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. Author manuscript; available in PMC: 2013 May 16.
Published in final edited form as: J Immunotoxicol. 2011 Dec 1;9(2):129–140. doi: 10.3109/1547691X.2011.631953

Asbestos activates CH12.LX B-lymphocytes via macrophage signaling

Devon L Rasmussen 1, Jean C Pfau 1
PMCID: PMC3655430  NIHMSID: NIHMS463640  PMID: 22133189

Abstract

The impact of asbestos exposure on the development and progression of autoimmunity is becoming increasingly recognized as a public health issue. Epidemiological studies have shown an association between exposure to airborne silicates, such as asbestos, and autoimmunity, but the etiology remains unresolved. B1a B-lymphocytes have been implicated in autoimmune responses in mice, and splenic B1a cell numbers are altered following asbestos exposure. The purpose of this study was to explore the possible role of B1a B-lymphocytes in the production of pathogenic autoantibodies by testing the hypothesis that B1a B-lymphocytes directly react with asbestos and increase production of antibodies. The B1a-like B-lymphocyte model, CH12.LX, was exposed to asbestos in vitro via direct and indirect mechanisms. The effect was determined of these exposures on the rate of proliferation and on production of various immunoglobulin classes. Direct exposure elicited no measurable response by the CH12.LX cells. Culturing the CH12.LX cells in media from asbestos-exposed RAW 264.7 macrophages, however, decreased the proliferation rate and stimulated the cells to increase production of the immunoglobulin isotypes IgG1, IgG3, and IgA. It was discovered that asbestos stimulated the macrophages to increase production of the cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α. Recombinant murine IL-6 caused similar results seen with the macrophage media, indicating a role of IL-6 in stimulating a response by the B1a B-lymphocytes to asbestos. In correlation with the in vitro data, it was determined ex vivo that exposure of peritoneal cells (from C57Bl/6 mice) to asbestos caused an increase in the expression of IL-6 and TNFα, as well as of surface expression of IgA on the peritoneal B1a B-lymphocytes. These data demonstrate that asbestos leads to immunologic changes consistent with activation of B1a B-lymphocytes. This study also provides a model for analyzing the critical steps that may be involved in asbestos-induced autoimmune responses.

Keywords: B1a B-lymphocytes, IL-6, TNFα

Introduction

Within the last decade, epidemiological studies have demonstrated a higher incidence of systemic autoimmune disorders (SAID), such as systemic lupus erythematosus (SLE), systemic scleroderma, and rheumatoid arthritis, in persons exposed to varying levels of asbestos when compared to unexposed populations (Powell et al., 1999; Pfau et al., 2005; Noonan et al., 2006). Exposure to the silicate compound asbestos remains a major concern as exposures continue to occur in a variety of occupations, including mining industries and building construction (Niklinski et al., 2004). In the town of Libby, MT, environmental asbestos exposure remains a concern as asbestos-contaminated vermiculite was not only extracted and processed nearby, but the vermiculite was also utilized in many ways within the community (Peipins et al., 2003) and shipped to processing sites throughout North America. The asbestos within the vermiculite belongs to the pathogenic amphibole family that includes the asbestiforms of tremolite, crocidolite, winchite, and amosite (Cunningham and Pontefract, 1973; Wylie and Verkouteren, 2000; Meeker et al., 2003).

The mechanisms behind the pathogenic immunological effects of asbestos exposure remain unresolved within the scientific and clinical communities. However, studies have demonstrated that small cohorts of asbestos- exposed individuals have increased immunological activity such as elevated immunoglobulin titers and Type III hypersensitivity (Lange, 1980; Zerva et al., 1989). Within the Libby, MT population, certain-exposed individuals not only had positive anti-nuclear antibody (ANA) tests, but they also manifested with elevated IgA titers (Pfau et al., 2005). In C57Bl/6 mice, asbestos instilled animals had positive ANA tests, as well as a decrease in regulatory T (CD4+CD25+)-lymphocytes and a change in the B1a B-lymphocyte population (Pfau et al., 2008).

B1a B-lymphocytes are a specialized subset of lymphocytes that arise during fetal development and reside within the pleural and peritoneal cavities. B1a B-lymphocytes replenish through self-renewal. These cells can be differentiated from the B2 subset of lymphocytes by their co-expression of the cell surface markers CD5 and IgM. B1a B-lymphocytes have been implicated in the development of autoimmune disorders as they secrete natural immunoglobulin molecules that may be idiotypic to self antigens; furthermore, B1a B-lymphocytes do not undergo central tolerance mechanisms (DeFranco et al., 1982; Hayakawa et al., 1984, 1986; Baumgarth et al., 2000; Baker and Ehrenstein, 2002; Berland and Wortis, 2003; Duan and Morel, 2006; Esplin et al., 2009). In addition, depletion of peritoneal B1a cells led to protection in mouse models of lupus and diabetes (Murakami et al., 1995; Kendall et al., 2004). To study the effects of asbestos on B1a B-lymphocytes in vivo is difficult as the peritoneal cell population is complex. Further, if any effects were seen, one would not be able to interpret the results as a response by the B1a B-lymphocytes directly or as a response triggered by interaction with other immune cells. Therefore, we utilized the immortalized B-lymphocyte line CH12.LX as a model of B1a B-lymphocytes to analyze the in vitro effects of direct and indirect exposure to asbestos (Arnold et al., 1983; Bishop et al., 1988).

