Interferon-╨Ю┬▒ priming promotes lipid uptake and macrophage-derived foam cell formationA novel link between interferon-╨Ю┬▒ and atherosclerosis in lupus.код для вставкиСкачать
ARTHRITIS & RHEUMATISM 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 expression. 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). 1 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@ yahoo.com.cn. 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 492 IFN␣ PRIMING PROMOTES LIPID UPTAKE 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 (20,21). 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 493 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 AND METHODS 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 ⫻ 494 105/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 LI ET AL 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 (TreeStar). 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 primer 5⬘-CGACGCGTATTGTAAAGGAAAGAGTGTGA 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 assay. 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, IFN␣ PRIMING PROMOTES LIPID UPTAKE 495 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. 496 LI ET AL 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 definitions. 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. RESULTS 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 IFN␣ PRIMING PROMOTES LIPID UPTAKE 497 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 498 LI ET AL 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 IFN␣ PRIMING PROMOTES LIPID UPTAKE 499 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). DISCUSSION 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 500 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 LI ET AL 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. IFN␣ PRIMING PROMOTES LIPID UPTAKE 501 ACKNOWLEDGMENTS 14. Banchereau J, Pascual V. Type I interferon in systemic lupus erythematosus and other autoimmune diseases. Immunity 2006; 25:383–92. 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. 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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. AUTHOR CONTRIBUTIONS 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. 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