Class B Scavenger Receptors CD36 and SR-BI Are Receptors for Hypochlorite-modified Low Density Lipoprotein
2003; Elsevier BV; Volume: 278; Issue: 48 Linguagem: Inglês
10.1074/jbc.m308428200
ISSN1083-351X
AutoresGunther Marsche, Robert Zimmermann, Seikoh Horiuchi, Narendra N. Tandon, Wolfgang Sattler, Ernst Malle,
Tópico(s)Vitamin K Research Studies
ResumoThe presence of HOCl-modified epitopes inside and outside monocytes/macrophages and the presence of HOCl-modified apolipoprotein B in atherosclerotic lesions has initiated the present study to identify scavenger receptors that bind and internalize HOCl-low density lipoprotein (LDL). The uptake of HOCl-LDL by THP-1 macrophages was not saturable and led to cholesterol/cholesteryl ester accumulation. HOCl-LDL is not aggregated in culture medium, as measured by dynamic light scattering experiments, but internalization of HOCl-LDL could be inhibited in part by cytochalasin D, a microfilament disrupting agent. This indicates that HOCl-LDL is partially internalized by a pathway resembling phagocytosis-like internalization (in part by fluid-phase endocytosis) as measured with [14C]sucrose uptake. In contrast to uptake studies, binding of HOCl-LDL to THP-1 cells at 4 °C was specific and saturable, indicating that binding proteins and/or receptors are involved. Competition studies on THP-1 macrophages showed that HOCl-LDL does not compete for the uptake of acetylated LDL (a ligand to scavenger receptor class A) but strongly inhibits the uptake of copper-oxidized LDL (a ligand to CD36 and SR-BI). The binding specificity of HOCl-LDL to class B scavenger receptors could be demonstrated by Chinese hamster ovary cells overexpressing CD36 and SR-BI and specific blocking antibodies. The lipid moiety isolated from the HOCl-LDL particle did not compete for cell association of labeled HOCl-LDL to CD36 or SR-BI, suggesting that the protein moiety of HOCl-LDL is responsible for receptor recognition. Experiments with Chinese hamster ovary cells overexpressing scavenger receptor class A, type I, confirmed that LDL modified at physiologically relevant HOCl concentrations is not recognized by this receptor. The presence of HOCl-modified epitopes inside and outside monocytes/macrophages and the presence of HOCl-modified apolipoprotein B in atherosclerotic lesions has initiated the present study to identify scavenger receptors that bind and internalize HOCl-low density lipoprotein (LDL). The uptake of HOCl-LDL by THP-1 macrophages was not saturable and led to cholesterol/cholesteryl ester accumulation. HOCl-LDL is not aggregated in culture medium, as measured by dynamic light scattering experiments, but internalization of HOCl-LDL could be inhibited in part by cytochalasin D, a microfilament disrupting agent. This indicates that HOCl-LDL is partially internalized by a pathway resembling phagocytosis-like internalization (in part by fluid-phase endocytosis) as measured with [14C]sucrose uptake. In contrast to uptake studies, binding of HOCl-LDL to THP-1 cells at 4 °C was specific and saturable, indicating that binding proteins and/or receptors are involved. Competition studies on THP-1 macrophages showed that HOCl-LDL does not compete for the uptake of acetylated LDL (a ligand to scavenger receptor class A) but strongly inhibits the uptake of copper-oxidized LDL (a ligand to CD36 and SR-BI). The binding specificity of HOCl-LDL to class B scavenger receptors could be demonstrated by Chinese hamster ovary cells overexpressing CD36 and SR-BI and specific blocking antibodies. The lipid moiety isolated from the HOCl-LDL particle did not compete for cell association of labeled HOCl-LDL to CD36 or SR-BI, suggesting that the protein moiety of HOCl-LDL is responsible for receptor recognition. Experiments with Chinese hamster ovary cells overexpressing scavenger receptor class A, type I, confirmed that LDL modified at physiologically relevant HOCl concentrations is not recognized by this receptor. The oxidation of low density lipoprotein (LDL) 