2,3,7,8-Tetrachlorodibenzo-p-dioxin Causes Alterations in Lymphocyte Development and Thymic Atrophy in Hemopoietic Chimeras Generated from Mice Deficient in ARNT2

Michael D. Laiosa*, Zhi-Wei Lai*, T. Scott Thurmond{dagger}, Nancy C. Fiore*, Charles DeRossi{ddagger}, Bernadette C. Holdener{ddagger}, Thomas A. Gasiewicz{dagger} and Allen E. Silverstone*,1

* Department of Microbiology and Immunology, State University of New York, Upstate Medical University, 750 East Adams Street, Syracuse, New York 13210; {dagger} Environmental Health Science Center, Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, New York 14642; and {ddagger} Department of Biochemistry and Cell Biology and the Institute for Cell and Developmental Biology, State University of New York at Stony Brook, Stony Brook, New York 11794

Received February 28, 2002; accepted May 31, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is well established that dioxins cause a variety of toxic effects and syndromes including alterations of lymphocyte development. Exposure to the prototypical dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to severe thymic atrophy in all species studied. It has been shown that most of this toxicity is due to TCDD binding to and activating the aryl hydrocarbon receptor (AHR). Upon activation, the AHR enters the nucleus, dimerizes with the AHR nuclear translocator (ARNT), and this heterodimer modulates a number of genes that mediate toxicity. The AHR and ARNT are members of the basic-helix-loop-helix-Per, ARNT, and Sim homology (bHLH-PAS) family of transcription factors. In this study, we wanted to determine if another bHLH-PAS transcription factor, ARNT2, which has high amino acid sequence identity to ARNT and has been shown to dimerize with the TCDD-activated AHR, is involved in mediating TCDD's effect on lymphocyte development. We determined by RT-PCR that ARNT2 is expressed at a low level in whole thymus, thymocytes, and bone marrow lymphocytes. We created hemopoietic chimeras by lethally irradiating C57BL/6 mice and reconstituting them with fetal liver stem cells that either have or are deficient in a portion of chromosome 7 that contains ARNT2. Regardless of whether chimeras possessed or lacked this chromosome fragment, equal sensitivity to TCDD-induced thymic atrophy was observed despite expression of ARNT2 in the thymus. Furthermore, the absence of ARNT2 (or any other genes found on this portion of chromosome 7) did not confer any protection against TCDD-induced alterations in bone marrow B-cell subsets. These data indicate that in this model system the effects of TCDD-induced thymic atrophy and alterations in B-cell maturation are not dependent on an AHR-ARNT2 heterodimer.

Key Words: TCDD; dioxin; ARNT2; thymus; bone marrow; lymphocyte development; bHLH-PAS; AHR.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The industrial and environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes thymic atrophy in every species studied (de Heer et al., 1994Go; Esser, 1994Go; Kerkvliet, 1994Go; Silverstone et al., 1994Go). In C57BL/6 (B6) mice, a single dose of 30 µg/kg TCDD produces a marked reduction in thymic weight and cellularity, reaching maximal atrophy 10–12 days postinjection. Full recovery of the thymus to normal size does not occur until 35–40 days postinjection (Murante and Gasiewicz, 2000Go; Silverstone et al., 1994Go; Staples et al., 1998aGo).

Most, if not all of the toxicological effects of TCDD are mediated by the activation of the aryl hydrocarbon receptor (AHR). The AHR is a member of the basic-helix-loop-helix-Per, ARNT, and Sim (bHLH-PAS) family of transcription factors (Hankinson, 1995Go; Rowlands and Gustafsson, 1997Go; Schmidt and Bradfield, 1996Go) and is known to be activated by binding a wide variety of planar halogenated organic hydrocarbons (PHAH) of which TCDD is the prototypical and most potent family member (Kerkvliet, 1995Go; Schmidt and Bradfield, 1996Go). In the nucleus, the ligand activated AHR heterodimerizes with another member of the bHLH-PAS family, the AHR nuclear translocator (ARNT; Hoffman et al., 1991Go; Swanson et al., 1995Go). The AHR-ARNT heterodimer activates a wide variety of genes that contain dioxin responsive elements, (DREs) in their promoter regions. The genes regulated by the ligand activated AHR-ARNT heterodimer belong to a number of different gene families and include detoxification enzymes, growth factors, cytokines, cell cycle regulatory genes, and transcription factors (Kolluri et al., 1999Go; Lai et al., 1996Go; Rowlands and Gustafsson, 1997Go). Ligand activation of the AHR and heterodimerization with ARNT has been implicated in mediating the majority of the toxic effects of TCDD including alterations in lymphocyte development (Rowlands and Gustafsson, 1997Go). Despite a fundamental understanding of the role of the AHR in TCDD toxicity, a biological function of the AHR remains elusive.

