Induction of monocyte differentiation and foam cell formation in vitro by 7-ketocholesterol
2002; Elsevier BV; Volume: 43; Issue: 1 Linguagem: Inglês
10.1016/s0022-2275(20)30183-8
ISSN1539-7262
AutoresJohn M. Hayden, Libuse Brachova, Karen M. Higgins, Lewis Obermiller, Alex Sevanian, Srikrishna Khandrika, Peter D. Reaven,
Tópico(s)Immune Cell Function and Interaction
ResumoOxidized LDL (OxLDL) is composed of many potentially proatherogenic molecules, including oxysterols. Of the oxysterols, 7-ketocholesterol (7-KC) is found in relatively large abundance in OxLDL, as well as in atherosclerotic plaque and foam cells in vivo. Although there is evidence that 7-KC activates endothelial cells, its effect on monocytes is unknown. We tested the hypothesis that 7-KC may induce monocyte differentiation and promote foam cell formation. THP-1 cells were used as a monocyte model system and were treated with 7-KC over a range of concentrations from 0.5 to 10 μg/ml. Changes in cell adhesion properties, cell morphology, and expression of antigens characteristic of differentiated macrophages were monitored over a 7-day period. 7-KC promoted cells to firmly adhere and display morphologic features of differentiated macrophages; this effect was time and dose dependent and was markedly more potent than cholesterol treatment (45% of cells became adherent after 7 days of treatment with 7-KC at 10 μg/ml vs. less then 5% for control cells, P < 0.01). Similar effects were obtained when LDL enriched with 7-KC or OxLDL were added to THP-1 cells. 7-KC-differentiated cells expressed CD11b, CD36, and CD68, phagocytized latex beads, and formed lipid-laden foam cells after exposure to acetylated LDL or OxLDL. In contrast to 7-KC, oxysterols with known cell regulatory effects such as 25-hydroxycholesterol, 7β-hydroxycholesterol, and (22R)-hydroxycholesterol did not effectively promote THP-1 differentiation. In conclusion, these results demonstrate for the first time that 7-KC, a prominent oxysterol formed in OxLDL by peroxidation of cholesterol, may play an important role in promoting monocyte differentiation and foam cell formation. These studies also suggest that 7-KC induces monocyte differentiation through a sterol-mediated regulatory pathway that remains to be characterized. —Hayden, J. M., L. Brachova, K. Higgins, L. Obermiller, A. Sevanian, S. Khandrika, and P. D. Reaven. Induction of monocyte differentiation and foam cell formation in vitro by 7-ketocholesterol. J. Lipid Res. 2002. 43: 26–35. Oxidized LDL (OxLDL) is composed of many potentially proatherogenic molecules, including oxysterols. Of the oxysterols, 7-ketocholesterol (7-KC) is found in relatively large abundance in OxLDL, as well as in atherosclerotic plaque and foam cells in vivo. Although there is evidence that 7-KC activates endothelial cells, its effect on monocytes is unknown. We tested the hypothesis that 7-KC may induce monocyte differentiation and promote foam cell formation. THP-1 cells were used as a monocyte model system and were treated with 7-KC over a range of concentrations from 0.5 to 10 μg/ml. Changes in cell adhesion properties, cell morphology, and expression of antigens characteristic of differentiated macrophages were monitored over a 7-day period. 7-KC promoted cells to firmly adhere and display morphologic features of differentiated macrophages; this effect was time and dose dependent and was markedly more potent than cholesterol treatment (45% of cells became adherent after 7 days of treatment with 7-KC at 10 μg/ml vs. less then 5% for control cells, P < 0.01). Similar effects were obtained when LDL enriched with 7-KC or OxLDL were added to THP-1 cells. 7-KC-differentiated cells expressed CD11b, CD36, and CD68, phagocytized latex beads, and formed lipid-laden foam cells after exposure to acetylated LDL or OxLDL. In contrast to 7-KC, oxysterols with known cell regulatory effects such as 25-hydroxycholesterol, 7β-hydroxycholesterol, and (22R)-hydroxycholesterol did not effectively promote THP-1 differentiation. In conclusion, these results demonstrate for the first time that 7-KC, a prominent oxysterol formed in OxLDL by peroxidation of cholesterol, may play an important role in promoting monocyte differentiation and foam cell formation. These studies also suggest that 7-KC induces monocyte differentiation through a sterol-mediated regulatory pathway that remains to be characterized. —Hayden, J. M., L. Brachova, K. Higgins, L. Obermiller, A. Sevanian, S. Khandrika, and P. D. Reaven. Induction of monocyte differentiation and foam cell formation in vitro by 7-ketocholesterol. J. Lipid Res. 2002. 