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• W1973622668 abstract "Cholesterol-laden monocyte-derived macrophages are phagocytic cells characteristic of early and advanced atherosclerotic lesions. Interleukin-6 (IL-6) is a macrophage secretory product that is abundantly expressed in atherosclerotic plaques but whose precise role in atherogenesis is unclear. The capacity of macrophages to clear apoptotic cells, through the efferocytosis mechanism, as well as to reduce cellular cholesterol accumulation contributes to prevent plaque progression and instability. By virtue of its capacity to promote cellular cholesterol efflux from phagocyte-macrophages, ABCA1 was reported to reduce atherosclerosis. We demonstrated that lipid loading in human macrophages was accompanied by a strong increase of IL-6 secretion. Interestingly, IL-6 markedly induced ABCA1 expression and enhanced ABCA1-mediated cholesterol efflux from human macrophages to apoAI. Stimulation of ABCA1-mediated cholesterol efflux by IL-6 was, however, abolished by selective inhibition of the Jak-2/Stat3 signaling pathway. In addition, we observed that the expression of molecules described to promote efferocytosis, i.e. c-mer proto-oncogene-tyrosine kinase, thrombospondin-1, and transglutaminase 2, was significantly induced in human macrophages upon treatment with IL-6. Consistent with these findings, IL-6 enhanced the capacity of human macrophages to phagocytose apoptotic cells; moreover, we observed that IL-6 stimulates the ABCA1-mediated efflux of cholesterol derived from the ingestion of free cholesterol-loaded apoptotic macrophages. Finally, the treatment of human macrophages with IL-6 led to the establishment of an anti-inflammatory cytokine profile, characterized by an increased secretion of IL-4 and IL-10 together with a decrease of that of IL-1β. Taken together, our results indicate that IL-6 favors the elimination of excess cholesterol in human macrophages and phagocytes by stimulation of ABCA1-mediated cellular free cholesterol efflux and attenuates the macrophage proinflammatory phenotype. Thus, high amounts of IL-6 secreted by lipid laden human macrophages may constitute a protective response from macrophages to prevent accumulation of cytotoxic-free cholesterol. Such a cellular recycling of free cholesterol may contribute to reduce both foam cell formation and the accumulation of apoptotic bodies as well as intraplaque inflammation in atherosclerotic lesions. Cholesterol-laden monocyte-derived macrophages are phagocytic cells characteristic of early and advanced atherosclerotic lesions. Interleukin-6 (IL-6) is a macrophage secretory product that is abundantly expressed in atherosclerotic plaques but whose precise role in atherogenesis is unclear. The capacity of macrophages to clear apoptotic cells, through the efferocytosis mechanism, as well as to reduce cellular cholesterol accumulation contributes to prevent plaque progression and instability. By virtue of its capacity to promote cellular cholesterol efflux from phagocyte-macrophages, ABCA1 was reported to reduce atherosclerosis. We demonstrated that lipid loading in human macrophages was accompanied by a strong increase of IL-6 secretion. Interestingly, IL-6 markedly induced ABCA1 expression and enhanced ABCA1-mediated cholesterol efflux from human macrophages to apoAI. Stimulation of ABCA1-mediated cholesterol efflux by IL-6 was, however, abolished by selective inhibition of the Jak-2/Stat3 signaling pathway. In addition, we observed that the expression of molecules described to promote efferocytosis, i.e. c-mer proto-oncogene-tyrosine kinase, thrombospondin-1, and transglutaminase 2, was significantly induced in human macrophages upon treatment with IL-6. Consistent with these findings, IL-6 