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

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.

Figure 1

Libby 6-mix induces apoptosis in murine macrophages. RAW264.7 cells were exposed to Libby amphibole asbestos or wollastonite fibers (62.5 µg/cm²) for 72 h or 1 µM staurosporine for 6 h. Apoptosis was quantified through Annexin V staining as described in Materials and Methods. Apoptosis was quantified as the percent of cells that were positive for Annexin V staining through flow cytometry. Results represent mean percentages ± SEM from two independent experiments. Asterisks indicate a significant increase compared to time matched controls (P < 0.05, n = 3).

Figure 2

Libby 6-mix exposure results in the cleavage of PARP. A) RAW264.7 cells were exposed to Libby 6-mix fibers at a final concentration of 62.5 µg/cm² for 72 h or to 1 µM staurosporine for 6 h (positive control). Cell lysates were prepared and analyzed through immunoblotting using an anti-PARP rabbit polyclonal that detects the full length protein (116 kDa) and the cleaved fragment (85 kDa). B) Quantification of full length PARP from RAW264.7 cells exposed to Libby 6-mix. Values signify the densitometric units determined for the immunoreactive bands of full length PARP divided by the densitometric units determined for the immunoreactive bands of the actin loading control and reported as the fold change compared to control.

Figure 2

Libby 6-mix exposure results in the cleavage of PARP. A) RAW264.7 cells were exposed to Libby 6-mix fibers at a final concentration of 62.5 µg/cm² for 72 h or to 1 µM staurosporine for 6 h (positive control). Cell lysates were prepared and analyzed through immunoblotting using an anti-PARP rabbit polyclonal that detects the full length protein (116 kDa) and the cleaved fragment (85 kDa). B) Quantification of full length PARP from RAW264.7 cells exposed to Libby 6-mix. Values signify the densitometric units determined for the immunoreactive bands of full length PARP divided by the densitometric units determined for the immunoreactive bands of the actin loading control and reported as the fold change compared to control.

Figure 3

The SSA/Ro52 autoantigen is not cleaved during apoptosis. RAW264.7 cells were exposed to Libby 6-mix fibers at a final concentration of 62.5 µg/cm² for 72 h or to 1 µM staurosporine for 6 h (positive control). Cell lysates were prepared and analyzed through immunoblotting using an anti-SSA/Ro52 rabbit polyclonal antibody and an antiactin rabbit polyclonal antibody.

Figure 4

Libby 6-mix exposure results in the redistribution of SSA/Ro52 to surface blebs of apoptotic cells. Representative confocal microcopy images of RAW264.7 cells untreated (Panels A–D) or exposed to Libby asbestos for 48 h (Panels E–L). The distribution of the SSA/Ro52 autoantigen was visualized through confocal microscopy using a rabbit polyclonal anti-SSA/Ro52 and an Alexa Fluor 488 conjugated goat anti-rabbit IgG secondary (Green in panels A, E and I). Nuclei were counterstained with propidium iodide (Red in panels B, F and J). Apoptotic nuclei are indicated by arrows. The plasma membrane was visualized with Alexa Fluor 647 cholera toxin subunit B conjugate (Magenta in panels C, G and K). Right panels (D, H and L) show merged images with arrows indicating apoptotic cell surface blebs enriched in SSA/Ro52.

Figure 5

Staurosporine treatment results in the redistribution of SSA/Ro52 to surface blebs of apoptotic cells. A representative confocal microcopy image of RAW264.7 cells treated with 1 µM staurosporine for 6 h. The distribution of the SSA/Ro52 autoantigen was visualized through confocal microscopy using a rabbit polyclonal anti-SSA/Ro52 and an Alexa Fluor 488 conjugated goat anti-rabbit IgG secondary (Panel A). Nuclei were counterstained with propidium iodide (Panel B). Apoptotic nuclei are indicated by arrows. The plasma membrane was visualized with Alexa Fluor 647 cholera toxin subunit B conjugate (Panel C). Panel D shows the merged image with an arrow indicating apoptotic cell surface blebs enriched in SSA/Ro52.

Figure 6

Autoantibodies from asbestos-exposed mice recognize apoptotic blebs enriched with the SSA/Ro52 autoantigen. Representative confocal microcopy images of RAW264.7 cells exposed to Libby asbestos for 48 h. The SSA/Ro52 autoantigen was visualized through confocal microscopy using a rabbit polyclonal anti-SSA/Ro52 antibody and an Alexa Fluor 488 conjugated goat anti-rabbit IgG secondary (Green in panels A, E and I). Nuclei were counterstained with propidium iodide (Cyan in panels B, F and J). Apoptotic nuclei are indicated by arrows. Binding of mouse autoantibodies were visualized with an Alexa Fluor 647 conjugated goat anti-mouse IgG secondary (Red in panels C, G and K). Panels C and G include AA staining from a mouse exposed to tremolite asbestos. Panel K includes AA staining from a mouse exposed to wollastonite fibers. Right panels (D, H and L) show merged images. Colocalization of SSA/Ro52 and autoantigens recognized by asbestos-induced mouse AAs is visualized in yellow in merged images and indicated by arrows. Colocalization was confirmed through Image J analysis.