Role of Death Receptor Pathway in Estradiol-Induced T-Cell Apoptosis in Vivo

Yoonkyung Do*, Seongho Ryu*, Mitzi Nagarkatti* and Prakash S. Nagarkatti{dagger},1

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

Received May 21, 2002; accepted July 26, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the current study we investigated the mechanism by which ß-estradiol-17-valerate (E2) induces apoptosis in T cells. To this end, C57BL/6 wild-type (+/+), Fas-deficient (C57BL/6-lpr/lpr), and FasL-deficient (C57BL/6-gld/gld) mice were treated with various concentrations of E2, including 75, 25, 5, 1, or 0.1 mg/kg body weight or the vehicle. The thymi from these mice were harvested on days 1, 4, or 7 following treatment, and cellularity and apoptosis were determined. Treatment with E2 caused a decrease in thymic cellularity at all doses except 0.1 mg/kg in all three groups of mice, particularly on days 4 and 7. Interestingly, however, the degree of thymic atrophy in C57BL/6-lpr/lpr and C57BL/6-gld/gld mice was significantly less than that seen in C57BL/6 wild-type mice. When thymocytes were analyzed for apoptosis, cells from C57BL/6-lpr/lpr and C57BL/6-gld/gld mice showed decreased levels of apoptosis. Moreover, cDNA array analysis of gene expression revealed that treatment with E2 upregulated several genes involved in apoptosis, including FasL, caspases, TRAIL, and iNOS, but not bcl-2 gene family. Reverse transcriptase-polymerase chain reaction data also demonstrated the increased expression of Fas and FasL genes following E2 treatment. Caspase 8 inhibitor blocked the E2-induced apoptosis of thymocytes in vitro. These data suggested that E2 may induce apoptosis by activating the death-receptor rather than the mitochondrial pathway. E2 treatment decreased the expansion of peripheral Vß3+ T cells to the bacterial superantigen SEA in vivo and their subsequent in vitro proliferative response to SEA, thereby suggesting increased induction of apoptosis in Vß3+ T cells. The current study suggests that E2 may trigger the death receptor pathway in vivo in T cells, thereby inducing apoptosis.

Key Words: estrogen; immunotoxicity; Fas; FasL; apoptosis; thymus.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is becoming increasingly clear in recent years that reproductive hormones such as estrogen exert pleiotropic effects on the immune system. Cells of the immune system express estrogen receptors (ERs) through which the estrogen may regulate the immune cell differentiation and functions (Couse and Korach, 1999Go). This is indicated by the defects in T-cell maturation seen in ER knockout mice (Yellayi et al., 2000Go). Furthermore, administration of estrogen has previously been shown to induce thymic atrophy and suppress lymphocyte functions (Hirahara et al., 1994Go; Silverstone et al., 1994Go).

It has been reported that several microbes and plants produce estrogens, such as enterolactone and coumestrol, respectively (Welshons et al., 1987Go). In addition, many environmental pollutants behave as estrogens by binding to the ER, and mediate reproductive and developmental as well as immunotoxicity (Degen and Bolt, 2000Go). For example, compounds such as bisphenol A and nonylphenol bind ER and induce transactivation of estrogen-responsive genes (Safe et al., 2001Go). Previous studies have shown that phytoestrogens and xenoestrogens bind ER, but may differ in their ability to induce cell proliferation and to trigger ER-regulated end products (Zava et al., 1997Go). Estrogen is used in various forms for contraception or for pharmacological purposes in the treatment of postmenopausal women to prevent osteoporosis, Alzheimer’s disease, and cardiovascular diseases (Ruggiero and Likis, 2002Go). In addition, diethylstilbestrol (DES), a synthetic estrogen, was used extensively in the United States and Europe between the 1940s and 1970s to treat pregnant women. An estimated 5–10 million Americans received DES during pregnancy or were exposed to the drug in utero during this period (Giusti et al., 1995Go). Such an exposure to DES has resulted in an increased risk for breast cancer in DES mothers and a lifetime risk of cervicovaginal cancers in DES daughters (Bornstein et al., 1987Go; Melnick et al., 1987Go). Pregnant women treated with DES may also be more susceptible to autoimmunity (Cutolo et al., 1995Go).

Estrogen induces thymic atrophy (Holladay et al., 1993Go; Silverstone et al., 1994Go; Staples et al., 1999Go). Previous studies from our laboratory demonstrated that in vivo administration of ß-estradiol-17-valerate (E2) into mice triggers apoptosis in thymocytes (Okasha et al., 2001Go). Similarly, addition of E2 to fetal thymic organ culture induces apoptosis (Barnden et al., 1997Go). The ability of E2 to induce apoptosis in fetal and adult thymocytes can lead to alterations in the T-cell repertoire in such a way that the immune system may be more skewed to react strongly toward self-antigens and weakly against foreign antigens. Such an immunomodulation can explain the increased susceptibility of E2-exposed individuals to autoimmunity and cancer.

