors by function are competent signal transduction
molecules, although individual receptors that mediate this effect have
not been well defined.5 Moreover, much less is known about
the effects of uptake of the various forms of modified LDL on the gene
expression, synthesis, and secretion of molecules in human
monocyte–derived macrophages (M).
Several lines of evidence indicate that nuclear factor-B (NF-B)
transcription factors may play an important role in
atherosclerosis. Homodimeric or heterodimeric
combinations of NF-B family proteins, such as p50, p52, p65, c-Rel,
and Rel B, mediate NF-B activity. NF-B is found in an inactive
form (I-B) in the cytoplasm. On stimulation, I-B is
phosphorylated, and thereby NF-B is released from
I-B, is translocated to the nucleus, and binds to the promoter
DNA.6 Activation of NF-B by inflammatory stimuli has
been demonstrated in M, endothelial cells, smooth
muscles cells, and T cells. All of these cell types have been reported
to play a major role in atherogenesis and display a distinct pattern of
gene expression in this condition.7 Recently, OxLDLs have
been implicated in modulating the expression of several
cytokines, growth factors, and lipopolysaccharide
(LPS)-induced molecules, which are regulated by
NF-B.8 9 The secretion of TNF- and IL-1ß by
isolated M in normal subjects has been measured with contradicting
results, but no such reports on human CD36–deficient M have been
available yet.
In view of the potential importance and unresolved issues regarding the
role of human CD36 in cytokine expression, this study was
designed to analyze the in vitro effect of OxLDL on the release
of cytokines from CD36-deficient M in comparison with that
from control M and to clarify the CD36-mediated intracellular
signaling stimulated by OxLDL. To approach this problem, we used human
M obtained from CD36-deficient patients and CD36-positive control
subjects and showed that CD36 is an important mediator for activation
of NF-B when stimulated with OxLDL.
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Methods |
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Study Subjects
Blood was obtained from 6 healthy, unrelated, CD36-positive control subjects and 6 different unrelated, CD36-deficient patients, identified by screening with specific antibodies and immunofluorescent flow cytometry, who were matched for age and risk factors. Their monocytes as well as platelets lacked CD36 on cell surfaces (data not shown).
Materials
Thrombospondin-1 (TSP-1) and monoclonal antibody to human
platelet TSP-1 used an ELISA that was obtained from Hematologic
Technologies, Inc.
Lipoprotein Preparation
LDLs (d=1.019 to 1.063 g/mL) were isolated by
sequential ultracentrifugation from the sera of 5
normolipidemic volunteers who had fasted overnight for 12 to 14 hours
as described previously.10 The protein content of LDL
was determined by the method of Lowry et al11 with bovine
serum albumin (BSA) as a standard. This represents the
native LDL used in the current study. Oxidation of LDL was performed in
a cell-free system. In brief, native LDL was diluted to 1 mg of protein
per milliliter with PBS and dialyzed at 4°C for 24 hours against PBS
in the absence of EDTA. The LDL was sterilized by ultrafiltration
through a 0.45-µm filter (Millipore), diluted to a concentration of
400 µg protein per milliliter with PBS, and incubated at 37°C in
the presence of 5 µmol/L CuSO4 for 8 hours
unless otherwise specified. Oxidation of LDL was confirmed by agarose
gel electrophoresis. The OxLDL was stored under nitrogen at 4°C and
used within 48 hours. LDL was acetylated with repeated
additions of acetic anhydride. Freshly prepared LDL, OxLDL, and
acetylated LDL were used for each experiment. To assess the
optimal secretion levels of cytokines in the presence or
absence of OxLDL, time course experiments were performed. (Please see
Figure
I, published online and available at http.) These peak levels
were evident after 6 hours of incubation.
