CD4+ T-Cell Activation and Induction of Autoimmune Hepatitis following Trichloroethylene Treatment in MRL+/+ Mice

Joseph M. Griffin*, Kathleen M. Gilbert{dagger}, Laura W. Lamps{ddagger} and Neil R. Pumford*,1

* Department of Pharmacology and Toxicology, {dagger} Department of Microbiology and Immunology, and {ddagger} Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205; and § Division of Agriculture, University of Arkansas, Fayetteville, Arkansas 72701

Received April 24, 2000; accepted June 29, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure to relatively high levels of trichloroethylene has recently been shown to accelerate the development of an autoimmune response in the autoimmune prone MRL+/+ mice. The trichloroethylene-induced autoimmune response was associated with an increase in activated CD4+ T cells, producing Th1-like cytokines. The present study was conducted to determine whether lower, more occupationally relevant doses of trichloroethylene could also promote autoimmunity, in MRL+/+ mice, and if so, to investigate the mechanism of this accelerated autoimmune response. In addition, histological studies were performed to determine if trichloroethylene was capable of producing pathological markers consistent with an autoimmune disease. Trichloroethylene was administered to mice in the drinking water at 0, 0.1, 0.5, and 2.5 mg/ml for 4 and 32 weeks. There was a significant increase above controls in serum antinuclear antibody (ANA) levels following 4 weeks of both 0.1 and 0.5 mg/kg/day of trichloroethylene. After 32 weeks of treatment, ANA levels were elevated and equal in all groups. The kinetics of the ANA response indicated that trichloroethylene accelerated the innate autoimmune response in the MRL+/+ mice. There was a dose-related increase in the percentage of activated CD4+ T cells in both the spleens and lymph nodes of mice treated for 32 weeks with trichloroethylene when compared to controls. CD4+ T cells isolated from MRL+/+ mice after either 4 or 32 weeks of treatment with trichloroethylene secreted inflammatory or Th1-like cytokines. Following 32 weeks of trichloroethylene treatment, there was a significant increase in hepatic mononuclear infiltration localized to the portal region, a type of hepatic infiltration consistent with autoimmune hepatitis. Taken collectively, these data suggest that exposure to occupationally relevant concentrations of trichloroethylene can accelerate an autoimmune response and can lead to autoimmune disease. The mechanism of this autoimmunity appears to involve, at least in part, activated CD4+ T cells that then produced inflammatory cytokines.

Key Words: antinuclear antibodies (ANA); inflammatory cytokines; dose-related response; hepatic mononuclear infiltration; scleroderma; systemic lupus erythematosus.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Several xenobiotics have been implicated in the development of autoimmune diseases such as systemic lupus erythematosus and systemic sclerosis. One such xenobiotic, trichloroethylene, is an industrial organic solvent most often used for cleaning and degreasing of fabricated metal parts. Approximately 3.5 million persons are occupationally exposed to trichloroethylene in the United States (ATSDR, 1994Go, 1999Go). Trichloroethylene is one of the most ubiquitous chemicals found in the environment and was present in over 355 National Priorities List (NPL) sites (ATSDR, 1999Go). Occupational exposure is primarily due to evaporation from degreasing operations (ATSDR, 1994Go, 1999Go). Occupational exposure to trichloroethylene has recently been linked to the development of scleroderma (Nietert et al., 1998Go). In addition, environmental exposure to trichloroethylene has been associated with systemic lupus erythematosus (Clark et al., 1994Go; Kilburn and Warshaw, 1992Go), systemic sclerosis (Flindt-Hansen and Isager, 1987Go; Lockey et al., 1987Go; Yanez Diaz et al., 1992Go) and fasciitis (Waller et al., 1994Go).

A model for studying the effects of trichloroethylene on the development of autoimmunity has been developed using lupus-prone MRL+/+ mice (Khan et al., 1995Go). Using this model, we have recently shown that trichloroethylene accelerates autoimmunity in MRL+/+ mice in association with the activation of CD4+ T cells, which express a Th1 cytokine profile (Griffin et al., 2000aGo). We have also shown that metabolic activation of trichloroethylene by cytochrome P450 2E1 is important for the activation of CD4+ T cells in the MRL+/+ mouse model (Griffin et al., 2000bGo).

