Evidence for Induction of Apoptosis in T Cells from Murine Fetal Thymus following Perinatal Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)

Iris A. Camacho*, Mitzi Nagarkatti* and Prakash S. Nagarkatti{dagger},1

* Department of Microbiology and Immunology and {dagger} Department of Pharmacology and Toxicology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298

1 To whom correspondence should be addressed at the Department of Pharmacology and Toxicology, PO Box 980613, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298-0613. Fax: (804) 828-0676. E-mail: pnagark{at}hsc.vcu.edu.

Received October 2, 2003; accepted November 12, 2003

ABSTRACT

Perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes thymic atrophy, but the precise mechanism of such toxicity remains unresolved. The current study investigated the role of apoptosis in TCDD-induced thymic involution following perinatal exposure to TCDD. To this end, C57BL/6 pregnant mice were injected intraperitoneally on gestational day (GD) 14 with a single dose of 10 µg/kg TCDD. Analysis of the thymus on GDs 15, 16, 17, and 18, and on postnatal day (PD) 1, showed a remarkable reduction in thymic cellularity 3-7 days post-TCDD exposure. TCDD treatment also caused marked changes in the proportions of T-cell subsets, particularly on GD 17 and GD 18 thymocytes. In vitro culture of thymocytes from mice exposed perinatally to TCDD showed increased apoptosis when compared to the controls, which peaked on day 3 post-TCDD exposure. Triple-color staining showed that TCDD induced apoptosis in all four subpopulations of T cells, with the double-positive T cells undergoing the highest level. Moreover, increased cleavage of caspase-3 was seen when TCDD-exposed GD 17 thymocytes were directly tested. Furthermore, apoptosis-associated phenotypic changes were found in thymocytes of mice perinatally exposed to TCDD, characterized by an increase in expression of CD3, {alpha}ßTCR, IL-2R, and CD44, and a decrease in CD4, CD8, and J11d markers. Finally, thymocytes from mice exposed perinatally to TCDD showed higher levels of Fas, TRAIL, and DR5 mRNA, but the levels of Bcl-2, Bcl-xL, and Bax were either unaltered or changed moderately. Taken together, these results suggest that TCDD-induced thymic atrophy following perinatal exposure may result, at least in part, from increased apoptosis mediated by death receptor pathway involving Fas, TRAIL, and DR5.

Key Words: TCDD; perinatal exposure; thymus; immunotoxicity; apoptosis.

The developing immune system is highly sensitive to the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The thymus has shown great sensitivity to the toxic effects of TCDD in all species examined (Vos et al., 1997Go). For instance, rodents perinatally exposed to low doses of TCDD have shown decreased thymic weights and altered expression of thymic differentiation markers (Blaylock et al., 1992Go; Faith and Moore, 1977Go; Fine et al., 1989Go; Gehrs et al., 1997Go; Gehrs and Smialowicz, 1997Go; Holladay et al., 1991Go; Luster et al., 1979Go). TCDD-induced immunotoxic effects in the fetal and/or neonatal thymus have been associated with negative repercussions on postnatal cell-mediated immune responses. In fact, animals perinatally exposed to TCDD can carry over the immunosuppression until adulthood (Faith and Moore, 1977Go; Gehrs et al., 1997Go; Gehrs and Smialowicz, 1999Go; Thomas and Hinsdill, 1979Go), suggesting that impairments in the developing immune system at an early age could alter the susceptibility to infections, cancer, and autoimmunity in adulthood. For example, perinatal TCDD exposure impairs host resistance against infectious agents or syngeneic tumor cells (Faith and Moore, 1977Go; Luster et al., 1979Go). In addition, perinatal TCDD exposure elicits suppression of cell-mediated immune functions, including T-cell mitogen responses (Luster et al., 1979Go), skin graft rejection times, graft-versus-host reactivity (Vos and Moore, 1974Go), delayed hypersensitivity (Blaylock et al., 1992Go; Faith and Moore, 1977Go; Gehrs et al., 1997Go; Gehrs and Smialowicz, 1997Go), and cytotoxic T-lymphocyte responsiveness (Holladay et al., 1991Go). Such studies strongly suggest that perinatal exposure to TCDD alters the developing immune system, which may subsequently creates deficiencies in postnatal immunity (Holladay and Smialowicz, 2000Go).

Several hypotheses have been proposed to account for TCDD-induced thymic atrophy. Some investigators have suggested that TCDD may cause thymic involution by affecting progenitor T cells in the bone marrow and fetal liver, which subsequently results in reduced thymic seeding (Fine et al., 1989Go). There is also evidence suggesting that TCDD may directly target thymocytes by either preventing the differentiation of CD4+CD8+ T cells (Blaylock et al., 1992Go; Fine et al., 1989Go; Gehrs et al., 1997Go; Gehrs and Smialowicz, 1997Go; Holladay et al., 1991Go; Kerkvliet and Brauner, 1990Go; Lundberg et al., 1990Go), by affecting thymic stroma (De Waal et al., 1992Go; Greenlee et al., 1985Go; Kremer et al., 1995Go; Nagarkatti et al., 1984Go) or by inducing thymic apoptosis (Kamath et al., 1997Go; Rhile et al., 1996Go; Zeytun et al., 2002Go). Given the fact that TCDD induces the expression of a large array of genes (Zeytun et al., 2002Go), it is reasonable to believe that multiple mechanisms may act in concert to produce thymic atrophy.

