The Oxidation of Lipoproteins by Monocytes-Macrophages

low density lipoprotein ceruloplasmin endothelial cell interferon lipoxygenase lipopolysaccharide myeloperoxidase superoxide anion oligodeoxyribonucleotide(s) nitrogen monoxide (nitric oxide) protein kinase C conventional PKC cytosolic phospholipase A2 phorbol myristate acetate smooth muscle cell opsonized zymosan The oxidation of lipoproteins has been proposed as a biological process that initiates and accelerates arterial lesion development (1Chisolm G.M. Penn M.S. Fuster V. Ross R. Topol E.J. Oxidized Lipoproteins and Atherosclerosis. Raven Press, New York1996: 129-149Google Scholar, 2Steinberg D. J. Biol. Chem. 1997; 272: 20963-20966Abstract Full Text Full Text PDF PubMed Scopus (1407) Google Scholar, 3Berliner J.A. Navab M. Fogelman A.M. Frank J.S. Demer L.L. Edwards P.A. Watson A.D. Lusis A.J. Circulation. 1995; 91: 2488-2496Crossref PubMed Google Scholar, 4Witztum J.L. Horkko S. Ann. N. Y. Acad. Sci. 1997; 811: 88-99Crossref PubMed Scopus (45) Google Scholar, 5Heinecke J.W. Atherosclerosis. 1998; 141: 1-15Abstract Full Text Full Text PDF PubMed Google Scholar). Oxidized lipoproteins accumulate in lesions (6Yla-Herttuala S. Curr. Opin. Lipidol. 1998; 9: 337-344Crossref PubMed Scopus (93) Google Scholar) and may form at other inflammatory sites. Whether the oxidized lipoprotein is an initiator or accelerator of disease is the subject of speculation, debate, and intensive study. Various cellular and biochemical mediators of lipoprotein oxidation in vivo have been proposed, but none has yet been proven to be responsible.Two decades ago we demonstrated that low density lipoprotein (LDL),1 the plasma level of which correlates with the risk of atherosclerosis, could injure endothelial cells (ECs) in culture (7Hessler J.R. Robertson Jr., A.L. Chisolm G.M. Atherosclerosis. 1979; 32: 213-229Abstract Full Text PDF PubMed Scopus (389) Google Scholar). The capacity of LDL to injure cells was directly related to the level of LDL oxidation, and we speculated on a possible role for oxidized LDL-mediated endothelial injury in atherogenesis (8Hessler J.R. Morel D.W. Lewis L.J. Chisolm G.M. Arteriosclerosis. 1983; 3: 215-222Crossref PubMed Google Scholar, 9Morel D.W. Hessler J.R. Chisolm G.M. J. Lipid Res. 1983; 24: 1070-1076Abstract Full Text PDF PubMed Google Scholar). Contemporaneously, Dr. Daniel Steinberg's group (10Henriksen T. Mahoney E.M. Steinberg D. Arteriosclerosis. 1983; 3: 149-159Crossref PubMed Google Scholar, 11Henriksen T. Mahoney E.M. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6499-6503Crossref PubMed Google Scholar) demonstrated that LDL exposed to cultured ECs was altered such that it became a ligand for scavenger receptors. In 1984, both Steinberg's group and ours (12Steinbrecher U.P. Parthasarathy S. Leake D.S. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3883-3887Crossref PubMed Google Scholar, 13Morel D.W. DiCorleto P.E. Chisolm G.M. Arteriosclerosis. 1984; 4: 357-364Crossref PubMed Google Scholar) demonstrated that the "EC-modified LDL" they had characterized and the "oxidized LDL" we had described were the same entity. Their report highlighted the macrophage recognition of the EC-oxidized lipoprotein, and ours the capacity of EC or smooth muscle cell (SMC)-oxidized LDL to injure cells. These papers introduced the concept that reactive oxygen species from vascular cells could transform LDL, causing it to exhibit dramatically altered composition and atherogenic properties. The first demonstration that certain leukocyte populations could oxidize LDL employed human neutrophils and activated populations of adherent human monocytes, cells well known to generate abundant reactive oxygen species in vitro and in vivo (14Cathcart M.K. Morel D.W. Chisolm G.M. J. Leukocyte Biol. 1985; 38: 341-350Crossref PubMed Scopus (405) Google Scholar).The identity of the cells responsible for the oxidation of LDL that accumulates in lesions is uncertain. Although it is well known that free ferrous or cupric ions catalyze lipid peroxidation reactions in vitro, the presence of free metal ions in vivo is doubted (15Dabbagh A.J. Frei B. J. Clin. Invest. 