enhanced the capacity of human macrophages to phagocytose apoptotic cells; moreover, we observed that IL-6 stimulates the ABCA1-mediated efflux of cholesterol derived from the ingestion of free cholesterol-loaded apoptotic macrophages. Finally, the treatment of human macrophages with IL-6 led to the establishment of an anti-inflammatory cytokine profile, characterized by an increased secretion of IL-4 and IL-10 together with a decrease of that of IL-1β. Taken together, our results indicate that IL-6 favors the elimination of excess cholesterol in human macrophages and phagocytes by stimulation of ABCA1-mediated cellular free cholesterol efflux and attenuates the macrophage proinflammatory phenotype. Thus, high amounts of IL-6 secreted by lipid laden human macrophages may constitute a protective response from macrophages to prevent accumulation of cytotoxic-free cholesterol. Such a cellular recycling of free cholesterol may contribute to reduce both foam cell formation and the accumulation of apoptotic bodies as well as intraplaque inflammation in atherosclerotic lesions. The retention and accumulation of modified LDL and apoptotic cells in the arterial intima represent critical steps in the formation of cholesterol-rich vulnerable atherosclerotic plaques; such plaques feature an immunoinflammatory process in which monocyte-derived macrophages play a central role (1Weber C. Zernecke A. Libby P. Nat. Rev. Immunol. 2008; 8: 802-815Crossref PubMed Scopus (666) Google Scholar, 2Daugherty A. Webb N.R. Rateri D.L. King V.L. J. Lipid Res. 2005; 46: 1812-1822Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Indeed, intimal macrophages endocytose modified LDL via scavenger receptors (SR-A, CD36), thereby favoring intracellular lipid accumulation with foam cell formation. Cholesterol-laden macrophages may equally result from phagocytosis of apoptotic cells via the process of efferocytosis (3Thorp E. Tabas I. J. Leukoc Biol. 2009; 86: 1089-1095Crossref PubMed Scopus (158) Google Scholar); however, such a massive influx of cholesterol may prove cytotoxic if cholesterol recycling is disrupted in macrophage phagocyte (4Schrijvers D.M. De Meyer G.R. Herman A.G. Martinet W. Cardiovasc. Res. 2007; 73: 470-480Crossref PubMed Scopus (218) Google Scholar). The ATP binding cassette A1 (ABCA1) 3The abbreviations used are: ABCA1ATP binding cassette A1HMDMhuman monocyte-derived macrophageacLDLacetylated LDLCtrlcontrolTHBS1thrombospondin-1TG2transglutaminase 2PPARperoxisome proliferator-activated receptorLXRliver X receptor. transporter plays a central role in maintaining macrophage cholesterol homeostasis by preventing cellular lipid accumulation as a result of its capacity to efflux cellular free cholesterol to apoAI (5Oram J.F. Heinecke J.W. Physiol. Rev. 2005; 85: 1343-1372Crossref PubMed Scopus (427) Google Scholar). Thus, ABCA1 may promote elimination of cholesterol derived from ingestion of either modified lipoproteins or apoptotic cells (6Kiss R.S. Elliott M.R. Ma Z. Marcel Y.L. Ravichandran K.S. Curr. Biol. 2006; 16: 2252-2258Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Recent studies clearly highlight the close link between inflammation and cholesterol homeostasis in macrophages through mechanisms in which ABCA1 appears to be a major actor (7Fitzgerald M.L. Mujawar Z. Tamehiro N. Atherosclerosis. 2010; 211: 361-370Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Thus, increased intracellular free cholesterol concentration in ABCA1 KO macrophages is accompanied by enhanced proinflammatory response upon LPS induction (8Francone O.L. Royer L. Boucher G. Haghpassand M. Freeman A. Brees D. Aiello R.J. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1198-1205Crossref PubMed Scopus (81) Google Scholar, 9Koseki M. Hirano K. Masuda D. Ikegami C. Tanaka M. Ota A. Sandoval J.C. Nakagawa-Toyama Y. Sato S.B. Kobayashi T. Shimada Y. Ohno-Iwashita Y. Matsuura F. Shimomura I. Yamashita S. J. Lipid Res. 2007; 48: 299-306Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 10Zhu X. Lee J.Y. Timmins J.M. Brown J.M. Boudyguina E. Mulya A. Gebre A.K. Willingham M.C. Hiltbold E.M. Mishra N. Maeda N. Parks J.S. J. Biol. Chem. 2008; 283: 22930-22941Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). Moreover, the enhanced Toll-like receptors signaling observed in ABCA1/ABCG1 double KO macrophages is associated with free cholesterol accumulation and up-regulated expression of proinflammatory genes (11Yvan-Charvet L. Welch C. Pagler T.A. Ranalletta M. Lamkanfi M. Han S. Ishibashi M. Li R. Wang N. Tall A.R. Circulation. 2008; 118: 1837-1847Crossref PubMed Scopus (348) Google Scholar). Reciprocally, activation of Toll-like receptors 3 and 4 inhibits the induction of LXR target genes, such as ABCA1, in macrophages and strongly reduces cholesterol efflux (12Castrillo A. Joseph S.B. Vaidya S.A. Haberland M. Fogelman A.M. Cheng G. Tontonoz P. Mol. Cell. 2003; 12: 805-816Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). Although those studies suggest that the inflammatory property of ABCA1 results from its ability to modulate free cholesterol levels and distribution in plasma membrane, recent studies propose that macrophage ABCA1 may act as an anti-inflammatory receptor through activation of Jak2/Stat3 pathway after apoAI binding (13Tang C. Liu Y. Kessler P.S. Vaughan A.M. Oram J.F. J. Biol. Chem. 2009; 284: 32336-32343Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 14Yin K. Deng X. Mo Z.C. Zhao G.J. Jiang J. Cui L.B. Tan C.Z. Wen G.B. Fu Y. Tang C.K. J. Biol. Chem. 2011; 286: 13834-13845Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). ATP binding cassette A1 human monocyte-derived macrophage acetylated LDL control thrombospondin-1 transglutaminase 2 peroxisome proliferator-activated receptor liver X receptor. Among the cytokines present in atherosclerotic tissue, interleukin-6 (IL-6) is prominent and, moreover, is abundantly produced by free cholesterol-loaded macrophages in advanced lesions (15Li Y. Schwabe R.F. DeVries-Seimon T. Yao P.M. Gerbod-Giannone M.C. Tall A.R. Davis R.J. Flavell R. Brenner D.A. Tabas I. J. Biol. Chem. 2005; 280: 21763-21772Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). Importantly, interleukin-6 has been identified as an independent risk factor for premature coronary artery disease (16Woods A. Brull D.J. Humphries S.E. Montgomery H.E. Eur. Heart J. 2000; 21: 1574-1583Crossref PubMed Scopus (200) Google Scholar), and moreover, elevated levels of IL-6 are associated with an increased risk for myocardial infarction in healthy men (17Ridker P.M. Rifai N. Stampfer M.J. Hennekens C.H. Circulation. 2000; 101: 1767-1772Crossref PubMed Scopus (2040) Google Scholar). The Offspring Cohort of the Framingham Heart Study indicated that IL-6 levels were associated with internal carotid artery intima-media thickness and stenosis (18Thakore A.H. Guo C.Y. Larson M.G. Corey D. Wang T.J. Vasan R.S. D'Agostino Sr., R.B. Lipinska I. Keaney Jr., J.F. Benjamin E.J. O'Donnell C.J. Am. J. Cardiol. 2007; 99: 1598-1602Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The relative contribution of IL-6 as a causative factor, as a consequence, or as a marker of atherosclerosis is unclear. Indeed, experimental evidence in genetically modified mouse models susceptible to atherosclerosis highlight opposite functions of IL-6 (19Kleemann R. Zadelaar S. Kooistra T. Cardiovasc. Res. 2008; 79: 360-376Crossref PubMed Scopus (504) Google Scholar). Indeed, treatment of mice with recombinant mouse IL-6 increased lesion size in both C57BL/6 and apoE−/− mice fed a high fat/cholate diet (20Huber S.A. Sakkinen P. Conze D. Hardin N. Tracy R. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2364-2367Crossref PubMed Scopus (451) Google Scholar). However, IL6−/− mice developed larger fatty streak lesions than control mice when fed an atherogenic diet for 15 weeks (21Van Lenten B.J. Wagner A.C. Navab M. Fogelman A.M. J. Biol. Chem. 2001; 276: 1923-1929Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Similar studies in IL-6/LDL receptor double KO mice failed to detect a significant effect of IL-6 deficiency on lesion size (22Song L. Schindler C. Atherosclerosis. 2004; 177: 43-51Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Finally, a potential atheroprotective role for IL-6 was described in IL-6/apoE double KO mice maintained for 1 year on a chow diet (23Elhage R. Clamens S. Besnard S. Mallat Z. Tedgui A. Arnal J. Maret A. Bayard F. Atherosclerosis. 2001; 156: 315-320Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 24Schieffer B. Selle T. Hilfiker A. Hilfiker-Kleiner D. Grote K. Tietge U.J. Trautwein C. Luchtefeld M. Schmittkamp C. Heeneman S. Daemen M.J. Drexler H. Circulation. 2004; 110: 3493-3500Crossref PubMed Scopus (245) Google Scholar). Taken together, these findings suggest that IL-6 may exert opposing actions in the inflammatory dimension of the atherosclerosis process. To evaluate this question further, we explored the impact of IL-6 on the capacity of human macrophages to regulate cholesterol homeostasis in the lipido-inflammatory context of atherosclerosis. We presently report that disruption of cellular lipid homeostasis leading to cholesterol accumulation in human macrophages was accompanied by an increased secretion of IL-6. Moreover, IL-6 reduced macrophage lipid accumulation derived from ingestion of either modified lipoproteins or apoptotic cells by stimulating ABCA1-mediated free cholesterol efflux through activation of the Jak-2/Stat3 signaling pathway. In addition, IL-6 enhanced the capacity of human macrophages to ingest apoptotic cells and attenuated the proinflammatory phenotype of human cholesterol-loaded human macrophages. Our findings, therefore, suggest that the autocrine action of macrophage-secreted-IL-6 may contribute to reduce the formation of inflammatory foam cells and apoptotic macrophages in atherosclerotic lesions. Monocytes were isolated from the blood of individual healthy normolipidemic donors (Etablissement Français du Sang) on Ficoll gradients (Ficoll-Paque PLUS, GE Healthcare) and subsequently differentiated into human monocyte-derived macrophages (HMDM) on plastic Primaria plates (Falcon) over a period of 10 days of culture in RPMI 1640 medium supplemented with 10% heat-inactivated human serum, 2 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 20 ng/ml human macrophage colony-stimulating factor. The human THP-1 monocytic cell line was obtained from American Type Culture collection (ATCC) and maintained in culture in 5% CO2 at 37 °C in RPMI medium containing 10% heat-inactivated fetal bovine serum, 2 mm l-glutamine, and 80 units/ml penicillin, 80 μg/ml streptomycin. THP-1 monocytes were seeded onto 24- or 6-well plates at density 1 × 106 or 4 × 106 cell/well, respectively, in the presence of 50 ng/ml phorbol 12-myristate 13-acetate for 3 days to induce differentiation into macrophage-like cells. Human macrophages (THP-1 and HMDM) were cholesterol-loaded with 50 μg/ml acetylated LDL (acLDL) labeled with 1 μCi/ml [3H]cholesterol for 24 h in the presence or in the absence of 50 ng/ml recombinant human IL-6 (R&D Systems) in an RPMI medium containing 2 mm glutamine, 50 mm glucose, and 0.2% BSA (RGGB). All cell culture reagents were certified as endotoxin-free by the manufacturers. As described in our previous studies (25Dentan C. Lesnik P. Chapman M.J. Ninio E. Eur. J. Biochem. 