Assessment of Autoimmune Responses Associated with Asbestos Exposure in Libby, Montana, USA

Center for Environmental Health Sciences, Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, Montana, USA: Assessment of Autoimmune Responses Associated with Asbestos Exposure in Libby, Montana, USA

• Introduction
• Materials and Methods
• Results
• Discussion
• Conclusion

Address correspondence to J.C. Pfau, Center for Environmental Health Sciences, Department of Biomedical and Pharmaceutical Sciences, Skaggs 154, University of Montana, Missoula, MT 59812 USA. Telephone: (406) 243-4529. Fax: (406) 243-2807. E-mail: jean.pfau@umontana.edu

We thank the subjects, families, and the Center for Asbestos Related Diseases Clinic in Libby, MT; R. Hamilton [Center for Environmental Health Sciences (CEHS) biostatistics], C. Noonan (CEHS epidemiology), and M. Fritzler, University of Calgary (rheumatology) for their expertise; and T. Larson, Centers for Disease Control and Prevention/Agency for Toxic Substances and Disease Registry, for Tremolite Asbestos Registry data.

This work was supported by National Institutes of Health grants ES-04804 (J.C.P.) and ES-11676 (E.A.P.) and National Research Service Award ES-11249 (J.C.P.).

The authors declare they have no competing financial interests.

Received 16 July 2004; accepted 30 September 2004.

Introduction

Asbestos-related lung disease (ARD), including fibrosis, pleural plaques, and cancer, continues to be a serious and significant problem despite increasing awareness of the health hazards of asbestos inhalation. Although the exact mechanisms leading to the progression of these conditions have not been fully explained, there is evidence that some of the lung pathologies seen with both asbestos and silica exposures are immunologically mediated (Hamilton et al. 1996; Holian et al. 1997; Perkins et al. 1993). Silica and asbestos exposures also both appear to exacerbate autoimmune responses. Epidemiologic studies have shown strong associations between silica exposure and several autoimmune diseases, including scleroderma, systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA) (Koeger et al. 1995; Parks et al. 1999; Powell et al. 1999; Steenland and Goldsmith 1995). Increased serum immunoglobulins, positive antinuclear antibody (ANA) tests, and immune complexes have been reported in small cohorts of individuals exposed to asbestos (Lange 1980; Nigam et al. 1993; Zerva et al. 1989), but to our knowledge no comprehensive study has been undertaken to assess the prevalence, specificity, and significance of autoantibodies associated with asbestos exposures. The population in Libby provides a unique research opportunity because of significant exposures that occurred as a result of the mining of asbestos-contaminated vermiculite near the community. Exposures have been documented not only in the miners but also in their family members, as well as anyone who used the vermiculite or played near the mine tailings. Therefore, the Libby asbestos exposures were both occupational and environmental throughout the community (Peipins et al. 2003).

In addition to the ARD in Libby, there have been anecdotal reports of an increased prevalence of systemic autoimmune disease (SAID), but verification of these diagnoses is still in progress. When the Centers for Disease Control and Prevention/Agency for Toxic Substances and Disease Registry (ATSDR) performed its screening in Libby during 2000-2001, 494 (6.7%) of 7,307 screening participants indicated that they had been diagnosed with either SLE, scleroderma, or RA (Larson T, personal communication). In contrast, a prevalence of < 1% for these three conditions combined would be expected based on pooled estimates from 43 prevalence studies (Jacobson et al. 1997). These data, along with extensive evidence of silica-associated autoimmunity, provided the impetus to initiate a multifaceted assessment of the impact of asbestos exposures on the population of Libby, Montana, including possible autoimmune responses.

In this article we report on a study designed to assess whether there were humoral alterations in serum samples from an asbestos-exposed population (Libby samples) that might indicate autoimmune responses, providing the rationale for future full-scale studies. Serum samples of subjects from Libby and from a Montana community with no reported asbestos exposure were assayed for a variety of immune parameters, including ANA, immunoglobulin A (IgA), rheumatoid factor (RF), and antibodies to extractable nuclear antigens (ENA).

