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Heterogeneity of cellular cholesteryl ester accumulation by human monocyte-derived macrophages

  • Sonia I. Skarlatos
    Affiliations
    Section of Experimental Atherosclerosis, Molecular Disease Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA
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  • Mustapha Rouis
    Affiliations
    INSERM U-321, Unité e recherches sur Les Lipoprotéines et l'Athérogénèse, Pavillon Benjamin Delessert, Hôpital de la Pitie, 83, Bd. de l'Hôpital 75651 Paris Cedex 13 France
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  • M.John Chapman
    Affiliations
    INSERM U-321, Unité e recherches sur Les Lipoprotéines et l'Athérogénèse, Pavillon Benjamin Delessert, Hôpital de la Pitie, 83, Bd. de l'Hôpital 75651 Paris Cedex 13 France
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  • Howard S. Kruth
    Correspondence
    Correspondence to: Dr. Howard S. Kruth, National Institutes of Health, Building 10 Room 5N-113, Bethesda, MD 20892, USA. Tel.: (301)-496-4826.
    Affiliations
    Section of Experimental Atherosclerosis, Molecular Disease Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA
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      Abstract

      We have studied cholesteryl ester accumulation in human monocyte-derived macrophages, which together with smooth muscle cells, represent the major cell types that accumulate cholesterol in atherosclerotic lesions. Monocyte-derived macrophages were incubated with either acetylated low density lipoprotein (AcLDL) or non-lipoprotein cholesterol and the question as to whether all of the cells, or specific cell subpopulations could accumulate cholesteryl ester was examined. We stained cholesteryl ester in monocyte-macrophages with the fluorescent probe filipin. Cholesteryl ester accumulated as lipid droplets that were widely dispersed in the cell cytoplasm. Interestingly, no more than 65% of monocytemacrophages accumulated cholesteryl ester during the 1st day of incubation with non-lipoprotein cholesterol. By 2 days of incubation, greater than 90% of cells displayed cholesteryl ester deposition. The cholesteryl ester which accumulated during the 2nd day of incubation was derived from unesterified cholesterol that had accumulated during the 1st day of incubation. This finding was substantiated by the following: (1) chemical measurements showed that the total cholesterol content of monocyte-macrophages did not increase further after the 1 st day of incubation, and (2) all monocyte-macrophages had accumulated fluorescent tagged cholesterol during the 1st day of incubation. In contrast to the results obtained with non-lipoprotein cholesterol, more than 90% of monocyte-macrophages incubated with AcLDL for 1 day accumulated cholesteryl ester in two experiments. However, less than 62% of monocyte-macrophages accumulated cholesteryl ester in two other experiments, thereby resembling results obtained with nonlipoprotein cholesterol. Again, the lack of cholesteryl ester accumulation with AcLDL was not due to a lack of uptake of AcLDL, as greater than 90% of monocyte-macrophages accumulated fluorescent tagged AcLDL. The observed heterogeneity in cholesterol esterification among human monocyte-macrophages suggests that functional subpopulations of these cells may exist with respect to cholesterol processing. However, heterogeneity in cholesteryl ester accumulation did not seem to correlate with expression of HLA-DR antigen, a marker of immunological activation of macrophages. Other sources of heterogeneity most likely result from inter-cellular variation at one or more levels of regulation of the cholesterol trafficking and esterification process.

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      References

        • Mazzone T.
        • Jensen M.
        • Chait A.
        Human arterial wall cells secrete factors that are chemotactic for monocytes.
        in: Proc. Natl. Acad. Sci. USA. 80. 1983: 5094
        • Gerrity R.G.
        • Naito H.K.
        • Richardson M.
        • Schwartz C.J.
        Dietary induced atherogenesis in swine. Morphology of the intima in prelesion stages.
        Am. J. Pathol. 1979; 95: 775
        • Brown M.S.
        • Goldstein J.L.
        Lipoprotein metabolism in the macrophage: Implications for cholesterol deposition in atherosclerosis.
        Annu. Rev. Biochem. 1983; 52: 223
        • Kruth H.S.
        • Fry D.L.
        Histochemical detection and differentiation of free and esterified cholesterol in swine atherosclerosis using filipin.
        Exp. Mol. Pathol. 1984; 40: 288
        • Kruth H.S.
        Localization of unesterified cholesterol in human atherosclerotic lesions. Demonstration of filipin positive, oil-red-O-negative particles.
        Am. J. Pathol. 1984; 114: 201
        • Kruth H.S.
        Filipin-positive, Oil red O-negative particles in atherosclerotic lesions induced by cholesterol feeding.
        Lab. Invest. 1983; 50: 87
        • Shio H.
        • Haley N.J.
        • Fowler S.
        Characterization of lipid-laden aortic cells from cholesterol-fed rabbits. III. Intracellular localization of cholesterol and cholesteryl ester.
        Lab. Invest. 1979; 51: 160
        • Mazzone T.
        • Gump H.
        • Diller P.
        • Getz G.S.
        Macrophage free cholesterol content regulates apolipoprotein E synthesis.