During thymocyte development, stem cells from either the fetal liver or bone marrow (Rebel et al., 1996Go; Sanhadji et al., 1992Go) enter the thymus where they become fully committed T-cell precursors (Tourigny et al., 1997Go). At the first stages of development, thymocytes lack the surface expression of CD4 and CD8 molecules and are referred to as double negative (DN) cells (CD4-CD8-; Robey and Fowlkes, 1998Go). As the DN cells mature they upregulate both the CD4 and CD8 molecules and are called double positive (DP) cells (Sebzda et al., 1999Go). DP thymocytes go through a process of thymic selection and then differentiate into either the CD4 or CD8 single positive (SP) lineages (Anderson et al., 1999Go).

Following exposure to TCDD, there is a significant increase in the proportion of DN thymocytes, a slight decrease in DP thymocytes, and a significant increase in the CD8 SP T cells when compared with thymocytes from vehicle-treated controls (Lai et al., 1998Go; Silverstone et al., 1994Go; Staples et al., 1998bGo). Although the mechanism underlying these effects on the thymus has been controversial for some time, we have shown that TCDD directly activates the AHR in hemopoietic cells in the thymus and bone marrow to cause these alterations of lymphocyte development (Staples et al., 1998bGo).

Along with the effects of TCDD on the thymus and bone marrow stem cells, B lymphocytes are also known to be sensitive targets of TCDD. TCDD depresses pre-B and immature B lymphocyte cell numbers in mouse bone marrow following TCDD treatment (Thurmond et al., 2000Go), and it has been shown that TCDD reduces the mRNA expression of the lymphocyte stem cell specific enzymes terminal deoxynucleotidyl transferase, (TdT) and the recombination activation gene-1, (RAG-1) in murine bone marrow (Fine et al., 1989Go; Silverstone et al., 1994Go). Despite a growing body of evidence demonstrating that TCDD affects lymphocyte development through direct activation of the AHR in developing lymphocytes, the potential role of other bHLH-PAS proteins (ARNT, ARNT2) in mediating TCDD's effect on lymphocyte development has not been fully investigated.

Efforts over the past decade to identify additional bHLH-PAS proteins have produced more than a dozen additions to this gene family (Carver and Bradfield, 1999Go) raising the possibility that the classical model of AHR activation by TCDD and binding to ARNT could be expanded to include the activity of some of these newly identified bHLH-PAS genes. When compared to ARNT, one bHLH-PAS family member, ARNT2, has 57% amino acid sequence identity overall (Drutel et al., 1996Go; Hirose et al., 1996Go) and 78% amino acid identity in the conserved bHLH and PAS regions (Drutel et al., 1996Go; Hirose et al., 1996Go). Furthermore, the AHR has been shown to heterodimerize with ARNT2 and transactivate DRE-containing genes (Hirose et al., 1996Go). ARNT2 expression was found by in situ hybridization and Northern blotting to be most pronounced in the kidney and brain (Drutel et al., 1996Go; Jain et al., 1998Go; Petersen et al., 2000Go), and it may play a role in neuronal cell survival (Drutel et al., 1999Go) and responses to hypoxia (Keith et al., 2001Go; Maltepe et al., 2000Go). The mechanism by which ARNT2 accomplishes these functions appears to be through heterodimerization with the bHLH-PAS transcription factors AHR, ARNT, and hypoxia inducible factors (HIF1-{alpha}, HIF2-{alpha}, and HIF3-{alpha}; Jain et al., 1998Go; Maltepe et al., 2000Go; Petersen et al., 2000Go). Despite extensive research on ARNT2 expression and function, little attention has been paid to any potential role it may have in TCDD toxicity.

