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. 1996 Dec 10;93(25):14833-8.
doi: 10.1073/pnas.93.25.14833.

Cell surface expression of mouse macrosialin and human CD68 and their role as macrophage receptors for oxidized low density lipoprotein

M P Ramprasad  1 , V Terpstra, N Kondratenko, O Quehenberger, D Steinberg
Affiliations

Cell surface expression of mouse macrosialin and human CD68 and their role as macrophage receptors for oxidized low density lipoprotein

M P Ramprasad et al. Proc Natl Acad Sci U S A. .

Abstract

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 FA/11 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 degrees C and uptake at 37 degrees C of 125I-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 MS/CD68 at steady-state 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.

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Figures

Figure 1
Figure 1
FACS analysis of MS surface expression on MPMs. Binding of FA/11 (bold line) compared with that of a rat isotype control IgG (dashed line) to intact resident MPMs (A) or thioglycollate-elicited MPMs (B) is shown.
Figure 2
Figure 2
Cell surface expression of MS on elicited MPMs. Thioglycollate-elicited macrophages were harvested and subjected to cell-surface-specific biotinylation at 4°C followed by detergent extraction of cells and immunoadsorption with DS4 antiserum (lane 1). Alternatively, cell extracts were biotinylated and then processed for immunoadsorption (lane 2) to quantify total cellular expression of MS. The immunoprecipitates were subjected to SDS/PAGE and electroblot transfer, and the nitrocellulose strips were probed with streptavidin-alkaline phosphatase.
Figure 3
Figure 3
Copurification of CD68 and the 130- to 150-kDa OxLDL binding activity from THP-1 monocytes. Membrane detergent extracts of THP-1 monocytes were subjected to purification on wheat germ agglutinin lectin and OxLDL columns, and 20-μl aliquots of fractions (20–80 μg of protein) were examined by Western blot analysis with KP-1 monoclonal CD68 antibody and for OxLDL-ligand blotting activities.
Figure 4
Figure 4
FACS analysis of CD68 surface expression on intact THP-1 cells. Binding of KP-1 (bold line) and EBM-11 (normal line) compared with that of a mouse isotype control IgG (dashed line) to intact unstimulated THP-1 monocytes (A) or to THP-1 macrophages after 3 days of stimulation with 100 nM PMA (B). In A the curves for KP-1 and EBM-11 are superimposed on one another. Only cells negative for binding of pancytokeratin were included in the analysis.
Figure 5
Figure 5
FACS analysis of CD68 cell surface and total cellular expression in PMA-stimulated THP-1 macrophages. Binding of KP-1 (bold line) and mouse isotype control IgG (dashed line) to THP-1 cells treated 3 days with PMA before permeabilization (A) and after permeabilization (B). In A, as in Fig. 4, the cells selected for analysis were those totally negative for pancytokeratin but after permeabilization (B), the entire cell population was positive for pancytokeratin.
Figure 6
Figure 6
Inhibition by CD68 mAbs of specific binding and uptake of 125I-labeled OxLDL (5 μg/ml) by THP-cells. THP-1 cells were treated with PMA for 4 days after which binding at 4°C (Left) and uptake at 37°C (Right) were measured in the absence or presence of a 40- to 50-fold excess unlabeled OxLDL, EBM-11 (n = 5), KP-1 (n = 4), or an isotype-matched control IgG. Cell-bound and cell-associated radioactivity was assessed after 4–5 hr and were ≈260 and ≈800 ng of OxLDL per mg of cell protein, respectively. Nonspecific OxLDL binding and uptake (≈131 and 360 ng/mg of cell protein, respectively) were subtracted from all values to yield specific binding values. Activities in the presence of control IgG are taken as 100% (see Results). Data represent mean ± SD.
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