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Immunopharmacology 46 Ž2000. 123–137 www.elsevier.comrlocaterimmpharm

Trichloroethylene accelerates an autoimmune response by Th 1 T cell activation in MRLq rq mice Joseph M. Griffin a , Sarah J. Blossom b, Stephanie K. Jackson b, Kathleen M. Gilbert b, Neil R. Pumford a,) a

Department of Pharmacology and Toxicology, DiÕision of Toxicology, UniÕersity of Arkansas for Medical Sciences, 4301 West Markham, Little Rock, AR 72205, USA b Department of Microbiology and Immunology, UniÕersity of Arkansas for Medical Sciences, Little Rock, AR 72205, USA Accepted 6 August 1999

Abstract Trichloroethylene Ž1,1,2-trichloroethene. is a major environmental contaminant. There is increasing evidence relating exposure to trichloroethylene with autoimmunity. To investigate potential mechanisms, we treated the autoimmune-prone MRL q rq mice with trichloroethylene in the drinking water at 0, 2.5 or 5.0 mgrml and sacrificed them at 4, 8 and 22 weeks. As early as 4 weeks of treatment, Western blot analysis showed a dose-dependent increase in the level of trichloroethylene-modified proteins, indicating that a reactive metabolite of trichloroethylene was formed. Significant increases in antinuclear antibodies ŽANA. and total serum immunoglobulins were found following 4–8 weeks of trichloroethylene treatment, indicating that trichloroethylene was accelerating an autoimmune response. Investigation into possible mechanisms of this autoimmune response revealed that trichloroethylene treatment dramatically increased the expression of the activation marker CD44 on splenic CD4q T cells at 4 weeks. In addition, splenic T cells from mice treated for 4 weeks with trichloroethylene secreted more IFN-g and less IL-4 than control T cells, consistent of a T-helper type 1 ŽTh 1 . type immune or inflammatory response. A specific immune response directed against dichloroacetylated proteins was found at 22 weeks of trichloroethylene treatment. Taken collectively, the results suggest that trichloroethylene treatment accelerated an autoimmune response characteristic of MRL q rq mice in association with nonspecific activation of Th 1 cells. In addition, long-term treatment with trichloroethylene led to the initiation of a trichloroethylene-specific immune response. q 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: Trichloroethylene; Th 1 T cell; MRL q rq mice

1. Introduction Trichloroethylene Ž1,1,2-trichloroethene. is a volatile organic compound that has been used exten-

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Corresponding author. Tel.: q1-501-686-5467; fax: q1-501686-8970; e-mail: [email protected]

sively in industry as a degreasing agent for metals, used to clean computer chips, a polymer precursor and a dry cleaning agent. The production of trichloroethylene in the United States has increased from approximately 260,000 lb in 1981 to 320,000,000 lb in 1991. The primary environmental releases of trichloroethylene are due to air emissions from metal degreasing plants, and wastewater from metal finish-

0162-3109r00r$ - see front matter q 2000 Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 2 - 3 1 0 9 Ž 9 9 . 0 0 1 6 4 - 2

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ing and rubber processing industries. Because of its widespread commercial use and improper waste disposal, trichloroethylene has become a major environmental pollutant, and it is one of the most abundant organic contaminants at many of the United States National Priority List sites ŽAgency for Toxic Substances and Disease Registry, 1994.. There have been an increasing number of reports associating trichloroethylene exposure with autoimmunity ŽSaihan et al., 1978; Flindt-Hansen and Isager, 1987; Lockey et al., 1987; Hansen and Isager, 1988; Yanez Diaz et al., 1992; Waller et al., 1994.. For example, trichloroethylene has been associated with a rapid fatal progressive systemic sclerosis following a single exposure ŽLockey et al., 1987.. Also, Flindt-Hansen and Isager reported several patients that developed systemic sclerosis after prolonged and intensive exposure to trichloroethylene in connection with metal cleaning ŽFlindt-Hansen and Isager, 1987.. In a recent publication, Nietert et al. reported evidence in humans of an increased risk of developing systemic sclerosis associated with trichloroethylene exposure ŽNietert et al., 1998.. Trichloroethylene exposure is not only associated with systemic sclerosis but also with systemic lupus erythematosis ŽSLE.. For example, it has been reported that humans with long-term low-dose exposure to well water contaminated primarily with trichloroethylene had significant increases in symptoms of SLE determined by the American Rheumatism Association ŽARA. score and increases in antinuclear antibody ŽANA. titers ŽKilburn and Warshaw, 1992.. In a separate study, a significant increase in the likelihood of having elevated ANA levels was reported in an region known to be contaminated with trichloroethylene when compared to a unpolluted area ŽClark et al., 1994., although there is disagreement regarding the validity these studies ŽWallace and Quismorio, 1995.. Byers et al. reported an association between chronic exposure to a domestic water supply contaminated with high levels of trichloroethylene and lymphocyte abnormalities and increases in ANA ŽByers et al., 1988.. The etiology of autoimmunity is thought to be multifactoral including both genetic and environmental factors. Genetically, susceptible animals are routinely used to study idiopathic autoimmune diseases and many of these genetically susceptible strains are

