Lupus-derived monoclonal autoantibodies against apoptotic chromatin recognize acetylated conformational epitopes.
OBJECTIVE
Nuclear components targeted by autoantibodies are a characteristic feature of the autoimmune disease systemic lupus erythematosus (SLE). The nucleosome, a major autoantigen, is released in patients with SLE as a result of a disturbed apoptosis and/or an insufficient clearance of apoptotic debris. During apoptosis the nucleosome is modified, thereby creating more immunogenic epitopes. Subsequently, epitope spreading will lead to the formation of autoantibodies against unmodified chromatin components. However, characterization of B cell epitopes specific for apoptotic chromatin modifications is hampered by the fact that the existing monoclonal antibodies (mAbs) were originally selected on non-apoptotic chromatin. Here, we describe a novel approach for generating mAbs from lupus mice that are specific for apoptosis-induced chromatin modifications.


METHODS
Hybridomas were generated from pre-diseased and diseased lupus mice using standard fusion methods. Selection occurred on isolated apoptotic chromatin. Antibodies were further characterized by ELISA, western blot and immunofluorescence staining with apoptotic and non-apoptotic chromatin/cells. In addition, reactivity was determined with subnucleosomal complexes and with nucleosomes treated with trypsin or DNase I. Finally, reactivity was determined with cells treated with the histone deacetylase inhibitor TSA.


RESULTS
Most generated mAbs appeared to be nucleosome specific with a clear preference for apoptotic nucleosomes compared to normal nucleosomes. Although the exact elucidation of the epitopes of these mAbs specific for apoptosis-associated nucleosome modifications remains a major challenge, the epitopes contain both DNA and histones, whereby the histone tails play a role in establishing the epitopes. Most importantly, the conformational epitopes of these nucleosome-specific antibodies seem to contain acetylated residues.


CONCLUSIONS
Our approach, yielding a new panel of anti-apoptotic-chromatin antibodies, should facilitate the discovery of more apoptosis-induced chromatin modifications and their identification as key autoantigens in the pathogenesis of SLE.
van Bavel
Casandra C
CC
Nephrology Research Laboratory, Nijmegen Centre for Molecular Life Sciences, Department of Nephrology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
Dieker
Jürgen W
JW
Tamboer
Wim P
WP
van der Vlag
Johan
J
Berden
Jo H
JH
D000107:Acetylation; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D017209:Apoptosis / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology*; D015153:Blotting, Western; D002843:Chromatin / Q000276:immunology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D018985:Epitopes, B-Lymphocyte / Q000276:immunology*; D005260:Female; D005455:Fluorescent Antibody Technique; D008180:Lupus Erythematosus, Systemic / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D019463:Mice, Inbred MRL lpr; D008814:Mice, Inbred NZB; D009707:Nucleosomes / Q000276:immunology
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D001324:Autoantigens; D002843:Chromatin; D018985:Epitopes, B-Lymphocyte; D009707:Nucleosomes