Abstract 13773: A Novel Immunoglobulin G Autoantibody Against a Cryptic Epitope in Low Density Lipoprotein Induces Macrophage Tumour Necrosis Factor Release
Introduction/Objective: Although IgG autoantibodies reacting with modified LDL have been associated with cardiovascular events, there is controversy as to whether such antibodies can be pathogenic. We set out to generate monoclonal autoantibodies reacting with LDL for further mechanistic studies.
Methods/Results: We fused LDL receptor deficient (Ldlr-/-) mouse splenocytes with Sp2/0 myeloma cells, and screened hybridoma supernatants for reactivity with LDL. We selected a monoclonal IgG2b antibody, designated LO9. Variable heavy and light chain sequencing revealed somatic mutations indicative of LO9 being part of an acquired immune response. LO9 showed greater binding to unmanipulated solid phase human LDL than to Cu++ oxidised-LDL or malondialdehyde-conjugated LDL. LO9 did not react with LDL captured by immobilised anti-apoB, and binding to solid phase LDL was not inhibited by fluid phase LDL. Expression of the LO9 epitope on adherent LDL was increased by activated macrophage supernatant and this was inhibited by pepstatin A. Together, these observations suggest that LO9 reacts with a cryptic epitope in LDL unmasked by adhesion and/or the action of macrophage-derived enzyme(s). LO9 epitope expression in vivo was confirmed by immunohistochemical staining of human and mouse atherosclerosis. Following intra-venous injection of LO9 labelled with VivoTag-S 750-MAL, fluorescence molecular tomography imaging showed LO9 uptake in the aortic arch of Ldlr-/- but not wild-type animals. Confocal microscopy confirmed that LO9 localised in atherosclerotic regions beneath endothelium and in the vicinity of macrophages. In functional in vitro assays, addition of macrophages to LO9 bound to adherent LDL led to significant TNF release, an effect that was blocked by inhibition of Fc gamma receptors, and which was not seen when macrophages were incubated with adherent LDL alone.
Conclusions: We believe LO9 is the first example of an IgG autoantibody that reacts with a cryptic epitope in LDL, revealed by adherence and/or by macrophage-derived enzyme(s). Binding of LO9-like IgG antibodies to LDL trapped on arterial matrix could lead to promotion of atherosclerosis via ligation of macrophage Fc gamma receptors and release of TNF and other inflammatory mediators.
Author Disclosures: R. Khamis: None. K. Woollard: None. J.J. Boyle: None. S. Chang: None. G. Hyde: None. M. Johns: None. D.O. Haskard: None.
- © 2014 by American Heart Association, Inc.