The American Conference for Governmental Industrial Hygienists recommends a threshold limit value of 269 mg/m3 or a time-weighted average of 50 ppm for trichloroethylene (ATSDR, 1994Go). This converts to a concentration of approximately 40 mg/kg/day, assuming a moderately active work environment. The concentrations of trichloroethylene used in the present study of 0, 0.1, 0.5, and 2.5 mg/ml calculate to 0, 21, 100, and 400 mg/kg/day, respectively. This calculation is assuming nearly complete absorption, which has been previously shown to be the case in oral administration of trichloroethylene, with absorption of 93–98% (Goeptar et al., 1995Go). The concentrations of trichloroethylene, previously reported to cause an accelerated autoimmune response in MRL+/+ mice between 325 mg/kg/day to over 750 mg/kg/day (Griffin et al., 2000aGo; Khan et al., 1995Go), were considerably higher than those used in the present study.

In the present study, we treat MRL+/+ mice with lower, more occupationally relevant, levels of trichloroethylene in order to further examine the effects and mechanisms of this xenobiotic on the development of autoimmunity, and to investigate whether trichloroethylene is capable of inducing an autoimmune disease as well as an autoimmune response.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Treatments
Four-week-old female MRL+/+ mice were purchased from The Jackson Laboratories (Bar Harbor, ME). Trichloroethylene (purity 99+%) was purchased from Aldrich Chemical Co., Inc. (Milwaukee, WI) and suspended in drinking water with 1% of the emulsifier, Alkamuls EL-620, from Rhone-Poulenc (Cranbury, NJ). The mice received trichloroethylene in the drinking water at 0, 0.1, 0.5, or 2.5 mg/ml. These doses calculated to an average daily dose of 21, 100, and 400 mg/kg, respectively. The doses were selected based on a previous dosing regimen with similar average daily doses (Tucker et al., 1982Go). The water was changed every 2 to 3 days to ensure maintenance of the dose, as previously described (Tucker et al., 1982Go). The mice were weighed once a week to monitor weight changes. Eight animals per treatment group were sacrificed at either 4 or 32 weeks of trichloroethylene exposure.

Serological Tests
The mice were anesthetized with CO2, and blood was collected from the retro-orbital plexus at each sacrifice. Blood was allowed to clot at room temperature for 1 h, then centrifuged at 1,000 x g for 30 min, and serum was collected. Serum was evaluated for antinuclear antibodies (ANA) using a screening enzyme-immunoassay (EIA) similar to the screening assay used by Homburger et al. (1998) and confirmed using immunohistochemical staining of Hep-2 cells (Dako, Carpinteria, CA) following manufacturer's instructions. Briefly, nucleated Hep-2 cells were added to 96-well plates at a concentration of 3.5 x 104 cells/well in EMEM supplemented with 25 mM L-glutamine, 10% FCS, and 1% penicillin/streptomycin (Biowhittaker, Walkersville, MD) and grown to confluence. The cells were fixed with Bouin's fixative and washed with phosphate-buffered saline (PBS)-0.05% Tween 20 (Fisher Scientific, Pittsburgh, PA). Non-specific binding was blocked with 3% bovine serum albumin (BSA)-PBS for 2 h. The plates were washed, and diluted serum (1:50) was incubated in duplicate wells overnight at 4°C. The presence of ANA was determined with an alkaline phosphatase-labeled anti-mouse IgG detection system, using p-nitrophenylphosphate as the substrate, with absorbance measured at 410 nm. Nuclear staining was determined using immunohistochemical staining of Hep-2 cells (Dako) following standard methods, according to manufacturer's instructions.

Serum levels of alanine aminotransferase and blood urea nitrogen were measured according to the manufacturer's instructions, using kits from Sigma Diagnostic Inc. (St. Louis, MO).