Fas is a death receptor expressed on immune cells. Ligation of this receptor by Fas ligand (FasL), a member of the tumor necrosis factor family, triggers the induction of apoptosis in cells (Ju et al., 1999Go). In the current study, we tested the hypothesis that E2 may upregulate the FasL and/or Fas expression, thereby triggering apoptosis in Fas+ thymocytes and causing thymic atrophy. We measured the levels of FasL gene expression using cDNA array and reverse transcriptase-polymerase chain reaction (RT-PCR), and used Fas-deficient (C57BL/6-lpr/lpr) and FasL-defective (C57BL/6-gld/gld) mice to study the role of Fas-FasL interactions in E2-induced apoptosis and thymic atrophy. The data suggested that upregulation of FasL by E2 may play a significant role in apoptosis induction in T cells and immunotoxicity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mice.
Female C57BL/6 wild-type, C57BL/6-lpr/lpr, and C57BL/6-gld/gld mice 6 weeks of age were purchased from the Jackson laboratory (Bar Harbor, ME) and bred under specific pathogen-free conditions in the animal facility of Virginia Commonwealth University (VCU). The use of mice was approved by Institutional Animal Care and Use Committee (IACUC) of VCU.

Estradiol exposure.
ß-estradiol-17-valerate (1,3,5 [10]-estratrine-3,17 ß-diol 17-valerate) (Sigma, St. Louis, MO) was dissolved in olive oil for preparation of a dosing solution. Mice received a single sc injection of E2 at various doses, such as 75, 25, 5, 1, and 0.1 mg/kg of body weight or the vehicle.

Cell preparation and determination of cellularity.
Mice were sacrificed, and thymi and spleens were removed. Single-cell suspension was prepared using a laboratory homogenizer (Stomacher, Tekmar, Cincinnati, OH) in RPMI-1640 supplemented with 10% fetal calf serum (FCS), 10 mM HEPES, 1 mM glutamine, and 50 µg/ml gentamycin. The red blood cells were lysed using ammonium chloride, and the cells were resuspended in complete medium after three washings. The viable cells were counted using trypan blue dye exclusion.

Detection of apoptosis using AnnexinV staining.
Apoptosis was detected using AnnexinV staining as described earlier (Okasha et al., 2001Go; Vermes et al., 1995Go). Cells (1 x 106/tube) were washed twice with PBS, resuspended in 100 µl labeling solution containing 2 µl AnnexinV, and incubated for 30 min at room temperature. The cells were then washed and analyzed using flow cytometry. Propidium iodide was used to exclude necrotic cells. In some experiments, caspase8 inhibitor (Z-IETD-FMK, 50 µM, R&D Systems Inc. Minneapolis, MN) was added to the cell culture, and apoptosis was detected as described above.

Total RNA preparation.
Thymocytes from either E2-treated or vehicle-treated mice were prepared as described above, and total RNA was extracted using commercial kit based on manufacturer’s protocol (RNeasy kit, QIAGEN, Stanford Valencia, CA). The concentration and purity of RNA was determined using UV spectrophotometer (280 nm/260 nm).

cDNA array.
Pathway-specific expression arrays were purchased from Super Array Inc. (Bethesda, MD). Each array contained a positively charged nylon membrane on which cDNA fragments from various genes had been immobilized in duplicate. cDNA probes were synthesized from 8–10 µg of RNA using Super Array’s GEA primer mix as reverse transcriptase primers. The probes were biotinylated during cDNA synthesis by the incorporation of biotin-16-dUTP. Denaturation of probes, hybridization, and detection were carried out as described by the manufacturer. Briefly, membranes were first incubated with 10 ml hybridization solution containing 100 µg/ml salmon sperm DNA at 68°C for 2–4 h, then with denatured probes in 5 ml hybridization solution at 68°C for 16 h in a rotating hybridization chamber (Fisher Scientific, Suwanee, GA). The membranes were washed twice with 2% SSC/1% SDS and 0.1% SSC/0.5% SDS at 68°C. Following the blocking of nonspecific binding, they were further incubated with AP-conjugated streptavidin for 1 h at room temperature, then immersed in 5 ml CDP-Star substrate solution. Membranes were exposed to films for 30 sec, 1, 2, 4, 8, and 10 min.

Each membrane was spotted with a negative control of pUC18 DNA and two positive control genes, ß-actin and GAPDH. The intensity of each spot was measured using densitometry (Molecular Dynamics, Madison, WI). Mean intensity for the genes was calculated based on the different exposure times. The level of expression or induction of a gene in control and E2-treated tissues was normalized by comparing them with the respective expression of ß-actin and GAPDH. The changes in gene expression following E2 treatment were presented as fold increase or decrease when compared with expression profiles seen in mice injected with the vehicle.

Detection of Fas and FasL expression by semiquantitative RT-PCR.
RT-PCR was conducted to detect Fas and FasL gene expression as described earlier (Kamath et al., 1999Go). The primers used were as follows: Fas primer, 5‘-GCACAGAAGGGAAGGAGTAC-3‘ and 5‘-GTCTTCAGC AATTCTCGGGA-3‘; FasL primer, 5‘-GAGCGGTTCCATATGT GTCTTCC-3‘ and 5‘-GGGCTCCTCCAGGGTCAGTT-3‘; ß-actin primer, 5‘-GCACTGTA GTTTCTCTTCG ACACGA-3‘ and 5‘-ATCCTGACCCTGAACTACCCCATT-3‘. PCR amplification products were visualized in 1% agarose gels after staining with ethidium bromide. The density of PCR products was compared and ß-actin was used as internal control.