A previous study involving RAW 264.7 macrophages demonstrated that these cells are capable of internalizing the Libby amphibole asbestos. Internalization of asbestos by the RAW 264.7 macrophages leads to an increase in production of both reactive oxygen species and super-oxide molecules (Blake et al., 2007). Macrophages and other lymphoid cells secrete interleukin-(IL)-6 (Kishimoto and Hirano, 1988); its pleiotropic effects have been implicated in the disease states of rheumatoid arthritis, systemic lupus erythematosus (SLE), and several other diseases (Guerne et al., 1989; Liang et al., 2006). It is known that IL-6, in SLE patients, is elevated and increases the production of pathogenic antibodies by promoting hyperactivity of B-lymphocytes (Linker-Israeli et al., 1991).

Within this study, we demonstrate in vitro a mechanism that may explain a pathway by which asbestos influences the immune response toward autoimmunity. We show that RAW 264.7 macrophages, following exposure to asbestos, increase their production of IL-6. The IL-6, in turn, causes a B1a B-lymphocyte model (i.e., CH12. LX) to differentiate into multiple sub-populations that secrete various immunoglobulin isotypes.

Materials and methods

RAW 264.7 murine macrophage cell line

RAW264.7 murine macrophages (ATCC-2091: American Type Culture Collection, Manassas, VA) were maintained in culture at 37°C in a 5% CO2 incubator (Thermo-Scientific, Waltham, MA) in complete media that contained RPMI with L-glutamine and 25 mM HEPES (Media-tech, Manassas, VA) supplemented with 10% fetal bovine serum (FBS; Thermo-Scientific), antibiotic (100 U penicillin/ml and 100 μg streptomycin/ml) solution (Thermo-Scientific, Logan, UT), and 0.1% 2-mercaptoethanol (Sigma, St. Louis, MO). Cells were grown to confluency in T75 flasks, scraped, and counted using a Z-series Coulter counter (Beckman Coulter, Hialeah, FL). The RAW264.7 cells were then seeded into 6-well plates at a concentration of 1 × 106 cells per well and allowed to adhere overnight prior to exposure to asbestos fibers. While the RAW264.7 cells were not directly studied except to ensure that the treatments did not cause cell death (Cell-Titer Blue assay, Promega, Madison, WI), the media collected from these cells following asbestos exposure was used in some studies to culture the CH12.LX cells.

CH12.LX murine B-lymphocyte line

The CH12.LX B-lymphocytes (kindly provided by Dr Norbert Kaminski, Michigan State University) were maintained in culture at 37°C in a 5% CO2 incubator in a modified RPMI medium which contained L-glutamine and 25 mM HEPES supplemented with 10% FBS, penicillin-streptomycin solution, 5 ml of 100X non-essential amino acid solution (Irvine Scientific, Santa Ana, CA), 5 ml of 100 mM sodium pyruvate solution (Thermo-Scientific), and 0.1% 2-mercaptoethanol. The cells were kept at 37°C in the 5% CO2 incubator and passed every 3 days. The CH12.LX cells were counted using the Z-series Coulter counter and placed in 6-well plates at a concentration of 1 × 106 cells/well. Amphibole asbestos was directly placed within three wells at a concentration of 35 μg/cm2. For some experiments, recombinant mouse IL-6 or recombinant mouse tumor necrosis factor (TNF)-α (R&D Systems, Minneapolis, MN) or lipopolysaccharide (LPS, Escherichia coli, Type Type 0111:B4, Sigma) was added to the cultures at designated levels. Treatment of the CH12.LX cells with 35 μg of asbestos/cm2, recombinant cytokines IL-6 and TNFα, or LPS at the indicated concentrations did not significantly reduce cell viability (data not shown).

C57BL/6 mice

C57Bl/6 mice were bred in-house (Idaho State University Department of Laboratory Animal Welfare) and used for experiments at 6–10 weeks-of-age. The mice were maintained in microisolator cages under specific pathogen-free conditions with a 12-h light/12-h dark cycle, constant temperature (22°C), and relative humidity (45%), and had access to food and water ad libitum. All protocols used herein were approved by the Institutional Animal Care and Use Committee (IACUC) at Idaho State University, and were in accordance with guidelines established by the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals.

