Monoclonal autoantibodies specific for oxidized phospholipids or oxidized phospholipid–protein adducts inhibit macrophage uptake of oxidized low-density lipoproteins
Sohvi Hörkkö, David A. Bird, Elizabeth Miller, Hiroyuki Itabe, Norbert Leitinger, Ganesamoorthy Subbanagounder, Judith A. Berliner, Peter Friedman, Edward A. Dennis, Linda K. Curtiss, Wulf Palinski, Joseph L. Witztum
Sohvi Hörkkö, David A. Bird, Elizabeth Miller, Hiroyuki Itabe, Norbert Leitinger, Ganesamoorthy Subbanagounder, Judith A. Berliner, Peter Friedman, Edward A. Dennis, Linda K. Curtiss, Wulf Palinski, Joseph L. Witztum
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Monoclonal autoantibodies specific for oxidized phospholipids or oxidized phospholipid–protein adducts inhibit macrophage uptake of oxidized low-density lipoproteins

Abstract

We recently cloned monoclonal IgM autoantibodies which bind to epitopes of oxidized low-density lipoprotein (OxLDL) from apoE-deficient mice (EO– autoantibodies). We now demonstrate that those EO– autoantibodies that were originally selected for binding to copper-oxidized low-density lipoproteins (CuOx-LDL), also bound both to the oxidized protein and to the oxidized lipid moieties of CuOx-LDL. The same EO– autoantibodies showed specific binding to products of oxidized 1-palmitoyl-2-arachidonoyl-phosphatidylcholine (OxPAPC) and to the specific oxidized phospholipid, 1-palmitoyl-2-(5-oxovaleroyl)-phosphatidyl-choline (POVPC), whereas oxidation of fatty acids (linoleic or arachidonic acid) or cholesteryl esters (cholesteryl-oleate or cholesteryl-linoleate) did not yield any binding activity. Those EO– autoantibodies that bound to oxidized phospholipids (e.g., EO6) inhibited the binding and degradation of CuOx-LDL by mouse peritoneal macrophages up to 91%, whereas other IgM EO– autoantibodies, selected for binding to malondialdehyde (MDA)-LDL, had no influence on binding of either CuOx-LDL or MDA-LDL by macrophages. F(ab′)2 fragments of EO6 were equally effective as the intact EO6 in preventing the binding of CuOx-LDL by macrophages. The molar ratios of IgM to LDL needed to maximally inhibit the binding varied from ∼8 to 25 with different CuOx-LDL preparations. Finally, a POVPC–bovine serum albumin (BSA) adduct also inhibited CuOx-LDL uptake by macrophages. These data suggest that oxidized phospholipid epitopes, present either as lipids or as lipid-protein adducts, represent one class of ligands involved in the recognition of OxLDL by macrophages, and that apoE-deficient mice have IgM autoantibodies that can bind to these neoepitopes and inhibit OxLDL uptake.

Authors

Sohvi Hörkkö, David A. Bird, Elizabeth Miller, Hiroyuki Itabe, Norbert Leitinger, Ganesamoorthy Subbanagounder, Judith A. Berliner, Peter Friedman, Edward A. Dennis, Linda K. Curtiss, Wulf Palinski, Joseph L. Witztum

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(a) Immunoassay showing binding of EO– autoantibodies and DLH3 to unoxid...
(a) Immunoassay showing binding of EO– autoantibodies and DLH3 to unoxidized PAPC liposomes, OxPAPC liposomes, or POVPC. The liposomes were plated at 10 μg/ml overnight (4°C), and POVPC (10 μg/ml) was plated onto the microtiter wells by evaporation with air for 15 min just before use. Antibodies (10 μg/ml) were then incubated with antigens for 1 h (room temperature). The amount of antibody bound was measured with alkaline phosphatase–labeled goat anti–mouse IgM antibody using chemiluminescent technique. (b) Binding of EO6 to increasing concentrations of PAPC, CuOx-LDL, or POVPC-BSA. Each point is the mean of triplicate determinations. OxPAPC, oxidized PAPC; PAPC, unoxidized L-α1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; POVPC, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine.