Effect of a single oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin on immune function in male NC/Nga mice.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces immunosuppression in humans and animals. However, the effect of TCDD on Th2-type immune responses such as allergic reactions has been unclear. Using NC/Nga mice that developed atopic dermatitis-like skin lesions with marked elevation in plasma of total IgE when bred under conventional conditions, we investigated the effects of a single oral dose of TCDD on immune responses. NC/Nga mice received a single oral dose (0 or 20 microg/kg body weight) of TCDD. On day 7, treatment with TCDD alone decreased the cellularity of thymus. However, treatment with TCDD modified the cellularity of spleens and mesenteric lymph nodes (MLNs) but not of the thymus on day 28. When NC/Nga mice received ip immunization with OVA and alum on the same day as the TCDD treatment (0, 5, or 20 microg/kg body weight), TCDD markedly suppressed the concentrations of Th2-type cytokines (e.g., IL-4 and IL-5) in culture supernatants of spleen cells, whereas IFN-gamma production significantly increased. TCDD exposure reduced anti-OVA and total IgE antibody titers in plasma and did not induce the development of atopic dermatitis-like lesions in the pinnae or dorsal skin of NC/Nga mice. These results suggest that in NC/Nga mice, exposure to TCDD may impair the induction of Th2-type immune responses.

The effects of perinatal exposure to low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin on immune organs in rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is revealed to exert diverse biological effects including immunotoxicity, mainly by inadvertently activating the transcription factor arylhydrocarbon receptor (AhR). In the present study, the developmental effects of perinatal exposure to low doses of TCDD on the major immune organs of offspring, thymus and spleen, were investigated focusing on weaning time (postnatal day (PND) 21), puberty (PND 49) and adulthood (PND 120) in male rats. Concurrently, TCDD contents in those organs were measured with a high-resolution gas chromatography-mass spectrometry (GC/MS). In the thymus and spleen, CYP1A1 mRNA induction, the sensitive reaction caused by activation of AhR, was also measured in order to examine whether perinatally administered TCDD can elicit gene expressions in these organs. When pregnant dams were administered a single oral dose of 12.5-800 ng TCDD/kg body weight on gestation day (GD) 15, the weights of the thymus and spleen of the offspring did not differ from those of control animals throughout the experiments. The thymus and spleen maternally exposed to 800 ng TCDD/kg contained 102.0 and 62.7 pg TCDD/g tissue on PND 21, respectively, and the amounts decreased thereafter. In the thymus, dose-dependent CYP1A1 mRNA induction was clearly observed by maternal exposure to 50-800 ng TCDD/kg on PND 5. The induction was gradually decreased on PND 21 and 49. On the other hand, CYP1A1 mRNA induction in the spleen was very weak. In these thymi, no reproducible change was observed by TCDD exposure in cell number and cellular population defined by CD4 and CD8 molecules at any time. In contrast, splenocyte number was shown to decrease by maternal exposure to 12.5-800 ng TCDD/kg in a dose-dependent manner on PND 49. The alteration in spleen cellularity by TCDD was not detected on PND 21 or 120. These results clarified that perinatal exposure to low doses of TCDD affects the immune organs, which is apparent in spleen around puberty and likely to be hardly relevant to AhR-dependent gene expressions.

Reduction of thymocyte numbers in transgenic mice expressing viral FLICE-inhibitory protein in a Fas-independent manner.

A viral FLIP (FLICE/caspase-8-Inhibitory Protein), equine herpesvirus type 2 E8 protein, has been shown to inhibit Death receptor-induced apoptosis by suppressing the activation of FLICE/caspase-8. We generated transgenic mice specifically expressing E8 in thymocytes under the control of lck-proximal promoter. Although E8-expressing thymocytes were resistant to Fas-mediated apoptosis, the total number of thymocytes in 4-8-week-old E8 transgenic mice was more than 3-fold less than that in control littermates. This reduction was also observed in E8 transgenic mice with a Fas-/- background suggesting the reduction to be independent of Fas. The thymocytes of the transgenic mice, however, could similarly respond to CD3-mediated stimulation, indicating that the reduction of thymocyte numbers might be independent of T cell receptor complex-mediated stimulation. Thus, the Death receptor-mediated signaling pathway is too complex to be regarded as only an executor for apoptosis.

