Oxidized Low Density Lipoprotein Blocks Lipopolysaccharide-induced Interferon β Synthesis in Human Macrophages by Interfering with IRF3 Activation
2004; Elsevier BV; Volume: 279; Issue: 27 Linguagem: Inglês
10.1074/jbc.m313207200
ISSN1083-351X
AutoresAngie Marson, Richard M. Lawn, Thomas Mikita,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoIn response to lipopolysaccharide (LPS) exposure, macrophages activate the transcription of a large number of pro-inflammatory genes by way of signaling pathways downstream of the LPS receptor, Toll-Like Receptor 4. Many of these genes are expressed sequentially in time, with early synthesis events resulting in the secretion of soluble factors that drive the transcription of genes expressed later in the activation cycle. In this study we show that human blood-derived macrophages pretreated with oxidized low density lipoprotein (OxLDL) fail to transcribe and secrete interferon beta (IFNβ) immediately following LPS stimulation. As such, the normal downstream activation of Stat1 is blocked, and numerous IFNβ/Stat1-activated genes, including the chemokines IP10 and ITAC, are weakly expressed or not expressed at all in these cells. Inspection of the LPS-induced activation state of several transcription factors known to play a prominent role in IFNβ transcription reveals that, although NFκB, c-Jun, and ATF-2 activation appears normal, the LPS-induced activation of IFNβ regulatory factor 3 (IRF3), as measured by DNA-binding activity and association with the coactivator CBP, is inhibited in the OxLDL pre-treated cells. These IRF3 activities have been shown to be essential for the initiation of transcription of the IFNβ gene, and the loss of these activities presumably accounts for the lack of LPS-induced IFN β transcription seen in the OxLDL pre-treated cells. In response to lipopolysaccharide (LPS) exposure, macrophages activate the transcription of a large number of pro-inflammatory genes by way of signaling pathways downstream of the LPS receptor, Toll-Like Receptor 4. Many of these genes are expressed sequentially in time, with early synthesis events resulting in the secretion of soluble factors that drive the transcription of genes expressed later in the activation cycle. In this study we show that human blood-derived macrophages pretreated with oxidized low density lipoprotein (OxLDL) fail to transcribe and secrete interferon beta (IFNβ) immediately following LPS stimulation. As such, the normal downstream activation of Stat1 is blocked, and numerous IFNβ/Stat1-activated genes, including the chemokines IP10 and ITAC, are weakly expressed or not expressed at all in these cells. Inspection of the LPS-induced activation state of several transcription factors known to play a prominent role in IFNβ transcription reveals that, although NFκB, c-Jun, and ATF-2 activation appears normal, the LPS-induced activation of IFNβ regulatory factor 3 (IRF3), as measured by DNA-binding activity and association with the coactivator CBP, is inhibited in the OxLDL pre-treated cells. These IRF3 activities have been shown to be essential for the initiation of transcription of the IFNβ gene, and the loss of these activities presumably accounts for the lack of LPS-induced IFN β transcription seen in the OxLDL pre-treated cells. Numerous studies support the concept that the oxidation of LDL is a pro-atherogenic process that contributes to a succession of localized arterial wall changes involving several cell types (1Glass C. Witztum J.L. Cell. 2001; 104: 503-516Abstract Full Text Full Text PDF PubMed Scopus (2654) Google Scholar, 2Lusis A. Nature. 