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The Journal of Immunology, 2003, 171: 4582-4591.
Copyright © 2003 by The American Association of Immunologists

Cell Proliferation Arrest within Intrathymic Lymphocyte Progenitor Cells Causes Thymic Atrophy Mediated by the Aryl Hydrocarbon Receptor 1

Michael D. Laiosa*, Amber Wyman{dagger}, Francis G. Murante{dagger}, Nancy C. Fiore*, J. Erin Staples*, Thomas A. Gasiewicz{dagger} and Allen E. Silverstone2,*

* Department of Microbiology and Immunology, State University of New York, Upstate Medical University, Syracuse, NY 13210; and {dagger} Environmental Health Science Center, Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, NY 14642


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Activation of the aryl hydrocarbon receptor (AHR), a basic helix-loop-helix transcription factor, in lymphocytes by the immunosuppressive environmental contaminant 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to cause thymic atrophy in every species studied. We set out to identify the specific hemopoietic cellular populations in which the AHR was activated to lead to thymic atrophy and to determine the effect of AHR activation in those cellular populations. Initially, we examined whether AHR activation in intrathymic dendritic cells could mediate TCDD-induced thymic atrophy. It was found that thymic atrophy occurred only when the AHR could be activated in the thymocytes but not hemopoietic-derived dendritic cells or other APCs. We next analyzed the effect of TCDD on the proliferation of thymocytes in vivo. There was a significant increase in the percentage of thymocytes in the G1 phase of the cell cycle and a significant decrease in the percentage of S plus G2/M thymocytes, especially in the CD4-CD8-CD3- triple-negative intrathymic progenitor cell population 24 h after exposure to 30 µg/kg TCDD. Furthermore, by 12 h after exposure to TCDD, we observed ~60% reduction of 5-bromo-2'-deoxyuridine incorporation in specific intrathymic progenitor cell populations. This reduction persisted for at least 6 days. These data indicate that intrathymic progenitor cells are direct targets of TCDD in the thymus and suggest that TCDD causes thymic atrophy by reducing entrance into cell cycle in these populations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The thymus, the central organ for T cell development, is a dynamic organ constantly losing cells to apoptosis and emigration. Overall thymus size and cellularity is maintained by replenishing the cell loss with progenitor populations from the bone marrow (1). Once in the thymus, these progenitors undergo a complex and highly regulated process of differentiation and proliferation.

Thymocyte development can be segregated into cells undergoing expansion and cells undergoing selection and maturation. The primary proliferating cells are those that lack expression of the CD4 and CD8 surface molecules and mature TCR. These double-negative (DN) 3 intrathymic lymphocyte progenitor cells differentially express the CD44, CD25, and CD24 (heat-stable Ag (HSA)) surface molecules. The developmental progression of these cells is CD44+CD25-->CD44+CD25+CD24+->CD44-CD25+CD24+->CD44-CD25-CD24+, and for simplicity, these populations have been referred to as DN1, DN2, DN3, and DN4, respectively (2). Collectively, the DN progenitor cells comprise only 1–3% of the total thymus cellularity, but their high rate of proliferation leads to the CD4, CD8 double-positive (DP) stage of development, which makes up >=80% of the total thymus cellularity.

The importance of cell proliferation in the intrathymic DN progenitor cell compartment for maintaining thymus homeostasis has been established, and the proportion of cycling cells at each stage of differentiation has been described (3, 4). Within the intrathymic progenitor cell compartment, the bulk of cell cycling is occurring in the DN2->DN4 stages of development (3, 4). It has been estimated that 3–4 days and 8–10 cell divisions are required for the DN2->DP transition (4, 5). It has also been demonstrated that, although proliferation within the intrathymic progenitor cell compartment is essential for maintaining thymus homeostasis, this compartment is non-self-renewing, and in the absence of continued input of progenitors from the bone marrow, the thymus will atrophy (1).

Thymus atrophy has been observed in every experimental animal species tested following exposure to the environmental contaminant 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) (6, 7, 8, 9, 10). TCDD is a planar aromatic hydrocarbon produced accidentally during industrial processes involving organochlorine molecules, including the production of Agent Orange, the herbicide used during the Vietnam War. In C57BL/6 mice, for example, there is a decrease in thymic weight and thymocyte cell number 3 days after treatment with a single dose of 30 µg/kg TCDD (~20% reduction). Maximal reduction occurs by 10 days after exposure (>=80% decrease in cell number), but recovery to control levels is not achieved until at least 30 days after exposure (8, 11, 12). Despite this extensive and persistent cell loss, we cannot detect an increase in thymocyte apoptosis (11, 13).

Thymic atrophy induced by TCDD is mediated by binding to and activation of the aryl hydrocarbon receptor (AHR) as shown in AHR-deficient mice (14). The AHR is a basic helix-loop-helix (bHLH) transcription factor possessing a Period/AHR nuclear translocator/Singleminded (PAS) domain. The PAS domain is named for its shared homology with the Drosophila proteins Period and Singleminded, and the human AHR and AHR nuclear translocator (15, 16). The normal physiological function of the AHR is currently unknown, although proteins with PAS domains may be involved in circadian rhythms (17). Additionally, observations in the AHR-/- mice suggest a possible role in normal development of the liver, immune system, aging processes, ovarian follicle numbers, mammary gland development, vascular remodeling, and cardiac physiology (18, 19, 20, 21, 22, 23). However, AHR activation by TCDD leads to the modulation of a broad array of genes, including detoxification enzymes, factors involved in cellular growth, proliferation, and cell death (24, 25, 26). The precise relationships between elicited gene modifications and AHR function in physiology and/or development of any tissue has not been established.

Previous studies, using chimeric mice with the AHR in either hemopoietic or stromal tissues, proved that thymic atrophy was secondary to AHR activation in hemopoietic cells, not stromal tissues (27). There are two potential cellular targets within the hemopoietic compartment, including the hemopoietic progenitor cells and hemopoietically derived dendritic cells (DCs). In this study, we wanted to further clarify the specific intrathymic cellular targets and the effects that lead to thymic atrophy following AHR activation.

