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T Cell-Specific Disruption of Arylhydrocarbon Receptor Nuclear Translocator (Arnt) Gene Causes Resistance to 2,3,7,8-Tetrachlorodibenzo-p-dioxin-Induced Thymic Involution ¹

Shuhei Tomita^2,*,,, Hou-Bo Jiang^*, Tomoo Ueno, Satoshi Takagi, Keiko Tohi, Shin-ichi Maekawa, Akira Miyatake^*, Aizo Furukawa^*, Frank J. Gonzalez^||, Junji Takeda^¶, Yoshiyuki Ichikawa^* and Yousuke Takahama^,

^* Department of Biochemistry, Kagawa Medical University, Kagawa, Japan; Department of Immune System Development, The Institute of Physical and Chemical Research, Research Center for Allergy and Immunology and Division of Experimental Immunology, Institute for Genome Research, University of Tokushima, Tokushima, Japan; Departments of Plastic Surgery and ^¶ Social Environmental Medicine, Osaka University Graduate School of Medicine, Osaka, Japan; and ^|| Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892

	Abstract

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The arylhydrocarbon receptor nuclear translocator (ARNT) is a member of the basic helix-loop-helix, PER-ARNT-SIM family of heterodimeric transcription factors, and serves as a dimerization partner for arylhydrocarbon receptor (AHR) and hypoxia-inducible factor-1. To assess the function of ARNT in T cells, we disrupted the Arnt gene specifically in T cells of mice by conditional gene targeting using T cell-specific p56^lck-Cre (Lck-Cre) transgenic Arnt-floxed mice. Thus generated, T cell-specific Arnt-disrupted mice (Lck-Cre;Arnt^flox/ transgenic mice) exhibited complete loss of the expression of ARNT protein only in T cells, and were viable and appeared normal. The Arnt-disrupted T cells in the thymus were phenotypically and histologically normal. The Arnt-deficient T cells in the spleen were capable of responding to TCR stimulation in vitro. However, unlike normal mice in which exposure to the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an AHR ligand, resulted in thymic involution, the thymus of Lck-Cre;Arnt^flox/ mice were resistant to TCDD treatment in vivo. In contrast, benzo(a)pyrene, another AHR ligand, still caused thymic involution in Lck-Cre;Arnt^flox/ mice. Finally, fetal thymus organ culture using Lck-Cre;Arnt^flox/ and K5-Cre;Arnt^flox/ (epithelial cell-specific Arnt-disrupted mice) showed that thymocytes rather than thymic epithelial cells are predominantly responsible for TCDD-induced thymic atrophy. Our results indicate that ARNT in T lineage cells is essential for TCDD-mediated thymic involution.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The arylhydrocarbon receptor nuclear translocator (ARNT) ³ is a member of basic helix-loop-helix, PER-ARNT-SIM family of heterodimeric transcription factor, which includes arylhydrocarbon receptor (AHR), hypoxia-inducible factor-1 (HIF-1), and Drosophila single-minded protein (SIM) (1, 2, 3, 4). ARNT forms heterodimeric transcription factors by the association with other basic helix-loop-helix, PER-ARNT-SIM members such as AHR, HIF-1, and SIM. The AHR/ARNT heterodimer regulates genes involved in the metabolism of xenobiotics, such as cyp1a1, cyp1a2, and cyp1b1 (5, 6), in response to environmental pollutants and potent halogenated aromatic agonists of AHR, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo(a)pyrene (B(a)P) (7, 8, 9), whereas the HIF-1/ARNT heterodimer regulates the response to oxygen deprivation (10). SIM/ARNT plays a pivotal role in the midline development of the CNS (11).