1995; 96: 1958-1966Crossref PubMed Google Scholar). Multiple mechanisms exist in vivo for binding free transition metal ions, rendering them redox-inactive (15Dabbagh A.J. Frei B. J. Clin. Invest. 1995; 96: 1958-1966Crossref PubMed Google Scholar, 16Aasa R. Malmstrom B.G. Saltman P. Vangard T. Biochim. Biophys. Acta. 1963; 75: 203-222Crossref PubMed Google Scholar, 17Thomas C.E. Biochim. Biophys. Acta. 1992; 1128: 50-57Crossref PubMed Google Scholar). In this minireview, we take the position that monocyte-derived macrophages are likely candidates to mediate the in vivo oxidation of lipoproteins, because they are prominent in arterial lesions, known to generate activation-dependent reactive oxygen species, and, unlike EC and SMC (12Steinbrecher U.P. Parthasarathy S. Leake D.S. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3883-3887Crossref PubMed Google Scholar, 18Heinecke J.W. Rosen H. Chait A. J. Clin. Invest. 1984; 74: 1890-1894Crossref PubMed Google Scholar), capable in vitro of LDL oxidation in media free of metal ion additives. In vitro LDL appears to be oxidized extracellularly without interaction with the LDL receptor (19Tangirala R.K. Mol M.J. Steinberg D. J. Lipid Res. 1996; 37: 835-843Abstract Full Text PDF PubMed Google Scholar, 20Cathcart M.K. Li Q. Chisolm G.M. J. Lipid Res. 1995; 36: 1857-1865Abstract Full Text PDF PubMed Google Scholar, 21Aviram M. Rosenblat M. J. Lipid Res. 1994; 35: 385-398Abstract Full Text PDF PubMed Google Scholar). There are multiple potential pathways through which monocytes-macrophages may promote extracellular LDL oxidation. In this review we evaluate cellular mechanisms (both enzymatic and non-enzymatic) for LDL oxidation. We use the term "monocyte-macrophage" as a shorthand reference to in vitro studies performed on isolated monocytes, macrophages, and monocyte-like cell lines.The Role of CeruloplasminThe presence of free copper or iron ions in vivo is unlikely, but an interesting concept in redox metal-dependent oxidation of LDL by phagocytes was recently introduced. The idea followed the surprising observation that the copper-containing acute phase plasma protein, ceruloplasmin (Cp), studied for years as an antioxidant, could act instead as a potent oxidant of LDL (22Ehrenwald E. Chisolm G.M. Fox P.L. J. Clin. Invest. 1994; 93: 1493-1501Crossref PubMed Google Scholar). The oxidant capacity of Cp may have been overlooked previously because of the difficulty in preserving Cp in an undegraded state during its purification (23Ehrenwald E. Fox P.L. Arch. Biochem. Biophys. 1994; 309: 392-395Crossref PubMed Scopus (35) Google Scholar). Cp is a 132-kDa glycoprotein that contains seven copper atoms per molecule and carries most of the copper in blood. We found that LDL exposed to Cp exhibited many characteristics of LDL oxidized in the presence of free cupric ion (22Ehrenwald E. Chisolm G.M. Fox P.L. J. Clin. Invest. 1994; 93: 1493-1501Crossref PubMed Google Scholar). This oxidant activity of Cp in vitro has been verified by other laboratories (24Van Lenten B.J. Hama S.Y. de Beer F.C. Stafforini D.M. McIntyre T.M. Prescott S.M. La Du B.N. Fogelman A.M. Navab M. J. Clin. Invest. 1995; 96: 2758-2767Crossref PubMed Google Scholar, 25Lamb D.J. Leake D.S. FEBS Lett. 1994; 338: 122-126Crossref PubMed Scopus (84) Google Scholar, 26Swain J.A. Darley-Usmar V. Gutteridge J.M. FEBS Lett. 1994; 342: 49-52Crossref PubMed Scopus (139) Google Scholar).To determine whether Cp could substitute as a "physiological" source of redox-active metal, the equivalent of normal (unevoked) plasma levels of Cp was added to cultures in which ECs or SMCs were exposed to LDL in RPMI 1640, a cell culture medium without transition metal ion additives. Cp markedly enhanced LDL oxidation by both cell types (27Mukhopadhyay C.K. Ehrenwald E. Fox P.L. J. Biol. Chem. 1996; 271: 14773-14778Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Proteolytic cleavage of Cp or removal of the one of its seven copper atoms that is near His426, prevented its oxidative action (27Mukhopadhyay C.K. Ehrenwald E. Fox P.L. J. Biol. Chem. 1996; 271: 14773-14778Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 28Mukhopadhyay C.K. Fox P.L. Biochemistry. 