Materials and Methods

Human samples. All samples were acquired according to approved University of Montana institutional review board protocols, protecting the well-being and confidentiality of all subjects. Appropriate informed consent was obtained from all subjects, and a questionnaire was administered regarding overall health, smoking status, asbestos exposure, age, sex, and recontact information.

Two sample pools were obtained through other studies at the Center for Environmental Health Sciences. Subjects were recruited through flyers and ads in Missoula, Montana, for a study of immune function (E. Putnam), and serum samples were collected at the same time for convenience. Missoula is similar to Libby in that it is located in a mountain valley subject to similar climatic conditions, including winter inversions, dry summers, and exposure to smoke from fall forest fires. Because prevailing winds in Missoula are from the west and Libby is well to the north, there would be no transfer of asbestos from the Libby airshed to the Missoula airshed. Therefore, on a relative basis, although one cannot exclude the possibility that there could have been some minimal asbestos exposure by the Missoula population, it is an acceptable reference population for the Libby subjects who were definitely exposed to asbestos. For this study, we selected 50 samples from subjects with no reported asbestos exposure from the Missoula pool, excluding any who had lived or worked in Libby. Concurrently, subjects were being recruited from Libby, Montana, for a genetic study of ARD susceptibility (E. Putnam), and the samples were drawn at the Center for Asbestos Related Diseases Clinic in Libby when subjects came for screening or responded to subject recruitment advertisements. From this pool, 50 Libby subjects were selected that were matched to the 50 Missoula subjects to give similar mean age and sex ratios for the two subject sets. Permission was obtained to acquire information about prescription drug use and ARD status from medical records, and these data were inserted into a coded database.

Both communities from which subjects were drawn are fairly homogeneous in terms of ethnicity; most residents are of northern European descent, with 94.7% white in Lincoln County (Libby) and 93.6% white in Missoula County (Missoula) according to Montana census data (U.S. Census Bureau 2004). The mean age for both sample sets was 55 years (Missoula, 54.8 ± 2.5 years; Libby, 55.0 ± 2.1 years), and the male:female ratio was 25:25 for both sets. An exclusion criterion was the use of medications strongly associated with drug-induced autoimmunity (Fritzler 1994). The presence of diagnosed autoimmune disease did not exclude individuals from either sample set but was noted on intake. None of the Missoula subjects recruited had diagnosed SAID. Of the Libby subjects recruited initially for the genetics study, the percentage with diagnosed SAID was 5.9%, and in the final Libby sample set of 50, there were two with SLE, 1 with multiple sclerosis, and 1 with RA. Because the individual with RA also had SLE, this means that three people in 50 had SAID (6%). These values are consistent with the ATSDR screening data for this community (Larson T, personal communication).

The lung diseases in this community have been previously described (Peipins et al. 2003) and include primarily pleural abnormalities (17.8%) and interstitial abnormalities (< 1%). In our Libby sample set, 12 (24%) had no reported abnormalities, 27 (54%) had pleural abnormalities, 8 (16%) had interstitial abnormalities, and 3 (6%) had a combination of pleural and interstitial abnormalities.

Sample and data collection. The blood samples were collected, and serum samples were obtained and frozen by standardized clinical methods to prevent differences due to handling. The samples were blinded with only sex and age noted, and stored at -80°C until assayed. After testing, coded information regarding disease status and exposure was obtained from the questionnaire and ATSDR screening data (ATSDR 2002).

ARD and asbestos exposure rankings. ARD status, based on data recorded in the database primarily as a result of the ATSDR screenings, was ranked on a scale of 0-3, as described in Table 1.

The rankings were intentionally simplified, based on radiographic evidence of single versus extensive plaques or interstitial abnormalities, as well as spirometry evidence of functional deficits. For example, a subject with a single pleural plaque and no functional deficit would be scored at 1, whereas a subject with bilateral plaques and effects on spirometry was scored at 3. To further break down the sample sets by disease types would have made the subsets too small for statistical analysis. Exposure status was ranked on a scale of 0-4, as described in Table 2.

These scores were also simplified in order to focus on duration of exposure and the existence of occupational and/or environmental exposure to asbestos. The rankings of the Libby subjects were performed independently by two of the researchers (J.C.P. and E.A.P.).