Exposure of animals to TCDD induces a spectrum of toxic responses, including thymic involution, teratogenesis, tumor promotion, wasting, and epithelial hyperplasia and metaplasia, as well as xenobiotic metabolism (12, 13, 14, 15). One of most drastic effects of TCDD in vivo is the reduction of thymocyte number followed by immunosuppression (16). Kremer et al. (17, 18) have reported that TCDD-induced thymic atrophy is caused by reduced proliferation of immature thymocytes, leading to the reduction of CD4⁺CD8⁺ double-positive (DP) thymocytes. They have also shown that TCDD accelerates T cell maturation and skews thymocyte differentiation towards mature CD4^-CD8⁺ single-positive cells. In contrast, Kamath et al. (19, 20, 21) have reported that apoptosis plays a major role in TCDD-induced thymic atrophy. What types of cell are responsible for mediating these TCDD effects in the thymus is also controversial; some reports have suggested thymic stromal cells, including thymic epithelial cells (17, 22, 23, 24), whereas others have suggested hemopoietic cells including T cell lineage thymocytes (19, 25, 26, 27, 28) and earlier T cell progenitors (29). Nonetheless, how TCDD causes thymus involution is unclear.

The generation of AHR-deficient mice has extended the understanding of the molecular mechanisms of the effects of TCDD in mammals (30, 31, 32). Using AHR-deficient mice, it has been shown that AHR-deficient mice are viable, and that AHR is essential for TCDD-induced thymic involution (33). In contrast, systemic ARNT-null mice were embryonic lethal, indicating that, unlike AHR, ARNT plays an essential role in mammalian development (34, 35). However, it was not clear whether ARNT may also be involved in thymic involution. In order to examine a role of ARNT in adult tissues, we have recently generated conditional Arnt knockout mice by the using Cre-loxP strategy (36). We have previously shown that ARNT is essential for AHR- and HIF-1-mediated signal transduction in the liver cells in vivo (37).

In the present study, we have generated T cell-specific ARNT knockout mice by ablating the Arnt-floxed allele using Cre recombinase driven by the T cell-specific p56^lck proximal promoter. We show the following: 1) ARNT in T lineage cells is dispensable for T cell development and TCR responses, and 2) ARNT in T cells is essential for TCDD-mediated thymic involution.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

Mice carrying three identically oriented lox-P in the Arnt gene have been previously reported (37). These mice were bred with EIIa-Cre transgenic mice, which carry the Cre transgene under the control of the adenovirus EIIa promoter that targets expression of the Cre recombinase to the early mouse embryo (38), to delete the PGK-neo cassette (PGK-neo) in vivo, leaving either the allele with two loxP sites upstream and downstream of the exon 6 (flox allele) or the allele with one single loxP site at disrupted exon 6 ( allele) (see Fig. 1A). Arnt^+/ mice were bred with T-cell-specific p56^lck-Cre transgene transgenic mice (39) to generate mice carrying lck-Cre and heterozygous Arnt (Lck-Cre;Arnt^+/) genes. Lck-Cre;Arnt^+/ mice were mated with Arnt^flox/flox mice to generate Lck-Cre;Arnt^flox/ and Lck-Cre;Arnt^flox/+ mice. Genotypings were performed by PCR as described previously (37, 39). K5-Cre;Arnt^flox/ mice were also established with K5-Cre transgenic mice (40, 41) in the same way as described above.

View larger version (32K): [in this window] [in a new window]   FIGURE 1. Generation of a T cell-specific Arnt knockout mouse. A, Construction of the targeting vector. a, Targeted allele containing three loxP sites and neo gene. Cre-mediated deletion with EIIa-Cre transgenic mice in vivo, would produce Arntflox allele (b) and Arnt allele (c). Lck-Cre transgenic mice carrying Arnt+/ were crossed with Arntflox/flox mice to produce mice containing the Arntflox/ gene and Lck-Cre transgene, in which the Arnt gene should be deleted specifically in T cells. The hatched triangles represent loxP sites. The small arrow above the PGK-neo cassette shows the direction of neo transcription that is opposite to the direction of Arnt transcription. The BamHI restriction fragments detected with the 200-bp probe located in intron 4 are denoted by the double-ended arrows. The restriction sites are abbreviated as follows: A, AvrII; B, BamHI; E, EcoRI; S, SacI; X, XhoI. B, Southern blot analysis of genomic DNA from different tissues from Lck-Cre;Arntflox/ mice (upper panel). PCR genotyping of Lck-Cre;Arntflox/ mice using primers a, b, and c in A (lower panel). C, Northern blot analysis of ARNT transcripts. To confirm phenotype of Arnt-deficient mice, total RNA (10 µg) of two independent thymocyte samples from Lck-Cre;Arnt+/ or Lck-Cre;Arntflox/ mice was subjected to Northern blot analysis. -Actin was used as a loading control. D, Western blot analysis of ARNT protein. Thymocyte homogenate (50 µg of protein) was subjected to Western blot analysis. The anti-ARNT Ab detects a band migrating to molecular mass of 95-kDa in the wild-type or Arnt heterozygous thymocytes. Heterozygous thymocytes express a smaller amount of the protein than control thymocytes. Lck-Cre;Arntflox/ thymocytes do not express a detectable amount of ARNT. Lower bands represent nonspecific immunoreaction.

