Elsevier

Toxicology

Volume 246, Issues 2–3, 18 April 2008, Pages 172-179
Toxicology

Autoantibodies from mice exposed to Libby amphibole asbestos bind SSA/Ro52-enriched apoptotic blebs of murine macrophages

https://doi.org/10.1016/j.tox.2008.01.008Get rights and content

Abstract

Asbestos exposure is associated with increased autoimmune responses in humans. For example, in Libby, MT where significant asbestos exposure has occurred due to an asbestos-contaminated vermiculite mine near the community, residents have developed increased autoimmune responses compared to an unexposed population. However, the exact mechanism by which Libby amphibole asbestos generates autoimmune responses is unclear. A murine model of amphibole asbestos-induced autoimmunity was recently established, and one of the targets of the autoantibodies (AAs) was the SSA/Ro52 autoantigen. The purpose of this study was to determine whether the SSA/Ro52 autoantigen is exposed at the surface of cells as a result of asbestos exposure as a possible mechanism leading to antigenicity. Our results indicate that Libby asbestos induces apoptosis in murine macrophages as determined by phosphatidylserine exposure, cleavage of poly(ADP-ribose) polymerase and morphological changes such as nuclear condensation. Moreover, asbestos-induced apoptosis results in the formation of apoptotic cell surface blebs enriched in SSA/Ro52 as determined by confocal microscopy. Most importantly, apoptotic cell surface blebs are recognized by AAs from mice exposed to amphibole asbestos suggesting that these cell surface structures may be antigenic when presented in a pro-inflammatory context. This study supports the hypothesis that the induction of apoptosis plays a key role in environmentally induced autoimmunity through cell surface exposure of a known autoantigen.

Introduction

Systemic autoimmune disease (SAID), such as rheumatoid arthritis, systemic lupus erythematosus (SLE) and scleroderma, occurs when self-reactive T and B cells escape the protective tolerizing safe guards of the immune system and cause tissue damage, resulting in a diversity of debilitating symptoms. Although it is widely accepted that environmental factors combine with genetic susceptibility to exacerbate the development of these diseases, critical knowledge gaps remain regarding the mechanisms involved. The association of SAID with exposure to inhaled environmental silicates, asbestos and silica, provides an important experimental framework needed to fill those gaps.

Exposure to environmental xenobiotics such as silica, mercury and vinyl chloride is associated with the production of autoantibodies (AAs) and the development of SAID (D’Cruz, 2000, Hess, 2002, Parks and Cooper, 2005). Increased serum immunoglobulins, positive antinuclear autoantibody (ANA) tests and immune complexes have also been reported in small cohorts of individuals exposed to asbestos, a naturally occurring crystalline silicate fiber. The ability of asbestos exposure to exacerbate autoimmune responses in humans is supported by studies of an asbestos-exposed population from Libby, MT. Residents of Libby have higher frequencies and titers of positive ANA tests compared to an unexposed control population (Pfau et al., 2005), as well as increased risk for SAID, which is dependent upon the routes of exposure (Noonan et al., 2006). Although the exact mechanisms that lead to the induction of SAID and AA production as a result of environmental exposure have not been established, evidence supports the role of apoptosis as a potential initiating stimulus (Casciola-Rosen et al., 1994, Rosen and Casciola-Rosen, 1999, Rosen and Casciola-Rosen, 2004). Considerable literature supporting a role for apoptosis in silica-induced autoimmunity was recently reviewed (Brown et al., 2004), emphasizing the ability of silica to induce apoptosis and to drive SAID. Because asbestos can also cause apoptosis (Hamilton et al., 1996), a similar mechanism may link asbestos with systemic autoimmune responses.

Therefore, a murine model of asbestos-induced autoimmunity was recently established (Pfau et al., in press). Asbestos-exposed mice develop positive antinuclear antibody tests and mild glomerulonephritis suggestive of a SLE-like disease. The asbestos-induced SLE-like disease is characterized by the production of AAs that recognize the SSA/Ro52 autoantigen. SSA/Ro52 is a newly characterized RING-finger-type E3 ubiquitin ligase (Espinosa et al., 2006, Wada and Kamitani, 2006), which in unstimulated cells localizes to the cytoplasm (Ohlsson et al., 2002). Autoantibodies against SSA/Ro52 are commonly found in patients with SLE (Hassan et al., 2002, Hoffman et al., 2004, Popovic et al., 2007, Routsias et al., 2006). Interestingly, SSA/Ro52 redistributes itself to apoptotic blebs in cardiac monocytes, epithelial cells, salivary gland cells and keratinocytes after exposure to various pro-apoptotic agents (Igarashi et al., 1995, McArthur et al., 2002, Miranda et al., 1998, Ohlsson et al., 2002). Because AAs target SSA/Ro52 during autoimmune responses, the clustering of SSA/Ro52 to small surface blebs of apoptotic cells may be important in the induction of autoimmunity generated by xenobiotics (Casciola-Rosen et al., 1994).