now being used to examine the potential for chemicals and drugs to induce or accelerate autoimmune diseases. The utility of using genetically susceptible strains instead of non-susceptible or resistant strains has been shown for several compounds. For example, mercuric chloride is known to induce ANA in Brown Norway rats and not in Lewis rats ŽHultman and Johansson, 1991; Gillespie et al., 1996.. In addition, the ability of mercuric chloride, gold salts and D-penicillamine to induce autoimmunity in some mouse strains while not in others has been linked to a genetic susceptibility or predisposition ŽRobinson et al., 1986; Johansson et al., 1998.. Trichloroethylene treatment of non-autoimmune prone mice does not lead to stimulation of the immune system ŽSanders et al., 1982; Wright et al., 1991.. In fact, trichloroethylene-treated non-autoimmune prone rodents led to a suppression of the immune system ŽSanders et al., 1982; Wright et al., 1991.. Since a genetic predisposition is considered necessary for the development of autoimmune disease, we utilized a mouse strain that is genetically predisposed to autoimmunity. Khan et al. have developed an animal model for acceleration of an autoimmune response using trichloroethylene and we wanted a sensitive mouse strain to investigate the mechanisms for this immune stimulation we chose to use the MRL q rq mouse. Khan et al. reported an increase in markers of autoimmunity such as increases in ANA and serum immunoglobulin levels in MRL q rq mice treated with trichloroethylene ŽKhan et al., 1995.. To explore the mechanisms involved in this increase in autoimmune response by trichloroethylene, MRL q rq mice were treated with trichloroethylene at 0, 2.5 or 5.0 mgrml in the drinking water, and we examined the cells and soluble mediators involved in this autoimmune response. Since the presence of activated IFN-g-secreting CD4q T cells have been shown to be crucial to the development of autoimmunity in MRL mice ŽTakahashi et al., 1996., we measured the activation state of CD4q T cells and the cytokine profile secreted by activated CD4q T cells. Serum levels of antibodies specific for trichloroethylene-modified proteins were monitored to determine if a trichloroethylene-specific immune response was initiated. Serum levels of ANA and total IgG and IgM were measured to follow the development of an autoimmune response.

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2. Materials and methods

2.1. Animals and treatments Four-week old female MRLq rq mice purchased from The Jackson Laboratories ŽBar Harber, ME. were housed four per cage and maintained on a 12-h light:dark cycle. The animals were provided lab chow and drinking water ad libitum and were acclimated for 1 week before treatment. Trichloroethylene Žpurity 99 q %. was purchased from Aldrich ŽMilwaukee, WI. and suspended in drinking water with 1% of the emulsifier Alkamuls EL-620 from Rhone-Poulenc ŽCranbury, NJ.. The animals were divided into groups of eight animals and received trichloroethylene in the drinking water at a concentration of 0, 2.5 or 5.0 mgrml. The water was changed every 2–3 days to ensure maintenance of the dose. Sanders et al. determined that less than 20% of the trichloroethylene was lost when water was changed twice a week ŽSanders et al., 1982.. The mice were weighed once a week to monitor weight changes. The average dose of trichloroethylene received during the treatment period was 0, 455 or 734 mg kgy1 dayy1 , respectively. The animals were sacrificed at 4, 8 and 22 weeks of treatment and an interim bleed was taken at 6 weeks. Mice were euthanized with CO 2 and liver, lung, spleens and mesenteric lymph nodes were immediately removed and weighed. A section of liver, lung and kidney were fixed in neutral buffered formalin and processed for hematoxylin and eosin staining. The other section was used for fraction preparation for Western blot analysis.

2.2. Western blotting analysis The presence of trichloroethylene-protein adducts were used as a measure of bioactivation to a reactive metabolite as previously described ŽHalmes et al., 1996.. Microsomes, 10,000 = g pellet Žpellet. and cytosol were prepared as previously described ŽHalmes et al., 1996.. Protein concentration was determined by Coomassie Protein Assay ŽPierce, Rockford, IL. using bovine serum albumin ŽBSA;

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Sigma, St Louis, MO. as the standard. Proteins from the microsomal, pellet and cytosolic fractions were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis ŽSDS-PAGE. under reducing conditions and then transferred to nitrocellulose as previously described ŽHalmes et al., 1996.. Adducts were detected with affinity-purified anti-dichloroacetyl serum that has previously been shown to recognize dichloroacetylated protein-adducts formed by a reactive metabolite of trichloroethylene ŽHalmes et al., 1996.. Adducts were detected using peroxidase labeled goat anti-rabbit antibody ŽGibco, Gaithersburg, MD. and the adducts were visualized using a peroxidase-enhanced chemiluminescence system purchased from Amersham Life Sciences ŽBuckinghamshire, England.. 2.3. Determination of total serum ANA leÕels ANA levels were determined following manufactures instructions using Hep-2 slides obtained from The Binding Site ŽBirmingham, England.. Briefly, the Hep-2 coated wells were incubated with serum diluted 1:1000 from individual animals. The 1:1000 dilution was predetermined to be the optimal serum dilution. Following incubation, the presence of ANA was determined via avidin–biotin peroxidase detection system purchased from Dako ŽCarpinteria, CA. with diaminobenzadine as the substrate. Slides were counterstained with hematoxylin and mounted. Nuclear staining was quantified on the Samba 4000 Cell Image Analysis System ŽImmuno Software Program, Imagining Products, Chantily, VA. and a labeling index was generated for each sample ŽSulh et al., 1996.. The labeling index for ANA was designated as intensity index and was defined as the staining intensity of nuclear region per cell. Two fields for each sample at =40 magnification were analyzed and the labeling index was normalized to the positive control on each slide. Results from treated groups were compared to controls as relative intensity index. 2.4. Determination of total serum IgG and IgM leÕels Total serum immunoglobulin levels were determined using standard sandwich enzyme-linked im-