43: 26–35. It is now widely recognized that monocytes play an integral role in the development and progression of atherosclerosis (1Ross R. Atherosclerosis is an inflammatory disease.Am. Heart J. 1999; 138: S419-S420Google Scholar, 2Navab M. Berliner J.A. Watson A.D. Hama S.Y. Territo M.C. Lusis A.J. Shih D.M. Van Lenten B.J. Frank J.S. Demer L.L. Edwards P.A. Fogelman A.M. The Yin and Yang of oxidation in the development of the fatty streak. A review based on the 1994 George Lyman Duff Memorial Lecture.Arterioscler. Thromb. Vasc. Biol. 1996; 16: 831-842Google Scholar). Monocytes are among the first cells present in lesion-prone areas, and they continue to accumulate during plaque formation. On exposure to a variety of regulatory signals, monocytes rapidly differentiate into tissue macrophages in the vascular intima. When activated, monocyte-macrophages may secrete multiple cytokines and free radicals that promote inflammation and atherosclerosis (1Ross R. Atherosclerosis is an inflammatory disease.Am. Heart J. 1999; 138: S419-S420Google Scholar, 3Henson P.M. Riches D.W. Modulation of macrophage maturation by cytokines and lipid mediators: a potential role in resolution of pulmonary inflammation.Ann. N.Y. Acad. Sci. 1994; 725: 298-308Google Scholar, 4Liu Y. Hulten L.M. Wiklund O. Macrophages isolated from human atherosclerotic plaques produce IL-8, and oxysterols may have a regulatory function for IL-8 production.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 317-323Google Scholar). Macrophages also express multiple scavenger receptors that facilitate internalization of modified lipoproteins, leading to the development of cholesterol-laden foam cells and plaque formation in arteries (5Edwards P.A. Ericsson J. Signaling molecules derived from the cholesterol biosynthetic pathway: mechanisms of action and possible roles in human disease.Curr. Opin. Lipidol. 1998; 9: 433-440Google Scholar, 6Yamada Y. Doi T. Hamakubo T. Kodama T. Scavenger receptor family proteins: roles for atherosclerosis, host defence and disorders of the central nervous system.Cell. Mol. Life Sci. 1998; 54: 628-640Google Scholar, 7Steinbrecher U.P. Receptors for oxidized low density lipoprotein.Biochim. Biophys. Acta. 1999; 1436: 279-298Google Scholar). The primary role of monocyte-macrophages in atherogenesis has been confirmed in gene knockout mice lacking monocyte chemoattractant protein or its receptor, CCR2. In these respective mouse models it was demonstrated that aortic atherosclerosis was significantly reduced in comparison with wild-type controls under experimental conditions that predispose to atherosclerosis (8Gosling J. Slaymaker S. Gu L. Tseng S. Zlot C.H. Young S.G. Rollins B.J. Charo I.F. MCP-1 deficiency reduces susceptibility to atherosclerosis in mice that overexpress human apolipoprotein B.J. Clin. Invest. 1999; 103: 773-778Google Scholar, 9Boring L. Gosling J. Cleary M. Charo I.F. Decreased lesion formation in CCR2−;/− mice reveals a role for chemokines in the initiation of atherosclerosis.Nature. 1998; 394: 894-897Google Scholar). Although the consequences of macrophage accumulation in lesion-susceptible regions of the artery wall are now evident, the regulatory signals that induce monocyte differentiation in this environment are less completely understood. Although it has been demonstrated that oxidized low density lipoprotein (OxLDL) can induce the differentiation of monocytes (10Frostegard J. Nilsson J. Haegerstrand A. Hamsten A. Wigzell H. Gidlund M. Oxidized low density lipoprotein induces differentiation and adhesion of human monocytes and the monocytic cell line U937.Proc. Natl. Acad. Sci. USA. 1990; 87: 904-908Google Scholar), the components of OxLDL that actually promote this process are not fully elucidated. Initial studies have suggested that oxidized phospholipids are remarkably proinflammatory, and at least one report has implicated specific oxidized phospholipids in the regulation of monocyte differentiation (11Rajavashisth T.B. Andalibi A. Territo M.C. Berliner J.A. Navab M. Fogelman A.M. Lusis A.J. Induction of endothelial cell expression of granulocyte and macrophage colony-stimulating factors by modified low-density lipoproteins.Nature. 