We set out to determine the expression of ARNT2 in the thymus and bone marrow and found that it is expressed in the thymus at low levels compared with the expression in the brain. Given the high amino acid sequence identity between ARNT and ARNT2 and given that the AHR can heterodimerize with ARNT2, we hypothesized that if ARNT2 was acting to mediate some of the effects of TCDD on lymphocyte development in wild type mice, then mice that lack ARNT2 should be less sensitive to the toxic effects of TCDD. To test this hypothesis we generated hemopoietic chimeras from the radiation-induced mutant c112K mouse line (Erickson et al., 1968Go; Gluecksohn-Waelsch, 1979Go; Russell et al., 1979Go), which lacks ARNT2 (Wines et al., 1998Go), and tested their response to TCDD by evaluating several lymphocyte developmental parameters, including thymic atrophy and bone marrow alterations. If the c112K mutant chimeras were less sensitive to TCDD-induced thymic atrophy, this would suggest that ARNT2 (or other genes within the c112K deletion) are mediating the effects of TCDD on lymphocyte development. Alternatively, if TCDD still caused full thymic atrophy in c112K chimeras, then we would be able to rule out ARNT2 and other genes associated with the c112K mutation as necessary for TCDD-induced thymic atrophy.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
The c112K mutation maps to the albino locus on the murine chromosome 7 (Erickson et al., 1968Go; Gluecksohn-Waelsch, 1979Go; Russell et al., 1979Go). Mutations in the albino locus were created nearly 50 years ago as part of the specific locus test to identify radiation-induced chromosomal rearrangements at visible loci in mice (Russell, 1951Go; Russell and Russell, 1959Go). The albino deletions were induced in the germ line of (101/R1 x C3HF/R1) males (Russell, 1951Go; Russell and Russell, 1959Go). The c112K mice used in our study originated at Oak Ridge National Laboratory and were maintained as heterozygotes over a cch allele from a noninbred St2A strain (Rikke et al., 1997Go). Mice homozygous for the c112K deletion die within 6 h of birth due to liver and kidney defects (Gluecksohn-Waelsch, 1979Go; Russell and Raymer, 1979Go; Russell et al., 1979Go). The genetic loci deleted on chromosome 7 associated with the c112K mutation include the genes Arnt2, Fah, Tyr, Fzd4, and Mod2 (DeRossi et al., 2000Go; Wines et al., 1998Go). We chose to use the c112K mutant in generating hemopoietic chimeras because the ARNT2 gene was mapped to this chromosome 7 deletion (Wines et al., 1998Go).

Timed pregnant c112K heterozygotes were bred at SUNY Stony Brook with identification of vaginal plugs considered gestational day (GD) 0.5. Pregnant dams were shipped to the Upstate Medical University between GD 13.5 and 16.5. Mice were housed in isolation and euthanized at GD 18.5 for production of hemopoietic chimeras.

RT-PCR.
Total RNA was extracted from bone marrow lymphocytes, thymocytes, whole thymus, or brain tissue using TRIzol reagent (Life Technologies Inc., Gaithersburg, MD) according to the manufacturer's instruction. Two µgs of isolated RNAs were treated with DNase I Amplification Grade (Life Technologies, Grand Island, NY) for 15 min at room temperature in a volume of 10 µl DEPC-H2O, followed by the addition of 2.5 mM EDTA and a 10 min incubation at 60°C. Reverse transcription was performed in final volume of 40 µl containing 1 µg of oligo polyd(T)15 primer (Pharmacia, Uppsala, Sweden), 20 units RNasin Ribonuclease Inhibitor (Promega, Madison, WI), 400 units M-MLV Reverse Transcriptase (Life Technologies), 1 x RT buffer (50 mM Tris–HCl, 75 mM KCl, 3 mM MgCl2), 10 mM DTT, and 1 mM each of deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP; Pharmacia). The reverse transcription reaction was carried out for 60 min at 37°C and then heated to 70°C for 10 min using a BIOMETRA TRIO-Thermoblock (Biometra Inc., Tampa, FL). Samples were cooled to 4°C and stored at –80°C until further analyzed. Before amplification of ARNT2, cDNA were checked by amplifying the house-keeping gene HPRT (data not shown).

