In Vitro Study of the Binding of Antiribonucleoprotein Antibodies to the Nucleus of Isolated Living Keratinocytes

Abstract

The general accepted dogma is that antibodies do not penetrate living cells. However, this has recently been challenged for anti-ribonucleoprotein antibodies (anti-RNPab). We have studied here the "penetration" of trypsin isolated keratinocytes in vitro, using indirect immuno-fluorescence and immuno-peroxidase techniques. 70% (+/- 22) of a living keratinocyte cell suspension showed nuclear speckled staining when incubated with anti-RNP sera but only 9.5% (+/- 4) were stained with an anti-DNA antiserum (homogeneous pattern). A suspension of dead keratinocytes gave similar percentages with both anti-sera (89.5 [+/- 8] and 76.6 [+/- 18] respectively). The penetration of anti-RNPab into the nuclei of living epidermal cells increased gradually during the first hour of incubation without a parallel increase in the deadh rate measured by trypan blue dye exclusion. There was still high percentage of stained cell even after high dilution (1/1000) of anti-RNP sera. However, the percentage markedly decreased after previous incubation of the cells with increasing concentrations of concanavalin A. No decrease was obtained with dead keratinocytes in the same conditions. After they had been incubated with anti-RNPab, the epidermal cells were still capable of adhering to culture flasks and of incorporating labeled precursors. Only 4% of the epidermal cells in suspension were able to form rosettes with antibody coated erythrocytes, and were thus bearing receptors for the Fc fragment of IgG. These results strongly suggest that anti-RNPab penetrated living epidermal cells, but not through Fc receptors as reported for mononuclear blood cells.