© 1999 American Heart Association, Inc.
Vascular Biology |
Transcriptional Inhibition by Interleukin-6 of the Class A Macrophage Scavenger Receptor in Macrophages Derived From Human Peripheral Monocytes and the THP-1 Monocytic Cell Line
From the Cardiovascular and Pulmonary Research Institute (H.-S.L., Y.-J.G.), Allegheny University of the Health Sciences, Pittsburgh, Pa; the Division of Clinical Nutrition (A.M., H.I.), National Institute of Health and Nutrition, Tokyo, Japan; the Faculty of Pharmaceutical Sciences (T.D.), Osaka University, Osaka, Japan; and the Department of Molecular Biology and Medicine (H.-S.L., M.H., T.K.), Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan.
Correspondence to Yong-Jian Geng, MD, PhD, Cardiovascular and Pulmonary Research Institute, Allegheny University of the Health Sciences, 8th Floor, South Tower, 320 E North Ave, Pittsburgh, PA 15212. E-mail ygeng{at}pgh.auhs.edu
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Abstract—Expression of the class A macrophage scavenger receptor (MSR) contributes to the uptake of modified low density lipoproteins (LDL) by macrophages and transformation of these cells into lipid-laden foam cells, which characterize atherosclerosis. Many environmental factors, in particular, proinflammatory cytokines and growth factors, can exert regulatory effects on MSR expression, whereas intracellular accumulation of cholesterol itself does not influence MSR levels to any considerable extent. In the present study, by using an in vitro model, we examined whether stimulation with interleukin-6 (IL-6), an immunoregulatory, multipotential cytokine, modulates the expression and activities of the MSR in macrophages. When treated with IL-6, macrophages derived from peripheral monocytes and phorbol 12-myristate 13-acetate (PMA)–differentiated THP-1 monocytic cells showed significantly reduced uptake and/or binding of the MSR ligand, acetylated LDL. This effect was paralleled by a reduction in the expression of MSR protein and mRNA. Analysis of MSR promoter activity in THP-1 cells transfected with an MSR promoter–reporter gene construct demonstrated decreased activity of the MSR promoter in IL-6–treated THP-1 macrophages. Electrophoretic mobility gel shift assay also showed a reduction in the binding of a transcription factor to the MSR promoter AP-1/ets elements in IL-6–treated cells. Thus, exposure to IL-6 may inhibit expression of the class A MSR in differentiated macrophages at transcriptional levels. This result suggests that this cytokine may modulate foam cell formation during atherogenesis.
Key Words: scavenger receptors • atherosclerosis • cytokines • foam cells • lipoproteins
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Introduction |
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Atherosclerosis is characterized by the intimal accumulation of lipids, mainly cholesterol and cholesterol esters, and the infiltration of inflammatory cells, particularly macrophages and T cells, in addition to migration and proliferation of medial smooth muscle cells. The macrophage scavenger receptor (MSR) pathway plays a major role in the internalization of chemically modified lipoproteins, such as oxidized and acetylated (AcLDL) LDL by macrophages, leading to the transformation of macrophages into lipid-laden foam cells.
Expression of the class A MSR is limited to tissue macrophages
and related cell types.1 2 3 Peripheral
monocytes express the class A MSR at low levels.4 5
However, on penetrating the arterial intima and
differentiating into macrophages, MSR expression dramatically
increases.4 6 7 Many environmental factors that influence
the development and differentiation of monocytes/macrophages
can regulate expression of the class A MSR,8 9 even
though, in contrast to native-LDL receptors, class A MSR expression is
not affected by intracellular cholesterol accumulation. It
has been reported that proinflammatory cytokines produced by
activated immune cells (eg, T cells and macrophages)
can modulate MSR expression. For example, macrophage
colony-stimulating factor enhances,9 but transforming
growth factor-ß1 (TGF-ß1), interferon-
(IFN-), and tumor
necrosis factor-
(TNF-) inhibit, MSR
expression.10 11 12 13 The molecular mechanisms underlying
cytokine regulation of MSR expression remain largely unclear.
