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Monoclonal Antibodies Against LDL Further Enhance Macrophage Uptake of LDL Aggregates John C. Khoo, Elizabeth Miller, Frederic Pio, Daniel Steinberg, and Joseph L. Witztum Self-aggregates of low density lipoprotein (LDL) are taken up and degraded more rapidly by macrophages than is native LDL. That enhanced uptake is attributable in part to phagocytosis via the LDL receptor pathway. However, arterial macrophages appear to express little LDL receptor activity. The present studies demonstrate an alternative mechanism by which LDL aggregates could contribute to foam cell formation. This could occur by the formation of large immune complexes that are taken up by macrophages via the Fc receptor. When immune complexes were formed with native, soluble LDL and MB47, a monoclonal antibody specific to the apoprotein B domain recognized by the LDL receptor, the subsequent uptake and degradation of the LDL by macrophages were inhibited 50-80% compared with native LDL alone. In contrast, when aggregated LDL was bound to MB47 at a similar molar ratio, the subsequent degradation of the insoluble immune complexes was two- to fivefold greater than that of aggregated LDL alone. The enhanced uptake was abolished when Fab or F(ab'>2 fragments of MB47 were substituted for the intact antibody, indicating that the increased uptake was via the Fc receptor pathway. Furthermore, the uptake of the immune complexes of aggregated LDL was reduced by competition for the Fc receptor with heat-aggregated immunoglobulin. There was also an increase in the rate of cellular cholesterol esterification and an increase in macrophage cholesteryl ester mass. Since aggregates of LDL as well as autoantibodies against modified LDL have been demonstrated in atherosclerotic lesions, it is possible that immune complexes of aggregates of modified LDL may be generated in the intima. The resultant antibody-LDL aggregates might then be even more effective in generating foam cells than the LDL aggregates themselves. (Arteriosclerosis and Thrombosis 1992;12:1258-1266) KEY WORDS • macrophages • monoclonal antibodies • low density lipoprotein aggregates immune complexes • MB47 • MB24 • Fc receptor • phagocytosis • atherosclerosis
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revious studies from this laboratory have shown that self-aggregates of low density lipoprotein (LDL) produced by vortexing (Vx-LDL) were taken up and degraded by cultured macrophages much more rapidly than monomeric native LDL.1 This uptake was shown to occur via phagocytosis, mediated in part by the LDL receptor. These findings were confirmed by Suits et al2 using an aggregated-LDL preparation produced by treatment with phospholipase C (PLC-LDL). Studies by Frank and Fogelman3 suggested that aggregates of LDL are present in the arterial intima of Watanabe heritable hyperlipidemic (WHHL) and cholesterol-fed rabbits. Recently, they have shown that 2 hours after an intravenous bolus injection of human LDL into a normal rabbit, aggregates of LDL can be seen in the aortic intima by using ultra-rapid freezing and freeze-etching techniques.4 From the Division of Endocrinology/Metabolism, Department of Medicine, University of California, San Diego, La Jolla, Calif. Supported by National Institutes of Health grants HL-34724 and HL-14197 and in part by the Rose and Sam Stein Institute for Research on Aging. Address for reprints: Joseph L. Witztum, MD, UCSD Department of Medicine 0682, 9500 Gilman Drive, La Jolla, CA 92093-0682. Received December 15, 1991; revision accepted July 24, 1992.
•
In cell culture, the LDL receptor activity of monocyte-derived macrophages is downregulated by exposure to serum or LDL. Although monocytes that have recently entered the intima may still express LDL receptor activity, under conditions of marked hypercholesterolemia or in advanced lesions, arterial macrophages appear to express little or no LDL receptor mRNA.5 Thus, for LDL aggregates to continue to contribute importantly to macrophage accumulation of cholesterol under these conditions, an alternative pathway for macrophage uptake may be needed. Antibody-antigen complexes are taken up by macrophages via the Fc receptor pathway, and this should not be downregulated as a result of cholesterol loading. Although soluble antigen-antibody complexes bind readily to Fc receptors on macrophages, large, insoluble, multimeric complexes are required for high-affinity binding and rapid internalization. We now report that a model system of LDL aggregates complexed with monoclonal antibodies against LDL are taken up and degraded by macrophages twoto fivefold faster than uncomplexed aggregates of LDL. Moreover, this uptake is phagocytic, being mediated by the Fc receptor via the zipper mechanism.6 Since aggregates of LDL and modified LDL are likely to occur in the intima and autoantibodies to epitopes of modified LDL are present in plasma,7-11 the formation of such
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Khoo et al Macrophage Uptake of Immune Complexes of LDL immune complexes may provide an additional pathway for macrophage uptake of LDL and could play a role in accelerating cholesteryl ester accumulation. Methods Cells Mouse peritoneal macrophages were elicited by intraperitoneal injection of 2 ml thioglycollate medium (DIFCO) 3 days before harvest. For degradation and esterification assays, macrophages were plated in 24-well clustered dishes (lxlO 6 cells per well) in Iscove's modified Dulbecco's medium (IMDM) (GIBCO) containing 10% fetal bovine serum. For experiments in which cellular cholesterol mass was measured, six-well clustered dishes were used (4xlO 6 cells per well). Two hours after plating, nonadherent cells were removed by washing with phosphate-buffered saline (PBS). The cells were then incubated overnight at 37°C in IMDM containing 5 mg/ml lipoprotein-deficient serum (LPDS). Lipoproteins LDL (d=1.019-1.063 g/1) and LPDS (d> 1.215 g/1) were isolated by preparative ultracentrifugation from fresh human plasma as described previously.1 LDL was radioiodinated by the method of Salacinski et al.12 