TCE Exposure in vivo Promoted Autoimmune Hepatitis in MRL^+/+ Mice

Young female MRL^+/+ mice were treated with TCE in the drinking water for 4 or 32 wk, with the expectation that this treatment would accelerate the development of a lupus-like disease in these mice. TCE was added to the water at concentrations of 0.1, 0.5, or 2.5 mg/ml. Water uptake was used to calculate that the mice were exposed to 21, 100, or 400 mg/kg/d of TCE. These doses encompass occupational exposure based on the current 8-hour Permissible Exposure Limit [established by the Occupational Safety and Health Administration (OSHA)] for TCE of 537 mg/m³, which is approximately 76 mg/kg/d.

After only 4 wk of exposure, concentrations of TCE as low as 21 mg/kg/d in the drinking water significantly increased the serum level of ANA, compared with mice treated with water alone (Griffin et al., 2000b). However, in contrast to expectations, chronic treatment with TCE did not accelerate the development of the lupus nephritis. Instead, TCE exposure for 32 wk was shown to generate histopathological evidence of autoimmune hepatitis. The livers in the TCE-treated mice revealed extensive infiltration with mononuclear cells subsequently identified as CD3⁺ T-lymphocytes. Based on histopathological scoring a Wilcoxon's Rank-Sum Test analysis revealed that the degree of mononuclear infiltration in livers of mice treated with 0.5 or 2.5 mg/ml TCE was significantly greater than in control mice.

The autoimmune hepatitis observed in the TCE-treated MRL^+/+ mice was associated with increased CD4⁺ T-lymphocyte production of pro-inflammatory cytokine IFNγ (Griffin et al., 2000b). In addition, TCE treatment for 32 wk expanded the percentage of splenic and lymph node CD4⁺ T-lymphocytes with an activated phenotype (CD44^hi, CD62L^lo). In contrast to CD4⁺ T-lymphocytes, the percentage of CD8⁺ T-lymphocytes or B-lymphocytes that expressed an activated phenotype was increased very little by TCE exposure. These results showed that exposure to occupationally relevant concentrations of TCE in vivo expanded the population of activated IFNγ-producing CD4⁺ T-lymphocytes in MRL^+/+ mice.

TCE Exposure in vivo Promoted Oxidative Stress and Adduct Formation

It has been proposed that adducts formed by certain chemicals on cell proteins may promote autoimmune disease by initiating an immune response against these chemically altered self-antigens. During its metabolism in the liver some TCE is converted to a TCE oxide reactive intermediate. The amino group of lysine on proteins reacts with intermediates formed during the hydrolysis of the TCE oxide forming N⁶-formyl lysine or N⁶-dichloroacetyllysine adducts. We have developed antibodies to the dichloroacetyllysine adducts (Halmes et al., 1996), and using immunochemical methods have detected dichloroacetyllysine adducts as stable neoantigens in the liver of TCE-treated MRL^+/+ mice (Griffin et al., 2000b). The predominant dichloro-acetyllysine adducted protein was found to be CYP2E1, the primary enzyme for TCE oxidative metabolism. The TCE adducts are capable of stimulating an immune response; adduct-specific antibodies have been detected in TCE-treated MRL^+/+ mice (Halmes et al., 1996). Thus, TCE promoted an immune response against modified liver proteins.

The fact that TCE exposure promoted antibody production against modified liver proteins led us to examine whether such exposure also stimulated the production autoantibodies specific for unmodified liver proteins. As shown in Figure 1, MRL^+/+ mice treated for 32 wk with 2.5 mg/ml TCE developed significant levels of autoantibodies against liver microsomal proteins. Thus, it appeared that TCE exposure could trigger an immune response against both unmodified and TCE modified liver proteins. Whether the antibodies generated recognize the same liver protein(s) in both adducted and unmodified form is not known. In any case, it appeared that TCE exposure promotes the presentation of liver proteins, both unmodified and chemically altered, in an immunogenic fashion to CD4⁺ T-lymphocytes and B-lymphocytes, the cellular components required for antibody production.