Several lines of evidence indicate that those cytokines may
exert their regulatory effects at transcriptional levels of the
MSR.13 14 Recent studies of the MSR gene promoter have
documented that at least 2 transcription factor families, AP-1/ets and
Pu1, may be critical for MSR transcription.15
It is known that interleukin-6 (IL-6), a multifunctional cytokine produced by immune cells as well as smooth muscle cells,16 17 can regulate the development and differentiation of monocytes/macrophages in vivo18 19 20 as well as in vitro.21 IL-6 has been found to serve as a survival factor for certain lymphoma and hybridoma cells, and therefore it contributes to the synthesis of immunoglobulins.19 22 23 Challenged by antigens and proinflammatory factors, immune cells can elaborate substantial amounts of IL-6, which in turn promotes production of several important acute-phase proteins. Although IL-6 exerts a regulatory effect on monomyeloid cell differentiation and acute-phase protein production,19 21 little is known about whether IL-6 can modulate expression of the MSR.
Atherosclerotic lesions with features of chronic inflammation are
composed of cells that are able to produce IL-6 and the MSR,
respectively.24 25 26 27 It is important to examine
whether IL-6 influences the macrophage-scavenging function, as
do other cytokines such as IFN-,12
TGF-ß1,10 and TNF-.13 In this study, we
employed an in vitro model to address (1) whether treatment with IL-6
affects MSR activity; (2) whether IL-6 regulates MSR gene expression;
and (3) which signaling transduction pathway or transcription
factor is involved in IL-6–mediated regulation of MSR expression.
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Methods |
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Reagents
Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma Chemical Co and dissolved in dimethyl sulfoxide in a stock concentration of 200 µmol/L. 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) AcLDL, DiI-LDL, native LDL, and AcLDL were purchased from Perlmmune, Inc. Recombinant IL-6 was obtained from R&D Systems. Actinomycin D was purchased from GIBCO-BRL. Human monoclonal antibody against MSR I (MH1) was prepared in Dr Kodama's laboratory, University of Tokyo, Tokyo, Japan.28 Antibodies against AP-1/Jun B and AP-1/c-Jun were obtained from Santa Cruz Laboratories.
Cell Culture and Cytokine Treatment
The human monocytic leukemia cell line THP-1 was obtained from
the American Type Culture Collection (Manassas, Va) and
maintained in RPMI-1640 (GIBCO-BRL) medium supplemented with
heat-inactivated 10% FBS (Multiser), 100 U/mL penicillin,
100 µg/mL streptomycin, and 2 mmol/L glutamine at 37°C under
5% CO2. THP-1 cells were treated with or without
PMA (200 nmol/L) or a combination of PMA (200 nmol/L) and IL-6 (20
ng/mL) for 48 hours when not otherwise stated in the text.
Isolation and Treatment of Monocytes From Peripheral Blood
Mononuclear cells were isolated from peripheral
blood by Ficoll-Paque centrifugation (Sigma) and
suspended in RPMI-1640 medium supplemented with 10% FBS and 10%
pooled human serum (Sigma). Monocytes were enriched by adherence during
a 1-hour incubation at 37°C in 90-mm polystyrene culture dishes.
Nonadherent lymphocytes were removed by washing the dishes 3 times with
PBS. Adherent monocytes were gently collected by scraping the
dishes with a rubber policeman and cultured in 6-well culture plates
for 72 hours in RPMI-1640 medium containing 10% FBS and 10% pooled
human serum (Sigma). The medium was changed after 72 hours, and IL-6
(20 ng/mL) was added at that time. After 48 hours, the cells were
incubated with DiI-AcLDL for uptake assays.
Flow Cytometry of MSR Ligand Uptake and Binding
Macrophage binding and uptake of AcLDL and of native LDL
were evaluated by flow cytometry with lipoproteins labeled with the
fluorescent probe DiI. For uptake experiments, THP-1 cells
treated with or without PMA or a combination of PMA and IL-6 were
incubated with various concentrations of DiI-AcLDL in medium containing
2% lipoprotein-deficient human serum for 3 hours. For binding assays,
cells were incubated for 30 minutes on ice with various concentrations
of DiI-AcLDL. Unlabeled AcLDL and native LDL in excess amounts
(>40-fold) were added together with the fluorescent
lipoproteins for competition assays. At the end of incubation, the
cells were washed with PBS and gently removed from the culture dishes
by scraping with a rubber policeman. The cells were resuspended in PBS,
fixed in 3% paraformaldehyde in PBS, and then
subjected to flow cytometric examination on a Becton Dickinson
FACScalibur cytometer. The DiI fluorescence was recorded on
channel FL2 and analyzed by the CellQuest program.
Immunoblotting
Total proteins extracted from THP-1 cells treated with or
without PMA (200 nmol/L) in the presence or absence of IL-6 (20 ng/mL)
were analyzed for expression of MSR proteins by
immunoblotting. In brief, the cells were lysed in lysis
buffer (5 mmol/L Tris-HCl at pH. 7.5, 2 mmol/L EDTA, 1%
Triton X-100, and 1 mmol/L PMSF). The protein concentrations were
determined by use of a bicinchoninic acid protein assay kit (Pierce).
Proteins (30 µg per lane) were separated by electrophoresis on 7.5%
SDS–polyacrylamide gel electrophoresis (PAGE) gels and
transferred to a polyvinylidene difluoride membrane (Micron
Separations). After transfer, the membrane was blocked in 4% fat-free
milk in PBS and then probed with a monoclonal antibody to the human
MSR. Bound primary antibody was detected with horseradish
peroxidase–conjugated goat anti-mouse IgG. The membrane was developed
by use of an Amersham enhanced chemiluminescence Western blotting
kit.
Analysis of mRNA
Total RNA was isolated from THP-1 cells treated with or without
PMA in the presence or absence of IL-6 by the acid guanidinium
thiocyanate–phenol-chloroform extraction method.29 The
MSR I– specific DNA fragment (278 bp, nucleotides 1106
to 1384 in the human MSR cDNA sequence) and the MSR II–specific DNA
fragment (229 bp, nucleotides 1433 to 1662 of the human MSR
cDNA sequence) were amplified by the polymerase chain reaction (PCR,
Perkin Elmer) from the human MSR cDNA clone.30 These
fragments were subcloned into a pCR II vector (Invitrogen) and
confirmed by sequencing. These plasmids containing MSR I– and MSR
II–specific DNA fragments were linearized, treated with proteinase K,
and extracted with phenol-chloroform–isoamyl alcohol (25:24:1,
vol/vol/vol) to remove RNase activity. The riboprobes were
prepared by in vitro transcription with the linearized plasmid
constructs [-32P]UTP (NEN Research
Products) and T7 or SP6 RNA polymerase with a Riboprobe Gemini kit
(Promega). The labeled transcripts were treated with DNase I and
purified by extraction with phenol-chloroform–isoamyl alcohol. The
RNase protection assay was performed with an RPA II kit (Ambion).
Levels of mRNA were quantified by estimation of the corresponding band
with a BAS-2000 densitometer (Fuji Film). The intensity of the bands
was normalized with respect to the radioactivities of
32P-labeled uridines in the protected fragments.
All experiments were carried out in triplicate, and the GAPDH mRNA
level was used as an internal standard. To evaluate the half-life
(t1/2) of MSR mRNA, THP-1 cells pretreated with
200 nmol/L PMA for 48 hours were washed twice with PBS, and actinomycin
D was added to the cultures at 5 µg/mL in the presence or absence of
IL-6 (20 ng/mL). Total RNA was isolated at multiple time points (0, 1,
2, 4, 8, and 16 hours) and analyzed for MSR mRNA. For
analysis of MSR mRNA in cultured human peripheral
monocytes treated with or without IL-6 (20 ng/mL), a reverse
transcription (RT)–PCR assay was performed with a set of primers for
MSR types I and II as reported previously.4 5 As the
internal control, ß-actin mRNA was assessed with a set of specific
primers for the actin cDNA. The resulting PCR products were
analyzed by electrophoresis on 2% agarose gels.