        J. Biol. Chem. 1987; 262: 11657
        • Lesnik P.
        • Rouis M.
        • Skarlatos S.
        • Kruth H.S.
        • Chapman M.J.
        Uptake of exogenous free cholesterol induces upregulation of tissue factor expression in human monocyte-derived macrophages.
        in: Proc. Natl. Acad. Sci. USA. 89. 1992: 10370
        • Boyum A.
        Isolation of lymphocytes, granulocytes and macrophages.
        Scand. J. Immunol. 1976; 5: 9
        • Havel R.J.
        • Eder H.A.
        • Bragdon J.H.
        The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.
        J. Clin. Invest. 1955; 34: 1345
        • Basu S.K.
        • Goldstein J.L.
        • Anderson R.G.W.
        • Brown M.S.
        Degradation of cationized LDL and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts.
        in: Proc. Natl. Acad. Sci. USA. 73. 1976: 3178
        • Pitas R.E.
        • Innerarity T.L.
        • Weinstein J.N.
        • Mahley R.W.
        Acetoacetylated lipoproteins used to distinguish fibroblasts from macrophages in vitro by fluorescence microscopy.
        Arteriosclerosis. 1981; 1: 177
        • Craig I.F.
        • Via D.P.
        • Mantulin W.W.
        • Pownall H.J.
        • Gotto Jr, A.M.
        • Smith L.C.
        Low density lipoproteins reconstituted with steroids containing the nitrobenzoxadiazole fluorophore.
        J. Lipid Res. 1981; 22: 687
        • Koren E.
        • Koscec M.
        • McConathy W.J.
        • Fugate R.D.
        Possible role of macrophages in regression of atherosclerosis.
        Prog. Lipid Res. 1991; 30: 237
        • Brown M.S.
        • Goldstein J.L.
        Suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and inhibition of growth of human fibroblasts by 7-ketocholesterol.
        J. Biol. Chem. 1974; 249: 7306
        • Lowry O.H.
        • Rosebrough N.J.
        • Farr A.L.
        • Randall R.J.
        Protein measurement with the folin phenol reagent.
        J. Biol. Chem. 1951; 193: 265
        • Folch J.
        • Lees M.
        • Sloane-Stanley G.H.
        A simple method for the isolation and purification of total lipids from animal tissues.
        J. Biol. Chem. 1957; 226: 497
        • Gamble W.
        • Vaughan M.
        • Kruth H.S.
        • Avigan J.
        Procedure for determination of free and total cholesterol in micro- or nanogram amounts suitable for studies with cultured cells.
        J. Lipid Res. 1978; 19: 1068
        • Kruth H.S.
        • Vaughan M.
        Quantification of low density lipoprotein binding and cholesterol accumulation by single human fibroblasts using fluorescence microscopy.
        J. Lipid Res. 1980; 21: 123
        • Kruth H.S.
        • Cupp J.E.
        • Khan M.A.
        Method for detection and isolation of cholesteryl ester-containing `foam' cells using flow cytometry.
        Cytometry. 1987; 8: 146
        • Goldstein J.L.
        • Ho Y.K.
        • Basu S.K.
        • Brown M.S.
        Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition.
        in: Proc. Natl. Acad. Sci. USA. 76. 1979: 333
        • Bocan T.M.A.
        • Guyton J.R.
        Human aortic fibrolipid lesions. Progenitor lesions for fibrous plaques, exhibiting early formation of the cholesterol-rich core.
        Am. J. Pathol. 1985; 120: 193
        • Buja L.M.
        • Kovanen P.T.
        • Bilheimer D.W.
        Cellular pathology of homozygous familial hypercholesterolemia.
        Am. J. Pathol. 1979; 97: 327
        • Trillo A.A.
        The cell population of aortic fatty streaks in African green monkeys with special reference to granulocytic cells.
        Atherosclerosis. 1982; 43: 259
        • Weller R.O.
        Ultrastructure and cytochemistry of lipid inclusions in atherosclerosis.
        in: Cervos-Navarro J. The Cerebral Vessel Wall. Raven Press, New York1976: 51
        • Adams C.W.M.
        • Bayliss O.B.
        Crystals in atherosclerotic lesions: real or artifact?.
        Atherosclerosis. 1975; 22: 629
        • Pitas R.E.
        Expression of the acetyl low density lipoprotein receptor by rabbit fibroblasts and smooth muscle cells. Up-regulation by phorbol esters.
        J. Biol. Chem. 1991; 265: 12722
        • Andreesen R.
        • Bross J.K.
        • Osterholz J.
        • Emmrich F.
        Human macrophage maturation and heterogeneity: Analysis with a newly generated set of monoclonal antibodies to differentiation antigens.
        Blood. 1986; 67: 1257
        • Esa A.H.
        • Noga S.J.
        • Donnenberg A.D.
        • Hess A.D.
        Immunological heterogeneity of human monocyte subsets prepared by counterflow centrifugation elutriation.
        Immunology. 1986; 59: 95
        • Raff H.V.