Vx-LDL was prepared by vortexing LDL (0.5 mg protein/ml PBS) for 60 seconds as previously described,1 and PLC-LDL was produced by incubating LDL (0.25 mg protein/ml PBS) with 1 unit/ml phospholipase C (type XI, from Bacillus cereus; Sigma) at 37°C for 1 hour.2 Antibodies Protein A-purified apoprotein (apo) B-specific monoclonal antibodies MB4713 and MB2414 against LDL were generously provided by Dr. Richard S. Smith from Johnson and Johnson Biotechnology Center, La Jolla, Calif. Both MB47 and MB24 were identified by double immunodiffusion as immunoglobulin G2a (IgG2a).13>14 A control mouse immunoglobulin (IgG2a) was purchased from Calbiochem Corp. Fab fragments of MB47 were prepared as previously described by digestion with immobilized papain, followed by chromatography on a column of immobilized protein A-agarose (Pierce) to remove the Fc portion.13 F(ab') 2 fragments of MB47 were prepared by using the Pierce Immunopure F(ab') 2 kit. The MB47 was digested with immobilized pepsin and separated on an immobilized protein A-agarose column. One-milliliter fractions were collected, and absorbance was measured at 280 nm. The fractions containing F(ab') 2 fragments were combined and concentrated to a final volume of 1 ml in a Centricon 10 microconcentrator (Amicon). Products were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Heat-aggregated IgG was prepared by heating the control mouse IgG (20 mg/ml) at 63°C for 20 minutes, according to the method described by Griffith et al.15 Preparation of LDL-Immune Complexes LDL-immune complexes were prepared by incubating monomeric native LDL, Vx-LDL, or PLC-LDL with the indicated concentration of antibody for 16 hours at 4°C. The molar ratios were calculated by assuming a
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molecular mass for apo B of 550 kd, for monoclonal and control IgG of 166 kd, for Fab fragments of 40 kd, and for F(ab') 2 fragments of 105 kd. The ability of MB47 and its Fab or F(ab') 2 fragments to inhibit the degradation of LDL by human skin fibroblasts was measured as described13 by using cells grown to near confluency on 24-well clustered dishes that were incubated overnight in 5 mg/ml LPDS before the addition of soluble LDLMB47 complexes. Other Methods The degradation of 125I-LDL by cells was determined using methods described by Goldstein et al.16 Each monolayer of thioglycollate-elicited mouse peritoneal macrophages received 0.5 ml IMDM containing 5 mg/ml LPDS and the indicated concentration of 125ILDL protein. After incubation for the specified time at 37°C, the medium was removed. The amount of apo B degraded was determined by the 125I-trichloroacetic acid (TCA)-soluble (noniodide) material present in the medium. The cells were washed with PBS and treated with 0.1% trypsin in saline to remove LDL bound to the surface of macrophages. The types of aggregated LDL used for the various experiments did not adhere to the plastic dishes. The remaining cells were solublized in 0.2 M NaOH, and aliquots were removed for counting as well as for protein determination by the method of Lowry et al.17 For these studies we have defined the amount of 125I released from the cells by trypsin as "bound," the total amount of 125I remaining in the cells at the end of the trypsinization as "cell content," and the amount of noniodide TCA-soluble material present in the medium as amount "degraded." "Total uptake" was defined as the sum of bound plus cell content plus amount degraded. To measure the effect of heat-aggregated IgG to compete for the degradation of LDL-immune complexes, a 200-fold protein excess of the aggregated IgG was added to the cell monolayers 1 hour before the addition of the 125I-LDL. The incorporation of [l4C]oleate into cellular cholesteryl [14C]oleate was determined by the method described by Goldstein et al.16 Briefly, the indicated concentrations of LDL protein/ml IMDM, containing 5 mg/ml LPDS, were incubated with mouse peritoneal macrophages for 6 hours at 37°C. One hour before harvest, 0.2 mM [14C]oleate/albumin (11,000 dpm/nmol) was added to each well. The monolayers were washed with PBS, and the total lipid was extracted in situ. To measure cellular sterol content, macrophage monolayers on 35-mm culture dishes were incubated with 2 5 100 fig LDL protein/ml medium for 24 hours in serumfree medium. After washing three times with PBS, treating with 0.1% trypsin for 15 minutes at 37°C, and rewashing three times with PBS, the cells were scraped into 1 ml distilled water and sonicated for 20 seconds. Aliquots were removed for protein determination, and the remainder was extracted for total lipids by the method of Folch et al.18 The mass of total and free cholesterol was determined by the enzymatic/fluorometric method of Gamble et al.19 Results The monoclonal antibody MB47 has been shown to bind to the LDL receptor-binding domain of apo B.13
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We speculate that such immune complexes could exist in vivo. Further studies are needed to determine if the LDL aggregates present in the intima contain oxidized LDL and if autoantibodies against oxidized LDL are associated with such aggregates. Uptake of antibodyaggregated (modified) LDL complexes by macrophages not only could lead to enhanced macrophage accumulation of cholesterol but also could result in the crosslinking of Fc receptors, which has been shown to trigger the release of pro-oxidants such as superoxide anion and hydrogen peroxide.36-37 This in turn could lead to the generation of still more oxidized LDL. 313839 Furthermore, it has been reported that uptake of insoluble LDL-immune complexes by human monocyte-derived macrophages, while leading to increased intracellular cholesteryl ester accumulation, paradoxically led to enhanced LDL receptor activity.30-40 All of these in vitro studies suggest that uptake of immune complexes containing LDL could significantly modify macrophage behavior and thereby influence the atherogenic process. Further studies are needed to determine the consequences for the uptake of such immune complexes by macrophages and whether this indeed occurs in vivo.