The first potential intrathymic cellular targets we examined were hemopoietic-derived DCs and/or APCs, which could mediate alterations of thymocyte selection after AHR activation. To do this, we created a model where the APCs (DCs and macrophages) were responsive to TCDD, but the thymocytes were not. Using this model, we found that thymic atrophy required that AHR activation occur in developing thymocytes directly.

Having determined that the AHR must be activated in developing lymphoid cells, and given the 80% reduction of thymocyte cellularity within 10 days after exposure (8, 11, 12), we hypothesized that AHR activation was causing a reduction in cell proliferation, and this reduction occurred in the intrathymic progenitor cell compartment. We determined that TCDD inhibits cell proliferation within 9–12 h after exposure. This inhibition of cell cycling was limited to specific subsets in the intrathymic lymphocyte progenitor cell compartment. We believe this to be the primary cause of AHR-mediated thymic atrophy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals

Mixed hemopoietic chimeras were generated by reconstituting C57BL/6J LY 5.1 congenic mice, originally obtained from Dr. E. A. Boyse (Memorial Sloan-Kettering Cancer Center, New York, NY) with bone marrow cells from Ly 5.1 (CD45.1) Ahr-/- and Ly 5.2 (CD45.2) recombinase-activating gene (Rag)1-/- donors. AHR-/- mice were originally obtained from Dr. F. J. Gonzalez (National Cancer Institute, Bethesda, MD) and were bred and backcrossed 10 generations onto the LY 5.1 C57BL/6J congenic background at State University of New York (SUNY) Upstate Medical University. RAG1-/- mice on the C57BL/6J background (Ahrb/b; TCDD-sensitive allele) were purchased from The Jackson Laboratory (Bar Harbor, ME), and housed in isolation at SUNY Upstate Medical University for 1 wk before euthanasia and harvesting of bone marrow stem cells.

For the time course studies, 4- to 5-wk-old male C57BL/6J mice were purchased from The Jackson Laboratory and were allowed to acclimate for 1 full week after arrival at the University of Rochester. Mice were maintained on a 12-h dark, 12-h light cycle and were provided food (5001 rodent diet; Purina Mills, St. Louis, MO) and water ad libitum. All mice were housed and cared for according to protocols approved by the animal welfare committees at both SUNY Upstate Medical University and University of Rochester.

Chemicals and treatment protocol

TCDD was obtained from Cambridge Isotopes (Andover, MA). A stock solution in the solvent p-dioxane (Sigma-Aldrich, Milwaukee, WI) was diluted to an appropriate concentration in olive oil (F. Berio, Lucca, Italy) to yield a treatment solution containing 6 µg/ml TCDD. For the mixed chimera studies, mice were injected i.p. with either 30 µg/kg TCDD in olive oil per kilogram of body weight, or olive oil alone in a volume of 0.1 ml per 20 g of body weight 10 days before euthanasia.

For the time course studies, mice were injected i.p. with either 30 µg/kg TCDD or olive oil alone at 3, 6, 9, 12, 24, 48, 96, or 144 h before sacrifice. 5-Bromo-2'-deoxyuridine (BrdU; Sigma-Aldrich) was diluted in a stock concentration of 10 mg/ml in PBS. All mice were injected with 1.0 mg of BrdU in 0.1 ml of PBS 2 h before sacrifice.

For the apoptosis study, mice were injected with oil, TCDD, or 5 mg/kg dexamethasone (Dex) in PBS 12 h before euthanasia. All mice were injected with 1.0 mg of BrdU in 0.1 ml of PBS 2 h before sacrifice.

Bone marrow lymphocyte isolation and production of hemopoietic chimeras

Bone marrow cells were isolated and prepared as previously described (12, 27). Briefly, the marrow cavity was flushed with MEM plus 100 U/ml penicillin and 0.1 mg/ml streptomycin (P/S; Life Technologies, Rockville, MD) and 5% FBS. Cells were deaggregated, and debris was eliminated by passing the cell suspension through a Pasteur pipette containing 80-gauge nylon mesh (Tetko, Kansas City, MO). Cells were pelleted and depleted of erythrocytes by treatment with ACK buffer (0.17 M NH4Cl, 10 mM KHCO3, and 1 mM EDTA). Cell yield and viability were enumerated with a Neubauer hemocytometer (Reichert, Buffalo, NY). Cell viability was determined to be >95% from all animals by trypan blue dye exclusion. Cells were suspended in MEM without FBS in an appropriate concentration for reconstitution.

Generation of irradiation-reconstitution chimeras was performed as previously described (27), with the following modifications: C57BL/6J Ly 5.1 were reconstituted with either 1 x 106 bone marrow cells in a 1:1 ratio of Rag1-/-:Ahr-/- cells or 5 x 106 bone marrow cells in a 9:1 ratio of Rag-/-:Ahr-/- cells. Mice were allowed to reconstitute for a minimum of 5 wk to allow for reconstitution of bone marrow-derived lymphocyte, myeloid, and DC populations.

Four weeks after reconstitution, chimeric mice were transferred to University of Rochester and allowed to acclimate for 1 full week before dosing with TCDD. Mice were injected with either 30 µg/kg TCDD or olive oil alone. All mice were euthanized 10 days after receiving the injection. A minimum of five mice was used per treatment group, and cells from each mouse were analyzed separately.

Thymocyte isolation

Mice were euthanized by CO2 asphyxiation at the appropriate time point after TCDD treatment. The thymi were removed, and individual cell suspensions were made in cold MEM, containing 5% FBS and P/S. Debris was eliminated as described above. Cell viability was determined to be >95% for all thymocyte preparations.

