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*
Department of Microbiology and Immunology, State University of New York, Health Science Center, Syracuse, NY 13210; and
Environmental Health Science Center, Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, NY 14642
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Unbound AhR is found in the cellular cytoplasm complexed with multiple proteins, including hsp90 (14) and a recently identified protein, the AhR-interacting protein (15). AhR-interacting protein has sequence homology to proteins that are necessary for cell cycle and mRNA synthesis. Once a ligand, such as TCDD, enters the cell and binds to the AhR, the ligand-receptor complex is translocated to the nucleus where it forms a heterodimer with another bHLH protein, the AhR nuclear translocator. This complex then binds to a specific sequence in various genes, called the dioxin response element (16, 17, 18). Dioxin response elements are found in the regulatory region of many genes, including cytokines and growth factors (19, 20).
TCDD, via the AhR, has been shown to have a variety of effects on T cell development and function, including decreasing the number of thymocytes and altering the effector functions of mature Th and T killer cells (2, 21, 22). Although a variety of studies have been performed to determine how TCDD is influencing T cells and the thymus, the mechanism and targets of its actions are still unclear. It has been suggested that in the thymus both the stromal cells (23, 24, 25, 26) and the hemopoietic cells, including the thymocytes (27, 28, 29, 30) and their precursors (31), are sensitive to TCDD. Since TCDD exposure has multiple consequences on the thymus, such as decreasing the number of thymocytes and shifting their phenotypic profile (22, 24, 32, 33), it is possible that TCDD might be targeting both thymocytes and stromal elements in different ways to account for its effects. Additionally, since thymocyte precursors in the bone marrow are known to exhibit sensitivity to TCDD (31), it is also possible that marrow resident hemopoietic cells and/or stromal cells represent targets for TCDD.
The thymus itself is a complex organ that is responsible for the maturation and education of most peripheral T cells. It consists of two main components: the thymocytes and the supporting stromal elements. Stromal elements are a heterogeneous group of cells, including radioresistant epithelial cells and radiosensitive, macrophage, and dendritic cells (DC) (34, 35). The epithelial cells are found throughout the thymus, and the current consensus is that their main function is to mediate positive selection of double-positive thymocytes (36, 37, 38). DC and macrophages are derived primarily from bone marrow hemopoietic precursor cells (35, 39, 40), although DC can arise from intrathymic thymocyte precursors (41, 42). While the exact roles of DC and macrophages in the thymus are still not fully understood, it has been shown, using various models, that DC are essential for negative selection of thymocytes (36, 43, 44), while macrophages have multiple roles, including phagocytosis and negative selection (34, 40).
We wanted to further determine the role of hemopoietic cells and stromal cells in TCDD-induced thymic atrophy. To accomplish this we constructed radiation chimeras using mice that lack the dioxin-binding receptor (AhR-/-) and are, therefore, unresponsive to the effects of TCDD even at high toxic doses (45). By taking bone marrow cells, which contain hemopoietic stem cells, from these AhR-/- mice and using them to reconstitute lethally irradiated B6Ly5.1 (AhR+/+) congenic mice, we effectively created mice that have stromal cells that are sensitive to the actions of TCDD, while the hemopoietic cells are insensitive. We then treated these chimeric mice with TCDD and compared the results from these mice to radiation chimeras that contained both sensitive stromal and hemopoietic elements. These studies were additionally validated by lethally irradiating AhR-/- mice, reconstituting them with AhR+/+ marrow, and treating them with TCDD. Our results show that TCDD targets hemopoietic elements directly to cause alterations in the thymus, including thymic atrophy and phenotypic shifts, and that the radioresistant stromal elements do not play a significant role in these processes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TCDD was obtained from Cambridge Isotopes (Cambridge, MA). A stock solution, in p-dioxane, was diluted to an appropriate concentration in olive oil (F. Berio, Hackensack, NJ) to yield a treatment solution containing 6 µg of TCDD/ml.
Experimental animals
Strains used. C57BL/6 Ly5.1 congenic mice were originally obtained from Dr. E. A. Boyse (Memorial Sloan-Kettering Cancer Center, New York, NY) and maintained at our own facility. The 129/SV x C57BL/6N AhR+/+ and AhR-/- mice were obtained from P. Fernandez-Salguero and F. Gonzalez (National Cancer Institute, National Institutes of Health, Bethesda, MD) (4). These mice have the B6 Ly5.2 phenotype and either lack the AhR gene (AhR-/-) or have the AhRb/b-sensitive allele (AhR+/+) from the B6 founder. These mice will be called B6Ly5.2 AhR+/+ or AhR-/- for brevity. All mice were housed and cared for according to The Guide for the Care and Use of Laboratory Animals (46). Specifically, the mice were housed in isolation cages at constant temperature and humidity on a 12-h light/dark cycle, with food and water provided ad libitum. Due to increased susceptibility to infection in the colony of B6Ly5.2 AhR mice (5), they were maintained prophylactically on acid water (pH 3.0) and oxytetracycline HCl (Terramycin, Pfizer, New York, NY).
