Model IgG monoclonal autoantibody-anti-idiotype pair for dissecting the humoral immune response to oxidized low density lipoprotein

Abstract

Increasing evidence implicates IgG autoantibodies against oxidized forms of low density lipoprotein (oxLDL) in the pathophysiology of atherosclerotic arterial disease. However, insufficient knowledge of their structure and function is a key gap. Using an elderly LDL receptor-deficient atherosclerotic mouse, we isolated a novel IgG3k against oxLDL (designated MAb LO1). LO1 reacts with copper-oxidized LDL, but minimally with native LDL. Further analysis showed that MAb LO1 also reacts in vitro with malondialdehyde-conjugated LDL (MDA-LDL), a known key epitope in copper-oxidized LDL preparations. By screening a phage library expressing single chain variable region antibodies (scFv), we selected an anti-idiotype scFv (designated H3) that neutralizes MAb LO1 binding to MDA-LDL. Amino acid substitutions between H3 and an irrelevant control scFv C12 showed that residues in the H3 CDRH2, CDRH3, and CDRL2 are all critical for MAb LO1 binding, consistent with a conformational epitope on H3 involving both heavy and light chains. Comparison of amino acids in H3 CDRH2 and CDRL2 with apoB, the major LDL protein, showed homologous sequences, suggesting H3 has structural similarities to the MAb LO1 binding site on MDA-LDL. Immunocytochemical staining showed that MAb LO1 binds epitopes in mouse and human atherosclerotic lesions. The MAb LO1-H3 combination therefore provides a very promising model for analyzing the structure and function of an individual IgG autoantibody in relation to atherosclerosis.

FIG. 1.

Initial characterization of MAb LO1. (A) Reactivity by ELISA of MAb LO1 culture supernatant and pooled sera from Ldlr^-/- mice (diluted 1:40) against Cu-oxLDL (white bars) or native LDL (black bars). (B) Competition ELISA showing binding of different concentrations of MAb LO1 in the presence and absence of fluid phase Cu-oxLDL. Values are mean + SD of triplicates. (C) Binding by ELISA of MAb LO1 to LDL modified by MDA for durations up to 5 h. Also shown is the lack of binding of control IgG3 HK-PEG-1 at 1 and 10 μg/mL (curves superimposed). Values are mean ± SD of triplicates. (D) The extent of carbonyl adduction to LDL following incubation with MDA was estimated using a carbonyl assay, with results expressed as the number of nmoles of DNP/mg of protein (nmol/mg).

FIG. 2.

Amino acid sequence of the MAb LO1 V_H and V_L. The panel shows the MAb LO1 V_H and V_L sequences and their similarity to the germline V1–54/JH2 and V6–20/J2 sequences. LO1 amino acids that differ from germline are shown in red.

FIG. 3.

Reactivity of anti-idiotype scFv H3. (A) Specific binding of MAb LO1 to scFv H3. Microtiter plates were coated with H3 or C12 and then tested for binding of MAb LO1 or control IgG3k HK-PEG-1 (each at 2 μg/mL). Values are expressed as mean and upper value of duplicates. (B) Neutralization of MAb LO1 binding to MDA-LDL by scFv H3. MAb LO1 (2 μg/mL) were incubated with plates coated with MDA-LDL (10 μg/mL) in the presence of varying concentrations of scFv H3 or control scFv C12. (C,D) Effects of changing salt concentration (C) and pH (D) on binding of MAb LO1 to H3 and MDA-LDL. (C,D) Data are expressed as percentage of the binding seen with PBS and percentage of maximal binding respectively.

FIG. 4.

Similarities of sequences of LO-1 binding scFv and apoB-100; the figure shows the sequence of the V_H and V_L regions of scFv H3. Amino acids at each of the 18 points at which the Tomlinson I scFv library is diverse are in red. ApoB peptide sequences with similarities to H3 sequences are shown, with amino acids identical to H3 (green). Amino acid numbering is taken from NCBI protein sequences AAA51752.1 and NP_033823.2 for human and mouse apoB respectively.

FIG. 5.

Effects of V_H and V_L domain swops between scFv H3 and C12: H3, C12, H3V_H/C12V_L, and H3V_L/C12V_H were coated at 10 μg/mL. Binding of MAb LO1 was tested by ELISA across a concentration range of 0.1–2000 ng/mL.

FIG. 6.

Immunocytochemical staining of Ldlr^-/- mouse aortic root with MAb LO1. (A) Aortic valve cusp from a Ldlr^-/- mouse stained with Giemsa, a general purpose tissue stain. (B) Region indicated by the arrow is stained with the macrophage-specific marker CD68-AlexaFluor 488 (green) and the DNA-binding dye TOPRO (blue), revealing the localized area of macrophage infiltration and the unstained media (Med). Diffuse staining of the whole valve cusp is seen with MAb LO1 (C) but not with control IgG3 (D). (E) No equivalent staining was seen with MAb LO1 applied to the aortic root of a wild-type mouse. Preincubation of MAb LO1 with MDA-LDL (F) or scFv H3 (G), but not with scFv C12 inhibited staining (H), demonstrating specificity of MAb LO1 binding. Blue, TOPRO counterstain; red, AlexaFluor 568 fluorescence showing specific binding of MAb LO1. Ldlr^-/- mice fed a low-fat diet to the age of 22 weeks were chosen because their lesions are composed almost exclusively of macrophages, simplifying interpretation. Med, tunica media (vascular smooth muscle cell layer); Le, lesion (composed of macrophages); open arrow points to lesion.

FIG. 7.

Immunocytochemical staining of human carotid atherosclerotic plaque with MAb LO1. (A) For orientation, low power view of a human carotid atherosclerotic plaque stained with Giemsa, with the area within the box magnified (B). (C–E) Confocal imaging of consecutive serial sections in the vicinity of the boxed area (B). Single stained with MAb LO1 (red AlexaFluor 568) (C), dual-stained by MAb LO1 (red AlexaFluor 568), and CD68-AlexaFluor 488 (green) with brilliant yellow-white indicating colocalization (D), and single stained with control IgG3 (E) with protocol and settings identical to (C). Lu, lumen; LC, lipid core; Med, media; Blue, TOPRO; nuclei (DNA); fine arrowheads, inflammatory cells at edge of lipid core; scale bars; distances are indicated.