Assays for MSR Promoter Activity
Plasmids with luciferase constructs containing the segments from
–630 to +50 bp and from –10 to +50 bp of the MSR promoter fragments
from our previous preparation were generated as previously
described.31 THP-1 cells were cotransfected with
luciferase constructs and the chloramphenicol acetyltransferase
(CAT)–control plasmid (as an internal control) by
electroporation.31 In brief, THP-1 cells at
5x105/mL were plated 16 to 24 hours before
transfection. Electroporation was then carried out at room temperature
under 960 µF and 350 V in 700 µL of the reaction mixture containing
2x107 cells, 30 µg of the indicated luciferase
construct, and 10 µg of CAT–control plasmid DNA in a 0.4-cm
electroporation cuvette (Bio-Rad). After electroporation, the cuvette
was placed on ice for 10 minutes. Transfected cells were divided into 3
dishes containing 5 mL of RPMI-1640 medium and incubated at 37°C
under 5% CO2. The cells were treated with or
without PMA in the presence or absence of IL-6 and then harvested 48
hours after treatment for the luciferase and CAT assays.31
Relative luciferase activity was determined in 20 µg of total protein
of cell extracts. All experiments were carried out in triplicate.
Electrophoretic Mobility Gel Shift Assay
Cell extracts were prepared from THP-1 cells treated with or
without PMA in the presence or absence of IL-6. THP-1 cells were
collected by centrifugation, washed twice with cold
PBS, and then suspended in 400 µL of cold lysis buffer A (10
mmol/L HEPES, pH 7.9; 10 mmol/L KCl; 0.1 mmol/L EDTA;
0.1 mmol/L EGTA; 1 mmol/L DTT; and 1 mmol/L PMSF). After
incubation for 15 minutes on ice, 25 µL of 10% NP-40 was added and
the mixture was vortexed for 15 seconds. These cell lysates were
collected by centrifugation at 10 000g for
10 minutes at 4°C. The cell nuclei were resuspended in 50 µL of
cold buffer C (20 mmol/L HEPES, pH 7.9; 0.42 mmol/L NaCl;
1 mmol/L EDTA; 1 mmol/L EGTA; 1 mmol/L DTT; and 1
mmol/L PMSF) and rotated for 30 minutes at 4°C. After the cell debris
was removed by centrifugation, the supernatants were
collected. The concentrations of nuclear proteins were determined by a
bicinchoninic acid protein assay kit (Pierce). Electrophoretic mobility
gel shift assay was performed as described
previously.31 Double-stranded
oligonucleotides coding for the consensus sequences of
nuclear factor (NF)-
B, activator protein (AP)-1,
AP-3, Oct-1, NF-1, cAMP response element binding protein (CREB)
cis-elements (Stratagene), and for the MSR promoter,
AP-1/ets-like cis-element
(5'-aacgcaggaatgtgtcatttcctttct-3') were used for the gel shift
experiments after radiolabeling with T4 kinase and
[-32P]ATP. The reaction mixtures containing
6-µg of nuclear proteins and 10 000 counts per minute of radioactive
probes were incubated for 1 hour at room temperature and then loaded
onto 6% polyacrylamide. After electrophoresis at a constant
150 V for 1.5 hours at room temperature, the gels were fixed in 10%
acetic acid and 10% methanol for 10 minutes, dried, and exposed to
Kodak film overnight at –80°C. For blocking assays, nuclear extracts
were incubated with 1 µL of the antibodies against AP-1/Jun B and
AP-1/c-Jun at room temperature for 30 minutes and reacted with the
radioactive probes.
Statistic Analysis
Data were analyzed by 1-way ANOVA, followed by the
assessment of differences by Duncan's multiple range tests.
Significant differences were established when probability value were
less than 0.05.
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IL-6 Inhibits Uptake and Binding of DiI-AcLDL
The MSR ligand uptake and binding activities were examined by flow cytometry of human THP-1 monocytic cells exposed to DiI-AcLDL. After incubation with DiI-AcLDL under baseline conditions, we observed no significant fluorescent signals for DiI-AcLDL in the cells (Figure 1
). However, treatment with PMA
induced differentiation of THP-1 cells into macrophages, which
was accompanied by a marked increase in the cellular
fluorescent intensity of DiI-AcLDL, indicating enhanced uptake
of the MSR ligand by the cells. The DiI-AcLDL uptake was receptor
mediated because it could be blocked by incubation with excess amounts
of unlabeled AcLDL but not native LDL in the PMA-differentiated THP-1
macrophages (Figure 1), indicating the specificity of
DiI-AcLDL uptake by these cells.