For tissue harvests, mice were euthanized by CO2 asphyxiation, consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association. Skin covering the peritoneum was pulled back and 10 ml of warm media was injected into the peritoneal cavity using a 27-gauge needle. After brief gentle agitation, the fluid was removed using an 18-gauge needle, and the resulting cell suspension was centrifuged and counted. Peritoneal cells from multiple mice were pooled for ex vivo exposures to amphibole, plated in 12-well plates using RPMI supplemented with 10% FBS and antibiotics as described above. After exposure (Libby amphibole at 35 μg/cm2) for 2 days, media was harvested for cytokine analysis; non-adherent cells (primarily lymphocytes) were harvested for flow cytometry.

Particulate matter

The single asbestiforms utilized in this study were samples from the Libby site obtained from the United States Geological Survey (Libby, MT). The Libby amphibole fibers have been chemically and physically characterized by several laboratories and the samples used in the study were found to be chemically comparable to the amphibole found within the Libby mine (Wylie and Verkouteren, 2000; Gunter et al., 2003). The Libby amphibole was collected from six sites within the vermiculite mine, and is therefore called Six-mix. In order to disperse the asbestos into culture, 1 mg of the Six-mix was suspended into 1 ml of sterile 0.01 M phosphate-buffered saline (PBS, pH 7.4). Subsequently, the asbestos-containing vial was vortexed and sonicated (Branson, Inc, Danbury, CT) for 2 min to minimize aggregates within the suspension.

Proliferation assay

For measuring the degree of proliferation of the CH12. LX cells stimulated by asbestos via direct and indirect manners the CyQUANT NF Cell Proliferation assay (Invitrogen, Eugene, OR) was utilized. To measure the effect that direct exposure to asbestos had on the CH12.LX cells, we cultured the cells in six well dishes, as described above, supplemented with 35 μg amphibole asbestos/cm2. Cells were maintained for 48 h at 37°C in a 5% CO2 incubator and processed according to the manufacturer’s instructions. In short, a pellet of the CH12.LX cells was re-suspended by the addition of 50 μl Hank’s Balanced-Salt Solution and transferred to a microcentrifuge tube. Subsequently, 50 μl CyQUANT NF dye reagent was added to each tube and the cells incubated at 37°C for 60 min. The total volume from each tube was transferred to an opaque 96-well plate (Fisher Scientific). Degree of proliferation was measured by fluorescence spectroscopy at an emission wavelength of 530 nm using a Synergy HT Microplate Reader (BioTek, Winooski, VT).

An indirect assay was performed by exposing RAW264.7 macrophages to 35 μg asbestos/cm2 for 48 h. The culture media from the macrophages was then collected and used to culture the CH12.LX cells as explained above. Cells were then processed using the protocol previously explained using the CyQUANT NF Proliferation Assay.

Isotype ELISA

To perform the isotype enzyme-linked immunosorbent assay (ELISA) kit (BD Biosciences, San Jose, CA) CH12.LX cell culture media from either the direct or indirect methods was utilized, according to the manufacturer’s instructions. Briefly, using a 96-well high-absorbance ELISA plate (Fisher Scientific), isotype-specific rat anti-mouse antibodies were diluted 1:5 with blocking buffer and added to each designated well. The plate was then washed with PBS-Tween. Subsequently, blocking buffer was added to each well and the plate incubated at room temperature (RT), followed by a washing step. The samples and controls were then added to designated wells, the plate was washed again, and a horseradish peroxidase (HRP)-conjugated secondary rat anti-mouse antibody was added to each well. The plate was then incubated at RT for 30 min before TMB (3,3,5,5-tetramethylbenzidine) substrate was added to individual wells and the reaction was allowed to proceed for 8 min. The reaction was terminated by addition of Stop Solution and the absorbance (at 450 nm) in each well then obtained in a Synergy HT Microplate Reader (BioTek).

Flow cytometry of CH12.LX B-lymphocytes

CH12.LX cells were grown to confluence and counted using the Z series Coulter counter. The cell preparations were then stained with anti-mouse antibodies (BD Biosciences) including anti-IgM-PerCP-Cy5.5, anti-CD5-APC, anti-CD23-PE, anti-IgA-FITC, anti-CD11b-PerCP-Cy5.5, anti-α4-integrin-PE, and anti-MHC-II-FITC. Following the wash steps in staining buffer, the cells were analyzed on a FACS Calibur flow cytometer using CellQuest software (BD Biosciences). All antibodies were used at 1 μg/106 cells. Appropriate isotype control antibodies from BD Biosciences were used to set regions for background staining.

