вход по аккаунту


Interferon-╨Ю┬▒ priming promotes lipid uptake and macrophage-derived foam cell formationA novel link between interferon-╨Ю┬▒ and atherosclerosis in lupus.

код для вставкиСкачать
Vol. 63, No. 2, February 2011, pp 492–502
DOI 10.1002/art.30165
© 2011, American College of Rheumatology
Interferon-␣ Priming Promotes Lipid Uptake and
Macrophage-Derived Foam Cell Formation
A Novel Link Between Interferon-␣ and Atherosclerosis in Lupus
Jia Li,1 Qiong Fu,2 Huijuan Cui,3 Bo Qu,3 Wen Pan,3 Nan Shen,1 and Chunde Bao2
Objective. An increased risk of premature atherosclerosis has been associated with systemic lupus erythematosus (SLE), and type I interferon (IFN) has been
shown to play a pathogenic role in human SLE. The aim
of this study was to determine whether IFN␣ is involved
in the development of atherosclerosis in patients with
SLE by promoting lipid uptake and macrophagederived foam cell formation, which is a crucial step in
early atherosclerosis.
Methods. The effects of IFN␣ on lipid uptake and
foam cell formation were determined by flow cytometry
and oil red O staining. Messenger RNA and protein
expression of scavenger receptors (SRs) was examined.
Promoter activity was detected by luciferase reporter
assay. Expression of macrophage SR class A (SR-A) and
IFN-inducible genes (IFIGs) was measured in peri-
pheral blood mononuclear cells obtained from 42 patients with SLE and 42 healthy donors.
Results. IFN␣ priming increased the uptake of
oxidized low-density lipoprotein and hence enhanced
foam cell formation by up-regulating SR-A expression.
IFN␣ increased SR-A expression via enhancing its
promoter activities. Examination using signaling inhibitors revealed that a phosphatidylinositol 3-kinase/Akt
signaling pathway appeared to be involved in this
process. Notably, SR-A messenger RNA was significantly increased in patients with SLE compared to
normal subjects and positively correlated with IFIG
Conclusion. Our data suggest that IFN␣ priming
up-regulated the expression of SR-A in human
monocyte/macrophages, leading to increased lipid uptake and foam cell formation. Activation of the IFN
signaling pathway may be linked to the risk of atherosclerosis by affecting plaque formation in patients with
SLE. These findings provide novel insights into the
mechanisms of and potential therapeutic approaches to
premature atherosclerosis in patients with SLE.
Supported by the Chinese Natural Science Foundation
(grants 81025016, 30571737, 30471582, and 30471631), the Program of
the Shanghai Commission of Science and Technology (grants
06JC14050, 07ZR14130, and 08JC1414700), and the Program of
Shanghai Subject Chief Scientist (07XD14021), Shanghai Key Disciplines Project (T0203), and Shanghai Clinical Research Resource
Sharing Platform Project (SHDC 12007205).
Jia Li, MD, PhD, Nan Shen, MD: Shanghai Institute of
Rheumatology, Renji Hospital, Molecular Rheumatology Laboratory
of Institute of Health Sciences, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences, and Shanghai JiaoTong
University School of Medicine, Shanghai, China; 2Qiong Fu, MD,
PhD, Chunde Bao, MD: Shanghai Institute of Rheumatology, Renji
Hospital, and Shanghai JiaoTong University School of Medicine,
Shanghai, China; 3Huijuan Cui, MD, Bo Qu, PhD, Wen Pan, PhD:
Molecular Rheumatology Laboratory of Institute of Health Sciences,
Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, and Shanghai JiaoTong University School of Medicine,
Shanghai, China.
Address correspondence to Nan Shen, MD, or to Chunde
Bao, MD, Department of Rheumatology, Renji Hospital, Shanghai
JiaoTong University School of Medicine, 145 Shan Dong Road
(Central), Shanghai 200001, China. E-mail: baochunde_1678@
Submitted for publication December 16, 2009; accepted in
revised form November 16, 2010.
Systemic lupus erythematosus (SLE) is a complex
multisystem autoimmune disease that involves multiple
organs as a result of autoimmune-mediated tissue damage. In recent years, it has been established that the
incidence of premature atherosclerosis (and hence cardiovascular morbidity and mortality) is increased in
patients with SLE (1–3). Although traditional risk factors such as hypertension, hypercholesterolemia, and
diabetes mellitus, which can be promoted by immune
dysregulation and glucocorticoid use, are thought to be
important in mediating this increased risk of atherosclerosis in SLE, they fail to adequately explain the increased incidence of atherosclerotic diseases in patients
with SLE (4,5). Indeed, SLE itself is an independent risk
factor for atherosclerosis, as reported in the past few
years (6,7). Thus, the increasing prevalence of atherosclerosis in SLE is likely attributable to a complex
interaction involving traditional risk factors, diseaserelated factors such as medications and disease activity,
and inflammatory and immunogenic factors (8,9). It was
recently reported that the level of proinflammatory
high-density lipoprotein (HDL) was elevated and correlated with subclinical atherosclerosis in patients with
SLE (10). More studies are certainly needed to define
the exact mechanisms leading to this complication.
The expression of proinflammatory cytokines and
chemokines is increased in SLE (11,12). Among these
cytokines and chemokines, type I interferon (IFN) has
been recognized to play a pathogenic role in human SLE
(13,14). Serum levels of type I IFNs, predominantly
IFN␣, are elevated in ⬃50% of patients with SLE (15),
and gene expression profiling has revealed that the
expression of IFN-inducible gene (IFIG) transcripts is
also up-regulated (16). The presence of this “interferon
signature” is positively associated with serologic and
clinical manifestations, disease activity, and disease severity in SLE (17,18). In animal experiments, a deficiency of type I IFN receptor significantly reduced
lupus-like disease in NZB mice (19). More importantly,
a murine model of pristane-induced lupus further confirmed the key role of the type I IFN pathway in lupus
IFN␣ has been reported to be involved in atherosclerosis through several different mechanisms. IFN␣
promoted endothelial progenitor cell deletion and endothelial dysfunction in lupus, leading to abnormal
vascular repair (22,23). Plaque-residing plasmacytoid
dendritic cell–produced IFN␣ combined with lipopolysaccharide increased the expression of Toll-like receptor
4 and enhanced the production of tumor necrosis factor
␣ (TNF␣), interleukin-12 (IL-12), and matrix metalloproteinase 9 (MMP-9), threatening the stability of atherosclerotic plaques (24). Additionally, IFN␣ enhanced
cytotoxic T cell activities that may also trigger plaque
disruption in atherosclerosis (25). Interestingly, lowdensity lipoprotein (LDL) receptor–deficient mice
showed significantly accelerated atherosclerosis accompanied by increased plasma levels of cholesterol and
triglycerides after receiving an injection of IFN␣ (26).
