Binding of nucleosomes to a cell surface receptor: redistribution and endocytosis in the presence of lupus antibodies

Eur J Immunol. 1996 Feb;26(2):472-86. doi: 10.1002/eji.1830260230.

Abstract

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.

MeSH terms

  • Animals
  • Antibodies, Antinuclear / metabolism*
  • Antibodies, Monoclonal / chemistry
  • Antigen-Antibody Complex / metabolism
  • Binding Sites / immunology
  • Carrier Proteins / immunology
  • Carrier Proteins / metabolism*
  • Cell Line
  • Chlorocebus aethiops
  • Cytoskeleton / immunology
  • DNA / immunology
  • Endocytosis / immunology*
  • Endopeptidases / pharmacology
  • Endosomes / immunology
  • Endosomes / metabolism
  • Fibroblasts
  • Fluorescent Antibody Technique, Indirect
  • HMGN Proteins*
  • Histones / immunology
  • Lupus Erythematosus, Systemic / immunology*
  • Nucleosomes / immunology
  • Nucleosomes / metabolism*
  • Precipitin Tests
  • Receptors, Cell Surface / metabolism*

Substances

  • Antibodies, Antinuclear
  • Antibodies, Monoclonal
  • Antigen-Antibody Complex
  • Carrier Proteins
  • HMGN Proteins
  • HMGN4 protein, human
  • Histones
  • Nucleosomes
  • Receptors, Cell Surface
  • DNA
  • Endopeptidases