American Journal of Respiratory Cell and Molecular Biology

Asbestos causes malignant tumors such as lung cancer and malignant mesothelioma (MM). To determine whether asbestos exposure causes reduction of antitumor immunity, we established an in vitro T-cell line model of low-dose and continuous exposure to asbestos using an human adult T-cell leukemia virus–1 immortalized human polyclonal T-cell line, MT-2, and revealed that MT-2 cells exposed continuously to asbestos showed resistance to asbestos-induced apoptosis. In addition, the cells presented reduction of surface CXCR3 chemokine receptor expression and IFN-γ production. In this study, to confirm that these findings are suitable for clinical translation, surface CXCR3 and IFN-γ expression were analyzed using freshly isolated human CD4+ T cells derived from healthy donors and patients with pleural plaque (PP) or MM. The results revealed that CXCR3 and IFN-γ expression in the ex vivo model were reduced in some cases. Additionally, CXCR3 expression in CD4+ T cells from PPs and MMs was significantly reduced compared with that from healthy donors, and CD4+ T cells from patients with MMs exhibited a marked reduction in IFN-γ mRNA levels after stimulation in vitro. Moreover, CD4+CXCR3+ T cells in lymphocytes from MMs showed a tendency for an inverse correlation with its ligand CXCL10/IP10 in plasma. These findings show reduction of antitumor immune function in asbestos-exposed patients and indicate that CXCR3, IFN-γ, and CXCL10/IP10 may be candidates to detect and monitor disease status.

In this study, we show that chrysotile asbestos has the potential to reduce chemokine receptor CXCR3 expression in human peripheral CD4+ T cells using an ex vivo model, and CXCR3 expression is decreased in peripheral CD4+ T cells from patients with asbestos-related diseases, such as pleural plaque or malignant mesothelioma. Our findings suggest that antitumor immunity might not function normally in patients with asbestos-related disease because the low expression of CXCR3 induces depressed chemotaxis and indicate that CXCR3 may be effective tool for the prediction of impaired antitumor immune status in these patients.

The cellular and molecular biologic effects of asbestos fibers on alveolar epithelial cells and pleural mesothelial cells have been investigated using various animal models and culture cells (15). Studies of fibrogenesis have shown that the initial recognition of asbestos by alveolar macrophages (AMs) is important, and AM-releasing cytokines, such as TNF-α, TGF-β, and IL-1β and IL-8, play a critical role in activating mesenchymal cells to induce the proliferation of collagen fibers (5, 6). Moreover, recent advances resulting from cellular and molecular investigations regarding activation of the nucleotide oligomerization domain–like receptor family, the pryin domain–containing 3 (NLRP3)-inflammasome caused by asbestos, are leading to a better understanding of this recognition (79). Reactive oxygen species and reactive nitrogen species play important roles in carcinogenesis (16), in which substances that damage DNA are generated through the activity of iron contained in the asbestos. Alveolar and mesothelial cells exposed to asbestos then develop apoptosis with activation of a mitochondrial pathway and a B-cell lymphoma 2 (Bcl-2)-associated x protein (Bax) dominant balance of the Bax/Bcl-2 complex, cytosomal release of cytochrome-c from mitochondria, and activation of caspase 9 and 3 (16).

The above findings are based on studies using animal models and cell culture experiments in which exposure conditions were temporary and involved high doses. In human asbestos-related diseases, the latency is quite long and may range from 10 to 30 years for fibrogenic changes known as asbestosis and around 40 years for malignant mesothelioma (MM) (1012).

To investigate the immunological effects of asbestos on human immunocompetent cells, we constructed an in vitro experimental T-cell line model of continuous asbestos exposure using a human adult T-cell leukemia virus type 1–immortalized human polyclonal T-cell line (MT-2) because this cell line is not tumor-cell derived and is reported to have a normal karyotype (1316). In our experiments, chrysotile was used as the asbestos because it may be important to compare this substance with silica (SiO2), which causes silicosis and alteration of autoimmunity (17, 18). In addition, the dose of chrysotile used in industries around the world is much higher than that of other fibers, and carcinogenicity is considered much lower for chrysotile (1012).