Environmental Contaminant Trichloroethylene Promotes Autoimmune Disease and Inhibits T-cell Apoptosis in MRL^+/+ Mice

All authors

Kathleen M. Gilbert, Neil R. Pumford & Sarah J. Blossom

https://doi.org/10.1080/15476910601023578

Published online:

09 October 2008

FIG. 1 Detecting liver-specific antibodies in TCE-treated mice. Female MRL^+/+ mice were given water alone (control) or water with TCE (0.1, 0.5 or 2.5 mg/ml) for 32 wk. Autoantibodies specific for liver microsomal proteins were determined in individual animals by using 10 mg/well purified liver microsomal proteins as capture antigen in an ELISA. A secondary anti-mouse IgG antibody was used to visualize absorbance. The data are plotted as mean and SEM OD and statistical significance (*) was determined by ANOVA with p ≤ 0.05.

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To further evaluate the mechanism by which MRL^+/+ mice chronically treated with TCE developed autoimmune hepatitis, we tested whether TCE stimulated oxidative stress in the liver, an event associated with liver inflammation and idiopathic autoimmune hepatitis. One marker of oxidative stress is peroxidase activity; heme proteins with a porphyrin ring (e.g., CYP2E1) that are oxidized by reactive oxygen species in the liver will acquire peroxidase activity that can be measured by Western blotting. Liver homogenates from MRL^+/+ mice treated for 4 weeks with TCE had increased levels of peroxidase activity (Figure 2). The fact that in vivo exposure to TCE for as little as 4 wk induced significant oxidative stress in the liver suggested that this physiological phenomenon may participate in TCE-induced autoimmune hepatitis.

Environmental Contaminant Trichloroethylene Promotes Autoimmune Disease and Inhibits T-cell Apoptosis in MRL^+/+ Mice

All authors

Kathleen M. Gilbert, Neil R. Pumford & Sarah J. Blossom

https://doi.org/10.1080/15476910601023578

Published online:

09 October 2008

FIG. 2 TCE induced oxidative stress in the liver. Female MRL^+/+ mice were treated with TCE (2.5 or 5 mg/ml) in their water for 4 wk. Equal amounts of liver protein from each treatment group were separated by SDS PAGE, transferred to nitrocellulose and developed for chemoluminesence using a substrate specific for peroxidase activity. A parallel gel was stained with Coomassie blue to confirm equal protein loading.

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TCAH Exposure in vivo Also Increased Activated CD4⁺ T-Lymphocytes, but Induced Alopecia Rather Than Autoimmune Hepatitis in MRL^+/+ Mice

To test whether the immunotoxicological effects of TCE were mediated through a metabolite, the pathological and immunological effects of TCAH were examined in MRL^+/+ mice. The female MRL^+/+ mice were treated for 4 or 40 wk with 0.1, 0.3 or 0.9 mg/ml TCAH in the drinking water. These concentrations of TCAH were chosen to encompass the molar equivalents of TCAH found in the mice exposed to TCE in concentrations of 0.1, 0.5 and 2.5 mg/ml. The time period was extended from 32 to 40 wk in case a more extensive exposure was needed to document toxicant-induced lupus nephritis. The TCAH treatment did not alter body weight or spleen size at either timepoint.

In terms of T-lymphocyte activation, a 4-wk exposure of MRL^+/+ mice to TCAH significantly increased the percentage of activated (CD62L^lo) splenic and lymph node CD4⁺ T-lymphocytes (from a mean of 36 ± 6.9% in mice treated with water alone to 60.3 ± 10.0% in mice treated with 0.9 mg/ml TCAH) (Blossom et al., 2004). Based on splenic cellularity, this translated into an average of 20.0 ± 5.7 × 10⁶ CD62L^lo CD4⁺ T-lymphocytes compared to 28.7 ± 9.1 × 10⁶ CD62L^lo CD4⁺ T-lymphocytes in the spleens of mice treated with water or TCAH respectively. Also similar to TCE treatment, TCAH treatment of MRL^+/+ mice for only 4 wk stimulated a dose-dependent increase in CD4⁺ T-lymphocyte production of IFNγ, but had little effect on CD8⁺ T-lymphocytes or B-lymphocytes.

Although TCAH treatment generated the same types of CD4⁺ T-lymphocyte alterations seen in TCE-treated mice, TCAH-treated MRL^+/+ mice did not develop autoimmune hepatitis or lupus nephritis. Instead, TCAH-treated MRL^+/+ mice developed a dose-dependent alopecia and skin inflammation (Blossom and Gilbert, 2006).