Cytokine bead array

Samples were obtained from media used to culture RAW264.7 macrophages that had been treated with Libby amphibole asbestos at a concentration of 35 μg/cm2 or left untreated in 6-well dishes. As per the manufacturer’s instructions, six cytokines, e.g., IL- 6 and -10, monocyte chemoattractant protein (MCP)-1, interferon (IFN)-γ, TNFα, and IL-12p70 were measured using a Mouse Inflammation Cytometric Bead Array (CBA) kit (BD Biosciences). Following incubation at RT for 60 min, the beads in each well were washed and analyzed on a FACS Calibur flow cytometer using the CBA analysis software (BD Biosciences).

Statistical analyses

All data points were analyzed by one-way analysis of variance (ANOVA) or an unpaired 2-tailed t-test where the hypothesis for an experimental design was non-directional. Mean values (± SEM) are reported and the α-level for statistical significance was assigned as p ≤ 0.05.

Results

Cell surface marker characterization of CH12.LX cells

As B1a B-lymphocytes characteristically express an IgM+CD5+CD23− cell surface marker phenotype, the CH12.LX B-lymphocytes were profiled (via flow cytometry) to determine their morphological likeness to natural B1a B-lymphocytes (Tarlinton et al., 1995; Mohan et al., 1998). The CH12.LX B-lymphocytes, as previously determined, expressed cell surface markers CD5 and IgM (Figure 1). The CH12.LX B-lymphocytes also expressed surface markers CD19, CD11b, α4-integrin, and Major Histocompatibility Complex II, and lacked expression of CD23; this is consistent with the surface proteins expressed by B1a B-lymphocytes (data not shown).

Figure 1.

Figure 1

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Flow cytometric analysis of dual staining of CH12.LX B-lymphocytes. Expression of CD5 and IgM was measured by co-staining with antibodies conjugated to APC or PerCP-Cy5.5, respectively.

Proliferation of CH12.LX cells cultured in media of asbestos-exposed RAW264.7 macrophages

We performed analyses on the effect asbestos had directly on CH12.LX cells. Exposure of these cells to the Libby amphibole asbestos did not elicit any change in proliferation rates (Figure 2a). Furthermore, direct exposure did not cause the cells to increase antibody production (Figure 2b); however, LPS (as positive control) stimulated the CH12.LX cells to significantly (p < 0.05) produce the isotypes IgA, IgG, and IgM. After exposing RAW264.7 macrophages to asbestos (35 μg/cm2) for 24 h and culturing them in the specified CH12.LX media, we collected the media and cultured the CH12.LX cells for an additional 24 h. The proliferation rate of the CH12.LX cells significantly (p < 0.05) decreased when cultured in macrophage media from RAW264.7 cells that were either unexposed or exposed to asbestos. Nevertheless, the asbestos-treated macrophage media group rate of proliferation decreased almost by half compared to the untreated macrophage media (Figure 3).

Figure 2.

Figure 2

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(a) Direct effect of exposure to the Libby Six-Mix asbestos on the proliferation rate of CH12.LX B-lymphocytes. Cells were exposed to 35 μg asbestos/cm2 or 1 μg LPS/ml (positive control) for 48 h and assayed by measurement of fluorescence at 530 nm. n = 6; Error Bars = Standard Error of the Mean (SEM). LPS as a positive control significantly (*p < 0.05) increased proliferation rate of the CH12.LX B-lymphocytes. (b) Direct effect of exposure to Libby Six-Mix asbestos on the isotype of antibody secreted by CH12.LX B-lymphocytes. Cells were exposed to 35 μg asbestos/cm2 for 72 h and then assayed by an isotype-specific ELISA. Data suggests that two populations of CH12.LX cells exist in the culture, i.e., these secrete either IgM or IgA. IgG is not expressed above the level of background; 1 μg LPS/ml (positive control) did increase production of each isotype measured. n = 3; Error Bars = SEM; *p < 0.05.

Figure 3.

Figure 3

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Effect on rate of CH12.LX cell proliferation when incubated in media from RAW264.7 macrophages treated with asbestos (35 μg/cm2) or left untreated. CH12.LX cells were incubated for 72 h and assayed by measurement of fluorescence at 530 nm. n = 6; error bar = SEM. (a) indicates that the No Treatment (Tx) Mac Media had a significant (*p < 0.05) effect on decreasing the proliferative events of CH12.LX B-lymphocytes when compared to cells only; (b) shows that the media from macrophages treated with 35 μg asbestos/cm2 significantly (*p < 0.05) decreased the proliferation rate of the CH12.LX B-lymphocytes as compared to the No Tx Mac Media.