1998; 37: 14222-14229Crossref PubMed Scopus (48) Google Scholar, 29Mukhopadhyay C.K. Mazumder B. Lindley P.F. Fox P.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11546-11551Crossref PubMed Scopus (114) Google Scholar). Cell-derived superoxide anion (O⨪2) was essential for the enhancement of LDL oxidation by Cp under these conditions.Because monocytes-macrophages express Cp mRNA and protein (30Yang F.M. Friedrichs W.E. Cupples R.L. Bonifacio M.J. Sanford J.A. Horton W.A. Bowman B.H. J. Biol. Chem. 1990; 265: 10780-10785Abstract Full Text PDF PubMed Google Scholar, 31Fleming R.E. Whitman I.P. Gitlin J.D. Am. J. Physiol. 1991; 260: L68-L74PubMed Google Scholar), the hypothesis that cell-derived Cp contributes to LDL oxidation by these cells was tested. A model system similar to that of Cathcart et al. (20Cathcart M.K. Li Q. Chisolm G.M. J. Lipid Res. 1995; 36: 1857-1865Abstract Full Text PDF PubMed Google Scholar, 32Cathcart M.K. Chisolm G.M. McNally A.K. Morel D.W. In Vitro Cell. Dev. Biol. 1988; 24: 1001-1008Crossref PubMed Scopus (55) Google Scholar) was adopted, which used the human myelomonocytic cell line, U937, activated by zymosan in RPMI 1640 medium. Exogenous Cp added to unactivated U937 cells caused LDL oxidation to an extent similar to that by zymosan in the absence of exogenous Cp (33Ehrenwald E. Fox P.L. J. Clin. Invest. 1996; 97: 884-890Crossref PubMed Google Scholar). Zymosan induced Cp mRNA and (after a 5–6-h lag following activation) Cp protein synthesis (33Ehrenwald E. Fox P.L. J. Clin. Invest. 1996; 97: 884-890Crossref PubMed Google Scholar). This lag coincided with the lag prior to measurable LDL oxidation previously reported (34Cathcart M.K. McNally A.K. Morel D.W. Chisolm G.M. J. Immunol. 1989; 142: 1963-1969PubMed Google Scholar,35Hiramatsu K. Rosen H. Heinecke J.W. Wolfbauer G. Chait A. Arteriosclerosis. 1987; 7: 55-60Crossref PubMed Google Scholar). A neutralizing antibody to Cp blocked LDL oxidation by activated U937 cells, as did antisense oligodeoxyribonucleotides (ODN) targeted against segments of the Cp mRNA (33Ehrenwald E. Fox P.L. J. Clin. Invest. 1996; 97: 884-890Crossref PubMed Google Scholar). These studies suggested a possible in vivo role for Cp in monocyte-macrophage-mediated LDL oxidation, but they also revealed that cell-derived factors in addition to Cp are required for optimal oxidation. Among the candidates are, for example, O⨪2 and lipoxygenase, both of which are increased by activation of monocyte-macrophage-related cells. The role of O⨪2 may be to reduce the Cp-bound "oxidant" copper (28Mukhopadhyay C.K. Fox P.L. Biochemistry. 1998; 37: 14222-14229Crossref PubMed Scopus (48) Google Scholar).Factors that stimulate monocyte-macrophage Cp production and could contribute to Cp accumulation in vivo include endotoxin (31Fleming R.E. Whitman I.P. Gitlin J.D. Am. J. Physiol. 1991; 260: L68-L74PubMed Google Scholar) and interferon (IFN)-γ (36Mazumder B. Mukhopadhyay C.K. Prok A. Cathcart M.K. Fox P.L. J. Immunol. 1997; 159: 1938-1944PubMed Google Scholar). The observation that a bacterial product is an agonist may be significant given the possible link between bacterial infection and atherosclerosis (37Libby P. Egan D. Skarlatos S. Circulation. 1997; 96: 4095-4103Crossref PubMed Google Scholar). Because T-cells, a major source of IFN-γ, populate arterial lesion sites, Cp accumulation could be enhanced by a local inflammatory response. However, IFN-γ also stimulates antioxidant production by monocytes-macrophages (38Christen S. Thomas S.R. Garner B. Stocker R. J. Clin. Invest. 