Chimeric mice.
Radiation bone marrow chimeras are designated bone marrow
donor
irradiated host. Four-week-old B6Ly5.1 or B6Ly5.2
AhR-/- mice were irradiated by a calibrated x-ray
source (Philips MGC 30 x-ray machine, Philips, Hamburg, Germany) in two
doses of 550 rad spaced at a 4-h interval. By splitting the dose we can
reduce the toxicity to the gut (47) while delivering a cumulative dose
of 1100 rad, which is effectively lethal for hemopoietic cells in this
strain of mice (48). One half-hour after their second irradiation the
mice were given 1 x 106 bone marrow cells from
B6Ly5.1 AhR+/+, B6Ly5.2 AhR+/+, or B6Ly5.2
AhR-/- by tail vein injection. B6Ly5.2 AhR+/+
or AhR-/- bone marrow was used to reconstitute the
B6Ly5.2 AhR-/- hosts to avoid a graft-vs-host reaction
that could have resulted from B6Ly5.1 bone marrow cells recognizing the
129/SV component of the B6Ly5.2 AhR-/- mice. The
reconstituted mice were allowed to recover for 4 wk before they were
treated to be sure the thymus was fully reconstituted (49) and
releasing mature cells of donor origin (50). The mice were weighed
twice a week after being irradiated, and mice that had significant or
continuing weight loss, a sign of incomplete reconstitution, were
excluded from the analysis. All chimeric mice were given acid water
with terramycin approximately 2 to 3 days before irradiation and were
maintained on it until they were killed. To ensure that the irradiation
of the host mice was successful, one irradiated mouse in each
experiment was not reconstituted. All nonreconstituted irradiated mice
died or were killed because of morbidity within 9 to 12 days after
irradiation.
Treatment protocol
Eight-week-old chimeric or B6Ly5.1 male mice were injected i.p. with either 30 µg/kg of TCDD in olive oil or olive oil only (0.1 ml/20 g). All mice were killed 10 days after receiving an injection. Each experiment was performed with randomized, age-matched litters (±3 days). A minimum of four mice were used per treatment group, although the chimera studies were performed with at least five mice per group. Mice were analyzed individually. Each experiment was performed at least twice.
Cell isolation
For cell counting and flow cytometry. Mice were euthanized by CO2 asphyxiation, and the thymi were removed and dissected free of lymph nodes and blood vessels. Spleens, along with femurs and tibias from both legs, were also removed. Individual thymi and spleens were weighed and then mashed with the flat end of a 1- or 5-ml syringe plunger, to release the cells, in cold MEM with Hanks’ salts (Life Technologies, Grand Island, NY; catalog no. 41200-072) containing 5% FBS (Life Technologies) and penicillin-streptomycin (P/S; 100 U/ml penicillin and 0.1 mg/ml streptomycin; Life Technologies). The tissues were triturated, and debris was eliminated by passing the cell suspension through a Pasteur pipette stuffed with 80-gauge nylon mesh (Tetko, Kansas City, MO). Bone marrow was processed similarly, with the bones first being cleaned of muscle and connective tissue, then <1 mm of the end of each bone was removed. The marrow cavity was flushed with MEM with 5% FBS and P/S using a 25-gauge needle. The marrow was then suspended by passing the cells successively through a 22-gauge needle and a 25-gauge needle. The suspension was finally passed through 80-gauge nylon mesh. All leukocyte preparations were then pelleted by centrifugation. After the initial pelleting, the splenocytes and bone marrow cells were resuspended in 1 ml of ACK buffer (0.17 M NH4Cl, 10 mM KHCO3, and 1 mM EDTA) and incubated for 4 min at room temperature to lyse the RBC. These cells were washed once and repelleted. Cells were resuspended in MEM, 5% FBS, and P/S for cell counting. The cell yield was enumerated by diluting the cells and counting at least two samples for each cell preparation with a Neubauer hemocytometer (Reichert, Buffalo, NY). Cell viability was determined to be >90% from all animals by trypan blue dye (0.08%) exclusion.
For reconstitution. Bone marrow cells were obtained as described above with the following exception. After the cells were counted, they were repelleted and resuspended in MEM and P/S without FBS at a concentration of 5 x 106 cells/ml; 0.2 ml (1 x 106 cells) was then injected via tail vein into the irradiated recipients.