TCDD or B(a)P was administered to 5-wk-old mice as a single i.p. injection in a total volume of 100 µl of olive oil. Lck-Cre;Arnt^flox/+ and Lck-Cre;Arnt^flox/ mice received a dose of TCDD or B(a)P corresponding to 50 µg/kg or 2 mg/ml total body weight, respectively (20, 21, 42). In the fetal thymus organ culture (FTOC) experiment, day-17 fetal thymus lobes were cultured in organ in the presence or absence of 50 nM TCDD (43) for 5 days.

FTOC

Fetal thymus lobes from day 17 pregnant mice were prepared and cultured in RPMI 1640-based culture medium containing 10% FBS, 50 µM 2-ME, 2 mM L-glutamine, 1x nonessential amino acids (Invitrogen, Carlsbad, CA), 10 mM HEPES, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin as described previously (44). Thymocytes were prepared after 5 days of culture by passing through nylon mesh and stained with Abs against CD4 and CD8.

T cell-stimulation in culture

Splenic lymphocytes were cultured in the RPMI 1640-based culture medium described above for 16 h. Where indicated, cultures contained indicated concentrations of Con A (Sigma-Aldrich, St. Louis, MO), anti-CD3, and anti-CD28 mAbs (R&D Systems, Minneapolis, MN), PMA (Sigma-Aldrich), and calcium ionophore ionomycin (Sigma-Aldrich).

Multi-color flow cytometry analysis

For two-color immunofluorescence staining, cells were stained with FITC-labeled Ab and PE-labeled Ab. Labeled mAbs were obtained from B.D. Pharmingen (San Diego, CA). The detected cells on the cytometric profiles were selected and shown as live lymphocytes by electronically gating forward scatter/side scatter and propidium iodide.

Western blotting analysis

Thymocytes was homogenized with lysis buffer containing 20 mM HEPES-KOH (pH 7.4), 25% glycerol, 420 mM NaCl, 0.2 mM EDTA, 1 mM DTT, 0.2 mM PMSF, one tablet of Complete protease inhibitor mixture (Roche, Basel, Switzerland)/50 ml of buffer and 0.5% NP-40. Twenty micrograms of protein homogenate were electrophored on a SDS-polyacrylamide gel, transferred to a nylon membrane, and blotted with anti-mouse ARNT Ab (Affinity Bioreagents, Golden, CO) followed by HRP-conjugated anti-rabbit Ig (Amersham-Pharmacia Biotech, Piscataway, NJ). Signals were visualized with an ECL system (Amersham-Pharmacia Biotech).