Media of asbestos-exposed RAW264.7 macrophages or peritoneal cells alters immunoglobulin isotypes secreted by CH12.LX cells and expressed on peritoneal B1a cells, respectively

Concurrently with the proliferation data, we determined whether culturing the CH12.LX cells in media from RAW264.7 macrophages would cause the cells to increase antibody production and undergo class-switch recombination (as evidenced by a change in isotype expression). By normalizing antibody production to a per cell ratio, we noted that media from asbestos-exposed macrophages induced changes in the antibody isotype secreted. Specifically, media from asbestos-exposed macrophages significantly increased the production of isotypes IgG (subclasses 1 and 3) as well as IgA (Figure 4). IgM production was not significantly changed. Interestingly, surface expression of IgA on peritoneal B1a B-lymphocytes increased when collected peritoneal cells were exposed to asbestos ex vivo for 2 days (Figure 5).

Figure 4.

Figure 4

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Isotypes secreted by CH12.LX cells incubated in media from RAW264.7 macrophages treated with asbestos (35 μg/cm2) or left untreated. Data was normalized to reflect amount of antibody produced by each cell. CH12.LX cells were incubated for 72 h and the concentrations of individual isotypes was then measured using an ELISA. MΦ Media w/o Asbestos connotes antibody production by CH12. LX cells cultured in media from unexposed macrophages; MΦ Media w/Asbestos connotes antibody production by CH12.LX cells cultured in media from macrophages exposed to asbestos. n = 3; error bars = SEM. Treatment with asbestos caused the CH12.LX B-lymphocytes to significantly (*p < 0.05) increase secretion of (a) IgG1, (b) IgG3, and (c) IgA, but not (d) IgM.

Figure 5.

Figure 5

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Percentage of peritoneal B1a B-lymphocytes that express IgA in response to ex vivo asbestos exposure. Peritoneal cells were incubated with asbestos (at 50 μg/well) overnight, and then stained with antibodies to IgM, CD5, and IgA for flow cytometry. n = 4 mice per group; Treatment with asbestos significantly increased the expression of IgA; *p < 0.05.

Asbestos exposure induces IL-6 and TNFα production by RAW 264.7 macrophages and cultured peritoneal cells

As the in vitro CH12.LX experiments were performed in the absence of a T-lymphocyte population, we postulated that asbestos exposure caused the RAW264.7 macrophages to stimulate the CH12.LX B-lymphocytes through cytokine signaling. With regard to the ex vivo experiment described above, we found that peritoneal cells—when exposed to asbestos—increased the production of two cytokines, IL-6 and TNFα (Figure 6). Therefore, we collected media from asbestos-treated and untreated RAW264.7 macrophages and similarly detected increased IL-6 and TNFα production. We found that asbestos-exposed media had a 40-fold increase in IL-6 concentration (230 pg/ml) when compared to the control media concentration (6 pg/ml); TNFα levels had increased 4.5-fold (to 14 μg/ml) compared to that with the untreated macrophage media (3 μg/ml) (Figure 7).

Figure 6.

Figure 6

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Measurement of cytokines produced by peritoneal cells exposed to asbestos ex vivo, using Cytometric Bead Array analyses. Cells were incubated with asbestos (at 50 μg/well) overnight, and media was then collected for analyses. n = 4 wells per group; Treatment with asbestos significantly increased the production of TNFα and IL-6; *p < 0.05.

Figure 7.

Figure 7

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Cytometric bead array analyses of (a) IL-6 or (b) TNFα secreted from RAW246.7 exposed to asbestos (35 μg/cm2) for 48 h. n = 3 wells; experiments were repeated three times; error bars = SEM; *Exposure to asbestos significantly increased RAW264.7 macrophage secretion of IL-6 and TNFa compared to that by the No Treatment (0 μg/cm2) group; p < 0.05.

Recombinant IL-6 and TNFα decrease the proliferation rate of CH12.LX B-lymphocytes

Using recombinant murine IL-6 (rIL-6) and TNFα (rTNFα), we attempted to simulate the possible conditions created by macrophages when treated with asbestos. To simulate untreated macrophage media, either 6 pg rIL-6/ml or 3 μg TNFα/ml were added to cultures containing only CH12.LX cells. Asbestos-treated macrophage media was simulated by addition of 230 pg IL-6/ml or 14 μg TNFα/ml. A dose of 230 pg IL-6/ml decreased the rate of CH12.LX proliferation similarly compared to the CH12.LX cells incubated with treated macrophage media (Figure 8a). Overall, TNFα decreased the rate of proliferation; however, there was no statistical difference between conditions that simulated the untreated/treated macrophage media (Figure 8b).

Figure 8.

