ABSTRACT
The arylhydrocarbon receptor (AhR), a ligand-activated transcription factor, is differentially distributed in tissues and abundant in the thymus epithelium. The activated AhR can induce the transcription of an array of genes, including genes of cell growth and differentiation. Neither the physiological function of the AhR nor its putative natural ligand is known. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a xenobiotic high-affinity activator of the AhR, and appears to be essential for most of the multifold toxic effects of TCDD. Activation of the AhR by even low doses of TCDD results in general immunosuppression and thymus hypoplasia. TCDD exposure interferes with thymocyte development; for instance, it reduces the proliferation rate of the very immature (CD4- CD8- and CD4- CD8+ HSA+) thymocytes, leads to preferential emigration of very immature cells, and drastically skews the differentiation of thymocyte subpopulations towards mature CD4- CD8+ alphabeta TCRhigh thymocytes. As shown here, in fetal thymi of AhR-deficient mice, thymocyte differentiation kinetics as defined by CD4 and CD8 surface markers, was comparable to AhR+/+ C57BL/6 mice. Also, the cell emigration characteristics were similar to AhR+/+ mice. These parameters were refractory to TCDD exposure in the AhR-/- mice, but not in the C57BL/6 mice. However, in AhR deficient mice at gestation day 15 more CD4- CD8- immature cells bore high amounts of the (alphabeta-T-cell receptor. Also, fetal thymocyte numbers were significantly lower, as compared to strain C57BL/6. Thus, the AhR is the mediator of thymotoxic effects of TCDD.
Subject(s)
Immunosuppressive Agents/toxicity , Polychlorinated Dibenzodioxins/toxicity , Receptors, Aryl Hydrocarbon/physiology , T-Lymphocyte Subsets/drug effects , Thymus Gland/pathology , Animals , Atrophy , Cell Differentiation/drug effects , Cell Division/drug effects , Female , Flow Cytometry , Fluorescent Antibody Technique, Indirect , Hyaluronan Receptors/analysis , Immunologic Deficiency Syndromes/chemically induced , Mice , Mice, Inbred C57BL , Organ Culture Techniques , Receptors, Aryl Hydrocarbon/deficiency , Receptors, Aryl Hydrocarbon/drug effects , Receptors, Aryl Hydrocarbon/genetics , Thymus Gland/drug effects , Xenobiotics/pharmacokinetics , Xenobiotics/toxicityABSTRACT
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) binds and activates the aryl hydrocarbon receptor (Ah-R), an endogenous transcription factor that is expressed in the thymus. TCDD exposure leads, among other effects, to thymus atrophy and immunosuppression. We previously analyzed the interference of TCDD with differentiation processes in fetal thymus organ cultures and found that in the presence of TCDD, the proliferation rate of immature (CD4- CD8- and CD4- CD8+ HSA+) thymocytes is inhibited, whereas the maturation along the CD4/CD8 path is accelerated. Moreover, the differentiation of thymocytes is skewed by TCDD at < or = 40% (compared with approximately 15% without TCDD) of the CD8 single-positive subset of future cytotoxic T cells, and apparently more cells audition for and pass positive selection. The fetal murine thymus expresses functional Ah-R mRNA, as shown by reverse transcription-polymerase chain reaction and TCDD-inducible CYP1A1 and CYP1B1 expression. Because the differentiation of thymocytes is to a considerable extent controlled by cytokines and many cytokine genes are potential targets of the Ah-R due to Ah-R-binding elements (xenobiotic response elements) in their promoters, we analyzed the cytokine expression in fetal thymus organ culture exposed to TCDD. Fetal thymi were cultured from gestation day 15 for < or = 8 days, thus covering ex vivo the period after population of the thymus anlage until birth. We show with semiquantitative reverse transcription-polymerase chain reaction that more interleukin (IL)-1beta, IL-2, IL-6, tumor growth factor (TGF)-beta3, and tumor necrosis factor-alpha are produced in TCDD-exposed thymi, whereas other cytokines (e.g., TGF-beta1, PAI-2, or IL-4) are only slightly up- and down-modulated during the culture period or not modulated at all (e.g., IL-1beta, IL-7, interferon-gamma, and TGF-beta2).
Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytokines/genetics , DNA-Binding Proteins , Fetus/drug effects , Polychlorinated Dibenzodioxins/toxicity , Receptors, Aryl Hydrocarbon/drug effects , Thymus Gland/drug effects , Animals , Aryl Hydrocarbon Receptor Nuclear Translocator , Blotting, Southern , Cytochrome P-450 CYP1A1/drug effects , Cytochrome P-450 CYP1A1/genetics , Cytochrome P-450 CYP1B1 , Female , Fetus/metabolism , Gene Expression Regulation/drug effects , Male , Mice , Mice, Inbred C57BL , Organ Culture Techniques , Polymerase Chain Reaction , Receptors, Aryl Hydrocarbon/genetics , Thymus Gland/metabolism , Transcription Factors/drug effects , Transcription Factors/geneticsABSTRACT
Preparation of intact, full-size RNA from tissues or cells requires stringent precautions against ubiquitous and rather stable RNases. Fluorescence-activated cell sorting (FACS) usually aims at the isolation of cells according to cell surface markers on living cells, from which RNA can be obtained by standard protocols. The separation of cells according to intracellular immunofluorescence markers, such as intranuclear, intracytoplasmic, or secreted molecules, requires permeation of the cell membrane for the staining antibodies, which is usually achieved by fixation. However, commonly used fixatives such as ethanol, methanol, or formaldehyde do not inactivate RNases completely, thereby hampering the analysis of complete RNA molecules from fixed cells. We report isolation of intact, full-size RNA suitable for Northern blotting from cells that were fixed by 95% ethanol/5% acetic acid containing RNase inhibitors, stained intracellularly, and sorted by FACS.