As we reported recently, our experimental models, which use the MT-2 original cell line and six sublines exposed to chrysotile independently and continuously, revealed that all of the sublines exhibited reduction of surface expression of CXC chemokine receptor 3 (CXCR3) and production of IFN-γ (19). It is well known that IFN-γ is important in antitumor immunity and antiviral immunity (2024). In addition, CXCR3 is thought to be involved in antitumor immune function. Some reports have investigated the state of chemokines surrounding mesothelioma tumor cells (2528). It has been found that the ligand of CXCR3, C-X-C motif chemokine 10 (CXCL10)/IFN-γ–induced protein 10 kD (IP10), is present in pleural fluid collected from patients with MM and that human mesothelioma tumors are abundantly infiltrated with CD4+ T cells (29). Furthermore, CXCL10/IP10 mRNA expression is significantly higher in MM compared with normal mesothelial cell lines and pleural mesothelium (30). CXCL10/IP10 inhibits tumor growth and metastasis through a decrease in tumor-associated angiogenesis and recruits Th1/effector T cells by binding the chemokine receptor CXCR3 (20, 31, 32). Therefore, it is important to regulate lymphocyte transport mediated by chemokine receptors to enhance antitumor immune responses because CXCR3-positive Th1/effector CD4+ T cells are recruited to CXCL10/IP10-enriched tumor sites, where IFN-γ is secreted and subsequent suppression of tumor growth is induced (33).

We examined CXCR3 and IFN-γ expression in CD4+ T cells co-cultured with chrysotile and showed the potential of chrysotile to inhibit CXCR3 and IFN-γ expression. In addition, we investigated CXCR3 and IFN-γ expression in CD4+ T cells from healthy donors (HDs) and asbestos-exposed patients with pleural plaque (PP) or MM and found low-level expression of CXCR3 in CD4+ T cells from patients with PPs and MMs and decreased mRNA levels of IFN-γ in cultured CD4+ T cells from patients with MMs. These findings highlight parameters that are effective in predicting the risk of impaired antitumor immune status in patients with PP and MM.

Ex Vivo Activation and Exposure to Asbestos on Human CD4+ T Cells

After receiving informed consent, blood samples were obtained from six HDs (mean age, 34.8 ± 9.7 yr; range, 25–50 yr; three men and three women), and peripheral blood mononuclear cells (PBMCs) were isolated using the Ficoll-Paque method. CD4+ T cells were isolated by positive selection using anti-CD4–coated beads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) in accordance with the manufacturer's instructions. Freshly isolated CD4+ T cells (2 × 105 cells/well) in 96-well, U-bottomed plates were stimulated with anti-CD3 (clone UCHT1) and anti-CD28 (clone CD28.2) antibodies (Beckman Coulter, Inc., Fullerton, CA) at 2 μg/ml and cultured in RPMI 1640 medium containing 10% FBS and recombinant IL-2 (rIL-2) (10 ng/ml) (PeproTech Inc., Rocky Hill, NJ). After 3 days, activated CD4+ T cells were transferred to 24-well plates at 1 × 106 cells/well and expanded in medium supplemented with 10 ng/ml rIL-2 for 1 week and further cultured in the absence or presence of 2 to 10 μg/cm2 of chrysotile-B. Chrysotile-B was kindly provided by the Department of Occupational Health, National Institute for Occupational Health, South Africa (34). Before analysis, cultured CD4+ T cells were released from chrysotile-B by the Ficoll-Paque method and cultured for 3 days. For intracellular staining, cultured CD4+ T cells (1 × 106 cells/ml) were stimulated with 5 ng/ml phorbol 12-myristate 13-acetate and 250 ng/ml ionomycin for 6 hours in the presence of monesin.

Flow Cytometry

Surface antigens were stained as reported previously (19) with the following antibodies: anti-CD4 (clone RPA-T4), CXCR3 (clone 1C6), and CC chemokine receptor 5 (CCR5) (clone 2D7) (BD Biosciences Pharmingen, San Diego, CA). Intracellular staining was performed using a Cytofix/Cytoperm kit (BD Biosciences) in accordance with the manufacturer's instructions. Cells were stained with anti–IFN-γ (clone 4S.B3) antibody (BD Biosciences Pharmingen) for 30 minutes at 4°C and analyzed on a flow cytometer (FACSCalibur; BD Biosciences, Franklin Lakes, NJ).