Apoptosis is an inherent process in the thymus occurring on those T cells that exhibit reactivity to self-antigens or fail to be positively selected (Palmer, 2003Go). As apoptosis contributes to deletion of the majority of T cells in the thymus, this active regulatory mechanism could be a potential target for immunotoxicants currently known to destroy thymic tissue. To this end, we have demonstrated in adult mice that TCDD may kill T cells in the thymus in vivo by increasing Fas-mediated apoptosis. Experiments using TCDD-exposed Fas or FasL mutant mice showed that their thymocytes failed to undergo increased apoptosis and such mice were more resistant to thymic atrophy when compared to the wild-type mice (Kamath et al., 1999Go). We also observed that FasL was strongly induced in the thymus of TCDD-exposed wild-type mice (Kamath et al., 1999Go).

In perinatally exposed rodents, TCDD-induced thymic atrophy is accompanied by an increase in the percentage of CD4-CD8- and CD4-CD8+ as well as a decrease in the percentage of CD4+CD8+ T cells (Blaylock et al., 1992Go; Fine et al., 1989Go; Gehrs et al., 1997Go; Gehrs and Smialowicz, 1997Go; Holladay et al., 1991Go). Similar observations have been made in vitro using fetal thymic organ cultures (Kronenberg et al., 2000Go; Lai et al., 1998Go). In contrast, TCDD-induced thymic atrophy in adult mice is accompanied by only minor or no significant alterations in the percentage of T-cell subsets (Kerkvliet and Brauner, 1990Go; Lundberg et al., 1990Go; Rhile et al., 1996Go). Such differences in the proportions of T-cell subsets in the thymus seen following prenatal or postnatal exposure to TCDD may be less consequential considering the fact that both treatments cause marked thymic atrophy. In the current study, we investigated whether perinatal exposure to TCDD triggers apoptosis in thymocytes and alters the expression of apoptosis-regulating genes.

MATERIALS AND METHODS

Animals.
Timed pregnant (vaginal plug = day 0) C57BL/6 mice were purchased from the National Institute of Health (Bethesda, MD). All animals were housed in polyethylene cages equipped with filter tops and wood shavings. Each animal cage had rodent chow and tap water ad libitum. Mice were housed in an environment of constant temperature (23 ± 2°C) and on a 12 h-light:12 h-dark lighting schedule.

TCDD exposure and sample collection.
TCDD was generously donated by Dr. K. Chae of NIEHS (Research Triangle Park, NC). TCDD was dissolved in acetone and diluted in corn oil. The solution was gently heated to evaporate the acetone (Rhile et al., 1996Go). Time kinetic studies were carried out in pregnant animals on gestational day (GD) 14 by treating them intraperitoneally (ip) with a single dose of 10 µg/kg TCDD or the vehicle control (corn oil). The experimental protocol consisted of injecting three pregnant mice either with TCDD or vehicle. From each pregnant mother, we obtained an average of 7–9 pups. To reduce the variability among the pups in each litter, we combined the three litters from each treatment group to generate a pool of 21–27 pups. Thymi from 4–5 pups were randomly pooled per sample due to low thymic cellularity, and 5–6 replicate pools were used for statistical analysis.

Cell preparations.
Organs were removed and placed in RPMI-1640 medium (Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Grand Island, NY), 10 mM Hepes, 1 mM glutamine, 40 µg/ml gentamicin sulfate, and 50 µM 2-mercaptoethanol. Single cell suspensions were prepared from thymi by gently homogenizing the tissue. Cells were pelleted by centrifugation (1300 rpm, 7 min, 4°C) and resuspended in 0.83% ammonium chloride (Sigma, St. Louis, MO) to lyse the erythrocytes. Cells were further washed twice in medium. Cell viability was determined on a hemacytometer by trypan blue dye using an inverted phase contrast microscope. For calculating thymic cellularity, the data were expressed as total number of thymocytes/pup. Thus, for statistical analysis, 5–6 replicate pools were compared from each treatment group and depicted as mean cellularity/pup ± SEM.