1990; 344: 254-257Google Scholar). Other components of OxLDL, such as oxysterols, have also been shown to promote a variety of potentially proatherogenic events. Of the oxysterols, 7-ketocholesterol (7-KC) has been found in relatively high concentrations in OxLDL produced in vitro (12Brown A.J. Leong S.L. Dean R.T. Jessup W. 7-Hydroperoxycholesterol and its products in oxidized low density lipoprotein and human atherosclerotic plaque.J. Lipid Res. 1997; 38: 1730-1745Google Scholar, 13Chang Y.H. Abdalla D.S. Sevanian A. Characterization of cholesterol oxidation products formed by oxidative modification of low density lipoprotein.Free Radic. Biol. Med. 1997; 23: 202-214Google Scholar, 14Patel R.P. Diczfalusy U. Dzeletovic S. Wilson M.T. Darley-Usmar V.M. Formation of oxysterols during oxidation of low density lipoprotein by peroxynitrite, myoglobin, and copper.J. Lipid Res. 1996; 37: 2361-2371Google Scholar) and is enriched in arterial foam cells and atherosclerotic plaque in vivo (15Hulten L.M. Lindmark H. Diczfalusy U. Bjorkhem I. Ottosson M. Liu Y. Bondjers G. Wiklund O. Oxysterols present in atherosclerotic tissue decrease the expression of lipoprotein lipase messenger RNA in human monocyte-derived macrophages.J. Clin. Invest. 1996; 97: 461-468Google Scholar, 16Brown A.J. Jessup W. Oxysterols and atherosclerosis.Atherosclerosis. 1999; 142: 1-28Google Scholar). Furthermore, 7-KC induces apoptosis in vascular cells (17Lizard G. Moisant M. Cordelet C. Monier S. Gambert P. Lagrost L. Induction of similar features of apoptosis in human and bovine vascular endothelial cells treated by 7-ketocholesterol.J. Pathol. 1997; 183: 330-338Google Scholar, 18Miyashita Y. Shirai K. Ito Y. Watanabe J. Urano Y. Murano T. Tomioka H. Cytotoxicity of some oxysterols on human vascular smooth muscle cells was mediated by apoptosis.J. Atheroscler. Thromb. 1997; 4: 73-78Google Scholar), stimulates adhesion molecule expression in endothelial cells (19Lemaire S. Lizard G. Monier S. Miguet C. Gueldry S. Volot F. Gambert P. Neel D. Different patterns of IL-1beta secretion, adhesion molecule expression and apoptosis induction in human endothelial cells treated with 7alpha-, 7beta-hydroxycholesterol, or 7-ketocholesterol.FEBS Lett. 1998; 440: 434-439Google Scholar), and inhibits cholesterol efflux in macrophages (20Gelissen I.C. Brown A.J. Mander E.L. Kritharides L. Dean R.T. Jessup W. Sterol efflux is impaired from macrophage foam cells selectively enriched with 7-ketocholesterol.J. Biol. Chem. 1996; 271: 17852-17860Google Scholar). At present, the effects of oxysterols on monocyte-macrophage differentiation have not been examined. Herein we demonstrate for the first time that 7-KC promotes differentiation of THP-1 cells and primary human monocytes, and enhances foam cell formation in vitro. THP-1 cells (American Type Tissue Culture Collection, Rockville, MD) were grown in RPMI 1640 containing 10% heat-inactivated FBS (Irvine Scientific, CA), 10 mM HEPES, 1 mM sodium pyruvate, sodium bicarbonate (1.5 g/l), 2 mM l-glutamine, 5 × 10−5 M 2-mercaptoethanol, penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37°C in 5% CO2. In each experiment, cells were plated at a density of approximately 2.0 × 105 cells/ml. Initial experimentation demonstrated that THP-1 cells were more predisposed to 7-KC-mediated differentiation after pretreatment with serum-free medium (containing 0.1% BSA); therefore, in all experiments described in this study THP-1 cells were treated with serum-free RPMI 1640 medium containing 0.1% BSA for 24 h before exposure to oxysterols. After pretreatment with serum-free medium, heat-inactivated FBS was added to a final concentration of 10% in the medium and oxysterols were added (0.5 to 10 μg/ml) to the cells for various periods (24 h to 7 days). All stock solutions of oxysterols {5-cholesten-3β-ol-7-one (7-KC); 5-cholestene-3β,7β-diol (7β-hydroxycholesterol); 5-cholestene-3β,22[R]-diol [22(R)-hydroxycholesterol], and 5-cholestene-3β,25-diol (25-hydroxycholesterol); Sigma, St. Louis, MO} were prepared in absolute ethanol. The majority of experiments were performed