The initiating force for the occurrence of atherosclerosis is the accumulation of cholesterol-laden foam
cells in the arterial wall. The role of IFN␣ in this aspect
of atherosclerosis remains unknown. In the early stage of
atherosclerosis, circulating monocytes infiltrate into the
subintima where they differentiate into macrophages.
Upon exposure and uptake of modified lipoproteins,
especially oxidized LDL (ox-LDL), the macrophages are
transformed into foam cells, which are the primary
components of the earliest atherosclerotic lesion. Macrophage scavenger receptor (SR) family proteins can
internalize substantial quantities of cholesteryl ester
from ox-LDL and play a leading role in lipid accumulation and foam cell formation (27). In this study, we
focused on investigating the effects of IFN␣ on lipid
uptake and foam cell formation, especially on the expression and activities of macrophage SRs.
We demonstrated that IFN␣ priming was able to
promote ox-LDL engulfment and foam cell formation by
up-regulating the expression of macrophage SR class A
(SR-A). Enhanced SR-A promoter activities and the
phosphatidylinositol 3-kinase (PI3K)/Akt pathway appeared to be involved in this process. In addition, we
observed that the expression of SR-A was significantly
increased in the peripheral blood mononuclear cells
(PBMCs) of patients with SLE and was positively correlated with IFN signaling activity.
Patients, healthy donors, and sample handling. A total
of 42 patients with lupus and 42 age- and sex-matched healthy
volunteers were recruited for the study. Prior to participation,
written informed consent was obtained from all subjects. All
studies were performed in accordance with the Declaration of
Helsinki. All patients with SLE were recruited from the Lupus
Clinic Center of Renji Hospital and met the American College
of Rheumatology revised criteria for the classification of SLE
(28,29). Healthy volunteers, all of whom had no clinical
manifestations of SLE, cardiovascular disease, or cerebrovascular disease, were selected from a pool of healthy volunteers
at Renji Hospital. (Demographic variables and risk factors for
cardiovascular diseases in both groups, as well as information
on the treatment and clinical features of patients with SLE, are
available from the corresponding author.) PBMCs from each
subject were isolated, and total RNA was extracted, using
TRIzol (Invitrogen) for messenger RNA (mRNA) detection.
The study was approved by the Research Ethics Board of Renji
Hospital, Shanghai JiaoTong University School of Medicine.
Isolation of PBMCs and cell culture. PBMCs were
isolated from healthy donors by density-gradient centrifugation with Ficoll-Paque Premium (GE Healthcare), according
to the instructions provided by the manufacturer. The cells
were resuspended in cold buffer (containing phosphate buffered saline [PBS], 0.5% bovine serum albumin, and 2 mM
EDTA) to further negatively select by magnetic cell sorting
with the human Monocyte Isolation Kit II (Miltenyi Biotec).
The purity of human monocytes was assessed by flow cytometry (FACSAria; Beckton Dickinson). Purified monocytes
(⬎93% CD14⫹) were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (FBS) at a density of 5 ⫻
105/ml. Monocytes differentiated into macrophages in the
presence of 25 ng/ml of recombinant human macrophage
colony-stimulating factor (R&D Systems) for 7 days. The
medium and cytokines were replaced every 2–3 days.
The human monocytic THP-1 cell line was obtained
from American Type Culture Collection. THP-1 cells were
grown in RPMI 1640 with 10% FBS, penicillin (100 units/ml)–
streptomycin (100 ␮g/ml) at 37°C in a 5% CO2 atmosphere, to
a density of 106/ml. THP-1 cells were plated in RPMI 1640
containing 0.5% FBS for 24 hours prior to the experiments,
then THP-1 cells were differentiated into macrophages by
incubation for 24 hours with 100 nM phorbol myristate acetate
(PMA; Sigma).
Measurement of Dil-labeled ox-LDL uptake using
confocal microscopy and flow cytometry. THP-1 cell–derived
macrophages and human monocyte–derived macrophages
were treated with Dil-labeled ox-LDL (20 ␮g/ml; Yuanyuan
Biotec Institute) for 24 hours at 37°C, in the presence or
absence of IFN␣ priming for 24 hours. Cells were incubated
with Dil-labeled ox-LDL (20 ␮g/ml) at 4°C in order to exclude
binding, and an excess of unlabeled ox-LDL (400 ␮g/ml) was
added into the medium with Dil-labeled ox-LDL as a negative
control. After washing with PBS, cells in the plates were
harvested by gentle scraping. Fluorescence was analyzed using
a BD Calibur flow cytometer (BD Biosciences) and FlowJo
software (TreeStar). For SR-A–blocking experiments, macrophages were preincubated with 5 ␮g/ml specific SR-A–blocking
antibody (anti-human SR-A monoclonal antibody, clone SRAC6; Cosmo Bio) or mouse IgG1 isotype control (eBioscience)
for 1 hour before Dil-labeled ox-LDL was loaded.
For analysis using fluorescence microscopy, macrophages were washed twice with PBS and fixed in 4% paraformaldehyde for 30 minutes. Cells were counterstained with
DAPI for 3 minutes, mounted with anti-fading mounting
medium, and detected by confocal laser microscopy (Leica).
Foam cell formation and lipid staining. Cellular lipids
were stained with oil red O. Briefly, THP-1 cell–derived
macrophages were pretreated with or without 100 units/ml
IFN␣ for 24 hours. Cells were then further incubated in the
starved medium (0.5% FBS, RPMI 1640) with 100 ng/ml
ox-LDL for an additional 24 hours. The cells were fixed with
4% paraformaldehyde for 30 minutes and stained with a
working solution of oil red O for 5 minutes. The cell nucleus
was stained with hematoxylin. Foam cell formation was observed under a light microscope (Nikon Eclipse 80i), and the
number of cells in which intracellular lipid droplets occupied
more than one-third of cytoplasm was calculated.
