The penetrating potential of autoantibodies into live cells in vitro coincides with the in vivo staining of epidermal nuclei

Abstract

We have previously demonstrated that IgG autoantibodies derived from SLE patients are capable of penetrating into nuclei of living COLO-16 cells, in vitro. To address the possible correlation in Lupus Erythematosus (LE) between the in vivo ANA binding to nuclei of epidermal cells and the presence of intranuclear penetrating antibodies in sera of those patients, 25 consecutive patients were studied. Out of 25 skin biopsies, 11 specimens (8 of SLE and 3 of DLE) showed by immunofluorescent microscopy extensive in vivo presence of IgG in epidermal nuclei, whereas all sera of these patients stained nuclei of living COLO-16 cells, in vitro. Such penetration was also observed in additional 6/25 sera of patients, but with in vivo negative biopsies. This in vitro nuclear binding, which was unrelated to clinical symptoms of patients or their serological autoantibody profile and titer, was reproduced following cross-linking of intracellular protein by PLP fixation. Likewise, western blotting (immunoblotting) analysis, demonstrated the intranuclear presence of IgG in all in vitro intranuclear IgG staining sera. Furthermore, this in vitro presence, which neither affects cell viability nor DNA synthesis, is time-dependent and of a transient nature: nuclear staining disappears within 48 h following removal of the penetrating sera from medium. In conclusion, since the COLO-16 in vitro assay mirrors exactly the in vivo situation, and because of its higher sensitivity, it provides an excellent tool for the study of non-degraded autoantibody penetration into the nuclei of living cells.