2000; 407: 233-241Crossref PubMed Scopus (4735) Google Scholar). Oxidized LDL (OxLDL) 1The abbreviations used are: OxLDL, oxidized low density lipoprotein; ATF, activating transcription factor; CBP, cAMP-responsive element-binding protein; EMSA, electrophoretic mobility shift assay; IFN, interferon; IRF, IFN regulatory factor; LPS, lipopolysaccharide; RT, reverse transcriptase; Stat, signal transducer and activator of transcription; IL, interleukin; TLR4, Toll-Like Receptor 4; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; JNK, c-Jun NH2-terminal kinase; RANTES, regulated on activation normal T cell expressed and secreted. has been shown to compromise endothelial cell function by triggering the secretion of chemokines and increasing the expression of leukocyte adhesion proteins (3Libby P. Nature. 2002; 420: 868-874Crossref PubMed Scopus (7017) Google Scholar). This facilitates the entry of monocytes, and later, other leukocytes into the arterial wall (1Glass C. Witztum J.L. Cell. 2001; 104: 503-516Abstract Full Text Full Text PDF PubMed Scopus (2654) Google Scholar, 4Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19370) Google Scholar). Soon after entry, monocytes differentiate into macrophages, which scavenge the OxLDL that has accumulated in the sub-endothelial spaces of the affected artery. As professional scavenger cells, macrophages internalize OxLDL along with resident cell debris. However, the process of scavenging OxLDL, which harbors numerous biologically active molecules, initiates many changes in cell signaling, gene expression, cellular appearance, and function (1Glass C. Witztum J.L. Cell. 2001; 104: 503-516Abstract Full Text Full Text PDF PubMed Scopus (2654) Google Scholar, 5Ricote M. Valledor A.F. Glass C.K. Arterioscler. Thromb. Vasc. Biol. 2003; 24: 230-239Crossref PubMed Scopus (142) Google Scholar, 6Brown M.S. Goldstein J.L. Annu. Rev. Biochem. 1983; 52: 223-261Crossref PubMed Google Scholar, 7Shiffman D. Mikita T. Tai J.T. Wade D.P. Porter G. Seilhamer J.J. Somogyi R. Liang S. Lawn R.M. J. Biol. Chem. 2000; 275: 37324-37332Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). These changes are believed to differ from macrophage responses to scavenging of normal cell debris (1Glass C. Witztum J.L. Cell. 2001; 104: 503-516Abstract Full Text Full Text PDF PubMed Scopus (2654) Google Scholar, 5Ricote M. Valledor A.F. Glass C.K. Arterioscler. Thromb. Vasc. Biol. 2003; 24: 230-239Crossref PubMed Scopus (142) Google Scholar, 8Fadok V.A. Bratton D.L. Konowal A. Freed P.W. Westcott J.Y. Henson P.M. J. Clin. Invest. 1998; 101: 890-898Crossref PubMed Scopus (2577) Google Scholar). Indeed, because the appearance of OxLDL-loaded macrophages is so altered, they are often referred to as foam cells (6Brown M.S. Goldstein J.L. Annu. Rev. Biochem. 1983; 52: 223-261Crossref PubMed Google Scholar). Macrophage foam cells are not the only leukocyte present in atherosclerotic lesions, but from the earliest “fatty streak” to the late stage lesion, they are often the most abundant (1Glass C. Witztum J.L. Cell. 2001; 104: 503-516Abstract Full Text Full Text PDF PubMed Scopus (2654) Google Scholar, 3Libby P. Nature. 2002; 420: 868-874Crossref PubMed Scopus (7017) Google Scholar). In addition, numerous animal studies have shown that specific gene expression changes associated with the foam cell phenotype are pro-atherogenic. Among these changes are the increased expression of the scavenger receptor CD36 (9Suzuki H. Kurihara Y. Takeya M. Kamada N. Kataoka M. Jishage K. Ueda O. Sakaguchi H. Higashi T. Suzuki T. Takashima Y. Kawabe Y. Cynshi O. Wada Y. Honda M. Kurihara H. Aburatani H. Doi T. Matsumoto A. Azuma S. Noda T. Toyoda Y. Itakura H. Yazaki Y. Kodama T. Nature. 1997; 386: 292-296Crossref PubMed Scopus (1014) Google Scholar, 10Febbraio M. Podrez E.A. Smith J.D. Hajjar D.P. Hazen S.L. Hoff H.F. Sharma K. Silverstein R.L. J. Clin. Invest. 