RNA isolation and analysis of SRs and IFIGs by
real-time polymerase chain reaction (PCR). Total RNA was
extracted using TRIzol reagent according to the manufacturer’s protocol. The quality and quantity of total RNA were
measured using a NanoDrop ND-1000 spectrophotometer
(NanoDrop Technologies). Up to 500 ng of total RNA was
reverse transcripted to complementary DNA with the PrimeScript RT reagent Kit in a final volume of 10 ␮l (Takara).
Messenger RNA expression for SR-A, CD36, lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1), CD68, and
IFIGs (myxovirus resistance 1 [MX-1] and 2⬘,5⬘-oligoadenylate
synthetase 1 [OAS-1]) was quantitated by real-time PCR using
SYBR Premix Ex Taq (Takara). Amplification assays were
performed in triplicate, with the expression of TATA binding
protein used as a normalized reference for each sample.
(Information regarding the primers used is available from the
corresponding author.)
The amplification consisted of an initial holding at
95°C for 15 seconds, followed by a 2-step PCR program: 95°C
for 5 seconds and 60°C for 30 seconds for 40 cycles. A melting
curve analysis was performed after amplification. Data were
collected and quantitatively analyzed on an ABI Prism 7900
Sequence Detection System (Applied Biosystems).
Protein extraction and Western blot analysis. Macrophages were treated with IFN␣ (100 units/ml) for 24 hours,
with or without 0.2 ␮g/ml recombinant B18R protein (Vaccinia
Virus-Encoded Neutralizing Type I Interferon Receptor;
eBioscience) preincubation for 1 hour. Chemical inhibitors
against JNK, p38 MAPK, MEK-1, and PI3K, respectively,
were added to the cells 1 hour prior to IFN␣ treatment,
including SP600125 (25 ␮M; Tocris), SB203580 (10 ␮M;
Tocris), SB202190 (10 ␮M; Tocris), PD98059 (25 ␮M; Tocris),
LY294002 (15 ␮M; Sigma), or appropriate vehicle controls
(DMSO). Total cell protein lysates was extracted with iced lysis
buffer, with the complement by Halt Protease and Phosphatase
Inhibitor Cocktail (Pierce). Equal amounts of proteins were
loaded and separated by 10% sodium dodecyl sulfate–
polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After incubation in 5% skim
milk for 2 hours at room temperature, membranes were
incubated with anti-human SR-A (1:500; Santa Cruz Biotechnology), anti–STAT-1 (1:1,000; Santa Cruz Biotechnology),
anti–pSTAT-1 (Tyr701; 1:1,000) (Santa Cruz Biotechnology),
and anti–␤-actin (1:4,000; Sigma) overnight at 4°C. Membranes were washed and then incubated with 1:10,000 dilution–
specific secondary antibodies (Amersham) for 1 hour at
room temperature. Antigen detection was performed with the
Amersham ECL Western Blotting System.
Immunofluorescence flow cytometry for CD36. To
detect the cell surface expression of CD36, human monocyte–
derived macrophages were treated with or without 100 units/ml
IFN␣ for 24 hours. The cells were then washed in cold
fluorescence-activated cell sorting (FACS) buffer (2% FBS,
0.1% sodium azide in PBS) and incubated with phycoerythrinconjugated mouse anti-human CD36 antibody (eBioscience)
or isotype control antibody for 30 minutes on ice, washed twice
with cold buffer, and resuspended in 200 ␮l of fixation buffer
(1% paraformaldehyde in FACS buffer) for analysis using a
flow cytometer. Data were analyzed with FlowJo software
Chimeric construct, cell transfection, and measurement of luciferase activity. A fragment of SR-A promoter
(from ⫺1564 to ⫹49) was achieved by PCR with forward
and reverse primer 5⬘-GAAGATCTTGTTTCAATAGCACTCTCATC. After confirmation by DNA sequencing,
the promoter fragment was cloned in the luciferase reporter
vector with pGL3-Basic (Promega) for the luciferase reporter
HeLa cells were seeded in a 96-well plate and transfected with human SR-A gene promoter fragment/luciferase
constructs using Lipofectamine 2000 Transfection Reagent
(Invitrogen). For each well, 300 ng of reconstructed plasmid
and 2 ng of Renilla luciferase were added to the medium and
incubated with the cells for 6 hours. The medium was changed,
Figure 1. Effects of interferon-␣ (IFN␣) priming on oxidized low-density lipoprotein (ox-LDL) uptake in
macrophages. A, Ox-LDL uptake measured by flow cytometry, using Dil-labeled ox-LDL, in THP-1 cell–derived
macrophages following priming with graded doses of IFN␣. Bars show the mean ⫾ SD. ⴱ ⫽ P ⬍ 0.02. B,
Representative histogram of Dil-labeled ox-LDL uptake in THP-1 cell–derived macrophages with or without
priming with 100 units/ml IFN␣, as determined by fluorescence-activated cell sorting analysis. C, Oxidized LDL
uptake, as shown by confocal microscopy, in THP-1 cell–derived macrophages. An excess of unlabeled ox-LDL
(400 ␮g/ml) was used as a specificity control (CTRL). Red fluorescence indicates Dil-labeled ox-LDL, whereas
nuclei of the cells stained with DAPI are shown in blue. D, Representative histograms of Dil-labeled ox-LDL
uptake in human monocyte–derived macrophages with (arrow B) or without (arrow A) IFN␣ priming (100
units/ml) and pretreatment with IFN␣-neutralizing protein B18R prior to IFN␣ priming (arrow C). All
experiments were performed in triplicate.
and IFN␣ was added into the medium 24 hours after transfection. Cells were harvested and lysed in Reporter Lysis Buffer
24 hours later. Luciferase activity was measured using a
Dual-Luciferase Reporter Assay Kit (Promega) and a luminometer (Applied Biosystems). The firefly luciferase–to–
Renilla luciferase ratio was obtained for each well.