Immune responsiveness to SEA.
To study the effect of E2 on the immune responsiveness to staphylococcal enterotoxin A (SEA), C57BL/6 wild-type mice received 75 or 25 mg/kg body weight of E2 or vehicle sc and SEA into the footpads (10 µg/footpad) at the same time. On days 1, 3, and 5, the draining popliteal lymph nodes (LNs) were harvested, and total cell number was determined using trypan blue dye exclusion. The lymphocytes were then stained with PE-conjugated anti-Vß3 TCR mAb to detect Vß3+ population as described elsewhere (Kamath et al., 1997Go). Briefly, the cells (1 x 106) were washed twice with PBS and the Fc receptors were blocked with Fc-Block reagent (BD Pharmingen, San Diego, CA) for 30 min on ice. The PE-conjugated anti-Vß3 TCR mAb was added to the cells and incubated for another 30 min on ice. The cells were washed and fixed with 4% paraformaldehyde for 30 min at room temperature. The fluorescence of the cells was determined using flow cytometry. The Vß3+ cellularity in the lymph node was calculated by taking into consideration the percentage of Vß3+ cells and the total cellularity of the draining lymph node.

Proliferative responsiveness of T cells.
SEA (10 µg/footpad) was injected into footpads of C57BL/6 wild-type mice that received 25 mg/kg body weight of E2 or vehicle subcutaneously and 3 days later, the draining LNs were collected. The lymphocytes (5 x 105) were restimulated with SEA (4 µg/ml) in vitro or cultured with medium for 48 or 72 h in 96-well tissue-culture plates. During the final 8 h, the cells were pulsed with 2 µCi [3H]thymidine, and its incorporation was determined by liquid scintillation counter.

Statistical analysis.
Each experiment was repeated at least three times, and each vehicle or E2-treated group consisted of 4–6 mice. Statistical comparisons of dose-dependent responses were made by one-way analysis of variance (ANOVA). Post hoc comparison was accomplished using Tukey’s HSD. The level of significance was based on p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of E2 on Thymic Cellularity in C57BL/6 Wild-Type Mice
C57BL/6 wild-type mice were injected with 75, 25, 5, 1, or 0.1 mg/kg body weight of E2 or vehicle; 1, 4, and 7 days later, the mice were sacrificed and thymic cellularity was determined. The total number of cells per thymus is depicted in Figure 1Go. E2 caused a decrease in thymic cellularity at all doses tested except at 0.1 mg/kg body weight on days 4 and 7. Thymic atrophy was not seen on day 1 in any concentration tested, except at the highest dose of 75 mg/kg. We failed to observe a dose-dependent decrease in thymic cellularity caused by E2.



View larger version (38K):
[in this window]
[in a new window]
 
FIG. 1. Estrogen-induced thymic atrophy in C57BL/6 wild-type mice. Groups of 4–6 C57BL/6 wild-type mice were treated with 75, 25, 5, 1, or 0.1 mg/kg body weight of estradiol (E2) or vehicle, and thymi were removed on days 1, 4, and 7 following treatment. Empty bars indicate cellularity of thymocytes from vehicle-treated mice and checked bars indicate cellularity of thymocytes from E2-treated mice. Vertical bars represent the mean ± SEM. *Denotes statistically significant difference when compared with vehicle-treated mice (p < 0.05).

 
Effect of E2 on Thymic Cellularity in C57BL/6-lpr/lpr and C57BL/6-gld/gld Mice
To study the role of Fas-FasL interactions in E2-induced thymic atrophy, we tested the effect of E2 in Fas-deficient C57BL/6-lpr/lpr mice and FasL-defective C57BL/6-gld/gld mice. C57BL/6-lpr/lpr and C57BL/6-gld/gld mice were injected with 75, 25, 5, 1, or 0.1 mg/kg body weight of E2 or vehicle; 1, 4, and 7 days later, the mice were sacrificed and thymic cellularity was determined. The data demonstrated that treatment with E2 caused a significant decrease in thymic cellularity in both C57BL/6-lpr/lpr (Fig. 2Go) and C57BL/6-gld/gld (Fig. 3Go) mice. Once again, E2 failed to exhibit a dose-dependent decrease in thymic cellularity. Thymic atrophy was seen at all doses tested except at 0.1 mg/kg, and it was seen mainly on days 4 and 7. These results were similar to that seen with C57BL/6 wild-type mice (Fig. 1Go), except that C57BL/6-lpr/lpr and C57BL/6-gld/gld mice were more resistant to thymic atrophy when compared with the C57BL/6 wild-type mice at 25, 5, and 1 mg/kg of E2 (Fig. 4Go). For example, 4 days following E2 treatment (25 mg/kg body weight), C57BL/6-lpr/lpr and C57BL/6-gld/gld mice showed 25% and 33% decrease in thymic cellularity, respectively, when compared with thymocytes from vehicle-treated mice. In contrast, the wild-type mice showed approximately 50% decrease following E2 treatment. Moreover, when 1 mg/kg body weight of E2 was given, wild-type mice on day 4 showed a significant thymic atrophy (around 40% decrease in thymic cellularity), whereas C57BL/6-lpr/lpr and C57BL/6-gld/gld mice showed approximately 16 and 20% decrease, respectively. Together, the results demonstrated that C57BL/6-lpr/lpr and C57BL/6-gld/gld mice were more resistant to E2-induced thymic atrophy when compared with the C57BL/6 wild-type mice at doses of 25 mg/kg or less, thereby suggesting the involvement of Fas and/or FasL in E2-induced thymic atrophy.