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munosorbant assay ŽELISA. techniques. Briefly, wells of 96 well culture plates were incubated overnight at 208C with 2.5 mgrml of either monoclonal rat–anti-mouse IgG antibody Žclones LO-Mg7, LO-MG1–2, LO-Mg3–7; Zymed, S. San Francisco, CA. or rat–anti-mouse IgM monoclonal antibody ŽmAb. Žclone LO–MM; Caltag, Burlingame, CA. in a binding buffer consisting of 0.05 M Tris and 0.15 M NaCl at pH 8.0. The plates were washed in phosphate buffered saline ŽPBS. –0.05% Tween-20 ŽFisher, Pittsburgh, PA.. Samples from individual animals were diluted 1:500 in 3% BSA–PBS–0.05% Tween-20 and added in triplicate to the wells. Serum immunoglobulins were detected with an alkalinephosphatase labeled detection system using a antipolyclonal mouse IgG antibody directed against mouse IgG, IgM and IgA ŽZymed.. Total IgG or IgM was determined using a standard preparation of IgG or IgM ŽZymed.. 2.5. Immunophenotypic analyses Spleens from each treatment group were isolated and single-cell suspensions were prepared in serumfree RPMI 1640 ŽGibco, Grand Island, NY.. The spleen cells and lymph node cells were then stained with biotinylated-anti-CD44 mAb Žclone 1 M7, rat IgG2b ; Pharmingen, La Jolla, CA. or biotinylatedanti-CD45RB mAb Žclone 16A, rat IgG2a ; Pharmingen. for 30 min on ice followed by fluorescein isothiocyanate ŽFI. –streptavidin and phycoerytherin ŽPE. –anti-CD4 mAb Žclone GK1.5, rat IgG2b ; Pharmingen.. Other spleen cells were stained with PE–anti-CD45RrB220 mAb Žclone RA3–6B2, rat IgG2a ; Pharmingen., and either FI–anti-I-Ak mAb Žclone 11–5.2, mouse IgG2b ; Pharmingen., or FI– anti-CD3e mAb Žclone 145–2c11, hamster IgG1; Pharmingen.. The phenotypic analysis of 10,000 events per group was performed using a FACScan ŽBecton-Dickinson, Mountain View, CA. and the data is presented as histograms of CD4q cells. Nonviable cells, based on low forward scatter and high side scatter, were excluded in each sample. Data analysis was performed with the use of WinMDI software Žkindly provided by Joe Trotter, The Scripps Research Institute, La Jolla, CA.. For all groups tested, isotype Ig controls were analyzed.

2.6. Cytokine profile analysis The level of cytokines present in the supernatant of stimulated T cells isolated from trichloroethylene treated and non-treated animals were determined by capture assay technique. The single-cell splenocyte suspension for each treatment group was enriched for CD4q T cells by negative selection. Briefly, the spleen cells were resuspended in cold erythrocyte-lysing solution made from 0.17 M Tris and 0.16 M NH 4 Cl, pH 7.2. The cells were washed, resuspended in RPMI 1640 q 10% fetal calf serum ŽFCS. and allowed to incubate in 125 ml polystyrene tissue culture flasks for 1 h at 378C. The non-adherent cells were resuspended in RPMI 1640 q 5% FCS with rat anti-mouse Iak ŽMHC class II. mAb purchased from Pharmingen and incubated 45 min on ice. Washed BioMag goat anti-rat IgG antibody beads ŽPerSpective Biosystems, Cambridge, MA. were added to the suspension and incubated 30 min on ice. To remove cells containing MHC class II, the beads were separated with a magnet and the supernatant containing CD4q T cells was collected. Wells of a 96-well plate were incubated with 10 ngrml anti-CD3e mAb ŽPharmingen. for 72 h at 208C. Following enrichment, 1 = 10 5 cells per well were incubated overnight at 378C in wells pretreated with anti-CD3e mAb to activate the T cells. Following stimulation, the supernatants were removed and tested for interleukin-4 ŽIL-4. and interferon-gamma ŽIFN-g . with a sandwich ELISA. Cytokine concentrations in the ELISA’s were determined by comparison to a concentration of a standard preparation of mouse recombinant IFN-g and IL-4 ŽR & D Systems, Minneapolis, MN.. It must be noted that the activity of the IFN-g recombinant standard deteriorated to the point that it was not detectable in the 8 and 22 week sacrifices, hence, relative absorbance is presented. Absorbance readings were carried out at 410 nm on a Dynatech MR5000 microspectrophotometer ŽChantilly, VA..