Figure 2. Effect of IFN␣ priming on foam cell formation. A, Foam cell formation in
THP-1 cell–derived macrophages. Arrows indicate foam cells with oil red staining. B,
Percentage of foam cells in which intracellular lipid droplets occupied more than
one-third of the cytoplasm. IFN␣ treatment prior to ox-LDL loading led to an
increased percentage of foam cells. Bars show the mean ⫾ SD. ⴱ ⫽ P ⫽ 0.02 versus
experiments without IFN␣. See Figure 1 for definitions.
Figure 3. Up-regulation of the expression of scavenger receptor class A (SR-A) but not CD36 by IFN␣ in
macrophages. Cells were incubated with or without IFN␣, and B18R was used to competitively block IFN␣
binding to demonstrate that the effects induced by IFN␣ were specific. A, IFN␣ significantly up-regulated SR-A
mRNA expression in macrophages and had no significant effects on CD36, CD68, and lectin-like oxidized
low-density lipoprotein receptor 1 (LOX-1) expression. ⴱ ⫽ P ⫽ 0.01 versus experiments without IFN␣. B,
Activation of the IFN␣ signaling pathway was shown by immunoblotting in whole cell extracts, as measured by
pSTAT-1. B18R specifically blocked activation of the IFN␣-signaling pathway. C, IFN␣ specifically up-regulated
SR-A protein expression in THP-1 cell–derived macrophages. D, IFN␣ did not alter CD36 protein expression, as
measured by fluorescence-activated cell sorting analysis. E, IFN␣ up-regulated the expression of SR-A protein in
human monocyte–derived macrophages. All experiments were performed in triplicate. See Figure 1 for other
Statistical analysis. Statistical analysis was performed
using GraphPad version 5.0 software. Data are expressed as
the mean ⫾ SD or the median and interquartile range (IQR).
Differences between groups were evaluated by unpaired t-test
for continuous parametric variables and by nonparametric
Mann-Whitney U test for skewed-distribution variables. Correlations between groups were analyzed by Spearman’s test.
Two-tailed P values less than 0.05 were considered significant.
Role of IFN␣ priming in ox-LDL uptake and
foam cell formation in human macrophages. It is known
that IFN␣ priming is involved in the cross-talk of
signaling pathways associated with the inflammatory
response (30). Therefore, we investigated whether IFN␣
priming could promote lipid uptake in THP-1 cell–
derived macrophages and human monocyte–derived
macrophages. Dil-labeled ox-LDL was used to measure
lipid endocytosis in macrophages that did or did not
undergo priming with IFN␣. IFN␣-treated macrophages
showed increased uptake of Dil-labeled ox-LDL compared with untreated cells, in an IFN␣ dose–dependent
manner (Figure 1A). The dose of 100 units/ml IFN␣ was
selected for further experiments. Similar to THP-1 cell–
derived macrophages (Figures 1B and C), human
monocyte–derived macrophages were also promoted to
take up more lipid with the same amount of IFN␣
treatment (Figure 1D). More importantly, the increased
endocytosis was able to be blocked by IFN␣-neutralizing
protein B18R (Figure 1D), verifying that the enhanced
lipid uptake was mediated by IFN␣.
Because enhanced lipid uptake could lead to
increased foam cell formation, we subsequently investigated whether IFN␣ priming could promote foam cell
formation in THP-1 cell–derived macrophages, which
have been applied widely to study foam cell formation
in vitro (31). As shown in Figure 2, THP-1 cells were first
differentiated into macrophages by incubation with
PMA and were induced to become foam cells in the
presence of ox-LDL. IFN␣ treatment prior to ox-LDL
loading led to an increased percentage of foam cells
(mean ⫾ SD 15.3 ⫾ 2.5% versus 9.7 ⫾ 2.3% without
priming; P ⫽ 0.02) (Figure 2B). Taken together, our
data provided evidence that IFN␣ was able to promote
foam cell formation by augmenting ox-LDL uptake in
human macrophages.
Up-regulation of SR-A mRNA and protein expression by IFN␣. It has been widely accepted that SRs
play major mediating roles in engulfing ox-LDL into
Figure 4. Impact of scavenger receptor class A (SR-A) blocking on oxidized low-density lipoprotein (ox-LDL)
uptake induced by interferon-␣ (IFN␣) treatment. The addition of specific anti–SR-A antibodies abrogated the
enhanced ox-LDL uptake caused by IFN␣ priming in THP-1 cell–derived macrophages, compared to the samples
with control (CTRL) antibody IgG1 treatment, as measured by fluorescence confocal microscopy (A) and
fluorescence-activated cell sorting (B).
macrophages (32). Among the many different SR molecules, SR-A and CD36 have the major critical role in
lipid uptake and foam cell formation (33,34). We thus
examined the effects of IFN␣ on the mRNA expression
of the representative SRs, including SR-A, CD36,
LOX-1, and CD68. Real-time PCR analysis indicated
that IFN␣ treatment significantly up-regulated SR-A
mRNA expression in THP-1 cell–derived macrophages
(Figure 3A). As shown in Figure 3B, STAT-1 tyrosine
phosphorylation was detected following IFN␣ stimulation, which is used as a surrogate marker for activation
of the IFN-signaling pathway (35). The immunoblot
analysis also revealed that the expression of SR-A
protein was increased in the presence of IFN␣ treatment
in THP-1 cell–derived macrophages (Figure 3C) and
human monocyte–derived macrophages (Figure 3E). In
contrast, there was no significant difference in CD36
expression at the mRNA and protein levels (Figures 3A
and D) following IFN␣ treatment. These results revealed that IFN␣ significantly up-regulated SR-A
mRNA and protein expression. This function was also
demonstrated by the abrogatory effect of neutralizing
protein B18R (Figure 3C).
Role of SR-A in the increased ox-LDL uptake
stimulated by IFN␣ treatment. Because IFN␣ priming
up-regulated SR-A expression, the possible role of SR-A
in mediating the effect of IFN␣-promoted ox-LDL
uptake was investigated. As shown in Figure 4, enhanced
ox-LDL uptake was abolished by treatment with specific
anti–SR-A–blocking antibodies, whereas isotypematched control IgG1 did not exert any inhibitory effect
in this process. These results indicated that SR-A has a
role in lipid uptake induced by IFN␣ treatment.