Figure 8

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(a) Effects of cytokine IL-6 or (b) TNFα on CH12.LX B-lymphocyte proliferation rate. n = 6; error bars = SEM; * IL-6 (at 230 pg rIL-6/ml) significantly decreased the proliferation rate of the CH12.LX cells when compared to Cells Only group; p < 0.05. * TNFα at the concentration simulating treated macrophage media did not induce any significant effects compared to that from the concentration simulating TNFα levels in untreated macrophage media.

Recombinant IL-6 increases the production of IgG and IgA by the CH12.LX cells

Using rIL-6 and rTNFα as previously stated, we determined the effectiveness of these cytokines in inducing the CH12.LX cells to increase antibody production and express other isotypes. TNFα did not cause CH12.LX cells to increase production of IgG, IgA, or IgM (Figure 9). After normalizing the data to the number of cells contained in each analyzed well (to correct for changes due to proliferation), we determined that the levels of IgG secreted increased in the presence of IL-6. Cells treated with 230 pg IL-6/ml released significantly more IgG compared to the cells treated with 6 pg IL-6/ml (Figure 10a). IgA levels increased overall when CH12.LX cells were treated with both concentrations of IL-6; however, a comparison of the IL-6-treated groups shows that the IgA levels secreted were not significantly different (Figure 10b). The expression levels of IgM did not change significantly (Figure 10c). This data correlates with the previous findings that demonstrated that CH12.LX cells cultured in macrophage media increased their production of IgG and IgA.

Figure 9.

Figure 9

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Isotype ELISA data depicting levels of antibody secreted by CH12.LX B-lymphocytes incubated for 48 h with TNFα. Data was normalized to reflect amount of antibody produced by each cell. TNFα was unable to stimulate the CH12.LX cells to increase production of the three isotypes (a) IgM, (b) IgG, or (c) IgA. n = 3; Error Bars = SEM.

Figure 10.

Figure 10

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Isotype ELISA data depicting the concentration of antibody secreted by CH12.LX B-lymphocytes incubated with IL-6 for 48 h. Data was normalized to reflect the amount of antibody produced by each cell. Concentration of (a) IgG, (b) IgA, and (c) IgM secreted by the CH12.LX cells. n = 3; Error Bars = SEM; * Value significantly differs from that of Cells Only group (p < 0.05); ** value significantly differs from that of the 6 pg/ml group (at p < 0.05).

Discussion

Previous work in this laboratory and by others has suggested that asbestos exposure in humans can lead to autoimmune responses and possibly increase the risk of systemic autoimmune disease (Zerva et al., 1989; Nigam et al., 1993; Noonan et al., 2006), but the mechanism by which this occurs remains unknown. This study used C57Bl/6 mice and the CH12.LX B-lymphocyte line as ex vivo and in vitro models, respectively, to begin to identify the primary players in the autoimmune response following asbestos exposure. Asbestos concentrations used in this study were based on (a) toxicological studies on CH12.LX and RAW macrophages (Blake et al., 2007) in which concentrations were chosen that did not induce significant cell death, and (b) literature evidence of significant peritoneal and pleural fiber deposition in animals and humans exposed to asbestos. When animals were exposed to occupationally relevant aerosol concentration of amphibole (250 μg/cm3), fiber burden of just the thoracic wall was as high as 960 fibers per mg of dry tissue (McConnell et al., 1999). In humans exposed to amphibole asbestos, pleural fiber burdens have been shown to be as high as 100 × 106 fibers/g dry tissue, and in fact as high as that found within the lung itself (Dodson et al., 1991; Tossavainen et al., 1994). Therefore, significant contact with asbestos could be expected for pleural or peritoneal macrophages and B1a cells.

The first critical gap in knowledge that is tested in this study is whether there is a direct or indirect effect of asbestos on B-lymphocytes, the antibody-producing cells, in terms of proliferation and antibody production. Specifically, we were interested in B1a B-lymphocytes due to their potential role in autoimmunity in other models and their location in areas affected by asbestos exposure. The results of our work demonstrate that B1a B-lymphocytes are not directly stimulated by asbestos and need additional soluble factors, such as cytokines, from other cellular constituents in order to become activated. While the data does not support the original hypothesis that asbestos directly activated B1a B-lymphocytes, our results provide a potential mechanism involving B1a B-lymphocytes in antibody production development following asbestos exposure.

As reviewed in Duan and Morel (2006), B1a B-lymphocytes reside within the peritoneal and pleural cavities and are characterized as a long-lived, self- renewing population that produce a majority of the circulating IgM that serves as an initial defense mechanism against bacterial invasion. Furthermore, these immunoglobulins may also be autoreactive, with a broad range of self-epitopes within various tissues (Hayakawa et al., 1984; Casali et al., 1987; Hardy et al., 1987; Murakami and Honjo, 1995; Hamano et al., 1998; Zhang et al., 2006; Zhang and Carroll, 2007). The deletion of B1a B-lymphocytes within the peritoneal cavity by hypertonic shock prevented autoimmune hemolytic anemia, lupus nephritis, and production of anti-nuclear antibodies in autoimmune-prone mice (Murakami et al., 1995). Studies in our laboratory have shown that intratracheal instillation of C57Bl/6 mice with asbestos increased the production and presence of anti-nuclear antibodies, and also expanded the B1a B-lymphocyte population (Pfau et al., 2008).