RNA and DNA analysis

Northern blots were usually performed on 10 µg of total RNA with ³²P-labeled cDNA or PCR-generated DIG (Roche)-labeled probe. Full-length cDNA probes for ARNT (37), AHR (32), HIF-1 (37), and CYP1A1 (5) were previously described. RT-PCR of mouse liver RNA was used for production of a 738-bp probe for the mouse CYP1B1 mRNA (F, 5'-GGC GTT CGG TCA CTA CTC TG-3'; R, 5'-AGG TTG GGC TGG TCA CTC AT-3') (45). Briefly, 1 µg of total RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen) as per the manufacturer’s instructions using the indicated reverse primer. Twenty percent of the cDNA synthesis reaction was used as a template for a PCR consisting of 2.5 U of Vent DNA polymerase (New England Biolabs, Beverly, MA), 2.5 mM MgCl₂, 200 µM each dNTP, and 1 µM each gene-specific primer in a final volume of 50 µl. The following thermal-cycling profile was used for all PCRs: 1 cycle of 5 min at 95°C; 30 cycles of 45 s at 95°C, 45 s at 60°C, and 45 s at 72°C; and 1 cycle of 72°C for 6 min. PCR products were cloned into the PCRII-Topo vector (Invitrogen), analyzed by gel electrophoresis, and sequenced to confirm identities. Probes were ³²P-labeled by the random-primer method using Ready-to-Go DNA labeling beads (Amersham-Pharmacia Biotech). Probes were added to the hybridization buffer (1 x 10⁶ cpm/ml) and incubation at 42°C was continued overnight. The blots were then washed once with 2x SSC/1% SDS at 42°C for 15 min, twice with 0.1x SSC/1% SDS at 65°C, and once with 0.1x SSC at room temperature.

To detect the extent of Cre-mediated recombination in different tissues of Lck-Cre;Arnt^flox/ mice, genomic DNA (10 µg) from each tissue was digested with BamHI and subjected to electrophoresis on a 0.8% agarose gel, transferred to nylon membrane, and hybridized with the ³²P-labeled SalI to XbaI fragment shown in Fig. 1A. The estimated sizes of the fragments containing the floxed allele with PGK-neo cassette, the allele lacking PGK-neo cassette, and the deleted exon 6-deleted allele are 8.0, 6.0, and 4.5 kb, respectively.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation ofLck-Cre;Arnt^flox/ mice

Inactivation of the Arnt gene in ES cells by systemic targeting led to early embryonic lethality and could not reveal its role in adult tissues (34, 35). Recently, we have reported successful generation of mice including the PGK-neo cassette, flanked by loxP sites and inserted into intron 5 of the Arnt gene (Fig. 1A) (37). To eliminate possible artifacts by the gene-targeting process, we deleted the selection cassette (PGK-neo) leaving two loxP sites upstream and downstream of the intact exon 6, by crossing with EIIa-Cre transgenic mice in vivo (Arnt^flox allele) (Fig. 1A). We have also generated an exon 6 disruption of the Arnt gene (Arnt allele) (Fig. 1A). The Cre transgene was to be expressed in mice carrying the Arnt^flox/ gene in order to increase the efficiency of Cre-mediated Arnt disruption and decrease Cre-mediated interchromosomal recombination. Indeed, to generate mice in which ARNT would be deficient in T cell-specific manner, Arnt^flox/flox mice were mated with Lck-Cre;Arnt^+/ mice that carried both the lck promoter-driven Cre transgene and the Arnt^+/ gene. Their offspring, Lck-Cre;Arnt^flox/+ and Lck-Cre;Arnt^flox/ mice, were used for further analyses. Arnt wild-type Lck-Cre;Arnt^+/+ and Arnt heterozygous Lck-Cre;Arnt^flox/+ mice showed the same normal phenotype as far as we have tested (data not shown). The lck promoter directs gene expression specifically in immature T cells, so that a floxed gene should be disrupted throughout immature and peripheral T cells (39, 46). Mice with Lck-Cre and the floxed-Arnt gene (Lck-Cre;Arnt^flox/+ and Lck-Cre;Arnt^flox/) were genotyped for Arnt gene, by genomic PCR using the primers in Fig. 1A.

