Asbestos-Induced HM Cell Death Is PARP Dependent.

PARP was not cleaved in HM undergoing cell death following asbestos exposure; instead the levels of full-length PARP were markedly increased with increasing amounts of asbestos exposure (Fig. 1A). Real-time PCR experiments indicated that PARP mRNA was also induced by asbestos (Fig. S2). PARP activity, measured as conversion of radiolabeled NAD substrate, or production of poly-ADP ribose (PAR), was induced by asbestos treatment (Fig. 1 B and C).

To verify that asbestos-caused HM cell death was PARP dependent, we pretreated HM with the PARP inhibitor 3-aminobenzamide (3-ABA) for 1 h before asbestos exposure. 3-ABA protected HM from asbestos-induced cell death. Viability of HM was significantly increased by pretreatment with 3-ABA before asbestos exposure (Fig. 2A).

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Fig. 2.

Asbestos-induced HM death is PARP dependent and causes ATP depletion. (A) PARP inhibitor 3-ABA decreases asbestos cytotoxicity. HM were incubated with or without 3-ABA (0.5 mM) for 1 h before asbestos exposure (5 μg/cm²). Representative results of three separate experiments are shown. Cytotoxicity was detected using the LDH assay. 3-ABA decreased asbestos cytotoxicity and protected HM from asbestos-induced cell death. *Significantly different compared with HM without asbestos exposure. **Significantly different compared with HM exposed to asbestos without 3-ABA pretreatment (P < 0.05). (B) Asbestos induces ATP depletion in HM. HM were treated with actinomycin D (0.1 μM, positive control for apoptosis), H₂O₂ (200 μM, positive control for necrosis) or with asbestos (0.5–10 μg/cm²) for 24 h. Cellular ATP was measured by a bioluminescence assay. Increasing amounts of asbestos significantly induced ATP depletion. Each column represents the average of three separate experiments.

We determined the levels of ATP in HM exposed to increasing doses of asbestos by bioluminescence assay. We found significant ATP depletion in HM exposed to asbestos and to H₂O₂ (positive control for necrosis, Fig. 2B). In contrast, ATP levels remained high in HM following actinomycin D treatment, which triggers apoptosis (Fig. 2B). These findings indicate that asbestos induces and activates PARP, which in turn promotes ATP depletion and cell necrosis.

Asbestos-Induced HM Death Causes HMGB1 Release and Is Proinflammatory.

To further test the hypothesis that asbestos-induced HM cell death was a regulated form of necrosis, we evaluated HMGB1 localization by immunofluorescence staining. Six hours after asbestos exposure, HMGB1 translocated from the nucleus into the cytosol, as observed in HM exposed to H₂O₂. Conversely, in actinomycin D–exposed HM cells, HMGB1 remained within the nucleus (Fig. 3A). To verify the results, whole-cell extracts and concentrated culture medium were analyzed by Western blot. After 24 h of asbestos exposure, HMGB1 was released into the media in concert with cell death (Fig. 3 B and C). Release of HMGB1 into the extracellular space was paralleled by an increase in HMGB1 transcripts (Fig. S3).

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Fig. 3.

HMGB1 is released from HM into the extracellular space after asbestos exposure. (A) HMGB1 translocates from the nucleus to the cytosol upon asbestos exposure. Top row: HMGB1 staining (FITC conjugated antibody, green). Middle row: DAPI staining (blue). Bottom row: Overlay of HMGB1 (green) and DAPI staining (blue). HM were exposed to crocidolite asbestos (5 μg/cm²), H₂O₂ (100 μM) or actinomycin D (0.1 μM) for 6 h before immunofluoresence staining. Nuclei were visualized by DAPI staining. HMGB1 is localized in the nuclei in controls and actinomycin D–treated cells. In HM exposed to crocidolite or treated with H₂O₂, HMGB1 translocates to the cytosol and is seen mainly outside the nuclei (“overlay” panels, bottom row, where HMGB1 and DAPI stains are overlayed to identify location of HMGB1). (B) HMGB1 is released from HM after asbestos exposure. HM were exposed to 5 μg/cm² asbestos for 0.5–24 h. Cell extracts were analyzed by Western blot. The amount of HMGB1 decreases 16 h after asbestos exposure when cell death becomes detectable. (C) HMGB1 is released from HM into the extracellular medium during asbestos exposure. HM were exposed to asbestos (5 μg/cm²) for 24 h. (Upper) cell extract. (Lower) Cell culture medium was collected, concentrated, and tested by Western blot with a HMGB1-specific antibody.