Detection of phenotypic markers on thymocytes.
Thymocytes (1 x 106) were washed with phosphate-buffered saline (PBS; Invitrogen, Grand Island, NY) and incubated for 30 min on ice with 0.5 µg of the following primary monoclonal antibodies (mAbs): FITC-anti CD4 (L3T4), PE-anti CD8 (Ly-2), FITC-CD3 ({epsilon}chain), FITC-CD8 (Ly-2), PE-CD44 (IM7), FITC-{alpha}ßTCR (H57-597), or FITC-IL-2R (7D4) (BD Pharmingen, San Diego, CA). For double-staining studies, directly conjugated mAbs were simultaneously added to the sample. Detection of J11d marker was done by staining cells with J11d mAb (BD Pharmingen, San Diego, CA) followed by FITC- anti-rat IgM mAbs. After incubation with the mAbs, cells were then washed once with PBS. Negative controls consisted of cells that were stained with appropriate isotype-specific antibodies. Cells were then fixed with 1% p-formaldehyde. Flow cytometric analysis was performed by a FACScan (Becton Dickinson, Franklin Lakes, NJ) using the Cyclops operating software (Cytomation, Ft. Collins, CO). Ten thousand cells were analyzed per sample. Dead cells, clumps, and debris were excluded electronically by gating on forward (FSC) versus side scatter (SSC). Flow cytometric data were analyzed as described by Kamath et al. (1998)Go. In general, CD4/CD8 phenotypic data were represented as percentage positive cells expressing the surface marker (Fig. 2). For analysis of phenotypic markers (Fig. 5), the mean intensity of fluorescence (MIF), which represents the density of expression of the surface marker, was determined for the control and TCDD-treated cells. Subsequently, MIF values were used to calculate the percent change in MIF as follows:

The data from 5-6 replicate pools were depicted as percentage change in MIF ± SEM.



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FIG. 2. Effects of perinatal exposure to TCDD on thymic T-cell subsets. C57BL/6 pregnant mice (gestational day = 14) were injected with 10 µg/kg TCDD or the vehicle. One to seven days post-TCDD treatment, thymocytes from fetuses and neonates were double-stained with FITC-anti-CD4 and PE-anti-CD8 mAbs. Representative dot-plots showing the percentage of T-cell subsets in the thymus are depicted in the left panels and data collected from 5-6 replicate pools have been plotted in the right panel. Asterisks (*) indicate statistically significant differences when T-cell subsets from TCDD-exposed thymocytes were compared to vehicle controls within each group (p < 0.05).

 


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FIG. 5. Effect of TCDD on the surface marker expression in neonatal thymocytes. C57BL/6 pregnant mice (gestational day = 14) were treated with 10 µg/kg TCDD or the vehicle. Seven days post-TCDD treatment, neonates were sacrificed and thymocytes stained with FITC- or PE-conjugated mAbs against CD3, {alpha}ßTCR, IL-2R, CD44, CD4, CD8, or J11d. Cells were then analyzed by flow cytometry. Representative histograms are shown for each experimental group (A). The empty histogram represents cells stained with control antibodies, and the filled histogram represents thymocytes stained for antibodies against various surface markers. The percentage positive cells and mean intensity of fluorescence (MIF) for stained cells have been depicted for each histogram. The MIF obtained with each marker in TCDD-treated mice was compared to the MIF of the controls, and the data were expressed as percentage change in MIF (B). The percentage change in MIF from 5-6 replicate pools was represented as the mean ± SEM per treatment group.

 
Detection of apoptosis using the TUNEL method.
Thymocytes were analyzed for apoptosis using the TUNEL assay, which employs FITC-dUTP nick-end labeling to detect apoptosis-related DNA fragmentation (Boehringer Mannheim, Indianapolis, IN). As described earlier (Camacho et al., 2001Go; Kamath et al., 1999Go), thymocytes (1 x 106) from vehicle- or TCDD-treated mice were cultured for 24 h in 10%-FBS medium. The cells were washed twice with PBS and fixed with 4%-paraformaldehyde for 30 min at room temperature. The cells were next washed with PBS, permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate on ice for 2 min, washed again with PBS, and finally incubated with FITC-dUTP for 1 h at 37°C. Fluorescence of ten thousand cells was measured by flow cytometry. In addition, the percent change of apoptosis for the TCDD-treated cells relative to the vehicle-treated cells was calculated as follows:

The data from 5-6 replicate pools were depicted as mean percent change in apoptosis ± SEM.

In addition, triple-staining studies were carried out to determine the amount of apoptosis in each of the thymic T-cell subpopulations. Similar to double-staining studies, thymocytes were cultured for 24 h, harvested, and washed with PBS. Cells were incubated with PE-CD8 (Ly-2) and Tricolor-CD4 mAbs for 30 min on ice followed by one wash with PBS. Next, cells were subjected to the TUNEL protocol as described above. Fluorescence of 50,000 cells was collected by flow cytometry. Electronic compensation for fluorochrome spectral overlap was performed during flow cytometric analysis of multi-color immunofluorescence stainings. Apoptosis was detected in each CD4/CD8 T-cell population by gating the particular T-cell subset and measuring the amount of TUNEL positivity.