Alveolar macrophages are the primary cells that interact with inhaled particles and function to clear particles from the lung. Our study utilizes RAW264.7 cells, a phagocytic murine cell line with characteristics similar to alveolar macrophages (Xia et al., 2006). We have previously shown that exposure to Libby amphibole asbestos induces oxidative stress in these cells (Blake et al., 2007). The results of this study extend these findings and indicate that Libby asbestos induces apoptosis in macrophages leading to the redistribution of SSA/Ro52 to apoptotic blebs. The fact that antibodies from asbestos-exposed mice recognize these surface blebs suggests that the antigen in the apoptotic blebs can be immunogenic in vivo, ultimately resulting in production of AAs. This model has great potential to (a) clearly establish direct evidence for a role of apoptosis in silicate-induced autoimmunity, including mechanisms of lost tolerance to apoptotic material, (b) explore anti-Ro52 as a possible early marker of developing SAID following exposures, and (c) reveal potential therapeutic targets to halt disease progression.

Section snippets

Cell culture conditions

Mouse macrophages, RAW264.7 cells, (ATCC-2091: American Type Culture Collection, Manassas, VA) were cultured at 37 °C in a 5% CO2 incubator in complete media, which contained DMEM media with 4.5 g/l glucose and l-glutamine supplemented with 1.5 mM sodium pyruvate, 20 mM HEPES, 55 μM 2-mercaptoethanol, 10% fetal bovine serum and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B) (Gibco BRL, Bethesda, MD). Confluent RAW264.7 cells were scraped from T75 flasks, counted

Libby 6-mix induces apoptosis in murine macrophages

We have previously reported that Libby 6-mix asbestos decreases cell viability in RAW264.7 cells after 24 h at a final concentration of 62.5 μg/cm2 (Blake et al., 2007). To determine whether the decrease in cell viability was a result of apoptosis, RAW264.7 cells were exposed to Libby 6-mix as previously described (Blake et al., 2007) and apoptosis was quantified through Annexin V staining. Untreated control cells maintained a basal level of apoptosis of 3–5% (Fig. 1). Exposure to Libby 6-mix

Discussion

Environmental exposure to crystalline silicates, such as silica and asbestos, generate the production of AAs and induce autoimmune phenotypes in humans and in mice. Exposure to silica exacerbates autoimmune responses in individuals in ‘dusty’ trades as well as autoimmune prone NZM 2410 mice (Brown et al., 2004, Parks and Cooper, 2005). In addition to AA production and disease pathology, silica-exposed NZM mice produce AAs that bind to macrophages undergoing silica-induced apoptosis (Pfau et

Acknowledgements

The authors thank Sheng’ai Li, University of Montana, for helpful discussions and technical assistance, Pamela Shaw in the CEHS Fluorescence Cytometry Core for assistance with the FACS analysis and Lou Herritt in the CEHS Histology and Imaging Core for assistance with the confocal microscopy analysis.

This work was funded by grants from the NIH COBRE P20 NCRR017670, R21 ES012956 and from the University of Montana Graduate School.

References (48)

  • A. Bondanza et al.

    Requirement of dying cells and environmental adjuvants for the induction of autoimmunity

    Arthritis Rheum.

    (2004)
  • J.M. Brown et al.

    Silica, apoptosis and autoimmunity

    J. Immunotoxicol.

    (2004)
  • J.M. Brown et al.

    Effects of rottlerin on silica-exacerbated systemic autoimmune disease in New Zealand mixed mice

    Am. J. Physiol. Lung Cell Mol. Physiol.

    (2005)
  • L.A. Casciola-Rosen et al.

    Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes

    J. Exp. Med.

    (1994)
  • C.A. Casiano et al.