1996; 236: 48-55Crossref PubMed Scopus (56) Google Scholar, 26Petit L. Lesnik P. Dachet C. Moreau M. Chapman M.J. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 309-315Crossref PubMed Scopus (60) Google Scholar), pyrogen-free commercial plastic was used at all critical steps during LDL isolation to prevent endotoxin contamination. Under these conditions, endotoxin content was <1 pg/μg LDL protein as monitored by the Limulus amebocyte lysate chromogenic assay (Biogenic) (26Petit L. Lesnik P. Dachet C. Moreau M. Chapman M.J. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 309-315Crossref PubMed Scopus (60) Google Scholar). After the acetylation procedure, the endotoxin content of acLDL was <0.5 pg/μg of acLDL protein (25Dentan C. Lesnik P. Chapman M.J. Ninio E. Eur. J. Biochem. 1996; 236: 48-55Crossref PubMed Scopus (56) Google Scholar), such endotoxin levels being not able to induce a detectable change in cytokine secretion, including that of IL-6 (27Zhong W.W. Burke P.A. Hand A.T. Walsh M.J. Hughes L.A. Forse R.A. Arch. Surg. 1993; 128: 158-164Crossref PubMed Scopus (64) Google Scholar). Human macrophages were then equilibrated in RGGB in the presence or absence of 50 ng/ml recombinant human IL-6 and 1 μm TO901317 (Sigma) for an additional 24-h period. Cellular cholesterol efflux to 5 μg/ml lipid-free apoA-I (Biodesign) or HDL (density = 1.063–1.21 g/ml; 15 μg/ml PL) isolated from normolipidemic plasma by preparative ultracentrifugation (28Chapman M.J. Goldstein S. Lagrange D. Laplaud P.M. J. Lipid Res. 1981; 22: 339-358Abstract Full Text PDF PubMed Google Scholar) was assayed in serum-free medium for a 4-h chase period in the presence or absence of 50 ng/ml recombinant human IL-6 and 1 μm TO901317. Finally, culture media were harvested and cleared of cellular debris by brief centrifugation. Cell-associated radioactivity was determined by extraction in hexane-isopropanol (3:2), evaporation of the solvent, and liquid scintillation counting (Wallac Trilux 1450 Microbeta). The percentage of cholesterol efflux was calculated as 100 × (medium cpm)/(medium cpm + cell cpm). ApoA-I-specific cholesterol efflux was determined by subtracting nonspecific cholesterol efflux occurring in apoA-I-free medium. When required, a cell-permeable Stat3 inhibitor peptide (100 μm Stat3-I, Calbiochem) or a selective inhibitor of either the Jak-2 protein-tyrosine kinase (25 μm AG490, Sigma) or the Jak-3 protein-tyrosine kinase (10 μm ZM39923, Sigma) or the Jak-2/Stat3 signaling pathway (2 μm cucurbitacin I, Sigma) was added together with IL-6. The masses of esterified and free cholesterol in human macrophages was quantified using the Amplex Red cholesterol assay kit (Molecular Probes) as previously described (29Le Goff W. Settle M. Greene D.J. Morton R.E. Smith J.D. J. Lipid Res. 2006; 47: 51-58Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Cell protein extracts from human THP-1 macrophages were prepared from 6-well plates pretreated or not with 50 ng/ml recombinant human IL-6 in the presence or the absence of 1 μm TO901317 for 24 h in RGGB medium. Total cell protein was extracted in M-PER mammalian protein extraction reagent (Thermo) containing 10% protease inhibitor mixture (Roche Applied Science). Protein concentration was determined using a BCA protein assay (Pierce). Equal amounts of protein (50 μg) were loaded onto a 3–8% Tris acetate polyacrylamide gel (Invitrogen) and transferred onto a nitrocellulose membrane using iBlot technology (Invitrogen). The membrane was blocked with 5% skim milk (in PBS, 0.1% Tween) for 2 h, and ABCA1 was detected by incubation overnight at 4 °C with a rabbit anti-human ABCA1 (Novus) at 1:500 and horseradish