Thymus and splenic DC enrichment

DCs were isolated as previously described (28, 29). Briefly, either intact spleens or thymi were injected with a 25-gauge needle filled with 100 Mandl U/ml of collagenase D (Boehringer Mannheim, Indianapolis, IN), diluted in HBSS (Life Technologies). After injecting ~1 ml of collagenase, the thymus or spleen was torn open, and a cell suspension was made. The cells in suspension were collected and held on ice for later manipulation. The remaining spleen and thymus fragments were incubated with 400 U/ml collagenase D at 37°C and 5% CO2 for 90 min. Next, the digested thymus and spleen fragments were filtered through a sterile screen, rinsed with Ca2+Mg2+-free HBSS, and added to the cells obtained from the initial suspension step. The collagenase-digested splenocyte or thymocyte suspensions were then pelleted and subsequently resuspended in a 35% BSA (Cohn fraction V; Intergen, Purchase, NY) solution, which was layered over 1.5 ml of RPMI 1640 in a 15-ml conical tube. Low-density collagenase-digested mononuclear cells were collected after 30 min of centrifugation at 900 x g by removing the cells at the interface of the RPMI 1640 and BSA layers using a Pasteur pipette. Low-density lymphocytes were washed once with 50 ml of RPMI 1640 and pelleted at 350 x g for 10 min. Low-density lymphocytes were resuspended in 0.5 ml of RPMI 1640 containing 5% FBS, P/S, 10 mM HEPES, and 50 µM 2-ME (supplemented RPMI 1640) per thymus or 1 ml of supplemented RPMI 1640 per spleen. The DCs were then enriched overnight using plastic adherence in 35-mm tissue culture plates. After incubation, the nonadherent DCs were removed with a Pasteur pipette, pelleted, counted, and prepared for flow cytometry staining. Each spleen yielded between 0.5 and 1 x 106 DCs, and each thymus yielded between 0.5 and 1 x 105 DCs (data not shown) as reported by others (28, 29).

Enrichment of intrathymic progenitor cells

For the time course studies, 5 x 107 thymocytes from each mouse were washed twice with 5 ml of label buffer (PBS containing 2 mM EDTA). Next, thymocytes were resuspended in 0.5 ml of label buffer containing 3.75 µg each of biotin-conjugated anti-CD8{alpha} (clone 53-6.7, rat IgG2a) and anti-CD4 (clone RM4-5, rat IgG2a) Abs. Cells were incubated on ice for 25 min with mixing approximately every 10 min and washed twice with 5 ml of label buffer. After the second wash, thymocytes were resuspended in 0.45 ml of label buffer, and 0.05 ml of MACS streptavidin coupled to magnetic microbeads (Miltenyi Biotec, Auburn, CA), and incubated for 25 min. Bead-coupled thymocytes were washed twice with label buffer and resuspended in 0.5 ml of PBS plus 2 mM EDTA plus 0.5% BSA. Enrichment of CD4-CD8- DN thymocytes was accomplished by running the labeled cell suspensions through the VarioMACS magnet using a BS type column (Miltenyi Biotec). Each individual mouse thymus yielded between 0.8 and 1.5 x 106 cells with >96% purity (data not shown). Mature DN TCR-expressing cells were eliminated during analysis using HSA (CD24) (30, 31).

Abs and lymphocyte staining for flow cytometry

The following mAbs were used at predetermined saturating levels for staining freshly isolated cells: anti-CD8{alpha} (clone 53-6.7, rat IgG2a) conjugated to either FITC, biotin, or allophycocyanin; anti-CD4 (clone RM4-5, rat IgG2a) conjugated to PE; anti-CD3{epsilon} (145-2C11, hamster IgG) conjugated to FITC or biotin; anti-CD11c (clone HL3, hamster IgG) conjugated to PE; anti-HSA (CD24; clone M1/69, rat IgG2b) conjugated to PE; anti-CD25 (clone 7D4, rat IgM) conjugated to biotin; anti-CD44 (clone IM7, rat IgG2b) conjugated to allophycocyanin or PE; anti-LY 5.1 (CD45.1; clone A20, mouse IgG2a) conjugated to FITC or biotin; anti-LY 5.2 (CD45.2; clone 104, mouse IgG2a) conjugated to FITC or biotin. All Abs were purchased from BD PharMingen (San Diego, CA).

Cells were first stained with FITC-, allophycocyanin-, and biotin-conjugated mAbs, washed with PBS containing 0.1% NaN3 and 0.5% BSA, and then stained with PE-conjugated mAbs and either streptavidin-Red 670 (Life Technologies) for acquisition on the FACStarPlus flow cytometer (BD Biosciences, Mountain View, CA) or streptavidin-PerCP (BD PharMingen) for acquisition on the FACSCalibur flow cytometer (BD Biosciences).

For the detection of BrdU incorporation in thymocytes, intranuclear staining was performed following the staining of cell surface Ags as previously described (4). Briefly, cells were fixed and permeabilized using the BD PharMingen Cytofix/Cytoperm buffer for 15 min. Cells were washed twice with the BD PharMingen Perm/Wash buffer and then stained for 20 min with a solution of anti-BrdU (clone B44) conjugated to FITC, containing DNase I (BD Biosciences). After a final wash, cells were fixed in 1% paraformaldehyde and stored in the cold and dark overnight before acquisition on the flow cytometer.

For cell cycle determination, cells were first stained with a mixture of anti-CD3, -CD4, and -CD8 FITC, followed by fixation with 1% paraformaldehyde for 15 min. Cells were subsequently permeabilized with 70% ethanol (-20°C) for at least 48 h. After permeabilization, thymocytes were treated with 1 mg/ml RNase, and then DNA was stained with 50 µg/ml propidium iodide (PI). Cell cycle analysis was performed using FlowJo software (Tree Star, San Carlos, CA).

Detection of apoptotic thymocytes by TUNEL was performed as previously described (11), with the exceptions that DN cells were enriched first using magnetic separation, and fixation and permeabilization was performed with the BD PharMingen Cytofix/Cytoperm kit. Briefly, TdT was used with digoxigenin-conjugated dUTP to label the ends of nicked DNA. The TdT is followed by fluorescein-conjugated antidigoxigenin Ab to visualize the cells with DNA breaks. A control tube omitting the TdT step was used to establish the background level for the anti-digoxigenin Ab.

At least 20,000–50,000 viable cells were collected from each sample using either a three-color FACStarPlus flow cytometer or a four-color FACSCalibur flow cytometer (BD Biosciences) and analyzed using CellQuest software (BD Biosciences).