Role of IFN␣ in modulating SR-A expression by
targeting the SR-A promoter and the PI3K pathway. To
determine whether IFN␣ could directly affect SR-A
gene transcription, we cloned an SR-A promoter fragment into the luciferase reporter vector pGL3-Basic and
constructed a pGL3-Basic/SR-A promoter recombinant
plasmid (pSR-A Luc). Because human SR-A promoters
functioned in the monocyte/macrophage cell line as well
as in HeLa cells (36), the effects of IFN␣ on SR-A
promoter activity were examined in HeLa cells due to
their high transfection efficiency. The pSR-A Luc and
Renilla plasmids were cotransfected into HeLa cells.
Compared to cells transfected with pGL3-Basic, cells
transfected with pSR-A Luc had significantly higher
luciferase activity, verifying that SR-A promoter was
activated in HeLa cells (P ⫽ 0.0004) (Figure 5A).
Furthermore, induced luciferase activity was 25% higher
after IFN␣ stimulation (P ⫽ 0.0092) (Figure 5A). Thus,
one of the mechanisms of IFN␣-induced SR-A gene
transcription was to directly activate SR-A promoter. It
has been reported that transcription factor PU.1 and a
Figure 5. Regulatory mechanisms of SR-A expression by IFN␣. A, A construct of SR-A promoter–controlled
reporter luciferase (pSR-A Luc) was made and transfected into HeLa cells. The reporter gene activities induced
by IFN␣ for 24 hours were represented as relative luminescence units (firefly:Renilla [F/R]) measured with an
illuminometer. B, Different kinase inhibitors were added 1 hour prior to IFN␣ treatment, and their effects on
IFN␣-promoted SR-A expression were detected by Western blotting. C, The phosphatidylinositol 3-kinase
(PI3K) inhibitor LY294002 inhibited IFN␣-induced and basal SR-A expression in a dose-dependent manner.
AP-1 ⫽ activator protein 1; ISRE ⫽ IFN-stimulated response element; IRF-1 ⫽ IFN regulatory factor 1 (see
Figure 4 for other definitions).
composite activator protein 1/Ets motif were involved in
cell-specific expression of the SR-A gene (36). Although
bioinformatics analysis predicted that the IFN regulatory factor 1 (IRF-1)/IRF-2 binding site may also exist at
the promoter region of the SR-A gene, no clear IFNstimulated response element (ISRE) motifs were identified by bioinformatics analysis.
There is accumulating evidence that type I IFNs
could activate the p38 MAPK and PI3K signaling cascades, which are responsible for the generation of
cellular responses to IFNs (35). Meanwhile, it has been
reported that both the PI3K and MAPK pathways could
involve SR-A expression and foam cell formation
(37,38). To further clarify the signaling pathways used by
IFN␣ to up-regulate SR-A expression, we investigated
their possible involvement, using their specific chemical
inhibitors. The effects on IFN␣-induced up-regulation
of SR-A expression were examined using p38 MAPK
inhibitors (SB203580 and SB202190), JNK inhibitor
(SP600125), MEK-1 inhibitor (PD98059), and PI3K
inhibitor (LY294002). As shown in Figures 5B and C,
only PI3K inhibitor LY294002 significantly repressed
both IFN␣-induced and basal expression of SR-A in a
dose-dependent manner. In contrast to the prominent
effect of the PI3K inhibitor, MAPK inhibition did not
suppress IFN-mediated SR-A expression. Therefore,
PI3K is likely one of the mediators of IFN␣-induced
up-regulation of SR-A expression.
Increased SR-A expression in PBMCs from SLE
patients and positive correlation with elevated IFIG
expression. In order to illustrate the clinical relevance of
our findings, we studied the association between the
levels of IFN␣ and the levels of SR-A by comparing
them in patients with SLE and healthy control subjects.
Previous studies showed that quantification of type I
IFN by standard enzyme-linked immunosorbent assay is
unreliable in patients with SLE (39). Instead, IFIG
expression measured by real-time PCR was used as a
surrogate for the serum level of type I IFN (22,39). Two
representative IFIGs, MX-1 and OAS-1, were selected
for the assay, and the levels of SR-A expression were
determined by real-time PCR in PBMCs, because the
expression of SR-A is known to be mainly confined to
monocytes and macrophages (36,40).
A total of 42 patients with SLE and 42 healthy
control subjects matched for both age and sex were
studied. The patients with SLE had significantly increased SR-A expression compared with control subjects
(median fold expression 4.23 [IQR 3.25–5.11] versus
2.86 [IQR 2.18–3.34], P ⬍ 0.0001) (Figure 6A) and
increased IFIG expression (for MX-1, median relative
expression 11.12 [IQR 10.05–11.91] versus 9.08 [IQR
8.64–9.82], P ⬍ 0.0001; for OAS-1, median relative
expression 11.07 [IQR 10.44–11.77] versus 9.75 [IQR
9.36–10.12], P ⬍ 0.0001) (Figure 6B). More importantly,
a positive correlation between SR-A and IFIGs was
Figure 6. Elevated scavenger receptor class A (SR-A) expression in peripheral blood mononuclear cells
(PBMCs) from patients with systemic lupus erythematosus (SLE) and positive association between SR-A
expression and interferon (IFN)–inducible gene (IFIG) expression. A, Expression of SR-A in PBMCs from 42
patients with SLE and 42 healthy control (CTRL) subjects. B, Expression of myxovirus resistance 1 (MX-1) and
2⬘,5⬘-oligoadenylate synthetase 1 (OAS-1) in PBMCs from patients with SLE and healthy donors. C, Positive
correlation between SR-A expression and expression of OAS-1 and MX-1, surrogates for type I IFN pathway
activation. Each symbol represents an individual patient or control subject, and horizontal lines represent the
medians. The Mann-Whitney U test was used for comparisons between groups, and correlations between groups
were analyzed by Spearman’s test.
observed (for MX-1, r ⫽ 0.4812, P ⬍ 0.0001; for OAS-1,
r ⫽ 0.5462, P ⬍ 0.0001) (Figure 6C). These results
indicated clearly that activation of the type I IFN
pathway in patients with SLE was positively associated
with increased SR-A expression.