To evaluate the efficiency and tissue specificity of the Cre-mediated deletion of the floxed gene, we performed Southern blot and PCR genotyping analyses of several tissues (thymocytes, kidney, brain, heart, intestine, spleen, liver, and lung) on Lck-Cre;Arnt^flox/ mice (Fig. 1B). The Arnt exon 6 was deleted completely and specifically in thymocytes, because the 6.0-kb band corresponding to Arnt-floxed allele was not detected by Southern blot analysis. The 6.0-kb band for the other tissues did not seem to be decreased, consistent with the previous reports that the specificity of Cre-mediated recombination was restricted in T lineage cells (39, 46). Thus, disruption of floxed Arnt in Lck-Cre;Arnt^flox/ mice was successful in terms of efficiency and specificity in thymocytes. Northern blot analysis demonstrated the absence of wild-type ARNT mRNA in thymocytes of Lck-Cre;Arnt^flox/ mice (Fig. 1C). Western blot analysis using an Ab specific for the C-terminal portion of ARNT also confirmed the absence of wild-type ARNT protein in thymocytes of Lck-Cre;Arnt^flox/ mice (Fig. 1D). These results indicate that Lck-Cre;Arnt^flox/ mice exhibit a complete dysfunction of ARNT in thymocytes.

Lck-Cre;Arnt^flox/ mice were born at Mendelian frequency, appeared healthy, and showed no increase in mortality after birth. No significant difference in body weight was observed between these mice and control littermates (data not shown). The mutant mice were fertile and produced normal-sized litters.

TCDD-induced gene expression in ARNT-deficient thymocytes

To examine ARNT function in Lck-Cre;Arnt^+/ mice, TCDD-mediated responses of CYP1A1 and CYP1B1 gene expression were analyzed by Northern blot analysis. Specific bands were observed for both isoforms of CYP1 genes in liver cells and isolated thymocytes from TCDD-treated Lck-Cre;Arnt^+/ mice (Fig. 2). However, the band of CYP1A1 mRNA was undetectable in thymocytes from TCDD-treated Lck-Cre;Arnt^flox/ mice, although this gene was induced in the liver from the same mice. CYP1B1 was also barely responsive to TCDD treatment in thymocytes from Lck-Cre;Arnt^flox/ mice (Fig. 2). These results indicate that ARNT is essential for TCDD-induced expression of xenobiotic-metabolizing enzymes, confirming the specific disruption of the Arnt gene in thymocytes.

View larger version (47K): [in this window] [in a new window]   FIGURE 2. Expression of CYP1A1 and CYP1B1 mRNA in thymocytes and liver from TCDD-treated Lck-Cre;Arntflox/ and Lck-Cre;Arnt+/ mice. Total RNA (10 µg) of the TCDD-treated or untreated thymocytes (indicated as T) and liver (indicated as L) from Lck-Cre;Arnt+/ or Lck-Cre;Arntflox mice were subjected to Northern blot analysis. Mice were injected with 50 µg/kg of body weight of TCDD or olive oil as a vehicle control. GAPDH was used as a loading control.

T cell development inLck-Cre;Arnt^flox/ mice

We then examined whether ARNT deficiency in T lineage cells might affect T cell development in the thymus. As shown in Fig. 3A, total nucleated lymphoid cell numbers in the thymus and spleen in Lck-Cre;Arnt^flox/ mice were comparable with those of Lck-Cre;Arnt^flox/+ mice. In addition, flow-cytometric analysis of the cells from these organs revealed that thymocyte numbers and CD4/CD8 ratios in T cells of Lck-Cre;Arnt^flox/ mice were not significantly different from those of Lck-Cre;Arnt^flox/+. These results indicate that ARNT in T cells is not essential for the development of T cells in mice.

View larger version (37K): [in this window] [in a new window]   FIGURE 3. Phenotypic analyses for the expression of surface markers and TCR responsiveness of T cells in Lck-Cre;Arntflox/ and Lck-Cre;Arnt+/ mice. A, Flow-cytometric analysis of cells from lymphoid organs of Lck-Cre;Arntflox/ and Lck-Cre;Arnt+/ mice. Thymocytes and spleen cells were obtained from 5-wk-old mice with the indicated genetic characteristics. Cell numbers of each CD4/CD8 subset were determined by FACS analyses. Three independent experiments were performed for each genotype. Data are the means ± SD of cell numbers for the indicated cell subsets. B, Initiation of TCR signals provoking early cellular responses. After 16 h in culture with either Con A (2.5 µg/ml) or combination of anti-CD3 and anti-CD28 Abs, spleen lymphocytes were two-color stained with PE-labeled anti-CD4 Ab and FITC-labeled anti-CD25 or anti-CD69 or normal IgG. The profiles of CD25 and CD69 expression are shown by electronically gated CD4+ cells. Numbers indicate the mean fluorescence intensity of CD25 and CD69 within the gates indicated by the bars.