Phenotypic analysis of splenic and lymph node cells.
Equal numbers of spleen or mesenteric lymph node cells from individual mice were isolated in RPMI 1640 (Gibco BRL, Grand Island, NY) and pooled according to group. Because of the relatively large number of cell groups to be tested and the number of molecules examined, it was not practical to examine the staining pattern of individual mice. The cells were stained with either biotinylated anti-CD44 (clone 1M7, rat IgG2b; Pharmingen, La Jolla, CA), biotinylated anti-CD45RB (clone 16A, rat IgG2a; Pharmingen, La Jolla, CA) or biotinylated anti-CD54/ICAM-1 (clone 3E2, hamster IgG, Pharmingen; La Jolla, CA) for 30 min on ice, followed by flourescein isothiocyanate (FI)-streptavidin and either phycoerytherin (PE)-anti-CD4 (clone GK1.5, rat IgG2b; Pharmingen, La Jolla, CA) or PE-anti-CD8 antibody (clone 53–6.7, rat IgG2a; Pharmingen, La Jolla, CA). The phenotypic analysis of 10,000 events per group was carried out using a FACScan (Becton-Dickinson; Mountain View, CA), and the data is presented in a table or as histograms of CD4+ cells. Non-viable cells, based on low-forward scatter and side scatter, were excluded in each sample. Data analysis was performed with the use of WinMDI software (kindly provided by Joe Trotter, The Scripps Research Institute, La Jolla, CA). For all groups tested, staining with isotype Ig controls was also examined.

Cytokine profile analysis.
Splenic CD4+ T cells were isolated using anti-mouse CD4 magnetic beads following manufacturer's insert (Dynal, Lake Success, NY). The purity of CD4+ T cells was determined by flow cytometry to be >= 95%. Following purification, CD4+ T cells (2 x 105/well) were incubated for 72 h at 37°C in a 96-well plate containing immobilized anti-CD3 mAb [(Hamster IgG1 clone 145–2C11; Pharmingen, La Jolla, CA) 50 µl of 10 ng/ml for 24 h at 20°C] and soluble anti-CD28 mAb [(Hamster IgG1 clone 37.51; Pharmingen, La Jolla, CA) 10 µg/ml]. After 72 h, the supernatants were removed and tested in an enzyme-linked immunosorbant assay (ELISA) for interleukin-4 (IL-4) and interferon-gamma (IFN-{gamma}). Cytokine concentrations in the ELISAs were determined by comparison to a standard curve generated using mouse recombinant IL-4 and IFN-{gamma} (R & D Systems, Minneapolis, MN).

Histopathology
Liver, lung, kidney, and skin were collected, fixed in 10% neutral-buffered formalin (Fisher, St. Louis, MO) for 24 h, and processed for sectioning. Tissue sections were cut at 4 µm and stained with hematoxylin and eosin (H&E) or Gomori's trichrome stain. The kidneys were examined for the presence or absence of fibrosis, inflammatory infiltrates, vascular lesions, and glomerular changes. The livers were examined by a hepatic pathologist for histological parameters, including portal and lobular inflammation, reactive hepatocellular changes, apoptotic hepatocytes, and the presence of necrosis, fibrosis, bile duct damage, and granulomas. The lungs and skin were primarily examined for the presence of fibrosis. The histological changes in the livers were numerically scored (from 0–3+, ranging from no change to severe, respectively), and statistically analyzed using Wilcoxon's rank-order analysis. Scores of 0–1, 1–2, or 2–3, were assigned the numbers 0.5, 1.5, and 2.5, respectively, for the Wilcoxon's rank-order analysis.

T-Cell Staining
Formalin-fixed, paraffin-embedded liver sections (4 µm) were stained for the presence of T cells, using a polyclonal rabbit anti-CD3 antibody (Code No. A0452) kit from Dako (Carpinteria, CA) as directed in the manufacturer's insert. Antigen retrieval was performed with Dako proteinase K. Positive staining was detected with the Dako LSAB system. Mouse tonsil and lymph node served as positive controls. T-cell infiltration was numerically scored (from 0–3+, ranging from no change to severe, respectively).

