IN-VIVO ANA IS A FIXATION ARTIFACT - NUCLEOSOME-COMPLEXED ANTINUCLEOSOME AUTOANTIBODIES BIND TO THE CELL-SURFACE AND ARE INTERNALIZED

It has been suggested that binding of anti-double-stranded DNA antibod ies to cell surfaces, followed by internalization and nuclear binding (so called in vivo ANA) is of pathophysiological significance for tiss ue damage in systemic lupus erythematosus. We have shown before that p athogenic 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 antinucleos ome monoclonal antibodies (mAb) that exhibit in vivo ANA as seen by im munofluorescence in mice inoculated intraperitoneally with the hybrido ma producing the mAb. The same mAb complexed to nucleosomal antigens a fter intravenous injection into mice induced in vivo ANA, in contrast to purified noncomplexed mAb. To study this In more detail, we incubat ed complexed mAb with various cell lines and found binding to the cell surface and subsequent internalization into cytoplasmic vesicles. How ever, no binding to the nucleus was observed by immunoelectron microsc opy (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 p erfusion 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 obtai ned without in vivo fixation we again observed in vivo ANA. However, a fter in vivo PLP perfusion fixation, no nuclear binding was found. in IEM, localization in cytoplasmic vesicles was seen. in conclusion, ant inucleosome antibodies complexed to nucleosomal antigens can bind to t he cell surface and are transported into the cytoplasm, but do not bin d to the nucleus. The reported nuclear localization of antinuclear ant ibodies is caused by a fixation artifact.