View larger version (39K):
[in this window]
[in a new window]
 
FIG. 2. Estrogen-induced thymic atrophy in C57BL/6-lpr/lpr mice. Mice were treated with 75, 25, 5, 1, or 0.1 mg/kg body weight of estradiol (E2) or vehicle, as described in Figure 1Go, and thymi were removed on days 1, 4, and 7 following treatment. Empty bars indicate cellularity of thymocytes from vehicle-treated mice and checked bars indicate cellularity of thymocytes from E2-treated mice. Vertical bars represent the mean ± SEM. *Denotes statistically significant difference when compared with vehicle-treated mice (p < 0.05).

 


View larger version (41K):
[in this window]
[in a new window]
 
FIG. 3. Estrogen-induced thymic atrophy in C57BL/6-gld/gld mice. Mice were treated with 75, 25, 5, 1, or 0.1 mg/kg body weight of estradiol (E2) or vehicle, and thymi were removed on days 1, 4, and 7 following treatment, as described in Figure 1Go. Empty bars indicate cellularity of thymocytes from vehicle-treated mice and checked bars indicate cellularity of thymocytes from E2-treated mice. Vertical bars represent the mean ± SEM. *Denotes statistically significant difference when compared with vehicle-treated mice (p < 0.05).

 


View larger version (28K):
[in this window]
[in a new window]
 
FIG. 4. Comparison of percentage reduction in the number of thymocytes in C57BL/6 wild-type, C57BL/6-lpr/lpr, and C57BL/6-gld/gld mice following E2 or vehicle treatment. Mice were injected with E2, as described in Figures 1–3GoGoGo. Percentage decrease in thymic cellularity following E2 treatment was calculated, compared with the vehicle controls, and depicted. Vertical bars represent the mean ± SEM. *Denotes statistically significant difference when C57BL/6-lpr/lpr and C57BL/6-gld/gld mice were compared with C57BL/6 wild-type mice (p < 0.05).

 
E2-Induced Apoptosis in Thymocytes of C57BL/6 Wild-Type, C57BL/6-lpr/lpr, and C57BL/6-gld/gld Mice
Induction of apoptosis in vivo is difficult to detect because of rapid clearance of apoptotic cells by phagocytic cells (Ashwell et al., 2000Go). We have shown previously that in vitro culture of thymocytes from mice exposed to E2 in vivo leads to detection of increased levels of apoptosis when compared with in vitro cultured thymocytes from vehicle-treated mice, as indicated by AnnexinV+ cells (Okasha et al., 2001Go). Using a similar approach, we injected C57BL/6 wild-type, C57BL/6-lpr/lpr, and C57BL/6-gld/gld mice with the vehicle or 5 or 25 mg/kg of E2 on day 4, the thymocytes were harvested and cultured with medium alone for 24 h in vitro. Next, the thymocytes were stained with AnnexinV to detect apoptosis. Figures 5A and 5BGoGo show the apoptosis staining histograms of thymocytes from mice exposed in vivo to 25 or 5 mg/kg body weight of E2, respectively, on day 4. The in vivo E2-exposed C57BL/6 wild-type thymocytes upon in vitro culture exhibited increased levels of apoptotic cells when compared with similarly cultured thymocytes from vehicle-treated C57BL/6 wild-type mice. We also detected an increased percentage of apoptotic cells in the thymocytes obtained from C57BL/6-lpr/lpr and C57BL/6-gld/gld mice when compared with similarly cultured thymocytes from the respective vehicle-treated controls. However, the levels of apoptotic cells in C57BL/6-lpr/lpr and C57BL/6-gld/gld mice were much less than those seen in C57BL/6 wild-type mice. For example, upon subtraction from vehicle controls, approximately 38.78% of the thymocytes from E2-treated (25 mg/kg) C57BL/6 wild-type mice underwent apoptosis, whereas only 11.98 or 16.65% of the thymocytes from E2-treated C57BL/6-lpr/lpr or C57BL/6-gld/gld mice underwent apoptosis, respectively. In addition, when the thymocytes obtained from C57BL/6-lpr/lpr and C57BL/6-gld/gld mice treated with 5 mg/kg body weight of E2 were analyzed on day 4, they also showed increased resistance to the induction of apoptosis when compared with similarly treated thymocytes from C57BL/6 wild-type mice (Fig. 5BGo). The lowest dose of E2 (0.1 mg/kg body weight) did not cause apoptosis in any of the three groups of mice following in vitro culture (data not shown). Together, these data suggested that the thymic atrophy caused by E2 may result from induction of apoptosis. Furthermore, Fas-FasL interactions may play a significant role in T-cell apoptosis induced by E2.