PCR reactions were performed in 50 µl reaction volumes containing 5 µl cDNA (see above), 1 unit rTth DNA polymerase XL, (Roche, Indianapolis, IN), 15.2 µl 3.3X XL Buffer II (Roche), 1.5 mM MgCl2, 0.2 mM of each dNTP and 0.25 µM of each 5` and 3` primer. Amplifications were carried out in a BIOMETRA TRIO-Thermoblock for 5 min at 94°C before the first cycle, followed by 1 min cycles of 94°C for denaturation, 60°C for primer annealing, 72°C for extension, and a final 10 min extension at 72°C after the last cycle. The cycles for ARNT2 were 32, 36, and 40. 15 µl of PCR products from each sample were run in a 1.5% agarose gel and bands were visualized with ethidium bromide. The following primer sequences were used: 5`-GCTGGTGAAAAGGACCTCT-3` and 5`-CACAGGACTAGAACACCTGC-3` for HPRT; 5`-ACCCGAAGAAGATGCTGATGTC-3` and 5`-TGCCTGCTGTTGCTGAAGTTG-3` for ARNT2.

Production of hemopoietic chimeras.
Four- to five-week-old male C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and allowed to acclimate for 1 week. As previously described (Staples et al., 1998bGo), recipient mice were lethally X-irradiated with 2 doses of 5.5 Gy with a 4 h interval between each dose of radiation. During the 4 h interval, pregnant dams were euthanized and c112K fetuses were removed. The fetal livers were removed and cell suspensions were made using the flat end of a 1 ml syringe. Five pooled livers from either c112K mutant embryos (ARNT2-/-) or a mixture of wild type and heterozygote embryos (henceforth will be referred to as control) were used for reconstitution. The c112K mutant mice were differentiated from the control littermates by the complete absence of pigmentation in the eyes of the c112K homozygous mice (Erickson et al., 1968Go; Russell and Raymer, 1979Go). Fetal liver cell suspensions were depleted of erythrocytes by resuspending in 1 ml of ACK buffer (0.17 M NH4Cl, 10 mM KHCO3, and 1mM EDTA) and incubating for 4 min at room temperature. The ACK treated cell suspensions were washed once with MEM, 5% FBS and P/S, and prepared and counted as described below. After the cells were counted, they were resuspended in Minimal Essential Media (MEM) containing Hanks salts (Life Technologies, Grand Island, NY), and penicillin-streptomycin (P/S; 100 U/ml penicillin and 0.1 mg/ml streptomycin; Life Technologies), at a concentration of 1 x 107 cells/ml; 0.2 mls (2 x 106 cells) were then injected via tail vein into the irradiated recipients 30 min after the second dose of radiation. Reconstitution was allowed for 6 weeks to assure full restoration of the bone marrow and thymus lymphocyte populations. A radiation control mouse not given bone marrow cells perished 10 days after radiation due to a wasting syndrome confirming successful radiation. Radiation chimeras are designated as fetal liver donor -> irradiated host. Hemopoietic chimeras were maintained on acid water plus terramycin and were housed in a conventional animal colony, maintained on a 12 h light/dark cycle. Five weeks after reconstitution, chimeras were transferred to the University of Rochester where they were allowed to acclimate for an additional week before dosing with TCDD. All mice were housed and cared for according to The Guide for the Care and Use of Laboratory Animals(1996).

Cell isolation and counting.
Chimeric mice were euthanized by CO2 asphyxiation and the thymi were removed and dissected free of lymph nodes, connective tissue, and blood vessels. The femurs and tibias from both posterior legs were also removed. Individual thymi were weighed and mashed with the flat end of a 1 ml syringe plunger. Cells were released in cold MEM, 5% FBS (Life Technologies), and P/S. Thymus and bone marrow cell suspensions were made as previously described (Murante and Gasiewicz, 2000Go; Staples et al., 1998bGo; Thurmond et al., 1999Go). After lymphocyte suspensions were made cells were resuspended in MEM, 5% FBS and P/S for cell counting. The cell yield was enumerated by preparing at least 2 dilutions from each sample and counting each preparation with a Neubauer hemocytometer (Reichert, Buffalo, NY). Cell viability was determined to be >= 95% from all animals by trypan blue dye (0.08% in PBS) exclusion.