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) PMA (200 nmol/L) in the presence (
) or
absence of IL-6 (20 ng/mL) for 48 hours. Cells were washed twice with
cold PBS and incubated with various concentrations of DiI-AcLDL (0 to
25 µg/mL) in RPMI-1640 medium containing 2% human
lipoprotein-deficient serum for 3 hours. Native LDL (
) and unlabeled
AcLDL (
) at 40-fold higher concentrations than DiI-AcLDL were added
for competitive assays. After incubation, the cells were washed twice
with PBS and fixed with 3% paraformaldehyde in PBS.
DiI fluorescence was analyzed by flow cytometry, and
specific fluorescent intensity was determined by subtracting
the mean fluorescent intensity of unlabeled cells
(autofluorescence) from that of DiI-AcLDL–incubated cells. The
data represent means of triplicates, with CVs <10%.
*Significantly different from PMA-treated cells
(P<0.05, ANOVA).
To determine whether IL-6 can affect MSR activity in PMA-differentiated
and undifferentiated THP-1 cells, we treated THP-1 cells with PMA in
the presence of IL-6 and then evaluated DiI-AcLDL uptake by the
cotreated cells with the use of a flow cytometer. We observed no
DiI-AcLDL uptake by undifferentiated THP-1 cells treated with IL-6
alone. However, in PMA-treated, differentiated THP-1
macrophages, exposure to the same concentrations of IL-6
reduced the uptake of DiI-AcLDL by nearly 40% in a
concentration-dependent manner (Figure 2). In contrast, no significant changes
in the uptake of DiI-LDL were found in the PMA-treated THP-1 cells, and
addition of IL-6 did not alter the uptake of DiI-LDL by these cells
either, indicating that IL-6 had little effect on expression of the
native LDL receptor (Figure 3).
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To confirm the inhibitory effect of IL-6, monocytes were
isolated from peripheral blood and induced to mature into
macrophages in vitro. As reported previously,4 5
freshly isolated monocytes expressed lower levels of MSR activity.
However, when they differentiate into macrophages, the cells
exhibit a marked enhancement of their MSR activities. To examine the
effects of IL-6 on MSR expression, we first examined the uptake of
DiI-AcLDL by human monocyte–derived macrophages. We observed
that treatment with IL-6 significantly reduced the uptake of DiI-AcLDL
by the cells (Figure 4).
Macrophages derived from both the THP-1 monocytic cell line and
normal monocytes responded to IL-6 by internalizing less of the MSR
ligand, AcLDL.
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To determine whether the inhibitory effect of IL-6 on the
uptake of DiI-AcLDL was the consequence of reduced AcLDL binding, an
assay for evaluating DiI-AcLDL binding was conducted in the
PMA-differentiated THP-1 cells at 4°C, because lowering the
temperature can inhibit endocytosis of MSR ligands. We observed by flow
cytometry that exposure to IL-6 significantly reduced the binding of
DiI-AcLDL to PMA-differentiated THP-1 cells (Figure 5). IL-6 treatment appeared to inhibit
both uptake and binding of DiI-AcLDL in the PMA-differentiated THP-1
macrophages.
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IL-6 Inhibits Expression of MSR Protein and mRNA
We further examined whether IL-6 treatment inhibits the expression
of MSR proteins and mRNA. Immunoblotting assay showed
little or no MSR protein in PMA-untreated THP-1 monocytes (Figure 6). However, in PMA-differentiated THP-1
macrophages, there was a marked increase in expression of the
MSR proteins (Figure 6). Treatment with IL-6 clearly reduced the
density of the MSR protein bands (Figure 6), indicating the
suppression of MSR protein by IL-6 treatment in the cells.
