Environmental contaminant and disinfection by-product trichloroacetaldehyde stimulates T cells in vitro

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Abstract

It had been shown previously that MRL+/+ mice exposed to occupationally relevant doses of the environmental contaminant trichloroethylene in their drinking water developed lupus-like symptoms and autoimmune hepatitis in association with activation of Interferon-γ (IFN-γ)-producing CD4+ T cells. Since trichloroethylene must be metabolized in order to promote the T-cell activation associated with autoimmunity, the present study was initiated to determine whether the immunoregulatory effects of trichloroethylene could be mimicked by one of its major metabolites, trichloroacetaldehyde (TCAA). At concentrations ranging from 0.04 to 1 mM TCAA co-stimulated proliferation of murine T-helper type 1 (Th1) cells treated with anti-CD3 antibody or antigen in vitro. TCAA at similar concentrations also induced phenotypic alterations commensurate with activation (upregulation of CD28 and downregulation of CD62L) in both cloned memory Th1 cells, as well as naı̈ve CD4+ T cells from MRL+/+ mice. TCAA-induced Th1 cell activation was accompanied by phoshorylation of activating transcription factor 2 (ATF-2) and c-Jun, two components of the activator protein-1 (AP-1) transcription factor. TCAA at higher concentrations was also shown to form a Schiff base on T cells, and inhibition of Schiff base formation suppressed the ability of TCAA to phosphorylate ATF-2. Taken together, these results suggest that TCAA promotes T-cell activation via stimulation of the mitogen-activated protein (MAP) kinase pathway in association with Schiff base formation on T-cell surface proteins. By demonstrating that TCAA can stimulate T-cell function directly, these results may explain how the environmental toxicant trichloroethylene promotes T-cell activation and related autoimmunity in vivo.

Introduction

Although little is known about the etiology of autoimmune disease, it is thought to encompass an interaction between an individual's genetic predisposition and their exposure to environmental factors. Among the environmental factors linked to the development of autoimmunity are heavy metals such as mercury, chlorinated pesticides containing chlordecone and methoxychlore, crystalline silica dust, and solvents such as trichloroethylene [1], [2], [3], [4]. Trichloroethylene is widely used as a decreasing agent; the National Toxicological Program has estimated that 3.5 million workers are exposed to trichloroethylene [5]. Because of its improper disposal, trichloroethylene is a common contaminant in surface and ground water; 60% of the hazardous waste sites proposed for inclusion on the U.S. EPA National Priorities List are contaminated with trichloroethylene [6]. The Agency for Toxic Substances and Disease Registry has indicated that trichloroethylene is the most frequently reported organic contaminant in groundwater, and has estimated that between 9% and 34% of the drinking water supplies tested in the United States may have some trichloroethylene contamination [6]. Exposure to trichloroethylene occupationally, or through contaminated water supplies, has been associated with the development of several human autoimmune diseases, including arthritis, systemic lupus erythematosus and systemic sclerosis [7], [8], [9], [10], [11].

Using a mouse model to more precisely define the immunological effects of trichloroethylene, it was shown that trichloroethylene administered to mice in the water supply at levels commensurate with occupational exposure promoted the development of an autoimmune response, eventually progressing to autoimmune disease [12]. The autoimmunity generated by exposure to trichloroethylene in vivo was associated with the activation of CD4+ T cells [13]. A subsequent study showed that the immunoregulatory effects of trichloroethylene were blocked when mice were concurrently treated with diallyl sulfide, an inhibitor of CYP 2E1 [14]. The major pathway of trichloroethylene metabolism is oxidation via the cytochrome P450 system, primarily CYP2E1, with trichloroacetaldehyde (TCAA) (also known as chloral) representing the major metabolite detected in liver microsomes from both humans and rodents [15], [16].

This study was initiated in part to test the likelihood that the immunoregulatory effects of trichloroethylene were mediated by its metabolite TCAA. The American Convergence for Governmental Industrial Hygienists recommends threshold limit value of 269 mg/m3 or a time weighted average of 50 ppm for trichloroethylene [11]. Assuming that over 85% of trichloroethylene is metabolized through TCAA [17], this converts to exposure levels of TCAA of approximately 42.5 mg/kg/day. This means that the average 70 kg man with total body water of 0.671 l/kg could attain concentrations of 64 μg/ml or approximately 0.39 mM. The concentrations used in the present study encompass this level of TCAA exposure. The mechanism by which TCAA induced T-cell stimulatory effects was also investigated. The mechanism tested was based on the finding that acetaldehyde, a chemical structurally similar to TCAA, has been shown to activate T cells through Schiff base formation [18]. Schiff base formation is characterized as a transient covalent chemical interaction between carbonyls on one molecule and amines on a second molecule, and is essential to a number of dynamic physiological processes including lipoprotein oxidation, selective ligand binding by enzymes, and regulation of rhodopsin phosphorylation [19], [20], [21]. Testing whether TCAA could activate T cells directly in association with Schiff base formation would provide important mechanistic information concerning the ability of environmental toxicants to promote the development of autoimmune disease.

Section snippets

Animals and reagents

Female MRL+/+ mice at 6 to 8 weeks of age were purchased from Jackson Laboratory (Bar Harbor, ME). Keyhole limpet hemocyanin (KLH) (Imject) was purchased from Pierce (Rockford, IL). The following antibodies were used: rabbit anti-dichloroacetyl (supplied by Dr. Neil Pumford, University of Arkansas, Fayetteville, AR), rabbit IgG (PharMingen, San Diego, CA), PE-anti-CD4 antibody (GK1.5 rat IgG2b, κ, PharMingen), PE-anti-CD8a antibody (54–6.7 rat IgG2a, κ, PharMingen), FITC-goat anti-rabbit IgG

TCAA costimulates T-cell proliferation

Trichloroethylene treatment in vivo caused an expansion of activated/memory CD4+ T cells [12], [13]. Consequently, experiments were conducted here to determine whether TCAA, as a metabolite of trichloroethylene, was able to stimulate proliferation of CD4+ Th1 cells in vitro. TCAA by itself was ineffective at stimulating Th1 cell proliferation. However, concurrent stimulation of the Th1 cells with TCAA and a low concentration of anti-CD3 antibody significantly enhanced the proliferative response

Discussion

TCAA, one of the primary oxidative metabolites of the environmental toxicant trichloroethylene, was shown here to activate CD4+ T cells in vitro using both functional and phenotypic assays. The functional assessment consisted of showing that TCAA promoted Th1 cell proliferation in conjunction with anti-CD3 antibody, as well as with antigen. In addition, TCAA was shown to induce a T-cell phenotype commensurate with activation, e.g. downregulation of CD62L and upregulation of CD28. TCAA induced

Acknowledgments

We thank Annick DeLoose for the excellent technical assistance. This work was supported by the EPA (MA0223) and the Arkansas Biosciences Institute.

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