        • Picker L.J.
        • Stobo J.D.
        Macrophage heterogeneity in man. A subpopulation of HLA-, DRbearing macrophages required for antigen-induced T cell activation, also contains stimulators for autologous reactive T cells.
        J. Exp. Med. 1980; 152: 581
        • Picker L.J.
        • Raff H.V.
        • Goldyne M.E.
        • Stobo J.D.
        Metabolic heterogeneity among human monocytes and its modulation by PGE2.
        J. Immunol. 1980; 124: 2557
        • Zuckerman S.H.
        • Ackerman S.K.
        • Douglas S.D.
        Long-term human peripheral blood monocyte cultures: establishment, metabolism and morphology of primary human monocyte-macrophage cell cultures.
        Immunology. 1979; 38: 401
        • Musson R.A.
        Human serum induces maturation of human monocytes in vitro. Changes in cytolytic activity, intracellular lysosomal enzymes, and nonspecific esterase activity.
        Am. J. Pathol. 1983; 111: 331
        • Johnson W.D.
        • Mei B.
        • Cohn Z.A.
        The separation, long-term cultivation, and maturation of the human monocyte.
        J. Exp. Med. 1977; 146: 1613
        • Steinman R.M.
        • Nogueira N.
        • Witmer M.D.
        • Tydings J.D.
        • Mellman I.S.
        Lympokine enhances the expression and synthesis of la antigens on cultured mouse peritoneal macrophages.
        J. Exp. Med. 1980; 152: 1248
        • Hansson G.K.
        • Jonasson L.
        • Holm J.
        • ClaessonWelsh L.
        Class II MHC antigen expression in the atherosclerotic plaque: smooth muscle cells express HLADR, HLA-DQ and the invariant gamma chain.
        Clin. Exp. Immunol. 1986; 64: 261
        • Van der Wal A.C.
        • Das P.K.
        • Tigges A.J.
        • Becker A.E.
        Macrophage differentiation in atherosclerosis. An in situ immunohistochemical analysis in humans.
        Am. J. Pathol. 1992; 141: 161
        • Gown A.M.
        Cell type and cell state specific antibodies in the analysis of early lesions of human atherosclerosis.
        Am. J. Hypertens. 1992; 5: 1145
        • Chang C.C.Y.
        • Doolittle G.M.
        • Chang T.Y.
        Cycloheximide sensitivity in regulation of acyl coenzyme A:cholesterol acyltransferase activity in chinese hamster ovary cells. 1. Effect of exogenous sterols.
        J. Biochem. 1986; 25: 1693
        • Tabas I.
        • Boykow G.C.
        Protein synthesis inhibition in mouse peritoneal macrophages results in increased acyl coenzyme A:cholesterol acyl transferase activity and cholesteryl ester accumulation in the presence of native low density lipoprotein.
        J. Biol. Chem. 1987; 262: 12175
        • Basheeruddin K.
        • Rawstorne S.
        • Higgins M.J.P.
        Reversible activation of rat liver acyl-CoA:cholesterol acyltransferase in vitro.
        Biochem. Soc. Trans. 1982; 10: 390
        • Gavey K.L.
        • Trujillo D.L.
        • Scallen T.J.
        Evidence for phosphorylation/dephosphorylation of rat liver acylCoA:cholesterol acyltransferase.
        in: Proc. Natl. Acad. Sci. USA. 80. 1983: 2171
        • Suckling K.E.
        • Stange E.F.
        • Dietschy J.M.
        Dual modulation of hepatic and intestinal acyl-CoA:cholesterol acyltransferase activity by (de-) phosphorylation and substrate supply in vitro.
        FEBS Lett. 1983; 151: 111
        • Kruth H.S.
        • Comly M.E.
        • Butler J.D.
        • Vanier M.T.
        • Fink J.K.
        • Wenger D.A.
        • Patel S.
        • Pentchev P.G.
        Type C Niemann-Pick disease. Abnormal metabolism of low density lipoprotein in homozygous and heterozygous fibroblasts.
        J. Biol. Chem. 1986; 261: 16769
        • Pentchev P.G.
        • Kruth H.S.
        • Comly M.E.
        • Butler J.D.
        • Vanier M.T.
        • Wenger D.A.
        • Patel S.
        Type C Niemann-Pick disease. A parallel loss of regulatory responses in both the uptake and esterification of low density lipoprotein-derived cholesterol in cultured fibroblasts.
        J. Biol. Chem. 1986; 261: 16775
        • Khoo J.C.
        • Miller E.
        • McLoughlin P.
        • Tabas I.
        • Rosoff W.J.
        Cholesterol esterification as a limiting factor in accumulation of cell cholesterol: a comparison of two J774 macrophage cell lines.
        Biochim. Biophys. Acta. 1989; 1012: 215
        • Randolph R.K.
        • Smith B.P.
        • St. Clair R.W.
        Cholesterol metabolism in pigeon aortic smooth muscle cells lacking a functional low density lipoprotein receptor pathway.
        J. Lipid Res. 1984; 25: 903