2000; 105: 1049-1056Crossref PubMed Scopus (831) Google Scholar), the lipid-binding protein FABP4 (11Makowski L. Boord J.B. Maeda K. Babaev V.R. Uysal K.T. Morgan M.A. Parker R.A. Suttles J. Fazio S. Hotamisligil G.S. Linton M.F. Nat Med. 2001; 7: 699-705Crossref PubMed Scopus (576) Google Scholar), and the chemokine IL8 (12Ito T. Ikeda U. Curr. Drug Targets Inflamm. Allergy. 2003; 2: 257-265Crossref PubMed Scopus (130) Google Scholar, 13Gerszten R.E. Garcia-Zepeda E.A. Lim Y.C. Yoshida M. Ding H.A. Gimbrone Jr., M.A. Luster A.D. Luscinskas F.W. Rosenzweig A. Nature. 1999; 398: 718-723Crossref PubMed Scopus (1070) Google Scholar). Because macrophages are inflammatory cells, as well as scavenger cells, it is believed that they contribute in important ways to vascular inflammation levels, a critical determinant of atherosclerotic lesion instability (3Libby P. Nature. 2002; 420: 868-874Crossref PubMed Scopus (7017) Google Scholar, 4Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19370) Google Scholar, 14Hansson G.K. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1876-1890Crossref PubMed Scopus (734) Google Scholar). Activated macrophages are a major source of the soluble factors that coordinate the infiltration and local inflammatory response of numerous leukocytes in various tissue settings (15Abbas A.K. Lichtman A.H. Pober J.S. Cellular and Molecular Immunology. W. B. Saunders and Co., Philadelphia, PA2000: 270-290Google Scholar). Dysregulation or imbalances in these responses can lead to unresolved or chronic inflammatory states (16Nathan C. Nature. 2002; 420: 846-852Crossref PubMed Scopus (2051) Google Scholar). Indeed, late stage atherosclerotic lesions often have features associated with sites of chronic inflammation, such as enrichment of inflammatory cells, persistent cell damage, local tissue remodeling, and fibrosis (1Glass C. Witztum J.L. Cell. 2001; 104: 503-516Abstract Full Text Full Text PDF PubMed Scopus (2654) Google Scholar, 3Libby P. Nature. 2002; 420: 868-874Crossref PubMed Scopus (7017) Google Scholar). Furthermore, increases in circulating inflammatory factors like C-reactive protein and IL6 have been shown to be independent predictors of future cardiovascular disease (13Gerszten R.E. Garcia-Zepeda E.A. Lim Y.C. Yoshida M. Ding H.A. Gimbrone Jr., M.A. Luster A.D. Luscinskas F.W. Rosenzweig A. Nature. 1999; 398: 718-723Crossref PubMed Scopus (1070) Google Scholar,17Blake G.J. Ridker P.M. J. Intern. Med. 2002; 252: 283-294Crossref PubMed Scopus (573) Google Scholar). Other studies have implicated a role for infectious agents, like Chlamydia pneumoniae, in the pathogenesis of atherosclerosis (14Hansson G.K. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1876-1890Crossref PubMed Scopus (734) Google Scholar, 18Kalayoglu M.V. Libby P. Byrne G.I. JAMA. 2002; 288: 2724-2731Crossref PubMed Scopus (260) Google Scholar, 19Espinola-Klein C. Rupprecht H.J. Blankenberg S. Bickel C. Kopp H. Rippin G. Victor A. Hafner G. Schlumberger W. Meyer J. Athero. Gene Investigators Circulation. 2002; 105: 15-21Crossref PubMed Scopus (304) Google Scholar). In addition, several receptors of the innate immune system are overexpressed in human lesions (20Edfeldt K. Swedenborg J. Hansson G.K. Yan Z.Q. Circulation. 2002; 105: 1158-1161Crossref PubMed Scopus (750) Google Scholar). A polymorphism in one of these receptors, the LPS receptor, Toll-like Receptor 4 (TLR4), has been shown to be associated with a reduced risk of atherosclerosis in humans (21Kiechl S. Lorenz E. Reindl M. Wiedermann C.J. Oberhollenzer F. Bonora E. Willeit J. Schwartz D.A. N. Engl. J. Med. 2002; 347: 185-192Crossref PubMed Scopus (944) Google Scholar), presumably because of the reduced inflammatory signaling associated with this receptor variant. Consequently, recent studies have begun to focus on ways in which the inflammatory response of arterial macrophages and macrophage foam cells may exacerbate the development of vascular disease and thus represent an area for therapeutic intervention (12Ito T. Ikeda U. Curr. Drug Targets Inflamm. Allergy. 