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

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.

Circulating plasma levels of nucleosomes in patients with systemic lupus erythematosus. Correlation with serum antinucleosome antibody titers and absence of clear association with disease activity

“…Histone gel analysis (28,29) of the mononucleosome preparations demonstrated the presence of the core made up of the 4 histones in equimolar amounts, indicating that this preparation yielded "intact" mononucleosomes (26).…”

Nucleosomes inhibit phagocytosis of apoptotic thymocytes by peritoneal macrophages from MRL+/+ lupus-prone mice

“…Both in vivo transfer experiments with nucleosomes complexed with lupus antibody [23,24] and GBM binding in vitro of sera from lupus-prone mice and SLE patients [25,26] have indicated that nucleosomes, either within circulat-ing immune complexes or present in situ on target tissues, mediate binding of lupus autoantibodies to glomerular antigens. Furthermore, there is also evidence that the nucleosome could exert direct effects on cell function, presumably through binding to specific surface receptors [27][28][29]. Polyclonal B cell activation by nucleosomes has been demonstrated both with murine and human lymphocytes [30,31] and interleukin-6 (IL-6) secretion has been observed after interaction of nucleosomes with murine spleen cells [32].…”

Enhanced tumor MR imaging with gadolinium‐loaded polychelating polymer‐containing tumor‐targeted liposomes

“…These antibodies (including the representative monoclonal 2C5 antibody, mAb 2C5) demonstrate nucleosome-restricted specificity and recognize various live cancer cells via the cancer cell surfacebound nucleosomes released from apoptotically dying cancer cells (18). This phenomenon stems from the fact that live cancer cells from various tumors and cell lines seem to carry specific binding sites for nucleosomes on their surface (19). Previous studies have clearly demonstrated that, in addition to their own anticancer activity (20), such antibodies can serve as tumor-targeting ligands.…”