Autoantibodies from mice exposed to Libby amphibole asbestos bind SSA/Ro52-enriched apoptotic blebs of murine macrophages
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)
- et al.
Automatic and quantitative measurement of protein-protein colocalization in live cells
Biophys. J.
(2004) Autoimmune diseases associated with drugs, chemicals and environmental factors
Toxicol. Lett.
(2000)- et al.
Increased sensitivity to asbestos-induced lung injury in mice lacking extracellular superoxide dismutase
Free Radic. Biol. Med.
(2006) Environmental chemicals and autoimmune disease: cause and effect
Toxicology
(2002)- et al.
Intracellular trafficking and surface expression of SS-A (Ro), SS-B (La), poly(ADP-ribose) polymerase and alpha-fodrin autoantigens during apoptosis in human salivary gland cells induced by tumour necrosis factor-alpha
Arch. Oral Biol.
(2002) - et al.
Silica-exposed mice generate autoantibodies to apoptotic cells
Toxicology
(2004) - et al.
Oxidative stress mediates cell surface expression of SS-A/Ro antigen on keratinocytes
Free Radic. Biol. Med.
(2002) - et al.
Autoantigen Ro52 is an E3 ubiquitin ligase
Biochem. Biophys. Res. Commun.
(2006) - et al.
Internalization of Libby amphibole asbestos and induction of oxidative stress in murine macrophages
Toxicol. Sci.
(2007) - et al.
A guided tour into subcellular colocalization analysis in light microscopy
J. Microsc.
(2006)
Requirement of dying cells and environmental adjuvants for the induction of autoimmunity
Arthritis Rheum.
Silica, apoptosis and autoimmunity
J. Immunotoxicol.
Effects of rottlerin on silica-exacerbated systemic autoimmune disease in New Zealand mixed mice
Am. J. Physiol. Lung Cell Mol. Physiol.
Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes
J. Exp. Med.
Selective cleavage of nuclear autoantigens during CD95 (Fas/APO-1)-mediated T cell apoptosis
J. Exp. Med.
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.
Alveolar macrophage cytokine and growth factor production in a rat model of crocidolite-induced pulmonary inflammation and fibrosis
J. Toxicol. Environ. Health
Cleavage of poly(ADP-ribose) polymerase: a sensitive parameter to study cell death
Biochem. Cell Biol.
The Sjogren's syndrome-associated autoantigen Ro52 is an E3 ligase that regulates proliferation and cell death
J. Immunol.
Anti-idiotype-mediated epitope spreading and diminished phagocytosis by a human monoclonal antibody recognizing late-stage apoptotic cells
Cell Death Differ.
Asbestos induces apoptosis in human alveolar macrophages
Am. J. Physiol.
A comparison of asbestos and urban particulate matter in the in vitro modification of human alveolar macrophage antigen-presenting cell function
Exp. Lung Res.
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.
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