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M Isolation and Culture
Mixed mononuclear cells from blood samples of fasting, healthy,
CD36-positive volunteers and CD36-deficient patients were isolated by a
density gradient centrifugation method and lymphocyte
separation solution (Nacalai Tesque). Twenty milliliters of blood
(anticoagulated with 10 U/mL sodium heparin) was layered over 15 mL of
Ficoll-Paque and centrifuged at
104g for 30 minutes. The mixed mononuclear
cell band was removed by aspiration, and the cells were washed twice
with serum-free RPMI-1640 medium. The cells were resuspended and seeded
at a density of 107 in 10-cm cell-culture dishes
(Primaria Labware, Becton Dickinson and Co) in serum-free medium with
0.1% low-endotoxin BSA. Monocytes were incubated for 2 hours at 37°C
in 5% CO2, and then adherent cells were washed
twice to remove loosely adherent cells. All experiments were performed
in serum-free medium supplemented with 0.1% BSA. These cells
represented monocytes in an early stage of differentiation
and therefore are referred to as monocyte-M in the text. Freshly
isolated mononuclear cells from whole blood were used in each
experiment.
Immunofluorescent Flow Cytometry
FITC-conjugated (Sigma Chemical Co) mouse monoclonal antibodies
against human CD36, OKM5 (Ortho Diagnostic Systems Inc) and
FA6-152 (Cosmo Bio Inc), were used for immunofluorescent flow
cytometric analysis. Mononuclear cells were isolated by density
gradient centrifugation as described above. Mononuclear
cells (2x105) were incubated with IgG1 or
FITC-conjugated OKM5 (final concentration 2.5 µg/mL) for 30 minutes
at 4°C. Then the cells were washed twice with PBS and assayed by flow
cytometry (FACScan, Becton Dickinson and Co). Data were
analyzed by using the Cell Quest software program, and cell
debris was excluded by appropriate 2-dimensional gating methods.
Specific fluorescence intensity was calculated by subtracting
FITC-IgG1 intensity from the mean fluorescent intensity of
FITC–monoclonal antibodies.
Cytokine Analysis
ELISA was used for analysis of TNF- and IL-1ß
levels. The assay is sensitive and reproducible and has been applied
for the measurement of TNF- and IL-1ß in plasma and tissue
culture. One milliliter of the medium was centrifuged for 5
minutes at 103 rpm. An aliquot (200 µL) from 3
independent samples of the supernatant was then added to TNF- and
IL-1ß immunoassay plates (Amersham). The plates were processed
according to the manufacturer’s protocol and read within 30 minutes at
450 nm.
Preparation of Total Cellular RNA and Ribonuclease Protection
Assay
For ribonuclease (RNase) protection assay experiments, the M
from CD36-deficient and control subjects were isolated as described
above. The cells were incubated overnight in serum-free medium
supplemented with 0.1% BSA, subsequently washed in Dulbecco’s PBS,
and incubated for an additional 6 hours in the presence or absence of
40 µg/mL OxLDL in serum-free medium supplemented with 0.1% BSA. For
neutralizing experiments, the M from control subjects were
stimulated with 40 µg/mL OxLDL in serum-free medium; human-specific
anti-CD36 antibody FITC-OKM5 or nonspecific mouse antibody FITC-IgG1
(final concentration 1 µg/mL) was included in each assay, and OxLDL
without addition of antibodies served as a control. Total RNA was
extracted with TRIzol reagent (GIBCO). An antisense cRNA was
transcribed by using T7 RNA polymerase in the presence of
[32P]UTP (3000 Ci/mmol, New England Nuclear).
Five micrograms of total RNA from the CD36-deficient and control M
was hybridized with the probe at 56°C and incubated for 16 hours.
Annealed products were digested with ribonuclease T1 at 37°C for
30 minutes. The protected fragments were precipitated and subjected to
6% polyacrylamide/urea gel. Results were analyzed by
autoradiography and quantified by laser densitometric
scanning (FUJI BAS 200).
Electrophoretic Mobility Shift Assay
M were incubated with either 10 µg/mL bacterial LPS
(Escherichia coli 0111:B4, Sigma) or 40 µg/mL OxLDL
or in their absence in serum-free RPMI-1640 medium supplemented with
0.1% BSA. Nuclear extracts were prepared according to Mufson et
al,12 with the additional step of washing the nuclear
pellets in low-salt buffer before high-salt buffer extraction of
nuclear proteins to remove any residual cytosolic contamination.
