Induction of Apoptosis in Murine Fetal Thymocytes Following Perinatal Exposure to Diethylstilbestrol

Abstract

Perinatal exposure to diethylstilbestrol (DES) is known to cause thymic atrophy in mice, although the precise mechanism remains unclear. In the current study the authors investigated whether perinatal exposure to DES would trigger apoptosis in thymocytes. To this end, C57BL/6 pregnant mice were injected intraperitoneally (i.p.) on gestational day (gd)-15 and -16 with 5 μg/kg DES. Analysis of thymi harvested from mice on gd-17, gd-19 and postnatal day (PD)-1, showed a significant reduction in thymic cellularity on gd-17 only. Additionally, DES treatment significantly altered the proportion and absolute number of T-cell subsets, particularly on gd-17. Apoptosis was increased in DES-treated thymocytes when compared to the controls and was seen only on gd-17. Moreover, DES-treated gd-17 thymocytes had increased Asp-Glu-Val-Asp (DEVDase) activity. Microarray analysis of 96 apoptotic genes in gd-17 thymocytes revealed that exposure to DES increased the expression of several apoptotic genes primarily belonging to tumor necrosis factor (TNF) and TNF receptor (TNFR) family. Taken together, these results suggest that DES-induced thymic atrophy following perinatal exposure may result, at least in part, from increased apoptosis mediated by death receptor pathway involving TNF family members.

Estrogens have been shown to mediate a wide range of toxic effects on the immune system (Ahmed 2000). Specifically, the thymus has been found to be sensitive to the toxic effects of estrogens (Okasha et al. 2001; Do et al. 2002). Previous studies from our laboratory and elsewhere have shown that estrogens can cause thymic atrophy in adult mice by inducing apoptosis in T cells involving Fas-FasL (Fas ligand) interactions (Mor et al. 2000, 2001; Do et al. 2002; Okasha et al. 2001). Diethylstilbestrol (DES) is a synthetic estrogen that was widely used to support pregnancy from 1941 to 1974. In the USA, an estimated 5 to 10 million women received DES. Exposure to DES in humans has been associated with an increased risk for breast cancer in “DES mothers” and a lifetime risk of cervicovaginal cancers in “DES daughters” (Giusti, Iwamoto, and Hatch 1995). Exposure to DES has also been linked to a wide range of abnormalities in DES sons and daughters, including immune system disorders, psychosexual effects, and reproductive disorders (Giusti, Iwamoto, and Hatch 1995).

Fetal thymus has been shown to be very sensitive to DES-induced toxicity, which may result, at least in part, from its effect on stem cells (Holladay et al. 1993). In murine fetal thymic organ cultures, DES has been demonstrated to block thymocyte development by cell cycle arrest and apoptosis, thereby suggesting a direct effect of DES on the thymus as well (Lai et al. 2000). Thus, prenatal exposure to DES may alter the T-cell differentiation in the thymus and T-cell repertoire in the periphery, which in turn could have significant immunological consequences in the adult. Studies in humans suggested that prenatal exposure to DES can trigger increased autoantibody production and altered delayed type hypersensitivity (DTH) reaction (Forsberg 2000; Noller et al. 1988).

Thymocytes, along with other immune cells, have been shown to express the estrogen receptor (ER). Although ER alpha has been shown to be required for thymic development as well as atrophy induced by estrogens, the role of ER beta remains unclear (Kuiper and Gustafsson 1997; Staples et al. 1999). ER beta is known to be expressed in humans and rat thymus but not in mouse thymic tissue (Couse et al. 1997)

DES has been found to induce a variety of genes depending on the tissue (Matsuno et al. 2004; Terasaka et al. 2004). Some of the induced genes have been found to have a putative estrogen response element (ERE) in the promoter. One could speculate that given the variety of genes affected by DES, thymic atrophy may also be under the regulation of many genes that are altered by DES, including those involved in the regulation of apoptosis. Thymocytes have been demonstrated to express high levels of Fas and their interactions with FasL expressed on stromal cells may play a critical role during development (Kishimoto, Surh, and Sprent 1998; Castro et al. 1996). An example of this occurrence is during thymic selection (Kurasawa, Hashimoto, and Iwamoto 1999). It is interesting to note that estrogen treatment increases the expression of FasL through the binding of ER to the ERE motif expressed on the FasL promoter (Mor et al. 2000). Thus, such a mechanism may trigger estrogen-induced thymic apoptosis and atrophy (Do et al. 2002).