2.7. Serum antibodies specific for trichloroethylene in MRL q rq mice To determine if long-term oral treatment of trichloroethylene could induce an immune response

J.M. Griffin et al.r Immunopharmacology 46 (2000) 123–137

directed against proteins modified by a reactive metabolite of trichloroethylene, serum was tested in an ELISA for the presence of antibodies specific for the dichloroacetylated moiety on proteins ŽHalmes et al., 1996.. The ELISA was performed similar to previously described assays for antibodies against the chemical-adducts or haptens ŽBird and Williams, 1989; Martin et al., 1990; Knight et al., 1994; Khan et al., 1995.. Briefly, wells of a 96-well microplate were coated with 1.25 mgrwell overnight at 48C with either rabbit serum albumin ŽRSA. or dichloroacetic anhydride derivatized RSA ŽRSADCAA.. The derivatization of RSA with DCAA results in dichloroacetylation of lysing groups on the albumin ŽHalmes et al., 1996.. Serum from individual animals were diluted 1:100 and incubated in duplicate on RSA- and RSA-DCAA-coated wells at room temperature in a humidified chamber for 90 min. The plates were washed five times with washing buffer consisting of 0.05% Nonidet-40 ŽFluka, Milwaukee, WI. in PBS. Antibody binding was detected using a goat anti-mouse IgG ŽH q L. alkaline phosphatase labeled antibody ŽZymed. with the subsequent addition of p-nitrophenylphosphate substrate from Bio-Rad ŽHercules, CA.. Absorbance was measured at 410 nm on a Dynatech MR5000 microspectrophotometer. The level of specific anti-dichloroacetyl antibodies in the individual serum preparations was determined by subtracting the response obtained in wells coated with RSA from that obtained in wells coated with equal concentrations of RSA-DCAA. The difference represents specific antibodies against the chemical-adduct or hapten. 2.8. Clinical chemistry Serum levels of alanine aminotransferase and sorbitol dehydrogenase were measured with kits from Sigma Diagnostic. Renal function was determined by measuring blood urea nitrogen levels in serum with a kit from Sigma Diagnostic. 2.9. Statistical analysis When appropriate statistical significance was determined using ANOVA with a predetermined significance level of p F 0.05. To determine statistical

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differences between groups, Student–Neuman–Keuls post-hoc test was used.

3. Results 3.1. Physical characteristics It was previously reported that subchronic treatment of CD-1 mice with trichloroethylene via the oral route at the doses used in this experiment caused no major toxicities ŽTucker et al., 1982.. Similar treatment of MRL q rq mice with trichloroethylene in our experiment caused no measurable hepatic or renal damage. Alanine aminotransferase and sorbitol dehydrogenase levels were measured and found to be unaffected by trichloroethylene treatment when compared to control levels Ždata not shown.. Similarly, the known marker of renal toxicity, blood urea nitrogen, was not affected by trichloroethylene treatment Ždata not shown.. Similar to the report of Tucker et al. ŽTucker et al., 1982., body weight, liver weight and spleen weight measurements in the present study revealed no significant physical changes associated with long-term trichloroethylene treatment Ždata not shown.. In addition, histological analysis of hematoxylin and eosin stained slides showed no histopathological changes in the liver, kidney or lung In the high-dose animals there was a slight, but not significant, increase in liver weight at 22 weeks Ždata not shown.. This slight increase in liver weight is consistent with the findings of Tucker et al. ŽTucker et al., 1982.. 3.2. Western blot analysis as an indicator of metabolic actiÕation of trichloroethylene It has been previously shown in our laboratory using specific anti-dichloroacetyl antisera in a Western blot, that liver tissue from mice acutely exposed to trichloroethylene i.p. contain cellular proteins modified by a reactive metabolite of trichloroethylene ŽHalmes et al., 1996.. We have also shown that trichloroethylene-modified proteins can be found in the liver and serum of rats following acute trichloroethylene treatment ŽHalmes et al., 1997a.. In MRL q rq mice treated p.o. with trichloroethylene

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for 4, 8 and 22 weeks, several covalently modified proteins were found in both liver and lung ŽFig. 1.. At 4 weeks of trichloroethylene treatment, there was a dose-responsive increase in the liver cytosolic fraction of primarily a 40 and 60 kDa protein. The microsomes contained immunoreactive proteins at 50 and 75 kDa and a similar pattern was observed in the 10,000 = g pellet which also contained a band at 85–90 kDa ŽFig. 1, left panel.. The most intense staining in the microsomes was a 50-kDa protein adduct. The staining intensity of all the modified proteins increased with duration of exposure throughout the 22 weeks of the experiment. By 22 weeks of exposure with trichloroethylene, there were a large number of additional trichloroethylene-modified peptides in the microsomes and cytosol with the most prominent target a 50-kDa microsomal protein. In the lungs, the primary trichloroethylene-protein adduct was in the cytosol at approximately 50 kDa ŽFig. 1, right panel.. This 50 kDa protein adduct increases in intensity in a dose-dependent manner. In the 10,000 = g pellet of the lung, two major trichloroethylene-protein adducts were observed Ž45 and 75 kDa.. 3.3. ANA as an indicator of autoimmunity Since high levels of ANA are an indicator of autoimmune diseases ŽTan, 1982., individual serum samples from control and treated mice were tested for the presence of ANA ŽFig. 2.. Serum from mice treated for 4 weeks with trichloroethylene produced very low levels of ANA staining and there was no marked difference from controls. However, by 6 and 8 weeks of trichloroethylene treatment, there was a significant increase in ANA staining when compared to controls. At 22 weeks of treatment, the levels of serum ANA in the control and treated groups were similar and elevated compared to the levels of ANA