Protein concentration was measured by the method of Lowry et
al.11 The NF-B binding protein detection system (Life
Technologies, Inc) was used according to the manufacturer’s
instructions. In brief, end labeling of a double-stranded
oligonucleotide containing 2 NF-B–binding elements
was performed by using [32P]ATP and T4
polynucleotide kinase. Nuclear extracts (5 µg of protein)
were incubated with 106 counts per minute of the
labeled oligonucleotide at room temperature for 20
minutes. The specificity of protein-DNA complexes was verified by
competition assays with excess unlabeled
oligonucleotides. Nuclear extracts from HeLa cells were
used as a positive control. For supershift analysis, 2 µg of
the indicated antibody, p50 or p65 (Santa Cruz Biotechnology), was
incubated with nuclear extracts for 30 minutes before addition of the
labeled probe. The samples were separated on a 6% nondenaturing
polyacrylamide gel.
Statistical Analysis
Values are given as mean±SD. Statistical difference between
groups was performed with an unpaired t test. A value of
P<0.05 was considered significant.
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Results |
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To assess the optimal secretion of TNF-
and IL-1ß into the
medium in the presence or absence of OxLDL, time course experiments
were performed. The peak levels of TNF- and IL-1ß concentration
were evident after 6 hours of incubation. The secretion of TNF- and
IL-1ß declined progressively thereafter, and by 24 hours after
stimulation, secretion had returned to baseline levels. To establish
the range of concentrations over which lipoproteins are effective, we
performed a dose titration of lipoprotein-induced cytokine
expression. OxLDL stimulated M in a dose-dependent manner, with a
maximal effect at a concentration of 40 µg/mL (data not shown).
To address the relevance of CD36 in the release of cytokines
into the medium and mRNA expression in M exposed to OxLDL, secretion
of these cytokines was analyzed by ELISA with the use
of conditioned medium from cultured M. The secretion of TNF- from
the control M treated with 40 µg/mL OxLDL almost doubled after 6
hours of incubation, whereas that of CD36-deficient M was remarkably
low (P<0.01; Figure 1A
).
Furthermore, the IL-1ß levels in conditioned medium from the
CD36-deficient M after the addition of 40 µg/mL OxLDL were also
significantly lower than those from the control M
(P<0.01; Figure 1B). There was no significant
difference in the amounts of TNF- and IL-1ß released from the
unstimulated M from CD36-deficient and normal control subjects 6
hours after replacement of the medium (Figures 1A and
1B). By contrast, acetylated LDL and native LDL under
the same experimental condition had no significant effect on TNF-
and IL-1ß secretion in both groups (data not shown). The close
correspondence between TNF- and IL-1ß values in both stimulated
and unstimulated conditioned media of M from the controls and
patients suggests a common mechanism in releasing these
cytokines. To exclude the possibility of the effect of
bacterial LPS as a potent inducer of cytokine release from
M, the concentrations of LPS were measured in all preparations. When
the lipoprotein preparations contained >0.006 ng/mL LPS, they were not
used in the study.
We further showed that the expression of CD36 was mandatory for
mediating the enhanced secretion of cytokines into the
conditioned medium by coincubating the control M stimulated by OxLDL
with anti-human CD36 monoclonal antibody OKM5 or nonspecific mouse IgG1
(final concentration 1 µg/mL). The M incubated with OKM5 showed a
significant reduction of TNF- and IL-1ß secretion, whereas those
incubated with mouse IgG1 had no significant change (P<0.05
and P<0.01, respectively; Figures 2A and 2B).
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To determine the effect of another CD36 ligand, TSP-1, on
cytokine release, we cultured M from the controls and
CD36-deficient subjects in the absence or presence of varying
concentrations of TSP-1 for 2 hours before collection of the
conditioned media. Incubation with TSP-1 for 2 hours was chosen on the
basis of previous reports.13 The secretion of TNF- from
the control M treated with TSP-1 (concentrations of 0.4 to 1
µg/mL) was significantly higher than that from the CD36-deficient
M. (Please see Figure II, available online at http.) When control
M were cultured in the presence of neutralizing antibodies against
human CD36, OKM5, or TSP-1, the activation of TNF- was significantly
abrogated (data not shown).