1The abbreviations used are: LDLlow density lipoproteinapoapolipoproteinCEcholesteryl esterCHOChinese hamster ovaryFCSfetal calf serumhCD36human CD36HOCl/OCl–hypochlorous acid/hypochloriteMPOmyeloperoxidase, mSR-AI, murine scavenger receptor class A, type ImSR-BImurine scavenger receptor class B, type ITBSTris-buffered salineDMEMDulbecco's modified Eagle's mediumPBSphosphate-buffered saline. has been proposed as a biological process that initiates and accelerates arterial lesion development (for review, see Refs. 1Chisolm III, G.M. Hazen S.L. Fox P.L. Cathcart M.K. J. Biol. Chem. 1999; 274: 25959-25962Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar and 2Lusis A.J. Nature. 2000; 407: 233-241Crossref PubMed Scopus (4735) Google Scholar). Oxidized LDL (ox-LDL) accumulates in lesions and is present at other inflammatory sites. Recent findings revealed that monocyte-derived macrophages are likely candidates to mediate the in vivo oxidation of LDL. There are multiple potential pathways through which monocytes/macrophages may promote extracellular oxidation of LDL, but in principal non-enzymatic and enzymatic cellular mechanisms of LDL oxidation do exist. Although formation of the superoxide anion radical and/or its dismutation product, hydrogen peroxide, by monocyte-derived macrophages appears to be essential for LDL oxidation, a cell-derived factor that does participate in phagocyte-dependent oxidation of LDL is myeloperoxidase (MPO). low density lipoprotein apolipoprotein cholesteryl ester Chinese hamster ovary fetal calf serum human CD36 hypochlorous acid/hypochlorite myeloperoxidase, mSR-AI, murine scavenger receptor class A, type I murine scavenger receptor class B, type I Tris-buffered saline Dulbecco's modified Eagle's medium phosphate-buffered saline. MPO is an abundant heme protein (for review, see Refs. 3Kettle A.J. Winterbourn C.C. Redox Rep. 1997; 3: 3-15Crossref PubMed Scopus (592) Google Scholar and 4Hampton M.B. Kettle A.J. Winterbourn C.C Blood. 1998; 92: 3007-3017Crossref PubMed Google Scholar) released during the oxidative burst by activated neutrophils and monocytes. A major function of MPO is to hold a central role in microbial killing, and recent findings revealed an association between MPO levels and the risk of coronary artery disease (5Zhang R. Brennan M.L. Fu X. Aviles R.J. Pearce G.L. Penn M.S. Topol E.J. Sprecher D.L. Hazen S.L. J. Am. Med. Assoc. 2001; 286: 2136-2142Crossref PubMed Scopus (772) Google Scholar). MPO is also present in tissue macrophages such as those in vascular lesions (6Daugherty A. Dunn J.L. Rateri D.L. Heinecke J.W. J. Clin. Invest. 1994; 94: 437-444Crossref PubMed Scopus (1134) Google Scholar, 7Malle E. Waeg G. Schreiber R. Gröne E. Sattler W. Gröne H.J. Eur. J. 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Alternatively, MPO-generated HOCl oxidizes free α-amino acids to aldehydes (13Anderson M.M. Hazen S.L. Hsu F.F. Heinecke J.W. J. Clin. Invest. 1997; 99: 424-432Crossref PubMed Scopus (360) Google Scholar), leading to advanced glycation products present in human lesion material (14Nagai R. Hayashi C.M. Xia L. Takkeya M. Horiuchi S. J. Biol. Chem. 2002; 277: 48905-48912Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). However, the most common reaction of HOCl is with free amino groups of (apolipo)proteins, leading to formation of chloramines. LDL modified by HOCl (HOCl-LDL) displays a number of pathophysiological effects on phagocytes and vascular cells, contributing to the initiation and maintenance of the inflammatory process during the early phase of atherosclerotic lesion development. HOCl-LDL induces chemokine release of monocytes and chemotactic migration of neutrophils (15Woenckhaus C. Kaufmann A. Bussfeld D. Gemsa D. Sprenger H. Gröne H.J. Clin. Immunol. Immunopathol. 1998; 86: 27-33Crossref PubMed Scopus (42) Google Scholar), initiates the respiratory burst of macrophages (16Nguyen-Khoa T. Massy Z.A. Witko-Sarsat V. Canteloup S. Kebede M. Lacour B. Drueke T. Descamps-Latscha B. Biochem. Biophys. Res. Commun. 