with ethanol at a concentration ≤0.1% (v/v). At the highest 7-KC concentration used in the dose-response experiments ethanol concentrations were ≤0.5% in culture. At this dose, ethanol did not affect any parameter of differentiation that was examined in the study. Ethanol alone or cholesterol (Sigma) dissolved in ethanol served as negative controls in all experiments. In addition, phorbol 12-myristate 13-acetate (PMA, 0.01 μg/ml) was added to THP-1 cells to serve as a positive control for differentiation. In addition, we confirmed that oxysterol stimulation of THP-1 cells did not result from potential endotoxin contamination (Limulus amebocyte lysate assay; Charles River Laboratories, Charleston, SC) in 7-KC stock solutions or cell culture reagents. Primary human monocytes were isolated from sodium EDTA-treated blood that was obtained from healthy volunteers. The blood was diluted 1:1 with Dulbecco's PBS (D-PBS) and layered on Histopaque-1077 (Sigma) and centrifuged at 400 g for 30 min at room temperature. The resulting monocyte-enriched layer was collected, washed with D-PBS, and plated in medium (THP-1 growth medium minus 2-mercaptoethanol) that was supplemented with 10% autologous serum. After 2 h the nonadherent cells were removed by gentle washing with D-PBS and the remaining adherent monocyte-enriched cells were provided complete growth medium for 2 days. Monocytes were then exposed to ethanol alone or to 7-KC (5 and 20 μg/ml) for 7 days before subsequent analyses. Under normal culture conditions THP-1 cells remain in suspension; however, after treatment with 7-KC a subpopulation of cells becomes adherent within 3 to 7 days. During this period, morphologic changes of the adherent cells were assessed by phase-contrast microscopy (×20 or ×40 objective). In addition, adherent cells were released from the plates by treatment with trypsin and directly counted with a hemacytometer (Hausser Scientific, Horsham, PA) or a particle counter (model Z1 D/T; Beckman Coulter, Miami, FL). In additional experiments relative cell adherence was also assessed by the quantification of total cellular protein concentration (MicroBCA; Pierce, Rockford, IL). Control cells and THP-1 cells that remained in suspension (undifferentiated cells) after 7-KC exposure were applied to slides by centrifugation (Cytospin-2; Shandon, Pittsburgh, PA) at 500 rpm for 5 min at room temperature. Cells that were applied to the slides, and cells that were induced to adhere to plastic by 7-KC, were fixed by immersion in ice-cold methanol for 10 min at −20°C. After fixation, the cells were washed several times with PBS and endogenous peroxidase activity was inhibited by incubation with 0.3% hydrogen peroxide diluted in 50% methanol-PBS 1:1 (v/v) for 5 min. The cells were then pretreated with either normal mouse serum (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) or 3% BSA (Sigma) for 1 h before incubation with primary monoclonal antibodies [CD11b and CD68, 1:50 and 1:100 dilution, respectively (Dako, Carpinteria, CA), and CD36, 1:50 dilution (Novocastra Laboratories, Ltd., Burlingame, CA)] for 2 h at room temperature. After additional washing, bound primary antibodies were detected by incubation with a diluted (1:50) biotinylated anti-mouse immunoglobulin antibody (Vector Laboratories), followed by diaminobenzidine (DAB) treatment as per standard ABC/DAB immunohistochemistry methodology (Vectastain ABC kit). To exclude the possibility of nonspecific binding of primary antibodies, additional cells were treated with a nonspecific monoclonal antibody (von Willebrand factor, diluted 1:50; Sigma) or with PBS (no primary antibody addition) before detection. To assess whether oxysterol-treated adherent THP-1 cells demonstrate the typical phagocytic activity of macrophages, latex beads (averaging 0.8 μm in diameter; Sigma) were added to cells for 1 h at 37°C and 5% CO2. The cells were then rinsed several times with D-PBS and internalized latex beads were examined by phase-contrast microscopy (×40 objective). To evaluate the potential cytotoxic effect of 7-KC on THP-1 cells, lactate dehydrogenase (LDH) released in the supernatant was determined (CytoTox 96; Promega, Madison, WI) from nonadherent cells and adherent cells treated up to 7 days with 7-KC. The percentage of cellular LDH release