    Selective cleavage of nuclear autoantigens during CD95 (Fas/APO-1)-mediated T cell apoptosis

    J. Exp. Med.

    (1996)
  • R.M. Clancy et al.

    Impaired clearance of apoptotic cardiocytes is linked to anti-SSA/Ro and -SSB/La antibodies in the pathogenesis of congenital heart block

    J. Clin. Invest.

    (2006)
  • K.E. Driscoll et al.

    Alveolar macrophage cytokine and growth factor production in a rat model of crocidolite-induced pulmonary inflammation and fibrosis

    J. Toxicol. Environ. Health

    (1995)
  • P.J. Duriez et al.

    Cleavage of poly(ADP-ribose) polymerase: a sensitive parameter to study cell death

    Biochem. Cell Biol.

    (1997)
  • A. Espinosa et al.

    The Sjogren's syndrome-associated autoantigen Ro52 is an E3 ligase that regulates proliferation and cell death

    J. Immunol.

    (2006)
  • R. Gandhi et al.

    Anti-idiotype-mediated epitope spreading and diminished phagocytosis by a human monoclonal antibody recognizing late-stage apoptotic cells

    Cell Death Differ.

    (2006)
  • Gunter ME, Dyar DM, Twamley B, Foit FF Jr, Cornelius S. Composition, 2003. Fe+3/Fe and crystal structure of...
  • R.F. Hamilton et al.

    Asbestos induces apoptosis in human alveolar macrophages

    Am. J. Physiol.

    (1996)
  • R.F. Hamilton et al.

    A comparison of asbestos and urban particulate matter in the in vitro modification of human alveolar macrophage antigen-presenting cell function

    Exp. Lung Res.

    (2004)
  • A.B. Hassan et al.

    Serial analysis of Ro/SSA and La/SSB antibody levels and correlation with clinical disease activity in patients with systemic lupus erythematosus

    Scand. J. Rheumatol.

    (2002)
  • Cited by (21)

    • Comparative health effects in mice of Libby amphibole asbestos and a fibrous amphibole from Arizona

      2017, Toxicology and Applied Pharmacology
      Citation Excerpt :

      While the rates of pulmonary fibrosis (asbestosis) and cancer (mesothelioma, pulmonary carcinoma) are significantly elevated among people exposed to the Libby amphibole asbestos (LAA), the predominant negative health outcomes include systemic autoimmunity and a progressive pleural fibrosis that may also be autoimmune in nature through production of mesothelial cell autoantibodies (MCAA) (Peipins et al., 2003; Pfau et al., 2005; Rohs et al., 2008; Sullivan, 2007; Szeinuk et al., 2016; U.S. Environmental Protection Agency, R, 2011; Whitehouse et al., 2008; Winters et al., 2012; Larson et al., 2010a; Gilmer et al., 2016; Hanson et al., 2016; Marchand et al., 2012; Serve et al., 2013). Using a wildtype mouse model (C57BL/6), we have corroborated these outcomes in mice, providing a critical tool for evaluation of the relative toxicity of other mineral fibers (Blake et al., 2008; Ferro et al., 2013; Gilmer et al., 2015; Pfau et al., 2013; Pfau et al., 2008; Zebedeo et al., 2014). LAA exposures occurred due to contamination of vermiculite, which was mined outside of Libby for decades and used throughout the community in buildings, gardens, and playgrounds.

    • Mesothelial cell and anti-nuclear autoantibodies associated with pleural abnormalities in an asbestos exposed population of Libby MT

      2012, Toxicology Letters
      Citation Excerpt :

      Fibers reaching the pleural cavity would cause damage to the mesothelial cells that line the cavity: over time, mesothelial debris could lead not only to production of ANA, but also to autoantibodies to mesothelial surface proteins. This mechanism, in which apoptotic debris provides the antigens for autoimmune responses, has been proposed for production of autoantibodies following exposure to silica or asbestos (Brown et al., 2004; Blake et al., 2008), and in diseases such as SLE and SSc (Muñoz et al., 2010; Kahaleh, 2008). The data reported here are admittedly indirect evidence that asbestos caused these autoantibodies, and also that they are now contributing to the pleural disease in these patients.

    • Silica, silicosis, and autoimmunity

      2016, Frontiers in Immunology
    View all citing articles on Scopus
    View full text