staining detected at previous weeks. Upon microscopic examination, there was a mixture of speckled and homogeneous staining patterns within the nucleus of the Hep-2 cells incubated with serum from animals treated for 6 and 8 weeks of trichloroethylene Ždata not shown.. 3.4. Trichloroethylene-associated increases in serum immunoglobulin To determine if long-term treatment with trichloroethylene in the drinking water induced increases in serum immunoglobulin levels, serum from individual mice was analyzed by ELISA for total IgM and IgG. Treatment for 4 weeks with trichloroethylene at either 2.5 and 5.0 mgrml significantly increased serum levels of IgM Ž956 " 123 and 905 " 103 mgrml, respectively. when compared to age matched controls Ž559 " 66 mgrml. ŽFig. 3.. This trichloroethylene-induced increase in serum IgM was still observed at 8 weeks in mice treated with 5 mgrml trichloroethylene. However, by 22 weeks all three groups had similar elevated levels of serum IgM. Treatment for 4 weeks with trichloroethylene at either 2.5 or 5.0 mgrml increased serum IgG levels greater than 200% compared to controls and at later time points there was no significant differences between groups ŽFig. 4.. 3.5. Trichloroethylene associated alterations in CD4 q Upregulation of membrane CD44 expression ŽBudd et al., 1987. and downregulation of CD45RB expression ŽErnst et al., 1990. on CD4q T cells has been used to monitor the transition from naive to effector state. To determine if trichloroethylene affected the activation state of T cells, splenic CD4q T and lymph node cells from mice treated with

Fig. 1. Detection of trichloroethylene protein-adducts in the liver and lung. Crude cellular fractions of pooled liver and lung homogenate from control and treated MRL q rq mice exposed to trichloroethylene for 4, 8 and 22 weeks Ž100 mg per lane. were separated by SDS-PAGE under reducing conditions. Following transfer of cytosolic, pellet and microsomal protein to nitrocellulose, the presence of trichloroethylene-modified protein was detected with an affinity-purified anti-dichloroacetyl serum followed by a peroxidase detection system. For each series of blots, lane numbers 1, 4 and 7 represent control group. Lane numbers 2, 5 and 8 represent 2.5 mgrml trichloroethylene treatment group. Lane numbers 3, 6 and 9 represent 5.0 mgrml trichloroethylene treatment group.

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Fig. 2. Serum levels of ANA following trichloroethylene treatment in MRLqrq mice. ANA in the serum from individual mice were determined and quantified using Hep-2 cell slides. Wells were coated with serum from individual animals and the presence of ANA was determined via avidin–biotin peroxidase detection system and visualized with diaminobenzidine. Mice were treated with vehicle ŽI., 2.5 mgrml Ž9. or 5.0 mgrml ŽB. trichloroethylene. To quantify changes in ANA levels, spectrophotometric image analysis was performed on each sample and normalized to a positive control sample on each slide. Results from treated groups were compared to controls as relative intensity per cell. The data is presented as means"S.E.M. and statistical differences ŽU . were determined by analysis of variance with pF 0.05. The Ž. represents the number of animals per group.

trichloroethylene for 4 weeks were analyzed for expression of CD44 and CD45RB ŽFig. 5.. There was an overall increase in the mean fluorescence intensity of CD44 expressed on all CD4q T cells from treated groups when compared to controls. The percentage of splenic CD44 hi –CD4q T cells increased in a dose-dependent manner following trichloroethylene treatment indicating T cell activation ŽFig. 5.. There was a 220% and 262% increase in of CD4q T cells that exhibited a CD44 hi phenotype in mice treated with 2.5 and 5.0 mgrml trichloroethylene, respectively, when compared to controls. Also, the percentage of splenic CD4q T cells expressing a CD45RB lo phenotype, a second indicator of T cell activation, in mice treated with 2.5 or 5.0 mgrml trichloroethylene increased 147% and 131%, respectively, when compared to controls. Interestingly, the trichloroethylene-induced increase in CD44 expression in the spleen was also observed on non-CD4q

cells Ždata not shown.. Other markers, including B220, CD3, CD62L, CD40L, CD25 and CD54 ŽICAM. were measured at various time points and no major changes were detected with trichloroethylene treatment Ždata not shown.. Unlike spleen cells, lymph node cells from trichloroethylene-treated mice did not exhibit increased expression of CD44 Ždata not shown.. In view of the dramatic upregulation in CD44 levels observed on splenic CD4q T cells from mice treated for 4 weeks with trichloroethylene, expression of CD44 on CD4q T cells was monitored throughout the course of the experiment. It was found that when compared to CD4q T cells from control mice, CD4q T cells from trichloroethylenetreated mice expressed more CD44 at 4 weeks, comparable levels of CD44 at 8 weeks and less CD44 at 22 weeks ŽFig. 6.. In addition to CD4q T cells, cells expressing CD45RrB220, a pan-B cell marker, were also examined. MHC class II did not increase on the CD45RrB220q cells, and there was no evidence of the expansion of the CD3qrB220q population of T cells that is characteristic of MRL lpr r lpr mice ŽHahn, 1997. Ždata not shown..