Chemicals and treatment protocol.
TCDD was obtained from Cambridge Isotopes (Andover, MA). A stock solution in the solvent p-dioxane (St. Louis, MO), was diluted to an appropriate concentration in olive oil (F. Berio, Hackensack, NY) to yield a treatment solution containing 6 µg of TCDD/ml. Ten-week-old B6 chimeric male mice were injected with either 30 µg TCDD per kg of body weight, or olive oil alone in a volume of 0.1 ml/20 g body weight. All mice were euthanized 10 days after receiving the injection. A minimum of 5 mice were used per treatment group and each mouse was analyzed separately.

Antibodies and lymphocyte staining for flow cytometry.
The following monoclonal antibodies (mAbs) were used at predetermined saturating levels for staining cells: biotin-conjugated anti-CD8{alpha} (clone 53–6.7, rat IgG2a), phycoerythrin (PE)-conjugated anti-CD4 (clone RM4-5, rat IgG2a), fluorescein isothiocyanate (FITC)-conjugated anti-B220 (clone RA3-6B2, rat IgG2a), and biotinylated anti-IgM (clone R6-60.2, rat IgG2a). All antibodies were purchased from Pharmingen, San Diego, CA. Cells were first stained with FITC- and Biotin-conjugated mAbs, washed with PBS containing 0.1% NaN3 and 0.5% BSA, and then stained with PE-conjugated mAbs and Streptavidin Red 670 (Life Technologies).

Flow cytometry.
Data from 10,000 to 100,000 cells were collected from each sample on either a FACStar Plus flow cytometer and analyzed using LYSYS II software (Becton Dickinson), or a FACScan flow cytometer and analyzed using Cell Quest software (Becton Dickinson).

Statistics.
Results are presented as the mean ± SEM with 5–6 mice per group unless otherwise noted. A one-way ANOVA was performed on each set of data using SPSS for Windows software (Chicago, IL). Comparisons between the means were performed using Tukey's t-test. Values of p <= 0.05 were considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ARNT2 Is Expressed in Thymus Tissue
In order to determine if ARNT2 was expressed in developing immune tissues, RT-PCR was performed on RNA obtained from whole thymus tissue, thymocytes, and bone marrow lymphocytes in C57BL/6 mice. As has been previously reported, expression of ARNT2 in brain tissue was very high and detectable with 32 cycles of PCR (Fig. 1Go; Drutel et al., 1996Go; Jain et al., 1998Go; Petersen et al., 2000Go). Interestingly, when we increased the PCR from 32 to 36 and 40 cycles we were able to detect a PCR product for ARNT2 not only in the thymus tissue but also very low expression in the thymocytes and bone marrow lymphocytes (Fig. 1Go). The PCR product was sequenced and confirmed that the band at 451 base pairs was indeed the ARNT2 mRNA (data not shown). The smaller weak band seen in the thymocyte and bone marrow lanes was also sequenced and determined to be nonspecific amplification (data not shown). It is likely that the expression of ARNT2 in the thymus has not been previously reported due to the use of techniques such as in situ hybridization and Western blotting that lack the sensitivity of RT-PCR, especially when searching for mRNAs with low abundance transcripts. It should be pointed out that the mRNA expression does not necessarily indicate protein expression. However, RT-PCR combined with electrophoretic mobility shift analysis has been used in bone marrow cells to demonstrate functional protein expression of the AHR when the expression of this proteins was below detection limits for western blotting (Lavin et al., 1998Go). The potential low level expression of ARNT2 in thymus tissue raises the possibility that it has a function both in thymus development, and TCDD toxicity. To determine if ARNT2 plays a role in the response to TCDD, we generated hemopoietic chimeras from donor mice that were either ARNT2 positive or ARNT2 negative.



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FIG. 1. Ethidium bromide stained 1.5% agarose gel for RT-PCR amplification of ARNT2 in whole thymus, thymocytes, or bone marrow (BM) lymphocytes. Each set of 3 lanes represent increasing number of cycles used for amplification for the appropriate tissue. Brain tissue was used as a positive control for ARNT2 expression (32 cycles only) and RNA without reverse transcription was used as a negative control. The top band of 451 base pairs was confirmed by DNA sequencing to be ARNT2, the lower band migrating around 350 base pairs was also sequenced and determined to be nonspecific amplification.