1994; 93: 2149-2158Crossref PubMed Google Scholar) and has been shown under certain conditions to inhibit LDL oxidation (39Fong L.G. Albert T.S. Hom S.E. J. Lipid Res. 1994; 35: 893-904Abstract Full Text PDF PubMed Google Scholar, 40Folcik V.A. Cathcart M.K. J. Lipid Res. 1994; 35: 1570-1582Abstract Full Text PDF PubMed Google Scholar).The studies of Cp in cell-mediated LDL oxidation suggest other protein-bound redox-active transition metals might also participate in extracellular oxidation events. Under physiological conditions globin degradation products such as hemin (41Camejo G. Halberg C. Manschik-Lundin A. Hurt-Camejo E. Rosengren B. Olsson H. Hansson G.I. Forsberg G.B. Ylhen B. J. Lipid Res. 1998; 39: 755-766Abstract Full Text Full Text PDF PubMed Google Scholar) and holo forms of iron binding proteins such as transferrin (25Lamb D.J. Leake D.S. FEBS Lett. 1994; 338: 122-126Crossref PubMed Scopus (84) Google Scholar) and ferritin (42Abdalla D.S.P. Campa A. Monteiro H.P. Atherosclerosis. 1992; 97: 149-159Abstract Full Text PDF PubMed Scopus (72) Google Scholar) may catalyze oxidation reactions. The reducing agents required for activity and the physiological significance of these protein-bound redox metals remain uncertain.The Role of O⨪2The role of O⨪2 in monocyte-macrophage LDL oxidation is as debated as that of metal ion, and the two controversies are interrelated. The dependence on O⨪2 is less in cell systems in which the culture medium contains free redox metal ions. Using metal ion-containing (Ham's F12) medium, Garner et al. (47Garner B. Dean R.T. Jessup W. Biochem. J. 1994; 301: 421-428Crossref PubMed Google Scholar) reported O⨪2-independent LDL oxidation by human monocytes-macrophages. Our early studies with monocytes-macrophages were conducted in RPMI 1640 in an effort to mimic the predicted absence of free metal ions in vivo. Our studies showed that LDL oxidation required activation of the cells by certain activators, including opsonized zymosan (Zop) or LPS; other activators were not effective (14Cathcart M.K. Morel D.W. Chisolm G.M. J. Leukocyte Biol. 1985; 38: 341-350Crossref PubMed Scopus (405) Google Scholar, 32Cathcart M.K. Chisolm G.M. McNally A.K. Morel D.W. In Vitro Cell. Dev. Biol. 1988; 24: 1001-1008Crossref PubMed Scopus (55) Google Scholar). (Addition of free metal ions to monocytes incubated in RPMI 1640 resulted in activation-independent LDL oxidation (20Cathcart M.K. Li Q. Chisolm G.M. J. Lipid Res. 1995; 36: 1857-1865Abstract Full Text PDF PubMed Google Scholar).) The activation dependence in RPMI 1640, the successful inhibition by numerous antioxidants (14Cathcart M.K. Morel D.W. Chisolm G.M. J. Leukocyte Biol. 1985; 38: 341-350Crossref PubMed Scopus (405) Google Scholar, 32Cathcart M.K. Chisolm G.M. McNally A.K. Morel D.W. In Vitro Cell. Dev. Biol. 1988; 24: 1001-1008Crossref PubMed Scopus (55) Google Scholar), and the well described enhancement of O⨪2 production by monocytes following activation were consistent with a requirement for O⨪2. We and others have found cell-derived, extracellular O⨪2 to be required, but not sufficient, for LDL oxidation by monocytes-macrophages (34Cathcart M.K. McNally A.K. Morel D.W. Chisolm G.M. J. Immunol. 1989; 142: 1963-1969PubMed Google Scholar, 35Hiramatsu K. Rosen H. Heinecke J.W. Wolfbauer G. Chait A. Arteriosclerosis. 1987; 7: 55-60Crossref PubMed Google Scholar). O⨪2-generating systems alone do not mediate LDL oxidation (43Lynch S.M. Frei B. J. Lipid Res. 1993; 34: 1745-1753Abstract Full Text PDF PubMed Google Scholar, 44Bedwell S. Dean R.T. Jessup W. Biochem. J. 1989; 262: 707-712Crossref PubMed Google Scholar, 45Bonnefont-Rousselot D. Gardès-Albert M. Lepage S. Delattre J. Ferradini C. Radiat. Res. 1992; 132: 228-236Crossref PubMed Scopus (29) Google Scholar). Others have speculated that superoxide dismutase could be inhibitory by acting as a metal chelator, rather than a O⨪2scavenger, giving the false impression of O⨪2 dependence (46Jessup W. Simpson J.A. Dean R.T. Atherosclerosis. 