Analysis of caspase-3 activity.
Three days post-perinatal exposure to TCDD, freshly isolated thymocytes were immediately assayed for caspase-3 protease activity using a colorimetric assay (R&D Systems, Minneapolis, MN). Briefly, thymocytes were lysed and protein content of the cell lysates was estimated using the BCA protein assay (Pierce Biotechnology Inc., Rockford, IL). Freshly isolated thymocytes (1 x 106/ml) treated in vitro with dexamethasone (10-7 M) for 6 h were used as positive control (Marchetti et al., 2003Go). Fifty micrograms of total protein were used to assess caspase-3 activity. Cell lysates were incubated overnight at 37°C with caspase substrates conjugated with the chromophore p-nitroanilide (pNA). Cleavage of DEVD-pNA substrate by caspase-3 releases pNA, which was then quantified spectrophotometrically at a wavelength of 405 nm. The level of caspase enzymatic activity in the cell lysate was directly proportional to the optical density units (OD) of the color reaction. The data from 5-6 replicate pools were depicted as mean activity ± SEM.

Detection of apoptotic genes by RT-PCR.
Three or seven days post-perinatal exposure to TCDD, 4–5 thymi per treatment group were pooled and used for RNA extraction. Total RNA was isolated from thymocytes using the TRIZOL reagent according to the manufacturer's instructions (Life Technologies, Grand Island, NY). Five hundred nanograms of RNA were subjected to cDNA synthesis using the Omniscript RT kit (Quiagen, Valencia, CA). The reverse transcriptase (RT) reaction was run at 37° C for 60 min. First strand cDNA was amplified in 50-µl final volume containing 5 µl of each primer (final concentration 0.2 µM), 0.5 µl of Taq DNA polymerase (5 U/µl; Invitrogen, Grand Island, NY), 2 µl of 50 mM MgCl2, 4 µl of 2.5 mM dNTP, 5 µl of 10XPCR buffer, 1 µl cDNA, and water. The amplification conditions were 2 min at 94°C for initial denaturation, 29 cycles at 94°C for 30 s, annealing temperature at 40 s, 72°C for 1 min, and one cycle at 72°C for 2 min. Ten microliters of ß-actin product and 20 µl of the other mouse gene products were combined with loading dye, run at 150 V and visualized on a 1.5% agarose gel containing 0.25 µg ethidium bromide/ml. The fluorescent images were photographed under UV light. Primer sequences were from published sources or chosen from the published cDNA sequences, and are listed in Table 1. Bands were quantified using Scion Image software available on the Internet at http://rsb.info.nih.gov/nih-image. To normalize RNA loading and PCR variations, the signals of target genes were corrected with the signals of ß-actin mRNA. Gene expression was checked in three replicate pools and representative data were depicted.


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TABLE 1 PCR Primer Pairs Used in This Study

 
Statistical analysis.
Results are presented as the mean ± SEM of 5-6 replicate pools per treatment group. Statistical analyses were performed using JMP IN statistical software (SAS Institute, Cary, NC). ANOVA was performed with a significance level of {alpha} = 0.05. Comparison among means was made using the Tukey-Kramer HSD test, with values of p < 0.05 considered to be statistically significant.

RESULTS

Alterations of Thymic Cellularity and T-Cell Subpopulations following Perinatal Exposure to TCDD
TCDD was administered as a single dose of 10 µg/kg into pregnant C57BL/6 mice on GD 14 and thymic cellularity was determined on GDs 15, 16, 17, 18, and on postnatal day (PD) 1 (Fig. 1) in fetal and neonatal mice. One or two days post-TCDD treatment, GD 15 and GD 16 fetuses did not exhibit decreased thymic cellularity. However, from GD 17 onwards, thymic atrophy was demonstrable in these mice including on PD 1.



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FIG. 1. Thymic cellularity in TCDD-exposed fetuses and neonates. C57BL/6 pregnant mice (gestational day = 14) were injected with 10 µg/kg TCDD or the vehicle. One to seven days post-TCDD treatment, the thymi from fetuses and neonates were removed to determine organ cellularity. Asterisks (*) indicate statistically significant differences in the mean cellularity of TCDD-exposed thymocytes when compared to the vehicle controls within each group (p < 0.05). Data from 5-6 replicate pools were represented as the mean thymic cellularity per pup ± SEM.

 
In the above experiments, we also examined the percentage of T cells expressing CD4/CD8 markers in the thymus of TCDD-exposed fetuses and newborns. The results from a representative experiment have been shown in the left panel of Figure 2 and data from multiple experiments have been plotted in the right panel. GD 15 thymocytes, which are mostly comprised of double-negative (CD4-CD8- or DN) T cells, did not show any significant alterations 24 h post-TCDD treatment. The earliest changes in thymic T-cell subsets were seen on GD 16 thymocytes, obtained two days post-TCDD treatment. At this time point, TCDD caused a decrease in the percentage of DN T cells and an increase in the percentage of single-positive CD8 (CD4-CD8+ or spCD8) T cells. On GD 16, the double-positive (CD4+CD8+ or DP) and single-positive CD4 (CD4+CD8- or spCD4) T cells had not differentiated completely as indicated by relatively small percentage of such cells in the vehicle-treated mice. As expected, a major proportion of DN T cells differentiated into DP T cells by GD 17 and GD 18 (Penit and Vasseur, 1989Go). At these developmental stages, TCDD had a greater impact on T-cell differentiation, inasmuch as TCDD treatment decreased the percentage of DP T cells and increased the percentage of spCD8 and DN T cells.