1999; 263: 804-809Crossref PubMed Scopus (74) Google Scholar), stimulates polymorphonuclear leukocytes to an enhanced production of superoxide anion radical and hydrogen peroxide, enhances neutrophil degranulation (17Kopprasch S. Leonhardt W. Pietzsch J. Kühne H. Atherosclerosis. 1998; 136: 315-324Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), and inactivates lysosomal proteases (18Carr A.C. Redox Rep. 2001; 6: 343-349Crossref PubMed Scopus (12) Google Scholar). HOCl-LDL further decreases nitric oxide-synthesis in endothelial cells (19Nuszkowski A. Gräbner R. Marsche G. Unbehaun A. Malle E. Heller R. J. Biol. Chem. 2001; 276: 14212-14221Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), causes endothelial leakage and stimulates leukocyte adherence to, and migration into, the subendothelial space (20Liao L. Aw T.Y. Kvietys P.R. Granger D.N. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 2305-2311Crossref PubMed Scopus (45) Google Scholar). HOCl-LDL enhances platelet reactivity and release reaction (21Opper C. Schüssler G. Sattler W. Malle E. Platelets. 1998; 9: 339-341Crossref PubMed Scopus (15) Google Scholar, 22Zabe M. Feltzer R.E. Malle E. Sattler W. Dean W.L. Cell Calcium. 1999; 26: 281-287Crossref PubMed Scopus (20) Google Scholar), and most importantly, HOCl converts LDL into a high uptake form for mouse peritoneal macrophages (23Hazell L.J. Stocker R. Biochem. J. 1993; 290: 165-172Crossref PubMed Scopus (294) Google Scholar), leading to the formation of cholesteryl ester (CE)-laden foam cells, which are the hallmark of fatty streaks and the earliest recognizable lesion of atherosclerosis. The presence of 3-chlorotyrosine in human atherosclerotic lesions (12Hazen S.L. Heinecke J.W. J. Clin. Invest. 1997; 99: 2075-2081Crossref PubMed Scopus (755) Google Scholar), the presence of HOCl-modified epitopes inside and outside monocytes/macrophages, endothelial cells, and smooth muscle cells in human and rabbit lesion material (7Malle E. Waeg G. Schreiber R. Gröne E. Sattler W. Gröne H.J. Eur. J. Biochem. 2000; 267: 4495-4503Crossref PubMed Scopus (218) Google Scholar, 24Malle E. Waeg G. Thiery J. Sattler W. Gröne H.J. Biochem. Biophys. Res. Commun. 2001; 289: 894-900Crossref PubMed Scopus (26) Google Scholar, 25Bräsen J.H. Hakkinen T. Malle E. Beisiegel U. Ylä-Herttuala S. Atherosclerosis. 2003; 166: 13-21Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), and the presence of HOCl-modified apolipoprotein (apo) B-100 extracted from advanced human atherosclerotic lesions (26Hazell L.J. Arnold L. Flowers D. Waeg G. Malle E. Stocker R. J. Clin. Invest. 1996; 97: 1535-1544Crossref PubMed Scopus (541) Google Scholar) supported the view that the MPO/hydrogen peroxide/chloride system converts LDL into an atherogenic form under in vivo conditions. However, the cellular mechanisms leading to HOCl-LDL uptake/processing by macrophages has not yet been addressed. We, therefore, put major emphasis on the clarification of whether scavenger receptors on differentiated macrophages are responsible for HOCl-LDL uptake. A variety of scavenger receptors expressed on macrophages has been cloned; however, previous studies demonstrated that scavenger receptors class A, e.g. SR-AI/II, and class B (e.g. CD36) are the principal receptors responsible for binding and uptake of modified LDL (for review see Refs. 27de Winther M.P. van Dijk K.W. Havekes L.M. Hofker M.H. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 290-297Crossref PubMed Scopus (197) Google Scholar and 28Febbraio M. Hajjar D.P. Silverstein R.L. J. Clin. Invest. 