Patients and Isolation of PBMCs

Blood from a different series of 19 HDs (mean age, 42.3 ± 12.8 yr; range, 29–70 yr; 9 men and 10 women), 13 patients with PP (mean age, 71.5 ± 5.3 yr; range, 58–79 yr; 11 men and 2 women), and eight patients with MM (mean age, 67.4 ± 8.5 yr; range, 59–79 yr; seven men and one woman) was collected in citrate phosphate dextrose. Plasma was collected from supernatants after centrifugation at 3,000 rpm for 5 minutes. PBMCs were isolated as described above and subjected to FACS analysis. For analysis of mRNA expression and cytokine production, CD4+ T cells were isolated as described above. Freshly isolated CD4+ T cells were cultured in RPMI 1640 medium containing 10% FBS at 2 × 104 cells/100 μl in 96-well, U-bottomed plates and stimulated with anti-CD3 and anti-CD28 antibodies at 2 μg/ml. After 5 days, the supernatants and cells were collected. Informed consent was obtained from all donors, and the project and procedures used were approved by the Institutional Ethics Committees of Kawasaki Medical School and Okayama Rosai Hospital.

Changes of CXCR3 Cell Surface Expression in CD4+ T Cells Cultured with Chrysotile-B

As reported recently, our in vitro T-cell line model of low-level and continuous exposure to chrysotile asbestos showed reduction of Th1-type cell surface expression of CXCR3 (19). Therefore, we tried to determine whether freshly isolated human peripheral CD4+ T cells show a similar alteration ex vivo when proliferation is maintained by IL-2–containing medium in the presence of chrysotile-B. After 40 days of co-culture, cell surface CXCR3 expression decreased in a dose-dependent manner (2–10 μg/cm2) (Figure 1A). Thus, we examined cell surface expression of CXCR3 and CCR5 in CD4+ T cells derived from six HDs because both receptors are preferentially expressed in Th1/effector T cells. The expression of CXCR3 was significantly reduced after exposure to 10 μg/cm2 of chrysotile for 28 days, although this difference seemed to depend on one case in which the expression decreased remarkably (Figure 2, upper panel). Even if the culture conditions for the CD4+ T cells was limited to a period of around 4 weeks, four of six HDs showed a decrease of CXCR3 expression to various degrees, and it might be concluded that asbestos exposure potentiates reduction of CXCR3 expression in CD4+ T cells. On the other hand, the expression of CCR5 varied among all HDs, and there were no significant changes after 7 and 28 days of co-culture with chrysotile-B (Figure 1B, lower panel), as shown previously by the cell line model (19). These results indicated that CXCR3 expression might be specifically reduced by asbestos exposure.

IFN-γ Expression under Ex Vivo Exposure Conditions

We also showed previously that IFN-γ production in the T-cell line model was decreased by chronic exposure to chrysotile (19). Thus, we examined intracellular expression of IFN-γ in CD4+ T cells exposed to chrysotile-B (10 μg/cm2) from three healthy subjects under the same ex vivo exposure conditions used for the analysis of surface CXCR3 expression. After 28 days of co-culture, mRNA expression of IFN-γ was reduced in all three cases, although there were no significant differences (P = 0.3226) (Figure 2A). In addition, intracellular staining showed that IFN-γ–positive cells tended to be reduced in CD4+ T cells from all three subjects when cultured with chrysotile-B (P = 0.0511) (Figure 2B).

These findings from the ex vivo study indicated that chronic exposure to asbestos reduced CXCR3 and IFN-γ expression in CD4+ T cells, although the reductions were not statistically significant.

Expression of Cell Surface CXCR3 in Human CD4+ T Cells from Patients with Asbestos-Related Disease

To investigate whether peripheral CD4+ T cells in people exposed to asbestos also show reduction of CXCR3, we analyzed cell surface CXCR3 and CCR5 expression in peripheral CD4+ T cells from patients with asbestos-related PP and MM by flow cytometry (Figure 3A). Given that the population of lymphocytes in MMs was significantly lower than that in the other groups (Figure 3B, left panel), gated lymphocytes were analyzed for CXCR3 and CCR5 expression in CD4+ T cells. The percentage of CD4+ T cells in lymphocytes revealed no differences among the three groups (Figure 3B, right panel).

