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We have previously identified a 94-to 97-kDa oxidized low density lipoprotein (LDL)-binding protein in mouse macrophages as macrosialin (MS), a member of the lamp family. Earlier immunostaining studies have shown that MS and its human homolog, CD68, are predominantly intracellular proteins. However, using sensitive techniques such as flow cytometry (FACS) and cell-surface-specific biotinylation, we now show that there is significant surface expression of these proteins. FACS analysis of intact cells using mAb FA͞11 showed small but definite surface expression of MS in resident mouse peritoneal macrophages but this was greatly enhanced with thioglycollate elicitation. Biotinylation of intact cells and detergent-solubilized cell preparations followed by immunoprecipitation revealed 10-15% of the total MS content of elicited macrophages on the plasma membrane. Similar results were obtained with untreated RAW 264.7 cells. FACS analysis of intact THP-1 monocytic cells showed minimal surface expression of CD68 on unactivated cells (4% of total cell content). Stimulation with phorbol 12-myristate 13-acetate increased both surface and total CD68 expression considerably. Furthermore, the specific binding at 4؇C and uptake at 37؇C of 125 I-labeled oxidized LDL by activated THP-1 cells was inhibited by 30-50% by CD68 mAbs KP-1 and EBM-11. Thus, although the surface expression of MS͞CD68 at steadystate represents only a small percentage of their total cellular content, these proteins can play a significant role in oxidized LDL uptake by activated macrophages in vitro and could contribute to foam cell formation in atherosclerotic lesions.

A new receptor for oxidized low-density lipoprotein (LDL), lectin-like oxidized LDL receptor-1 (LOX-1), has recently been cloned from bovine endothelial cells and human lung. A limited tissue-distribution study suggested that the protein was mainly produced by the vascular endothelium. In the present study we demonstrate that LOX-1 is also expressed in macrophages, where it may function as a scavenger receptor. LOX-1 was not detected in undifferentiated THP-1 cells or in freshly isolated human blood monocytes. However, mature human monocyte-derived macrophages and differentiated THP-1 cells showed high levels of LOX-1 transcripts. Consistent with these results, immunofluorescence staining and FACS analysis demonstrated that LOX-1 protein is expressed on the plasma membrane of macrophages. Western-blot analysis of membranes from macrophages (but not those from monocytes) identified a single band, with an apparent molecular mass of about 40 kDa, that displayed oxidized LDL-binding activity. These results suggest that differentiation induces the expression of LOX-1 in macrophages, where it may play a role as a scavenger receptor and/or a receptor for oxidized LDL.

Abstract-Fully oxidized LDL (OxLDL) is believed to contribute to atherogenesis in part by virtue of uptake into macrophages via specific scavenger receptors. This phenomenon results in the formation of cholesterol-loaded foam cells, a major component of atherosclerotic lesions. The present study is directed at examining the effects of OxLDL and minimally oxidized LDL (MM-LDL) on scavenger receptor expression and activity in mouse peritoneal resident macrophages. Macrophages were preincubated with MM-LDL or OxLDL at concentrations of 25 or 50 g/mL for 24 to 48 hours, after which their ability to bind and take up 125 I-OxLDL or 125 I-acetylated LDL (AcLDL) was determined. MM-LDL pretreatment induced a clear increase of cell association and degradation of 125 I-OxLDL and 125 I-AcLDL. Pretreatment with OxLDL also enhanced scavenger receptor activity, but to a lesser degree. Neither native LDL nor AcLDL had any effect. Scatchard analysis showed that preincubation with 50 g/mL MM-LDL for 48 hours increased the B max of 125 I-OxLDL and 125 I-AcLDL by 139% and 154%, respectively, without significantly changing their affinity. Lipids extracted from MM-LDL also significantly induced scavenger receptor activity, but to a lesser extent than did intact MM-LDL. MM-LDL pretreatment increased both mRNA levels and protein levels of scavenger receptor A, CD36, and macrosialin. On the other hand, OxLDL pretreatment increased expression of macrosialin only. These results, showing that MM-LDL can upregulate scavenger receptor expression in mouse resident peritoneal macrophages, suggest that clearance of OxLDL by macrophages in lesions is more effective, in part because the OxLDL precursor, MM-LDL, primes the macrophage for foam cell generation. (Arterioscler Thromb Vasc Biol. 1998;18:794-802.) Key Words: minimally oxidized low density lipoprotein Ⅲ scavenger receptor A Ⅲ CD36 Ⅲ macrosialin Ⅲ mouse peritoneal resident macrophages S ubstantial evidence demonstrates that OxLDL can contribute to atherogenesis. [1][2][3][4] Many studies indicate that macrophages in the artery wall take up OxLDL via scavenger receptors, leading to cholesterol ester accumulation and resulting in the formation of foam cells, the hallmark of the arterial fatty streak, which is recognized as the earliest atherosclerotic lesion.

The binding and uptake of oxidatively modified low density lipoprotein (OxLDL) by mouse peritoneal macrophages occurs, in part, via the well characterized acetyl LDL receptor. However, several lines of evidence indicate that as much as 30-70%Yo of the uptake can occur via a distinct receptor that recognizes OxLDL with a higher affinity than it recognizes acetyl LDL. We describe the partial purification and characterization of a 94-to 97-kDa plasma membrane protein from mouse peritoneal macrophages that specifically binds OxLDL. This receptor is shown to be distinct from the acetyl LDL receptor as well as from two other macrophage proteins that also bind OxLDLthe Fc'yRII receptor and CD36. We suggest that this OxLDL-binding membrane protein participates in uptake of OxLDL by murine macrophages and also represents a receptor responsible for macrophage binding and phagocytosis of oxidatively damaged cells.

Macrosialin, the mouse homolog of human CD68, is a heavily glycosylated transmembrane protein found almost exclusively in macrophages. Its function remains uncertain. It has a high affinity for oxidized low-density lipoprotein (LDL) in ligand blots and antibodies against the human homolog, CD68, inhibit the binding of oxidized LDL to a human monocyte-derived cell line (THP-1). However, there is still controversy as to whether macrosialin, found predominantly in late endosomes, is expressed at all on the plasma membrane. The present studies, done in thioglycollate-elicited peritoneal macrophages, confirm that macrosialin is predominantly intracellular but show clearly that 10-15% of it is expressed on the cell surface. Exchange with intracellular pools occurs at an extremely high rate. The results are compatible with a surface function, including internalization of bound ligands or adhesion to surfaces. J. Leukoc. Biol. 67: 104-108; 2000.

Macrophage binding of oxidatively damaged red blood cells (OxRBC) and apoptotic thymocytes correlates in many instances with a loss of phospholipid bilayer asymmetry, i.e., with an increase in expression of phosphatidylserine on the outer leaf let of the plasma membrane. Oxidatively modified LDL (OxLDL) can compete for the binding of these ligands to macrophages. However, the receptor(s) responsible remains to be identified. The present studies show that mouse peritoneal macrophages totally lacking scavenger receptor A (SRA) bound OxRBC just as effectively as wildtype macrophages, whereas their binding and uptake of acetyl LDL was reduced by more than 80%. Binding of apoptotic thymocytes and binding of OxLDL were also reduced, but only by 20-30%. We conclude that SRA is not involved in the recognition of phosphatidylserine-rich membranes but contributes to the binding of OxLDL and apoptotic thymocytes. The binding of OxRBC was almost totally calcium-dependent, whereas the binding of apoptotic thymocytes was not, suggesting that the mechanisms involved in their uptake by macrophages under these conditions were different.

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