* Department of Microbiology and Immunology, State University of New York, Upstate Medical University, 750 East Adams Street, Syracuse, New York 13210;
Environmental Health Science Center, Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, New York 14642; and
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
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ABSTRACT |
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Key Words: TCDD; dioxin; ARNT2; thymus; bone marrow; lymphocyte development; bHLH-PAS; AHR.
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INTRODUCTION |
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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, 1995; Rowlands and Gustafsson, 1997
; Schmidt and Bradfield, 1996
) 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, 1995
; Schmidt and Bradfield, 1996
). In the nucleus, the ligand activated AHR heterodimerizes with another member of the bHLH-PAS family, the AHR nuclear translocator (ARNT; Hoffman et al., 1991
; Swanson et al., 1995
). 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., 1999
; Lai et al., 1996
; Rowlands and Gustafsson, 1997
). 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, 1997
). 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., 1996; Sanhadji et al., 1992
) enter the thymus where they become fully committed T-cell precursors (Tourigny et al., 1997
). 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, 1998
). As the DN cells mature they upregulate both the CD4 and CD8 molecules and are called double positive (DP) cells (Sebzda et al., 1999
). 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., 1999
).
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., 1998; Silverstone et al., 1994
; Staples et al., 1998b
). 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., 1998b
).
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., 2000), 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., 1989
; Silverstone et al., 1994
). 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, 1999) 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., 1996
; Hirose et al., 1996
) and 78% amino acid identity in the conserved bHLH and PAS regions (Drutel et al., 1996
; Hirose et al., 1996
). Furthermore, the AHR has been shown to heterodimerize with ARNT2 and transactivate DRE-containing genes (Hirose et al., 1996
). ARNT2 expression was found by in situ hybridization and Northern blotting to be most pronounced in the kidney and brain (Drutel et al., 1996
; Jain et al., 1998
; Petersen et al., 2000
), and it may play a role in neuronal cell survival (Drutel et al., 1999
) and responses to hypoxia (Keith et al., 2001
; Maltepe et al., 2000
). 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-
, HIF2-
, and HIF3-
; Jain et al., 1998
; Maltepe et al., 2000
; Petersen et al., 2000
). 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., 1968; Gluecksohn-Waelsch, 1979
; Russell et al., 1979
), which lacks ARNT2 (Wines et al., 1998
), 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.
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MATERIALS AND METHODS |
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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 TrisHCl, 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., 1998b), 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., 1968
; Russell and Raymer, 1979
). 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, 2000; Staples et al., 1998b
; Thurmond et al., 1999
). 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 (clone 536.7, 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 56 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.
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RESULTS |
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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 1
).
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DISCUSSION |
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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., 2000). 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 3
, 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., 2000
). 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., 2000). The smaller thymus in the c112K mutant likely result from a defect in cardiac neural crest development (DeRossi et al., 2000
). 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., 1996
). 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., 1997
; Greenlee et al., 1985
). 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., 1998b
). 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, 1995; Rowlands and Gustafsson, 1997
). The AHR-ARNT heterodimer then activates transcription of genes containing DREs (Hankinson, 1995
; Rowlands and Gustafsson, 1997
). Given the high amino acid sequence identity between ARNT and ARNT2 (Drutel et al., 1996
; Hirose et al., 1996
), 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., 1990
; Lai et al., 1998
; Staples et al., 1998b
). 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., 2000
), may help to clarify the significance and function of ARNT2 in vivo and the role these transcription factors play in TCDD-induced thymic atrophy.
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ACKNOWLEDGMENTS |
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NOTES |
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