The percentage of CD4+CXCR3+ T cells in lymphocytes from PPs and MMs were significantly lower than that of HDs, and MMs showed the lowest percentage compared with the other groups (Figure 3C, left panel). In contrast, the percentage of CD4+CCR5+ T cells in lymphocytes showed no differences (Figure 3C, right panel). Furthermore, the expression of CXCR3 in CD4+ T cells from PPs and MMs was significantly lower than that of HDs (Figure 3D, left panel), although CCR5 expression in CD4+ T cells showed no differences (Figure 3D, right panel).

To confirm whether there is an aging effect concerning decreased expression of CXCR3, 19 HDs were divided into two groups: younger (mean age, 34.9 ± 5.8 yr; range, 25–45 yr; n = 13) and older (mean age, 58.3 ± 7.6 yr; range, 51–70 yr; n = 6). The difference between these groups regarding age was significant (Figure 4A). However, our findings indicated that there were no significant differences in the percentage of CD4+CXCR3+ T cells in lymphocytes (Figure 4B, upper panel) and CXCR3 expression in CD4+ T cells (Figure 4B, lower panel) between the two groups. Thus, these results confirmed that aging is not associated with a reduction of CXCR3 expression and that decreased expression of CXCR3 in peripheral CD4+ T cells occurs in people exposed to asbestos.

IFN-γ mRNA Levels in Stimulated Peripheral CD4+ T Cells from Patients with Asbestos-Related Diseases

Real-time RT-PCR was used to detect the mRNA levels of IFN-γ in stimulated peripheral CD4+ T cells from 13 HDs, 13 PPs, and 7 MMs. mRNA expression levels of IFN-γ relative to GAPDH were significantly decreased only in MMs compared with HDs and PPs (Figure 5A). Additionally, the Spearman's rank correlation test showed that there was no significant correlation between levels of CXCR3 expression in peripheral CD4+ T cells and IFN-γ mRNA levels of stimulated CD4+ T cells in all three groups (Figures 5B–5D), although HDs showed a tendency for a positive correlation (P = 0.1295) and PPs showed a tendency for a negative correlation (P = 0.0502). Unlike suppressed CXCR3 expression, these results suggested that decreased mRNA expression of IFN-γ was dependent on the occurrence of mesothelioma and was not necessarily associated with asbestos exposure.

IFN-γ and Production of other Cytokines in Human CD4+ T Cells from Patients with Asbestos-Related Disease

The cytometric bead array assay was performed to measure the concentration of Th1/Th2/Th17 cytokine (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17A) in the supernatants from cultured peripheral CD4+ T cells. There were no significant differences in IFN-γ production from cultured peripheral CD4+ T cells (Figure 6), although there were statistically significant differences in IFN-γ mRNA expression (Figure 5A). Thus, the expected result for IFN-γ–producing activity was not obtained under these conditions. The results for other cytokines showed that the IL-6 concentration in PPs and MMs was significantly higher than that of HDs (Figure 6). Although the IL-4 concentration in MMs was significantly higher than that of HDs, the level might be too low for the cytokine to exert its physiological function (Figure 6).

Correlation between the Percentage of CD4+CXCR3+ T Cells in Lymphocytes and Levels of Plasma CXCL10/IP10

Because the ligand for CXCR3, CXCL10/IP10, is secreted not only from monocytes, endothelial cells, and fibroblasts (20, 31, 32) but also from mesothelial cells and transformed (mesothelioma) cells (30), the plasma levels of CXCL10/IP10 in HDs, PPs, and MMs were measured by CBA assay. The levels of plasma CXCL10/IP10 had no correlation with CXCR3 expression in CD4+ T cells (data not shown). Therefore, the correlation between the percentage of CD4+CXCR3+ T cells in lymphocytes and plasma chemokine levels was determined using Spearman's rank correlation test. The plasma concentration of CXCL10/IP10 in PPs or MMs tended to be higher when compared with that of HDs, although there were no significant differences (Figure 7A). Therefore, there was no significant correlation between the percentage of CD4+CXCR3+ T cells and CXCL10/IP10 concentration in all three groups (Figures 7B–7D), although MMs showed a tendency for an inverse correlation (P = 0.081) in comparison with HDs (P = 0.927) and PPs (P = 0.578).

