Thymus atrophy is induced by a variety of chemicals, including environmental contaminants and is used as a sensitive index to detect their adverse effects on lymphocytes. In the present study we adopted a toxicogenomics approach to identify the pathways that mediate the atrophy induced by arsenite. We also analyzed gene expression changes observed in the course of thymus atrophy by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), dexamethasone (DEX), and estradiol (E2), to determine whether arsenite induces atrophy by activating an arsenite-specific pathway or the same pathways as other chemicals. These compounds were intraperitoneally administered to C57BL/6 mice at doses that reduce thymus weight by approximately 30% within 3 days, and gene expression changes in the thymus 24 h after the administration were analyzed by using microarrays and real-time PCR. The microarray analysis showed that arsenite specifically downregulates a variety of E2F target genes that are involved in cell cycle progression. The same genes were also downregulated when mouse B-cell lymphoma A20 cells were exposed to arsenite. Arsenite exposure of the A20 cells was confirmed to induce cell cycle arrest, mainly in the G(1) phase, and reduce cell number. Cell cycle arrest in the G(1) phase was also confirmed to occur in the thymocytes of the arsenite-exposed mice. These results indicate that arsenite induces thymus atrophy through E2F-dependent cell cycle arrest. The results of this study also show that analysis of gene expression in thymuses is a useful method of obtaining clues to the pathways that mediate the effects of atrophy-inducing chemicals.

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Activation of aryl hydrocarbon receptor (AhR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in T-cells is required for TCDD-induced suppression of the allogeneic CTL response and for induction of CD25(hi)CD62L(low) adaptive regulatory T-cells. Here, the ability of a constitutively-active AhR (CA-AhR) expressed in T-cells alone to replicate the effects of TCDD was examined. The response of CA-AhR-expressing B6 donor T-cells in B6xD2F1 mice was compared to the response of wild-type B6 donor T-cells in B6xD2F1 mice given a single dose of TCDD. Expression of CA-AhR in donor T-cells enhanced the down-regulation of CD62L on Day 2 after injection, similar to a single oral dose of TCDD, but did not induce up-regulation of CD25 on Day 2 or affect CTL activity on Day 10. This suggests that activation of AhR in T-cells alone may not be sufficient to alter T-cell responses in this acute graft-versus-host (GvH) model. Since host APC are responsible for activating the donor T-cells, we examined the influence of the F1 host's AhR on donor T-cell responses by creating an AhR(-/-) B6xD2F1 host that had a greatly diminished AhR response to TCDD compared to wild-type F1 mice. As in AhR(+/+) B6xD2F1 mice, the CTL response in AhR(-/-) B6xD2F1 mice was completely suppressed by TCDD. This suggests that either CA-AhR dose not fully replicate the function of TCDD-activated AhR in suppression of the CTL response, or that minimal activation of AhR in host cells is required to combine with activation of AhR in T-cells to elicit the immunosuppressive effects of TCDD.

The ligand-dependent transcription factor aryl hydrocarbon receptor (AhR) has been implicated in various immune functions. Our previous studies have shown that AhR activation by exposure of ovalbumin (OVA)-immunized mice to the potent ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases immunization-induced IFN-gamma production in the spleen and suppresses the production of T(h)2 cytokines and OVA-specific antibodies. In the present study, we used transgenic (Tg) mice that express a constitutively active mutant of aryl hydrocarbon receptor (CA-AhR) specifically in T-lineage cells to clarify the role of AhR activation in T cells in these reactions. The results of this study clearly demonstrated that AhR activation only in the T cells augments IFN-gamma production upon OVA immunization. By contrast, production of T(h)2 cytokines and antibodies were not significantly suppressed by CA-AhR in the T cells. These results suggest that suppression of T(h)2 cytokines and antibodies production require AhR activation not only in T cells but also in other cell types as caused by TCDD exposure. Alternatively, these results may indicate that IFN-gamma augmentation and T(h)2 cytokines and antibodies suppression depend on different ways of functions of AhR in the T cells and that CA-AhR does not replicate the suppressive effect of TCDD-activated AhR on T(h)2 cytokines and antibodies. Expression of CA-AhR in the T cells was also shown to increase the percentage of CD25(+) cells among CD4(+) cells in the thymus and spleen. Thus, studies using T-cell-specific CA-AhR Tg mice provide a way to dissect the role of AhR in individual cell types and how the AhR functions.