* Department of Pharmacology and Toxicology,
Department of Microbiology and Immunology, and
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
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ABSTRACT |
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Key Words: antinuclear antibodies (ANA); inflammatory cytokines; dose-related response; hepatic mononuclear infiltration; scleroderma; systemic lupus erythematosus.
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INTRODUCTION |
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A model for studying the effects of trichloroethylene on the development of autoimmunity has been developed using lupus-prone MRL+/+ mice (Khan et al., 1995). 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., 2000a
). 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., 2000b
).
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, 1994). 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 9398% (Goeptar et al., 1995
). 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., 2000a
; Khan et al., 1995
), 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.
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MATERIALS AND METHODS |
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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 536.7, 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 1452C11; 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-
). Cytokine concentrations in the ELISAs were determined by comparison to a standard curve generated using mouse recombinant IL-4 and IFN-
(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 03+, ranging from no change to severe, respectively), and statistically analyzed using Wilcoxon's rank-order analysis. Scores of 01, 12, or 23, 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 03+, 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., 1996). 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.
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RESULTS |
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CD4+ T Cells from Trichloroethylene-Treated Mice Secreted Increased Levels of IFN-
Because IFN--secreting CD4+ T cells are involved in the development of autoimmune disease pathology in the genetically similar MRLlpr/lpr strain (Balomenos et al., 1998
; Haas et al., 1997
; Takahashi et al., 1996
), 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-
and IL-4. There was a dose-responsive and significant increase in the levels of IFN-
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. 3
). The increased ability of CD4+ T cells from mice treated with 0.5 or 2.5 mg/ml trichloroethylene to secrete IFN-
was still observed after 32 weeks of treatment. In contrast to IFN-
, 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., 2000a
), and demonstrate that trichloroethylene-treatment promotes a Th1 cell-like immune response in the MRL+/+ mice.
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DISCUSSION |
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Previously we have shown acceleration of an autoimmune response at high doses of trichloroethylene (Griffin et al., 2000a). 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., 1998; Yagi et al., 1992
). CD4+ T cells are also critical in the process of chemically-induced autoimmunity, especially in diseases associated with heavy metals (Goldman et al., 1991
; Pelletier et al., 1988
; Schuhmann et al., 1990
; Stiller-Winkler et al., 1988
). 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., 2000a
). 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., 2000b
). 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, 1990). 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., 1991
), 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., 1998
). 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., 1998
).
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., 1991), which are thought to be critical for disease development (Takahashi et al., 1996
). A Th1-like cytokine profile is characterized by an increase in secreted IFN-
, 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-
than CD4+ T cells from control mice. The importance of IFN-
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-
or IFN-
receptor have been deleted (Balomenos et al., 1998
; Haas et al., 1997
). Although the exact role of INF-
in the development of lupus remains to be delineated, it is thought that IFN-
-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, 1997
; Wuthrich et al., 1990
). Taken together, the results suggest that the ability of trichloroethylene to promote INF-
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., 1998; Zimmerman et al., 1984
) and minocycline (Angulo et al., 1998
). 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-
-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., 2000b). 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, 1985
; Goeptar et al., 1995
). 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. 5
). 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.
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ACKNOWLEDGMENTS |
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NOTES |
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