2001; 108: 785-791Crossref PubMed Scopus (937) Google Scholar). Both type I and type II SR-A bind a diverse array of macromolecules, including bacterial surface lipids (endotoxin and lipoteichoic acid), β-amyloid fibrils, protein modified by advanced glycation (advanced glycation end products), and modified lipoproteins, e.g. acetylated LDL (ac-LDL) or copper-oxidized-LDL (Cu-ox-LDL), respectively. In vivo CD36 is involved in diverse processes as recognition of senescent or apoptotic cells, fatty acid transport, cell-matrix interaction, and antiangiogenic actions. CD36 mediates lipid accumulation and macrophage foam cell formation in vitro and in vivo. SR-BI, another class B (type I) scavenger receptor, has also a multiligand specificity for various forms of native and modified (lipo)proteins (for review, see Ref. 29Krieger M. Annu. Rev. Biochem. 1999; 68: 523-558Crossref PubMed Scopus (460) Google Scholar). Although SR-BI in comparison to SR-A and CD36 is less abundant in atherosclerotic lesions, its mRNA expression pattern during differentiation of human macrophages is similar to SR-AI and CD36, and both Cu-ox-LDL and ac-LDL may up-regulate its expression (30Hirano K. Yamashita S. Nakagawa Y. Ohya T. Matsuura F. Tsukamoto K. Okamoto Y. Matsuyama A. Matsumoto K. Miyagawa J. Matsuzawa Y. Circ. Res. 1999; 85: 108-116Crossref PubMed Scopus (144) Google Scholar). Previous studies suggested that the primary routes for entry of ligands by macrophages are coated pits or caveolin-dependent endocytosis and/or phagocytosis. However, ligands can also be internalized non-concentration-dependent via fluid-phase uptake. Larger pinosomes termed macropinosomes can also internalize fluid-phase macromolecules via macropinocytosis (31Seastone D.J. Harris E. Temesvari L.A. Bear J.E. Saxe C.L. Cardelli J. J. Cell Sci. 2001; 114: 2673-2683PubMed Google Scholar). We here provide evidence that HOCl-LDL is internalized by receptor-mediated endocytosis via class B scavenger receptor CD36 and SR-BI and partly via a mechanism resembling phagocytotic ingestion. At physiologically relevant HOCl concentrations HOCl-LDL is not recognized by SR-AI. NaOCl, organic solvents, potassium bromide, and egg yolk lecithin were obtained from Sigma. Radiochemicals were purchased from PerkinElmer Life Sciences. DMEM, RPMI, and Ham's-F-12K medium were from Invitrogen, and fetal calf serum (FCS) was obtained from Roche Applied Science. Plastic ware used for tissue culture was obtained from Costar (Vienna). All other chemicals were obtained from Merck except where indicated. LDL (d = 1.035–1.065 g/ml) was isolated by ultracentrifugation as described previously (32Malle E. Ibovnik A. Leis H.J. Kostner G.M. Verhallen P.F. Sattler W. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 377-384Crossref PubMed Scopus (36) Google Scholar). The protein of the final LDL preparation consisted of 96–98% apoB-100 as measured immunochemically. Lipoprotein concentrations are expressed in mg or μg of protein/ml and were determined according to Lowry and coworkers (33Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using bovine serum albumin as a standard or calculated from total cholesterol, determined by the CHOD-PAP method (Roche Applied Science). Before modification, native LDL was desalted, and the preservatives were removed by dialysis or size exclusion chromatography on Econopac 10-DG columns (Bio-Rad). HOCl-LDL was prepared as described (34Malle E. Hazell L. Stocker R. Sattler W. Esterbauer H. Waeg G. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 982-989Crossref PubMed Scopus (131) Google Scholar). Briefly, 1 mg of LDL protein per ml of phosphate-buffered saline, pH 7.4, was incubated with HOCl solution at 4 °C for up to 1 h at pH 7.4. For LDL incubated between 0.2 and 1.6 mm final HOCl concentration this resulted in an oxidant:lipoprotein molar ratio in between 100:1 and 800:1. The modified LDL preparations were passed over a PD10 column to remove unreacted NaOCl and used immediately for cell culture experiments. Cu-ox-LDL (relative electrophoretic mobility = 2) and ac-LDL (relative electrophoretic mobility = 2.8) were prepared as described (32Malle E. Ibovnik A. Leis H.J. Kostner G.M. Verhallen P.F. Sattler W. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 377-384Crossref PubMed Scopus (36) Google Scholar). Aliquots of native and NaOCl-modified LDL (450 μg of protein) were lyophilized in 5-ml ampoules and purged with nitrogen before hydrolysis in constant boiling 6 n HCl (24 h, 120 °C). Amino acid analysis was performed on a Biotronics analyzer as described (35Panzenboeck U. Raitmayer S. Reicher H. Lindner H. Glatter O. Malle E. Sattler W. J. Biol. Chem. 1997; 272: 29711-29720Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Lipids were extracted from native or modified LDL as described (36Marsche G. Hammer A. Oskolkova O. Kozarsky K.F. Sattler W. Malle E. J. Biol. Chem. 2002; 277: 32172-32179Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Briefly, HCl was added to the LDL preparations to a final concentration of 10 mm, and the lipids were extracted using chloroform-methanol 2:1 (v/v). The chloroform phase was removed and evaporated under nitrogen, and the lipids were resuspended in Tris-buffered saline (TBS, 0.01 m Tris HCl, 0.001 m EDTA, 0.15 m NaCl, pH 7.4) and sonicated (to obtain a microemulsion of small lipid droplets). LDL Labeling with 125INa—Iodination of LDL and HOCl-LDL was performed as described (37Artl A. Marsche G. Lestavel S. Sattler W. Malle E. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 763-772Crossref PubMed Scopus (213) Google Scholar) using N-bromosuccinimide as the coupling agent. Routinely, 500 μCi of 125INa was used to label 2 mg of LDL/HOCl-LDL-protein. This procedure resulted in specific activities between 200 and 300 cpm/ng of protein with less than 3% lipid-associated activity. No cross-linking or fragmentation of apoB-100 due to the iodination procedure could be detected by SDS-PAGE and subsequent autoradiography. LDL Labeling with [3H]CE—LDL and HOCl-LDL were labeled with [cholesteryl-1,2,6,7-3H]palmitate by cholesteryl ester transfer protein-catalyzed transfer from donor liposomes as described (37Artl A. Marsche G. Lestavel S. Sattler W. Malle E. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 763-772Crossref PubMed Scopus (213) Google Scholar). This labeling procedure resulted in specific activities of 5–9 cpm/ng of protein. Macrophages—The human monocytic THP-1 cell line was maintained in RPMI 1640 medium supplemented with 2 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% FCS. To induce differentiation, THP-1 cells were cultured for 72 h in the presence of 160 nm phorbol myristate acetate. Chinese Hamster Ovary (CHO) Cells—Stable transfectant CHO cells expressing murine SR-AI (CHO[mSR-AI]) were maintained in Ham's-F-12K medium supplemented with 3% lipoprotein-deficient serum, 250 μm mevalonate, 40 μm mevinolin, 3 μg/ml ac-LDL, 2 mm glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin (38Ashkenas J. Penman M. Vasile E. Acton S. Freeman M. Krieger M. J. Lipid Res. 1993; 34: 983-1000Abstract Full Text PDF PubMed Google Scholar). Experiments with CHO[mSR-AI] were performed on confluent cell monolayers in Ham's F-12 medium containing 3% lipoprotein-deficient FCS. Vector-transfected CHO cells cultured in Ham's F-12 medium containing 5% FCS, 2 mm glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin were used as controls. Stable transfectant CHO cells expressing murine SR-BI (ldlA[mSRBI]) were maintained in Ham's-F-12K medium containing 5% (v/v) fetal bovine serum, 2 mm glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin (39Acton S.L. Scherer P.E. Lodish H.F. Krieger M. J. Biol. Chem. 1994; 269: 21003-21009Abstract Full Text PDF PubMed Google Scholar, 40Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (2011) Google Scholar) containing 0.5 mg/ml G-418. LdlA cells (clone 7, an LDL receptor-deficient CHO cell line) used as controls were cultured in medium without G-418. Stable transfectant CHO cells expressing human CD36 (CHO[hCD36]) (41Ohgami N. Nagai R. Ikemoto M. Arai H. Kuniyasu A. Horiuchi S. Nakayama H. J. Biol. Chem. 2001; 276: 3195-3202Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar) were cultured in Ham's-F-12K medium containing 5% (v/v) FCS, 2 mm glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin were maintained in medium containing 0.5 mg/ml G-418. Cell Association and Degradation Studies—Association studies of LDL and HOCl-LDL to cells were performed at 4 and 37 °C with increasing amounts of 125I-labeled CE- and/or [3H]CE-labeled lipoproteins in the absence (total cell-association) or presence of 1 mg of protein/ml (nonspecific cell association) of unlabeled autologous lipoprotein particles or specific polyclonal anti-human CD36 antiserum (42Tandon N.N. Ockenhouse C.F. Greco N.J. Jamieson G.A. Blood. 