In addition, monocytes from 12 patients and 9
healthy individuals were freshly isolated and incubated
with Dil-labeled ox-LDL to detect lipid uptake. The
results demonstrated a tendency toward increased oxLDL uptake in monocytes from patients with SLE
compared with that in monocytes from control subjects
(additional information is available from the corresponding author).
It is a well-known clinical phenomenon that
patients with SLE have an increased risk of atherosclerosis, but the mechanisms of this occurrence have not
been clarified. We proposed this study based on the fact
that IFN␣ activities are prominently enhanced in SLE,
and the presence of IFN␣ activities is required for the
development of SLE in animal models (20,21). IFN␣ has
been reported to be correlated with atherosclerosis via
different mechanisms (14,22,23,41). Activation of the
IFN pathway was significantly associated with carotid
intima-media thickness and with carotid plaque in a
recent lupus cohort study (42). Therefore, increasing
attention is being paid to the roles of IFN␣ in premature
atherosclerosis in patients with lupus (43). However, no
studies on the role of IFN␣ in foam cell formation have
been performed.
In the present study, we demonstrated for the
first time that IFN␣ priming promoted lipid uptake and
macrophage-derived foam cell formation in vitro. Mechanistically, up-regulated SR-A expression by IFN␣ was
associated with enhanced uptake of modified lipids and
an increased number of foam cells. Moreover, the
expression of SR-A mRNA was significantly increased in
PBMCs from patients with SLE and was positively
correlated with activation of the type I IFN pathway.
Besides, monocytes from patients with SLE showed a
tendency toward augmented lipid uptake. Therefore, we
believe that the premature atherosclerosis observed in
patients with SLE may be a consequence of increased
activities of type I IFN.
Macrophage-derived foam cells have been identified as one of the main components of early atherosclerotic lesions (44). The balance of lipid influx and
cholesterol efflux in macrophages is strictly controlled in
normal organisms by modulating the expression of SRs
in macrophages, which is essential for preventing atherosclerosis. Previous studies demonstrated that the
expression of SR-A was increased in macrophage-rich
areas of human atherosclerotic lesions and played a
proatherogenic role in plaque formation (34,45). It has
been reported that targeted disruption of SR-A mainly
decreased modified LDL uptake. Moreover, SR-A–
deficient mice showed a decreased tendency for the
development of atherosclerosis (33,45,46). These results
support the contribution of SR-A to the generation of
atherosclerotic plaques and the development of atherosclerosis. Furthermore, a recent study demonstrated that
SR-A polymorphisms were associated with the incidence
of atherosclerosis (47). Consistent with these studies, we
observed that IFN␣ activated the SR-A gene and upregulated its expression but did not have that effect on
CD36, although CD36 appears to be another important
SR responsible for the uptake of modified LDL (33).
Enhanced SR-A promoter activities appeared to
be involved in IFN␣-induced SR-A expression. However, no definite ISRE motifs were identified in the
SR-A promoter by bioinformatics analysis. Therefore, it
is not clear presently which transcription factor or
factors or unknown ISRE sequences are involved in
activation of the SR-A promoter by IFN␣ treatment.
IFN␣ can activate the MAPK and PI3K signaling
pathways in a STAT-independent manner; these cascades are known to be responsible for IFN␣-induced
biologic responses such as IFN-driven gene transcription
(35). Our data suggested that the PI3K/Akt pathway but
not the MAPK pathway was necessary for IFN␣-induced
SR-A expression. In contrast to a previous report that
LY294002 had no effect on basal SR-A expression in
RAW 264.7 cells (37), our data showed that LY294002
inhibited not only IFN␣-induced SR-A expression but
also basal SR-A expression in human macrophages. One
of the possible reasons for these different results may be
the different cell lines used. However, because it remains controversial whether STAT-1 can modulate
SR-A expression (48,49), more data will be needed to
clarify this issue.
High SR-A gene expression in PBMCs provided
a predictive marker for cardiovascular events (50). SR-A
was expressed at low levels in circulating monocytes and
was remarkably up-regulated during the process of
monocyte differentiation into macrophages (36). It has
been reported that SR-A gene expression was specifically increased in PBMCs from patients with acute
coronary syndrome (50). In our investigation, SR-A
mRNA expression was significantly increased in PBMCs
from patients with SLE and positively correlated with
activation of the type I IFN pathway. To our knowledge,
this is the first study to reveal this point. Elevated
expression of SR-A could promote cellular adhesion and
therefore increase monocyte recruitment, facilitating
their entry into the subendothelial space, as well as
enhance modified LDL uptake (40). Meanwhile, preliminary results indicated that monocytes from patients with
SLE had a tendency toward the uptake of more lipids
compared with monocytes from healthy subjects, even
though the difference was not statistically significant,
which may be attributable to the small sample size and
the variation in primary cells. This result was consistent
with the observation that IFN␣ priming was able to
facilitate cholesterol uptake.
All these activities together would likely be responsible for the increased incidence of atherosclerosis
in patients with SLE. However, factors other than IFN␣
cannot be excluded in the explanation of increased SR-A
expression in patients with SLE. We noticed that other
types of cytokines, such as TNF␣, IL-6, IFN␥, and
monocyte chemotactic protein 1, may also have promoting effects on atherosclerosis (41). Considering the
disorder of cytokine and chemokine production in SLE,
cytokines other than type I IFN may also be involved in
the initiation and progression of atherosclerosis in SLE
(11,12). In this study, we examined the effect of IFN␣ on
lipid uptake and SR-A expression, but further studies
are certainly needed to explore the detailed mechanisms
underlying the role of IFN␣ in atherosclerosis. An animal
model and a longitudinal multiple-cohort study will be
beneficial to further clarify these points.
In summary, our study provides new evidence
that IFN␣, as the pathogenic factor in SLE, promoted
lipid uptake and macrophage-derived foam cell formation by up-regulating SR-A expression, which was essential in the process of plaque formation and progress of
atherosclerosis. These findings should be helpful in
enhancing our understanding of the mechanisms of
atherogenesis, especially in the setting of autoimmune
disease. Furthermore, our findings may provide potential therapeutic targets for the prevention and treatment
of premature atherosclerosis in patients with SLE.