2003; 2: 257-265Crossref PubMed Scopus (130) Google Scholar, 22Cascieri M.A. Nat. Rev. Drug Discov. 2002; 1: 122-130Crossref PubMed Scopus (34) Google Scholar). In an earlier report, we described the results of a DNA microarray study that characterized the altered gene expression responses of OxLDL pre-treated THP1 macrophages to LPS stimulation (23Mikita T. Porter G. Lawn R.M. Shiffman D. J. Biol. Chem. 2001; 276: 45729-45739Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). This study revealed numerous early and late changes in the LPS-induced inflammatory cascade, often involving inappropriate cytokine and chemokine transcription levels. In particular, a group of late expressed genes, which included the chemokines IP10 and ITAC, were no longer seen to be induced by LPS treatment in these OxLDL pre-treated cells. In the current study we show that human blood-derived macrophages pre-treated with OxLDL fail to transcribe and secrete interferon β (IFNβ) immediately following LPS stimulation. As such, the downstream activation of Stat1 is blocked, and numerous IFNβ/Stat1-activated genes, like IP10 and ITAC, are weakly expressed or not expressed at all in these cells. OxLDL inhibits IFNβ transcription by apparently blocking the activation of the transcription factor IRF3, a step that has previously been shown to be essential for the initiation of transcription from the IFNβ promoter (24Suhara W. Yoneyama M. Kitabayashi I. Fujita T. J. Biol. Chem. 2002; 277: 22304-22313Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 25Yang H. Lin C.H. Ma G. Orr M. Baffi M.O. Wathelet M.G. Eur. J. Biochem. 2002; 269: 6142-6151Crossref PubMed Scopus (38) Google Scholar). Cell Culture—Human monocyte-derived macrophages were isolated as described previously (26Soutar A.K. Knight B.L. Biochem. J. 1982; 204: 549-556Crossref PubMed Scopus (22) Google Scholar). Briefly, buffy coats from individual blood donors were adjusted to 75 ml with PBS containing 1 mm disodium EDTA and layered onto Ficoll-Paque (Amersham Biosciences). Following centrifugation at room temperature for 30 min at 400 × g the layer of mononuclear cells was removed, diluted with three volumes of PBS/EDTA, and centrifuged at 800 × g for 10 min at 4 °C. The cells were washed three times with PBS/EDTA by centrifugation at 4 °C for 10 min at 100 × g and once with RPMI 1640 medium (Cellgro, Mediatech) without serum. The cells were then resuspended in RPMI 1640 medium without serum and plated at 5 × 106 cells per well in 6-well tissue culture trays or at 12 × 106 cells per T-25 flask. After 2 h, non-adherent cells were removed by washing with RPMI 1640, and the medium was replaced with RPMI 1640 containing 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. This medium was changed two to three times over the course of 12 days of growth and differentiation. On day 12 the medium was replaced again by medium with or without 100 μg/ml human oxidized LDL (prepared by CuSO4 oxidation of LDL; Intracel Corp., Rockville, MD), and the cells were allowed to grow for 3 more days. The cells were then treated with 0.5 μg/ml LPS to activate the macrophages. In some experiments, 3000 units/ml human IFNβ (BIOSOURCE International; units are those of supplier) was used to activate the macrophages. Depending on the experiment, RNA, supernatants, whole cell lysates, and/or nuclear extract samples were then harvested according to procedures listed below and at the