View larger version (21K):
[in this window]
[in a new window]
 
FIG. 5. Estrogen-induced apoptosis in thymocytes from C57BL/6 wild-type, C57BL/6-lpr/lpr, and C57BL/6-gld/gld mice. Thymocytes from mice treated with 25 mg/kg (A) or 5 mg/kg (B) were harvested on day 4. The cells were cultured for 24 h in medium and stained with AnnexinV. The fluorescence was determined using flow cytometry. The percentage of AnnexinV+ cells, which represent apoptotic cells, has been depicted in each histogram. The increased percentage of apoptotic cells following E2 treatment compared with the vehicle controls was calculated and depicted in parenthesis for each group.

 
Use of cDNA Array to Detect the Upregulation of Genes Involved in Apoptosis following E2 Injection
To further corroborate that E2 may induce the expression of FasL and to analyze the possible genes involved in E2-induced thymic apoptosis, we used a pathway-specific cDNA array. C57BL/6 wild-type mice were injected with 75 mg/kg body weight of E2 or vehicle and 1 day later the thymocytes were obtained. Total RNA was extracted, converted into cDNA, then hybridized to the membrane spotted with genes regulating apoptosis. The characterization and function of these genes are shown in Table 1Go. The data shown in Figure 6Go demonstrate that E2 treatment upregulated several apoptotic genes. FasL gene expression was upregulated over five times when compared with that of vehicle-treated mice and caspases 1, 2, 3, and TRAIL were also significantly increased. We did not see significant induction of bcl-2 gene family. Of particular interest was the dramatic induction of iNOS gene following E2 treatment. These data were reproducible in two independent experiments. These data together suggested that E2 treatment can induce several genes involved in apoptosis and that E2 may trigger death-receptor pathway of apoptosis rather than the mitochondrial pathway.


View this table:
[in this window]
[in a new window]
 
TABLE 1 List of Genes Analyzed Involved in Apoptosis
 


View larger version (18K):
[in this window]
[in a new window]
 
FIG. 6. cDNA array analysis showing upregulation in the expression of several apoptotic genes following E2 treatment. C57BL/6 wild-type mice were treated with 75 mg/kg body weight of E2 or vehicle, as described in Figure 1Go. One day later, thymi were collected and analyzed for gene expression profile using cDNA array, as described in Materials and Methods. The data were expressed as fold increase seen in E2-treated mice compared with vehicle controls.

 
Use of RT-PCR to Detect the Upregulation of Fas and FasL Genes following E2 Injection
To further validate the cDNA array data, we investigated the expression of Fas and FasL genes in E2-treated thymocytes using RT-PCR. Figure 7Go shows increased Fas and FasL gene expression in thymocytes following E2 (1 mg/kg) treatment when compared with vehicle-treated cells, thereby suggesting that E2-induced upregulation of Fas and FasL genes may regulate apoptotic death in thymocytes. We also detected upregulated Fas and FasL gene expression at other concentrations of E2 when compared with the vehicle controls (data not shown).



View larger version (32K):
[in this window]
[in a new window]
 
FIG. 7. RT-PCR analysis showing upregulation in the expression of Fas and FasL genes following E2 treatment. C57BL/6 wild-type mice were treated with 1 mg/kg body weight of E2 or vehicle. Eighteen hours later, thymi were collected. Total RNA was extracted and converted into cDNA using reverse transcriptase. The cDNA was amplified by PCR with primers specific for Fas, FasL, and ß-actin. A photograph of ethidium bromide-stained amplicons is depicted.

 
The Involvement of Caspase 8 in E2-Induced Apoptosis
Apoptosis triggered through the death receptor pathway is mediated through the activation of caspase 8 (Hengartner, 2000Go). Although we could not detect an increase in caspase 8 mRNA expression following E2 treatment (Fig. 6Go), this experiment did not rule out the involvement of caspase 8. To further study the role of caspase 8 in E2-induced apoptosis, various concentrations of caspase 8 inhibitors were added to in vitro culture of thymocytes from mice exposed to vehicle or 25 mg/kg of E2 (Fig. 8Go). As before, an increased percentage of apoptotic cells was detected in thymocytes obtained from E2-treated mice when compared with similar cells obtained from vehicle-treated mice. The addition of 50 µM caspase 8 inhibitor blocked the apoptosis induced by E2 (Fig. 8Go). These data suggest the participation of caspase 8 in E2-induced apoptosis.



