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-nuclea r antibodies bound to nucleosomes released from dead cells play an imp ortant role in the pathogenesis of lupus nephritis. However, the natur e and composition of apoptotic nucleosomes still remain elusive. Since large amounts of nucleosomes are released from cells undergoing apopt osis in hybridoma cell cultures, we used hybridomas secreting anti-DNA and anti-nucleosome antibodies grown in protein-free medium to genera te nucleosome/anti-DNA and /anti-nucleosome immune complexes, as well as an irrelevant antibody hybridoma to generate free, nor.-complexed a poptotic nucleosomes. Hybridoma supernatants were fractionated by size -exclusion gel chromatography and eluted fractions with a ratio of A26 0/A280 >1.2 were pooled and analysed for DNA and histone profiles by g el electrophoresis and immunoblotting. When run on a 'native' gel,'int act' apoptotic nucleosomes, free or within anti-nucleosome immune comp lexes, showed a strikingly reduced size compared with 'standard' nucle osomes prepared in vitro by endonuclease digestion of cell nuclei. Nuc leosomal DNA (extracted from either free or complexed apoptotic nucleo somes) appeared as a major band of 160-180 bp, and had the size of 'st andard' mononucleosome DNA, suggesting degradation of the histone moie ty of apoptotic nucleosomes. Histone immunoblotting revealed degradati on of histones H3 and H4, which was dramatically enhanced when apoptot ic nucleosomes were complexed with an anti-nucleosome antibody. Our re sults provide direct evidence for abnormal histone composition of apop totic nucleosomes and suggest that the fine specificity of the complex ing antibody has an influence on complexed nucleosome composition. (C) 1998 Academic Press Limited.