times indicated. These samples were then assayed as described below for individual experiments. RNA Isolation and mRNA Quantitation—Total RNA extraction, cDNA synthesis, and real-time, quantitative RT-PCR were performed as previously described (23Mikita T. Porter G. Lawn R.M. Shiffman D. J. Biol. Chem. 2001; 276: 45729-45739Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). ELISA Measurements—Blood-derived macrophages were isolated and allowed to differentiate as described above. The cells were then pre-treated with or without OxLDL for an additional 3 days and then treated with LPS, as described. Culture media was collected at 0, 1, 3, 6, and 9 h following LPS stimulation. Supernatants were then assayed by ELISA for the presence of IFNβ, IP10, and ITAC using reagents and protocols of the supplier (BIOSOURCE International for IFNβ and IP10, and R&D Systems for ITAC). Western Blots and Co-immunoprecipitations—SDS Western blots were run following standard procedures. The blots were probed using the following antibodies. Stat1, Stat1-p(Tyr-701), c-Jun, c-Jun-p(Ser-73), ATF2, ATF2-p(Thr-71), p38, p-p38(Thr-180/Tyr-182), and p-NFκB p65 (Ser-536) antibodies were from Cell Signaling Technology. NFκB p50(H119) and NFκB p65(A) antibodies were from Santa Cruz Biotechnology. IRF3 antibodies were from Active Motif. Co-immunoprecipitations were done as follows. Blood-derived macrophages were isolated and allowed to differentiate as described above. The cells were then pre-treated with or without OxLDL for an additional 3 days and then treated with LPS, as described. The cells were harvested at 0, 45, and 120 min post LPS treatment, in an Nonidet P-40 lysis buffer (50 mm Tris/Cl, pH 8.0, 1% Nonidet P-40, 150 mm NaCl), supplemented with 1 mm phenylmethylsulfonyl fluoride, 100 μg/ml leupeptin, 1 mm sodium orthovanadate) and then vortexed to produce the whole cell extract. Insoluble material was removed by centrifugation (14,000 rpm, 10 min). 0.2–0.4 ml of lysate (containing ∼200–400 μg of protein) was mixed with 10 μl of IRF3 antibody and kept on ice for 30 min. Then 25 μl of protein G-agarose beads (Pierce) were added to the mix, which was then rotated for 1–2 h at 4 °C. The beads were then spun down and washed five times with 0.6 ml of Nonidet P-40 lysis buffer. The bead-bound protein was then extracted in SDS sample buffer and analyzed by standard SDS-PAGE. Following transfer, the blots were probed with CBP(A-22) antibodies (Santa Cruz Biotechnology). Nuclear Extracts and EMSAs—Nuclear extract preparation, generation of probes, and DNA binding conditions for gel shift mobility assays followed standard protocols that have been described previously (27Osborn L. Kunkel S. Nable G.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2336-2340Crossref PubMed Scopus (1373) Google Scholar). The following oligonucleotide and its complement were used to generate the NFκB-specific probe: 5′-AGTTGAGGGGACTTTCCCAGGC-3′. Pull-down Assay—We followed a procedure similar to that reported by Sgarbanti et al. (28Sgarbanti M. Borsetti A. Moscufo N. Bellocchi M.C. Ridolfi B. Nappi F. Marsili G. Marziali G. Coccia E.M. Ensoli B. Battistini A. J. Exp. Med. 2002; 195: 1359-1370Crossref PubMed Scopus (80) Google Scholar). Briefly, a biotinylated oligonucleotide corresponding to the human ISG15 ISRE (5′-GATCCATGCCTCGGGAAAGGGAAACCGAAACTGAAGCC-3′) was synthesized and annealed to its compliment in a standard annealing buffer (10 mm Tris-HCl, pH 7.5, 50 mm NaCl, 2 mm EDTA). 