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Examination of MSR mRNA levels by an RNase protection assay
demonstrated that stimulation with IL-6 also reduced the levels of both
MSR I and II mRNAs in the PMA-differentiated THP-1 macrophages
(Figure 7). The levels of MSR I and II
mRNAs were simultaneously reduced by 50% to 60% by
addition of IL-6 (Figure 7), indicating that IL-6 might decrease
the expression of the MSR at the transcriptional level. The
inhibitory effect of IL-6 was independent of PMA, because
14 hours after PMA withdrawal, the cells could respond to IL-6
stimulation by a reduction in MSR transcripts (data not shown). In
contrast to undifferentiated THP-1 cells, freshly isolated
peripheral monocytes expressed lower but significant levels
of MSR type II mRNA as determined by RT-PCR (Figure 8). However, similar to differentiated
THP-1 cells, monocyte-derived macrophages produced MSR type I
as well as type II mRNA, and IL-6 treatment also inhibited expression
of both MSR mRNA isoforms in the normal monocyte-derived
macrophages (Figure 8). We evaluated the
t1/2 of MSR mRNA in the cells treated with
the transcription inhibitor actinomycin D. The MSR I mRNA
t1/2 was estimated to be 16.4±3.8 hours in
the cells treated with actinomycin D and 15.8±4.5 hours with
actinomycin D plus IL-6. Similarly, the MSR II mRNA
t1/2 was 14.5±2.2 hours with actinomycin D
and 14.6±4.6 hours with actinomycin D plus IL-6. No difference in the
t1/2 of both MSR I and II transcripts was
observed between IL-6–treated and untreated THP-1 cells in the
presence of actinomycin D, indicating that IL-6 has little effect on
posttranscriptional modulation of MSR mRNA stability.
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IL-6 Inhibits MSR Promoter Activity
To determine whether the IL-6–induced reduction in expression of
MSR mRNA in PMA-differentiated THP-1 macrophages resulted from
an attenuation of MSR gene promoter activity, we analyzed the
luciferase activity driven by a promoter of the MSR gene in THP-1 cells
transiently transfected with MSR promoter–luciferase reporter
constructs by electroporation. Two forms of the MSR-luciferase
constructs were generated, which contained the sequence from –630 to
+50 bp and from –10 to +50 bp, with most of the
cis-elements responsible for transcription of the MSR gene
(Figure 9). After transfection,
luciferase activity was measured in THP-1 cells treated with or without
PMA in the presence or absence of IL-6. In untreated THP-1 cells, only
low luciferase activity was observed, but treatment with PMA markedly
increased luciferase activity in the cells transfected with both longer
and shorter forms of the luciferase reporter constructs, indicating
that both sequences in the MSR promoter contain the responsive elements
to PMA stimulation. This finding is consistent with that by
Moulton et al,32 who showed that the short promoter
in the human MSR gene promoter has an AP-1/ets–like binding site.
Treatment with IL-6 reduced luciferase activities in PMA-differentiated
THP-1 cells transfected with either the long or short form of the MSR
promoter–luciferase construct (Figure 9). Deletion of the
sequence from –630 to –10 nucleotides of the MSR (long
construct) reduced the luciferase activity but did not appear to
abolish the inhibitory effect of IL-6 (Figure 9),
suggesting that the AP-1/ets element in the short-form construct might
be responsible for IL-6 inhibition of MSR transcription.
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To characterize the transcription factors responsible for IL-6–induced
suppression of MSR promoter activity in PMA-differentiated THP-1
macrophages, we performed the electrophoretic mobility gel
shift assay with double-stranded oligonucleotides for
various nuclear transcription factors. We found that stimulation with
IL-6 increased the binding activity of AP-3 and NF-B (Figure 10a) while reducing the binding
activity of AP-1, CREB, NF-1, and Oct-1 (Figure 10b). AP-1/ets
and Pu1 were reported to be required for transcription of the MSR
gene.32 We therefore determined the effect of IL-6 on the
binding activity of AP-1/ets–like transcription factors by using a
corresponding cis-element of AP-1/ets in the human MSR
promoter. We observed little AP-1/ets–like activity in untreated,
MSR-negative, THP-1 cells. During PMA-induced differentiation of THP-1
cells into macrophages, the activity of AP-1/ets–like
transcription factors dramatically increased (Figure 11). However, addition of IL-6 to the
cultures significantly inhibited PMA-induced AP-1/ets–like
transcription factor activity (Figure 11). The specificity of
the AP-1/ets–like transcription factor was determined by using
antibodies against AP-1/c-Jun and AP-1/Jun-B. We observed that the
addition of anti–c-Jun or anti–Jun-B in the reaction mixtures of
nuclear proteins and the radioactive probe partially blocked the
binding of PMA-induced complex to its cis- formation of the
complexes between the transcription factor and the probe for the
AP-1/ets domain (Figure 11). There was no supershifted band
appearing in the gel shift assay, suggesting that the binding sites for
the AP-1/ets probe were blocked by the antibodies, in agreement with a
finding by Moulton et al.32 These results indicated
that IL-6–mediated reduction in transcription of the MSR gene might be
at least partially due to a reduction in activity of a transcription
factor that interacts with the AP-1/ets cis-elements.