 
TCDD Induces Thymic Atrophy in Hemopoietic Chimeric Mice Lacking ARNT2
The c112K deletion completely removes the ARNT2 gene (Wines et al., 1998Go), and therefore provides a good source of ARNT2 deficient stem cells. However, because the c112K mutants die within 6 h of birth (Gluecksohn-Waelsch, 1979Go; Grompe et al., 1993Go; Kelsey et al., 1993Go), we chose to make hemopoietic chimeras using the fetal liver stem cells of the c112K homozygous mutants (or control littermates) transplanted into lethally irradiated B6 mice. After 6 weeks of recovery, a time more than sufficient to get >= 98% reconstitution of bone marrow and thymus cell populations as shown by Staples et al.(1998b), we dosed the mice with 30 µg/kg TCDD or with vehicle ip to test whether adult chimeras which lack the ARNT2 gene in their hemopoietic stem cells and lymphocytes would be sensitive to the effects of TCDD. Ten days after dosing, the mice were euthanized and analyzed for signs of TCDD-induced thymic atrophy. The dose of TCDD and the duration of time after dosing was previously determined to be the dose and time of maximal thymic atrophy induced by TCDD in B6 mice (Silverstone et al., 1994Go). Since the donor mice used for reconstitution were on an outbred noncongenic background, graft versus host (GVH) pathology was a concern. However, at necropsy there was no gross evidence of GVH pathology such as weight loss, inflammation of the liver or kidney, or spleen/lymph node enlargement.

To determine if ARNT2 affects thymocyte development, we compared the thymic weights, cellularity, and phenotype of the vehicle-treated c112K -> B6 and control -> B6 chimeras. There was no statistical difference in the overall thymic weight between the c112K mutant and control treated chimeras, indicating that stem cells from the c112K and control donors are equally capable of reconstituting a lethally irradiated thymus (Table 1Go).


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TABLE 1 Thymic Weight and Cell Number in Control -> B6 and c112K -> B6 Chimeras Treated with TCDD
 
In order to determine if the c112K mutation provided protection against TCDD-induced immune alterations, we characterized the effects of TCDD on thymocyte development using changes in thymic weight, cellularity, and thymocyte subpopulations as markers. A single injection with 30 µg/kg body weight TCDD induced a significant decrease in thymic weight and thymic cellularity in both the control -> B6 and in the c112K -> B6 chimeras when compared with vehicle-treated controls (p < 0.001; Table 1Go). TCDD treatment also produced a statistically significant increase in the percentage of DN cells in both the control -> B6 and c112K -> B6 chimeras compared with vehicle controls (p < 0.01; Table 2Go). In addition, there was a significant decrease in the percent of DP thymocytes and a significant increase in the percentage of both CD4 and CD8 SP thymocytes (p < 0.01) in both types of chimeras.


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TABLE 2 Phenotypic Alterations in the Thymocyte Profile of Control -> B6 and c112K -> B6 Chimeras Treated with TCDD
 
The Alterations of Bone Marrow B Lymphocyte Populations Induced by TCDD Are Equivalent in Both the c112K Mutant and WT Chimeras
In addition to the effects of TCDD on the thymus, it has also been established that TCDD alters bone marrow B-cell maturation profiles in male C57BL/6 mice (Thurmond et al., 2000Go). To determine whether these alterations would also occur in our chimeric models we analyzed the effects of TCDD on B-cell development. B-cell development in the bone marrow can be followed using flow cytometry to analyze expression of specific maturation stage-related cell surface markers. Three stages of B lymphocyte maturation were analyzed using criteria initially established by Hardy et al.(1991) and based upon the cell surface expression of CD45R/B220 (B220) and IgM; pro/pre-B cells (B220l°/IgM-), immature B cells (B220l°/IgM+) and mature B cells (B220hi/IgM+; Fig. 2Go). The relative cell numbers for the pro/pre-B, immature B, and mature B cell populations are shown for the vehicle and TCDD-treated control -> B6 and c112K -> B6 chimeras in Figure 3Go. These cell numbers represent the total numbers of cells within each subpopulation found within the viable cell gate (Fig. 2Go). As shown in Figure 3Go, and as previously observed in B6 mice (Thurmond et al., 2000Go), TCDD exposure produces a reduction in cell number of all 3 bone marrow B lymphocyte maturation stages. This reduction is equivalent in both the c112K -> B6 and control -> B6 chimeras. It should be noted that the number of bone marrow cells recovered from the vehicle-treated animals were not significantly different when interchimeric group comparisons were made, indicating that bone marrow reconstitution was comparable for both chimeras (data not shown).