100 pmol of biotinylated DNA were then mixed with 500–1000 μg of nuclear extract (prepared as described above) in a standard binding buffer (20 mm Tris-HCl, pH 7.5, 75 mm KCl, 1 mm dithiothreitol, 20% glycerol, 10 μg/ml bovine serum albumin, and 0.3 μg/ml poly(dI-dC), and incubated at room temperature for 20 min. This DNA·protein mixture was then added to a 50-μl slurry of pre-washed streptavidin-agarose beads (Sigma-Aldrich) and allowed to mix by rotation for 40 min at 4 °C, followed by further mixing at room temperature for an additional 10 min. At this point, the DNA·protein·bead complex was spun down, the supernatant was removed, and the beads were washed four times in 400 μl of binding buffer. Protein was eluted from the beads into SDS sample buffer, separated on 7.5% SDS-PAGE, then immunoblotted with antibody against IRF3 (Active Motif). Human blood-derived macrophages were isolated and allowed to differentiate in culture for 12 days, following standard procedures for the establishment of blood-derived macrophage cultures (26Soutar A.K. Knight B.L. Biochem. J. 1982; 204: 549-556Crossref PubMed Scopus (22) Google Scholar). On day 12, the cells were treated with OxLDL for an additional 3 days, to lipid load the cells and establish a foam cell-like phenotype based on increased levels of oil red-o staining of neutral lipids, and the increased expression of several marker genes, including ABCA1, ABCG1, Adipophilin, and FABP4 (as determined by RT-PCR, data not shown). Control cells were maintained in culture for the same length of time with no OxLDL treatment. The cells were then treated with LPS, and supernatants and RNA were collected at various times. Fig. 1 shows the results of ELISA measurements that were made to determine the amount of IP10 and ITAC being secreted into the media by these cells. As the data in this figure show, the LPS-inducible secretion of both IP10 (Fig. 1A) and ITAC (Fig. 1B) is dramatically reduced in the OxLDL pretreated blood-derived macrophages. RT-PCR analysis of mRNA isolated from these same cells showed that inhibition of LPS-induced ITAC and IP10 production occurred at the level of transcription (data not shown). Results from other groups have shown that both of these genes can be induced in macrophages by a number of stimuli, including IFNβ (29Toshchakov V. Jones B.W. Perera P.Y. Thomas K. Cody M.J. Zhang S. Williams B.R. Major J. Hamilton T.A. Fenton M.J. Vogel S.N. Nat. Immunol. 2002; 3: 392-398Crossref PubMed Scopus (682) Google Scholar, 30Widney D.P. Xia Y.R. Lusis A.J. Smith J.B. J. Immunol. 2000; 164: 6322-6331Crossref PubMed Scopus (75) Google Scholar). Additional studies have shown that LPS treatment of macrophages immediately activates the transcription and secretion of IFNβ (29Toshchakov V. Jones B.W. Perera P.Y. Thomas K. Cody M.J. Zhang S. Williams B.R. Major J. Hamilton T.A. Fenton M.J. Vogel S.N. Nat. Immunol. 2002; 3: 392-398Crossref PubMed Scopus (682) Google Scholar, 31Doyle S. Vaidya S. O'Connell R. Dadgostar H. Dempsey P. Wu T. Rao G. Sun R. Haberland M. Modlin R. Cheng G. Immunity. 2002; 17: 251-263Abstract Full Text Full Text PDF PubMed Scopus (734) Google Scholar). Accordingly, we investigated whether defects in LPS-induced IFNβ production could account for the lack of IP10 and ITAC synthesis in the OxLDL pre-treated cells. As the RT-PCR data in Fig. 2A show, LPS induces a rapid, but transient, increase in IFNβ transcription in the control cells. In contrast, the OxLDL pre-treated cells show an almost total absence of LPS-induced IFN β transcription over the course of 9 h of observation. As the ELISA data in Fig. 2B show, LPS induces secretion of IFNβ in the control cells starting after 1 h. In contrast, very little IFNβ secretion is measured in the OxLDL pre-treated cells. These data show that LPS-induced IFNβ production is blocked in OxLDL pretreated cells and that the inhibition occurs at the level of transcription. The RT-PCR data in Fig. 2C are included to show that the LPS-induced transcription of another immediate