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The MSR is a membrane glycoprotein responsible for macrophage uptake of an unusually diverse array of ligands, including modified lipoproteins and bacterial lipopolysaccharides. This receptor has been shown to represent a marker for monocyte-to-macrophage differentiation.1 33 34 Progress in understanding the structure and expression of the MSR has provided important information on how it is involved in the pathogenesis of atherogenesis. Recent work by Suzuki et al35 36 has shown that targeted disruption of the MSR-A gene can reduce the size of atherosclerotic lesions in mice that are deficient in both apolipoprotein E and the MSR, indicating the significance of this gene for atherogenesis.
In this study, we utilized an in vitro model to address whether IL-6, a
well-defined cytokine in immune and inflammatory responses,
exerts a regulatory effect on expression of the class A MSR in
macrophages derived from human peripheral monocytes
and the THP-1 monocytic cell line. The observations that IL-6 inhibited
MSR ligand binding and uptake activities, protein expression, and mRNA
transcription strongly suggest that IL-6 might serve as a negative
regulator of MSR gene expression. Apparently, IL-6 shares several
features with other cytokines in terms of downregulation of
class A MSR transcription. Previous reports indicate that treatment
with IFN-12 and TGF-ß110 reduces MSR
mRNA expression as well as MSR-mediated uptake and binding of AcLDL.
Hsu et al13 reported that TNF- decreased transcription
of both the MSR and MSR mRNA steady-state levels in
PMA-differentiated THP-1 macrophages. The
inhibitory effects of IL-6 are similar to those of
IFN-,12 TNF-,13 and
TGF-ß111 on class A MSR gene expression. Such similarity
indicates that activation of macrophages by these
cytokines may actually lead to attenuation of MSR activities
and the reduction of lipid accumulation in the cells.
This in vitro model has several limitations. For instance, data from the in vitro studies may not precisely reflect the situation observed in vivo, particularly in atherosclerotic lesions where macrophages express high levels of the MSR, despite the presence of proinflammatory cytokines. This controversial phenomenon may be explained by an imbalance between MSR-promoting and -inhibiting factors in the microenvironment. This concept of how IL-6 affects in vivo expression of the MSR deserves further study. Additionally, IL-6 treatment does not appear to completely block uptake and binding of DiI-AcLDL by PMA-differentiated THP-1 cells, even though reductions in MSR mRNA and protein are substantial. One explanation for this is that in addition to the class A MSR, other membrane proteins expressed in differentiated monocytic cells may mediate endocytosis of DiI-AcLDL as well. Recently, 1 new type of MSR, structurally different from the class A MSR, has been cloned.37 Macrophages from mice lacking the class A MSR also maintain to a certain degree the capacity to take up and bind AcLDL.38 Future studies are needed to clarify whether IL-6 stimulation affects expression of other forms of the MSR. Nonetheless, the reduced AcLDL uptake and binding in IL-6–treated cells with decreased expression of the class A MSR mRNA and protein point to a role for IL-6 in the regulation of MSR-mediated degradation of chemically modified lipoproteins in macrophages.