Autoantibody testing. A clinical test for nuclear antigens (ANA assay), used to screen for antibodies commonly seen in SAID, was performed at a screening dilution of the sera. All serum samples were diluted 1:40 in phosphate-buffered saline (PBS) and tested by indirect immunofluorescence (IIF) on a single lot of commercially prepared and fixed HEp-2 cells (ImmunoConcepts Inc., Sacramento, CA), according to manufacturer's instructions. The staining pattern and relative fluorescence intensity were compared with known positive and negative controls using a Zeiss fluorescence microscope with 40X objective and recorded as positive (1+ to 4+) or negative (0). The staining pattern was also noted and recorded. The same microscope and settings were used for all samples, and the slides were read by two independent readers. Samples showing homogeneous staining patterns were reevaluated using the Crithidia luciliae substrate (ImmunoConcepts), which specifically detects antibodies to double-stranded DNA (dsDNA), and by enzyme-linked immunosorbent assay (ELISA) to detect antibodies to chromatin (INOVA Diagnostics, San Diego, CA), both according to the manufacturers' instructions. Samples with positive ANAs were also evaluated using a modified ANA test to determine whether any of the anti-ANA antibodies were of the IgA isotype. The samples were tested on HEp-2 slides as above, but instead of the anti-human IgG fluorescein isothiocyanate (FITC) conjugate included with the slides, we used goat anti-human IgA FITC conjugate (Southern Biotech, Birmingham, AL). The slides were read as described above.

IgA ELISA. For detection of serum IgA, the mucosal antibody isotype, 96-well polysorb plates (Nunc, Rochester, NY) were coated with 1 µg/mL of anti-human kappa light chain (Southern Biotech) in carbonate coating buffer overnight at 4°C. Wells were then blocked with PBS containing 1% bovine serum albumin (BSA) for 1 hr. Subject samples were diluted 1:4,000, 1:8,000, and 1: 16,000 in diluent buffer (PBS, 1% BSA, 0.1% Tween-20). Samples, standards, and blanks were added to wells to give 100 µL/well. After 1 hr, plates were washed with three changes of PBS containing 0.1% Tween-20. The detection antibody [goat anti-human IgA alpha chain coupled to horseradish peroxidase (HRP); Caltag, Burlingame, CA] was added and the plates were incubated for 1 hr at room temperature. The plates were washed again and developed using HRP-tetramethylbenzidine (TMB) substrate (Zymed, San Francisco, CA). The reaction was stopped with 2N H₂SO₄, and the plate was read on a SpectraMax plate reader (Molecular Devices, Sunnyvale, CA). All data were evaluated against a standard curve, using human IgA (Sigma, St. Louis, MO).

RF ELISA. RF in the subjects' serum was measured with an ELISA kit according to the manufacturer's protocol (INOVA Diagnostics). The plates were read on the SpectraMax plate reader. Optical density (OD) values were compared with known controls provided with the kit and rated as negative or positive (marginal, moderate, or high).

ENA array. Analyses of antibodies to five extractable nuclear proteins commonly seen in SAID (Sm, RNP, SS-A, SS-B, and Scl-70) were performed using an addressable bead array kit (QuantaPlex ENA-5; INOVA Diagnostics) according to the manufacturer's instructions, on a Luminex multiplex system (MiraiBio, Alameda, CA). The values were compared by using MasterPlex software (MiraiBio) to negative and graduated positive control reagents provided with the kit, and determined to be low, moderate, or high positive, or negative.

Statistics. In this study we included analysis of several different types of data, including percentages/frequencies (e.g., ANA frequencies), ordinal (e.g., disease status assessed on a 4-point scale), and scale (e.g., mg/mL IgA) data. Consequently, we used the following statistical methods: a) differences in percentages were tested using raw frequencies with Fisher's exact test; b) contingency tables with 4- and 5-point ordinal level frequency comparisons were made via the chi-square test; and c) independent sample t-tests were used for scale measures. In the Libby samples, comparisons (correlations) between ANA levels and disease and exposure rankings were made using the nonparametric Spearman rank correlation. In all analyses we used two-tailed, unpaired analyses, and reported 0.05 type I error levels. Data reported in the text are mean ± SEM.

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