Treatment of Mice with 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Leads to Aryl Hydrocarbon Receptor-Dependent Nuclear Translocation of NF-κB and Expression of Fas Ligand in Thymic Stromal Cells and Consequent Apoptosis in T Cells

Absence of AhR does not alter the phenotype and function of thymocytes. Thymocytes from untreated AhR^+/+ and AhR KO mice were double-stained with FITC-anti-CD4 and PE-anti-CD8 mAbs (A), or single-stained with PE-conjugated anti-Fas or FITC-conjugated anti-CD69 mAbs (B). Cells were then analyzed by flow cytometry. Representative dot plots or histograms are shown on the left side of A and B, respectively. The empty histograms in B represent cells stained with control Abs, and the filled histograms represent thymocytes stained for Fas or CD69. B, Also depicts the percentage of positive cells and MIF for stained cells in each histogram. On the right side of A and B, data from four to six individual experiments were collected and expressed as mean ± SEM. In C, thymocytes from untreated AhR^+/+ and AhR KO mice were cultured for 48 h with either medium alone or in the presence of anti-CD3 mAbs (5 μg/ml). Cellular proliferation was measured with a [³H]thymidine incorporation assay in four independent experiments. Data were represented as the mean cpm ± SEM of triplicate cultures of the combined experiments.

Effect of TCDD on phenotypic markers in the thymus of AhR^+/+ and AhR KO mice. AhR^+/+ and AhR KO mice were injected with 50 μg/kg TCDD or the vehicle. Five days posttreatment, thymocytes were stained with FITC- or PE-conjugated mAbs against CD3, αβTCR, IL-2R, CD44, CD4, CD8, or J11d. Cells were then analyzed by flow cytometry. Representative histograms are shown for each experimental group on the left side of the figure. The empty histograms represent cells stained with control Abs, and the filled histograms represent thymocytes stained for Abs against various surface markers. The percentage of positive cells and MIF for stained cells have been depicted for each histogram. On the right side of the figure, data from four to six individual experiments were collected and expressed as mean MIF ± SEM. Asterisks (∗) indicate statistically significant differences in TCDD-exposed thymocytes when compared with the vehicle controls within each group (p < 0.05).

TCDD increases the expression of FasL on thymic stromal cells. A, Stromal cells from the thymi were stained with anti-CD45 mAbs and anti-TSA Abs. The CD45⁻ cells were gated and analyzed for TSA expression. The empty histogram represents cells stained with control Abs, and the filled histogram represents cells stained with anti-TSA Abs. B, The stromal cells exposed to TCDD or vehicle as described above were stained with anti-CD45 Abs and anti-FasL Abs. Then, the CD45⁻ cells were gated and analyzed for FasL. The percent FasL⁺ cells has been depicted. The histogram in red shows cells stained with Abs against FasL and the dark histogram represents cells stained with isotype control Abs. C, Thymocytes were stained with anti-CD45 mAbs and anti-FasL Abs. The CD45⁺ cells were gated and analyzed for FasL expression. The empty histogram represents cells stained with control Abs, and the filled histogram represent cells stained with anti-FasL Abs. Results are representative of at least three separate experiments.

TCDD-induced FasL on stromal cells causes enhanced apoptosis in Fas⁺ target cells. A, Thymic stromal cells (Thy1.2⁺) were isolated 24 h posttreatment of wild-type mice with 50 μg/kg TCDD or the vehicle and cocultured overnight with Thy 1.1⁺ thymocytes, either in the presence of control Abs or anti-FasL Abs (Kay-10). Cells were then double-stained with PE-conjugated anti-Thy1.1 mAb and FITC-dUTP for TUNEL. The Thy1.1⁺ cells were gated and analyzed for apoptosis. B, Thy 1.2⁺ thymic stromal cells from (a) gld/gld, (b) wild-type, or (c) AhR KO mice exposed to 50 μg/kg TCDD or the vehicle, were cultured for 24 h with thymocytes from untreated wild-type (Thy1.1⁺) mice (a and c) or from lpr/lpr (Thy1.2⁺) mice (b). Cells were then double-stained with PE-conjugated anti-Thy1.1 (a and c) or PE-conjugated anti-Fas mAbs (b), and FITC-dUTP for TUNEL. Next, the Thy1.1⁺ (a and b) or Fas⁻ cells (b) were gated and analyzed for apoptosis. The right panels in A and B show pooled data from four to six experiments. Data were expressed as mean percent apoptosis ± SEM. Asterisks (∗) indicate statistically significant differences in the mean of TCDD-exposed thymocytes when compared with the vehicle controls within each group (p < 0.05).