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FIG. 2. Dot plot representation of gates used for analysis of B lymphocytes. Left panel shows the viable cell gate established based upon forward and side scatter parameters. Right panel indicates the gates used for analysis of the B-lymphocyte subpopulations based upon cellular expression of CD45R/B220 and IgM.

 


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FIG. 3. TCDD treatment of the control -> B6 or c112K mutant -> B6 chimeras produces the same changes in the B-lymphocyte subpopulations. Data is presented as the mean ± SEM with 5–6 mice per group. The total cell numbers in the viable FSC by SSC gate for each B-lymphocyte subpopulation are shown. *p <= 0.05 compared to the vehicle-treated population in the same chimeric group.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We used the c112K mutant mouse hemopoietic chimera to determine whether ARNT2 plays a role in mediating TCDD-induced alterations in lymphocyte development. From the results, we observed that TCDD induced significant atrophy of the thymus in both the control and c112K mutant chimeras. This, despite the observation that ARNT2 is expressed in the thymus (Fig. 1Go). If ARNT2 (or any other gene in the c112K deletion) was playing a role in mediating TCDD-induced changes then we would expect to see little or no alteration in the lymphocyte populations in the TCDD treated c112K mutants when compared with lymphocytes from the vehicle-treated controls. From our data we conclude that ARNT2 is not essential for TCDD-induced thymic atrophy. Further, we did not observe any significant differences in the percentage of cells in each thymocyte subpopulation between the control -> B6 and c112K -> B6 vehicle-treated chimeras indicating that the stem cell reconstitution of the thymus is normal. It should be noted however, that there were slight differences in cell number between the control and c112K -> C57BL/6 chimeras (Tables 1 and 2GoGo). Since we were reconstituting mice with a mixture of ARNT2+/- and +/+ bone marrow, it is at least possible that this difference is due to reduced protein expression (either of ARNT2 and/or another c112K associated gene) in the +/- cells. Had we exclusively used +/+ mice as donors the difference in cell number may have been more pronounced; however, analysis of the B-lymphocyte cell number did not reveal even slight differences between the 2 chimeric groups in any of the 3 populations (Fig. 3Go). This might indicate the mixture of ARNT2+/- and +/+ cells had little effect on lymphocyte development, or, thymus development is more sensitive to changes in ARNT2 expression.

Examination of the changes produced by TCDD in the bone marrow B-lymphocytes showed that there is no difference in the B-cell maturation profile between the 2 chimera groups following TCDD treatment, indicating ARNT2 is not required for mediating alterations in this cell type. In previous work we determined that the AHR needed to be present in both the bone marrow hemopoietic and stromal components for normal B-cell maturation to occur (Thurmond et al., 2000Go). In the c112K chimera model any effect of ARNT2 on B-lymphocyte development should have been reflected as changes in the relative cell number between the vehicle-treated control -> B6 and c112K -> B6 chimeras. As shown in Figure 3Go, the B-lymphocyte subpopulations for the control chimeras were identical between the 2 groups, and TCDD treatment induced equivalent B cell subset reductions in both sets of chimeras. It should be noted the cell numbers of the Pro/pre B and mature B cell populations observed in this study were larger than those observed in the AHR+/+ -> AHR+/+ chimeras described in our previous report (Thurmond et al., 2000Go). This difference could be due to a number of possibilities including differences in strain (St2A versus the B6x129 used previously), the difference between fetal liver reconstitution and the bone marrow reconstitution we used in our previous studies, or the length of time allowed for reconstitution before TCDD exposure (6 weeks in this report versus 4 weeks in the previous report).