Fig. 3. Serum levels of total IgM following trichloroethylene treatment in MRLqrq mice. Total serum IgM from individual mice was determined by sandwich ELISA, using IgM-specific capture and detecting antibodies. Absorbance was measured spectrophotometrically at 410 nm. Mice were treated with vehicle ŽI., 2.5 mgrml Ž9. or 5.0 mgrml ŽB. trichloroethylene. The data is presented as means"S.E.M. and statistical significance ŽU . was determined by ANOVA with pF 0.05. The Ž. represents the number of animals per group.

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3.6. Cytokine profile analysis

Fig. 4. Serum levels of total IgG following trichloroethylene treatment in MRLqrq mice. Total serum IgM from individual mice was determined by sandwich ELISA, using IgG-specific capture and detecting antibodies. Absorbance was measured spectrophotometrically at 410 nm. Mice were treated with vehicle ŽI., 2.5 mgrml Ž9. or 5.0 mgrml ŽB. trichloroethylene. The data is presented as means"S.E.M. and statistical significance ŽU . was determined by ANOVA with pF 0.05. The Ž. represents the number of animals per group.

The profile of cytokines secreted by activated CD4q T cells is often measured as an indicator of the type of immune response that is induced by a given stimulus. This profile can be used to predict whether the stimulus promotes a T-helper type 1 ŽTh 1 . type response that reflects macrophage activation and inflammation or promotes a T-helper type 2 ŽTh 2 . type response that most likely leads to antibody production. To measure the type of immune response initiated by trichloroethylene exposure, cytokine levels from activated CD4q T cells were measured using capture ELISA techniques. It must be noted that the enrichment process from total spleen cells leads to a mixed population of primarily CD4q T cells with some CD8q T cells. The cytokine profile secreted by activated T cells from mice treated with the low dose of trichloroethylene for 4 weeks was consistent with a Th 1 type response, with an increase in IFN-g Ž121 " 0.05 ngrml. com-

Fig. 5. Expression of CD44 and CD45RB by CD4q T cells from mice treated for 4 weeks with trichloroethylene. Single cell suspensions of splenocytes from MRLq rq mice treated for 4 weeks with 2.5 or 5.0 mgrml trichloroethylene or vehicle alone were stained with PE-anti-CD4 mAb and biotinylated-anti-CD44 and CD45RB followed by FI–streptavidin. Data is presented as FI-histograms representing the gated sub-population of CD4q T cells. The percent of CD4q T cells expressing a CD44 hi or CD45RB lo phenotype are identified for each treatment group. The unshaded area represents Ig isotype control.

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Fig. 6. Comparison of CD44 expression by CD4q T cells from mice treated for 4, 8 or 22 weeks with trichloroethylene. Spleen cells from the control group Žthin line., or the group treated with 2.5 mgrml trichloroethylene Žbold line. for 4, 8 or 22 weeks were double stained with PE-anti-CD4 and FI–anti-CD44 mAb and analyzed by flow cytometry. Shown are the FI histograms for CD4q cells. The results generated using CD4q T cells from the 5.0 mgrml trichloroethylene dose were similar to those treated with 2.5 mgrml trichloroethylene dose and are not presented. The shaded area represents Ig isotype control.

pared to activated T cells from control mice Ž65.5 " 0.12 ngrml. and a decrease in IL-4 Ž658 " 0.03 pgrml. when compared to controls Ž1725 " 0.03 pgrml. ŽFig. 7.. The cytokine levels from the high dose treatment group were not measured at this time

point because the T cells from mice exposed to the high dose of trichloroethylene had apparently died following anti-CD3 stimulation. At 8 weeks, the levels of IFN-g and the IL-4 secreted by cells from the treated groups were similar to control levels.

Fig. 7. Cytokine levels from anti-CD3 activated CD4q T cells from MRLq rq mice treated with trichloroethylene. Spleen cells from control mice and mice treated with vehicle ŽI., 2.5 mgrml Ž9. or 5.0 mgrml ŽB. trichloroethylene were isolated, grouped and enriched by negative selection for CD4q T cells. CD4q T cells were stimulated and the supernatants were tested for IFN-g or IL-4 with a sandwich ELISA using monoclonal antibodies specific for IFN-g and IL-4 and an alkaline phosphatase detection antibody. Absorbance was measured spectrophotometrically at 410 nm. Data presented represents mean " standard deviation of triplicate absorbance readings taken for each treatment group and statistical differences ŽU . were determined by ANOVA with p F 0.05.