Alterations of thymocyte development, thymic emigrants and peripheral T cell population in rats exposed to 2,3,7, 8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exerts diverse biological effects by activating the cytosolic transcription factor, arylhydrocarbon receptor (AhR), which translocates to nuclei by TCDD binding and induces gene expressions. Among the well known-adverse effects of TCDD is thymus atrophy. In thymus atrophy, TCDD alters the proliferation as well as the differentiation of immature thymocytes. Previous studies on the effects of TCDD on thymocyte development were primarily carried out with high doses of TCDD. The present study investigates the effects of lower doses of TCDD (1 or 2 microg TCDD/kg by gavage) on thymocyte development, and furthermore, their sequential consequences on the peripheral T cell repertoire. Seven days after treatment with 1 or 2 microg TCDD/kg, the expression of CYP1A1 mRNA, one of the sensitive responses caused by the binding of TCDD to AhR, was detected in the thymus of rats. Thymus weights and thymus cell numbers decreased in TCDD-treated rats in a dose-dependent manner. The ratios of CD4 single-positive (SP) cells/CD8 SP cells were significantly reduced by TCDD exposure, indicating that the maturation of CD4(+)CD8(+) double-positive (DP) cells was skewed toward CD8 SP cells. These changes in the thymus were parallel to those previously observed with high doses of TCDD exposure. However, the specific reduction of DP cells reported in previous studies with high doses of TCDD was not detected in the present study. On the other hand, the skewing of mature CD4/CD8 T cell ratio in thymocytes by TCDD was not reflected in mesenteric lymph node (LN) lymphocytes, where the proportion of CD8 T cells was rather lowered by TCDD with a significant difference at 1 microg TCDD/kg. In LN lymphocytes, the percentage of recent thymic emigrants (RTEs), defined by the surface markers of Thy1(+)CD45RC(-), was shown to be significantly reduced by exposure to 1 and 2 microg TCDD/kg. T cell supply from the thymus has a crucial role in keeping the diversity of the T cell repertoire. The results of the present study indicated that lower doses of TCDD affect thymocyte development, especially differentiation, and reduce the proportion of RTE in LN, which may cause immunosuppression by reducing the variety of the T cell receptor repertoire.

Requirement of cooperative functions of two repeated death effector domains in caspase-8 and in MC159 for induction and inhibition of apoptosis, respectively.

BACKGROUND: The death effector domain (DED), which functions as a domain for a homophilic protein interaction, plays a role in death receptor-mediated apoptosis. Two tandemly repeated DEDs in the prodomain of caspase-8 (Casp8NC-DED) and those in MC159 (viral FLIP) have been shown to positively and negatively regulate apoptosis, respectively, by binding to caspase-8 and/or Fas-associated death domain (FADD). However, characteristics of each DED in Casp8NC-DED and those in MC159 have not been well examined. RESULTS: We analysed deletion and chimera mutants of DEDs derived from Casp8NC-DED and MC159, and found that MC159 and Casp8NC-DED require the combined effects of the two repeated DEDs to exert their binding and biological activities. The carboxy-terminal DED of Casp8NC-DED (Casp8C-DED) has the potential to induce apoptosis, and the amino-terminal DED of MC159 showed a dominant inhibitory effect on apoptosis when combined with Casp8C-DED. In addition, the two repeated DEDs in Casp8NC-DED and MC159 were shown to regulate the activities of caspase differently from the caspase recruitment domain (CARD) in the prodomains of caspase-2, -9 and Apaf-1. CONCLUSIONS: Although each of the DEDs in Casp8NC-DED and MC159 has the potential to stimulate or inhibit apoptosis, the combination of the two-repeated DEDs is necessary for the DED-containing proteins to stimulate or inhibit apoptosis.

Molecular cloning and characterization of mouse caspase-8.

Fas (APO-1/CD95) is a transmembrane receptor protein which induces apoptosis upon activation. In apoptosis triggered by Fas, a subset of cysteine proteases designated caspases is activated, playing a central role as effector molecules. Among these caspases, human caspase-8 (FLICE/MACH/Mch5) has been isolated and shown to be indispensable for Fas-mediated apoptotic signaling. In this study, we isolated the mouse homologue to human caspase-8 from a BaF3 cell cDNA library. This molecule conserved the death effector domain (DED) and protease domain as detected in human caspase-8, and was capable of inducing apoptosis in KB and Rat-1 cells when overexpressed. Expression of caspase-8 was detected in the various tissues of adult mouse and in embryos at 9.5 days and 17.5 days of development by Northern-blot analysis. Further, we isolated a chromosomal gene for caspase-8 from a mouse genomic library and analyzed the genomic structure of the isolated gene. This gene consisted of eight exons and seven introns spanning about 26 kb in the coding region.

A simple screening for mutant DNA binding proteins: application to murine transcription factor PEBP2alpha subunit, a founding member of the Runt domain protein family.

Mouse transcription factor PEBP2 (polyomavirus enhancer-binding protein (2) is composed of two distinct subunits alpha and beta. The alpha subunit has an ability to bind the specific DNA sequences, which is enhanced by formation of a heterodimer with the beta subunit. The DNA binding and heterodimerization activities of the alpha subunit are both localized within a 128-amino-acid (aa) region termed as the Runt domain for its homology to the Drosophila segmentation gene runt. To characterize the molecular determinants for these activities, the Runt domain was randomly mutagenized and produced in E. coli as a secreted form. Using E. coli culture supernatant, the DNA binding and heterodimerization of mutant Runt domains were analyzed by gel retardation assay. Nine randomly picked single-aa substitution mutants showed various functional alterations in DNA binding and heterodimerization either separately or simultaneously. This observation suggests that the structure of Runt domain is highly ordered and is quite sensitive to modulations in its primary structure. The method presented here provides a simple and quick method to characterize a large number of mutant DNA binding proteins.