Next we investigated whether the induced release of TNF- and IL-1ß
observed after incubation with OxLDL was associated with activation of
mRNA expression. We performed an RNase protection assay with multiprobe
template sets in RNA derived from unrelated CD36-deficient patients as
well as CD36-positive controls. We observed that after 6 hours of
incubation with OxLDL, mRNAs of a variety of cytokines were
significantly lower from CD36-deficient patients than those from
control M (Figures 3A and
3B). These observations were highly reproducible in
repeated experiments. To determine whether changes in mRNA levels can
be ascribed, at least in part, to the difference in CD36 expression,
control M stimulated with OxLDL were incubated with either the
anti-CD36 antibody OKM5 or IgG1. Interestingly, when the control M
stimulated with OxLDL were treated with OKM5, the intensity of the
TNF-–retarded band was significantly diminished. By contrast, when
incubated with nonspecific mouse IgG1, no significant change in the
intensity of the OxLDL-enhanced band was noted (Figure 4). A similar result was observed
with IL-1ß (data not shown). This finding suggests that the
expression of CD36 might be a key factor in the release of these
cytokine mRNAs after stimulation with OxLDL. Resting M
expressed little to no mRNA of human TNF- and human IL-1ß in both
control and CD36-deficient subjects (data not shown).
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Because NF-B is involved in the control of a variety of genes that
are activated on inflammation, NF-B may play a central role
in atherogenesis. Other reports have shown that a rapid activation of
NF-B involving dissociation from its inhibitor and
translocation to the nucleus occurs in response to a variety of agents
that include OxLDL, LPS, and cytokines.14 15 To
further search for the role of CD36 in NF-B activation, we used
nuclear proteins harvested from the M of unrelated CD36-deficient
and control subjects, incubated with or without either OxLDL or LPS,
and explored the possibility that activation of NF-B
represents an important step in the regulation of
cytokine expression by an electrophoretic mobility shift
assay (EMSA) with a radiolabeled oligonucleotide
containing the NF-B binding sequence. The EMSA revealed that NF-B
activities were increased after incubation with OxLDL in the control
M, whereas the M from CD36-deficient patients showed a defective
response to OxLDL. Interestingly, the addition of bacterial LPS to the
medium induced a significant stimulative response in M from both the
controls and patients, suggesting that LPS-mediated signaling was
conserved in the patients’ M. The EMSA reaction specificity was
verified by competition assays with a 20-fold excess of unlabeled
oligonucleotide, which resulted in the disappearance of
the bands established by OxLDL in control M (Figure 5A). To confirm the specificity of the
protein-DNA interaction, we performed a gel supershift assay with
antibodies for the p50 and p65 subunits of NF-B. When incubated with
the nuclear extracts, a specific DNA binding complex of NF-B
interacted with antiserum p65 (Figure 5B). Thus, NF-B complex
was likely to contain dominantly homodimers of p65. We took a step
further to determine whether CD36 was directly mediating the
OxLDL-enhanced NF-B DNA binding activity by coincubating
OxLDL-treated M from control subjects with human-specific anti-CD36
OKM5 (1 µg/mL) or mouse IgG. When incubated with OKM5, the intensity
of the gel-retarded band was significantly diminished, although a
degree of activation was observed. In contrast, in the M incubated
with control IgG, no change in the intensity of the OxLDL-enhanced band
was observed (Figure 5C). Although the precise molecular
mechanism involving NF-B–OxLDL activation has yet to be fully
established, the principal effects seem to involve CD36. These findings
confirm previous reports implicating the involvement of CD36 in
OxLDL-induced NF-B activation and hence, cytokine
regulation.
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Discussion |
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This study examined the role of CD36 in cytokine secretion by using M
from CD36-deficient patients and CD36-positive control
individuals. We also evaluated the CD36-mediated intracellular
signaling stimulated by OxLDL. CD36 is a cell surface receptor that has
been shown to interact with a variety of ligands, including TSP,
collagen, Plasmodium falciparum–infected erythrocytes,
apoptotic neutrophils, modified LDLs, anionic phospholipids,
and long-chain fatty acids. A number of these CD36 ligands elicit the
transduction of intracellular signals involved in cell activation and
internalization of bound ligands.16 17 Recently, we
have reported that this molecule is expressed on M of
atherosclerotic lesions from human aorta.18 Furthermore,
we demonstrated that in monocyte-derived M from CD36-deficient
patients, the uptake of OxLDL was reduced by 50% compared with that
in control M, suggesting that CD36 may also play a significant role
in OxLDL uptake.19 The present findings provide
additional insight that in monocyte-derived M from CD36-deficient
subjects, neither TNF- nor IL-1ß secretion into the medium was
significantly induced by OxLDL. Furthermore, CD36-deficient M
stimulated with OxLDL had significantly reduced levels of a variety of
cytokine mRNAs, including genes encoding IL-1Ra, IL-1ß, IL-6,
TNF- and -ß, and IFN-1 and -ß, in comparison with normal
control M.