1991; 78: 2809-2813Crossref PubMed Google Scholar) in DMEM/RPMI medium without FCS. After this incubation, the medium was aspirated, and the cells were rinsed 2 times with TBS (containing 5% (w/v) bovine serum albumin) followed by 2 washes with TBS only. Cells were lysed with 0.3 n NaOH. The radioactivity and protein content of the cell lysate were measured in the same aliquot. Specific cell association was calculated as the difference between total and nonspecific cell association. Degradation of 125I-labeled lipoprotein particles by CHO cells was estimated by measuring the non-trichloroacetic acid-precipitable radioactivity in the medium as described (37Artl A. Marsche G. Lestavel S. Sattler W. Malle E. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 763-772Crossref PubMed Scopus (213) Google Scholar, 43Marsche G. Levak-Frank S. Quehenberger O. Heller R. Sattler W. Malle E. FASEB J. 2001; 15: 1095-1097PubMed Google Scholar). Specific degradation was calculated as the difference between total and nonspecific degradation (presence of blocking antibodies or control cells). The specificity of HOCl-LDL binding sites was examined by competition experiments. The association of 10 μg of [3H]CE-labeled lipoprotein/ml was competed for by increasing concentrations (up to 200 μg of protein or protein eq/ml) of indicated unlabeled competitors. Determination of Cholesterol Content in THP-1 Macrophages—Lipids of THP-1 cells were extracted with hexane-isopropanol (3:2) and dried under argon. The free and total cholesterol content was measured with an enzymatic kit according to the manufacturer's instructions (Wako Chemicals GmbH). The CE content was determined by subtracting free cholesterol content from total cholesterol content. Measurement of Fluid-phase Endocytosis—Fluid-phase endocytosis was determined by incubating THP-1 macrophages with 1 μCi/ml [3H]sucrose in the presence of LDL or HOCl-LDL. After the incubations, the cells were rinsed 2 times with TBS (containing 5% (w/v) bovine serum albumin) followed by 2 washes with TBS only. Cells were lysed with 0.3 n NaOH. The radioactivity and protein content of the cell lysate was measured in the same aliquot. Cell-free wells were incubated with [3H]sucrose in parallel incubations and gave a background count less than 5% of the [3H]sucrose radioactivity detected in macrophages (44Kruth H.S. Huang W. Ishii I. Zhang W.Y. J. Biol. Chem. 2002; 277: 34573-34580Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Amino acid analysis of apoB-100 revealed that cysteine and methionine are the most susceptible amino acids to oxidation by HOCl (Table I). Tyrosine and histidine are consumed dose-dependently up to 20 and 10%, respectively, at the highest HOCl concentrations. Whereas the ϵ-amino group of arginine is not prone to be modified, the ϵ-amino group of lysine represents a preferential target for HOCl attack. The increase in the anionic charge of LDL, a result from modification of the ϵ-amino group of peptidyl lysine residues from apoB-100, is further reflected by an increased electrophoretic mobility of the modified LDL particle. In line with previous reports (23Hazell L.J. Stocker R. Biochem. J. 1993; 290: 165-172Crossref PubMed Scopus (294) Google Scholar) no significant increase of lipid peroxidation products was detected as a function of increasing oxidant:LDL molar ratio ranging between 100:1 and 800:1 (data not shown).Table ICharacterization of native and HOCl-modified LDLLDLHOCl-LDL100:1200:1400:1800:1mol/molCys0.7NDNDNDNDMet3.1NDNDNDNDTyr17.416.515.41413.8Lys31.429.928.126.219.1His10.510.210.09.49.5Arg15.715.715.815.615.5REM11.151.351.752.1 Open table in a new tab Based on in vitro experiments HOCl concentrations at sites of acute inflammation were calculated to be in the range of 340 μm or above (45Weiss S.J. Test S.T. Eckmann C.M. Roos D. Regiani S. Science. 