Our current observation indicates that IL-6 inhibited MSR promoter
activity by reducing the binding of the AP-1/ets–like transcription
factor to the MSR promoter. Comparison of sequences upstream from the
5' ends of bovine, human, and murine MSR genes shows a high similarity
in the 300 bp proximal to the start site of transcription, but it does
not appear to have a popular TATA box in the region 
30 bp upstream
from the transcription start site.30 Moulton et
al32 have shown that AP-1/ets and Pu1 elements located in
the MSR promoter regions are critical for expression of the class A MSR
gene in PMA-differentiated THP-1 cells. Our data indicate that new
elements located from –504 to –399 bp in the human MSR gene promoter
are also required for the greatest expression of the MSR gene in murine
macrophages.31 These results indicate that the
proximal promoter regions at <600 bp from the initial site play a
critical role in the regulated expression of the MSR gene. When THP-1
cells differentiate into macrophages on exposure to PMA, the
luciferase activity of both –630 to +50 bp and –10 to +50 bp
constructs was markedly enhanced. The results indicate that the
responsive element to PMA is located at –10 to +50 bp in the promoter
region, in agreement with the findings by Wu et al15 and
Moulton et al.32 Our current data indicate that IL-6
treatment can inhibit by 30% and 40%, respectively, the promoter
activities of the 2 constructs. These findings suggest that IL-6
inhibits transcription of MSR mRNA by decreasing the promoter activity
of the MSR gene.
It is unclear which signal transduction pathway is involved in
IL-6–mediated downregulation of MSR expression. Binding of
transcription factors to cis-elements triggers transcription
of many different genes, including the MSR gene. So far, no
IL-6–responsible cis-elements have been identified in the
human MSR gene promoter regions. Therefore, we examined whether IL-6
affects other transcription factors that may bind to the
cis-elements. Examination of the binding activities of
well-characterized transcription factors such as NF-B, AP-1, AP-3,
and CREB by electrophoretic mobility gel shift assays demonstrated that
IL-6 increased the binding activity of NF-B and AP-3 but decreased
that of CREB, Oct-1, NF-1, and AP-1. There are several AP-1/ets
cis-elements similar to the popular AP-1
cis-element in the human MSR
promoter.15 32 The anti–c-Jun and Jun B antibodies
have been shown to inhibit the formation of a complex between
transcription factors and AP-1/ets
cis-elements.32 This result is
consistent with our current observation (Figure 11
)
that anti–c-Jun and Jun-B antibodies blocked the binding of a nuclear
transcription factor to the AP-1/ets probe. Our observation that IL-6
inhibited the activity of an AP-1/ets–like transcription factor(s)
suggests that a transcription factor recognizing AP-1/ets elements may
be at least partially responsible for the inhibitory effect
of IL-6 on MSR gene transcription. This notion is also in agreement
with the recent finding by Horvai et al,14 who showed that
IFN- inhibits transcription of the MSR gene by antagonizing the
Ras-dependent activities of AP-1 and cooperating ets domain
transcription factors, apparently as a result of competition between
AP-1/ets factors and activated STAT (signal transducers and
activators of transcription)-1, leading to a limitation in
the amounts of CREB binding protein and p300.
MSR-mediated lipid loading into macrophages and transformation
of these cells into foam cells represent a critical event
during the pathogenesis of atherosclerosis. The results
of our current study shed new insight on the biological function of
IL-6, pointing to complex interactions between lipid-carrying proteins,
inflammatory cells, and vascular cells. The MSR is expressed
predominantly by macrophages, and IL-6 is produced by smooth
muscle cells and macrophages in atherosclerotic lesions.
Locally produced IL-6, together with other cytokines such as
IFN-, TNF-, and TGF-ß1, may exert regulatory effects on the
formation of lipid-laden foam cells during the development of
atherogenesis.
|
Acknowledgments |
|---|
This study was supported by National Institutes of Health (Bethesda, Md) grant HL-59249-01 (to Y.G.), by an American Heart Association, Pennsylvania Affiliate, Grant-In-Aid award (to Y.G.), in part by a grant from the Human Health Sciences Foundation (to T.K.), and by a grant for research on aging and heart from the Ministry of Health and Welfare (to T.K.), Japan. We thank Tamaral Pittman and Qi Wu for their technical assistance.
Received October 2, 1998; accepted January 19, 1999.
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