Trichloroethylene-Protein Adduct Staining
Liver sections were immunochemically stained for protein adducts using antiserum that was previously shown to recognize cellular macromolecules covalently modified by a reactive metabolite of trichloroethylene (Halmes et al., 1996Go). Briefly, following depariffinization and rehydration, antigen retrieval was performed using a 0.1% trypsin solution for 30 min at room temperature. The sections were blocked with Dako Protein Block (Dako, Carpinteria, CA), stained with anti-dichloroacetyl antiserum, and positive staining detected with the DAKO LSAB system. The sections were counter-stained with Gills hematoxylin (Fisher Scientific, Pittsburgh, PA).

Statistical Analysis
Statistical significance was determined when appropriate, using one-way analysis of variance with a predetermined significance level of p < 0.05. To determine statistical differences between groups, the Student-Neuman-keuls post-hoc test was used.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Trichloroethylene Accelerates an Increase in Antinuclear Antibodies
In order to determine whether the low doses of trichloroethylene used in the present study promoted autoimmunity, individual serum samples from control and treated MRL+/+ mice were tested for the presence of ANA (a marker for systemic autoimmune disease (Tan, 1982Go)). There was a dose-related and significant increase in serum ANA in mice treated with 0.1 and 0.5 mg/ml trichloroethylene for 4 weeks, compared to control mice treated with saline for 4 weeks (Fig. 1Go). The ANA levels in mice treated with 2.5 mg/ml trichloroethylene were also higher than controls, albeit not significantly. The lack of significance may in part be due to competing toxicity at the higher dose. After 32 weeks, there was no difference in serum ANA levels, with all treatment groups exhibiting similar elevated levels of ANA when compared to groups at 4 weeks (Fig. 1Go). Using Hep-2 cell slides, immunohistochemical analysis of serum samples was performed to visualize the nuclear staining pattern of the ANA. Serum collected following 4 weeks of trichloroethylene treatment induced a nucleolar pattern within the nucleus of the Hep-2 cells, while control serum induced a lighter, more homogeneous staining. A homogeneous nuclear staining pattern was induced by serum collected after 32 weeks from either trichloroethylene-treated or control mice (data not shown).



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FIG. 1. MRL+/+ Mice treated for 4 weeks with trichloroethylene exhibited increased serum levels of ANA. ANA were determined and quantified using nucleated Hep-2 cells grown to confluence in 96-well plates and fixed. Serum (1:50) from control or trichloroethylene-treated mice was incubated in duplicate wells, and the presence of ANA was determined using an alkaline phosphatase-labeled goat anti-mouse IgG detection system. The data is presented as means ± standard error of the mean. Statistical differences (*) were determined by analysis of variance with p < 0.05.

 
Trichloroethylene Exposure Activates CD4+ T Cells
Cell surface expression of CD44, CD45RB, and CD54/ICAM-1 on lymph node and splenic CD4+ T cells was measured as an indication of T-cell activation in trichloroethylene-treated MRL+/+ mice. Four weeks of treatment with trichloroethylene did not induce any changes in T-cell expression of CD44, CD45RB, and CD54 (data not shown). In contrast, trichloroethylene treatment for 32 weeks induced a dose-related increase in the number of lymph node CD4+ T cells that expressed a high level of CD44 (CD44hi) (Fig. 2Go). There were 11, 61, and 156% increases in lymph node CD4+ T cells that expressed a CD44hi phenotype in the 0.1, 0.5, and 2.5 mg/ml trichloroethylene treatment groups, respectively, when compared to lymph node CD4+ T cells from controls (Fig. 2Go, Table 1Go). There was a corresponding increase in the percentage of lymph node CD4+ T cells that expressed a low level of CD45RB (CD45RBlo) (Table 1Go).