Inasmuch 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. In the current study, we investigated the ability of perinatal DES exposure to induce apoptosis and alter regulatory apoptotic genes in thymocytes. We provide direct evidence that prenatal exposure to DES induces thymic atrophy that correlates with increased apoptosis induction in thymocytes.

MATERIAL AND METHODS

Animals

All animal procedures were approved by the university Institution Animal Care and Use Committee (IACUC). 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 at a constant temperature (23°C ± 2°C) with a 12-h light:12-h dark lighting schedule.

DES Exposure and Sample Collection

DES was obtained from Sigma, St. Louis, MO. DES was dissolved and diluted in corn oil. Time kinetic studies were carried out on pregnant mice treated intraperitoneally (i.p.) with 5 μg/kg DES or the vehicle control (corn oil) on gestational day (gd)-15 and -16. To this end, two pregnant mice were injected either with DES or vehicle. From each pregnant mouse, we obtained an average of 8 to 10 pups. Thymi from pups were harvested and viable cellularity enumerated.

Cell Preparations

Thymi 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 using homogenization. Cells were pelleted by centrifugation (1300 rpm, 7 min, 4°C) and resuspended in 0.83% ammonium chloride lysis buffer (Sigma) to lyse the erythrocytes. Cells were again washed twice in medium. Viability of cells was determined on a hemocytometer by trypan blue dye exclusion, using an inverted phase contrast microscope. The data on thymic cellularity were depicted as the total number of viable thymocytes/pup.

Detection of T-Cell Subsets by Flow Cytometry

Harvested thymocytes (1 × 10⁶) were washed with phosphate-buffered saline (PBS) (Invitrogen, Grand Island, NY). In double-staining studies, 0.5 μg of fluorescein isothiocyanate (FITC)–anti-CD4 and phycoerythrin PE-anti-CD8 monoclonal antibodies (mAbs) (BD Pharmingen, San Diego, CA) were simultaneously added to each sample. Cells were then fixed with 1% paraformaldehyde and flow cytometric analysis was performed by a FACScan (Becton Dickinson, Franklin Lakes, NJ) using the Cyclops operating software (Cytomation, Ft. Collins, CO).

Detection of Apoptosis Using the TUNEL Assay

Thymocytes were analyzed for apoptosis using the TUNEL assay, in which FITC-dUTP nick-end labeling is used to detect apoptosis-related DNA fragmentation (Boehringer Mannheim, Indianapolis, IN). As described earlier (Kamath et al. 1997), thymocytes (1 × 10⁶) from vehicle- or DES-treated mice were cultured for 24 h in RPMI with 10% FBS. This approach allows for better detection of drug-induced apoptosis in the thymus because apoptosis-committed cells can die without being rapidly cleared by phagocytic cells (Kamath et al. 1997; Camacho, Nagarkatti, and Nagarkatti 2004). The cells were washed twice with PBS and fixed with 4%-paraformaldehyde for 30 min at room temperature. The cells were again washed with PBS, permeabilized 2 min on ice with 0.1% Triton X-100 in 0.1% sodium citrate, washed again with PBS, and incubated with FITC-dUTP for 1 h at 37°C. Fluorescence was measured by flow cytometry.

Analysis of DEVDase Activity

Perinatal samples from pups were obtained at gd-17, -19, and postnatal day (PD)1 following DES treatment of the pregnant mother. Freshly isolated thymocytes were assayed for DEVDase activity following in vitro culture using the ApoOne Homogeneuos Caspase 3/7 Assay obtained from Promega (Madison, WI). Manufacture’s protocol was used to determine activity. The data from replicate pools were depicted as mean activity ± SEM.

Microarray Analysis

Microarray analysis was performed as described in detail elsewhere (Fisher, Nagarkatti, and Nagarkatti 2004). Briefly, thymi from gd-17 and -19 pups exposed to DES or vehicle were harvested and RNA was extracted using TRIzol reagent (GibcoBRL, Carlsbad, CA). Apoptotic Q series microarrays from SuperArray (Bethesda, MD) were used to determine apoptotic gene induction. Manufacturer’s protocol was used to perform the assay. The list of 96 genes screened is provided at the manufacturer’s website: http://www.superarray.com/product_q.php

Statistical Analysis

We used two pregnant mice per treatment group. The pups from each pregnant mouse were individually analyzed for thymic cellularity. The data were expressed as mean cellularity per pup ± SEM. We analyzed all pups and did not select. Statistical analyses were performed using the Student’s t test and p <.05 was considered to be statistical significant. For flow cytometry, a representative experiment was depicted in the figures. Microarray data were analyzed as follows: The gene expression profiles in DES-treated mice were compared to the vehicle-treated controls and expressed as fold increase/decrease. The mean ± SE of the change in gene expression seen in DES-treated samples when compared to vehicle controls was calculated from two independent experiments, with each experiment carried out in duplicate and analyzed statistically using Student’s t test. Based on the variability, a twofold or greater change in gene expression was found to be statistically significant (p < .05) for most genes and was used as an indicator of DES-induced alteration.