1986; 234: 200-203Crossref PubMed Scopus (210) Google Scholar, 46Katrantzis M. Baker M.S. Handley C.J. Lowther D.A. Free Radic. Biol. Med. 1991; 10: 101-109Crossref PubMed Scopus (66) Google Scholar). Assuming LDL plasma concentrations of ≈2 μm (and it is conceivable that subendothelial concentrations could be lower) one would yield a minimal estimate HOCl:LDL molar ratio of ∼200:1. Therefore, an oxidant:LDL molar ratio of 100:1 up to 400:1 was considered suitable for further experiments. Previous reports suggest that exposure of LDL to reagent HOCl results in LDL aggregation due to the formation of chloramines from apoB-100 lysine amino groups (23Hazell L.J. Stocker R. Biochem. J. 1993; 290: 165-172Crossref PubMed Scopus (294) Google Scholar, 47Hazell L.J. van den Berg J.J. Stocker R. Biochem. J. 1994; 302: 297-304Crossref PubMed Scopus (246) Google Scholar). Because aggregation could be reversed by the addition of methionine or lysine (leading to reversion of apoB-100 chloramines), we studied whether HOCl-LDL aggregates are stable in culture medium (RPMI). Therefore, native and HOCl-LDL (1 mg of protein/ml of phosphate-buffered saline, oxidant:LDL molar ratio 400:1) were dialyzed against phenol red-free RPMI and analyzed by dynamic light scattering experiments as described (35Panzenboeck U. Raitmayer S. Reicher H. Lindner H. Glatter O. Malle E. Sattler W. J. Biol. Chem. 1997; 272: 29711-29720Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Under these conditions no HOCl-LDL aggregates were detected to be present in RPMI. The hydrodynamic radius of HOCl-LDL only moderately increased from 12.7 ± 1.3 to 16.6 ± 2.8 nm, indicating that no aggregation occurs under the conditions applied. To investigate binding properties of HOCl-LDL to THP-1 cells at 4 °C, the protein moiety of the lipoprotein particle was labeled with 125INa. Non-linear regression analysis revealed a significant increase in binding affinity (Kd values) and saturable binding characteristics for HOCl-LDL when compared with native LDL (Fig. 1A, Table II). This indicates that HOCl-LDL is recognized by receptor(s) or binding protein(s). THP-1 macrophages were further incubated with HOCl-LDL for 24 h, and cellular cholesterol content was estimated. Incubation of cells led to a significant accumulation of free (Fig. 1B) and esterified cholesterol from HOCl-LDL (Fig. 1C). It is important to note that under the conditions described LDL only marginally increased cellular CE content (Fig. 1C). In contrast to binding experiments at 4 °C, cell association of HOCl-LDL at 37 °C is not saturable (Fig. 1D), indicating involvement of a pathway resembling phagocytic ingestion. Therefore, cell experiments were performed in the presence of cytochalasin D, a microfilament-disrupting agent (48Poussin C. Foti M. Carpentier J.L. Pugin J. J. Biol. Chem. 1998; 273: 20285-20291Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) known to block phagocytosis and macropinocytosis. About 35–46% of cell association of LDL and HOCl-LDL (at concentrations higher than 100 μg of protein/ml), respectively, could be inhibited by cytochalasin D (Fig. 1D). Association of vortex-aggregated LDL, which is internalized through a phagocytic mechanism (49Khoo J.C. Miller E. McLoughlin P. Steinberg D. Arteriosclerosis. 1988; 8: 348-358Crossref PubMed Google Scholar), was inhibited by >90% by cytochalasin D treatment of cells (data not shown). Previous findings revealed that modified LDL particles are internalized by macrophages in part via macropinocytosis, a phagocytic mechanism that leads to ingestion of fluid-filled macropinosomes (50Jones N.L. Willingham M.C. Anat. Rec. 19
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