14. Banchereau J, Pascual V. Type I interferon in systemic lupus
erythematosus and other autoimmune diseases. Immunity 2006;
15. Baechler EC, Gregersen PK, Behrens TW. The emerging role of
interferon in human systemic lupus erythematosus. Curr Opin
Immunol 2004;16:801–7.
16. Baechler EC, Batliwalla FM, Karypis G, Gaffney PM, Ortmann
WA, Espe KJ, et al. Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus. Proc
Natl Acad Sci U S A 2003;100:2610–5.
17. Dall’era MC, Cardarelli PM, Preston BT, Witte A, Davis JC Jr.
Type I interferon correlates with serological and clinical manifestations of SLE. Ann Rheum Dis 2005;64:1692–7.
18. Kirou KA, Lee C, George S, Louca K, Papagiannis IG, Peterson
MG, et al. Coordinate overexpression of interferon-␣–induced
genes in systemic lupus erythematosus. Arthritis Rheum 2004;50:
19. Santiago-Raber ML, Baccala R, Haraldsson KM, Choubey D,
Stewart TA, Kono DH, et al. Type-I interferon receptor deficiency
reduces lupus-like disease in NZB mice. J Exp Med 2003;197:
20. Reeves WH, Lee PY, Weinstein JS, Satoh M, Lu L. Induction of
autoimmunity by pristane and other naturally occurring hydrocarbons. Trends Immunol 2009;30:455–64.
21. Nacionales DC, Kelly-Scumpia KM, Lee PY, Weinstein JS, Lyons
R, Sobel E, et al. Deficiency of the type I interferon receptor
protects mice from experimental lupus. Arthritis Rheum 2007;56:
22. Lee PY, Li Y, Richards HB, Chan FS, Zhuang H, Narain S, et al.
Type I interferon as a novel risk factor for endothelial progenitor
cell depletion and endothelial dysfunction in systemic lupus erythematosus. Arthritis Rheum 2007;56:3759–69.
23. Denny MF, Thacker S, Mehta H, Somers EC, Dodick T, Barrat FJ,
et al. Interferon-␣ promotes abnormal vasculogenesis in lupus: a
potential pathway for premature atherosclerosis. Blood 2007;110:
24. Niessner A, Shin MS, Pryshchep O, Goronzy JJ, Chaikof EL,
Weyand CM. Synergistic proinflammatory effects of the antiviral
cytokine interferon-␣ and Toll-like receptor 4 ligands in the
atherosclerotic plaque. Circulation 2007;116:2043–52.
25. Niessner A, Sato K, Chaikof EL, Colmegna I, Goronzy JJ, Weyand
CM. Pathogen-sensing plasmacytoid dendritic cells stimulate cytotoxic T-cell function in the atherosclerotic plaque through
interferon-␣. Circulation 2006;114:2482–9.
26. Levy Z, Rachmani R, Trestman S, Dvir A, Shaish A, Ravid M, et
al. Low-dose interferon-␣ accelerates atherosclerosis in an LDL
receptor-deficient mouse model. Eur J Intern Med 2003;14:
27. Hartvigsen K, Chou MY, Hansen LF, Shaw PX, Tsimikas S,
Binder CJ, et al. The role of innate immunity in atherogenesis. J
Lipid Res 2009;50 Suppl:S388–93.
28. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield
NF, et al. The 1982 revised criteria for the classification of systemic
lupus erythematosus. Arthritis Rheum 1982;25:1271–7.
29. Hochberg MC, for the Diagnostic and Therapeutic Criteria Committee of the American College of Rheumatology. Updating the
American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus [letter]. Arthritis
Rheum 1997;40:1725.
30. Sharif MN, Tassiulas I, Hu Y, Mecklenbrauker I, Tarakhovsky A,
Ivashkiv LB. IFN-␣ priming results in a gain of proinflammatory
function by IL-10: implications for systemic lupus erythematosus
pathogenesis. J Immunol 2004;172:6476–81.
31. Osto E, Kouroedov A, Mocharla P, Akhmedov A, Besler C,
Rohrer L, et al. Inhibition of protein kinase C ␤ prevents foam cell
formation by reducing scavenger receptor A expression in human
macrophages. Circulation 2008;118:2174–82.
We thank Y. L. Dai, X. P. Chen, Lan Yin, Y. J. Tang,
Fang Du, X. B. Luo, H. B. Zhou, Xia Zhao, S. J. Wang, and
Ping Ye. We also thank all of the patients, healthy volunteers,
and rheumatologists in the Department of Rheumatology of
Renji Hospital who participated in this study.
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Shen had full access to all of the
data in the study and takes responsibility for the integrity of the data
and the accuracy of the data analysis.
Study conception and design. Shen, Bao.
Acquisition of data. Li, Fu, Cui, Qu, Pan.
Analysis and interpretation of data. Li, Fu, Shen, Bao.
1. Thompson T, Sutton-Tyrrell K, Wildman RP, Kao A, Fitzgerald
SG, Shook B, et al. Progression of carotid intima-media thickness
and plaque in women with systemic lupus erythematosus. Arthritis
Rheum 2008;58:835–42.
2. Salmon JE, Roman MJ. Subclinical atherosclerosis in rheumatoid
arthritis and systemic lupus erythematosus. Am J Med 2008;121(10
Suppl 1):S3–8.
3. Roman MJ, Shanker BA, Davis A, Lockshin MD, Sammaritano L,
Simantov R, et al. Prevalence and correlates of accelerated
atherosclerosis in systemic lupus erythematosus. N Engl J Med
4. Bessant R, Duncan R, Ambler G, Swanton J, Isenberg DA,
Gordon C, et al. Prevalence of conventional and lupus-specific risk
factors for cardiovascular disease in patients with systemic lupus
erythematosus: a case–control study. Arthritis Rheum 2006;55:
5. Esdaile JM, Abrahamowicz M, Grodzicky T, Li Y, Panaritis C, du
Berger R, et al. Traditional Framingham risk factors fail to fully
account for accelerated atherosclerosis in systemic lupus erythematosus. Arthritis Rheum 2001;44:2331–7.
