Modulation of THP-1 Macrophage and Cholesterol- Loaded Foam Cell Apolipoprotein E Levels by Glycosphingolipids Brett Garner, 1 Howard R. Mellor, Terry D. Butters, Raymond A. Dwek, and Frances M. Platt Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom Received December 26, 2001 Macrophages synthesize and secrete apolipoprotein E (apoE) constitutively. This process is upregulated under conditions of cholesterol loading. The response to cholesterol is antiatherogenic as it is believed to promote cholesterol efflux from the artery wall. The concentration of lactosyl ceramide (LacCer), a glyco- sphingolipid recently discovered to regulate cellular signaling, proliferation, and expression of adhesion molecules, is also increased in atherosclerotic tissues. Here we have investigated the effect of exogenous LacCer on macrophage apoE levels. We show that in- creasing macrophage LacCer levels sevenfold led to reductions in cellular and secreted apoE (15 and 30%, respectively, over a 24-h period) as determined by enzyme-linked immunosorbent assay. A similar effect was also induced by glucosyl ceramide (GlcCer) but not by ganglioside species. When macrophages were converted to cholesterol-loaded foam cells by incuba- tion with acetylated LDL, the resulting increase in cellular apoE levels was inhibited by 26% when the cells were subsequently enriched with LacCer. After metabolic labeling of cellular glycosphingolipids with [ 14 C]palmitate, we also discovered that high-density lipoprotein (HDL) stimulates the efflux of glycosphin- golipids from foam cells. These data imply that LacCer and GlcCer may be proatherogenic due to the suppres- sion of macrophage apoE production. Furthermore, the efflux of glycosphingolipids from macrophage foam cells to HDL could indicate a potential pathway for their removal from the artery wall and subsequent delivery to the liver. © 2002 Elsevier Science (USA) Key Words: macrophage; foam cell; apoE; glycosphin- golipid; atherosclerosis; cholesterol metabolism; LDL; HDL; LacCer; GlcCer. Apolipoprotein E (apoE) is a 34-kDa glycoprotein that circulates in plasma as a surface component of chylomicrons, very low density lipoproteins, and high density lipoproteins (HDL). In this context, apoE plays an important role in the receptor-mediated endocytosis of lipoproteins by the liver (1). ApoE is also constitu- tively produced by macrophages and its synthesis is upregulated under conditions of cholesterol loading to form foam cells that are a feature of atherosclerotic lesions (2). Several transcription factors that affect apoE gene expression have been identified but the mechanisms underlying cholesterol induced apoE syn- thesis have not yet been elucidated (3, 4). ApoE syn- thesis and secretion plays a crucial role in the efflux of cholesterol from macrophage foam cells (5). In accor- dance with this, apolipoprotein AI and HDL, the major extracellular acceptors of cholesterol from peripheral tissues, markedly stimulate macrophage apoE secre- tion (6). Macrophage derived apoE may also exert a number of effects not related to cholesterol metabo- lism; including the regulation of smooth muscle cell (SMC) proliferation (7), provision of antioxidant activ- ity (8) and the suppression of T-cell activation (9). Because these pathways also contribute to atherogen- esis, it is important to understand the factors that regulate macrophage apoE production. In addition to the accumulation of cholesterol in the atherosclerotic artery wall, certain glycosphingolipids (GSLs) accumulate (10, 11). The neutral GSL, LacCer, is increased in concentration by fivefold (when ex- pressed per milligram of cholesterol) in atherosclerotic lesions (11). This increase is due to increased cellular LacCer and to the presence of extracellular GSL- enriched vesicles (10). Plasma levels of the major neu- Abbreviations used: 2-AB, 2-amino benzamide; acLDL, acetylated LDL; apoE, apolipoprotein E; CTH, ceramide trihexoside Gal1- 4Gal1-4Glc-Cer; ELISA, enzyme-linked immunosorbent assay; GlcCer, glucosyl ceramide; GSL, glycosphingolipid; HDL, high- density lipoprotein; HP-TLC, high-performance thin-layer chroma- tography; LacCer, lactosyl ceramide Gal1-4Glc-Cer; Glob, globoside GalNAc1-3Gal1-4Gal1-4Glc-Cer; LDL, low-density lipoprotein; NP-HPLC, normal-phase high-performance liquid chromatography. 1 To whom correspondence and reprint requests should be ad- dressed. Fax: 44-1865-275216. E-mail: brett@glycob.ox.ac.uk. Biochemical and Biophysical Research Communications 290, 1361–1367 (2002) doi:10.1006/bbrc.2002.6356, available online at http://www.idealibrary.com on 1361 0006-291X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.