TCR responsiveness ofLck-Cre;Arnt^flox/ T cells in vitro

As shown in Fig. 3B, T cells from Lck-Cre;Arnt^flox/ mice exhibited normal increases in CD25 (IL-2R) and CD69 upon stimulation with either the combination of anti-CD3 and anti-CD28 Abs, or Con A, indicating that T cells lacking ARNT can normally transmit TCR signals provoking early cellular responses. Thus, ARNT-deficient T cells can initiate TCR engagement.

Effects of TCDD on the thymuses

Thymic atrophy associated with TCDD exposure is generally characterized by severe regression of the cortex (23, 47, 48). To investigate the role of ARNT in TCDD-induced thymic involution, we next administered TCDD into Lck-Cre;Arnt^flox/ or Lck-Cre;Arnt^+/ mice. Interestingly, thymuses of Lck-Cre;Arnt^flox/ mice upon the treatment of TCDD were not atrophic, although severe thymic atrophy was observed in TCDD-treated Lck-Cre;Arnt^+/ mice (Fig. 4A). Thymuses obtained from TCDD-treated Lck-Cre;Arnt^flox/ mice did not exhibit any cortical atrophy (Fig. 4B, g and h vs e and f), whereas control mice expressing functional ARNT in thymocytes (Lck-Cre;Arnt^+/) experienced a significant reduction in size and cellularity of thymic cortical areas by the exposure to TCDD (Fig. 4B, c and d vs a and b).

View larger version (75K): [in this window] [in a new window]   FIGURE 4. Morphological analysis of TCDD-treated thymuses from Lck-Cre;Arntflox/ and Lck-Cre;Arnt+/ mice. Macroscopic (A) and microscopic (B) appearances of thymuses from 5-wk-old Lck-Cre;Arntflox/ and Lck-Cre;Arnt+/ mice treated with or without TCDD. Mice were injected with 50 µ/kg of body weight of TCDD or olive oil as a vehicle control. Thymuses were obtained 3 days after treatment with TCDD. Histological analysis was performed using H&E staining. Bar, 200 µm. M, Thymic medullar region; C, thymic cortex region.

flox/

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View larger version (41K): [in this window] [in a new window]   FIGURE 5. Effect of TCDD and B(a)P on T cell development in Lck-Cre;Arntflox/ and Lck-Cre;Arnt+/ mice. A and B, Effects of TCDD (A and B) on thymocyte numbers (A) and T cell subsets (B). Five-week-old mice were injected with 50 µg/kg of body weight of TCDD or olive oil as a vehicle control. Data in bar graphs are the means ± SE of thymocyte number in Lck-Cre;Arntflox/ (flox/+) or Lck-Cre;Arnt+/ (flox/) mice. Flow-cytometric analyses of T cell subsets in the thymuses were performed 3 days after TCDD treatment. Thymocytes from the treated mice were two-color stained with FITC-labeled CD4 Ab (x-axis) and PE-labeled CD8 antibody (y-axis), to analyze T cell subsets according to the expression of CD4/CD8. The data are representative dot blots. Numbers in each box indicate the frequency of cells within that box. The experiments were performed independently three times, and the results regarding each T cell subset and the cell ratio are shown in each graph. * and **, p < 0.05 and p < 0.01, respectively. C and D, Effect of B(a)P on thymocyte numbers and T cell subsets. Mice were injected with 2 mg/kg of body weight B(a)P or olive oil as a vehicle control. Flow-cytometric analysis of T cell subsets (CD4/CD8) in thymuses were performed described above. * and **, p < 0.05 and p < 0.01, respectively. E, Dex-induced thymic involution in Lck-Cre;Arntflox/ (flox/+) and Lck-Cre;Arnt+/ (flox/) mice. Thymocytes from Dex-treated mice were analyzed in the same manner as described above. Three independent experiments were performed.