peroxidase-conjugated goat ant-rabbit secondary antibody at 1:5000. The signal was revealed with an enhanced chemiluminescence Immobilon Western substrate (Millipore). Quantification of Western blots was performed using a Kodak Image Station 440 CF with Kodak 1D Image Analysis software. Quantification of ABCA1 protein levels was normalized to matching annexin1 (Zymed Laboratories Inc.) levels. Free cholesterol-induced apoptotic THP-1 macrophages were produced by incubation with 100 μg/ml acLDL and 10 μg/ml concentrations of an acyl-CoA:cholesterol acyltransferase inhibitor (S58035, Sigma) for 24 h in serum-free medium as previously described (30Cui D. Thorp E. Li Y. Wang N. Yvan-Charvet L. Tall A.R. Tabas I. J. Leukoc. Biol. 2007; 82: 1040-1050Crossref PubMed Scopus (56) Google Scholar). UV-induced apoptotic human Jurkat cells were generated by UV exposition (312 nm, 6 × 15 Watts) for 15 min followed by a 4-h incubation at 37 °C in a 5% CO2 atmosphere. Apoptosis was quantified by both annexin V and propidium iodide (PI) staining (Beckman Coulter) using flow cytometry (Beckman FC500). The degrees of apoptosis in Jurkat apoptotic cells were ∼74% in early apoptosis (annexin V-positive only), <7% in late apoptosis (annexin V + PI-positive), and <2% in necrosis (PI-positive only). Apoptotic Jurkat cells were labeled with 5 μm calcein-AM (Invitrogen) and added to THP-1 macrophage phagocyte cells (Ratio 3:1) previously treated in the presence or absence of 50 ng/ml recombinant human IL-6 ± 1 μm TO901317. After 30 min of contact, which has been reported to be a sufficient period to completely engage and internalize the apoptotic cells (6Kiss R.S. Elliott M.R. Ma Z. Marcel Y.L. Ravichandran K.S. Curr. Biol. 2006; 16: 2252-2258Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), non-ingested apoptotic cells were removed by extensive washing with PBS. Subsequently, THP-1 phagocytes were detached from the plates and then subjected to flow cytometry (Beckman FC500) to quantify positive fluorescent phagocytic cells that had ingested calcein-AM-labeled apoptotic Jurkat cells. Positive fluorescent phagocytic cells were visualized by fluorescence microscopy to ensure that the detected fluorescence was localized within the phagocytes. Data are expressed as a phagocytic index corresponding to the mean fluorescence in positive phagocytes. As originally described by Cui et al. (30Cui D. Thorp E. Li Y. Wang N. Yvan-Charvet L. Tall A.R. Tabas I. J. Leukoc. Biol. 2007; 82: 1040-1050Crossref PubMed Scopus (56) Google Scholar) in mouse peritoneal macrophages, monolayers of [3H]cholesterol-labeled apoptotic THP-1 macrophages were extensively washed with PBS, detached from 100-mm bacterial dishes, and added to THP-1 phagocytes (Ratio 5:1) for 30 min at 37 °C in medium containing 10 μg/ml S58035. Non-ingested apoptotic cells were removed by extensive washing with PBS, and efflux of [3H]cholesterol from THP-1 phagocytes, derived from the ingestion of [3H]cholesterol-labeled apoptotic cells, was assayed in the presence of lipid-free apoAI for 4 h in a medium containing 10 μg/ml S58035 as described above. Silencing of ABCA1 expression was performed by application of siRNA oligonucleotides (Dharmacon) targeted to the cDNA sequence of the human ABCA1 gene (GenBankTM #NM_005502). THP-1 macrophages were grown in 24-well plates and transfected with 50 nm control siRNA (Dharmacon) or siRNA targeting human ABCA1 using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer's instructions. Human THP-1 macrophages were incubated with or without 50 ng/ml recombinant human IL-6 in the presence or the absence of 1 μm TO901317 for 24 h at 37 °C. Cells were then washed twice with cold PBS, and total RNA was extracted using a NucleoSpin RNA II kit (Macherey-Nagel) according