TCDD induces activation of NF-κB in thymic stromal cells. AhR^+/+ and AHR KO mice were injected with 50 μg/kg TCDD or the vehicle. Stromal cells were isolated 6 h posttreatment and used for the analysis of CD3 (A) expression and intracellular localization of NF-κB p65 (green) and p50 (red) (B) in stromal cells (∗) and T cells by confocal microscopy. Cell morphology (Nomarski), colocalization of p50/p65 (yellow), and Hoechst-33258 nuclear staining (blue) are also shown. Three independent samples were evaluated and representative data were depicted.

Effect of NF-κB inhibitor on the nuclear translocation of NF-κB subunits after TCDD treatment of stromal cells in vitro. Stromal cells from untreated AhR^+/+ mice were cultured overnight with the following treatments: vehicle (DMSO), 1 nM TCDD, 1 nM TCDD + 20 μM TPCK, or 20 μM TPCK as described in Materials and Methods. Intracellular localization of NF-κB p65 (green) and p50 (red) in stromal cells (∗) and T cells was visualized by confocal microscopy. Cell morphology (Nomarski), colocalization of p50/p65 (yellow), and Hoechst-33258 nuclear staining (blue) are also shown. Three independent samples were evaluated and representative data were depicted.

TCDD regulates FasL promoter activity through NF-κB. A, Sequence of a 689-bp mouse FasL promoter upstream of the FasL translational start site has been depicted. NF-κB-binding sites in the FasL promoter are underlined. B, EL-4 cells were analyzed for AhR expression using RT-PCR. C, EL-4 cells were transiently transfected with various luciferase constructs (pGL-3-FasL720, pGL-3 control) and pcDNA3.1 with pCMV-β-galactosidase. Two days posttransfection, the transfected cells were left untreated or treated with DMSO, 100 nM/ml TCDD, 100 nM/ml TCDD + 100 ng/ml α-naphthoflavone (TCDD + AN), or 100 ng/ml α-naphthoflavone alone (AN). The luciferase activity was normalized to β-galactosidase activity and expressed as normalized fold induction. The vertical bars represent mean ± SEM from three independent experiments. D, EL-4 cells were transiently transfected with normal NF-κB luciferase construct (pGL-3-FasL720) or with various NF-κB mutant luciferase constructs of FasL promoter (pGL-3-FasL720-NF-κB1 mutant, pGL-3-FasL720-NF-κB2 mutant, pGL-3-FasL720-NF-κB1 and B2 mutants). Two days posttransfection, the transfected cells were left untreated or treated with DMSO or 100 nM/ml TCDD. Vertical bars represent mean ± SEM from three independent experiments.

Effect of TCDD on activation of members of the death receptor and the mitochondrial pathway. Wild-type mice were injected with 50 μg/kg TCDD (T) or the vehicle (V). Cell lysates were isolated from thymocytes 12 or 18 h posttreatment. A and B, Proteins (50 μg/lane) were resolved by SDS-PAGE gel electrophoresis and probed with Abs against anti-caspase-8, caspase-9, caspase-2, bid, bax, bcl-x_L, cytochrome c (cyt C), and Smac. As a loading control, blots were stripped and reprobed with β-actin Abs. C, Bands were quantified, normalized to β-actin, and expressed as the percentage change of the band density of the TCDD sample relative to the vehicle control sample.

Effect of TCDD on the Δψ_m of thymocytes. Thymocytes were obtained from wild-type mice treated with 50 μg/kg TCDD (T) or the vehicle (V) 6, 12, or 18 h posttreatment, and then stained with DIOC₆ to evaluate Δψ_m. Empty histograms represent thymocytes from vehicle-treated mice, whereas filled histograms represent thymocytes from TCDD-treated mice (A). Δψ_m was also measured in thymocytes exposed in vivo for 12 h with different doses of TCDD (1, 5, 10, 25, or 50 μg/kg) (B). Results are representative of at least three separate experiments.

Role of Bid in TCDD-induced apoptosis in the thymus. Wild-type (Bid^+/+) and Bid-deficient mice were injected with 50 μg/kg TCDD or the vehicle. Three days posttreatment, thymocytes were collected and used to determine thymic cellularity (A) and apoptosis (B). The bar diagrams represent data from five to six individual mice. Asterisks (∗) indicate statistically significant between TCDD-exposed thymocytes when compared with the vehicle controls within each group (p < 0.05).