Statistics

For each experiment, we individually analyzed three to five mice per treatment group. Each time point was performed on at least two separate occasions. Results are presented as the mean ± SEM with the number of mice per group appropriately noted. A one-way ANOVA was performed on each set of data using Statview SAS (Cary, NC) software for Macintosh. Comparisons between the means were performed using Tukey’s t test. Values of p <= 0.05 were considered statistically significant.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of the AHR in thymocytes is required for mediating TCDD-induced thymic atrophy

TCDD-induced thymic atrophy is due to the activation of the AHR, and this activation must occur strictly in the hemopoietic compartment (27). However, it is possible that bone marrow-derived DCs or myeloid APCs could mediate the inhibitory effects of TCDD on T cell development through an indirect mechanism, such as increased negative selection of thymocytes or failure to support positive selection. To address this possibility, we generated mixed hemopoietic chimeras by reconstituting lethally irradiated C57BL/6J Ly 5.1 mice with a mixture of bone marrow from Rag1-/- Ly 5.2 mice on the C57BL/6J background and Ahr-/- Ly 5.1 mice. Although T cell development is blocked in the Rag-/- mice, previous studies have shown that development of the intrathymic dendritic and myeloid lineage is unaffected (32, 33). Therefore, the Rag1-/- bone marrow was used as a source of Ahr+/+ DCs and APCs that could potentially mediate TCDD-induced thymic atrophy indirectly, by affecting the TCDD-insensitive Ahr-/- thymocytes (14, 27). We generated a 1:1 and 9:1 mixture of Rag1-/-:Ahr-/- bone marrow radiation-reconstitution chimeras and treated each set with either oil or 30 µg/kg TCDD.

To verify that donor cells reconstituted the lethally irradiated thymus in the proportions we expected, we analyzed LY 5.1 expression in both total thymocytes and bone marrow-derived DC/myeloid-enriched cells. As shown for the 9:1 mixed chimeras in Fig. 1A, high and low peaks of LY 5.1 fluorescence can be observed. The LY 5.1high cells represent thymocytes derived from the Ahr-/- donor (and perhaps a small proportion of residual radio-resistant host cells; <=5%), which make up ~90% of the total thymocyte population. The LY 5.1low cells represent the Rag-/--derived donor thymocytes. The LY 5.1low cells were independently confirmed to express LY 5.2 (data not shown). As expected, the LY 5.1low cells are blocked in the DN stage of thymocyte development (Fig. 1A, middle dot plot). In comparison to the Rag-/- derived thymocytes, the LY 5.1high (Ahr-/--derived cells) exhibit normal proportions of thymocytes in each major subset (Fig. 1A, right dot plot), indicating that competition with an excess of Rag-/--derived bone marrow progenitors does not inhibit development of the Ahr-/- thymocytes. In contrast to the thymocyte compartment, where there are ~10 times the number of Ahr-/--derived thymocytes to Rag-/--derived thymocytes, the low-density plastic adherent cells (29) enriched for DC and myeloid lineages are skewed toward the LY 5.1low- or RAG-/--derived donors (Fig. 1B). Using HSA expression for the thymic DCs, or CD11c for splenic DCs (34, 35), reveals the predicted 9:1 ratio of LY 5.1low to LY 5.1high cells corresponding to the input of bone marrow progenitors from the Rag-/- and Ahr-/- donors (Fig. 1B, lower two panels).



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  		FIGURE 1. Reconstitution of thymocyte and enriched dendritic/myeloid cell populations in 9 Rag1-/-:1 Ahr-/- mixed chimeras. A, Five weeks after reconstitution, thymi were removed from mixed chimeras and stained for LY 5.1, CD4, and CD8. The percentage of LY 5.1high (Ahr-/-) or LY 5.1low (Rag-/-) thymocytes is shown above the gates in the representative histogram. The phenotypic distribution of Ahr-/- or RAG-/- thymocytes was obtained by analyzing the CD4 vs CD8 distribution within LY 5.1low (middle dot plot) or LY 5.1high (right dot plot) thymocytes. The proportion of viable lymphocyte-sized cells as determined by forward light scatter and side light scatter discrimination for each thymocyte subpopulation is shown in the upper left quadrant of each CD4 vs CD8 dot plot. B, DC/myeloid cells from pooled thymi or individual spleens were enriched as described in Material and Methods. DCs in the thymi and spleen were stained with Abs against HSA or CD11c, and LY 5.1. DC enrichment was confirmed by first gating on viable cells (by forward light scatter vs side light scatter) and then either HSA (thymic DCs; left histogram) or CD11c (splenic DCs; right histogram). Reconstitution by each donor was determined by analyzing the ratio of LY 5.1high (AHR-/- derived) to LY 5.1low (Rag1-/-) expression within the respective gates.

 
If Ahr+/+ DCs contribute to TCDD-induced thymic atrophy in an indirect manner, then in the mixed chimeras, there would be a reduction in the number of Ahr-/- thymocytes after TCDD exposure. As shown in Table I, the total number of Ahr-/--derived cells was the same in both the vehicle- and TCDD-treated mixed chimeras. This was true whether the 1:1 or 9:1 Rag1-/-:Ahr-/- ratio was used. In comparison, the number of Rag1-/--derived thymocytes was reduced in both sets of chimeras by >=60%, and this reduction achieved statistical significance in the 9:1 group (p <= 0.01). When mixed chimeras are generated with RAG1-/-:Ahr+/+ bone marrow, TCDD induced a >70% reduction in the number of the AHR+/+-derived thymocytes (Table I). Moreover, when the absolute number of DN, DP, and CD4 and CD8 single-positive thymocytes are determined for each mixed chimera, the only reductions observed following TCDD exposure are in cells possessing the AHR (either Rag-/- derived or Ahr+/+ derived; Table II). Taken together, these data indicate TCDD induces thymic atrophy by directly affecting lymphocytes and/or lymphocyte precursors.