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Vx-LDL (Mg protein/ml) FIGURE 9. Line plots showing concentration-dependent binding (panel A), internalization (panel B), degradation (panel C), and degradation as a percentage of total uptake (panel D) of vortexed (Vx) low density lipoprotein (LDL) and immune complexes of Vx-LDL by mouse peritoneal macrophages. Increasing concentrations of l25I-Vx-LDL were preincubated for 16 hours at 4°C in the absence ( o ) or presence ( • ) of equal concentrations ofMB47 and MB24 at a molar ratio of 5:1 (total antibodies: apoprotein B). I25I-VxLDL and immune complexes of l25I-Vx-LDL were added to monolayer cultures of mouse peritoneal macrophages at the indicated LDL protein concentrations. After incubation at 37°C for 5 hours, the media were removed and analyzed for 12S I-trichloroacetic acid-soluble (noniodide) material. The cells were treated with trypsin, washed with phosphatebuffered saline, and solublized in 0.2 M NaOH. Each point represents the mean of triplicate determinations±SD. Gisinger et al30 demonstrated that when soluble and insoluble immune complexes of LDL were adsorbed to erythrocytes, they were avidly taken up by macrophages. Monoclonal antibodies bind to specific epitopes on the LDL particle and form soluble immune complexes. In our model studies, we used these monoclonal antibodies to prepare highly defined immune complexes of LDL.
References 1. Khoo JC, Miller E, McLoughlin P, Steinberg D: Enhanced macrophage uptake of low density lipoprotein after self-aggregation. Arteriosclerosis 1988;8:348-358 2. Suits AG, Chait A, Aviram M, Heinecke JW: Phagocytosis of aggregated lipoprotein by macrophage: Low density lipoprotein receptor-dependent foam-cell formation. ProcNatlAcad Sci USA 1989;86:2713-2717 3. Frank JS, Fogelman AM: Infrastructure of the intima in WHHL and cholesterol-fed rabbit aortas prepared by ultra-rapid freezing and freeze-etching. J Lipid Res 1989;30:967-978 4. Nievelstein PFEM, Fogelman AM, Mottino G, Frank JS: Lipid accumulation in rabbit aortic intima 2 hours after bolus infusion of low density lipoprotein: A deep-etch and immunolocalization study of ultrarapidly frozen tissue. Arterioscler Thromb 1991;11: 1795-1805 5. Yla-Herttuala S, Rosenfeld ME, Parthasarathy S, Sigal E, Sarkioja T, Witztum JL, Steinberg D: Gene expression in macrophage-rich human atherosclerotic lesions: 15-Lipoxygenase and acetyl LDL receptor messenger RNA colocalize with oxidation specific lipid— protein adducts. J Clin Invest 1991;87:1146-1152 6. Griffin FM, Griffin JA, Leider JE, Silverstein SC: Studies on the mechanism of phagocytosis: I. Requirements for circumferential attachment of particle-bound ligands to specific receptors on the macrophage plasma membrane. / Exp Med 1975;142:1263-1282 7. Palinski W, Rosenfeld ME, Yla-Herttuala S, Gurtner GC, Socher SS, Butler SW, Parthasarathy S, Carew TE, Steinberg D, Witztum JL: Low density lipoprotein undergoes oxidative modification in vivo. Proc NatlAcad Sci USA 1989;86:1372-1376 8. Rosenfeld ME, Palinski W, Yla-Herttuala S, Butler S, Witztum JL: Distribution of oxidation specific lipid-protein adducts and apolipoprotein B in atherosclerotic lesions of varying severity from WHHL rabbits. Arteriosclerosis 1990;10:336-349 9. Parums DV, Brown DL, Mitchinson MJ: Serum antibodies to oxidized low density lipoprotein and ceroid in chronic periaortitis. Arch Pathol Lab Med 1990;l 14:383-387 10. Witztum JL, Steinbrecher UP, Kasaniemi YA, Fisher M: Autoantibodies to glucosylated protein in the plasma of patients with diabetes mellitus. Proc NatlAcad Sci USA 1984;81:3204-3208 11. Orekhov AN, Tertov VV, Kabakov AE, Adamova IY, Pokrovsky SN, Smirnov VN: Autoantibodies against modified low density lipoprotein. Arterioscler Thromb 1991;ll:316-326 12. Salacinski PRP, McLean C, Sykes JEC, Clement-Jones VV, Lowry PJ: Iodination of proteins, glycoproteins, and peptides using a solid-phase oxidizing agent, l,3,4,6-tetrachloro-3a, 6a-diphenyl glycoluril (Iodogen). Anal Biochem 1981;117:136-146 13. Young SG, Witztum JL, Casal DC, Curtiss LK, Bernstein S: Conservation of the low density lipoprotein receptor-binding domain of apoprotein B: Demonstration by a new monoclonal antibody, MB47. Arteriosclerosis 1986;6:178-188
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14. Young SG, Smith RS, Hogle DM, Curtiss LK, Witztum JL: Two new monoclonal antibody-based enzyme-linked assays of apolipoprotein B. Clin Chem 1986;32:1484-1490 15. Griffith RL, Virella GT, Stevenson HC, Lopes-Virella MF: Low density lipoprotein metabolism by human macrophages activated with low density lipoprotein immune complexes. J Exp Med 1988; 168:1041-1059 16. Goldstein JL, Basu SK, Brown MS: Receptor-mediated endocytosis of low density lipoprotein in cultured cells. Methods Enzymol 1983;98:241-260 17. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. / Biol Chem 1951;193: 265-275 18. Folch J, Lees M, Sloane Stanley GH: A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 1957;226:497-509 19. Gamble WM, Vaughan M, Kruth HS, Avigan J: Procedure for determination of free and total cholesterol in micro- or nanogram amounts suitable for studies with cultured cells. / Lipid Res 1978; 19:1068-1070 20. Hoff HF, O'Neil J: Lesion-derived low density lipoprotein and oxidized low density lipoprotein share a lability for aggregation, leading to enhanced macrophage degradation. Arterioscler Thromb 1991;ll:1209-1222 21. Hoff HF, O'Neil J, Chisolm GM, Cole TB, Quehenberger O, Esterbauer H, Jurgens G: Modification of low density lipoprotein with 4-hydroxynonenal induces uptake by macrophages. Arteriosclerosis 1989;9:528-549 22. Tertov VV, Sobenin IA, Gabbasov ZA, Popov EG, Orekhov AN: Lipoprotein aggregation as an essential condition of intracellular lipid accumulation caused by modified low density lipoproteins. Biochem Biophys Res Commun 1989;163:489-494 23. Hurt E, Camejo G: Effect of arterial proteoglycans on the interaction of LDL with human monocyte-derived macrophages. Atherosclerosis 1987;67:115-126 24. Vijayagopal P, Srinivasan SR, Jones KM, Radhakrishnamurthy B, Berenson GS: Complexes of low-density lipoproteins and arterial proteoglycan aggregates promote cholesteryl ester accumulation in mouse macrophages. Biochim Biophys Ada 1985;837:251—261 25. Mora R, Lupu F, Simionescu N: Prelesional events in atherogenesis: Colocalization of apoprotein B, unesterified cholesterol and extracellular phospholipid liposomes in the aorta of hyperlipidemic rabbit. Atherosclerosis 1987;67:143-154 26. Falcone DJ, Salisbury BG: Fibronectin stimulates macrophage uptake of low density lipoprotein-hepann-collagen complexes. Arteriosclerosis 1988;8:263-273 27. Beaumont J-L, Beaumont V: Autoimmune hyperlipidemia./ir/ierosclerosis 1977;26:405-418
28. Szondy E, Horvath M, Mezey Z, Sizekely J, Lengyel E, Fust G, Gero S: Free and complexed anti-lipoprotein antibodies in vascular diseases. Atherosclerosis 1983;49:69-77 29. Klimov AN, Denisenko AD, Popov AV, Nagornev VA, Pleskov VM, Vinogradov AG, Denisenko TV, Magracheva EY, Kheifes GM, Kuznetzov AS: Lipoprotein-antibody immune complexes: Their catabolism and role in foam cell formation. Atherosclerosis 1985;58:1-15 30. Gisinger C, Virella GT, Lopes-Virella MF: Erythrocyte-bound low-density lipoprotein immune complexes lead to cholesteryl ester accumulation in human monocyte-derived macrophages. Clin Immunol Immunopathol 1991;59:37-52 31. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL: Beyond cholesterol: Modifications of low-density lipoprotein that increase its atherogenecity. NEnglJMed 1989;320:915-924 32. Steinbrecher UP, Fisher M, Witztum JL, Curtiss LK: Immunogenicity of homologous low density lipoprotein after methylation, ethylation, acetylation, or carbamylation: Generation of antibodies specific for derivatized lysine. J Lipid Res 1984;25:1109-1116 33. Salonen JT, Yla-Herttuala S, Yamamoto R, Butler S, Korpela H, Salonen R, Nyyssonen K, Palinski W, Witztum JL: Autoantibody against oxidised LDL and progression of carotid atherosclerosis. Lancet 1992;339:883-886 34. Yla-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D: Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest 1989;84:1087-1095 35. Yla-Herttuala S, Butler S, Picard S, Palinski W, Steinberg D, Witztum JL: Rabbit and human atherosclerotic lesions contain IgG that recognizes MDA-LDL and copper-oxidized LDL. (abstract) Arterioscler Thromb 1991;! 1:1426a 36. Aida Y, Onoue K: Triggering of the superoxide generation of macrophages by cross-linking of Fc-y receptor. J Biochem 1984;95: 1067-1072 37. Nathan CF, Root RK: Hydrogen peroxide release from mouse peritoneal macrophages: Dependence on sequential activation and triggering. J Exp Med 1977;146:1648-1662 38. Hiramatsu K, Rosen H, Heinecke JW, Wolfbauer G, Chait A: Superoxide initiates oxidation of low density-lipoprotein by human monocytes. Arteriosclerosis 1987;7:55-60 39. Cathcart MK, McNally AK, Morel DW, Chisolm GM III: Superoxide anion participation in human monocyte-mediated oxidation and conversion of low-density lipoprotein to a cytotoxin. J Immunol 1989;142:1963-1969 40. Lopes-Virella MF, Griffith RL, Shunk KA, Virella GT: Enhanced uptake and impaired intracellular metabolism of low density lipoprotein complexed with anti-low density lipoprotein antibodies. Arterioscler Thromb 1991;11:1356-1367
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Monoclonal antibodies against LDL further enhance macrophage uptake of LDL aggregates. J C Khoo, E Miller, F Pio, D Steinberg and J L Witztum Arterioscler Thromb Vasc Biol. 1992;12:1258-1266 doi: 10.1161/01.ATV.12.11.1258 Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1992 American Heart Association, Inc. All rights reserved. Print ISSN: 1079-5642. Online ISSN: 1524-4636
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Monoclonal Antibodies Against LDL Further Enhance Macrophage Uptake of LDL Aggregates John C. Khoo, Elizabeth Miller, Frederic Pio, Daniel Steinberg, and Joseph L. Witztum Self-aggregates of low density lipoprotein (LDL) are taken up and degraded more rapidly by macrophages than is native LDL. That enhanced uptake is attributable in part to phagocytosis via the LDL receptor pathway. However, arterial macrophages appear to express little LDL receptor activity. The present studies demonstrate an alternative mechanism by which LDL aggregates could contribute to foam cell formation. This could occur by the formation of large immune complexes that are taken up by macrophages via the Fc receptor. When immune complexes were formed with native, soluble LDL and MB47, a monoclonal antibody specific to the apoprotein B domain recognized by the LDL receptor, the subsequent uptake and degradation of the LDL by macrophages were inhibited 50-80% compared with native LDL alone. In contrast, when aggregated LDL was bound to MB47 at a similar molar ratio, the subsequent degradation of the insoluble immune complexes was two- to fivefold greater than that of aggregated LDL alone. The