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Abstract-The subendothelial accumulation of macrophage-derived foam cells is one of the hallmarks of atherosclerosis. The recruitment of monocytes to the intima requires the interaction of locally produced chemokines with specific cell surface receptors, including the receptor (CCR2) for monocyte chemoattractant protein-1 (MCP-1). We have previously reported that monocyte CCR2 gene expression and function are effectively downregulated by proinflammatory cytokines. In this study we identified low density lipoprotein (LDL) as a positive regulator of CCR2 expression. Monocyte CCR2 expression was dramatically increased in hypercholesterolemic patients compared with normocholesterolemic controls. Similarly, incubation of human THP-1 monocytes with LDL induced a rapid increase in CCR2 mRNA and protein. By 24 hours the number of cell surface receptors was doubled, causing a 3-fold increase in the chemotactic response to MCP-1. The increase in CCR2 expression and chemotaxis was promoted by native LDL but not by oxidized LDL. Oxidized LDL rapidly downregulated CCR2 expression, whereas reductively methylated LDL, which does not bind to the LDL receptor, had only modest effects on CCR2 expression. A neutralizing anti-LDL receptor antibody prevented the effect of LDL, suggesting that binding and internalization of LDL were essential for CCR2 upregulation. The induction of CCR2 expression appeared to be mediated by LDL-derived cholesterol, because cells treated with free cholesterol also showed increased CCR2 expression. These data suggest that elevated plasma LDL levels in conditions such as hypercholesterolemia enhance monocyte CCR2 expression and chemotactic response and potentially contribute to increased monocyte recruitment to the vessel wall in chronic inflammation and atherogenesis. (Arterioscler Thromb Vasc Biol. 1998;18:1983-1991.)

There is growing evidence that CD36 has an important physiological function in the uptake of oxidized low density lipoprotein (OxLDL) by macrophages. However, the ligand specificity and the nature of the ligands on OxLDL that mediate the binding to CD36 remain ill defined. Results from recent studies suggested that some of the macrophage scavenger receptors involved in the uptake of OxLDL recognized both the lipid and the protein moieties of OxLDL, but there was no conclusive direct evidence for this. The present studies were undertaken to test whether a single, well characterized OxLDL receptor, CD36, could bind both the lipid and protein moieties of OxLDL. COS-7 cells transiently transfected with mouse CD36 cDNA bound intact Ox-LDL with high affinity. This binding was very effectively inhibited (ϳ50%) both by the reconstituted apoB from OxLDL and by microemulsions prepared from OxLDL lipids. The specific binding of both moieties to CD36 was further confirmed by direct ligand binding analysis and by demonstrating reciprocal inhibition, i.e. apoB from OxLDL inhibited the binding of the OxLDL lipids and vice versa. Furthermore, a monoclonal mouse antibody that recognizes oxidation-specific epitopes in OxLDL inhibited the binding of intact OxLDL and also that of its purified protein and lipid moieties to CD36. This antibody recognizes the phospholipid 1-palmitoyl 2-(5-oxovaleroyl) phosphatidylcholine. This model of an oxidized phospholipid was also an effective competitor for the CD36 binding of both the resolubilized apoB and the lipid microemulsions from OxLDL. Our results demonstrate that oxidized phospholipids in the lipid phase or covalently attached to apoB serve as ligands for recognition by CD36 and, at least in part, mediate the high affinity binding of OxLDL to macrophages.

No abstract

We have previously shown that CD36 recognizes oxidation products of phospholipids on oxidized LDL (OxLDL) such as 1-palmitoyl-2-(5 -oxovaleroyl)-sn -glycero-3-phosphocholine (POVPC). The current study was designed to examine whether the phosphocholine (PC) headgroup in POVPC constitutes an obligatory binding target for CD36. To examine the contribution of PC in the binding of POVPC to CD36, we used well-defined synthetic oxidized phospholipids (OxPLs) cross-linked to BSA or to a hexapeptide. The OxPL adducts were then tested for their ability to bind to CD36-transfected cells and for their ability to inhibit OxLDL binding to CD36. Mounting evidence implicates oxidized low density lipoprotein (OxLDL) in the pathogenesis of atherosclerosis. The oxidative modifications are presumed to occur after the entry of plasma LDL into the intima and are catalyzed by the cellular constituents of the arterial wall. Although clearance of the highly cytotoxic OxLDL may protect the surrounding tissue from damage, the unregulated uptake of OxLDL by macrophages within the arterial wall leads to the formation of lipid-laden foam cells and the development of the fatty streak, the hallmark of early atherosclerotic lesions. The recognition and uptake of OxLDL by macrophages is mediated by specific cell surface scavenger receptors, including CD36, a class B scavenger receptor (1). CD36 is a heavily glycosylated protein with broad ligand specificity, a characteristic of pattern recognition receptors. It binds long-chain fatty acids (2), anionic phospholipids (3), ␤ -amyloid (4), advanced glycation end products (5), and OxLDL (1). It also functions in the recognition and removal of Plasmodium falciparum -infected erythrocytes (6) and apoptotic cells (7). It serves as the primary receptor for platelet adhesion to collagen (8) and may mediate the antiangiogenic activity of thrombospondin (9).Recent data derived from CD36-deficient mice support an important role of CD36 in foam cell formation and atherogenesis. A significant decrease in binding and degradation of OxLDL was observed in macrophages from null mice compared with those from control mice (10). Similarly, macrophages from CD36-deficient patients were less capable of binding and degrading OxLDL and accumulated less cholesteryl ester than macrophages from control subjects (11). The relative contribution of scavenger receptors in the uptake of OxLDL was further addressed in mice lacking both scavenger receptor A and CD36 (12). Binding and uptake studies demonstrated that scavenger receptor A and CD36 were the principal macrophage receptors responsible for the binding of OxLDL and the accumulation of cholesteryl ester derived from modified lipoproteins. Consistent with a principal pathogenic role of scavenger receptors in atherogenesis, the targeted disrup-