early gene, TNFα, proceeds normally in the OxLDL pre-treated cells. Further support linking the early inhibition of IFNβ synthesis and secretion with the late inhibition of IP10 and ITAC synthesis was found in an additional set of experiments. Human blood-derived macrophages were isolated and allowed to differentiate in culture as before. The cells were then pretreated with OxLDL for 3 days, followed by treatment with IFNβ. Control cells were treated with IFNβ only. RNA was collected at various times and analyzed. As the RT-PCR data in Fig. 3 show, treatment with IFNβ stimulated an increase in IP10 transcription in the OxLDL pre-treated cells that approached that of the control cells. Similar results were obtained for IFNβ-induced ITAC expression (data not shown). These data strongly suggest that it is the absence of LPS-induced IFNβ production that is responsible for the lack of IP10 and ITAC expression seen in the OxLDL/LPS-treated cells. Because IFNβ is known to act in an autocrine/paracrine fashion to activate a second wave of transcription, largely through the activation of the transcription factor Stat1 (29Toshchakov V. Jones B.W. Perera P.Y. Thomas K. Cody M.J. Zhang S. Williams B.R. Major J. Hamilton T.A. Fenton M.J. Vogel S.N. Nat. Immunol. 2002; 3: 392-398Crossref PubMed Scopus (682) Google Scholar), we also checked the level of LPS-induced Stat1 activation in blood-derived macrophages. Cells were isolated, cultured, and treated with OxLDL and LPS as before. Cell lysates were prepared at various times following LPS treatment and analyzed on protein gels. As the data in Fig. 4 show, there is an equivalent level of Stat1 in both the control and OxLDL pretreated cells, but only in the LPS-treated control cells does the active form of Stat1 appear. These results are thus consistent with the levels of IFNβ production seen in both the control and OxLDL pre-treated cells. We also examined the expression levels of other IFNβ- and Stat1-activated genes identified from the literature. As before, macrophage cells were isolated, cultured, and treated with OxLDL for 3 days, followed by LPS treatment. Control cells were treated with LPS only. RNA was collected at various times and analyzed. As the RT-PCR data in Fig. 5 show, the LPS-induced expression levels of the chemokines, RANTES and MCP2; the tryptophan-catabolizing enzyme, indoleamine 2,3-dioxygenase; the antiviral protein, ISG54; and the IFN regulatory factors, IRF2 and IRF7b, were all found to be inhibited in the OxLDL pre-treated macrophages. Because these genes have all been shown to be either IFNβ- or Stat1-responsive (32Marie I. Durbin J.E. Levy D.E. EMBO J. 1998; 17: 6660-6669Crossref PubMed Google Scholar, 33Nakaya T. Sato M. Hata N. Asagiri M. Suemori H. Noguchi S. Tanaka N. Taniguchi T. Biochem. Biophys. Res. Commun. 2001; 283: 1150-1156Crossref PubMed Scopus (150) Google Scholar, 34Struyf S. Van Collie E. Paemen L. Put W. Lenaerts J.P. Proost P. Opdenakker G. Van Damme J. J. Leukocyte Biol. 1998; 63: 364-372Crossref PubMed Scopus (76) Google Scholar, 35Hida S. Ogasawara K. Sato K. Abe M. Takayanagi H. Yokochi T. Sato T. Hirose S. Shirai T. Taki S. Taniguchi T. Immunity. 2000; 13: 643-655Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 36Robinson C.M. Shirey K.A. Carlin J.M. J. Interferon Cytokine Res. 2003; 23: 413-421Crossref PubMed Scopus (109) Google Scholar, 37Cremer I. Ghysdael J. Vieillard V. FEBS Lett. 2002; 511: 41-45Crossref PubMed Scopus (22) Google Scholar), these data are consistent with the lack of IFNβ production and Stat 1 activation seen in the OxLDL/LPS-treated cells. The next question we addressed was how OxLDL pre-treatment interferes with LPS-induced IFNβ transcription in human blood-derived macrophages. Previous work