enhanced uptake was abolished when Fab or F(ab'>2 fragments of MB47 were substituted for the intact antibody, indicating that the increased uptake was via the Fc receptor pathway. Furthermore, the uptake of the immune complexes of aggregated LDL was reduced by competition for the Fc receptor with heat-aggregated immunoglobulin. There was also an increase in the rate of cellular cholesterol esterification and an increase in macrophage cholesteryl ester mass. Since aggregates of LDL as well as autoantibodies against modified LDL have been demonstrated in atherosclerotic lesions, it is possible that immune complexes of aggregates of modified LDL may be generated in the intima. The resultant antibody-LDL aggregates might then be even more effective in generating foam cells than the LDL aggregates themselves. (Arteriosclerosis and Thrombosis 1992;12:1258-1266) KEY WORDS • macrophages • monoclonal antibodies • low density lipoprotein aggregates immune complexes • MB47 • MB24 • Fc receptor • phagocytosis • atherosclerosis
P
revious studies from this laboratory have shown that self-aggregates of low density lipoprotein (LDL) produced by vortexing (Vx-LDL) were taken up and degraded by cultured macrophages much more rapidly than monomeric native LDL.1 This uptake was shown to occur via phagocytosis, mediated in part by the LDL receptor. These findings were confirmed by Suits et al2 using an aggregated-LDL preparation produced by treatment with phospholipase C (PLC-LDL). Studies by Frank and Fogelman3 suggested that aggregates of LDL are present in the arterial intima of Watanabe heritable hyperlipidemic (WHHL) and cholesterol-fed rabbits. Recently, they have shown that 2 hours after an intravenous bolus injection of human LDL into a normal rabbit, aggregates of LDL can be seen in the aortic intima by using ultra-rapid freezing and freeze-etching techniques.4 From the Division of Endocrinology/Metabolism, Department of Medicine, University of California, San Diego, La Jolla, Calif. Supported by National Institutes of Health grants HL-34724 and HL-14197 and in part by the Rose and Sam Stein Institute for Research on Aging. Address for reprints: Joseph L. Witztum, MD, UCSD Department of Medicine 0682, 9500 Gilman Drive, La Jolla, CA 92093-0682. Received December 15, 1991; revision accepted July 24, 1992.
•
In cell culture, the LDL receptor activity of monocyte-derived macrophages is downregulated by exposure to serum or LDL. Although monocytes that have recently entered the intima may still express LDL receptor activity, under conditions of marked hypercholesterolemia or in advanced lesions, arterial macrophages appear to express little or no LDL receptor mRNA.5 Thus, for LDL aggregates to continue to contribute importantly to macrophage accumulation of cholesterol under these conditions, an alternative pathway for macrophage uptake may be needed. Antibody-antigen complexes are taken up by macrophages via the Fc receptor pathway, and this should not be downregulated as a result of cholesterol loading. Although soluble antigen-antibody complexes bind readily to Fc receptors on macrophages, large, insoluble, multimeric complexes are required for high-affinity binding and rapid internalization. We now report that a model system of LDL aggregates complexed with monoclonal antibodies against LDL are taken up and degraded by macrophages twoto fivefold faster than uncomplexed aggregates of LDL. Moreover, this uptake is phagocytic, being mediated by the Fc receptor via the zipper mechanism.6 Since aggregates of LDL and modified LDL are likely to occur in the intima and autoantibodies to epitopes of modified LDL are present in plasma,7-11 the formation of such
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Khoo et al Macrophage Uptake of Immune Complexes of LDL immune complexes may provide an additional pathway for macrophage uptake of LDL and could play a role in accelerating cholesteryl ester accumulation. Methods Cells Mouse peritoneal macrophages were elicited by intraperitoneal injection of 2 ml thioglycollate medium (DIFCO) 3 days before harvest. For degradation and esterification assays, macrophages were plated in 24-well clustered dishes (lxlO 6 cells per well) in Iscove's modified Dulbecco's medium (IMDM) (GIBCO) containing 10% fetal bovine serum. For experiments in which cellular cholesterol mass was measured, six-well clustered dishes were used (4xlO 6 cells per well). Two hours after plating, nonadherent cells were removed by washing with phosphate-buffered saline (PBS). The cells were then incubated overnight at 37°C in IMDM containing 5 mg/ml lipoprotein-deficient serum (LPDS). Lipoproteins LDL (d=1.019-1.063 g/1) and LPDS (d> 1.215 g/1) were isolated by preparative ultracentrifugation from fresh human plasma as described previously.1 LDL was radioiodinated by the method of Salacinski et al.12 Vx-LDL was prepared by vortexing LDL (0.5 mg protein/ml PBS) for 60 seconds as previously described,1 and PLC-LDL was produced by incubating LDL (0.25 mg protein/ml PBS) with 1 unit/ml phospholipase C (type XI, from Bacillus cereus; Sigma) at 37°C for 1 hour.2 Antibodies Protein A-purified apoprotein (apo) B-specific monoclonal antibodies MB4713 and MB2414 against LDL were generously provided by Dr. Richard S. Smith from Johnson and Johnson Biotechnology Center, La Jolla, Calif. Both MB47 and MB24 were identified by double immunodiffusion as immunoglobulin G2a (IgG2a).13>14 A control mouse immunoglobulin (IgG2a) was purchased from