Next, we tested whether the release of HMGB1 was linked to an inflammatory response. The cell medium was collected, concentrated, and added to macrophages in tissue culture. Macrophage activation was measured by the secretion of the proinflammatory cytokine TNF-α, which promotes asbestos carcinogenesis (14). We found that the cell culture medium from asbestos-exposed HM promoted macrophage secretion of TNF-α. Similar results were obtained using the culture medium from HM in which necrosis was induced by H₂O₂. The culture medium from HM exposed to actinomycin D (positive control for apoptosis) did not induce TNF-α secretion (Fig. 4). To confirm that the induction of TNF-α secretion by macrophages was mediated through the HMGB1 released by necrotic HM, we incubated the culture medium of HM exposed to asbestos with Box A, a fragment of HMGB1 with antagonist activity (33, 34). Box A significantly reduced TNF-α secretion by macrophages treated with culture medium from asbestos-exposed HM (P < 0.05) (Fig. 4). These findings indicate that the release of HMGB1 from asbestos-exposed HM triggers the release of TNF-α and the inflammatory response associated with asbestos carcinogenesis.

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Fig. 4.

Asbestos-induced cell death and the release of HMGB1 promote TNF-α secretion by macrophages. HM were exposed to asbestos (5 μg/cm²), treated with H₂O₂ (1 mM) or actinomycin D (0.5 μM) for 1 h. Drugs were washed away and cells were fed with fresh media. After 24 h, cell culture media were collected, concentrated by ultrafiltration, and added to 10⁵ macrophages in tissue culture with or without preincubation with Box A. The amount of TNF-α released by macrophages was measured 24 h later. TNF-α secretion is induced by culture medium from asbestos-exposed HM and from H₂O₂-treated HM but not by the medium from actinomycin D–treated HM. TNF-α secretion is significantly inhibited by preincubation of medium with HMGB1 antagonist Box A (100 ng/mL) (P < 0.05).

HMGB1 Is Linked to Pathogenesis of Asbestos-Related Disease.

We investigated the possible involvement of HMGB1 in the pathogenesis of asbestos-related disease in our mouse and hamster asbestos experimental models (35). Six hamsters and 10 BALB/c mice were injected with crocidolite asbestos intraperitoneally (i.p.). Chronic inflammation and mesothelial hyperplasia were detected in the peritoneum and tunica vaginalis of all of the injected hamsters and mice around areas of asbestos deposits. Histology showed that in these areas, the asbestos fibers were associated with cells with morphology consistent with macrophages, some of these cells multinucleated (foreign body–type histiocytic cells). Several of these cells contained asbestos fibers or part of these fibers in their cytoplasm (phagocytosis). We observed the same phagocytic response when HM or macrophages were grown in tissue culture in the presence of asbestos. Unlike other cell types, such as fibroblasts, we observed that both HM and macrophages phagocytized asbestos. Immunohistochemistry (performed on mouse tissues) showed that most of the mononuclear and the multinucleated cells found around asbestos deposits were positive for the macrophage marker, F4/80. In addition, there were scattered lymphocytes (CD4⁺ and CD8⁻), plasma cells and HM (pan-cytokeratin+) (Fig. S4). The inflammatory reaction formed irregular nodules around asbestos deposits. These nodules were situated in the loose connective tissue below the mesothelial covering (Fig. 5 and Fig. S4A). The mesothelial cells above these nodules showed focal hyperplasia. HMGB1 and TNF-α staining were specifically localized in these areas. Representative staining of mice specimens are shown in Fig. 5 and S4. HMGB1 was localized in both the nuclei and in the cytoplasm of reactive mesothelial and inflammatory cells and in the nearby extracellular space, consistent with HMGB1 release from cells undergoing necrosis. Away from areas of asbestos deposits, no chronic inflammation or HMGB1 cytoplasmic staining was detected (Fig. 5 A and B, Middle). These findings were very specific and consistently observed in all specimens from mice and hamsters (Fig. S5). TNF-α expression was detected in the same areas (Fig. 5 A and B, Right), consistent with our in vitro data showing that HMGB1 release leads to TNF-α secretion by inflammatory cells (Fig. 4).

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Fig. 5.