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FIG. 2. Trichloroethylene treatment of MRL+/+ mice increased the number of lymph node CD4+ T cells with a memory/activated phenotype. Lymph node cells from MRL+/+ mice treated for 32 weeks with different concentrations of trichloroethylene in the drinking water were double stained with PE-anti-CD4 mAb, and either FI-anti-CD44 mAb or FI-anti-CD45RB mAb, and analyzed by flow cytometry. Shown are the FI histograms of the PE+ cells. The open histograms represent the PE+ cells stained with FL-labeled control Ig.

 

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TABLE 1 Expression of Activation Markers on CD4+ T Cells from MRL+/+ Mice Treated for 32 Weeks with Trichloroethylene
 
Upregulation of CD44 and downregulation of CD45RB are both indicators of CD4+ T-cell activation (Chu et al., 1994Go). Trichloroethylene exposure also induced a similar pattern of CD44 and CD45RB expression in splenic CD4+ T cells following 32 weeks of treatment (Table 1Go). An additional indicator of CD4+ T-cell activation is upregulation of the intercellular adhesion molecule CD54 (ICAM)-1 (Springer, 1990Go). Trichloroethylene treatment induced a dose-dependent increase in the percentage of lymph node and splenic CD4+ T cells that express a CD54hi phenotype when compared to CD4+ T cells isolated from controls (Table 1Go). In contrast to CD4+ T cells, the percentage of CD8+ T cells that expressed an activated/memory phenotype was increased very little in either the lymph nodes or spleens of trichloroethylene-treated MRL+/+ mice (data not shown). Taken together, the phenotypic analysis revealed that long-term treatment with low doses of trichloroethylene increased the percentage of activated CD4+ T cells, but not the CD8+ T cells, in the spleens and lymph nodes.

CD4+ T Cells from Trichloroethylene-Treated Mice Secreted Increased Levels of IFN-{gamma}
Because IFN-{gamma}-secreting CD4+ T cells are involved in the development of autoimmune disease pathology in the genetically similar MRLlpr/lpr strain (Balomenos et al., 1998Go; Haas et al., 1997Go; Takahashi et al., 1996Go), the profile of cytokines secreted by CD4+ T cells from trichloroethylene-treated mice was measured. Culture supernatants from splenic CD4+ T cells stimulated in vitro were tested in an ELISA for the presence of IFN-{gamma} and IL-4. There was a dose-responsive and significant increase in the levels of IFN-{gamma} secreted by CD4+ T cells isolated from MRL+/+ mice treated for 4 weeks with 0.5 and 2.5 mg/ml trichloroethylene (1.29 ± 0.1 and 1.7 ± 0.14 µg/ml, respectively) when compared to controls (0.48 ± 0.1 µg/ml) (Fig. 3Go). The increased ability of CD4+ T cells from mice treated with 0.5 or 2.5 mg/ml trichloroethylene to secrete IFN-{gamma} was still observed after 32 weeks of treatment. In contrast to IFN-{gamma}, IL-4 production by CD4+ T cells was not increased when the mice were treated for either 4 or 32 weeks with trichloroethylene. These results are in accordance with our previous findings (Griffin et al., 2000aGo), and demonstrate that trichloroethylene-treatment promotes a Th1 cell-like immune response in the MRL+/+ mice.



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FIG. 3. IFN-{gamma} and IL-4 levels secreted by CD4+ T cells from MRL+/+ mice treated with trichloroethylene. CD4+ T cells isolated from the spleens of MRL+/+ mice treated for 4 or 32 weeks with trichloroethylene were stimulated with anti-CD3e mAb, and the culture supernatants were tested for the presence of IFN-{gamma} and IL-4 using ELISA's. The data is presented as means ± standard error of the mean and statistical differences (*) were determined by analysis of variance with p < 0.05.