Abs
The following mAbs were used at predetermined saturating levels:
FITC- and biotin-conjugated anti-CD8
(clone 53-6.7, rat IgG2a);
FITC- and PE-conjugated anti-CD4 (clone RM4-5, rat IgG2a); FITC-,
PE-, and biotin-conjugated anti-CD3
(clone 145-2C11 and clone
500A2, hamster IgG); PE- and biotin-conjugated anti-B220 (clone
RA3-6B2, rat IgG2a); PE-conjugated anti-CD44 (clone IM7, rat
IgG2b); biotin-conjugated anti-CD25 (IL-2R, clone 7D4, rat IgM);
FITC-conjugated anti-CD45.1 (Ly5.1, clone A20, rat IgG2a);
biotin-conjugated anti-TER-119 (clone TER-119, rat IgG2b);
biotin-conjugated anti-Gr-1 (Ly-6G, clone RB6-8C5, rat IgG2b);
biotin-conjugated anti-Mac-1 (CD11b, clone M1/70, rat IgG2b);
FITC-conjugated anti-c-Kit (CD117, clone 2B8, rat IgG2b); and
PE-conjugated anti-Sca-1 (Ly-6A/E, clone E13-161.7, rat IgG2a;
PharMingen, San Diego, CA). Biotin-conjugated anti-CD45.2 was
made at our facility using Ly5.2 hybridoma clone, A-20 1.7 (mouse
IgG2a) obtained from Dr. Shoji Kimura (Memorial Sloan-Kettering Cancer
Center).
Flow cytometric staining and analysis
Freshly isolated thymocytes and splenocytes were pelleted by centrifugation and washed in PBS containing 0.5% BSA and 0.1% sodium azide. One hundred thousand cells were then incubated with FITC and biotin-conjugated mAb in 50 µl for 30 min at 4°C. Cells were washed twice with 1 ml of PBS-0.5% BSA and 0.1% sodium azide and were then incubated with PE-conjugated mAb and streptavidin-conjugated Red 670 (Life Technologies) for 30 min at 4°C. After two more washes, the cells were fixed in 1% paraformaldehyde in PBS. Ten thousand or more fixed cells were analyzed on a Becton Dickinson (Mountain View, CA) FACStar Plus flow cytometer using the Becton Dickinson LYSYS II program. Single and dual stainings of fresh thymocytes from untreated young adults were used in assorted combinations to set compensation. Negative staining was performed with either isotype or total Ig controls stained appropriately.
Freshly isolated bone marrow cells were pelleted by centrifugation and
washed in HBSS with Ca2+ and Mg2+ (Life
Technologies, catalog no. 14025-092) containing 0.2% BSA (HBSS-0.2%
BSA). Aliquots of 8 x 106 cells were then incubated
for 30 min in a mixture of biotin-conjugated mAbs (anti-Mac-1,
anti-Gr-1, anti-TER-119, anti-CD3, anti-CD8, and
anti-B220) diluted with HBSS-0.2% BSA. Additionally, the mixture
contained PE-conjugated anti-Sca-1 mAb and FITC-conjugated
anti-c-Kit mAb. The cells were washed once with HBSS-0.2% BSA and
were then incubated for 30 min in Dulbecco’s PBS containing 1% BSA
and streptavidin-conjugated Red 670. After one rinse in HBSS-0.2% BSA,
the cells were fixed in 1% paraformaldehyde in PBS. Data on the fixed
bone marrow cells were acquired on a Becton Dickinson FACScan flow
cytometer using the Becton Dickinson LYSYS II program and were analyzed
with Becton Dickinson CellQuest software. A mean linear fluorescence-3
value was initially determined for each sample from a data file of
25 x 103 fixed bone marrow cells, and a
fluorescence-3 intensity equal to 1/10th of the mean fluorescence-3
value was then used to define a low fluorescence-3 gate. A second data
file of 50 x 103 fixed bone marrow cells that
satisfied the predetermined low fluorescence-3 gate for each sample was
acquired. Single staining of vehicle-treated bone marrow cells with
FITC-anti-c-Kit, PE-anti-Sca-1, or biotinylated Ab mixture and
streptavidin-Red 670 was used to set compensation. Spleen and bone
marrow cells were preblocked with anti-Fc
III/IIR (clone 2.4G2,
rat IgG2b; PharMingen) to reduce nonspecific binding.
RNA analysis for CYP1A1 induction
Total RNA was isolated from liver sections using TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. Twenty micrograms of total RNA was separated by electrophoresis through a 1% agarose formaldehyde gel and transferred to Hybond-N nylon membrane (Amersham, Arlington Heights, IL) by capillary action. RNA was cross-linked to the membrane using a UV Stratalinker 1800 (Stratagene, La Jolla, CA). Duplicate blots were prehybridized for 1 to 4 h and hybridized overnight at 42°C in hybridization solution (50% formamide, 5x Denhardt’s solution, 5x SSPE, 0.5% SDS, 20 mg/ml salmon sperm DNA, and 32P-labeled cDNA probe) for CYP1A1 expression. Blots were also probed with 32P-labeled L32 DNA (51) for normalization of lanes. Blots were washed twice for 10 min each time with 2x SSPE/0.1% SDS at room temperature and twice for 15 min each time with 1x SSPE/0.1% SDS at 65°C. Specific bands were visualized by autoradiography. Results shown represent duplicate analyses.