On GD 16 and GD 17, spCD4+ T cells had not yet differentiated. Thus, the increased proportion of spCD8+ T cells found in vehicle-treated mice during these stages of gestation (Fig. 2) may represent immature rather than mature T cells. To confirm this, we carried out further analysis of such cells by double-staining the thymocytes with antibodies against CD3 and CD8 or {alpha}ß TCR and CD8. Such an analysis of thymocytes on GD 17 revealed that majority of the spCD8+ T cells seen in Figure 2 were indeed immature cells because they were CD3-CD8+ or {alpha}ßTCR-CD8+ (Table 2). Moreover, exposure to TCDD caused a significant increase in proportion of the immature spCD8+ T cells when compared to the vehicle controls (Table 2). Interestingly, by PD 1, the percentage of T-cell subsets in TCDD-treated groups had been restored to normal levels with the exception of a persistent increase in spCD8 T cells. At every time point tested, spCD4 T cells were unaffected by TCDD treatment. These data showed that perinatal exposure to TCDD induces profound thymic atrophy and changes in T-cell subsets in the fetal thymus.


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TABLE 2 Percentage of Immature CD8 T Cells in the Thymus following Perinatal Exposure to TCDD

 
Detection of Apoptosis in the Developing Thymus following Perinatal Exposure to TCDD Using TUNEL Assay
Thymi from TCDD-exposed fetuses and newborns were evaluated for apoptosis using the TUNEL method (Fig. 3). In this assay, freshly isolated thymocytes were incubated for 24 h in vitro with medium prior to TUNEL staining. This in vitro approach allows better detection of TCDD-induced apoptotic cells because apoptosis-committed cells can ultimately die without being rapidly engulfed by phagocytic cells (Kamath et al., 1997Go). The results from a representative experiment have been shown in Figure 3A and data from multiple experiments have been plotted in Figure 3B. The thymocytes from TCDD-treated groups consistently showed significantly higher percentage of apoptosis at all stages of gestation and on PD 1, when compared to the vehicle-treated groups. Maximal change in apoptosis was detected on GD 17 thymocytes 3 days post-TCDD treatment (Fig. 3B).



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FIG. 3. Induction of thymic apoptosis in the fetus and the neonate following TCDD treatment. C57BL/6 pregnant mice (gestational day = 14) were injected with 10 µg/kg TCDD or the vehicle. One to seven days post-TCDD treatment, thymocytes from fetuses and neonates were isolated and cultured for 24 h at 37°C and then stained with FITC-dUTP for apoptosis detection. Percentage of apoptotic cells is represented in each representative histogram (A). The percentage of apoptotic cells obtained in the TCDD-treated thymocytes was compared to that seen in controls, and the data were expressed as percentage change in apoptosis (B). The percent change in apoptosis from 5-6 replicate pools was represented as the mean ± SEM per treatment group.

 
Detection of Caspase-3 in Thymocytes following Perinatal Exposure to TCDD
Because caspase cleavage is an early event and measurement of caspase-3 protease activity is a reliable indicator of caspase-dependent apoptosis (Gurtu et al., 1997Go), we also evaluated caspase-3 activity following perinatal exposure to TCDD. We limited the analysis of caspase-3 activity to GD 17 thymocytes three days post-perinatal exposure to TCDD because this time point showed the highest level of apoptosis. Also, in these experiments, unlike the TUNEL assay, we used freshly isolated prenatal thymocytes from vehicle or TCDD-exposed embryos without further in vitro culture. The data shown in Figure 4 suggested a statistically significant 5-fold increase in caspase-3 enzymatic activity in TCDD-exposed thymocytes when compared to those exposed to vehicle only. In addition, this assay included a positive control, which consisted of treating thymocytes with dexamethasone in vitro. Dexamethasone-treated thymocytes exhibited a strong induction of caspase-3 activity, which was inhibited by incubating the cell lysates with caspase-3 inhibitor (DEVD-CHO), thereby confirming the specificity.



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FIG. 4. Perinatal exposure to TCDD increases caspase-3 activity in the thymus. C57BL/6 pregnant mice (gestational day = 14) were treated with 10 µg/kg TCDD or the vehicle. Three days post-TCDD treatment, fetuses were sacrificed and thymocytes isolated to assess caspase-3 activity as described in the Materials and Methods section. As a positive control, cell lysates from thymocytes cultured with dexamethasone for 6 h were used. As a specificity control, cell lysates from dexamethasone-treated thymocytes pre-incubated with caspase-3 inhibitor were also included. Data from 5-6 replicate pools were represented as the mean activity ± SEM.