by others has identified the binding sites for numerous transcription factors within the IFNβ promoter (38Agalioti T. Lomvardas S. Parekh B. Yie J. Maniatis T. Thanos D. Cell. 2000; 103: 667-678Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar, 39Falvo J.V. Parekh B.S. Lin C.H. Fraenkel E. Maniatis T. Mol. Cell. Biol. 2000; 20: 4814-4825Crossref PubMed Scopus (115) Google Scholar). Prominent among these factors are NFκB, c-Jun, ATF2, and IRF3 (40Wathelet M.G. Lin C.H. Parekh B.S. Ronco L.V. Howley P.M. Maniatis T. Mol. Cell. 1998; 1: 507-518Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar). We thus investigated the LPS-induced activation state of these four transcription factors in the control and OxLDL pre-treated cells. Cells were isolated, cultured, and treated with OxLDL and LPS as before. Nuclear extracts or whole cell lysates were prepared at various times following LPS stimulation. Transcription factor activity was assessed in these extracts and lysates as described below. The EMSA result in Fig 6A shows the LPS-induced NFκB DNA binding signal in both the control and OxLDL pre-treated cell nuclear extracts. NFκB is similarly induced by LPS in both the control and OxLDL pre-treated cells. The NFκB activity induced consists of both p65 and p50 subunits, because antibodies against both NFκB forms supershift the LPS-inducible bands (data not shown). The stimulus-induced phosphorylation of NFκB at serine 536 is a modification known to augment NFκB transactivation potential (41Yang F. Tang E. Guan K. Wang C.Y. J. Immunol. 2003; 170: 5630-5635Crossref PubMed Scopus (336) Google Scholar), but here too we observed no difference in LPS-induced phosphorylation intensity between the control and OxLDL pre-treated cells (data not shown). Fig. 6B shows a Western blot measuring the amount of phosphorylated c-Jun, the active form of the transcription factor, induced by LPS in both control and OxLDL pre-treated whole cell lysates. The active form of c-Jun is similarly induced by LPS in both control and OxLDL pre-treated cells, although there is some level of activation seen in the OxLDL pre-treated cells prior to LPS addition (lane 4). Finally, Fig. 6C shows a Western blot measuring the amount of phosphorylated ATF2, the active form of the transcription factor, induced by LPS in both the control and OxLDL pre-treated whole cell lysates. The active form of ATF2 is similarly induced by LPS in both control and OxLDL pre-treated cells. The LPS-induced phosphorylation state of P38, an upstream activating kinase of ATF2, was also found to be comparable in the control and OxLDL pretreated cells (data not shown). Thus, the LPS-induced activation of NFκB, c-Jun, and ATF2, three transcription factors known to be essential for the activation of IFNβ transcription (39Falvo J.V. Parekh B.S. Lin C.H. Fraenkel E. Maniatis T. Mol. Cell. Biol. 2000; 20: 4814-4825Crossref PubMed Scopus (115) Google Scholar, 40Wathelet M.G. Lin C.H. Parekh B.S. Ronco L.V. Howley P.M. Maniatis T. Mol. Cell. 1998; 1: 507-518Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar), were found to be comparable in both the control and OxLDL pre-treated cells. These figures were representative of data obtained from similarly treated macrophages isolated from several different blood donors. We next measured the LPS-inducible DNA-binding activity of IRF3 in a standard pull-down assay. In this experiment, nuclear extracts are combined with a biotinylated DNA duplex that contains an IRF3 binding site. After a period of incubation, any DNA·protein complexes that form are pulled down with streptavidin-coated agarose beads via centrifugation. Bound protein is identified by SDS-PAGE, followed by immunoblotting with antibodies for IRF3. Th
Referência(s)