6. Hahn BH. Systemic lupus erythematosus and accelerated atherosclerosis. N Engl J Med 2003;349:2379–80.
7. Roman MJ, Salmon JE. Cardiovascular manifestations of rheumatologic diseases. Circulation 2007;116:2346–55.
8. Agarwal S, Elliott JR, Manzi S. Atherosclerosis risk factors in
systemic lupus erythematosus. Curr Rheumatol Rep 2009;11:
9. Selzer F, Sutton-Tyrrell K, Fitzgerald SG, Pratt JE, Tracy RP,
Kuller LH, et al. Comparison of risk factors for vascular disease in
the carotid artery and aorta in women with systemic lupus
erythematosus. Arthritis Rheum 2004;50:151–9.
10. McMahon M, Grossman J, Skaggs B, Fitzgerald J, Sahakian L,
Ragavendra N, et al. Dysfunctional proinflammatory high-density
lipoproteins confer increased risk of atherosclerosis in women with
systemic lupus erythematosus. Arthritis Rheum 2009;60:2428–37.
11. McMahon M, Hahn BH. Atherosclerosis and systemic lupus
erythematosus: mechanistic basis of the association. Curr Opin
Immunol 2007;19:633–9.
12. Hahn BH, Grossman J, Chen W, McMahon M. The pathogenesis
of atherosclerosis in autoimmune rheumatic diseases: roles of
inflammation and dyslipidemia. J Autoimmun 2007;28:69–75.
13. Pascual V, Farkas L, Banchereau J. Systemic lupus erythematosus:
all roads lead to type I interferons. Curr Opin Immunol 2006;18:
32. Moore KJ, Freeman MW. Scavenger receptors in atherosclerosis:
beyond lipid uptake. Arterioscler Thromb Vasc Biol 2006;26:
33. Kunjathoor VV, Febbraio M, Podrez EA, Moore KJ, Andersson
L, Koehn S, et al. Scavenger receptors class A-I/II and CD36 are
the principal receptors responsible for the uptake of modified low
density lipoprotein leading to lipid loading in macrophages. J Biol
Chem 2002;277:49982–8.
34. Matsumoto A, Naito M, Itakura H, Ikemoto S, Asaoka H,
Hayakawa I, et al. Human macrophage scavenger receptors:
primary structure, expression, and localization in atherosclerotic
lesions. Proc Natl Acad Sci U S A 1990;87:9133–7.
35. Platanias LC. Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat Rev Immunol 2005;5:375–86.
36. Moulton KS, Semple K, Wu H, Glass CK. Cell-specific expression
of the macrophage scavenger receptor gene is dependent on PU.1
and a composite AP-1/ets motif. Mol Cell Biol 1994;14:4408–18.
37. Li K, Yao W, Zheng X, Liao K. Berberine promotes the development of atherosclerosis and foam cell formation by inducing
scavenger receptor A expression in macrophage. Cell Res 2009;
38. Xu WY, Wang L, Wang HM, Wang YQ, Liang YF, Zhao TT, et
al. TLR2 and TLR4 agonists synergistically up-regulate SR-A in
RAW264.7 through p38. Mol Immunol 2007;44:2315–23.
39. Zhuang H, Narain S, Sobel E, Lee PY, Nacionales DC, Kelly KM,
et al. Association of anti-nucleoprotein autoantibodies with upregulation of type I interferon-inducible gene transcripts and
dendritic cell maturation in systemic lupus erythematosus. Clin
Immunol 2005;117:238–50.
40. De Winther MP, van Dijk KW, Havekes LM, Hofker MH.
Macrophage scavenger receptor class A: a multifunctional receptor in atherosclerosis. Arterioscler Thromb Vasc Biol 2000;20:
41. Galkina E, Ley K. Immune and inflammatory mechanisms of
atherosclerosis (*). Annu Rev Immunol 2009;27:165–97.
42. Zhao W, Somers EC, McCune WJ, Kaplan MJ. Type I interferon
gene signatures are associated with vascular risk and atherosclerosis in systemic lupus erythematosus [abstract]. Arthritis Rheum
2009;60 Suppl 10:582.
43. Crow MK. Developments in the clinical understanding of lupus.
Arthritis Res Ther 2009;11:245.
44. Gerrity RG, Naito HK, Richardson M, Schwartz CJ. Dietary
induced atherogenesis in swine: morphology of the intima in
prelesion stages Am J Pathol 1979;95:775–92.
45. Linton MF, Fazio S. Class A scavenger receptors, macrophages,
and atherosclerosis. Curr Opin Lipidol 2001;12:489–95.
46. Suzuki H, Kurihara Y, Takeya M, Kamada N, Kataoka M, Jishage
K, et al. A role for macrophage scavenger receptors in atherosclerosis and susceptibility to infection. Nature 1997;386:292–6.
47. Durst R, Neumark Y, Meiner V, Friedlander Y, Sharon N, Polak
A, et al. Increased risk for atherosclerosis of various macrophage
scavenger receptor 1 alleles. Genet Test Mol Biomarkers 2009;13:
48. Grewal T, Priceputu E, Davignon J, Bernier L. Identification of a
gamma-interferon-responsive element in the promoter of the
human macrophage scavenger receptor A gene. Arterioscler
Thromb Vasc Biol 2001;21:825–31.
49. Agrawal S, Febbraio M, Podrez E, Cathcart MK, Stark GR,
Chisolm GM. Signal transducer and activator of transcription 1 is
required for optimal foam cell formation and atherosclerotic
lesion development. Circulation 2007;115:2939–47.
50. Nakayama M, Kudoh T, Kaikita K, Yoshimura M, Oshima S,
Miyamoto Y, et al. Class A macrophage scavenger receptor gene
expression levels in peripheral blood mononuclear cells specifically
increase in patients with acute coronary syndrome. Atherosclerosis
Без категории
Размер файла
439 Кб
atherosclerosis, formation, foam, cells, lupus, promote, macrophage, link, uptake, novem, priming, derived, lipid, interferon
Пожаловаться на содержимое документа