Primary Peripheral T Cells Become Susceptible to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin-Mediated Apoptosis in Vitro upon Activation and in the Presence of Dendritic Cells

Abstract

Although the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on T cells in vivo have been well characterized, attempts to reproduce these findings in vitro have not been successful. In the current study, we examined whether activation or the presence of dendritic cells (DCs) would make primary naive T cells from C57BL/6 mice susceptible to TCDD-induced apoptosis in vitro. Although nonactivated primary T cells cultured with 10 to 1000 nM TCDD were relatively resistant to apoptosis, they became sensitive to apoptosis upon activation with concanavalin A (ConA). Moreover, ConA-activated T cells cultured in the presence of DCs showed highest levels of TCDD-induced apoptosis. Likewise, primary T cells from OT.II.2a mice cultured with specific ovalbumin peptide and syngeneic DCs showed higher levels of apoptosis compared with similar nonactivated T cells. T-cell activation led to up-regulation of aryl hydrocarbon receptor (AhR), Fas, and Fas-ligand (FasL) expression. In addition, DC maturation and culture with TCDD caused significant induction of FasL. TCDD-mediated apoptosis in activated peripheral T cells was AhR-dependent. Analysis of why nonactivated T cells are more resistant, whereas activated T cells are sensitive to TCDD-induced apoptosis revealed that TCDD treatment of activated but not nonactivated T cells led to down-regulation of cellular FLICE inhibitory protein (c-FLIP), an inhibitor of apoptosis. Moreover, down-regulation of c-FLIP using small interfering RNA in nonactivated T cells made them sensitive to TCDDinduced apoptosis. The current study demonstrates for the first time that TCDD can induce apoptosis in vitro in peripheral T cells upon activation and in the presence of DCs and that this may be mediated by down-regulation of c-FLIP.2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental pollutant that mediates toxicity through activation of the aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor and member of the basic helix-loop-helix-Per/Arnt/Sim (periodicity/aryl hydrocarbon receptor nuclear translocator/simple-minded) gene family (Marlowe and Puga, 2005;Bock and Kohle, 2006;Harper et al., 2006). TCDD binds and activates cytosolic AhR attached to 90-kDa heat shock protein; and upon activation, AhR dissociates from 90-kDa heat shock protein, migrates to the nucleus, dimerizes with AhR nuclear translocator (ARNT), and forms AhR/ARNT heterodimer. This in turn binds to dioxin-response elements (DRE), present as cis-acting elements in the regulatory regions of dioxin-responsive genes, and it up-regulates a large set of genes that directly or indirectly participate in TCDD-induced toxicity (Schmidt and

Differential Proteomics Analysis Reveals a Role for E2F2 in the Regulation of the Ahr Pathway in T Lymphocytes

“…Previous studies have implicated the Ahr pathway in the generation of TH17ϩ inflammatory cells (50,51). Furthermore, several reports have shown that T cells in the thymus and periphery are highly sensitive to TCDD-induced apoptosis (47,48,52).…”

“…It has been shown that TCDD induces apoptosis in T cells involving Fas/FasL interactions (48). We investigated whether the increased susceptibility of activated E2F2 Ϫ/Ϫ ; three independent experiments).…”

Exposure to Diethylstilbestrol during Pregnancy Modulates MicroRNA Expression Profile in Mothers and Fetuses Reflecting Oncogenic and Immunological Changes

“…PCR was performed using mouse FasL-or Fas-specific sets of forward and reverse primers as described earlier (Singh et al, 2008). The PCR products, generated from mouse Fas and FasL primer pairs, were normalized against mouse-specific 18S PCR products as described previously (Singh et al, 2008). The intensity of PCR products was determined using the ChemiDoc image analysis system (Bio-Rad).…”

“…In brief, single cell suspension of thymic cells post-VEH or post-DES treatment was prepared as described above. Expression of Fas and FasL was determined by RT-PCR as described previously (Singh et al, 2008). For RT-PCR, total RNAs from VEH-or DES-treated groups were isolated using the RNeasy mini kit following the protocol of the company (QIAGEN, Germantown, MD).…”

“…cDNA synthesis was performed in a 20 ml reaction mixture containing 1 mg total RNA using the iScript kit and following the protocol of the manufacturer (Bio-Rad, Hercules, CA). PCR was performed using mouse FasL-or Fas-specific sets of forward and reverse primers as described earlier (Singh et al, 2008). The PCR products, generated from mouse Fas and FasL primer pairs, were normalized against mouse-specific 18S PCR products as described previously (Singh et al, 2008).…”