View larger version (11K):
[in this window]
[in a new window]
 
FIG. 8. Caspase 8 inhibitor blocks the apoptosis induced by E2 treatment. Thymocytes from C57BL/6 wild-type mice treated with 25 mg/kg of E2 or vehicle were harvested on day 4. The cells were cultured for 24 h in medium, as described in Figure 5Go, in the presence or absence of caspase 8 inhibitor (50 µM), and stained with AnnexinV. Fluorescence was determined using flow cytometry. The percentage of AnnexinV+ cells, which represent apoptotic cells, has been depicted in each histogram.

 
E2 Suppresses the T-Cell Response to SEA
Injection of superantigens such as SEA in vivo triggers the activation of T cells bearing certain Vß specificities such as Vß3, which rapidly expand and undergo activation-induced cell death (AICD; Aroeira and Martinez, 1999Go). In the current study, therefore, we tested whether E2 would enhance AICD in the periphery.

To this end, we injected C57BL/6 wild-type mice with 75 mg/kg body weight of E2 or vehicle, and at the same time we injected superantigen SEA into the footpads. On days 1, 3, and 5 following E2 treatment, the draining popliteal lymph nodes were harvested, and the presence of Vß3+ T cells was enumerated by flow cytometry. The total number of Vß3+ T cells in the draining LNs was also calculated. The data shown in Figure 9Go indicate that number of Vß3+ T cells peaked on day 3, thereby suggesting that SEA injection led to rapid expansion of Vß3+ T cells and a subsequent decline on day 5 suggestive of AICD. E2 treatment did not significantly alter the numbers of Vß3+ T cells on day 1. However, on days 3 and 5, there was a significant decrease in Vß3+ T cells, thereby suggesting that E2 was enhancing the AICD. To further confirm that treatment with E2 caused enhanced depletion of Vß3+ cells, we injected C57BL/6 wild-type mice with 75 mg/kg body weight of E2 or vehicle, and SEA into the footpads. On day 3, we collected the lymphocytes from draining lymph nodes and cultured the cells in vitro with medium or SEA. The proliferative responsiveness was measured after 48-h or 72-h incubation. Figure 10Go shows that restimulation of draining lymphocytes with SEA in vitro caused a significant decrease in proliferative responsiveness of cells from E2-treated mice when compared with the proliferation of cells from vehicle-treated mice. In summary, E2 treatment increased the depletion of SEA-activated T cells, resulting possibly from increased AICD.



View larger version (13K):
[in this window]
[in a new window]
 
FIG. 9. Estrogen-induced decrease in Vß3+ T-cell numbers following SEA injection. C57BL/6 wild-type mice were injected with 75 mg/kg body weight of E2 or vehicle and at the same time, they were injected with SEA (10 µg/footpad) into the footpads. On days 1, 3, and 5, the draining lymph nodes were harvested, and total cellularity was calculated using trypan blue dye exclusion. The cells (1 x106) were stained with anti-Vß3 TCR mAb, and fluorescence was determined using flow cytometry. The Vß3+ cell numbers were then calculated by using the percentage of Vß3+ T cells and the total cellularity. Empty bars represent the Vß3+ lymphocytes from vehicle-treated mice and checked bars represent the Vß3+ lymphocytes from E2-treated mice. Vertical bars represent the mean ± SEM. *Denotes statistically significant difference when compared with vehicle-treated mice (p < 0.05).

 


View larger version (19K):
[in this window]
[in a new window]
 
FIG. 10. Effect of E2 on SEA-activated T cells restimulated in vitro with SEA. C57BL/6 wild-type mice were injected with 75 mg/kg body weight of E2 or vehicle, and at the same time, they were injected with SEA (10 µg/footpad) into the footpads. Three days later, the draining lymph nodes were collected. The cells (5 x 105) were cultured in 96-well plates with SEA (4 µg/ml) or medium alone for 48 (A) or 72 h (B). During last 8 h, [3H]thymidine was added to the cultures, and incorporation was measured using a liquid scintillation counter. Vertical bars represent the mean cpm ± SEM of triplicate cultures. *Denotes statistically significant difference between E2-treated versus vehicle-treated mice (p < 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies have shown that treatment with estrogens triggers thymic atrophy (Holladay et al., 1993Go; Silverstone et al., 1994Go; Staples et al., 1999Go). However, the precise mechanism remains unclear, and many hypotheses have been proposed. Studies have suggested that estrogens may induce thymic atrophy by affecting prethymic stem cells in bone marrow or fetal liver (Holladay et al., 1993Go; Silverstone et al., 1994Go). In contrast, other studies have demonstrated that estrogen alters intrathymic T-cell development (Brunelli et al., 1992Go; Rijhsinghani et al., 1996Go; Screpanti et al., 1989Go). It has also been suggested that E2 might affect lymphocytes indirectly by mediating estrogenic effects on thymic epithelial cells, which have higher expression of ER (Luster et al., 1984Go). Studies from our laboratory and elsewhere also suggested that E2 may induce apoptosis in thymocytes (Okasha et al., 2001Go). A recent study investigated the relationship between the level of FasL expression and thymus cell number using ovariectomized female rats, and suggested that estrogen-induced thymic atrophy may be mediated by estrogen-induced FasL expression (Mor et al., 2001Go). Our studies using Fas- and FasL-deficient mice are consistent with these studies and, moreover, extend these results by showing that E2-induced apoptosis in thymocytes was markedly decreased in C57BL/6-lpr/lpr and C57BL/6-gld/gld mice. In the current study, we demonstrated that E2-induced thymic atrophy and apoptosis may be regulated by Fas-FasL interactions. This was supported by the observations that treatment with E2 upregulated the expression of Fas and FasL. Moreover, mice deficient in Fas or FasL were more resistant to apoptosis as well as thymic atrophy induced by E2.