Our assumption that CD36 plays a principal role in the release of
cytokines was reinforced by the fact that the M from
CD36-deficient subjects stimulated by TSP-1, a natural ligand for CD36,
also secreted a lesser amount of TNF- into the culture medium in
comparison with controls. (Please see Figure II, available online at
http.) Further confirmation that the association of OxLDL or TSP-1 with
CD36 on M was vital in the activation of TNF- and IL-1ß was the
finding that when OKM5 or TSP-1 antibody was added to the medium in the
presence of TSP-1, there was significant attenuation of the effects of
TSP-1 in activating M to release TNF- or IL-1ß (data not
shown). These findings led us to seriously consider the possibility
that activation occurs at the cell surface of activated M
and requires the expression of CD36.
Our data show that CD36-deficient patients have significantly lower
levels of CD36 mRNA expression and release of TNF- and IL-1ß in
response to OxLDL in comparison with control subjects; however, CD36
OxLDL-dependent induction of TNF- and IL-1ß release into the
medium and mRNA expression were in part inhibited by incubation of
control M with the human CD36–specific antibody OKM5 (Figures
2 and 4). The high levels of mRNA seen in the control
M indicate that these cells were in an activated state.
Previous studies have shown that monocyte adhesion is associated with
the activation of cytokine mRNA
production.20 Hence, in the intima, M will
release cytokines on their adhesion to the extracellular
matrix, and this might be further induced if M are exposed to OxLDL
trapped within the matrix. We have observed that the M obtained from
CD36-deficient patients adhere poorly to collagen-coated culture dishes
(M.J. et al, unpublished observations, 2000). Furthermore, OxLDL
significantly enhanced NF-B–like activity in nuclear extracts from
controls, whereas no significant activation was observed in the
patients’ nuclear extracts. The activation of NF-B DNA binding
activity was manifested, at least in part, by the p65 subunit, which
was detected with a supershift gel assay. Besides p65 and p50, other
proteins may bind NF-B target DNA, whose electrophoretic mobility
would not be affected by p65 or p50 subunit–specific antibody. This
concept supports our earlier studies that OxLDL itself upregulates the
expression of CD36 in human M and that this action occurs at the
transcription level.21
We observed some degree of induction of NF-B in the presence of CD36
antibody (Figure 5C), but the mechanism of induction was not
very clear. There are several possible explanations for this
phenomenon: (1) In this series of experiments, M were coincubated
for 6 hours in the culture medium in the presence of OKM5. The
incubation for 6 hours was possibly sufficient for antibody-bound CD36
to internalize and for de novo synthesis of CD36. (2) The role of other
receptors such as scavenger receptor type A was not accounted
for in the present series of studies. (3) Presumably sequestration
of TNF- and IL-1ß occurs on the cell surface via CD36; hence, the
presence of antibody may lead to the displacement of cytokines
from the cell surface and proceed to the activation of transcription
factor NF-B. (4) The interaction of CD36 with its specific
antibodies has been reported to lead to an oxidative burst in
M.22 Studies to determine whether any of these events
result in activation of NF-B after stimulation of CD36 by OxLDL are
underway.
Above all, the low LPS concentrations in the test samples led us to
speculate that the M activation observed in the present study
was possibly in part mediated through CD36 and could not be attributed
to LPS contamination, suggesting a distinct signaling pathway and the
growing influence that CD36, as an individual receptor, has on the
process of foam cell formation. However, in a separate series of
experiments, stimulation of M by LPS led to an increase in NF-B
activity in both controls and patients, suggesting that LPS-mediated
signaling was conserved in the patients’ M, most likely via
CD14.23 These results indicate that the LPS-dependent
activation pathway of cytokine secretion is different from that
of the CD36-dependent activation pathway.