There are few publications regarding the effects of asbestos on the general immune system, particularly antitumor immunity. However, the mechanisms by which local immune cells, such as alveolar macrophages, recognize asbestos fibers have been investigated, particularly the discovery of the NLRP3–inflammasome and its ability to recognize and process asbestos fibers and activate IL-1β and caspase 1 (79). Asbestos is a mineral silicate that contains magnesium, iron, and calcium. Patients exposed to silica particles may develop a disease known as silicosis, which is often complicated with autoimmune diseases such as rheumatoid arthritis (known as Caplan's syndrome), systemic sclerosis, and anti–neutrophil cytoplasmic autoantibody–related vasculitis/nephritis (3537). However, the most important complication resulting in asbestos-exposed patients is the occurrence of cancer, such as lung cancer and MM (1012). In particular, MM is known to be caused by low-level and long-term exposure to asbestos (1012).

Therefore, asbestos fibers may affect the human immune system, particularly antitumor immunity. In fact, we have been reporting impaired NK cell function with reduced expression of the NK-activating receptor NKp46 (38, 39). In addition, we attempted to establish an in vitro T-cell model of continuous and low-level exposure to asbestos and reported that the MT-2 subline exhibited resistance to asbestos-induced apoptosis with activating Src-family kinases, up-regulation of IL-10, phosphorylation of STAT3, and subsequent up-regulation of Bcl-2 (15, 16). Furthermore, six independent sublines of MT-2 original cells continuously exposed to chrysotile showed reduced Th1-related IFN-γ production, CXCL10/IP10 production, and cell surface expression of the CXCL10/IP10 receptor CXCR3 (19).

If these findings obtained using the in vitro cell line model are compatible with those pertaining to asbestos-exposed patients, such as those possessing PPs and MMs, these molecules may represent clinical markers for asbestos exposure, disease progression, and even therapeutic targets. Thus, we tried to confirm alteration of surface CXCR3 expression and IFN-γ production using freshly isolated human CD4+ T cells derived from HDs and patients with PP and MM. To use the ex vivo model of low-dose and long-term exposure to chrysotile asbestos, these CD4+ T cells should be cultured for several months. However, because IL-2–dependent proliferation of CD4+ T cells is not permanent, our experiment was limited to culture for several weeks in the presence of IL-2 after stimulation of anti-CD3/CD28 antibodies. The results showed that four of six cases exhibited reduced CXCR3 expression within the limitation of the ex vivo model. Moreover, intracellular IFN-γ expression was clearly reduced in this ex vivo experimental situation, although the number of applied cases was small. These findings encouraged us to examine CXCR3 expression and IFN-γ production using the samples derived from asbestos-exposed patients, such as those possessing PPs and MMs.

Analysis of the case samples revealed that the expression of CXCR3 in CD4+ T cells from PPs and MMs was decreased when compared with that of HDs, even though another Th1-type chemokine receptor, CCR5, did not differ between the three groups. Because CXCR3 is implicated in the migration of Th1/effector T cells, decreased CXCR3 expression in CD4+ T cells by asbestos exposure may reduce normal Th1/effector T-cell recruitment to tumor sites, resulting in impairment of antitumor immune functions. Meanwhile, asbestos is accumulated in several lymph nodes (4042). Thus, it seems that CXCR3 expression in CD4+ T cells is inhibited by contact with asbestos in the lymph nodes of patients with PP and MM. In addition, CD4+CXCR3+ T cells in lymphocytes were significantly reduced in MMs rather than PPs because the population of lymphocytes was significantly decreased in MMs compared with PPs. This suggests that CD4+CXCR3+ T cells are gradually decreased and that they depend not only on asbestos exposure but also on disease status, such as the occurrence of mesothelioma.

IFN-γ mRNA levels in CD4+ T cells derived from MMs stimulated in vitro were lower than those of HDs or PPs, whereas there were no significant differences in IFN-γ production. These results suggest that CD4+ T cells from MMs already possess a reduced capacity to produce IFN-γ, which is important for activation of antitumor immune function due to long-term asbestos exposure and the presence of tumor. A correlation test showed that mRNA levels of IFN-γ in PPs tended to increase in stimulated CD4+ T cells with decreasing surface CXCR3 expression levels, although mRNA levels of IFN-γ in HDs were positively correlated with CXCR3 expression. Given that asbestos exposure is partially associated with autoimmune responses (43), asbestos exposure in PPs might change the character of CD4+ T cells to enhance antitumor immune function, and IFN-γ mRNA expression might not be suppressed in patients with PP who do not have MM.