Calbiochem Corp. Fab fragments of MB47 were prepared as previously described by digestion with immobilized papain, followed by chromatography on a column of immobilized protein A-agarose (Pierce) to remove the Fc portion.13 F(ab') 2 fragments of MB47 were prepared by using the Pierce Immunopure F(ab') 2 kit. The MB47 was digested with immobilized pepsin and separated on an immobilized protein A-agarose column. One-milliliter fractions were collected, and absorbance was measured at 280 nm. The fractions containing F(ab') 2 fragments were combined and concentrated to a final volume of 1 ml in a Centricon 10 microconcentrator (Amicon). Products were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Heat-aggregated IgG was prepared by heating the control mouse IgG (20 mg/ml) at 63°C for 20 minutes, according to the method described by Griffith et al.15 Preparation of LDL-Immune Complexes LDL-immune complexes were prepared by incubating monomeric native LDL, Vx-LDL, or PLC-LDL with the indicated concentration of antibody for 16 hours at 4°C. The molar ratios were calculated by assuming a
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molecular mass for apo B of 550 kd, for monoclonal and control IgG of 166 kd, for Fab fragments of 40 kd, and for F(ab') 2 fragments of 105 kd. The ability of MB47 and its Fab or F(ab') 2 fragments to inhibit the degradation of LDL by human skin fibroblasts was measured as described13 by using cells grown to near confluency on 24-well clustered dishes that were incubated overnight in 5 mg/ml LPDS before the addition of soluble LDLMB47 complexes. Other Methods The degradation of 125I-LDL by cells was determined using methods described by Goldstein et al.16 Each monolayer of thioglycollate-elicited mouse peritoneal macrophages received 0.5 ml IMDM containing 5 mg/ml LPDS and the indicated concentration of 125ILDL protein. After incubation for the specified time at 37°C, the medium was removed. The amount of apo B degraded was determined by the 125I-trichloroacetic acid (TCA)-soluble (noniodide) material present in the medium. The cells were washed with PBS and treated with 0.1% trypsin in saline to remove LDL bound to the surface of macrophages. The types of aggregated LDL used for the various experiments did not adhere to the plastic dishes. The remaining cells were solublized in 0.2 M NaOH, and aliquots were removed for counting as well as for protein determination by the method of Lowry et al.17 For these studies we have defined the amount of 125I released from the cells by trypsin as "bound," the total amount of 125I remaining in the cells at the end of the trypsinization as "cell content," and the amount of noniodide TCA-soluble material present in the medium as amount "degraded." "Total uptake" was defined as the sum of bound plus cell content plus amount degraded. To measure the effect of heat-aggregated IgG to compete for the degradation of LDL-immune complexes, a 200-fold protein excess of the aggregated IgG was added to the cell monolayers 1 hour before the addition of the 125I-LDL. The incorporation of [l4C]oleate into cellular cholesteryl [14C]oleate was determined by the method described by Goldstein et al.16 Briefly, the indicated concentrations of LDL protein/ml IMDM, containing 5 mg/ml LPDS, were incubated with mouse peritoneal macrophages for 6 hours at 37°C. One hour before harvest, 0.2 mM [14C]oleate/albumin (11,000 dpm/nmol) was added to each well. The monolayers were washed with PBS, and the total lipid was extracted in situ. To measure cellular sterol content, macrophage monolayers on 35-mm culture dishes were incubated with 2 5 100 fig LDL protein/ml medium for 24 hours in serumfree medium. After washing three times with PBS, treating with 0.1% trypsin for 15 minutes at 37°C, and rewashing three times with PBS, the cells were scraped into 1 ml distilled water and sonicated for 20 seconds. Aliquots were removed for protein determination, and the remainder was extracted for total lipids by the method of Folch et al.18 The mass of total and free cholesterol was determined by the enzymatic/fluorometric method of Gamble et al.19 Results The monoclonal antibody MB47 has been shown to bind to the LDL receptor-binding domain of apo B.13
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We speculate that such immune complexes could exist in vivo. Further studies are needed to determine if the LDL aggregates present in the intima contain oxidized LDL and if autoantibodies against oxidized LDL are associated with such aggregates. Uptake of antibodyaggregated (modified) LDL complexes by macrophages not only could lead to enhanced macrophage accumulation of cholesterol but also could result in the crosslinking of Fc receptors, which has been shown to trigger the release of pro-oxidants such as superoxide anion and hydrogen peroxide.36-37 This in turn could lead to the generation of still more oxidized LDL. 313839 Furthermore, it has been reported that uptake of insoluble LDL-immune complexes by human monocyte-derived macrophages, while leading to increased intracellular cholesteryl ester accumulation, paradoxically led to enhanced LDL receptor activity.30-40 All of these in vitro studies suggest that uptake of immune complexes containing LDL could significantly modify macrophage behavior and thereby influence the atherogenic process. Further studies are needed to determine the consequences for the uptake of such immune complexes by macrophages and whether this indeed occurs in vivo.