1993; 99: 107-120Abstract Full Text PDF PubMed Google Scholar). However, further evidence for the requirement for O⨪2 in this system is derived from recent studies using in vitro knockout of p47phox. O⨪2 and LDL oxidation were concomitantly ablated when this requisite component of the NADPH oxidase complex was inhibited by antisense ODN. 2E. Bey and M. K. Cathcart, submitted for publication.Xing et al. (48Xing X. Baffic J. Sparrow C.P. J. Lipid Res. 1998; 39: 2201-2208Abstract Full Text Full Text PDF PubMed Google Scholar) provided evidence that systems using zymosan as an activator may in fact be metal ion-dependent because the yeast cell walls, like transferrin and ferritin, can carry iron in an oxidized (Fe3+) state. Their results are consistent with Zop-bound Fe3+ being reduced to Fe2+ by cell-derived O⨪2, which then catalyzes LDL oxidation, thus putting the Zop-activated cell system into a category parallel with the concept presented above: that activated monocytes-macrophages oxidize LDL by a pathway requiring O⨪2 to reduce bound transition metal (e.g. Cp-borne cupric or ferritin-, transferrin-, or hemin-borne ferric).The formation of O⨪2 and/or its dismutation product, H2O2, by monocytes-macrophages appears to be essential for LDL oxidation in vitro, whether the process is Cp-, myeloperoxidase- (MPO) (see below), or Zop-dependent. Accordingly, considerable effort has been devoted to elucidate signaling pathways regulating the generation of O⨪2. Multiple approaches have demonstrated that the pathway for optimal oxidation of LDL by Zop-activated monocytes-macrophages involved calcium via both influx and release from intracellular stores (50Li Q. Tallant A. Cathcart M.K. J. Clin. Invest. 1993; 91: 1499-1506Crossref PubMed Google Scholar). Using PKC inhibitors, down-regulation of PKC expression by PMA, and antisense ODN, the requirement for activation of the calcium-dependent cPKC isoenzyme, PKCα, was convincingly shown (51Li Q. Cathcart M.K. J. Biol. Chem. 1994; 269: 17508-17515Abstract Full Text PDF PubMed Google Scholar, 52Li Q. Subbulakshmi V. Fields A.P. Murray N.R. Cathcart M.K. J. Biol. Chem. 1999; 274: 3764-3771Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Selective inhibition of cytosolic phospholipase A2 (cPLA2) using antisense ODN and pharmacological inhibitors, revealed cPLA2 activity to be another essential step in the signaling sequence for O⨪2 production and LDL oxidation (53Li Q. Cathcart M.K. J. Biol. Chem. 1997; 272: 2404-2411Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Addition of arachidonic acid, the product of cPLA2, restored both the production of O⨪2 and LDL oxidation (53Li Q. Cathcart M.K. J. Biol. Chem. 1997; 272: 2404-2411Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Recent data, mentioned above, using p47phoxantisense ODN suggest that the enzymatic source of O⨪2 is NADPH oxidase. PKC activation may be involved in phosphorylation of various protein components of this enzyme complex.The Role of 15-LipoxygenaseAre there other cell-derived factors required for optimal monocyte-macrophage oxidation of LDL? Several have been studied; these include not only Cp but also LO and MPO. LOs are non-heme iron-containing enzymes found in various cells, including reticulocytes, monocytes-macrophages, and certain endothelial cells. They catalyze the direct insertion of molecular oxygen into polyenoic fatty acids, forming hydroperoxides. There are a number of ways in which LOs could participate in LDL oxidation. LO could oxidize cellular fatty acid, cholesteryl ester, or phospholipid substrates, and the hydroperoxide products could transfer to LDL, making LDL more susceptible to oxidation (54Parthasarathy S. Wieland E. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1046-1050Crossref PubMed Google Scholar). If LO could come in contact with LDL, it could use phospholipid or cholesteryl ester as substrate (55Belkner J. Stender H. Kuhn H. J. Biol. Chem. 1998; 273: 23225-23232Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), leading to lipid peroxidation. LO products could also participate in signal transduction pathways regulating other monocyte-macrophage functions involved in oxidation.In monocyte-macrophage systems, 5-LO has been as a in LDL oxidation V.A. Cathcart M.K. J. Lipid Res. 1993; 34: Full Text PDF PubMed Google Scholar, W. Darley-Usmar V. S. Biochem. J. 1991; PubMed Scopus Google Scholar). Sparrow et al. C.P. Parthasarathy S. Steinberg D. J. 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