The study at hand extends previous observations within our laboratory by characterizing the direct and indirect effects of asbestos on B1a B-lymphocyte activity. B-lymphocytes are activated through either the B-cell receptor (BCR) or via pattern recognition receptors that react with specific molecular patterns that are associated with various pathogens. As the crocidolite asbestiform specifically binds to scavenger receptors produced by macrophages (Resnick et al., 1993), we initially hypothesized that direct recognition of the Libby amphibole asbestos by B1a B-lymphocytes and subsequent activation would occur via pattern recognition receptors, such as the Toll-like receptors, which are constitutively expressed on the cell surface. Clearly, the data demonstrated that asbestos did not directly activate the CH12.LX B-lymphocyte line in terms of proliferation or antibody production, despite the fact that these cells do express a variety of Toll-like receptors (North et al., 2010).

Without a direct effect, and due to the large population of macrophages that also reside in the pleural and peritoneal cavities, we hypothesized that asbestos causes an indirect effect on B1a B-lymphocytes through macrophage-induced cytokines. Because previous work by Blake et al. (2007) demonstrated that RAW264.7 macrophages react with the Libby amphibole asbestos, we determined the effects of media from macrophages on the physiological activities of the CH12.LX B-lymphocytes. Macrophage media caused the CH12.LX B-lymphocytes to increase the production of various non-IgM isotypes as well as decrease the rate of proliferation. IgA and IgG3 were the predominant sub-classes produced by the CH12.LX B-lymphocytes. IgG3 is typically produced by B-lymphocytes in response to a TH1-type immune response. The IgG3 sub-class, if autoreactive, has demonstrated the potential of developing diseases ranging from glomerulonephritis to SLE as complement has a strong affinity towards the constant region of all IgG3 sub-classes (Fulpius et al., 1993; Baudino et al., 2006). The production of IgA by this B1a B-lymphocyte model has been reported in response to various cytokines or LPS (Bao et al., 1999; Kuzin et al., 2004), and is consistent with the fact that B1a B-lymphocytes are responsible for producing the majority of IgA within the gut and lamina propria (Kroese et al., 1989, 1993). This further supports the use of CH12.LX cells as a model for studying the antigen-independent signaling responses by B1a B-lymphocytes that result in immunoglobulin production.

In order to determine what macrophage signal might be eliciting this response, a cytokine bead array was performed. We demonstrated that in vitro exposure to asbestos induces production of the cytokines IL-6 and TNFα by RAW 264.7 macrophages. Interestingly, using peritoneal cells from C57Bl/6 mice, we further discovered that the cytokines TNFα and IL-6 were significantly elevated from peritoneal cells cultured in the presence of Libby amphibole asbestos. The TNFα most likely was derived from the peritoneal macrophage population, while the IL-6 could have been produced by macrophages or B-lymphocytes. The data also demonstrated that surface IgA expression on the peritoneal B1a B-lymphocytes significantly increased. As expected, surface expression of other isotypes did not increase, since B1a cells and particularly CH12.LX cells are known to express surface IgM and IgA, but to secrete IgG (Whitmore et al., 1991; Nakamura et al., 1996). Taken together, these data suggested that possible signals affecting B1a B-lymphocytes include IL-6 and TNFα.

The production of IL-6 and TNFα is consistent with a TH1 or pro-inflammatory immune response and correlates with the production of IgG3 by the CH12.LX B-lymphocytes. Both cytokines are implicated in SAID (Sabry et al., 2006), but their specific effects on B-lymphocytes continue to challenge a clear picture. Even less is known about their effects specifically on B1a B-lymphocytes. TNFα may affect autoimmunity through phenotypic changes consistent with regulatory B-lymphocytes, including increased expression of CD5 on circulating B-lymphocytes (Karampetsou et al., 2011, Schioppa et al., 2011). A single study has shown that both TNFα and IL-6 increased proliferation in a plaque-forming assay using either B2 or B1a cells, suggesting that they can both elicit proliferation (Jones, 1996). Interestingly, the CH12.LX cells responded to recombinant TNFα with a decrease in proliferation. The major difference between the two studies is that the former used primary peritoneal B-lymphocytes, used a mix culture of cells, and pre-stimulated the cells with pokeweed antigen and Staphylococcus. Therefore, our data is the only study we are aware of that looks at the direct effect of TNFα on a monoculture of B1a-like B-lymphocytes that are already actively dividing. The decrease in proliferation indicates a phenotypic change in these cells, but treatment with TNFα did not induce changes in antibody production (Figure 9).