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There was a substantial decrease in IL-4 secretion by T cells in the control group at 8 weeks when compared to the control levels at 4 weeks. Long-term treatment with trichloroethylene appears to suppress the capacity of the T cells to secrete either IFN-g or IL-4, and is consistent with the decreased expression of CD44 measured on T cells in these mice ŽFig. 6.. 3.7. Serum antibodies specific for trichloroethylene in MRL q rq mice The ability of trichloroethylene to induce a specific immune response directed against proteins modified by a reactive metabolite was measured using an ELISA. RSA and RSA-DCAA in equal concentration were used as solid phase antigens to determine serum antibodies specific for dichloroacetylated-adducts. The difference between absorbance of RSADCAA and RSA can be attributed to specific antibodies against the chemical hapten. MRL q rq mice treated for 22 weeks with trichloroethylene were

Fig. 8. Serum antibodies specific for trichloroethylene in MRLq rq mice. Specific anti-dichloroacetyl antibody response was determined in individual animals by measuring the difference between reactivity towards RSA and DCAA-RSA in an ELISA. The presence of antibody binding was visualized via an alkaline phosphatase detection system with absorbance measured at 410 nm. The difference represents specific antibodies against the dichloroacetyl group. The data are plotted as the anti-dichloroacetyl response for individual samples with average and S.E.M. also plotted and statistical significance ŽU . was determined by ANOVA with pF 0.05. The line on the graph represent q2 standard deviations from the mean of the controls.

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found to have serum antibodies that recognized the chemical bound to protein ŽFig. 8.. The level of antibodies specific for dichloroacetylated protein-adducts in mice treated with 2.5 mgrml of trichloroethylene were significantly Ž p F 0.05. increased when compared to controls and three mice treated with trichloroethylene had anti-chemical antibody levels 2 standard deviations above controls. Linear regression analysis revealed a significant association between trichloroethylene treatment and the presence of serum antibodies that recognize dichloroacetylated proteins with a p F 0.05 Ždata not shown..

4. Discussion The etiology of autoimmune disease, like SLE and systemic sclerosis, is believed to be associated with both genetic and environmental components. An environmental component of autoimmunity may be exposures to infectious agents or exposure to environmental chemicals like the chlorinated ethylene, trichloroethylene. Trichloroethylene is one of the most abundant environmental contaminants. An association exists between trichloroethylene exposure and the development of an autoimmune response andror disease in not only humans ŽKilburn and Warshaw, 1992; Clark et al., 1994; Nietert et al., 1998. but also in the autoimmune prone MRL q rq mice ŽKhan et al., 1995.. Khan et al. ŽKhan et al., 1995. observed an increase in antinuclear autoantibodies and total serum IgG in MRL q rq mice treated with trichloroethylene or its reactive metabolite dichloroacetyl chloride. This model takes into account the genetic component as well as the environmental component apparently required for the development of autoimmunity. The significant increases in ANA and total serum immunoglobulins following trichloroethylene treatment in this study confirm work by Ansari and coworkers that trichloroethylene exacerbates an autoimmune response in MRL q rq mice ŽKhan et al., 1995.. The route of trichloroethylene administration used in the present study, via drinking water, may better reflect environmental exposure compared

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to i.p. injection used by Khan et al. The increase in ANA seen at 6 and 8 weeks with trichloroethylene treatment that precedes the overall increases in control and treated levels of ANA seen at 22 weeks indicates that trichloroethylene treatment is accelerating the inherent autoimmune response in MRL q rq mice. The early increases in serum IgG and IgM found in our study also indicate that activation of the immune system is occurring in association with trichloroethylene treatment. The mechanism by which trichloroethylene accelerates an autoimmune response is not known but may involve activation of CD4q T cells. The MRL q rq mice used in this experiment were developed in conjunction with the MRL lprrlpr strain by Murphy and Roths ŽAndrews et al., 1978.. The MRL lpr r lpr mice develop a spontaneous lupus-like autoimmune disease early in life largely mediated by the lpr mutation of the Fas apoptosis gene ŽWatanabeFukunaga et al., 1992.. In contrast, the MRLq rq mice are often used as negative controls for experiments involving the MRL lpr r lpr strain, since the MRL q rq mice, although autoimmune prone, do not usually develop lupus-like symptoms until late in life and do not have a defect in Fas-mediated apoptosis ŽWatanabe-Fukunaga et al., 1992.. In MRL lprr lpr mice, there are increases in the percentage of CD4q T cells which express high levels of CD44 Žalso referred to as Pgp-1. and these increases may accompany the autoimmune syndrome seen in this strain ŽBudd et al., 1991.. The increased expression of CD44 in the present study by the splenic CD4q T cells in MRL q rq mice treated for 4 weeks with trichloroethylene may be similarly related to the development of an autoimmune response. The dominant T cell in the MRL lpr r lpr strain express the surface phenotype CD3q, CD4y Žor weakly q., CD8y and B220q ŽHahn, 1997.. This CD3qrB220q subset of T cells is thought to provide the T cell help necessary for the high levels of serum immunoglobulins and ANA level characteristic of the lpr strain. In the present study, there was no evidence of an increase in the CD3qrB220q T cell subset in the MRL q rq strain following trichloroethylene treatment. This suggests that the apparent acceleration of autoimmunity by trichloroethylene in the MRL q rq strain is not related to a lymphoproliferation of the CD3qrB220q