Estrogen has recently been shown to induce apoptosis in nonlymphoid tissues (Hughes et al., 1996Go; Robertson et al., 1996Go; Shevde and Pike, 1996Go), but whether estrogen induces thymic involution by triggering apoptosis is not clear. Staples et al. (1998)Go failed to detect apoptosis in thymocytes following in vivo administration of E2. However, this laboratory subsequently reported that DES, a synthetic estrogen, induced apoptosis in fetal thymic organ cultures (Lai et al., 2000Go). In an earlier study, we could not detect apoptosis in freshly isolated thymocytes following E2 treatment (Okasha et al., 2001Go). Therefore, we proposed a novel strategy to detect apoptosis in which in vivo chemical-exposed thymocytes are cultured in vitro with medium for an additional 12–24 h. Using such a strategy, we could detect increased apoptosis induced by chemicals such as dioxin and dexamethasone, as well as E2, when compared with vehicle controls (Kamath et al., 1997Go, 1998Go, 1999Go; Okasha et al., 2001Go). In this study, we used a similar approach to investigate the mechanism by which E2 triggers thymic involution, and found that upon in vitro culture, E2-exposed thymocytes underwent increased levels of apoptosis when compared with vehicle controls.

Apoptosis is triggered mainly through two pathways: the death receptor pathway and the mitochondrial pathway (Hengartner, 2000Go). In the death receptor pathway, ligation of a receptor belonging to the tumor necrosis factor (TNF) receptor family such as Fas and TRAIL receptors leads to induction of apoptosis through activation of caspase 8. In contrast, in the mitochondrial pathway, there is activation and recruitment of members of the bcl-2 family, leading to activation of caspase 9. It has been demonstrated that overexpression of bcl-2, an antiapoptotic protein, could not protect the thymus from E2-induced atrophy (Staples et al., 1998Go). In the current study, we did not observe any significant changes in the expression of proapoptotic members of the bcl-2 gene family, although we observed an increase in the expression of Fas and FasL. Thus, E2-induced thymic atrophy may primarily involve the death receptor pathway. This was further supported by the upregulation of TRAIL, a member of the death receptor family.

It should be noted that in the current study, the Fas- and FasL-deficient mice were not completely resistant to E2-induced thymic atrophy and apoptosis. In addition, at the higher dose tested (75 mg/kg), these mice were as sensitive as the C57BL/6 wild-type mice. These data suggested that alternative mechanisms exist that regulate thymic atrophy. Thus E2, particularly at higher doses, may affect the bone marrow stem cells and prevent them from seeding the thymus (Silverstone et al., 1994Go). This may also explain why in the current study we did not see any dose-dependent effect on the thymus with E2 acting on multiple sites. E2 caused upregulation of other genes involved in apoptosis such as TRAIL, which may also play a role in E2-induced apoptosis. We also found a dramatic increase in the mRNA expression of inducible nitric oxide synthase (iNOS). iNOS has been previously shown to be produced in the thymus and associated with the induction of apoptosis through the expression of Fas and FasL (Esaki et al., 2000Go; Tai et al., 1997Go). Thus, it is possible that E2-induced iNOS may play a role in Fas-and FasL-mediated apoptosis in thymocytes. In the current study, we observed an increase in the mRNA expression of caspase 1, 2, and 3, but not caspase 8 following E2 treatment. However, we observed that caspase 8 inhibitor could prevent apoptosis induced by E2. These data suggest that treatment with E2 may not induce caspase 8, but may induce cleavage of procaspase 8 into caspase 8, which in turn triggers apoptosis.

SEA is a superantigen that triggers a rapid clonal expansion of T cells bearing Vß3 when injected in vivo. Such cells also undergo apoptosis immediately thereafter, leading to downregulation of T-cell response (Aroeira and Martinez, 1999Go). Because apoptosis is difficult to detect in vivo, we used this established model to study whether E2 would affect the responsiveness of SEA-activated T cells. Our results showed that E2 did not affect the initial expansion of Vß3+ T cells on day 1. However, on days 3 and 5, there was a marked decrease in their number. Furthermore, when the lymph node cells were restimulated with SEA in vitro, they showed decreased proliferative response. These data together suggest that E2 may facilitate AICD, thereby deleting SEA-activated T cells. It should be noted that ER{alpha} is expressed on all lymphocyte subsets, including T cells (Tornwall et al., 1999Go). In contrast, ERß is expressed in low levels in thymic T cells. Thus, it is likely that E2 mediates its effect on T cells through ER{alpha}.

