Receptors for oxidized low-density lipoprotein on elicited mouse peritoneal macrophages can recognize both the modified lipid moieties and the modified protein moieties: implications with respect to macrophage recognition of apoptotic cells

Abstract

It has been shown previously that the binding of oxidized low-density lipoprotein (OxLDL) to resident mouse peritoneal macrophages can be inhibited (up to 70%) by the apoprotein B (apoB) isolated from OxLDL, suggesting that macrophage recognition of OxLDL is primarily dependent on its modified protein moiety. However, recent experiments have demonstrated that the lipids isolated from OxLDL and reconstituted into a microemulsion can also strongly inhibit uptake of OxLDL (up to 80%). The present studies show that lipid microemulsions prepared from OxLDL bind to thioglycollate-elicited macrophages at 4 degrees C in a saturable fashion and inhibit the binding of intact OxLDL and also of the apoB from OxLDL. Reciprocally, the binding of the OxLDL-lipid microemulsions was strongly inhibited by intact OxLDL. A conjugate of synthetic 1-palmitoyl 2(5-oxovaleroyl) phosphatidylcholine (an oxidation product of 1-palmitoyl 2-arachidonoyl phosphatidylcholine) with serum albumin, shown previously to inhibit macrophage binding of intact OxLDL, also inhibited the binding of both the apoprotein and the lipid microemulsions prepared from OxLDL. Finally, a monoclonal antibody against oxidized phospholipids, one that inhibits binding of intact OxLDL to macrophages, also inhibited the binding of both the resolubilized apoB and the lipid microemulsions prepared from OxLDL. These studies support the conclusions that: (i) at least some of the macrophage receptors for oxidized LDL can recognize both the lipid and the protein moieties; and (ii) oxidized phospholipids, in the lipid phase of the lipoprotein and/or covalently linked to the apoB of OxLDL, likely play a role in that recognition.

Figure 1

Binding of DiO-labeled OxLDL lipid microemulsions to mouse macrophages in the presence of unlabeled native LDL lipids (■), OxLDL lipids (●), intact native LDL (□), or OxLDL (○). Elicited mouse macrophages were incubated in the presence of 10 μg/ml DiO-labeled OxLDL lipid microemulsions for 2 h at 4°C with or without competitors added (expressed as μg of phospholipid per ml for lipid microemulsions and μg of protein for intact LDL). The cells were then washed, scraped, and the mean fluorescence intensity of 30,000 events measured by using flow cytometry and cell quest software.

Figure 2

Binding of ¹²⁵I-apoB from OxLDL to mouse macrophages in the presence of native LDL lipids (■) or OxLDL lipids (●). Elicited mouse macrophages were incubated with native LDL lipid or OxLDL lipid microemulsions for 1.5 h at 4°C. After being washed to remove the unbound lipids, the cells were then incubated with 4 μg/ml of ¹²⁵I-apoB for an additional 1.5 h at 4°C. Binding was quantitated by measuring the (¹²⁵I) radioactivity per mg of cell protein as described in Methods.

Figure 3

Inhibition by monoclonal antibody EO6 of the binding of ¹²⁵I-apoB from OxLDL (open bars) and DiO-labeled OxLDL lipid (closed bars). Binding assays for ¹²⁵I-apoB (at 4 μg/ml) and DiO-labeled OxLDL lipids (at 10 μg/ml) were performed as described for Figs. 1 and 2, respectively, in the presence of EO6 or EO14 at 100 μg/ml. The values represent the means ± SD (n = 3). ∗, P < 0.005 compared with incubation with EO14, a control IgM.

Figure 4

(A) Binding of DiO-labeled OxLDL lipid microemulsions to mouse macrophages in the presence of liposomes containing PAPC (■) or oxidized PAPC (●). Elicited mouse macrophages were incubated in the presence of 10 μg/ml DiO-labeled OxLDL lipid microemulsions and PAPC-containing liposomes for 2 h at 4°C. Binding was quantitated as described for Fig. 1. (B) Binding of ¹²⁵I-apoB from OxLDL to mouse macrophages in the presence of liposomes containing PAPC (■) or oxidized PAPC (●). Elicited mouse macrophages were incubated with PAPC liposomes for 1.5 h at 4°C. After being washed to remove the unbound lipids, the cells were then incubated with 4 μg/ml of ¹²⁵I-OxApoB for an additional 1.5 h at 4°C. Binding was quantitated as described for Fig. 2.

Figure 5

Macrophage binding of ¹²⁵I-apoB (open bars) and of DiO-labeled OxLDL lipids (solid bars) in the presence of unlabeled ligands (15-fold excess) and in the presence of three different concentrations of POVPC-BSA.