 
Phenotypic Changes in TCDD-Exposed Thymocytes Characteristic of Apoptosis
Another direct approach to detect apoptosis in the thymus is to study changes in the expression of certain surface markers. Kishimoto et al. (1995) reported that apoptotic thymocytes from adult C57BL/6 mice exhibited downregulation of CD4, CD8, and J11d markers as well as upregulation of CD3, {alpha}ßTCR, IL-2R, and CD44. Previous studies from our laboratory used adult C57BL/6 mice and demonstrated that such changes in thymic surface markers can be used as an indicator of TCDD-induced apoptosis (Kamath et al., 1998Go). In the current study, we investigated whether perinatal exposure to TCDD would cause apoptosis-associated phenotypic changes in PD-1 thymocytes. We excluded the evaluation of surface markers on the more immature thymocytes (GD 15 to GD 18) because of absence or low expression of some of these markers at these gestational stages. Figure 5A illustrates representative histograms for each surface marker and data from multiple experiments have been pooled and presented as % change in MIF in Figure 5B. Treatment with TCDD did not significantly alter the percentage of T cells bearing the various surface markers; however, marked changes were noticed in the density of expression of markers as shown by changes in MIF. TCDD increased the expression of CD3, {alpha}ßTCR, IL-2R, and CD44 when compared to that in vehicle-treated thymocytes. On the other hand, TCDD treatment reduced the density of expression of CD4, CD8, and J11d on PD-1 thymocytes. These phenotypic changes were suggestive of apoptotic thymocytes (Kamath et al., 1998Go; Kishimoto et al., 1995Go).

Detection of Apoptosis in T Cell Subpopulations following TCDD Treatment
We next studied which subpopulations of thymic T cells were sensitive to TCDD-induced apoptosis. In these experiments, GD 17 thymocytes exposed to TCDD as described earlier, were harvested, cultured for 24 h in medium, and triple-stained with TUNEL and mAbs against CD4 and CD8 markers. The four subpopulations tested showed variable degrees of spontaneous apoptosis as seen in vehicle-treated groups (Fig. 6A). However, upon TCDD treatment, all subpopulations showed increased levels of apoptosis (Fig. 6A). When data from several experiments were pooled (Fig. 6B), TCDD-induced increase in apoptosis in each T-cell subset was statistically significant when compared to their respective control groups. Interestingly, the DP T cells showed ~3-fold increase in apoptosis following exposure to TCDD, whereas the other subpopulations were less sensitive to apoptosis, exhibiting ~1.6-fold increase in apoptosis.



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FIG. 6. Detection of apoptotic T-cell subsets following perinatal exposure to TCDD. C57BL/6 pregnant mice (gestational day = 14) were treated with 10 µg/kg TCDD or the vehicle. Three days post-TCDD treatment, fetuses were sacrificed and thymocytes triple-stained with PE-CD8, Tricolor-CD4, and FITC-dUTP after 24 h culture in medium. Each T-cell subpopulation was next gated and the percentage of apoptotic cells in each group was determined and depicted by histograms (A). In addition, the mean percentage of apoptosis for each treatment group is depicted and was calculated from 5-6 replicate pools (B). Asterisks (*) indicate statistically significant differences in the mean percentage of apoptosis in the TCDD-exposed thymocytes when compared to the vehicle controls (p < 0.05).

 
Expression of Apoptotic Genes in the Developing Thymus following Perinatal Exposure to TCDD
We investigated whether TCDD altered the expression of genes in the thymus associated with apoptosis regulation. The approach consisted of extracting RNA from TCDD- or vehicle-exposed thymocytes on GD 17 and PD 1. RT-PCR was carried out with primers specific to death receptors (Fas and DR5), death ligands (TRAIL), and members of the Bcl-2 family (Bcl-2, Bcl-xl, Bax). The data from a representative experiment have been shown in Figure 7. Both GD 17 and PD 1 thymocytes expressed higher mRNA transcripts for Fas, TRAIL, and DR5 following TCDD exposure. The expression of Bax, another pro-apoptotic gene, was elevated in TCDD-exposed thymocytes on GD 17, but not at PD 1. We could not visualize alterations in Bcl-xl gene expression in TCDD-exposed thymocytes when compared to the controls. However, slight downregulation of anti-apoptotic Bcl-2 was seen in TCDD-exposed PD-1 thymocytes. The RT-PCR analysis was carried out in 3 replicate pools with consistent results and representative data were depicted. Taken together, these data showed that the expression of several apoptotic genes is upregulated in thymocytes perinatally exposed to TCDD.