was calculated as the portion of LDH detected in the conditioned medium divided by total LDH from conditioned medium plus LDH from the cell lysate. In additional experiments, trypan blue (0.4% solution) staining was used to document the viability of suspension and adherent THP-1 cells after exposure to 7-KC. All cells (stained and unstained) were counted as described above. LDL (1.021–1.063 g/ml) was isolated from plasma provided by healthy volunteers by sequential gradient ultracentrifugation as described previously (21Reaven P.D. Grasse B.J. Tribble D.L. Effects of linoleate-enriched and oleate-enriched diets in combination with alpha-tocopherol on the susceptibility of LDL and LDL subfractions to oxidative modification in humans.Arterioscler. Thromb. 1994; 14: 557-566Google Scholar). 7-KC-loaded or vehicle (ethanol)-treated LDL were also prepared from plasma that was first incubated at 37°C for 4 h with a small volume of 7-KC (2.4 mM final concentration of 7-KC) or ethanol (same volume as used for 7-KC) (20Gelissen I.C. Brown A.J. Mander E.L. Kritharides L. Dean R.T. Jessup W. Sterol efflux is impaired from macrophage foam cells selectively enriched with 7-ketocholesterol.J. Biol. Chem. 1996; 271: 17852-17860Google Scholar). The amount of 7-KC in LDL was determined by gas chromatography as described previously (13Chang Y.H. Abdalla D.S. Sevanian A. Characterization of cholesterol oxidation products formed by oxidative modification of low density lipoprotein.Free Radic. Biol. Med. 1997; 23: 202-214Google Scholar). After isolation, all LDL were dialyzed against PBS containing 75 μM EDTA, pH 7.4, and stored in the dark at 4°C until use. Before use, LDL was further dialyzed against D-PBS alone. Acetylated LDL (AcLDL) was prepared by the repetitive addition of acetic anhydride to LDL (22Goldstein J.L. Ho Y.K. Basu S.K. Brown M.S. Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition.Proc. Natl. Acad. Sci. USA. 1979; 76: 333-337Google Scholar) and OxLDL was prepared by treatment with CuSO4 at 37°C for 24 h as described previously (21Reaven P.D. Grasse B.J. Tribble D.L. Effects of linoleate-enriched and oleate-enriched diets in combination with alpha-tocopherol on the susceptibility of LDL and LDL subfractions to oxidative modification in humans.Arterioscler. Thromb. 1994; 14: 557-566Google Scholar). The extent of LDL oxidation was assessed by thiobarbituric acid-reactive substance (TBARS) formation (23Yagi K. A simple fluorometric assay for lipoperoxide in blood plasma.Biochem. Med. 1976; 15: 212-216Google Scholar). In addition, oxidation and acetylation of LDL samples were also confirmed by comparison with native LDL by electrophoretic mobility (1% agarose gels; Helena Laboratories, Beaumont, TX). In these assays LDL bands were visualized by staining with Fat Red 7B (Sigma). Differentiated THP-1 cells (after 7 days of 7-KC treatment at 5 μg/ml) were treated with trypsin and replated in 24-well plates at a density of 2.5 × 105 cells/well for 24 h. The readherent cells were then incubated with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI)-labeled AcLDL (5 μg of protein per ml; Biomedical Technologies, Stoughton, MA) with or without excess unlabeled competitors [AcLDL or native LDL (nLDL), 10–200 μg of protein per ml, for 4 h at 37°C and 5% CO2]. The cells were then washed with D-PBS and fluorescence was measured at 520 nm (excitation) and 575 nm (emission) with a Victor2 multilabel counter (EG&G Wallac, Turku, Finland) at 37°C. The specific fluorescence intensity resulting from each treatment was corrected by subtracting autofluorescence intensity obtained from nontreated THP-1 cells. To assess the effect of 7-KC exposure on foam cell formation, THP-1 cells were treated with 7-KC (5 μg/ml) for 7 days. At this time, the nonadherent cells were removed, and the adherent cells remaining were washed and growth medium was replenished with AcLDL or OxLDL (50 μg of protein per ml) for 24 h. The cells were then washed with D-PBS, fixed with 4% paraformaldehyde, and stained with oil red O (Sigma). Stained neutral intracellular lipids were then examined by light microscopy (×40 objective). Whole cell lysates were collected 7 days after treatment with 7-KC, PMA, or vehicle as described above. The cells were lysed in PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, phenylmethylsulfonyl fluoride (100 μg/ml), aprotinin (30 μl/ml; Sigma, A6279) and 1 mM sodium orthovanadate. The protein content of each sample was determined by the bicinchoninic acid method (BCA; Pierce) before storage at −70°C. Protein samples (50 to 100 μg/lane) were separated by electrophoresis (4 to 20% acrylamide gradient gels; Invitrogen/Novex, Carlsbad, CA), and transferred to nitrocellulose (Hybond ECL; Amersham Pharmacia Biotech, Piscataway, NJ) by standard electroblotting procedures with use of a buffer containing 0.25 M Tris, 1.92 M glycine, 0.04% sodium dodecyl sulfate, and 20% methanol. The blots were pretreated with a solution containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 5% nonfat dried milk for at least 1 h before the addition of the primary antibodies (dilution range varying from 1:500 to 1:1000) for CD68 (Dako), or CD36 (Novocastra Laboratories, Ltd.). After incubation overnight, the primary antibodies were removed and appropriate horseradish peroxidase-conjugated secondary antibodies (Boehringer Mannheim, Indianapolis, IN) were added in a dilution range of 1:2500 to 1:5000 for 1 h at room temperature. Proteins were detected by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech) and by exposure to X-ray film (Hyper ECL; Amersham Pharmacia Biotech) for various times. Quantification of the protein bands was then accomplished by densitometry (ChemiImager 4400; Alpha Innotech, San Leandro, CA). THP-1 and primary monocytes were treated with 7-KC and PMA for 7 days as described above. Total RNA was extracted from the cells by the TRIzol method (GIBCO-BRL, Grand Island, NY). After acid-phenol-chloroform extraction and isopropyl alcohol precipitation, the resulting RNA pellet was washed with ice-cold 80% ethanol and redissolved in nuclease-free water. The quantity of RNA was then determined by absorbance at 260 nm before storage at −70°C. The effect of 7-KC on CD68, CD36, and macrophage scavenger receptor class A-1 (SRA-1) mRNA abundance was determined by reverse transcription-polymerase chain reaction procedures (Access RT-PCR system; Promega Corp., Madison WI). Briefly, this system supports synthesis and amplification of cDNA products in a single tube by a one-step reaction that provides a buffer system allowing for optimal activity of combined avian myeloblastosis virus reverse transcriptase and Thermus flavus (Tfl) DNA polymerase. Total RNA (0.1 to 1.0 μg) was added in the system along with forward and reverse primers for each of the following genes: CD68, 5′-CGTCACAGTTCATCCAACAAGC-3′ and 5′-TTGGGGTTCAGTACAGAGATGC-3′ (332-bp product); CD36, 5′-GCTGAGGACAACACAGTCTC-3′ and 5′-GCTGATGTCTAGCACACCAT-3′ (674-bp product); and SRA-1, 5′-TCTCATTGGAATAGTGGCAGC-3′ and 5′-TATTGGACCTGGAAATCCTCG-3′ (699-bp product). For each set of analyses, genomic β-actin transcripts (331-bp product) were also amplified and examined as an internal control (24Hayden J.M. Strong D.D. Baylink D.J. Powell D.R. Sampath T.K. Mohan S. Osteogenic protein-1 stimulates production of insulin-like growth factor binding protein-3 nuclear transcripts in human osteosarcoma cells.Endocrinology. 1997; 138: 4240-4247Google Scholar). Reverse transcription was per formed at 48°C for 45 min, and the cDNA amplification program consisted of an initial denaturation step (94°C for 2 min) followed by 35 amplification cycles of denaturation (94°C) for 30 s, annealing (59°C for CD36 and 63°C for CD68 and SRA-1) for 1 min, and extension (68°C) for 7 min. Each amplification reaction was analyzed by electrophoresis (10 μl/lane) in gels containing 1.5% agarose and stained with ethidium bromide. Analysis of variance was conducted to examine whether significant (P < 0.05) main treatment and time effects occurred. Additional post hoc comparisons of treatment means were conducted by using the Dunnett's t-test (treatments vs. controls) and Bonferroni t-test (selected comparisons) as indicated. Data given represent means ± standard deviation. Initial studies examining the effect of 7-KC on THP-1 cell differentiation demonstrated that these cells, originally in suspension, began to aggregate in clusters of 7 to 15 cells after 24 h of 7-KC treatment. With longer duration of exposure to this oxysterol (~3 days), a population of the cells began to adhere. Within 