 
Trichloroethylene Treatment Induced Histopathological Changes Similar to Autoimmune Hepatitis
Histopathological analyses of liver sections showed no histological alterations following 4 weeks of trichloroethylene treatment (data not shown). However, following 32 weeks of trichloroethylene treatment, histopathological analysis revealed extensive hepatic mononuclear cell infiltration. A representative liver section from a control and a treated animal is presented in Figure 4Go. Portal tracts and focal areas within the lobule contained a mixed inflammatory infiltrate consisting of plasma cells and lymphocytes. Staining with an antibody specific for CD3, a molecule found only on T cells, confirmed that the lymphocytes were a mixture of CD3+ and CD3 lymphocytes (data not shown). Based on histopathological scoring, the degree of mononuclear infiltration in mice treated with 0.5 and 2.5 mg/ml trichloroethylene was significantly greater than that found in control animals (Table 2Go). In addition, the livers from all the groups of mice treated with trichloroethylene contained significantly more hepatocyte reactive changes (defined as the presence of multinucleated hepatocytes, variations in hepatocyte nuclear morphology and hepatocytes in mitosis), when compared to livers from control mice (Table 2Go). Unlike the liver, the kidney, skin, and lung of trichloroethylene-treated mice revealed no histopathological changes by analysis of H&E and Gomori's trichrome-stained slides (data not shown).



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FIG. 4. Liver histopathology following treatment of MRL+/+ mice with trichloroethylene. Liver sections were fixed in 10% neutral buffered formalin and stained with H&E. Panel A is a representative liver section from a control mouse examined at 32 weeks and panel B is a representative liver section from a mouse treated with 2.5 mg/ml trichloroethylene for 32 weeks. The arrows indicate areas of intense mononuclear infiltration; magnification x 140. PV, portal vein; CV, central vein.

 

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TABLE 2 Liver Histopathological Scoring of H&E Liver Sections
 
Immunohistochemical Localization of Trichloroethylene-Protein Adducts
Metabolism of trichloroethylene is involved in CD4+ T-cell activation (Griffin et al., 2000bGo). To determine if metabolic activation of trichloroethylene was occurring, and the hepatic localization of the covalently modified protein adducts, immunohistochemical staining was performed using anti-dichloroacetyl antibody. Representative liver sections from a control mouse and a mouse treated with 2.5 mg/ml of trichloroethylene for 32 weeks are presented in Figure 5Go. No positive staining was apparent in the livers of control mice. In contrast, high levels of trichloroethylene-protein adducts were found in the liver of the trichloroethylene-treated mice. The anti-dichloroacetyl staining was most intense in hepatocytes of the centrilobular zone, with bridging staining to the portal zone, which contained very low levels of adducts.



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FIG. 5. Immunohistochemical analysis of liver tissue for the presence of trichloroethylene-protein adducts. Liver sections were developed with anti-dichloroacetyl antiserum followed by Dako LSAB detection system and the sections were counterstained with Gills hematoxylin (magnification x 170). Panel A is a representative liver section from a control mouse examined at 32 weeks, and panel B is a representative liver section from a mouse treated with 2.5 mg/ml trichloroethylene for 32 weeks. PV, portal vein; CV, central vein.

 
Trichloroethylene Treatment Induced No Overt Manifestations of Toxicity
Blood urea nitrogen levels were unaffected by trichloroethylene treatment throughout the 32-week period. However, there was a slight, but significant, increase in serum levels of alanine aminotransferase in animals treated with 0.5 mg/ml trichloroethylene for 32 weeks when compared to controls (38.2 ± 25.21 and 20.0 ± 8.04 IU/L, respectively). There was no trichloroethylene-induced increase in liver function tests at any other time or dose. Body, liver, and spleen weights revealed no significant physical changes associated with trichloroethylene treatment (data not shown).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The etiology of autoimmunity is believed to involve both genetic and environmental components. Infectious agents and industrial chemicals are environmental agents that may contribute to the development of autoimmune disease. One of the industrial chemicals often linked with the development of autoimmunity, in both humans and autoimmune-prone mice, is trichloroethylene (Clark et al., 1994Go; Goldman, 1996Go; Griffin et al., 2000aGo; Khan et al., 1995Go; Kilburn and Warshaw, 1992Go; Nietert et al., 1998Go).