We did not evaluate specific CD36 features required for the
OxLDL-mediated enhancement of NF-B DNA binding activity. However, it
has been shown that OxLDL-mediated enhancement of NF-B DNA binding
activity after activation of CD36-producing Chinese hamster ovary cells
required an intact carboxyl-terminal cytoplasmic segment of
CD36.24 Furthermore, Yehualaeshet et al25
showed that activation of rat alveolar M transforming growth
factor-ß1 required interaction of CD36 ligands and its cell surface
receptor, CD36. We are therefore confident that the OxLDL activation of
NF-B binding in M observed herein required expression of CD36.
This was further confirmed by the finding that NF-B activity was
inhibited in part by coincubation of M with OKM5, suggesting a
direct effect of OxLDL on CD36 but not on other receptors.
The physiological function of CD36 has not been
well characterized, although this molecule has been proposed to mediate
cytoadherence of P falciparum–parasitized erythrocytes. It
will be important to know whether such adherence could also transduce a
transmembrane signal that is able to modulate transcription factor(s)
in a similar way to what we observed here. Tontonoz et
al26 showed that expression of CD36 was directly
induced by the transcription factor peroxisome
proliferator–activated receptor-, which has a role in
atherogenesis, adipogenesis, and insulin signaling. Furthermore, CD36
functions in a feedback loop as a receptor for OxLDL, in which the
lipid components of OxLDL activate peroxisome
proliferator–activated receptor-, thereby inducing CD36
expression. Therefore, it will also be of great interest to determine
whether this link is involved in CD36-mediated signal transduction by
OxLDL, hence modulating NF-B activity and leading to
cytokine regulation.
We and others have extensively clarified the pathophysiological significance of CD36 as a major receptor for OxLDL. Since we discovered the CD36-deficient subjects, we have identified 3 types of gene mutations.19 Some of our CD36-deficient patients in this study have not shown any clinical symptoms of coronary artery disease (CAD), while others have signs and symptoms of it. It remains an interesting question whether such low levels of cytokines are protective against atherosclerosis and/or whether they delay the process of atheroma formation. Previous reports have shown evidence that the circulating levels of cytokines, under different conditions, can exhibit favorable or unfavorable effects. The unfavorable effects may occur when concentrations are high or when low concentrations persist for longer periods.27 Recently, we have found that the human CD36 deficiency is relatively frequent in the Japanese general population and appears proatherogenic in association with the clustering of multiple risk factors. We analyzed 26 patients with type 1 CD36 deficiency and clarified the clinical characteristics of these patients, especially with respect to atherogenicity. These patients had multiple risk factors for CAD, such as hyperlipidemia, high blood pressure, and insulin resistance. Furthermore, we found that the frequency of type 1 CD36 deficiency was 3-fold higher (0.9%) in patients with CAD than in the general population (0.3%; K. Miyaoka, personal communication, 1999).
In addition, it has recently been reported that 17% of Japanese
patients with CAD have CD36 deficiency, suggesting a significant
relative risk attributable to this genetic defect.28
Furthermore, Hwang et al29 showed that of 7 patients with
CD36 deficiency who underwent biochemical tests, 3 had type 2 diabetes
(1 with insulin resistance and 1 with
hypertriglyceridemia), 1 had hypertension,
and 1 had CAD. In summary, the biological plausibility of CD36 and the
phenotype of patients with CD36 deficiency suggest that CD36
may play an important part in the pathogenesis of
atherosclerosis, at least in populations in which the
prevalence of CD36 deficiency is rather high.
With the limited number of patients with this abnormality, our current
results do not permit any firm conclusions, although long-term clinical
follow-up in cohorts of our patients is underway to resolve the exact
mechanism involved. However, so far, both human and rodent results
suggest that CD36 deficiency may play an important part in the
pathogenesis of CAD and the human insulin resistance syndrome. Taken
together, this study demonstrates that in addition to reduced OxLDL
uptake, CD36-mediated intracellular signaling is altered in
CD36-deficient patients, leading to a defective response of
OxLDL-induced NF-B activation and subsequent cytokine
secretion.
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Acknowledgments |
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This research was partly supported by a grant-in-aid from the Japanese Ministry of Education, Culture, Sports and Welfare (07557074). We gratefully acknowledge the technical assistance of Eiko Okuda, Atsuko Ohya, Chiaki Ikegami, and Yuri Fuse.
Received September 6, 1999; accepted April 20, 2000.
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