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FIG. 7. Expression of apoptosis-related genes following perinatal exposure to TCDD. C57BL/6 pregnant mice (gestational day = 14) were treated with 10 µg/kg TCDD (T) or the vehicle (V). Three or seven days post-TCDD treatment, fetuses and neonates were sacrificed and thymocytes used for RNA extraction and reverse transcription. The cDNA samples were subjected to PCR amplification using primers for mouse Fas, TRAIL, DR5, Bcl-2, Bcl-xl, or ß-actin. The PCR products were visualized on a 1.5% agarose gel containing ethidium bromide. Depicted values represent band densities relative to ß-actin. Gene expression profiles were confirmed by carrying out two additional independent experiments.

 
DISCUSSION

Perinatal exposure to TCDD induces thymic atrophy in all species tested thus far and occurs at doses that are well below those that cause maternal and fetal toxicity (Vos et al., 1997Go). Such sensitivity has led to the belief that prenatal exposure to TCDD may result in thymic atrophy in humans. Some differences in the nature of alterations in the T-cell subpopulations have been noticed in the thymus following perinatal and postnatal exposure to TCDD (Blaylock et al., 1992Go; Kerkvliet and Brauner, 1990Go; Rhile et al., 1996Go). In addition, exposure to TCDD during early development of the immune system can result in immune dysfunction carried over into adulthood (Faith and Moore, 1977Go; Gehrs et al., 1997Go; Gehrs and Smialowicz, 1999Go; Thomas and Hinsdill, 1979Go). The precise mechanism by which prenatal exposure to TCDD leads to thymic atrophy and T-cell dysfunction is unknown. Thus, while apoptosis plays a crucial role in TCDD-induced thymic atrophy in the adult mouse (Kamath et al., 1997Go), whether this phenomenon also regulates thymic atrophy following perinatal exposure to TCDD has not been previously investigated. To this end, the current study conclusively demonstrates that TCDD induces enhanced levels of apoptosis in thymocytes following perinatal exposure.

Developing thymocytes sequentially rearrange their TCR {alpha} and ß chain genes to generate the T-cell repertoire. Such T cells undergo a meticulous selection process that eliminates autoreactive and nonfunctional cells from this repertoire, and apoptosis plays a crucial role in this selection process (Palmer, 2003Go). In the present study, we performed a systematic analysis of the effect of TCDD on T-cell differentiation at various stages of gestation. Our results showed that when mice are exposed to TCDD on GD 14, it takes approximately three days before significant thymic atrophy can be observed in the embryos. Interestingly, however, apoptosis in thymocytes could be detected on all days including GD 15-18 and PD 1. The reason as to why TCDD-induced apoptosis can be detected on GD 15 and GD 16 whereas thymic atrophy is seen from GD 17 onwards is unknown, although we can speculate that, at earlier time points, the apoptosis in the thymus may be compensated by the increased seeding of progenitor T cells from the fetal stem cells. However, on day 3, when TCDD-induced apoptosis was at its peak, this seeding may not be sufficient to compensate for the loss of thymocytes, ultimately leading to thymic atrophy. It is also possible that at later stages, TCDD may act on progenitor T cells in the bone marrow thereby decreasing their ability to seed the thymus as suggested by others (Fine et al., 1989Go; Luster et al., 1980Go).

Our studies also noted that in control GD 15 fetuses, the majority of the cells were DN T cells. On GD 16, a significant percentage of spCD8+ T cells and a small percentage of DP T cells started to appear in the thymus. The spCD8+ T cells seen at this stage may represent an intermediate stage between DN and DP T cells (Blaylock et al., 1992Go), because at this stage, we did not see significant levels of mature spCD4+ T cells. On GD 17 onwards, significant proportions of DP T cells as well as mature spCD4+ and spCD8+ T cells started to appear in the thymus. Also noticed was the varied effect of TCDD on T-cell subpopulations during the different stages of gestation. While TCDD did not have any effect on T-cell subpopulations on GD 15, it caused marked increase in the percentage of spCD8 T cells on GD 16 and GD 17 and a decrease in the percentage of DN T cells on GD 16. The increase in spCD8 T cells on GD 17 resulted from an enrichment of immature CD8 cells lacking the CD3 and {alpha}ßTCR markers (Table 2). It was proposed that these immature spCD8 cells belong to the intermediate stage between the DN and DP stages (Blaylock et al., 1992Go). On GD 17 and GD 18, TCDD caused an identical change with an increase in the percentage of spCD8 and DN T cells and a decrease in the percentage of DP T cells. It was interesting to observe that TCDD did not have any effect on the percentage of T-cell subpopulations by PD1, except for the effect on spCD8 T cells. Together, these data suggested that the T-cell subpopulations are differentially susceptible to TCDD during T-cell ontogeny. Further studies analyzed the individual subsets of T cells undergoing apoptosis, with the objective to test whether TCDD exposure altered the proportions of T cells by inhibiting T-cell differentiation at various stages or by inducing apoptosis in various subpopulations. Our results showed that all four subpopulations of T cells were sensitive to TCDD-induced apoptosis on GD 17. However, the DP T cells were the most sensitive population, undergoing a 3-fold increase in TCDD-induced apoptosis, whereas the others showed marginal increases (<1.6-fold). This may explain why the proportion of DP T cells decreased at GD 17 with a consequent increase in the percentages of spCD8 and DN T cells.

