title: DNA-histone complexes as ligands amplify cell penetration and nuclear targeting of anti-DNA antibodies via energy-independent mechanisms.
abstract: We have generated three monoclonal cell-penetrating antibodies (CPAbs) from a non-immunized lupus-prone (NZB × NZW)F1 mouse that exhibited high anti-DNA serum titres. These CPAbs are polyreactive because they bind to DNA and other cellular components, and localize mainly in the nucleus of HeLa cells, albeit with a distinct nuclear labelling profile. Herein, we have examined whether DNA-histone complexes (DHC) binding to CPAbs, before cell entry, could modify the cell penetration of CPAbs or their nuclear staining properties. By applying confocal microscopy and image analysis, we found that extracellular binding of purified CPAbs to DHC significantly enhanced their subsequent cell-entry, both in terms of percentages of positively labelled cells and fluorescence intensity (internalized CPAb amount), whereas there was a variable effect on their nuclear staining profile. Internalization of CPAbs, either alone or bound to DHC, remained unaltered after the addition of endocytosis-specific inhibitors at 37° or assay performance at 4°, suggesting the involvement of energy-independent mechanisms in the internalization process. These findings assign to CPAbs a more complex pathogenetic role in systemic lupus erythematosus where both CPAbs and nuclear components are abundant.
authors.affiliation: Immunology Laboratory, Immunology Department, Hellenic Pasteur Institute, Athens, Greece.
authors.forename: Markella
authors.initials: M
authors.lastname: Zannikou
authors.affiliation: Division of Biomedical Research, Foundation of Research and Technology-Hellas, Institute of Molecular Biology & Biotechnology, University Campus, Ioannina, Greece.|Department of Informatics and Telecommunications Engineering, University of Western Macedonia, Kozani, Greece.
authors.forename: Sofia
authors.initials: S
authors.lastname: Bellou
authors.affiliation: Immunology Laboratory, Immunology Department, Hellenic Pasteur Institute, Athens, Greece.
authors.forename: Petros
authors.initials: P
authors.lastname: Eliades
authors.affiliation: Immunology Laboratory, Immunology Department, Hellenic Pasteur Institute, Athens, Greece.
authors.forename: Aikaterini
authors.initials: A
authors.lastname: Hatzioannou
authors.affiliation: Laboratory of Biological Chemistry, Medical School, University of Ioannina, Ioannina, Greece.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Mantzaris
authors.affiliation: Division of Endocrinology, Faculty of Medicine, Memorial University of Newfoundland, St John's, NL, Canada.
authors.forename: George
authors.initials: G
authors.lastname: Carayanniotis
authors.affiliation: Immunology Laboratory, Immunology Department, Hellenic Pasteur Institute, Athens, Greece.
authors.forename: Stratis
authors.initials: S
authors.lastname: Avrameas
authors.affiliation: Immunology Laboratory, Immunology Department, Hellenic Pasteur Institute, Athens, Greece.
authors.forename: Peggy
authors.identifier: http://orcid.org/0000-0002-4693-789X
authors.initials: P
authors.lastname: Lymberi
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D000974:Antibodies, Antinuclear / Q000276:immunology / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D002467:Cell Nucleus / Q000276:immunology / Q000378:metabolism*; D057846:Cell-Penetrating Peptides / Q000378:metabolism*; D002843:Chromatin / Q000276:immunology / Q000378:metabolism*; D004247:DNA / Q000276:immunology / Q000378:metabolism*; D004195:Disease Models, Animal; D004705:Endocytosis; D006367:HeLa Cells; D006657:Histones / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008180:Lupus Erythematosus, Systemic / Q000235:genetics / Q000276:immunology*; D008297:Male; D008814:Mice, Inbred NZB; D018613:Microscopy, Confocal
chemical_list: D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D057846:Cell-Penetrating Peptides; D002843:Chromatin; D006657:Histones; D008024:Ligands; D004247:DNA
keywords: cell-nucleus penetration; cell-penetrating antibodies; lupus-prone mice; nuclear molecules; polyreactive anti-DNA antibodies