Binding of nucleosomes to a cell surface receptor: redistribution and endocytosis in the presence of lupus antibodies.
In the present study, we sought evidence for a surface nucleosome receptor in the fibroblastic cell line CV-1, and questioned whether anti-double-stranded (ds) DNA and/or anti-histone autoantibodies could recognize and influence the fate of cell surface-bound nucleosomes. 125I-labeled mononucleosomes were shown to bind to the cell layer in a specific, concentration-dependent and a saturable manner. Scatchard analysis revealed the presence of two binding sites: a high-affinity site with a Kd of approximately 7nM and a low-affinity site (Kd approximately 400 nM) with a high capacity of 9 x 10(7) sites. Visualization of bound mononucleosomes by fluorescence revealed staining on both the cell surface and the extracellular matrix (ECM). Purified mononucleosome-derived ds DNA (180-200 bp) was found to complete for binding of 125I-mononucleosomes on the low-affinity site, to stain exclusively the ECM in immunofluorescence, and to precipitate three specific proteins of 43, 180 and 240 kDa from 125-I-labeled cell lysates. Nucleosomes were found to precipitate not only the 180-kDa ds DNA-reactive component, but also a unique protein of 50 kDa, suggesting that this protein is a cell surface receptor for nucleosomes on these fibroblasts. Once bound on the cell surface, mononucleosomes were recognized and secondarily complexed by lupus anti-ds DNA or anti-histone antibodies (i.e. anti-nucleosome antibodies), thus forming immune complexes in situ. The presence of these complexing auto-antibodies was found dramatically to enhance the kinetics of mononucleosome internalization. Following the internalization of the nucleosome-anti-nucleosome complexes by immunofluorescence, we observed the formation of vesicles at the edge of the cells by 5-10 min which moved toward the perinuclear region by 20-30 min. By means of double-fluorescence labeling and proteolytic treatment, these fluorescent vesicles were shown to be in the cytoplasm, suggesting true endocytosis of nucleosome-anti-nucleosome immune complexes. As shown by confocal microscopy, at no stage of this endocytic process was there any indication that coated pits or coated vesicles participated. Co-distribution of the endocytic vesicles with regions rich in actin filaments and inhibition of endocytosis of nucleosome-anti-nucleosome complexes by disruption of the microfilament network with cytochalasin D suggest a mechanism mediated by the cytoskeleton. Taken together, our data provide evidence for the presence of a surface nucleosome receptor. We also show that anti-ds DNA and anti-histone antibodies can form nucleosome-anti-nucleosome immune complexes in situ at the cell surface, and thus dramatically enhance the kinetics of nucleosome endocytosis.
Koutouzov
S
S
INSERM U 25, Hôpital Necker, Paris, France.
Cabrespines
A
A
Amoura
Z
Z
Chabre
H
H
Lotton
C
C
Bach
J F
JF
D000818:Animals; D000974:Antibodies, Antinuclear / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000936:Antigen-Antibody Complex / Q000378:metabolism; D001665:Binding Sites / Q000276:immunology; D002352:Carrier Proteins / Q000276:immunology / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D003599:Cytoskeleton / Q000276:immunology; D004247:DNA / Q000276:immunology; D004705:Endocytosis / Q000276:immunology*; D010450:Endopeptidases / Q000494:pharmacology; D011992:Endosomes / Q000276:immunology / Q000378:metabolism; D005347:Fibroblasts; D019084:Fluorescent Antibody Technique, Indirect; D024001:HMGN Proteins; D006657:Histones / Q000276:immunology; D008180:Lupus Erythematosus, Systemic / Q000276:immunology*; D009707:Nucleosomes / Q000276:immunology / Q000378:metabolism*; D011233:Precipitin Tests; D011956:Receptors, Cell Surface / Q000378:metabolism*
D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002352:Carrier Proteins; D024001:HMGN Proteins; C028567:HMGN4 protein, human; D006657:Histones; D009707:Nucleosomes; D011956:Receptors, Cell Surface; D004247:DNA; D010450:Endopeptidases