Statistics
Two-tailed Student’s t test for paired and unpaired variables was used to evaluate differences between treatment groups and their respective vehicle-treated controls. Results were considered statistically significant at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To create receptor chimeric mice, we irradiated B6Ly5.1 mice and
reconstituted them with bone marrow cells from various donors. Since
radiation is known to have an adverse effect on many cells and cellular
systems, it was possible that the cells remaining after irradiation in
the host mouse had an altered sensitivity to TCDD. To rule out this
possibility, we took 4-wk-old B6Ly5.1 male littermates and divided them
into two groups (Table I
, groups 1 and
2). One group of mice was irradiated and reconstituted with bone marrow
cells from another B6Ly5.1 mouse (designated
5.1+5.1+), while the second group of
mice (designated 5.1+) was not irradiated or reconstituted.
At 8 wk of age, half of each group was treated with 30 µg/kg of TCDD,
while the other half received vehicle alone.
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). In
5.1+5.1+ mice treated with TCDD, thymic
weight and thymic cell number were 37 and 18% of control values,
respectively, while in 5.1+ mice the values were 35 and
21%, respectively. Similar alterations were seen in their thymocyte
phenotypes with respect to CD4, CD8, and CD3 expression in response to
TCDD, as was shown previously (31).
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Construction and validation of AhR chimeric mice
Once we found that irradiation and reconstitution did not alter
the sensitivity of the chimeric mice to TCDD, we developed hemopoietic
chimeras to test whether stromal elements, hemopoietic elements, or
both were involved in TCDD-induced changes in the thymus (Table I
,
groups 3 and 4). To do this we took bone marrow cells from a B6Ly5.2
AhR-/- mouse and used them to reconstitute
irradiated B6Ly5.1 (AhR+/+) mice. In doing this we
effectively created mice that have stromal elements that are sensitive
to TCDD, while the hemopoietic elements are insensitive, B6Ly5.2
AhR-/-B6Ly5.1 (designated
5.2-5.1+). Simultaneously, we reconstituted
another set of irradiated B6Ly5.1 mice with bone marrow from a B6Ly5.2
AhR+/+ mouse derived from the same heterozygous cross that
led to the B6Ly5.2 AhR-/- mice. The B6Ly5.2
AhR+/+B6Ly5.1 mice (designated
5.2+5.1+), in contrast to the
AhR-/- reconstituted mice, have both stromal and
hemopoietic elements that are sensitive to TCDD.
To validate that there was successful radiation-induced ablation of the
host immune cells and reconstitution with donor cells in the chimeric
mice, we performed two-color flow cytometry using Ly5.1 (host) vs Ly5.2
(donor) markers. On the average, <1% of the cells in the thymus (Fig. 2A), bone marrow (Fig. 2B), and B cell compartment of the spleen (not shown)
contained Ly5.1 on their surface. Further analysis of the
5.1- thymocytes showed that >98% were labeled with the
donor phenotype, Ly5.2 (Fig. 2C).
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shows that while there was no
detectable CYP1A1 mRNA in the oil-treated controls, there was induction
in the liver of CYP1A1 in both 5.2+5.1+ and
5.2-5.1+ chimeric mice. Furthermore, the
induction obtained in 5.2-5.1+ chimeric
mice was similar to that in 5.2+5.1+
chimeric mice.
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5.1+ mice are unresponsive to
TCDD’s ability to induce thymic atrophy
As shown in Figure 4, A
and B, there was significant thymic atrophy in the
5.2+5.1+ chimeric mice treated with TCDD,
with respect to both thymic weight (36% control) and thymic cell
number (18% control). These results are consistent with those in
5.1+5.1+ mice and 5.1+ mice
treated with TCDD. A small, but significant (p
< 0.03), decrease in the weight of the spleen of treated mice was also
seen, but a similar decline in splenocyte numbers was not seen when the
leukocytes were counted.
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5.1+ chimeras were treated with TCDD,
no alterations were seen in thymic weight or thymic cell number (Fig. 4, C and D). Other parameters, such as
body weight and splenic weight, did not show any significant difference
from the controls.
Phenotypic profile of various immune compartments is unaltered in
5.2-5.1+ mice
Given that the roles of stromal cells in hemopoietic tissues are to support the maturation, differentiation, and, in the thymus, selection of thymocytes, it is possible that TCDD could be acting on the stromal elements to cause shifts in the phenotypic profile of the hemopoietic cells independently of thymic atrophy. Three-color flow cytometry was performed to assess phenotypic shifts among the thymocytes, splenocytes, and bone marrow cells from chimeric mice treated with TCDD.