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  		Table I. An excess of AHR+/+ DC and myeloid lineage cells cannot mediate a reduction of AHR-/- thymocytes after TCDD exposurea

 

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  		Table II. TCDD causes a reduction in AHR+/+ thymocytes directlya

 
TCDD causes an accumulation of thymocytes in the G1 stage of the cell cycle

Given that thymocytes are sensitive targets of TCDD, we set out to identify what effect AHR activation has on these cells and to identify which stages of thymocyte differentiation are immediately affected. Previous studies had demonstrated an inhibitory effect of TCDD on cell proliferation (13, 36, 37, 38). However, the exact mechanism in vivo and the specific thymocyte cell populations where alterations of cell cycle progression are occurring have yet to be elucidated. To determine whether TCDD inhibits entry into the cell cycle in vivo, we injected 4- to 5-wk-old C57BL/6J mice with 30 µg/kg TCDD. Twenty-four hours after injection, we analyzed the distribution of either total or CD8/CD4/CD3 triple-negative (TN) thymocytes within the cell cycle. We analyzed TN thymocytes by electronic gating and determined PI fluorescence by applying the Dean and Jett algorithm to determine the percentage of thymocytes in G1, S, and G2/M phase of the cell cycle (39).

In total thymocytes from TCDD-treated mice, there was a slight but significant increase (3% increase; p <= 0.05) in the percentage of total thymocytes in the G1 phase of the cell cycle and ~80% decrease in both the percentage and number of cells in the S plus G2/M phase (p <= 0.01). In TN thymocytes, which have a higher proportion of cycling cells, there is also a significant decrease in the proportion and number of cells within the S plus G2/M phase of the cell cycle. As shown in Fig. 2, C and D, after exposure to TCDD for 24 h, the proportion of cells in S plus G2/M declines from ~15 to 7%, and there is a ~64% reduction in the absolute number of cells possessing a DNA content of >2 N (p <= 0.01).



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  		FIGURE 2. Distribution of thymocytes in the cell cycle after exposure to 30 µg/kg TCDD. C57BL/6J mice were injected with 30 µg/kg TCDD, and 24 h after injection, mice were euthanized, and thymocyte suspensions were made, stained with a mixture of FITC-conjugated anti-CD8/CD4 and CD3, permeabilized, and stained with PI. Cells were subsequently acquired on a FACSCalibur flow cytometer, and data were analyzed using Dean and Jett cell cycle algorithms for determining the percentage of cells in G1, S, and G2/M. A distribution of total thymocytes in the cell cycle obtained from mice treated either with oil (left panel) or TCDD (right panel). The percentages of cells in each phase of the cell cycle are shown in the top right corners of each representative histogram and are expressed as means ± SEM with four mice per group. B, Representative dot plots showing the gates used for analyzing cell cycle distribution of TN thymocytes in oil- or TCDD-treated mice. C, Distribution of TN thymocytes in the cell cycle. D, The absolute numbers of thymocytes (left bar graph) or TN thymocytes (right bar graph) in each phase of the cell cycle are shown. Cell number is expressed x107 for the total population and x106 for the TN thymocytes. Cell numbers were obtained by multiplying the percentage of cells in each phase of the cell cycle by the total number of thymocytes. TN cell number was obtained by first multiplying the percentage of CD4-CD8-CD3- thymocytes by the total cellularity followed by multiplying this number by the percentage of cells in each phase of the cell cycle. *, Statistically significant with p <= 0.01.

 
Identification of the developmental stages where cell proliferation is inhibited

After determining that TCDD-induced atrophy is due to a direct effect on thymocytes, and that the primary effect is a reduction of TN cells in the S plus G2/M phase of the cell cycle, we set out to determine whether inhibition of cell proliferation is more pronounced in a specific DN stage of thymocyte development. To identify which cell population(s) in the thymus were most susceptible to the effects of TCDD on cell proliferation, we analyzed BrdU incorporation in each DN progenitor cell population.

As shown in Fig. 3, viable-sized lymphocytes were first electronically gated using forward and side light scattered parameters. The lymphocyte-sized cells expressing HSA (CD24; Fig. 3A, middle histogram) were gated and then analyzed for CD44 and CD25 expression (A, top right dot plot). HSA was used as a marker for all populations, except the DN1 (CD44+CD25-) population. The earliest DN1 cells have previously been reported to have low surface expression of HSA and are cells that undergo a low rate of cell cycling. In contrast, DN1 HSA+ cells are in the process of expressing CD25 on their surface as they mature into the DN2 population (2, 4). HSA expression was used for staging the DN2 through DN4 populations, because it confirms that these cells are primarily immature thymocytes and not mature CD4-CD8- {gamma}{delta} or {alpha}{beta} T cells that would not be eliminated using our depletion protocol (30, 31). For these studies, we further separated the DN4 cells into a DN4A (HSA+CD44-CD25int) and DN4B (HSA+CD44-CD25low) subset based on the difference in BrdU incorporation observed in cells expressing intermediate levels of CD25 when compared with the CD25low cells in the control-treated mice. As shown in Fig. 3A, an electronic gate was set surrounding the DN2 through DN4B populations for analysis of BrdU incorporation. A separate analysis was performed for determination of cell proliferation in the DN1 population by removing the electronic HSA gate (R2).



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  		FIGURE 3. TCDD inhibits BrdU incorporation in the DN3->DN4B intrathymic lymphocyte progenitor cell populations. C57BL/6J mice were injected with 30 µg/kg TCDD or oil 24 h before sacrifice. Two hours before sacrifice, all mice were injected with 1 mg of BrdU. Individual thymi were removed, and cell suspensions were made, counted, and labeled with CD4 and CD8 Abs. DN cells were enriched by magnetic separation and stained with fluorochrome-labeled Abs directed against HSA, CD44, CD25, and BrdU. Mice were analyzed individually, with four mice per group, and each time point was repeated at least once (minimum of eight mice per time point per treatment). A, In the top left dot plot, a representative forward by side scatter distribution for enriched DN thymocytes is shown, and viable lymphocyte-sized cells are electronically gated (R1) and further analyzed for HSA expression (R2) as shown in the middle histogram. Within HSA+ thymocytes, the CD44 vs CD25 distribution was determined, and BrdU incorporation was measured in each major stage of development within the DN compartment (with the exception of DN1 cells, which were not gated on HSA+ thymocytes) as shown in the top right dot plot. B–D, Representative histograms measuring BrdU incorporation in oil treatment (left panels) compared with TCDD treatment (right panels) in various thymic progenitor cell populations 24 h after TCDD treatment. Shown are the DN3 intrathymic progenitor cells (B), the DN4A intrathymic progenitor cells (C), and the DN4B intrathymic progenitor cells (D). There were no changes in BrdU incorporation in the DN1 or DN2 populations at 24 h after TCDD treatment.