RESULTS

Altered Thymic Cellularity and T-Cell Subsets Following Perinatal Exposure to DES

A dose of 5 μg/kg DES was administered into pregnant C57BL/6 mice on gd-15 and -16 and thymic cellularity was determined on gd-17, -19, and on postnatal day (PD)-1 in fetal and neonatal mice (Figure 1). Thymic atrophy was demonstrated on gd-17 but not on gd-19 and PD-1. In fact, on gd-19, the DES-treated group had significantly increased numbers of thymocytes when compared to the vehicle-treated mice. We also examined the percentage of T cells expressing CD4/CD8 markers in the thymus of DES-exposed fetuses and newborns. The results from a representative experiment (Figure 2A ) and data from multiple experiments (Figure 2B ) are presented. The double-negative (CD4 –CD8– or DN) T cells, which form the predominant subset at gd-17, showed no significant alteration post-DES treatment. However, the percentage of the double-positive (CD4 + CD8+ or DP) T cells and single-positive CD4 (CD4 + CD8– or SPCD4+) T cells decreased significantly post-DES treatment. The percentage of single-positive CD8 (CD4 – CD8+ or SPCD8) T cells remained unchanged following DES treatment on gd-17. On gd-19, DES-exposure caused an increase in the percentage of SPCD8 T cells, whereas other T-cell subsets were not altered. On PD-1, DES failed to mediate alterations in the proportions of T-cell subsets. When the overall cellularity for each T-cell subset was calculated (Figure 3), it was reduced in DES-treated groups for DP, DN, and SPCD4, but not SPCD8 T cells on gd-17. On gd-19, an increase absolute cellularity was noted in SPCD8 but not in DP, DN, and SPCD4 T-cells. On PD-1, no significant alterations were observed in total cellularity for all T-cell subsets following DES exposure when compared to the vehicle control (Figure 3). Together, these data indicated that most significant DES-induced changes can be observed on gd-17. It should be noted that on gd-19 post DES treatment, the total number of thymocytes increased significantly when compared to the controls. This can be explained by the increase in SPCD8 T-cell subset.

Detection of Apoptosis in Developing Thymus after Perinatal DES Exposure

Thymi from mice exposed to DES perinatally were assayed for apoptosis using the TUNEL method. In this assay, freshly isolated thymocytes were incubated in medium for 24 h in vitro then stained by TUNEL. This approach allows greater detection of DES-induced apoptotic cells because apoptosis-committed cells can die without being rapidly cleared by phagocytic cells (Kamath et al. 1997). The results from a representative experiment have been shown in Figure 4. Thymocytes from gd-17 but not gd-19 or PD-1 mice, exposed to DES showed significantly higher percentage of apoptosis, when compared to the vehicle-treated groups. These data correlated well with DES-induced thymic atrophy that was seen only on gd-17, but not on other days.

Detection of DEVDase in Thymocytes Following Perinatal Exposure to DES

Caspase activation is an early event in apoptosis, marked by classic caspase-3 activation (Gurtu, Kain, and Zhang 1997). We therefore determined caspase-3/-7 activity following perinatal DES treatment. As shown in Figure 5, significant increases in caspase-3/-7 activity in gd-17, but not gd -19 or PD-1 thymocytes were seen following perinatal exposure to DES. These data were consistent with apoptosis induction seen on gd-17 but not on other days.

Expression of Apoptotic Genes in the Developing Thymus Following Perinatal Exposure to DES

We determined if DES could alter the expression of apoptotic genes in the thymus. To this end, microarray analysis of 96 apoptotic genes was carried out following DES treatment on thymocytes from gd-17 and -19 mice. On gd-17 following DES treatment, several tumor necrosis factor (TNF) and tumor necrosis factor receptor (TNFR) family members were up-regulated, as shown in Table 1. However on gd-19, we observed that the mRNA profile was no different from control, as indicated by no significant increase in gene expression (data not shown). Taken together, these data showed that the expression of several apoptotic genes was up-regulated in thymocytes on gd-17.