Immunohistochemical analyses show strong HMGB1 and TNF-α staining around areas of asbestos deposits in two representative murine specimens. (A) (Left) H&E staining shows nodular area of chronic inflammation (CI) around asbestos deposits under peritoneum, top left quadrant. P, pancreas. HMGB1 (Center) and TNF-α (Right) are present in area with chronic inflammation and not in pancreas (some staining is seen also along connective tissue that divides pancreatic lobes that contains lymphatic vessels). (Original magnification: top row, 100×; lower row, 400×.) (B) Top row. (Left) H&E staining. Testis (T) is shown on left half of figure. Tunical albuginea separate testis from tunical vaginalis (i.e., peritoneum), where chronic inflammation (CI) has formed around areas containing asbestos fibers. HMGB1 (Center) and TNF-α (Right) stain area with chronic inflammation (some staining can be seen also along the connective tissue, right portion of the panel, which contains lymphatic vessels with inflammatory cells). (Original magnification: top row, 100×; lower row, 400×.). H&E staining: Arrows point to asbestos fibers surrounded by inflammatory cells, macrophages, giant cells, and lymphocytes. HMGB1 and TNF-α are detected in cytoplasm and extracellular space.

Materials.

Actinomycin D was obtained from Calbiochem, z-VAD-fmk from R&D Systems, and H₂O₂, and TPA and 3-ABA from Sigma. Box A was provided by HMGBiotech. Crocidolite asbestos was obtained from the Union Internationale Contre le Cancer (UICC) and processed as previously described (14). Fiber concentration was 5 μg/cm² or as indicated.

Western Blotting.

Western blot assays were performed as previously described (14) with anti-HMGB1 and anti-PAR (BD Bioscience Pharmingen), anti-PARP, anti-caspase-3 (Santa Cruz), and anti-GAPDH (Chemicon International). To assess HMGB1 release by HM, media were concentrated with Amicon Centrifugal Filter (Millipore).

Cytotoxicity.

HM cytotoxicity was analyzed 24 h after exposure to crocidolite, with or without 0.5 mM of the PARP inhibitor 3-ABA, by the LDH-cytotoxicity detection kit (Roche) according to the manufacturer's instructions.

PARP Activity.

PARP activity was determined in HM exposed to crocidolite, by using ³²P-NAD (Amersham) and Poly (ADP-ribose) Polymerase Assay Kit (Trevigen). ³²P incorporation in ribosylated proteins was evaluated with a scintillation counter.

Determination of ATP.

ATP levels were determined by the bioluminescence somatic cell assay kit according to the instructions of the manufacturer (Sigma) in HM treated with actinomycin D (0.1 μM), H₂O₂ (200 μM), or different doses of crocidolite (0.5, 1.0, 2.5, 5.0, or 10 μg/cm²) for 24 h.

Immunofluorescence.

HM were exposed to crocidolite, H₂O₂ (100 μM) or actinomycin D (0.1 μM) for 6 h. After fixation and permeabilization, cells were incubated with HMGB1 antibody (BD Bioscience Pharmingen) and a fluorescein-conjugated secondary antibody (Chemicon, Temecula). Nuclei were stained with DAPI.

TNF-α Secretion.

HM were exposed to crocidolite or treated with H₂O₂ (1 mM) or actinomycin D (0.5 μM) for 1 h. After 24 h, culture media were concentrated by ultrafiltration, and TNF-α production by macrophages was measured by the Quantikine human TNF-α ELISA kit (R&D Systems). HMGB1 was antagonized by adding Box A at 100 ng/mL.

Animal Experiments.

Animal experiments were performed as described (35). Briefly, six female Syrian hamsters and 10 BALB/c mice that were 21-d-old (to test for interspecies variability) were injected intraperitoneally with 0.4 mg crocidolite in PBS every 2 wk for 10 wk, for a total amount of 4 mg. Control groups (six hamsters and 10 mice) were injected with PBS. Animals were killed after 4 mo. All of the major organs were evaluated and studied histologically.

Immunohistochemistry.

Immunohistochemical analyses of hamster and mouse tissues were performed according to standard procedures (SI Materials and Methods).

ELISA for HMGB1.

The human HMGB1 ELISA kit (Shino-Test Corporation) was used to measure the level of serum HMGB1. Specimens were tested in duplicate.

Serum.

Serum samples were collected from 20 individuals with a documented history of asbestos exposure (38) and from unexposed controls (SI Materials and Methods). Participants provided informed consent, and procedures and protocols were approved by the institutional review board (IRB).

We thank Francesco De Marchis for quality control of Box A. We thank Dr. Giovanni Gaudino for the critical review. This work was supported by National Cancer Institute (NCI) Grants R01 and P01 (to M.C.); the Mesothelioma Applied Research Foundation (MARF), the Hawaii Community Foundation’s Leahi fund, the Riviera United-4 a CURE (to H.Y.); Grant NCI R01 (to G.F.); the Associazione Italiana Ricerca sul Cancro (M.E.B.); and the Early Detection Research Network (EDRN)-NCI (H.I.P.).