Previously we have shown acceleration of an autoimmune response at high doses of trichloroethylene (Griffin et al., 2000aGo). The occupationally relevant doses used in the present study were shown to induce significant increases in ANA, indicating that trichloroethylene is capable of accelerating an autoimmune response, even at exposure levels relevant to humans.

The development of many spontaneous systemic autoimmune diseases appears to be dependent on CD4+ T cells (Adachi et al., 1998Go; Yagi et al., 1992Go). CD4+ T cells are also critical in the process of chemically-induced autoimmunity, especially in diseases associated with heavy metals (Goldman et al., 1991Go; Pelletier et al., 1988Go; Schuhmann et al., 1990Go; Stiller-Winkler et al., 1988Go). Along these lines, we recently reported that an accelerated autoimmune response in trichloroethylene-treated MRL+/+ mice was associated with an increase in activated CD4+ T cells expressing a Th1 cytokine profile (Griffin et al., 2000aGo). The increase in Th1 cytokine production by the CD4+ T cells appears to be dependent on the metabolism of trichloroethylene by CYP 2E1 to an active metabolite (Griffin et al., 2000bGo). Taken together, the results suggest that metabolites of trichloroethylene may activate CD4+ T cells to express Th1 cytokines, which may, in turn, promote the development of autoimmunity.

To determine if the CD4+ T lymphocytes in the MRL+/+ mice treated with low doses of trichloroethylene exhibited an activated phenotype, cell surface expression of CD44 and CD45RB was measured. The observation that splenic and lymph node CD4+ T cells, isolated from MRL+/+ mice treated for 32 weeks with trichloroethylene, exhibited increased expression of CD44 and decreased expression of CD45RB when compared to CD4+ T cells isolated from control animals, indicated the presence of CD4+ T-cell activation in the treated mice. In addition, trichloroethylene induced a dose-related increase in the percentage of CD4+ T cells that expressed a high level of CD54/ICAM-1, another indicator of T-cell activation (Springer, 1990Go). This apparent polyclonal expansion of activated/memory CD4+ T cells following trichloroethylene treatment mimics the activity of CD4+ T cells observed in the genetically similar MRLlpr/lpr mice. In MRLlpr/lpr mice, spontaneous autoimmune disease progression is accompanied by an increase in the percentage of CD4+ T cells expressing high levels of CD44 (Budd et al., 1991Go), and it has been suggested that these CD44hi T cells contribute to the lupus-like syndrome seen in this strain. An increase in lymphocyte CD44 expression has been shown to be a marker of autoimmune disease activity in other systems as well (Estess et al., 1998Go). The mechanism by which high levels of CD44 contribute to autoimmunity is not known, but CD44 has been shown to be important in T-cell extravasation into sites of inflammation (Estess et al., 1998Go).

In addition to an increased expression of CD44 on CD4+ T cells, spontaneous autoimmune disease in MRLlpr/lpr mice is associated with increased levels of Th1-like cytokines (Budd et al., 1991Go), which are thought to be critical for disease development (Takahashi et al., 1996Go). A Th1-like cytokine profile is characterized by an increase in secreted IFN-{gamma}, with a corresponding decrease or at least no increase in IL-4 secretion. As shown in the present study CD4+ T cells from MRL+/+ mice treated with low doses of trichloroethylene secreted significantly higher amounts of IFN-{gamma} than CD4+ T cells from control mice. The importance of IFN-{gamma} in promoting autoimmunity is emphasized by the finding that autoimmune disease development is severely diminished in MRLlpr/lpr mice in which the genes for IFN-{gamma} or IFN-{gamma} receptor have been deleted (Balomenos et al., 1998Go; Haas et al., 1997Go). Although the exact role of INF-{gamma} in the development of lupus remains to be delineated, it is thought that IFN-{gamma}-induced upregulation of MHC class II molecules and ICAM-1 promotes lupus nephritis in MRLlpr/lpr mice by augmenting the interaction of T-cell interaction with renal tissue (Fan and Wuthrich, 1997Go; Wuthrich et al., 1990Go). Taken together, the results suggest that the ability of trichloroethylene to promote INF-{gamma} production by CD4+ T cells in MRL+/+ mice may help accelerate the development of autoimmunity.