 
As shown in Fig. 3B, BrdU incorporation in the DN3 thymocyte population was reduced by >60% 24 h after TCDD exposure compared with DN3 thymocytes from vehicle-treated mice (p < 0.001). There were also statistically significant decreases in BrdU incorporation in the DN4A and DN4B populations from TCDD-treated mice (Fig. 3, C and D; p < 0.001). The reduction of BrdU incorporation in the DN3, DN4A, and DN4B thymocyte progenitor cells indicates that these cells are sensitive targets for AHR activation.

Kinetics of the effects on DN cell proliferation

For AHR activation to cause significant reductions in total cell number as soon as 3 days after exposure and for the reductions to gradually increase in severity over time (8, 11, 12), AHR activation must result in the inhibition of proliferation in the progenitor cell populations within 1–2 days, and the inhibition must be persistent. As shown in Fig. 4A, BrdU incorporation in DN1 cells was unaffected until 144 h after TCDD treatment, at which point a slight increase was observed. However, the observed increase in BrdU incorporation in DN1 cells was not statistically significant. There were also no statistical differences between TCDD- and vehicle-treated mice in the DN2 population (Fig. 4B). However, 12 h after TCDD exposure, there were statistically significant reductions in BrdU incorporation in the DN3 and DN4A populations (Fig. 4, C and D; p < 0.01). Even earlier reductions in BrdU incorporation were observed at 9 h (p < 0.01) and at 12 h after TCDD exposure in the DN4B population (Fig. 4E; p < 0.05). Moreover, this decrease in BrdU incorporation at these early time points (<=12 h) precedes the time where any cell loss is observed. BrdU incorporation remained inhibited for at least 144 h in the DN3, DN4A, and DN4B stages of thymocyte development.



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  		FIGURE 4. The effect of TCDD on entry into the cell cycle over time. Mice were injected with 30 µg/kg TCDD or oil at various time points before euthanasia and BrdU 2 h before sacrifice. Individual thymi were removed, and DN cells were enriched, stained for CD44, CD25, HSA, and BrdU, and analyzed as shown in the previous figure. Data are presented as means ± SEM with eight mice per group (four mice per experiment and data pooled from two experiments). •, Oil-treated mice; {blacktriangleup}, TCDD-treated mice. *, Statistically significant data compared with controls, p <= 0.001. A–E, The percentage of cells incorporating BrdU within a particular cell population is compared with time after TCDD exposure. A, DN1. B, DN2. C, DN3. D, DN4A. E, DN4B.

 
It should be noted that the slight reduction observed in both the control- and TCDD-treated mice at the earliest time points in all sets was not statistically significant and is thought to be due to a stress effect caused by the two injections, because equivalent reductions were observed in both oil- and TCDD-treated mice.

Effects on the total number of proliferating cells

Following analysis of BrdU incorporation in each DN progenitor cell population, the kinetics of cell loss was determined. Absolute cell number for each DN population was calculated by multiplying the total thymic cellularity at each time point after TCDD exposure by the percentage of CD4-CD8- cells (determined by flow cytometry), and by the percentage of HSA+ cells in each progenitor cell compartment (or HSA- for DN1 only) (data not shown). The number of BrdU+ cells was calculated by multiplying the total DN cell number by the percentage of cells demonstrating BrdU incorporation in that population at each time point. There were no statistical differences in cell number at any time point after injection with the vehicle, and therefore, the average from all control-treated mice was used for comparisons with TCDD-treated mice.

Twenty-four hours after injection, total thymic cellularity (data not shown) and the absolute cell number for each DN thymocyte population in TCDD-treated mice do not differ significantly from those in vehicle-treated mice (Fig. 5A). However, 48 h after TCDD treatment (still at a time point before any significant total cell loss), there is ~3-fold reduction in the number of DN3 thymocytes compared with the same population in vehicle-treated mice (p <= 0.01). This reduction in the DN3 population remains essentially constant through the first 144 h after TCDD exposure. In the DN4A and DN4B populations, the cell numbers are reduced starting at 48 h postexposure, and this reduction reaches maximal levels by 96 h (p < 0.01). In comparison, the absolute cell numbers for the DN1 and DN2 populations remain unchanged at all time points evaluated.



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  		FIGURE 5. Absolute number of DN cells or BrdU+ DN cells in each stage of differentiation within the intrathymic lymphocyte progenitor compartment. *, Statistically significant compared with oil-treated mice, with p <= 0.01. Data are presented as the mean of eight mice per group ± SEM. A, The absolute number of DN cells at various time points after exposure to 30 µg/kg TCDD. Cell numbers were obtained by multiplying the percentage of cells within each stage of differentiation by the total number of DN cells at each time point. B, The absolute number of BrdU+ DN cells at each time point. To determine the number of BrdU-positive cells, the percentage of BrdU+ cells was multiplied by the number of cells in each DN population shown in A.

 
As shown in Fig. 5B, there was a 3.6-fold reduction in the absolute number of BrdU+ cells in the DN3 population 24 h after exposure, and the number of BrdU+ cells remained reduced for each successive time point (p < 0.01). TCDD also reduced the number of BrdU+ cells 24 h after treatment in the DN4A and DN4B populations by ~3-fold. The reduction in the number of BrdU+ cells in the DN4A and DN4B populations became even more pronounced as the time after TCDD treatment progressed, causing a >15-fold reduction in the number of cycling cells 144 h after exposure (for DN4A, cell numbers decreased from 3.23 ± 0.31 to 0.31 ± 0.06; for the DN4B, cell numbers decreased from 0.46 ± 0.08 to 0.03 ± 0.01; p < 0.01; all numbers are x105). These trends of decreasing numbers of BrdU+ cells following exposure support our hypothesis that TCDD-induced thymic atrophy is caused by inhibiting the DN3, DN4A, and DN4B thymic progenitor cell populations from entering into the cell cycle.