Figure 5A shows the phenotypic
alterations seen in the thymus when chimeric mice containing both
sensitive stroma and hemopoietic elements
(5.2+5.1+) were treated with TCDD. The
phenotypic results of all mice from all experiments are averaged in
Table II. Slight, but statistically
significant, shifts in the percentage of
CD4+CD8+, CD4-CD8+,
and CD4-CD8- cells were seen. The cells were
also stained with CD3, and there was a modest increase in cells with a
high level of CD3 on their surface (14.5 to 19.8%), as we would have
expected from the increases seen with the single-positive CD4 and CD8
cells.
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5.1+ mice, there were significant
decreases in the percentage and the number of CD25+ cells
in the triple-negative population. There was also a significant
increase in the percentage of CD44+ cells, although the
absolute number of cells was reduced (Table III). These results suggest that TCDD is
affecting the DN population at a specific stage of maturation.
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5.1+) were treated
with TCDD, no major alterations were seen in the CD4 and CD8 phenotypes
of their thymocytes compared with the controls (Fig. 5B). There was, however, a slight decline in the
percentage of CD4+ cells (Table II), but this was not a
consistent finding among all the experiments. Expression levels of CD44
and CD25 in the triple-negative population were not altered
significantly when these mice were treated with TCDD (Table III). These
phenotypic results indicate that the radioresistant stromal cells were
not responsible for alterations seen in the double-positive
(CD4+CD8+), single-positive CD8 cells, or DN
populations of 5.2+5.1+ TCDD-treated
mice.
Analysis of the splenocytes failed to show any significant alterations
in CD4, CD8, CD3, or B220 expression for either chimera,
5.2+5.1+ or
5.2-5.1+, treated with TCDD (data not
shown). Therefore, even when both the stromal and hemopoietic elements
were sensitive to TCDD, there were no significant alterations in spleen
lymphocyte phenotypes.
Since TCDD is known to impair the thymic reconstitution capacity of
bone marrow prothymocytes (31) and reduce the expression of
lymphocyte-specific markers in the bone marrow (31, 53), it is possible
that changes in bone marrow contribute to TCDD-induced thymic
alterations in the AhR chimeric mice. To determine this, we analyzed
the bone marrow of AhR chimeric mice for phenotypic shifts in the
putative hemopoietic stem cell populations. Although there is
continuing controversy over the exact correlation of phenotypic markers
in the bone marrow with T-lineage commitment (54, 55, 56, 57, 58, 59), there is some
general agreement about which bone marrow populations contain cells
that have thymic reconstituting ability (60, 61, 62). Efforts to isolate
and characterize stem cells by surface phenotype have found that the
murine lineage-negative (lin-), low Thy-population is
enriched for cells with long term multilineage reconstitution activity
and day 12 CFU spleen and thymic repopulating ability (60). Cells in
this compartment that stain for Sca-1 have more stem cell potential
than those lacking this marker (60, 61). Other studies showed that
c-kit+Sca-1+lin- cells contained
more stem cell activity than the c-kit- set (61, 62).
Based on these data, we examined the consequence of TCDD exposure on
the c-kit and Sca-1 bone marrow populations in the chimeric mice.
Figure 6A shows phenotypic
alterations to the lin- fraction of bone marrow cells with
respect to Sca-1 and c-kit expression when chimeric mice containing
both sensitive stroma and hemopoietic elements
(5.2+5.1+) were treated with TCDD. By
contrast, Figure 6B shows a lack of effect on these
populations when stroma is sensitive but hemopoietic elements are not.
Phenotypic results for lin- populations from chimeras with
positive stroma are summarized in Table IV. Statistically significant increases
were seen in the lin-c-kit+Sca-1+
and the
lin-c-kit-Sca-1+
populations, while a significant decrease was seen in the
lin-c-kit+Sca-1- set. Chimeras
lacking the AhR in their hemopoietic compartment did not show
statistically significant alterations. These results suggest that TCDD
is altering the hemopoietic stem cell population through activation of
the AhR in hemopoietic cells and not through radioresistant stromal
cells.
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5.2- mice are responsive to
TCDD’s ability to induce thymic atrophy and phenotypic shifts
To verify our findings that the hemopoietic elements, not the
radioresistant stromal elements, are the targets in TCDD-induced thymic
atrophy, we constructed chimeric mice that had sensitive hemopoietic
elements but insensitive stromal elements
(5.2+5.2-; Table I, group 5). Given
our results with the 5.2+5.1+ and
5.2-5.1+ chimeras, we expected to see TCDD
induce thymic atrophy in these mice to a similar degree as mice with
both sensitive hemopoietic and stromal elements
(5.2+5.1+ mice). Concurrently, we
constructed 5.2-5.2- mice (Table I, group
6) to further substantiate that AhR-/- chimeras are
completely unresponsive to the effects of TCDD in the thymus and that
the process of irradiation/reconstitution did not alter their
unresponsiveness.