Alternatively, the producible capacities of IL-6 in stimulated CD4+ T cells tended to be higher in PPs and MMs compared with HDs, suggesting that asbestos exposure enhances inflammatory cytokine producibility in CD4+ T cells. We showed previously that the plasma concentration of IL-6 in MM was higher than that of HDs and PPs (44). Given that the levels of plasma IL-6 were not high in PPs, it seems that plasma IL-6 in MMs is mainly derived from mesothelioma cells (4547). Furthermore, IL-6 inhibits Th1-cell differentiation and promotes Th2-cell differentiation (48). Thus, plasma IL-6 might induce decreased IFN-γ mRNA expression and increased IL-4 production in stimulated CD4+ T cells from MMs. On the other hand, although there were no significant differences in IL-10 and IL-17A production under these experimental conditions, there are several reports regarding the relationship between IL-10/IL-17 and cancer status, which indicates that IL-10 may suppress antitumor immune function and that IL-17 may promote inflammation in cancer (49, 50). Future studies should therefore investigate the pathophysiological roles of IL-10 and IL-17 in asbestos-exposed patients such as those with PPs or MMs.

Finally, we found that the plasma concentration of CXCL10/IP10 tended to be higher for PPs and MMs than HDs. Furthermore, there tended to be an inverse correlation between the population of CD4+CXCR3+ T cells in lymphocytes and the plasma concentration of CXCL10/IP10 in MMs but not in HDs or PPs. These results indicated that antitumor immune function in MMs may be impaired after reduction of CD4+CXCR3+ T cells in lymphocytes because CXCL10/IP10 from monocytes, endothelial cells, fibroblasts, mesothelial cells, and mesothelioma in the region of existing tethered asbestos and a cancerous lesion attracts antiinflammatory or antitumor T cells.

In conclusion, this study revealed that reduction of CXCR3 expression in human peripheral CD4+ T cells occurred after chronic exposure to chrysotile. This finding suggests that antitumor immunity might not function normally in patients with asbestos-related disease because the low expression of CXCR3 induces depressed chemotaxis. Additionally, it seems that IFN-γ expression is inhibited by the occurrence of mesothelioma rather than asbestos exposure, and the weak production of IFN-γ leads to suppression of antitumor activity. Further research of chemokine-mediated migration dependent on CXCR3 expression is required to determine the mechanism of immune dysfunction via chemokine receptors after exposure to asbestos and to investigate the use of the chemokine receptor CXCR3 as a key molecule in immunotherapy strategies for asbestos-related disease. In addition, these molecules may be effective tools for the detection of asbestos exposure, considering our previous findings of enhanced Bcl-2 expression in CD4+ T cells from MMs (16) and higher plasma concentration of IL-6, IL-10, and TGF-β (44). For example, higher plasma IL-10 and TGF-β with lower INF-γ expression may be interpreted as an enhanced regulatory T-cell activity overcoming antitumor T-cell activity in MMs.

Further studies regarding the size and function of regulatory T cells in MMs and the in vitro model using the cell line should be performed to better understand immunological status among asbestos-exposed patients with or without mesothelioma. Moreover, a device to evaluate total antitumor activities in these patients using the degree of reduced expression in CD4+ T cells of an NK cell–activating surface receptor such as NKp46, including CXCR3 and IFN-γ expression, may assist in the diagnosis of MMs and reveal good prognostic factors for these patients.

The authors thank Ms. Tamayo Hatayama, Minako Kato, Naomi Miyahara, Shoko Yamamoto, Misao Kuroki, Keiko Kimura, Yoshiko Yamashita, and Tomoko Sueishi for technical help.

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Correspondence and requests for reprints should be addressed to Megumi Maeda, Ph.D., Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology, Okayama University, 1-1-1 Tsushima-naka Kita-ku, Okayama 700-8530, Japan. E-mail:

This study was supported in part by Special Coordination Funds for Promoting Science and Technology (H18–1-3–3-1, Comprehensive approach on asbestos-related diseases); grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (18390186, 19659153, 19790411, 20390178 and 22700933); and Kawasaki Medical School Project Grants (18–209T, 19–205Y, and 20–210O).

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Originally Published in Press as DOI: 10.1165/rcmb.2010-0435OC on February 25, 2011

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