Isolation and characterization of apoptotic nucleosomes, free and complexed with lupus autoantibody generated during hybridoma B-cell apoptosis.

Increasing evidence suggests that immune complexes made of anti-nuclear antibodies bound to nucleosomes released from dead cells play an important role in the pathogenesis of lupus nephritis. However, the nature and composition of apoptotic nucleosomes still remain elusive. Since large amounts of nucleosomes are released from cells undergoing apoptosis in hybridoma cell cultures, we used hybridomas secreting anti-DNA and anti-nucleosome antibodies grown in protein-free medium to generate nucleosome/anti-DNA and /anti-nucleosome immune complexes, as well as an irrelevant antibody hybridoma to generate free, non-complexed apoptotic nucleosomes. Hybridoma supernatants were fractionated by size-exclusion gel chromatography and eluted fractions with a ratio of A260/A280 >1.2 were pooled and analysed for DNA and histone profiles by gel electrophoresis and immunoblotting. When run on a 'native' gel, 'intact' apoptotic nucleosomes, free or within anti-nucleosome immune complexes, showed a strikingly reduced size compared with 'standard' nucleosomes prepared in vitro by endonuclease digestion of cell nuclei. Nucleosomal DNA (extracted from either free or complexed apoptotic nucleosomes) appeared as a major band of 160-180 bp, and had the size of 'standard' mononucleosome DNA, suggesting degradation of the histone moiety of apoptotic nucleosomes. Histone immunoblotting revealed degradation of histones H3 and H4, which was dramatically enhanced when apoptotic nucleosomes were complexed with an anti-nucleosome antibody. Our results provide direct evidence for abnormal histone composition of apoptotic nucleosomes and suggest that the fine specificity of the complexing antibody has an influence on complexed nucleosome composition.