Figure 7, A and B,
shows that there was significant thymic atrophy in the
5.2+5.2- chimeric mice treated with TCDD
with respect to both thymic weight and thymic cell number. By cell
number there was an average 69% reduction in all TCDD-treated thymi
from 5.2+5.2- mice compared with the
control value. This was slightly less than the 82% reduction seen in
5.2+5.1+ mice, but this difference was not
significant since some of the individual
5.2+5.2- experiments had just as much, if
not more, atrophy. Body weight, splenic weight, and splenic cell counts
were not notably altered in the 5.2+5.2-
mice treated with TCDD.
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, C and D, shows that there were no
alterations in the 5.2-5.2- mice with
regard to body, thymic, and splenic weights or thymic and splenic cell
numbers. This finding concurs with the finding that
AhR-/- mice are unresponsive to TCDD.
Table V shows phenotypic shifts in the
CD4/CD8 thymic populations of 5.2+5.2- mice
treated with TCDD. These shifts are very similar to those seen in
5.2+5.1+ mice (Table II) with regard to both
percentages and cell numbers. Cell surface staining of
CD4-CD8-CD3- cells yielded a
similar change in CD44/CD25 cell populations in the
5.2+5.2- mice (Table VI) compared with that
in the 5.2+5.1+ mice (Table IV). The bone marrow data from these mice
also paralleled those from the 5.2+5.1+
treated mice with an increase in the
c-kit+Sca-1+ phenotype from 5.5 ± 0.5 to
11.9 ± 3.9 (p < 0.01), an increase in
the c-kit-Sca-1+ phenotype from 4.2 ±
1.3 to 6.0 ± 1.6 (p < 0.01), and a
decrease in the c-kit+Sca-1- phenotype from
58.6 ± 7.5 to 38.6 ± 9.9. In comparison, phenotypic results
for both the thymus (data not shown) and the bone marrow cells of
TCDD-treated 5.2-5.2- mice failed to show
any statistically significant difference from control values (10.4
± 3.9 to 8.9 ± 2.1 for c-kit+Sca-1+,
4.6 ± 0.9 to 4.3 ± 1.0 for
c-kit-Sca-1+, and 42.5 ± 7.9 to
44.5 ± 4.5 for c-kit+Sca-1-).
Splenocytes from treated 5.2+5.2- and
5.2-5.2- mice showed no difference from
controls in CD3, CD4, CD8, and B220 populations (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results indicate that hemopoietic elements of the immune system
play the major, if not the exclusive, role in mediating the effects of
TCDD on the thymus. When chimeric mice containing unresponsive
(AhR-/-) hemopoietic elements and responsive
(AhR+/+) stromal elements were treated with TCDD, no signs
of thymic atrophy were seen (Fig. 4, C and
D). In addition, there were no significant
alterations in the phenotype of the thymus (Fig. 5B and
Table II). This lack of atrophy and phenotypic alterations were also
seen in chimeras containing both insensitive stromal and insensitive
hemopoietic elements (Fig. 7, C and D). In
contrast, chimeric mice containing sensitive hemopoietic and stromal
elements and mice that had only sensitive hemopoietic elements had
similar and significant declines in thymic weight and cell numbers
after TCDD treatment (p < 0.001; Fig. 4, A and B, and Fig. 7, A and
B). Both these chimeras had similar phenotypic
alterations, including a reduction in the percentage of
CD4+CD8+ and an increase in the percentage of
CD4-CD8- and
CD4-CD8+ cells compared with the control
percentages (Fig. 5A and Tables II and V). These
observations agree with those reported by us and other investigators in
other mouse strains after TCDD treatment (22, 24, 32, 33, 53).
Since there were alterations in the percentage and number of DN cells
in TCDD-treated chimeras containing sensitive hemopoietic cells, we
analyzed this population using CD44 and CD25 markers (52). After TCDD
treatment, there was a decrease in the percentage of more mature
CD25+ cells in this set and a relative increase in the
percentage of immature CD44+ cells. A calculation of the
actual numbers of cells in each population compared with controls
suggested that this change was due to less of a decline in
CD44+ cells compared with that in the CD25+
cells (Tables III and VI). These results suggest that TCDD is
interfering with the development and/or the proliferation of DN cells
by either blocking maturation of CD44+ cells or potentially
killing CD25+ cells. Furthermore, there were no changes in
the CD44/CD25 profile in the 5.2-5.1+
chimeras (Table III), suggesting that TCDD must be directly targeting
hemopoietic cells to cause this early thymocyte developmental
block.