7 days of culture, the adherent THP-1 cells displayed hypertrophy, developed vacuoles in the cytoplasm, and formed extended processes; all characteristics of mature macrophages (Fig. 1B). Incontrast, THP-1 cells treated with vehicle alone (Fig. 1A) and cells treated with cholesterol (results not shown) did not develop these aforementioned morphological changes. We also directly counted the adherent THP-1 cells resulting from exposure to 7-KC (range, 0.5 to 10 μg/ml) for 7 days. The number and percentage of adherent cells increased in proportion to increasing concentration and time of exposure to 7-KC (ANOVA, P < 0.0001; linear effect, P < 0.001). 7-KC at 10 μg/ml induced nearly 45% of the initial cell population to become adherent (Fig. 2). To more accurately assess the effects of low doses of 7-KC on THP-1 cell adherence, total protein in cell lysates taken from each well was determined. The results from this procedure showed that total protein content of adherentcells also increased after 7-KC treatment in a dose-dependent manner (P < 0.001; data not shown). In addition, a strong correlation between cell number and protein concentration of adherent cells (r = 0.98; P < 0.001) was present in these experiments, indicating that the increase in adherent cell protein was not due to an increase in cellular hypertrophy, but actually resulted from an increase in cell number. On the basis of these results, the amount of adherent cell protein was used in the majority of subsequent experiments to assess effects of 7-KC treatment on cell adherence. Similar experiments were performed with LDL that was isolated from plasma exposed to vehicle only or 7-KC before isolation. Vehicle-treated LDL (TBARS = 6 nmol/mg protein, 7-KC concentration = 15.5 nmol/mg protein) and LDL loaded with 7-KC (TBARS = 4 nmol/mg protein, 7-KC concentration = 283 nmol/mg protein) were added to THP-1 cells in a concentration that ranged between 12.5 and 50 μg of LDL protein per ml for 7 days. Whereas vehicle-treated LDL had little effect on THP-1 cell adherence, LDL that was enriched with 7-KC induced marked changes in cell adherence and morphology that are characteristic of differentiation. These effects occurred in a dose-related fashion (data not shown) and maximal cell adherence for the 7-KC-loaded LDL treatment occurred at 50 μg of protein per ml (Fig. 3). In addition, the effect on cell adherence produced by 7-KC-enriched LDL matched those induced by 7-KC that was solubilized in ethanol. In separate experiments, OxLDL (TBARS = 80 to 100 nmol/mg protein) also induced cell adherence; however, this effect appeared slightly less than that of 7-KC treatment alone. Interestingly, the effect of 7-KC-loaded LDL on THP-1 cell adherence occurred between days 3 and 7, a time frame that is comparable to induction of monocyte differentiation by OxLDL or 7-KC in solution. Because previous reports have shown that oxysterols may produce cytotoxicity in other cell types, we examinedwhether the dose range of 7-KC used in the present study may induce THP-1 cell death. Experiments examining the extent of trypan blue staining in THP-1 cells remaining in suspension after 7-KC treatment demonstrated that concentrations of 7-KC ranging from 0.5 to 5.0 μg/ml did not induce significant cell death when compared with control cells. In contrast, exposure of THP-1 cells to 7-KC at 10 μg/ml for 7 days significantly increased the number of trypan blue-stained nonadherent cells (~34% nonviable cells vs. 2.4% for controls, P < 0.05). In contrast to the THP-1 cells remaining in suspension, adherent cells did not stain positive for trypan blue even when this population of cells was exposed to levels of 7-KC up to 25 μg/ml. The release of LDH from THP-1 cells exposed to 7-KC followed a similar trend as trypan blue staining. Only the 10 μg/ml dose of 7-KC significantly increased membrane leakage and release of LDH in the total population of THP-1 cells after 7 days of treatment (3.9-fold increase in LDH vs. nontreated controls, P < 0.01). In contrast, in a separate set of experiments, LDH release was not induced by 7-KC treatment (up to 25 μg/ml) in the adherent population of THP-1 cells. On the basis of these data, the concentration of 7-KC used in subsequent cell
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