Recombinant IL-6 (rIL-6) also reduced proliferation of the CH12.LX cells and increased the secretion of immunoglobulins, particularly IgA. This is consistent with the role of IL-6 in the phenotypic change of B-lymphocytes to antibody-producing cells (McGinnes and Paige, 1991). Within B-lymphocytes, IL-6 increases the expression of the recombination enzyme RAG, which is responsible for V(D)J recombination events during B-lymphocyte maturation (Hillion et al., 2007). This step has been thought to be essential for subsequent class switch recombination. Therefore, signaling through IL-6 may explain the increased production of isotypes other than IgM by the CH12.LX cells.

In these experiments, CH12.LX cells were treated with levels of cytokines approximating what had been measured from the macrophage media; effects were induced even by media from untreated macrophages. This may be due to the baseline level of IL-6 and TNFα released by RAW 264.7 cells, even without stimulation, but it also may suggest that other factors produced by macrophages are involved, and there may be additive or synergistic effects that remain to be discovered. This idea is further supported by the fact that the level of individual non-IgM isotypes produced when CH12.LX B-lymphocytes were incubated in media from asbestos-exposed macrophages is elevated compared to the levels produced by the B-lymphocytes treated with rIL-6 alone. As the Ig molecules produced by the B1a B-lymphocytes are considered to be polyreactive with weak affinity, the production of IL-6 may aid in increasing the affinity of natural antibodies, through antibody maturation, upon asbestos exposure and, therefore, aid in developing and exacerbating the autoimmune disease states.

A previous dogma in B-lymphocyte biology suggested that mature, terminally differentiated B-lymphocytes were incapable of increasing affinity of the BCR due to down-regulation of the RAG gene products and recombination machinery. However, Hillion et al. (2007) have demonstrated that IL-6 is capable of inducing upregulation of RAG and permitting secondary rearrangements of the BCR in mature B2 lymphocytes. As B1a B-lymphocytes are mature, terminally-differentiated B-lymphocytes it may be plausible to speculate that IL-6 activity may allow for affinity maturation of the polyreactive antibodies secreted by B1a B-lymphocytes. In turn, this affinity maturation could increase the pathogenicity of the antibodies and therefore drive an autoimmune process. IL-6 is elevated in many autoimmune disorders and therapeutics designed to decrease its levels are currently used in decreasing the severity of the disease (Hata et al., 2004; Gigante et al., 2011; Grivennikov and Karin, 2011; Snir et al., 2011).

In conclusion, we have shown that asbestos stimulates both peritoneal macrophages and RAW264.7 cells to increase production of IL-6 and TNFα, which is consistent with previous studies. Media from cultured macrophages stimulated changes in proliferation and immunoglobulin secretion in a B1a B-lymphocyte line, and addition of recombinant IL-6 suggests that this cytokine is at least one of the macrophage factors driving these changes. Although we showed increased IgA expression on peritoneal B1a cells following asbestos exposure, the experiment did not specifically demonstrate which factors were involved, although IL-6 and TNFα were dominant cytokines in the peritoneal cell culture, supporting our hypothesis indirectly.

Nevertheless, in studies currently being pursued in our laboratory, we are striving to decipher the significance of the role of IL-6 and the possibility that other macrophage factors contribute. This pursuit will also be accomplished through studies with neutralizing antibodies towards IL-6 and various other cytokines and chemokines, or possibly using siRNA to knock down specific macrophage cytokines in the cultures. The neutralizing experiments will allow us to determine if IL-6 performs a primary or a secondary role in signaling the CH12.LX cells to perform class-switch recombination in order to secrete antibodies such as IgA. Further study is also needed to determine additional signals present in the peritoneal or pleural cavity following asbestos exposure that may drive activation of B1a B-lymphocytes. In addition, in vivo studies will be needed to determine the activation of B1a B-lymphocytes following exposure to asbestos, and whether those responses truly contribute to the pathologies of autoimmunity seen following amphibole asbestos exposures.

Acknowledgments

We gratefully acknowledge Dr Norb Kaminiski, Michigan State University, for generously providing the CH12.LX B-lymphocytes for the study. We also acknowledge the Center for Environmental Health Sciences, The University of Montana, for providing the Libby Six-mix asbestos, as well as the Molecular Core Research Facility, Idaho State University, for the use of their instrumentation.

Footnotes

Declaration of interest

This work was supported by INBRE Grant #P20 RR016454, University of Washington ITHS Pilot Award, and ISU GSRSC Grant #F09-26 (DR). The authors report no conflicts of interest.

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