To further address the specificity of cellular targets of TCDD effects in the thymus, FTOC was performed using epithelial cell-specific Arnt-disrupted mice (K5-Cre;Arnt^flox/) as well as T cell-specific Arnt-disrupted Lck-Cre;Arnt^flox/ mice. K5-Cre;Arnt^flox/ mice, whose epithelial cells lack functional ARNT, were born in a Mendelian manner. However, these mice died within 24 h after birth due to a severe dehydration caused by water loss from skin surfaces (S. Takagi, H. Tojo, S. Tomita, S. Sano, S. Itami, M. Hara, S. Inoue, K. Horie, G. Kondoh, K. Hosokawa, F. Gonzalez, and J. Takeda, manuscript in preparation). Nonetheless, FTOC of these mice was useful to examine the role of ARNT in thymic epithelial cells. As shown in Fig. 6A, flow-cytometric analysis of Lck-Cre;Arnt^flox/ and K5-Cre;Arnt^flox/ mice revealed that CD4/CD8 distribution in FTOC was normal in these mutant mice, indicating that ARNT in T lineage cells or thymic epithelial cells is not essential for T cell development in the thymus. Interestingly, TCDD treatment decreased the numbers of total thymocytes and CD4⁺CD8⁺ DP cells in K5-Cre;Arnt^flox/ FTOC, although no significant decrease was observed in those in Lck-Cre;Arnt^flox/ FTOC (Fig. 6B). It should be noted that the number of thymocytes in Lck-Cre;Arnt^flox/ FTOC was consistently lower than that of control Lck-Cre;Arnt^flox/+ FTOC in the absence of TCDD, perhaps due to the lack of HIF-1/ARNT-mediated cell survival signals in FTOC (see Discussion). CD4/CD8 phenotype in FTOC from Lck-Cre;Arnt^flox/ mice was not altered by TCDD, whereas CD4⁺CD8⁺ thymocytes in FTOC from K5-Cre;Arnt^flox/ mice were markedly susceptible to TCDD (Fig. 6A). These results indicate that TCDD-induced thymic involution is dependent on ARNT-mediated signals in thymocytes rather than thymic epithelial cells.

View larger version (59K): [in this window] [in a new window]   FIGURE 6. Effects of TCDD in FTOC derived from Lck-Cre;Arntflox/ and K5-Cre;Arntflox/ mice. Day-17 fetal thymus lobes from Lck-Cre;Arntflox/ and K5-Cre;Arntflox/ mice were cultured for 5 days as intact organs in the absence or presence of 50 nM TCDD. A, Thymocytes from FTOC were two-color stained with FITC-labeled anti-CD4 Ab (x-axis) and PE-labeled anti-CD8 Ab (y-axis). Indicated data are representative dot blots from three independent measurements. Numbers in each box indicate the frequency of cells within that box. B, Alteration of the numbers of thymocytes for total cells and CD4/CD8 subsets by the treatment of TCDD. Thymocytes from FTOC were measured for viable cell numbers using trypan blue dye exclusion method. Individual lines indicate the data from thymus lobes from one fetus, in the absence or presence of TCDD.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

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Our results showing that ARNT in T cells is essential for TCDD-mediated thymic involution support the possibility that AHR/ARNT-dependent signal in T lineage cells is essential for mediating TCDD toxicity to the thymus, suggesting that thymocytes are the direct target cells for TCDD action to the thymus. Our results further show that T cell-specific ARNT-deficient FTOC from Lck-Cre;Arnt^flox/ mice was resistant to TCDD treatment, whereas epithelial cell-specific ARNT-deficient FTOC from K5-Cre;Arnt^flox/ mice was susceptible to TCDD treatment. These data argue that TCDD-mediated thymic involution is mediated by TCDD stimulation of the AHR/ARNT signal pathway in thymocytes rather than thymic stromal cells including epithelial cells.