Besides examining the thymus, we also analyzed the effects of TCDD on
spleen and bone marrow in these chimeras. Both
5.2+5.1+ and
5.2-5.1+ chimeras manifested loss in spleen
weight, although the decline only achieved significance in the
5.2+5.1+ mice (Fig. 4, A and
C). This decline in weight, however, did not
translate into a decline in leukocyte cell number (Fig. 4, B
and D). A feasible explanation for this discrepancy
could be a loss of RBC, which has been reported previously (64).
Analysis of spleen cell phenotypes did not reveal differences in the
major T cell classes or in total B220+ B cells in any of
the AhR chimeras studied. This, too, has been reported for nonchimeric
mice of other strains (32).
Bone marrow data from chimeric mice with sensitive (AhR+/+)
hemopoietic elements showed an increase in
lin-c-kit+Sca-1+ and
lin-c-kit-Sca-1+ and a decrease
in lin-c-kit+Sca-1- populations
regardless of whether the radioresistant stromal elements were
AhR+/+ or AhR-/- (Fig. 6, Table IV, and
text). Since the total number of bone marrow cells isolated and the
percentage of lin- cells obtained from TCDD-treated
chimeras were not significantly different from the control values,
these results suggest that shifts are occurring within the putative
hemopoietic stem cell populations, including those cells that could
give rise to thymocytes. These results supplement our previous studies
that demonstrated that bone marrow cells from mice exposed to TCDD had
reduced thymic reconstituting potential (31) and a decided reduction in
the expression of lymphocyte-specific markers such as terminal
deoxynucleotidyl transferase protein (31) and terminal deoxynucleotidyl
transferase and recombinase-activating gene-1 mRNA (53). Since there is
still a fair amount of controversy about which cell markers define the
potential T cell progenitors or pluripotent hemopoietic stem cells in
the bone marrow (57, 58, 59, 60, 61, 62, 65, 66), it is difficult to correlate our
current findings of lin-c-kit/Sca-1 alterations to our
previous findings of a reduction in lymphoid stem cell activity and
markers. Nevertheless, the results from the chimeric mice suggest that
TCDD is directly affecting putative hemopoietic stem cell populations
in the bone marrow independent of the bone marrow stromal elements. In
addition, these data are consistent with the possibility that
TCDD-induced thymic alterations may be mediated at least in part by
effects of TCDD directly on the hemopoietic compartment in the bone
marrow.
Although we did not see significant alterations in the thymus or bone
marrow of TCDD-treated 5.2-5.1+ chimeras,
we did see induction of CYP1A1 RNA in liver from these animals. The
degree of induction in the 5.2-5.1+
chimeras was similar to that of 5.2+5.1+
chimeras (Fig. 3), confirming that AhR-/- hemopoietic
cell-containing mice treated with TCDD produce a normal hepatocyte
response. In preliminary observations we also noted that there were
fewer significant lesions in the livers of TCDD-treated
5.2-5.1+ chimeras than in the livers of
TCDD-treated 5.2+5.1+ chimeras, which had
focal necrosis with infiltration of immune cells in the perivascular
regions T.S. Thurmond, manuscript in preparation). This observation
suggests that some liver lesions induced by TCDD may be a result of
immune cell activation. TCDD treatment has been shown by others to
potentiate the response of lymphoid cells to anti-CD3 T cell
activation (67). Therefore, these chimeras could be used to determine
whether the effects of TCDD and congeners are due to a direct effect on
a tissue or an indirect effect mediated by activation of immune
cells.
Our results show that TCDD-induced thymic atrophy is mediated by hemopoietic cells, but these studies do not allow us to determine the specific cell type(s) that is the direct target(s) for mediating this effect. Both DC and macrophages of the thymic stroma are believed to be sensitive to irradiation (39) and should therefore be ablated in recipients during the construction of the chimeras. Since these elements are bone marrow derived (40, 68), they are reconstituted with either AhR-/- or AhR+/+ cells depending on the donor. These hemopoietic elements of stroma might be the cellular targets for TCDD activation of the AhR, rather than developing lymphoid cells. However, both DC (6, 43, 44) and macrophages (34, 40) have been shown to be actively involved in negative selection, and since TCDD does not seem to alter negative selection (33, 69), it is less likely that nonlymphoid hemopoietic cells are the decisive cellular targets. This evidence again suggests that the hemopoietic elements are the main target of TCDD action. Previous work by others has also provided some evidence that hemopoietic elements mediate TCDD suppression of the T cell-dependent primary Ab response (70). Additionally, we have provided evidence that some of the lymphoid targets for the effects of TCDD could include T cell progenitors in the bone marrow (31, 53).