In the present study, accelerated autoimmune response induced by treatment with occupationally relevant doses of trichloroethylene for 32 weeks was accompanied by an increase in serum ALT levels, indicating low-level hepatotoxicity. Increased ALT levels were not seen in the previous studies that employed higher doses of trichloroethylene for a shorter time-period. In addition to the release of ALT from damaged hepatocytes, there was a massive hepatic mononuclear infiltration within the portal tracts and focal areas of the lobule following 32 weeks of trichloroethylene treatment in MRL+/+ mice. This type of infiltration, along with the hepatotoxicity, is associated with autoimmune hepatitis induced by a number of therapeutic agents including tienilic acid (Manns et al., 1998Go; Zimmerman et al., 1984Go) and minocycline (Angulo et al., 1998Go). Thus, it appears that long-term treatment with low doses of trichloroethylene promotes more than an autoimmune response, and actually induces histopathology consistent with an autoimmune disease. It is possible that low-level T-cell activation following short-term trichloroethylene exposure results in a skewing of the T-cell response toward Th1 cells, and promotes the development of ANA, while long-term exposure to trichloroethylene leads to widespread T-cell activation, and this expansion of CD44hi T cells promotes the extravasation of IFN-{gamma}-secreting T cells into the liver, ultimately resulting in autoimmune hepatitis.

We recently reported that metabolism of trichloroethylene may be required for the accelerated autoimmune response observed in MRL+/+ mice treated with trichloroethylene (Griffin et al., 2000bGo). In addition, it has been shown by others that metabolic activation of trichloroethylene is a prerequisite for the hepatotoxicity observed following exposure to the xenobiotic (Buben and O'Flaherty, 1985Go; Goeptar et al., 1995Go). In our present study of MRL+/+ mice treated with low concentrations of trichloroethylene, we used immunohistochemical methods to determine if metabolic activation of trichloroethylene was occurring, and if so, where this metabolic activity was taking place. With an antibody that recognizes proteins covalently modified by a reactive metabolite of trichloroethylene, we were able to localize the covalently modified protein adducts within the central lobular region of the liver (Fig. 5Go). The differences in the localization of the protein adducts and the infiltrated lymphocytes may indicate that protein adducts are not important in the mechanism of trichloroethylene-induced autoimmune hepatitis.

Based on the results obtained here, we hypothesize that the accelerated autoimmune response in MRL+/+ mice exposed to trichloroethylene is associated with activated CD4+ T cells. These activated T cells secrete a Th1-like cytokine profile capable of inducing an inflammatory response. In association with this inflammatory cytokine profile, increases in ANA, and ultimately, autoimmune hepatitis, are induced by trichloroethylene.


    ACKNOWLEDGMENTS
 
This work was supported in part by the United States Environmental Protection Agency (N.R.P., R826409–01–0), American Cancer Society (K.M.G., 271115115011464) and the United States Department of Energy (N.R.P., DE-FG01-92EW50625).


    NOTES
 
1 To whom correspondence should be addressed at the Center of Excellence for Poultry Science, POSC O-214, University of Arkansas, Fayetteville, AR 72701. Fax: (501) 575-3026. npumford{at}uark.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adachi, Y., Inaba, M., Sugihara, A., Koshiji, M., Sugiura, K., Amoh, Y., Mori, S., Kamiya, T., Genba, H., and Ikehara, S. (1998). Effects of administration of monoclonal antibodies (anti-CD4 or anti-CD8) on the development of autoimmune disease in (NZW x BXSB) F1 mice. Immunobiology 198, 451–464.[ISI][Medline]

Angulo, J. M., Sigal, L. H., and Espinoza, L. R. (1998). Coexistent minocycline-induced systemic lupus erythematosus and autoimmune hepatitis. Semin. Arthritis Rheum. 28, 187–192.[ISI][Medline]

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