TCDD does not lead to an increase in thymocyte apoptosis

One of the earliest studies investigating the mechanism of TCDD-induced thymic atrophy concluded that there was an increase in Ca2+-dependent endonucleases leading to increased thymocyte apoptosis (40). Subsequent studies attempting to support this finding in vivo have reported an increase in thymocyte apoptosis, but only at a very high dose of TCDD and only after overnight culture of the thymocytes in vitro (41). However, in a recent review by Robertson and Orrenius (42) of toxicological agents known to induce apoptosis, the authors conclude that the recent data do not support apoptosis as a primary mechanism for TCDD-induced thymic atrophy.

To address the question of whether a loss of BrdU incorporation was a result of increased thymocyte apoptosis, particularly in the DN compartment, we compared DNA fragmentation in oil-, TCDD-, and Dex-treated mice using the TUNEL assay 12 h after exposure. As shown in Fig. 6A, BrdU incorporation is unaffected in DP thymocytes 12 h after exposure to either oil or TCDD. In comparison, BrdU incorporation in DP thymocytes from mice treated with 5 mg/kg Dex is reduced by ~5-fold. Fig. 6B shows that, when assaying for apoptotic DP thymocytes, only 1–2% of the cells in both oil- and TCDD-treated mice are apoptotic, whereas ~15% of the DP thymocytes from the mice treated with Dex are TUNEL positive. Importantly, there are no TUNEL+ DN3 or DN4 thymocytes in either the oil- or TCDD-treated mice, whereas there is a small population of TUNEL+ cells in the DN3 and DN4 cells from the Dex-treated mice (Fig. 6C). These data support a model where the earliest effects of TCDD in the thymus are a decrease in intrathymic progenitor cell proliferation with no detectable increase in apoptosis.



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  		FIGURE 6. TCDD does not cause an increase in thymocyte apoptosis. Mice were injected with oil, 30 µg/kg TCDD, or 5 mg/kg Dex, and all mice were injected with 1 mg of BrdU 2 h before euthanasia. Twelve hours after oil, TCDD, or Dex injection, mice were euthanized, thymi were removed, and cell suspensions were made as described above for the time course studies. A representative histogram from four mice per group is shown. A, DP thymocytes were gated and analyzed for BrdU incorporation. The percentage of BrdU+ cells is shown above the analysis gate for each treatment, and the treatment is listed above each column of histograms. B, DP thymocytes were gated and the percentage of TUNEL+ cells is shown above the analysis gate for each treatment. A control tube lacking TdT was used to determine the background and is shown as the shaded overlay. C, TUNEL staining in enriched DN thymocytes gated on the DN3 and DN4 cell populations from oil-, TCDD-, or Dex-treated mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well established that TCDD has potent suppressive effects on T cell development (6, 7, 8, 38, 43). However identification of the direct cellular and molecular targets of AHR activation in the thymus and bone marrow that results in altered lymphocyte development have only started to emerge (12, 27). Recently, we demonstrated that TCDD-induced thymic atrophy is dependent on activation of the AHR exclusively in hemopoietic cells (27). However, the possibility that bone marrow-derived DCs or other APCs are mediating TCDD-induced thymic atrophy had yet to be ruled out. To determine whether the primary cellular target in the thymus was a thymocyte or other hemopoietic cell, we generated mixed chimeras by reconstituting lethally irradiated hosts with bone marrow from both Rag1-/- and Ahr-/- mice as donors. The Rag1-/- progenitor cells would provide DCs and macrophages but not thymocytes beyond the DN stage, whereas the Ahr-/- bone marrow progenitor cells provided thymocytes insensitive to TCDD. Using the Ahr:Rag1-/- mixed chimeras, we determined that the reduction in thymocyte cellularity observed in TCDD-treated mice is due to a direct effect of TCDD on thymocytes and/or committed lymphocyte precursors and not DCs.

We also demonstrated that TCDD causes an apparent increased accumulation of TN thymocytes in the G1/G0 phase of the cell cycle and a 60% reduction in the percentage and number of thymocytes in the S plus G2/M phase 24 h after exposure in intact mice. Additionally, a significant reduction in BrdU incorporation within developing thymocytes was observed. Inhibition of BrdU incorporation was cell stage specific, occurring in the DN3 and DN4 stages of T cell development. It should be noted that there was a slight decrease in BrdU incorporation even in the vehicle-treated mice in the first three time points examined. Although this decrease was not statistically significant, it might be due to elevated stress/glucocorticoid levels in the mice after receiving two i.p. injections (oil and BrdU in PBS). Because the oil-alone effect was not statistically significant, it would be inappropriate to go beyond this without undue speculation. Nevertheless, the decrease in BrdU incorporation in TCDD-treated mice was ultimately much more severe and achieved statistical significance as early as 9 h after exposure in the DN4B population and 12 h after exposure in the DN3 and DN4A subsets. This reduction in BrdU incorporation at these early time points occurs before measurable cell loss is observed. Moreover, because BrdU was administered 2 h before sacrifice, our results suggest AHR activation causes alterations in the DNA replication machinery by 7 h after exposure in vivo. The 60% reduction in BrdU incorporation can be directly correlated to the net reduction in the total number of cells synthesizing DNA in the DN3, DN4A, and DN4B thymocyte populations 24 h after TCDD exposure, and this reduction is persistent for at least 6 days after exposure. These data suggest that the mechanism behind TCDD-induced thymic atrophy is mainly due to the inhibition of cell proliferation in the DN progenitor cell compartment and is not due to apoptosis as suggested by other investigators (40, 41). Moreover, we cannot detect an increase in thymocyte apoptosis in either DN or DP thymocytes by the TUNEL assay in vivo. It is possible that apoptotic cells are rapidly cleared by resident macrophages (44); however, we can detect TUNEL+ cells 12 h after exposure to Dex, but not TCDD (Fig. 6). It is also possible that TCDD causes a delayed apoptosis where first cell cycle is inhibited, and this is rapidly followed by activation of death pathways. However, we have shown both in vitro and in vivo that lck proximal promoter-driven Bcl-2 transgenic mice are sensitive to TCDD-induced thymic atrophy but are protected from Dex-induced atrophy (11). In comparison to the Bcl-2TG mice, it has been shown in fetal thymic organ cultures that mice deficient in the G1->S cell cycle regulator, p27KIP, are partially protected from TCDD-induced thymic atrophy, adding further support that TCDD activation of the AHR results in modulation of the cell cycle to cause atrophy (36).