Although our results support a direct action on developing thymocytes or lymphoid stem cells by TCDD, they contradict the results of others (24, 25, 26) who have argued that epithelial cells mediate the action of TCDD in the thymus. These previous investigations, however, were performed using higher doses of TCDD or in vitro culture systems. For example, De Waal et al. (26) saw changes in the cortical-epithelial ultrastructure of the rat thymus following the administration of 150 µg/kg of TCDD. However, at 50 µg/kg such epithelial changes were not detectable, although there was significant atrophy. We observed atrophy induction at an even lower dose, so the epithelial effects observed by De Waal et al. might be due to additional toxic effects of TCDD at higher doses.
Greenlee (25) showed that thymocytes cultured on thymic epithelial layers incubated with TCDD had reduced responses to T cell mitogens. This result does not explain thymic atrophy induction, since the main responders in the mitogen assay should be the mature population in the thymus, and the mature cells, according to our data, do not appear to be the target population. Kremer et al. (24) used fetal thymic organ cultures to show that epithelial elements exposed to TCDD failed to support proliferation of early thymocytes. This difference from our in vivo results has precedent (21), possibly because factors present in serum of the cultures or interactions of TCDD with serum factors (71) could be creating in vitro-specific effects. The in vitro systems are also unable to evaluate the contributions of bone marrow precursors, thymic ultrastructure, and neuroendocrine signals (72). One might argue that fetal thymic organ cultures address some of these deficiencies, but it is hard to make a direct connection between the in vitro effects of TCDD on an immature (gestational day 15) thymic structure and a block in development to TCDD effects on the adult organ that clearly involve a loss of cells. For example, DC are not prominent in the thymus until gestational day 17 (73), and in the developing organ there could be multiple targets (74).
Our overall conclusions are the following. 1) TCDD induces thymic
atrophy by directly targeting the T cell progenitors (31, 53). This
leads to a reduction of cells entering the thymus and a decline in the
absolute number of DN cells (Tables II and V). 2) TCDD exposure
inhibits the development of cells in the DN compartment through a
direct effect on hemopoietic cells. This causes a decline in the number
of maturing DN CD25+ cells (Tables III and VI), which, in
turn, leads to a decline in the number of cells leaving the DN
compartment and contributing to the double-positive compartment. 3) The
shifts seen in double-positive, CD8 single-positive, and DN cells in
the thymus after TCDD treatment are also caused by a direct effect on
hemopoietic cells as chimeras, with AhR-/- hemopoietic
elements do not show these shifts (Table II).
Finally, our results further support the idea that the AhR plays a role in the immune system, as suggested by the development of AhR knockout mice. It was reported that AhR-/- mice, in which exon 1 of the AhR gene is knocked out, developed perturbations in peripheral T and B cell populations in the spleen at various times during development (4). These mice also had an increased susceptibility to infection, with lymphocytic infiltrates being found in various organs, such as gut, lung, and urinary tract, in mice that die suddenly (5). We have also noted problems with the AhR-/- mice as host mice in the irradiation/reconstitution experiments. Unlike the AhR+/+ host, the AhR-/- host were more likely to die after the procedure, and their reconstituted thymi were smaller and more variable in size. Some of these immunologic deficiencies were not observed in a second AhR-/- mouse in which exon 2 of the AhR gene was knocked out instead of exon 1 (7). In the second AhR-/- mouse, however, changes in splenic architecture and in splenic mononuclear cells were reported.
Although our results show that activation of the AhR by TCDD leads to effects on T cell development, the receptor does not seem to play a key role in the establishment of a normal T cell compartment. The thymi of lethally irradiated B6Ly5.1 mice reconstituted with AhR-/- or AhR+/+ bone marrow are similar in size and phenotype. Given that the AhR does not play a significant role in the establishment of the thymus but when activated can invoke changes in T cell development, we suggest that the receptor may be involved in a stress response. For instance, during a microbial infection, when there is a need for mature CD8+ cells and less development of cells that could be tolerized, the AhR may be activated to produce this result. Such suggestions for the normal function of the AhR are limited by the caveat that the natural ligand might not bind with the high affinity and persistence seen with TCDD, an exogenous ligand for the receptor. Further studies of normal mice with AhR-/- hemopoietic systems might provide insight into the normal immune system role for the AhR.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Allen E. Silverstone, Department of Microbiology and Immunology, State University of New York Health Science Center, 750 East Adams St., Syracuse, NY 13210. E-mail address:
3 Abbreviations used in this paper: AhR, aryl hydrocarbon receptor; bHLH, basic helix-loop-helix; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; hsp90, heat shock protein 90; DC, dendritic cells; P/S, penicillin/streptomycin; PE, phycoerythrin; CYP1A1, cytochrome P4501A1; DN, double-negative CD4-CD8- cells; lin-, lineage-negative cells, i.e., CD3-, CD8-, B220-, Gr-1-, Mac-1-, and TER119-negative cells.
Received for publication August 25, 1997. Accepted for publication December 22, 1997.
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