Circulating autoantibodies to phospholipids (aPLs), such as cardiolipin (CL), are found in patients with antiphospholipid antibody syndrome (APS). We recently demonstrated that many aPLs bound to CL only after it had been oxidized (OxCL), but not to a reduced CL analogue that could not undergo oxidation. We now show that the neoepitopes recognized by some aPLs consist of adducts formed between breakdown products of oxidized phospholipid and associated proteins, such as beta2 glycoprotein 1 (beta2GP1). Addition of human beta2GP1, polylysine, native low-density lipoprotein, or apolipoprotein AI to OxCL-coated wells increased the anticardiolipin antibody (aCL) binding from APS sera that first had been diluted so that no aCL binding to OxCL could be detected. No increase in aCL binding was observed when these proteins were added to wells coated with reduced CL. The ability of beta2GP1, polylysine, or low-density lipoprotein to be a "cofactor" for aCL binding to OxCL was greatly reduced when the proteins were methylated. Incubation of beta2GP1 with oxidized 1-palmitoyl-2-linoleyl-[1-14C]-phosphatidylcholine (PC), but not with dipalmitoyl-[1-14C]-PC, led to formation of covalent adducts with beta2GP1 recognized by APS sera. These data suggest that the reactive groups of OxCL, such as aldehydes generated during the decomposition of oxidized polyunsaturated fatty acids, form covalent adducts with beta2GP1 (and other proteins) and that these are epitopes for aCLs. Knowledge that the epitopes recognized by many aPLs are adducts of oxidized phospholipid and associated proteins, including beta2GP1, may give new insights into the pathogenic events underlying the clinical manifestations of APS.

Background

We recently suggested that many anticardiolipin antibodies bind only to oxidized cardiolipin (OxCL) and/or to OxCL-beta(2)-glycoprotein 1 (beta(2)GP1) adducts but not to a "reduced" cardiolipin that is unable to undergo oxidation. To test this hypothesis, we investigated 24 sera, 4 protein A-purified IgG fractions, and 3 human monoclonal antibodies that were all isolated from patients with antiphospholipid antibody syndrome (APS); testing was also performed in 7 controls. Two monoclonal antibodies (IS3 and IS4) were selected for binding to CL and one was selected for binding to beta(2)GP1 (LJB8).

Methods and results

By chemiluminescent immunoassay, all APS sera samples bound only to OxCL and not to reduced CL, and the binding was inhibited >95% by OxCL but not reduced CL. All purified IgG fractions bound to beta(2)GP1 but only when the beta(2)GP1 was plated on microtiter wells coated with OxCL. All 3 monoclonal antibodies bound only to OxCL. On Western blots, IS4 and LJB8 bound to beta(2)GP1 as well as to delipidated apoB of oxidized LDL but not to native apoB. IS3 also bound to oxidized apoB on Western blot. Covalent modification of beta(2)GP1 with oxidation products of CL made it more antigenic for APS serum samples, for purified IgG fractions, and for the monoclonal antibodies.

Conclusions

These data support the hypothesis that oxidation of CL is needed to generate epitopes for many anticardiolipin antibodies and that some of these epitopes are covalent adducts of OxCL with beta(2)GP1 or apoB.