Microparticles as antigenic targets of antibodies to DNA and nucleosomes in systemic lupus erythematosus

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Abstract

Systemic lupus erythematosus is a prototypic autoimmune disease characterized by antibodies to DNA and other nuclear molecules. While these antibodies can form immune complexes, the mechanisms generating the bound nuclear antigens are not known. These studies investigated whether microparticles can form complexes with anti-DNA and other anti-nucleosomal antibodies. Microparticles are small membrane-bound vesicles released from dead and dying cells; these particles contain a variety of cellular components, including DNA. To assess antigenicity, microparticles generated in vitro from apoptotic cell lines were tested using murine monoclonal anti-DNA and anti-nucleosomal antibodies as well as plasma from lupus patients. Antibody binding was assessed by flow cytometry. As these studies showed, some but not all of the monoclonal antibodies bound to microparticles prepared from apoptotic HL-60, THP-1 and Jurkat cells. For HL-60 cells, both staurosporine and UV radiation led to the production of antigenically active particles. For the anti-DNA antibody with high particle binding, prior treatment of DNase reduced activity. With plasma from patients with SLE, antibody binding to microparticles was present although a clear relationship with anti-DNA antibody levels was not observed. To determine whether lupus plasma contains immune complexes with particle properties, particle preparations were tested for bound IgG by flow cytometry. These studies indicated that lupus plasma contains particles with IgG binding, with numbers correlated with anti-DNA levels. Together, these findings indicate that microparticles display DNA and nucleosomal molecules in an antigenic form and could represent a source of immune complexes in SLE.

Introduction

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by the production of antibodies to the cell nucleus (antinuclear antibodies or ANA) [1], [2]. These antibodies bind to components of chromatin, including nucleosomal antigens such as DNA, histones and complexes of these molecules. Among these antibodies, antibodies to DNA (anti-DNA) are the serological hallmark of SLE and serve as markers of diagnostic and prognosis significance [3]. The expression of anti-DNA is directly linked to nephritis, with glomerular deposition of immune complexes inciting inflammation. Since anti-DNA antibodies are essentially unique to SLE, elucidation of their properties can provide fundamental insight into immune disturbances underlying autoimmunity [4], [5], [6].

Anti-DNA antibodies are part of the spectrum of anti-nucleosomal antibodies whose production exemplifies autoreactivity to nuclear molecules. Thus, for both patients with SLE as well as murine models, the variable region sequences of monoclonal autoantibodies display features of selection by DNA antigen [7], [8], [9]. In autoantibody induction, however, DNA may not behave as a conventional antigen because of its intrinsic immunological properties (alone or in immune complexes) and its ability to stimulate toll-like receptor (TLR) and non-TLR nucleic acid sensors to induce cytokine production [10], [11], [12], [13], [14], [15], [16], [17].

While antibodies to DNA and other nucleosomal molecules have been the focus of intense investigation, much less is known about the properties of the nuclear antigens that induce these responses or form immune complexes in vivo. In general, nuclear antigens impacting on the immune system have been considered to be the products of cell death, especially apoptosis [18]. In SLE, the amount of this antigenic material may increase because of excessive cell death or impaired clearance of dead and dying cells. For nuclear antigens to form complexes, however, they must migrate from the cell nucleus in a form that allows extracellular release [19], [20], [21], [22].

In the current studies, we have investigated the nature of extracellular DNA and other nucleosomal molecules, focusing on microparticles as a source. Microparticles (MPs) are small membrane-bound vesicles that circulate in the blood and serve as intercellular signaling elements to stimulate immunity and promote thrombosis [23], [24], [25], [26]. In the context of SLE, microparticles may be related to blebs that form during cell death, although blebs are usually visualized as intracellular components [23]. MP release, however, can also occur during activation and involve cells such as platelets, which do not undergo classical apoptosis. Like blebs, microparticles can contain both cytoplasmic and nuclear components such as DNA and RNA [27]. Microparticles differ from apoptotic bodies, which are larger and include the compacted remains of an apoptotic cell.

To investigate microparticles as a source of nuclear antigens, we have used murine monoclonal anti-nucleosomal antibodies to assess the antigenic properties of microparticles generated in vitro from cell lines undergoing apoptosis. In addition, we have tested the blood of patients with SLE for their ability to bind microparticles and for their content of particles displaying bound IgG. As results present herein indicate, microparticles generated in vitro can display DNA and other nucleosomal structures in an antigenic form. Furthermore, our findings indicate that the blood of SLE patients can contain particles with bound IgG, suggesting a type of immune complexes that may contribute to pathogenesis.

Section snippets

Cell lines and production of microparticles

HL-60, Jurkat and THP-1 human cell lines were obtained from the Duke University Comprehensive Cancer Center Cell Culture Facility and cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT) and 20 μg/ml gentamicin (Invitrogen) at 37 °C and 5% CO2. Microparticles were generated by making cells (107 cells/ml) apoptotic by treatment with 1 μM staurosporine (STS) (Sigma, St Louis, MO) for 20–24 h; in some experiments, apoptosis was

Microparticles from apoptotic cells as a source of DNA and other nucleosomal antigens that bind antibodies

These studies investigated whether microparticles can bind anti-DNA and other anti-nucleosomal antibodies, using as a source of antigen microparticles from cell lines induced to undergo apoptosis in vitro. For this purpose, microparticles were isolated by differential centrifugation and their identity as microparticles established by flow cytometry by previously described methods [27]. As illustrated in Fig. 1, this purification procedure provided preparations that lacked significant

Discussion

Results of these studies indicate that microparticles released from apoptotic cells display nucleosomal molecules in an antigenically active form. For a panel of monoclonal anti-DNA and anti-nucleosomal antibodies from lupus mice, the binding to particles occurred with microparticles derived from 3 different cell lines undergoing apoptosis in vitro. Since the binding of some of these antibodies was sensitive to DNase, the identity of the target antigen as a DNA or a DNA-histone complex was

Acknowledgements

This work was supported by a VA Merit Review grant and NIH Grant R21A1083923.

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