T cell subset that is the case in the MRL lpr r lpr strain. In addition to an increase in expressed CD44 on T cells, autoimmune disease in the MRL lpr r lpr mice is also accompanied by the activation of CD4q T cells secreting a Th 1 type cytokine profile ŽTakahashi et al., 1996.. This type of Th 1 response is marked by an increased secretion of IFN-g with a simultaneous decrease in the secretion of the cytokine IL-4. The importance of IFN-g in the development of lupus in MRL lpr r lpr mice is underlined by the finding that disease development is severely diminished in lpr mice in which the genes for IFN-g or IFN-g receptor have been deleted ŽHaas et al., 1997; Balomenos et al., 1998.. The type of cytokine profile measured at 4 weeks with 2.5 mgrml trichloroethylene treatment in the present study was consistent with a Th 1 type response. Similar to the increase in CD44 expression, IFN-g production by the T cells from mice treated with trichloroethylene for 22 weeks was diminished when compared to controls. Collectively, the results show that treatment with trichloroethylene accelerates an initial activation of IFN-g-secreting CD4 q T cells. As treatment continues, this increase in activation is lost. This may be due to a compensatory mechanism or may be a factor in the progression of autoimmunity in the control mice. Although the metabolic activation of trichloroethylene to a reactive intermediate is considered a prerequisite for toxicity to occur ŽBruckner et al., 1989., the requirement of activation of trichloroethylene for the development of autoimmunity has not yet been established. We have developed a method by which metabolic activation of trichloroethylene can be measured using an antibody that recognizes cellular protein covalently modified by a reactive metabolite of trichloroethylene. We have previously shown in rats ŽHalmes et al., 1997a., mice ŽHalmes et al., 1996. and human hepatocytes ŽGriffin et al., 1998. that trichloroethylene treatment leads to the production of one or more covalently modified hepatic proteins, and that these modified proteins can be detected in the serum of rats ŽHalmes et al., 1997a.. In the present study, using the antibody specific for trichloroethylene-modified proteins, we showed that long-term oral exposure to trichloroethylene in MRL q rq mice led to the formation of several protein

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adducts in the liver and lung. These adducts increased as a function of dose and duration of exposure. Several of these adducts were similar in molecular weight to our previously reported adducts, particularly the 50-kDa adduct ŽHalmes et al., 1996; Halmes et al., 1997a.. Although the pathogenic role of protein adducts formed following trichloroethylene exposure is not known, it is possible that the modified self-proteins may be seen by the immune system as foreign, thereby, leading to an autoimmune response. Along these lines, it has been reported that the primary target for trichloroethylene is cytochrome P450 2E1 ŽHalmes et al., 1997b., and autoantibodies directed against cytochrome P450s have been identified in several autoimmune diseases including drug-induced hepatitis ŽBeaune et al., 1994; Bourdi et al., 1996.. Also, the presence of a specific immune response directed against cellular proteins modified by chemicals has been demonstrated in the case of halothane hepatitis ŽBird and Williams, 1989; Martin et al., 1990; Pohl, 1990; Knight et al., 1994.. Until now, there has been no reported evidence of a hapten-specific immune response in mice treated with trichloroethylene. Khan et al. found that MRLq rq mice exposed to dichloroacetyl chloride, a metabolite of trichloroethylene, developed dichloroacetyl chloride-specific antibodies, but they did not, however, find trichloroethylene-specific antibodies in MRL q rq mice exposed to trichloroethylene ŽKhan et al., 1995.. In the present studies, MRL q rq mice treated with 2.5 mgrml trichloroethylene for 22 weeks were found to have significantly higher levels of serum antibodies that recognize dichloroacetylated proteinŽs. when compared to control mice. There were three treated animals with antibody levels at least 2 standard deviations above the controls, and a significant correlation between the dose of trichloroethylene and serum antibody levels. It is possible that the differences between our study and Khan’s study may be due to the length of the exposure to trichloroethylene, or due to the different route of administration. The data presented here, indicating a lack of hepatic or renal toxicity with the doses of trichloroethylene used in this experiment, is consistent with previously reported data ŽTucker et al., 1982.. This finding suggests that the immunomodulation reported with trichloroethylene treatment in

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the present study is not due to any direct toxicity in the liver or kidney. Our findings showed no splenomegaly following exposure, whereas Kahn et al. found a significant increase in spleen weight and spleen to body weight ratio following 6 weeks of treatment ŽKhan et al., 1995.. However, since the route of administration of trichloroethylene was i.p. in the Khan study, not oral as was used in this experiment, the difference in the route of administration may account for the discrepancies. Based on the results, we hypothesize that the acceleration of an autoimmune response in MRL q rq mice exposed to trichloroethylene may be associated with an early activation of CD4q T cells. These activated T cells secrete a Th 1-like cytokine profile capable of inducing an inflammatory response. Perhaps in association with this initial inflammatory cytokine profile, high levels of ANA are induced, and eventually anti-hapten specific antibodies are developed. With long-term treatment, trichloroethylene appears to lead to a hyporesponsiveness of CD4q T cells with an immunosuppressive action on the cytokines secreted, and the expression of T cell activation markers. The mechanism by which CD4q T cells become activated is likely a critical step in the trichloroethylene-accelerated autoimmune response. Further study is needed to determine the mechanisms of this CD4q T cell activation, and also to determine if the covalent modification of cellular proteins in the liver and lung by trichloroethylene may play a role in the acceleration of autoimmunity.

Acknowledgements This work was supported in part by the United States Environmental Protection Agency ŽR82640901-0. and the United States Department of Energy ŽDE-FG01-92EW50625..

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