to the manufacturer's instructions. Then, 1500 ng of RNA was reverse-transcribed with 75 ng of random hexamer using 200 units of Moloney murine leukemia virus reverse transcriptase. An initial denaturation step for 5 min at 68 °C was followed by an elongation phase of 1 h at 42 °C; the reaction was completed by a 5-min incubation at 68 °C. Real time quantitative PCR was performed using a LightCycler LC480 (Roche Applied Science). The reaction contained 2.5 ng of reverse transcribed total RNA, 150 pmol of forward and reverse primers, and 5 μl of Master Mix SYBR Green in a final volume of 10 μl. Samples underwent the standard PCR protocol. Crossing point values for genes of interest were normalized to housekeeping genes (human δ-aminolevulinate synthase and human α-tubulin). Expression data were based on the crossing points calculated with the software for LightCycler data analysis and corrected for PCR efficiencies of the target and the reference gene. Data were expressed as a -fold change in mRNA expression relative to control values. Human THP-1 macrophages were incubated in serum-free media with 50 ng/ml human recombinant IL-6 for 24 h, and secreted cytokines in the culture media were quantified using a semiautomated Biochip Array Technology analyzer (Evidence Investigator, Randox). Data are shown as the mean ± S.E. Experiments were performed in triplicate, and values correspond to the mean from at least three independent experiments. Comparisons of two groups were performed by a two-tailed Student's t test, and comparisons of three or more groups were performed by analysis of variance with the Newman-Keuls post-test. All statistical analyses were performed using Prism software from GraphPad (San Diego, CA). Analysis of secreted IL-6 levels by human macrophages (Fig. 1) revealed that the secretion of IL-6 by cholesterol-loaded human THP-1 macrophages (16.9 ± 0.3 versus 12.9 ± 0.4 μg of cholesterol/mg of protein in cholesterol-loaded and control cells, respectively, p < 0.005) was ∼10-fold (p < 0.01) more elevated than that observed in non-loaded macrophages, thereby suggesting that foam cells contribute to a major degree to the determination of IL-6 amounts produced in human atherosclerotic plaques. Interestingly, disruption of lipid homeostasis in cholesterol-loaded macrophages through silencing of ABCA1 expression using RNA interference (ABCA1 KD) (supplemental Fig. 1) led to an exacerbated secretion of IL-6 (8.4-fold, p < 0.001) as compared with control cholesterol-loaded cells (Ctrl). Taken together, those results indicate that the lipid accumulation in human macrophages is accompanied by a marked elevation of secreted IL-6 amounts. To determine whether this increased IL-6 production may constitute a compensatory response from human macrophages to cholesterol loading, we next examined the effect of IL-6 on the capacity of human macrophages to eliminate the excess of cholesterol resulting from either the uptake of modified LDL or the ingestion of apoptotic cells. To determine whether IL-6 may modulate cellular free cholesterol efflux from cholesterol-loaded human macrophages after incubation with modified LDL (acLDL), we analyzed the impact of IL-6 on cholesterol efflux from HMDM and from THP-1 macrophages to both lipid-poor apoAI and to HDL particles. As shown in Fig. 2, ABCA1-mediated cholesterol efflux to apoAI from THP-1 (Fig. 2A) and HMDM (Fig. 2B) was stimulated upon incubation with IL-6 (2.2- and 2.8-fold, respectively, p < 0.05). When human macrophages were stimulated with a synthetic LXR agonist (TO901317), which markedly induced ABCA1-mediated cholesterol efflux to apoAI subsequent to marked up-regulation of ABCA1 mRNA expression (31Rowe A.H. Argmann C.A. Edwards J.Y. Sawyez C.G. Morand