Previous studies from our laboratory have shown that adult animals treated with TCDD exhibit apoptosis in thymocytes and activated T cells only after in vitro culture, a procedure that prevents the rapid phagocytosis of apoptotic cells, as known to occur in vivo (Camacho et al., 2001Go; Kamath et al., 1997Go; Pryputniewicz et al., 1998Go). In the current study, we made a similar observation using perinatally exposed thymocytes. In addition, we used several alternative strategies to corroborate induction of apoptosis in TCDD-exposed thymocytes directly following their isolation. By measuring caspase-3 activity, which is a biomarker of caspase-dependent apoptosis, we demonstrated that TCDD-exposed GD 17 thymocytes exhibited higher levels of caspase-3 activity when compared to the vehicle-treated thymocytes. Moreover, the phenotypic changes such as upregulation of TCR, CD3, IL-2R, CD44 and downregulation of CD4, CD8, and J11d were characteristic of apoptotic thymocytes as shown previously (Kamath et al., 1998Go; Kishimoto et al., 1995Go). Lastly, RT-PCR data showed that TCDD-treated thymocytes had higher induction of several genes regulating apoptosis including Fas, TRAIL, and DR5, and these results correlated well with the increase in caspase-3 activity.

Fas has been shown to play a critical role in negative selection of T cells in the thymus. For example, Castro et al. (1996) reported that blockade of Fas-FasL interactions in vivo can inhibit apoptosis of thymocytes. Also, increased expression of FasL in T cells as seen in transgenic mice, was found to induce increased apoptosis and decreased thymic cellularity (Cheng et al., 1997Go). According to Fleck et al. (1998), there is a window of susceptibility during murine thymocyte development from GD 15 to GD 17, during which substantial Fas-mediated apoptosis could take place. Our data showing increased Fas expression in TCDD-exposed thymocytes suggested that this pathway, at least in part, may contribute towards apoptosis of developing thymocytes. We have detected the presence of dioxin responsive elements (DREs) in the Fas gene promoter, using the MatInspector program (Quandt et al., 1995Go), which may explain the mechanism of its upregulation on thymocytes following TCDD exposure. If Fas expression is significantly enhanced by TCDD treatment, then the interactions between Fas+ thymocytes and FasL-expressing thymic stromal cells (French et al., 1997Go) may result in strong induction of apoptosis. Although we have shown in the past that FasL expression is increased in the thymus from TCDD-treated adult mice (Kamath et al., 1999Go), the current investigation could not detect FasL mRNA in thymocytes from either vehicle- or TCDD-exposed embryos and neonates (data not shown).

The current study also suggested the involvement of other members of the death receptor family in TCDD-induced apoptosis, such as TRAIL and DR5, an observation also seen in thymocytes from TCDD-exposed adult mice (Zeytun et al., 2002Go). It is thought that the expression of TRAIL receptors in the thymus may contribute to the regulation of negative selection (Lamhamedi-Cherradi et al., 2003Go). We also evaluated other apoptosis-related molecules belonging to the mitochondrial pathway, such as Bax, Bcl-2, and Bcl-xl, but TCDD either modestly altered or did not significantly change their mRNA expression levels. Although our findings cannot rule out the involvement of Bcl-2 pro-apoptotic members in TCDD-induced apoptosis, the data clearly suggested that TCDD might mediate cell death through the death receptor pathway, possibly involving Fas and TRAIL. In this regard, it is noteworthy that thymocytes from Bcl-2 transgenic mice were sensitive to TCDD-mediated toxicity, thereby ruling out a role for Bcl-2 in TCDD-mediated thymic atrophy (Lai et al., 2000Go).

T cells that mature in the thymus carefully undergo positive and negative selection so that those that respond to self-antigens are clonally deleted, while those responding to non-self (foreign) antigens are selected and sent to the periphery (Palmer, 2003Go). The fact that prenatal exposure to TCDD leads to thymic apoptosis and atrophy suggests that TCDD may interfere with the T-cell selection processes in the thymus, ultimately altering the T-cell repertoire. Because T cells are long-lived, this effect may influence the immune response postnatally as well. It is reasonable to expect that the impact of TCDD on thymic selection processes may be more profound in the fetus and the neonate than in the adult because adults have an established immune system, with fewer immune cells undergoing selection (Holladay, 1999Go). Alterations in the fetal immune environment may predispose the highly sensitive fetal immune system to loss of tolerance to self-antigens and subsequent increased risk for autoimmune disease. Alternatively, altered T-cell repertoire to foreign antigens may cause the neonate to become more susceptible to infections.

ACKNOWLEDGMENTS

This work was funded in part by grants from National Institutes of Health AI 053703, ES 09098, DA 014885, HL058641, DA016545, and F31ES11562.

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