From the decrease in BrdU incorporation detected in the DN3, DN4A, and DN4B populations 7–10 h after TCDD exposure, we conclude that these populations are the primary cellular targets in the thymus. If AHR activation was targeting an earlier progenitor cell population, it is possible that this earlier population might continue to differentiate but fails to proliferate once it reaches the DN3 or DN4 stage of maturation. However, it is been estimated that the average murine thymocyte doubling time is between 10 and 12 h, and that it can require two cellular divisions for cells in the DN2 population to differentiate into the DN3 population (4, 45). Therefore, because we observed a decrease in BrdU incorporation in the DN3 and DN4 populations 10 h after TCDD exposure, it is highly likely that the vast majority of cells in the DN3, DN4A, and DN4B populations have decreased DNA synthesis directly. This reduction is most likely dependent on activation of the AHR, because in preliminary studies using Ahr-/- mice, there was no difference in BrdU incorporation within any DN cell population in either control- or TCDD-treated mice (data not shown).

These data demonstrating that TCDD-induced thymic atrophy is caused by inhibition of cell cycle within the intrathymic progenitor population complement earlier studies by Frey et al. (1) where they demonstrated that, in the absence of bone marrow input, the thymus begins to undergo profound thymic atrophy within 1 wk after the intrathymic progenitor population is exhausted. We have found that TCDD-induced thymic atrophy can be due to inhibition of bone marrow reconstitution of the thymus (43, 46) and inhibition of cell proliferation within the intrathymic progenitor population. In fact, based on our data, one might expect an accumulation of DN3 cells in the thymus if they are not dying and if there is continual input from the bone marrow. A consideration of the present and previous data (43, 46) would indicate that a lack of accumulation is due to the finding that TCDD also inhibits bone marrow progenitor reconstitution of the thymus. The result of AHR activation in both the bone marrow and intrathymic progenitors is a statistically significant reduction in overall thymocyte cellularity beginning 3 days after exposure, reaching maximal levels (80% reduction) by day 10, and persisting for at least 24 days after exposure to TCDD at this dose (11, 12, 27).

The reduction of cell proliferation in specific stages of thymocyte development by AHR activation raises some intriguing possibilities for the biological role of the AHR at these particular developmental checkpoints. Specifically, the stages of development where inhibition seems to be most profound (DN3 and DN4) are normally characterized by RAG expression, TCR gene recombination, and pre-TCR signaling. Additionally, genetic studies have demonstrated critical roles for several bHLH transcription factors, including HEB and Id3 (47, 48, 49). It is tempting to speculate that the AHR may play some unknown role at this stage of development in coordination with one or more of these other factors. This role may be disrupted in the presence of TCDD, leading to the observed atrophy following AHR activation. However, studies in the Ahr-/- mouse have not revealed any obvious deficiencies in this particular stage of thymopoeisis (18, 27), indicating a possible redundancy with other bHLH proteins during normal development.

An alternative mechanism for the observed inhibition of cell proliferation in immature thymocytes comes from studies demonstrating that TCDD-induced AHR activation results in the up-regulation of the gelsolin-like actin binding protein adseverin (50, 51). Adseverin induction occurred exclusively in the DN3 and DN4 stages of development, but not in earlier or later stages of thymocyte development (52). Perhaps adseverin induction by the AHR interferes with actin polymerization, which could mediate a block or reduction in cell cycle progression in the DN3 and DN4 thymocytes.

In conclusion, the primary cellular targets in which the AHR is activated in the thymus are DN3, DN4A, and DN4B intrathymic lymphocyte progenitor cells. The effect of TCDD in these cell populations is an arrest of cell proliferation, which can account for the observed thymic atrophy.


   Acknowledgments
 
We thank Dr. Nicholas Gonchoroff (SUNY Upstate Medical University) and Dr. Peter Keng (University of Rochester) for technical assistance and advice with the flow cytometry, and Dr. Pedro Fernandez-Salguero (University of Extremadura, Extremadura, Spain) for the gift of AHR-/- founder mice and for initial discussions on use of the RAG-/- in mixed chimeras. We also thank Dr. Ellen Rothenberg (California Institute of Technology, Pasadena, CA), Dr. Edward Barker (SUNY Upstate Medical University), and Dr. Karen Vikstrom (SUNY Upstate Medical University) for critical reading of this manuscript.


   Footnotes
 
1 This work was supported by National Institute of Environmental Health Sciences Grant ES07216 (to A.E.S.), Hendrick’s Fund (to A.E.S.), Grant ES04862 (to T.A.G.), Center Grant ES01247 (University of Rochester), and Training Grant ES07026 (University of Rochester). Back

2 Address correspondence and reprint requests to Dr. Allen E. Silverstone, Department of Microbiology and Immunology, State University of New York, Upstate Medical University, 766 Irving Avenue, Syracuse, NY 13210. E-mail address: silversa{at}upstate.edu Back

3 Abbreviations used in this paper: DN, double negative; DP, double positive; TN, triple negative; int, intermediate; HSA, heat-stable Ag; TCDD, 2,3,7,8,-tetrachlorodibenzo-p-dioxin; AHR, aryl hydrocarbon receptor; bHLH, basic helix-loop-helix; PAS, Period/AHR nuclear translocator/Singleminded; DC, dendritic cell; RAG, recombinase-activating gene; BrdU, 5-bromo-2'-deoxyuridine; Dex, dexamethasone; PI, propidium iodide. Back

Received for publication April 21, 2003. Accepted for publication August 25, 2003.


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 Introduction
 Materials and Methods
 Results
 Discussion
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