O.H. Hegele R.A. Huff M.W. Circ. Res. 2003; 93: 717-725Crossref PubMed Scopus (82) Google Scholar), elevation in rates of cholesterol efflux to apoAI in response to IL-6 was also observed (THP-1, +38%; HMDM, +42%, p < 0.05). However, IL-6 did not affect cellular cholesterol efflux to HDL from both THP-1 macrophages and HMDM treated in the presence or in the absence of LXR agonists (Fig. 2, C and D), thereby indicating that the effect of IL-6 on cellular cholesterol efflux mechanisms is specific to ABCA1-mediated cholesterol efflux. Consistent with the induction of ABCA1-mediated cholesterol efflux to apoAI, cellular cholesterol mass was significantly reduced (−14%, p < 0.001) in cholesterol-loaded human macrophages when incubated with IL-6 (Fig. 2E). We next determined whether activation of ABCA1-mediated cholesterol efflux by IL-6 may result from stimulation of ABCA1 gene expression in human macrophages. As shown in Fig. 3A, IL-6 significantly up-regulated ABCA1 mRNA levels in THP-1 macrophages when acting alone (+84%, p < 0.01) or in combination with the synthetic LXR agonist TO901317 (+94%, p < 0.001); such induction of ABCA1 by IL-6 was confirmed at the protein level (Fig. 3B). By contrast, mRNA levels of other genes known to modulate cellular cholesterol efflux mechanisms (ABCG1, ABCA7, Cla-1, apoE, LXRα, Retinoid X Receptor α, PPARα,γ) were not affected by IL-6 (Table 1). Those findings, therefore, suggest that stimulation of ABCA1-mediated cholesterol efflux to apoAI by IL-6 likely results from induction of ABCA1 gene expression.TABLE 1Gene expression profile in human macrophages upon stimulation with interleukin-6Human macrophagesTHP-1HMDM-Fold increase (IL-6 vs. control)p-Fold increase (IL-6 vs. control)pLipid homeostasisABCG11.2 ± 0.2NS1.1 ± 0.1NSABCA71.1 ± 0.3NS1.0 ± 0.2NSCla-11.0 ± 0.2NS1.1 ± 0.3NSApoE1.0 ± 0.1NS0.9 ± 0.1NSLPL0.9 ± 0.3NS1.0 ± 0.2NSCEH1.0 ± 0.2NS0.8 ± 0.1NSACAT-11.1 ± 0.3NS1.0 ± 0.0NSHMG-CoA receptor1.1 ± 0.2NS1.1 ± 0.1NSLDL receptor1.1 ± 0.3NS1.2 ± 0.1NSSR-A0.9 ± 0.2NS1.1 ± 0.2NSCD361.0 ± 0.2NS1.2 ± 0.1NSLRP1.0 ± 0.1NS0.9 ± 0.1NSGene regulationPPARα1.2 ± 0.1<0.051.0 ± 0.1NSPPARδ1.4 ± 0.2<0.051.9 ± 0.1<0.00005PPARγ1.2 ± 0.2NS0.9 ± 0.2NSRXRα1.0 ± 0.1NS1.0 ± 0.1NSLXRα0.9 ± 0.2NS0.9 ± 0.1NSBcl-32.3 ± 0.8<0.052.5 ± 0.5<0.05Cell survivalBcl-21.2 ± 0.2NS1.0 ± 0.2NSBcl-xL1.1 ± 0.1NS1.0 ± 0.1NSJak/Stat pathwayStat11.1 ± 0.1NS1.2 ± 0.1NSStat31.7 ± 0.2<0.00051.7 ± 0.2<0.0005SOCS33.1 ± 1.1<0.059.4 ± 1.4<0.0005Oxidative stressHO-11.1 ± 0.1NS0.9 ± 0.1NS Open table in a new tab As IL-6 has been described to exert its cellular action by activation of the Jak/Stat pathway (32Levy D.E. Lee C.K. J. Clin. Invest. 2002; 109: 1143-1148Crossref PubMed Scopus (758) Google Scholar), we analyzed the effect of the inhibition of the Jak/Stat signaling pathway on stimulation of cholesterol efflux to apoAI by IL-6 using specific inhibitors targeting either Jak-2 or Jak-3 or Jak-2/Stat3. As shown in Fig. 4, the inhibition of the Jak-2 protein-tyrosine kinase by a specific inhibitor (AG490) completely abolished the induction of cholesterol efflux to apoAI in response to IL-6 in both non-stimulated (Fig. 4A) and TO901317-stimulated human macrophages (Fig. 4B), whereas the inhibition of the Jak-3 protein-tyrosine kinase (ZM39923) was without e" @default.
• W1973622668 created "2016-06-24" @default.
• W1973622668 creator A5000346574 @default.
• W1973622668 creator A5005734283 @default.
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• W1973622668 date "2011-09-01" @default.
• W1973622668 modified "2023-09-30" @default.
• W1973622668 title "Interleukin-6 Protects Human Macrophages from Cellular Cholesterol Accumulation and Attenuates the Proinflammatory Response" @default.
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