B(a)P is another AHR ligand that mediates teratogenic and carcinogenic effects (42), and it has been shown that B(a)P can also cause severe thymic involution (49). Interestingly, our results show that B(a)P-mediated thymic involution was still observed in Lck-Cre;Arnt^flox/ mice, in which thymuses were completely resistant to TCDD. In contrast, the thymuses in AHR-deficient mice were resistant to B(a)P treatment in vivo (S. Maekawa, S. Tomita, and Y. Takahama, unpublished data). These results indicate that, unlike TCDD, B(a)P-mediated thymic involution is dependent on AHR, but can occur, at least partially, independent of ARNT in T lineage cells.

It should be noted that FTOC from Lck-Cre;Arnt^flox/ mice were markedly smaller than control FTOC, even though these FTOC thymuses were resistant to TCDD treatment. We have noticed that thymocyte numbers of freshly isolated fetal thymuses from Lck-Cre;Arnt^flox/ mice were comparable to those from fetal thymuses of age-matched normal mice (data not shown). Thus, we think that the reduction of thymocyte numbers likely occurred during the 5-day organ culture condition. During the development of fetal thymus in the organ culture condition, the number of thymocytes and the volume of thymus lobes are indeed increased in culture (as shown in Fig. 6B). However, the cells in the central core region of the cultured thymus lobes have been observed to be dead or necrotic, suggesting that oxygen supply in the conventional organ culture condition is not good enough to support the cells in the central core region of the thymus lobes, and that the cells in the central core region may die by hypoxia in the organ culture condition (50). By the lack of HIF-1/ARNT-mediated hypoxic responses in ARNT-deficient thymocytes in the thymus lobes, the cells in the FTOC may not be able to survive efficiently, thereby resulting in the reduced numbers of thymocytes after the organ culture.

ARNT is abundantly expressed in immune tissue including the thymus (51, 52, 53, 54). However, the present results reveal no role for ARNT in the development of functional thymus or T cells. Nonetheless, it should be interesting to note that the IL-2 gene has been shown to be induced directly by the TCDD-liganded AHR/ARNT complex via the xenobiotic responsive element sequence that is located upstream of the IL-2 gene in mouse lymphocytes (55). There are an additional three E-box core sequences, to which ARNT homodimer could bind (56), and the xenobiotic responsive element sequence that is at a distal region (-1300 to -800) of the mouse IL-2 promoter (57). These reports suggest that the AHR/ARNT heterodimer and/or ARNT homodimer could regulate IL-2 transcription and are involved in the activation of T cells. In vivo and in vitro analyses of T cell functions in the T cell-specific ARNT-deficient mice are currently in progress.

In conclusion, in the present study, we established a T cell-specific ARNT-deficient mouse, by which we could identify that ARNT in T cells is essential for TCDD-induced thymic involution. These mice appear normal in terms of T cell development in the thymus. We have identified that TCDD-induced thymic atrophy is mediated by ARNT in T cells rather than in thymic stromal cells including thymic epithelial cells. These mice should serve as a useful model system to understand how environmental pollutants, including TCDD and B(a)P, cause thymic involution and subsequently compromise the immune system.

	Acknowledgments

 
We thank Dr. N. Ueda at Kagawa Medical University for reviewing the manuscript.

	Footnotes

 
¹ This work was supported in part by grant-in-aid from the Ministry of Education, Science, Sports and Culture.

² Address correspondence and reprint requests to Dr. Shuhei Tomita, Division of Experimental Immunology, Institute for Genome Research, University of Tokushima, 3-18-15 Kuramoto, Tokushima 770-8503, Japan. E-mail address: tomita{at}genome.tokushima-u.ac.jp

³ Abbreviations used in this paper: ARNT, arylhydrocarbon receptor nuclear translocator; AHR, arylhydrocarbon receptor; HIF-1, hypoxia-inducible factor-1; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; Lck-Cre, T cell-specific p56^lck-Cre transgene; FTOC, fetal thymus organ culture; Dex, dexamethasone; DP, double positive; B(a)P, benzo(a)pyrene.

Received for publication March 17, 2003. Accepted for publication August 6, 2003.

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