DISCUSSION

Perinatal exposure to DES in humans is know to cause significant alterations in the immune system leading to increased susceptibility to autoimmunity and infections (Adam et al. 1985; Noller et al. 1988). Previous studies in experimental animals have suggested that prenatal exposure to DES causes thymic atrophy and that this may result from toxicity against stem cells (Holladay et al. 1993). Using fetal thymic organ cultures, it was shown that DES inhibits thymocytes at different stages of development as well as induces apoptosis (Lai et al. 2000). However, whether in vivo prenatal exposure to DES would trigger apoptosis in thymocytes has not been reported previously. To this end, the current study demonstrates that DES promotes significant apoptosis induction in developing thymocytes, which in turn may be responsible for causing thymic atrophy.

During thymic development, T cells undergo a selection process to produce cells that are self–MHC (major histocompatibility complex) restricted and self-tolerant, involving DN, DP, and SP stages of differentiation (von Boehmer et al. 1989; Kisielow and von Boehmer 1995). In the current study, we analyzed the effect of DES on T-cell differentiation at different stages of development. Our data showed that following DES exposure on gd-15 and -16, significant thymic atrophy could be detected at gd-17. Additionally, apoptosis of thymocytes could be detected only at gd-17 but not on gd-19 or PD-1. These data indicate that thymocytes are more sensitive during the early selection process. This finding is consistent with other studies that have found that during later stages of T-cell development, the effects of DES are less pronounced (Holladay et al. 1993). Our studies also show that at gd-17, approximately 30% of the thymocytes are DP in the control groups. DN T cells represented approximately half of the thymocytes and SPCD8 approximately 20%. Very few SPCD4 thymocytes were observed. Following exposure to DES, gd-17 thymocytes were altered most significantly in the percentages of DP and SPCD4 populations that were reduced significantly. Moreover, on gd-17, the absolute numbers of all T-cell subsets except SPCD8 cells were reduced following DES exposure. On gd-19, no significant DES-induced effects were observed except for an increase in SPCD8 T cells, whereas no significant alterations in T-cell subsets were noted on PD-1. Thus, it appears that the DES effect was short-lived and that the thymic cellularity returned to normal levels by PD-1. However, it should be noted that in our studies, we exposed the mice to DES on gd-15 and -16 only. It is likely that exposure of pregnant mice beyond gd-16 to DES may trigger immuntoxic effects that would be evident in the pups even after birth.

Previous studies have shown that the ability to detect drug-induced apoptosis in thymocytes in vivo is difficult due to rapid clearance of apoptotic cells by phagocytes (Camacho, Nagarkatti, and Nagarkatti 2004; Kamath et al. 1997). We have shown that in vitro culture allows for the detection of apoptosis without interference of phagocytosis of apoptotic cells (Kamath et al. 1997). In addition, the ability of DES to induce apoptosis in thymocytes was corroborated by demonstrating that gd-17 thymocytes had increased levels of DEVDase activity.

Microarray analysis of apoptotic genes at gd-17 showed that DES-treated thymocytes up-regulate several TNF and TNFR family members. Interestingly, FasL was among one of the up-regulated transcripts. At the gd-19 stage, the gene profile was not significantly different from that of control. These results correlated well with the induction of apoptosis at gd-17 and lack of at gd-19. Fas-FasL interactions play a critical role in the thymus during T-cell development (Castro et al. 1996). Fas has been observed during specific developmental stages of thymocytes such as the DN stage (Ogasawara et al. 1993). Given this, it is clear that there are specific developmental stages that are sensitive to Fas-mediated apoptosis (Fleck et al. 1998). Our data suggested that increased FasL expression in DES-exposed thymocytes may play a role in DES-induced apoptosis in thymocytes. Given that the estrogen responsive element (ERE) has been found in the FasL gene promoter (Mor et al. 2000), it can be speculated that DES may act through ERE to induce FasL gene expression. These findings are consistent with previous studies from our laboratory demonstrating that Fas-FasL interactions play a critical role in estrogen-induced apoptosis in thymocytes (Do et al. 2002).

T cells that mature in the thymus undergo positive and negative selection. These processes select self–MHC-restricted T cells and delete autoreactive cells, thereby allowing tolerant cells to be sent to the periphery (Palmer 2003; Sebzda et al. 1999; Saito 1998; Saito and Watanabe 1998). The finding that prenatal exposure to DES can lead to thymic atrophy through apoptosis prior to gd-19 suggests that DES may interfere with the T-cell selection processes in the thymus, which could alter the T-cell repertoire in the periphery. Such alterations during development may influence postnatal immune responses particularly because T cells are long-lived. Moreover, continuous exposure to DES during the entire gestation period may have more pronounced effect on T-cell differentiation in the thymus and consequently in the periphery that could continue to persist in postnatal life.