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It has been suggested that binding of anti-double-stranded DNA antibodies to cell surfaces, followed by internalization and nuclear binding (so called in vivo ANA) is of pathophysiological significance for tissue damage in systemic lupus erythematosus. We have shown before that pathogenic antinuclear antibodies complexed to nucleosomal antigens can bind to heparan sulfate in the glomerular basement membrane in vivo. Because nucleosomes are also reported to bind to the cell surface, we hypothesized that in vivo ANA is a property of antinuclear antibodies bound to nucleosomal antigens. Therefore, we studied three antinucleosome monoclonal antibodies (mAb) that exhibit in vivo ANA as seen by immunofluorescence in mice inoculated intraperitoneally with the hybridoma producing the mAb. The same mAb complexed to nucleosomal antigens after intravenous injection into mice induced in vivo ANA, in contrast to purified noncomplexed mAb. To study this In more detail, we incubated complexed mAb with various cell lines and found binding to the cell surface and subsequent internalization into cytoplasmic vesicles. However, no binding to the nucleus was observed by immunoelectron microscopy (IEM) and confocal laser microscopy. Noncomplexed mAb did not bind to the cell surface, Next, from mice bearing the hybridomas producing the mAb intraperitoneally, a small part of the kidney was snap frozen in liquid N-2, fixed with acetone, and studied In immunofluorescence, whereas the remaining part of the kidney was fixed In vivo by renal perfusion with a mixture of 0.01 M sodium periodate, 0.075 M lysine HCI, 0.0375 M Na2HPO4, and 2% paraformaldehyde (PLP) and studied in both immunofluorescence and IEM. In the acetone-fixed kidney sections obtained without in vivo fixation we again observed in vivo ANA. However, after in vivo PLP perfusion fixation, no nuclear binding was found. in IEM, localization in cytoplasmic vesicles was seen. in conclusion, antinucleosome antibodies complexed to nucleosomal antigens can bind to the cell surface and are transported into the cytoplasm, but do not bind to the nucleus. The reported nuclear localization of antinuclear antibodies is caused by a fixation artifact.