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Vx-LDL (Mg protein/ml) FIGURE 9. Line plots showing concentration-dependent binding (panel A), internalization (panel B), degradation (panel C), and degradation as a percentage of total uptake (panel D) of vortexed (Vx) low density lipoprotein (LDL) and immune complexes of Vx-LDL by mouse peritoneal macrophages. Increasing concentrations of l25I-Vx-LDL were preincubated for 16 hours at 4°C in the absence ( o ) or presence ( • ) of equal concentrations ofMB47 and MB24 at a molar ratio of 5:1 (total antibodies: apoprotein B). I25I-VxLDL and immune complexes of l25I-Vx-LDL were added to monolayer cultures of mouse peritoneal macrophages at the indicated LDL protein concentrations. After incubation at 37°C for 5 hours, the media were removed and analyzed for 12S I-trichloroacetic acid-soluble (noniodide) material. The cells were treated with trypsin, washed with phosphatebuffered saline, and solublized in 0.2 M NaOH. Each point represents the mean of triplicate determinations±SD. Gisinger et al30 demonstrated that when soluble and insoluble immune complexes of LDL were adsorbed to erythrocytes, they were avidly taken up by macrophages. Monoclonal antibodies bind to specific epitopes on the LDL particle and form soluble immune complexes. In our model studies, we used these monoclonal antibodies to prepare highly defined immune complexes of LDL.
References 1. Khoo JC, Miller E, McLoughlin P, Steinberg D: Enhanced macrophage uptake of low density lipoprotein after self-aggregation. Arteriosclerosis 1988;8:348-358 2. Suits AG, Chait A, Aviram M, Heinecke JW: Phagocytosis of aggregated lipoprotein by macrophage: Low density lipoprotein receptor-dependent foam-cell formation. ProcNatlAcad Sci USA 1989;86:2713-2717 3. Frank JS, Fogelman AM: Infrastructure of the intima in WHHL and cholesterol-fed rabbit aortas prepared by ultra-rapid freezing and freeze-etching. J Lipid Res 1989;30:967-978 4. Nievelstein PFEM, Fogelman AM, Mottino G, Frank JS: Lipid accumulation in rabbit aortic intima 2 hours after bolus infusion of low density lipoprotein: A deep-etch and immunolocalization study of ultrarapidly frozen tissue. Arterioscler Thromb 1991;11: 1795-1805 5. Yla-Herttuala S, Rosenfeld ME, Parthasarathy S, Sigal E, Sarkioja T, Witztum JL, Steinberg D: Gene expression in macrophage-rich human atherosclerotic lesions: 15-Lipoxygenase and acetyl LDL receptor messenger RNA colocalize with oxidation specific lipid— protein adducts. J Clin Invest 1991;87:1146-1152 6. Griffin FM, Griffin JA, Leider JE, Silverstein SC: Studies on the mechanism of phagocytosis: I. Requirements for circumferential attachment of particle-bound ligands to specific receptors on the macrophage plasma membrane. / Exp Med 1975;142:1263-1282 7. Palinski W, Rosenfeld ME, Yla-Herttuala S, Gurtner GC, Socher SS, Butler SW, Parthasarathy S, Carew TE, Steinberg D, Witztum JL: Low density lipoprotein undergoes oxidative modification in vivo. Proc NatlAcad Sci USA 1989;86:1372-1376 8. Rosenfeld ME, Palinski W, Yla-Herttuala S, Butler S, Witztum JL: Distribution of oxidation specific lipid-protein adducts and apolipoprotein B in atherosclerotic lesions of varying severity from WHHL rabbits. Arteriosclerosis 1990;10:336-349 9. Parums DV, Brown DL, Mitchinson MJ: Serum antibodies to oxidized low density lipoprotein and ceroid in chronic periaortitis. Arch Pathol Lab Med 1990;l 14:383-387 10. Witztum JL, Steinbrecher UP, Kasaniemi YA, Fisher M: Autoantibodies to glucosylated protein in the plasma of patients with diabetes mellitus. Proc NatlAcad Sci USA 1984;81:3204-3208 11. Orekhov AN, Tertov VV, Kabakov AE, Adamova IY, Pokrovsky SN, Smirnov VN: Autoantibodies against modified low density lipoprotein. Arterioscler Thromb 1991;ll:316-326 12. Salacinski PRP, McLean C, Sykes JEC, Clement-Jones VV, Lowry PJ: Iodination of proteins, glycoproteins, and peptides using a solid-phase oxidizing agent, l,3,4,6-tetrachloro-3a, 6a-diphenyl glycoluril (Iodogen). Anal Biochem 1981;117:136-146 13. Young SG, Witztum JL, Casal DC, Curtiss LK, Bernstein S: Conservation of the low density lipoprotein receptor-binding domain of apoprotein B: Demonstration by a new monoclonal antibody, MB47. Arteriosclerosis 1986;6:178-188
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14. Young SG, Smith RS, Hogle DM, Curtiss LK, Witztum JL: Two new monoclonal antibody-based enzyme-linked assays of apolipoprotein B. Clin Chem 1986;32:1484-1490 15. Griffith RL, Virella GT, Stevenson HC, Lopes-Virella MF: Low density lipoprotein metabolism by human macrophages activated with low density lipoprotein immune complexes. J Exp Med 1988; 168:1041-1059 16. Goldstein JL, Basu SK, Brown MS: Receptor-mediated endocytosis of low density lipoprotein in cultured cells. Methods Enzymol 1983;98:241-260 17. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. / Biol Chem 1951;193: 265-275 18. Folch J, Lees M, Sloane Stanley GH: A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 1957;226:497-509 19. Gamble WM, Vaughan M, Kruth HS, Avigan J: Procedure for determination of free and total cholesterol in micro- or nanogram amounts suitable for studies with cultured cells. / Lipid Res 1978; 19:1068-1070 20. Hoff HF, O'Neil J: Lesion-derived low density lipoprotein and oxidized low density lipoprotein share a lability for aggregation, leading to enhanced macrophage degradation. Arterioscler Thromb 1991;ll:1209-1222 21. Hoff HF, O'Neil J, Chisolm GM, Cole TB, Quehenberger O, Esterbauer H, Jurgens G: Modification of low density lipoprotein with 4-hydroxynonenal induces uptake by macrophages. Arteriosclerosis 1989;9:528-549 22. Tertov VV, Sobenin IA, Gabbasov ZA, Popov EG, Orekhov AN: Lipoprotein aggregation as an essential condition of intracellular lipid accumulation caused by modified low density lipoproteins. Biochem Biophys Res Commun 1989;163:489-494 23. Hurt E, Camejo G: Effect of arterial proteoglycans on the interaction of LDL with human monocyte-derived macrophages. Atherosclerosis 1987;67:115-126 24. Vijayagopal P, Srinivasan SR, Jones KM, Radhakrishnamurthy B, Berenson GS: Complexes of low-density lipoproteins and arterial proteoglycan aggregates promote cholesteryl ester accumulation in mouse macrophages. Biochim Biophys Ada 1985;837:251—261 25. Mora R, Lupu F, Simionescu N: Prelesional events in atherogenesis: Colocalization of apoprotein B, unesterified cholesterol and extracellular phospholipid liposomes in the aorta of hyperlipidemic rabbit. Atherosclerosis 1987;67:143-154 26. Falcone DJ, Salisbury BG: Fibronectin stimulates macrophage uptake of low density lipoprotein-hepann-collagen complexes. Arteriosclerosis 1988;8:263-273 27. Beaumont J-L, Beaumont V: Autoimmune hyperlipidemia./ir/ierosclerosis 1977;26:405-418
28. Szondy E, Horvath M, Mezey Z, Sizekely J, Lengyel E, Fust G, Gero S: Free and complexed anti-lipoprotein antibodies in vascular diseases. Atherosclerosis 1983;49:69-77 29. Klimov AN, Denisenko AD, Popov AV, Nagornev VA, Pleskov VM, Vinogradov AG, Denisenko TV, Magracheva EY, Kheifes GM, Kuznetzov AS: Lipoprotein-antibody immune complexes: Their catabolism and role in foam cell formation. Atherosclerosis 1985;58:1-15 30. Gisinger C, Virella GT, Lopes-Virella MF: Erythrocyte-bound low-density lipoprotein immune complexes lead to cholesteryl ester accumulation in human monocyte-derived macrophages. Clin Immunol Immunopathol 1991;59:37-52 31. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL: Beyond cholesterol: Modifications of low-density lipoprotein that increase its atherogenecity. NEnglJMed 1989;320:915-924 32. Steinbrecher UP, Fisher M, Witztum JL, Curtiss LK: Immunogenicity of homologous low density lipoprotein after methylation, ethylation, acetylation, or carbamylation: Generation of antibodies specific for derivatized lysine. J Lipid Res 1984;25:1109-1116 33. Salonen JT, Yla-Herttuala S, Yamamoto R, Butler S, Korpela H, Salonen R, Nyyssonen K, Palinski W, Witztum JL: Autoantibody against oxidised LDL and progression of carotid atherosclerosis. Lancet 1992;339:883-886 34. Yla-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D: Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest 1989;84:1087-1095 35. Yla-Herttuala S, Butler S, Picard S, Palinski W, Steinberg D, Witztum JL: Rabbit and human atherosclerotic lesions contain IgG that recognizes MDA-LDL and copper-oxidized LDL. (abstract) Arterioscler Thromb 1991;! 1:1426a 36. Aida Y, Onoue K: Triggering of the superoxide generation of macrophages by cross-linking of Fc-y receptor. J Biochem 1984;95: 1067-1072 37. Nathan CF, Root RK: Hydrogen peroxide release from mouse peritoneal macrophages: Dependence on sequential activation and triggering. J Exp Med 1977;146:1648-1662 38. Hiramatsu K, Rosen H, Heinecke JW, Wolfbauer G, Chait A: Superoxide initiates oxidation of low density-lipoprotein by human monocytes. Arteriosclerosis 1987;7:55-60 39. Cathcart MK, McNally AK, Morel DW, Chisolm GM III: Superoxide anion participation in human monocyte-mediated oxidation and conversion of low-density lipoprotein to a cytotoxin. J Immunol 1989;142:1963-1969 40. Lopes-Virella MF, Griffith RL, Shunk KA, Virella GT: Enhanced uptake and impaired intracellular metabolism of low density lipoprotein complexed with anti-low density lipoprotein antibodies. Arterioscler Thromb 1991;11:1356-1367
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Monoclonal antibodies against LDL further enhance macrophage uptake of LDL aggregates. J C Khoo, E Miller, F Pio, D Steinberg and J L Witztum Arterioscler Thromb Vasc Biol. 1992;12:1258-1266 doi: 10.1161/01.ATV.12.11.1258 Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1992 American Heart Association, Inc. All rights reserved. Print ISSN: 1079-5642. Online ISSN: 1524-4636
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