Exposure to TCE or TCAH in vivo Inhibited Activation-Induced Apoptosis in CD4⁺ T-Lymphocytes from MRL^+/+ Mice

It seemed possible that expansion of activated CD4⁺ T-lymphocytes in MRL^+/+ mice treated with TCE or its metabolite TCAH was due to a toxicant-induced decrease in activation-induced apoptosis. To test this possibility, CD4⁺ T-lymphocytes from TCE-and TCAH-treated MRL^+/+ mice were examined for their susceptibility to activation-induced apoptosis in vitro. Almost 88% of the activated CD4⁺ T-lymphocytes isolated from control MRL^+/+ mice at the 4 wk time period were induced to undergo activation-induced apoptosis in vitro. In contrast, only 55% of CD4⁺ T-lymphocytes from mice exposed to 0.5 mg/ml of TCE for 4 wk underwent apoptosis. Similarly, only 61% of CD4⁺ T-lymphocytes from mice exposed to 0.1 mg/ml of TCAH in vivo underwent apoptosis. This percentage was decreased even further, to 52%, in mice treated with 0.9 mg/ml of TCAH (Blossom et al., 2004). It should be noted that CD4⁺ T-lymphocytes from TCE-treated or TCAH-treated MRL^+/+ mice were not exposed to TCE or TCAH during the 5-d culture period required to induce activation-induced apoptosis. Therefore, exposure to TCE or TCAH in vivo induced resistance to activation-induced apoptosis in CD4⁺ T-lymphocytes that was retained even after the cells were no longer exposed to the toxicant.

Since susceptibility to activation-induced apoptosis can be mediated by alterations in the expression of Fas and FasL on CD4⁺ T-lymphocytes, the effect of TCAH exposure in vivo on these two molecules were examined at the 40-wk timepoint. FasL expression was inhibited on CD4⁺ T-lymphocytes from TCAH-treated mice, while Fas expression was not (Blossom and Gilbert, 2006). Thus, CD4⁺ T-lymphocytes from MRL^+/+ mice exposed to TCE or TCAH for 4 wk were resistant to activation-induced apoptosis and expressed lower levels of FasL, but not Fas.

Fas-mediated CD4⁺ T-lymphocyte apoptosis is believed to comprise at least one method by which the host protects itself against repeated stimulation and expansion of self-reactive CD4⁺ T-lymphocytes (Green et al., 2003; Tripathi and Hildeman, 2004). Our results suggested that TCE and TCAH inhibited apoptosis in CD4⁺ T-lymphocytes from MRL^+/+ mice by decreasing FasL expression. FasL expression on the cell surface can be regulated by cleavage of membrane-bound FasL into soluble FasL (sFasL) by MMP-7 and MMP-3 (Tanaka et al., 1998). Because sFasL is much less efficient than membrane-bound FasL at inducing Fas-mediated apoptosis in T-lymphocytes, any mechanism that promotes FasL shedding can dramatically decrease FasL bioactivity (Oyaizu et al., 1997; Tanaka et al., 1998) thereby decreasing Fas-mediated apoptosis and promoting CD4⁺T-lymphocyte-mediated autoimmunity. The findings described in this study suggest that TCE and TCAH promote expansion of activated CD4⁺ T-lymphocytes in MRL^+/+ mice by decreasing FasL expression, thus enabling activated CD4⁺ T-lymphocytes to escape Fas-mediated deletion but retain effector function.

The decrease in FasL on CD4⁺ T-lymphocytes in TCE-and TCAH-treated MRL^+/+ mice was associated with increased serum levels of MMP-7, a matrix metalloproteinase shown by others to cleave FasL from the cell surface (Vargo-Gogola et al., 2002). Most MMPs are not constitutively expressed in normal tissue, but can be induced by a variety of stimuli, including certain xenobiotics. For example, exposure of endothelial cells to cigarette smoke condensate up-regulated the genes for MMP-9, MMP-8, and MMP-1 (Nordskog et al., 2003). It appears that TCE and TCAH can be added to the list of toxicants that can increase MMP levels, at least in certain strains of mice.