In the hemopoietic chimeras we observed normal proportions of DN, DP, CD4, and CD8 single positive thymocytes and T cells indicating the lymphoid regenerative capacity of the fetal liver stem cells from the c112K mutant is not compromised. Although ARNT2 does not have an apparent role in TCDD-induced thymic atrophy, previous studies have shown that c112K homozygote fetuses have smaller thymi (DeRossi et al., 2000Go). The smaller thymus in the c112K mutant likely result from a defect in cardiac neural crest development (DeRossi et al., 2000Go). Additionally, the smaller thymus in c112K mutants was shown to be due to a defect in the thymic stromal environment. The thymic stroma are the cells necessary for producing factors that facilitate stem cell homing to the thymus, growth in the thymus, and thymocyte selection (Anderson et al., 1996Go). A defect in the stroma could have an effect on growth and differentiation of the thymocytes resulting in a smaller thymus. Interestingly, the expression pattern seen in the ARNT2 RT-PCR indicates that the majority of ARNT2 expression in the thymus is restricted to the stromal and nonlymphoid tissue. Alterations in the stromal cells induced by TCDD had long been thought to be responsible for TCDD-induced thymic atrophy (De Waal et al., 1997Go; Greenlee et al., 1985Go). However, using chimeras produced from AHR-/- and AHR+/+ animals our lab has previously shown that the stromal elements do not play any role in mediating TCDD-induced thymic alterations (Staples et al., 1998bGo). Therefore, any role ARNT2 or any other deleted gene in the c112K mutation plays in the development and function of thymic stromal cells should not be considered as an important factor in mediating TCDD-induced thymic atrophy.

The current model for TCDD-induced toxicity is the activation of the AHR, causing its translocation to the nucleus where it heterodimerizes with ARNT (Hankinson, 1995Go; Rowlands and Gustafsson, 1997Go). The AHR-ARNT heterodimer then activates transcription of genes containing DREs (Hankinson, 1995Go; Rowlands and Gustafsson, 1997Go). Given the high amino acid sequence identity between ARNT and ARNT2 (Drutel et al., 1996Go; Hirose et al., 1996Go), it is possible that the AHR could heterodimerize with ARNT2 to mediate some of TCDD's toxic effects. However, with respect to lymphocyte development we find no evidence in these studies to suggest ARNT2 is a major contributor to the mechanism of TCDD-induced thymic atrophy. Previous work has demonstrated that both the bone marrow and thymus are sensitive targets of TCDD-induced immune suppression, and suppression is dependent on activation of the AHR (Fine et al., 1990Go; Lai et al., 1998Go; Staples et al., 1998bGo). The lack of a protective effect in the c112K -> B6 mutants could be due to the extremely low expression of ARNT2 in thymocytes and lymphocyte stem cells as determined by RT-PCR. However, in this study we cannot exclude the possibility that ARNT2 may play a role in TCDD-induced thymic atrophy, but in its absence ARNT can compensate. Furthermore, a study by Keith et al.(2001) demonstrated that ARNT and ARNT2 possess both distinct and overlapping functions during development. Possible future studies investigating the function of ARNT2 in the inducible Cre-loxP ARNT-/- system (Tomita et al., 2000Go), may help to clarify the significance and function of ARNT2 in vivo and the role these transcription factors play in TCDD-induced thymic atrophy.


    ACKNOWLEDGMENTS
 
We thank Dr. Nicholas Gonchoroff (Department of Pathology, SUNY Upstate Medical University) for technical assistance and advice with the flow cytometry, the Upstate Medical University Department of Lab and Animal Resources for their care and help with the mice, Denise Hahn for technical help, and Dr. Edward Barker for critical reading of this manuscript. This work was supported by National Institute of Environmental Health Sciences Grants ES07216 (to A.E.S.), ES05774 (to T.S.T.), ES04862 (to T.A.G.), Center Grant ES01247 (to University of Rochester), National Institute of Health Grant HD53964 (to B.C.H.), and National Science Foundation Grant 96-04530 (to B.C.H.).


    NOTES
 
1 To whom correspondence should be addressed. Fax: (315) 464-7652. E-mail: silversa{at}upstate.edu. Back


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