In the current study, we used several doses of E2 from as high as 75 mg/kg to as low as 0.1 mg/kg body weight. These dose ranges are similar to those used in earlier studies that demonstrated thymic atrophy and T-cell subset alterations (Rijhsinghani et al., 1996Go; Screpanti et al., 1989Go; Staples et al., 1999Go). Furthermore, estrogen at 0.1–1.0 mg/mouse has been considered to be a pharmacological dose and compares well with the doses used in the current study (Okuyama et al., 1992Go). A previous study demonstrated that estrogen, when injected into mice at a dose of 0.01 mg/mouse every 4 days for 30 days, led to serum estrogen levels of 300 pg/ml, whereas the serum estradiol levels in normal female mice ranged from 47 to 66 pg/ml. In humans, estradiol levels are present in the range of 118 ± 80 pg/ml in women between 20 and 39 years of age (Giglio et al., 1994Go).

The current study has significant clinical impact in understanding the effect of E2 on the immune system. Previous studies have shown that E2 may enhance susceptibility to autoimmune disease (Cutolo et al., 1995Go). Furthermore, pregnant women treated with synthetic estrogens such as DES are more susceptible to autoimmunity and cancer (Cutolo et al., 1995Go). Moreover, the daughters exposed in utero to DES are at a higher risk for developing cervical cancers caused by human papilloma viruses (Bornstein et al., 1987Go; Melnick et al., 1987Go). Our studies shed new light on how apoptosis induced by E2 in the thymus and periphery may alter the T-cell repertoire such that the individual may respond differentially to self and nonself antigens, which may cause increased susceptibility to autoimmunity and cancer.


    ACKNOWLEDGMENTS
 
We thank Dr. W. Hans Carter, Jr., for his help in statistical analysis of the data.


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


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aroeira, L. S., and Martinez, A. C. (1999). The role of IL-4 in the staphylococcal enterotoxin B-triggered immune response: Increased susceptibility to shock and deletion of CD8Vß8+ T cells in IL-4 knockout mice. Eur. J. Immunol. 29, 1397–1405.[ISI][Medline]

Ashwell, J. D., Lu, F. W., and Vacchio, M. S. (2000). Glucocorticoids in T cell development and function. Annu. Rev. Immunol. 18, 309–345.[ISI][Medline]

Barnden, M. J., Heath, W. R., and Carbone, F. R. (1997). Down-modulation of CD8 beta-chain in response to an altered peptide ligand enables developing thymocytes to escape negative selection. Cell. Immunol. 175, 111–119.[ISI][Medline]

Bornstein, J., Kaufman, R. H., Adam, E., and Adler-Storthz, K. (1987). Human papillomavirus associated with vaginal intraepithelial neoplasia in women exposed to diethylstilbestrol in utero. Obstet. Gynecol. 70, 75–80.[Abstract]

Brunelli, R., Frasca, D., Baschieri, S., Spano, M., Fattorossi, A., Mosiello, L. F., D‘Amelio, R., Zichella, L., and Doria, G. (1992). Changes in thymocyte subsets induced by estradiol administration or pregnancy. Ann. N. Y. Acad. Sci. 650, 109–114.[ISI][Medline]

Couse, J. F., and Korach, K. S. (1999). Estrogen receptor null mice: What have we learned and where will they lead us? Endocr. Rev. 20, 358–417.[Abstract/Free Full Text]

Cutolo, M., Sulli, A., Seriolo, B., Accardo, S., and Masi, A. T. (1995). Estrogens, the immune response and autoimmunity. Clin. Exp. Rheumatol. 13, 217–226.[ISI][Medline]

Degen, G. H., and Bolt, H. M. (2000). Endocrine disruptors: Update on xenoestrogens. Int. Arch. Occup. Environ. Health 73, 433–441.[ISI][Medline]

Esaki, T., Hayashi, T., Muto, E., Kano, H., Kumar, T. N., Asai, Y., Sumi, D., and Iguchi, A. (2000). Expression of inducible nitric oxide synthase and Fas/Fas ligand correlates with the incidence of apoptotic cell death in atheromatous plaques of human coronary arteries. Nitric Oxide 4, 561–571.[ISI][Medline]

Giglio, T., Imro, M. A., Filaci, G., Scudeletti, M., Puppo, F., De Cecco, L., Indiveri, F., and Costantini, S. (1994). Immune cell circulating subsets are affected by gonadal function. Life Sci. 54, 1305–1312.[ISI][Medline]

Giusti, R. M., Iwamoto, K., and Hatch, E. E. (1995). Diethylstilbestrol revisited: A review of the long-term health effects. Ann. Intern. Med. 122, 778–788.[Abstract/Free Full Text]

Hengartner, M. O. (2000). The biochemistry of apoptosis. Nature 407, 770–776.[ISI][Medline]

Hirahara, H., Ogawa, M., Kimura, M., Iiai, T., Tsuchida, M., Hanawa, H., Watanabe, H., and Abo, T. (1994). Glucocorticoid independence of acute thymic involution induced by lymphotoxin and estrogen. Cell. Immunol. 153, 401–411.[ISI][Medline]

Holladay, S. D., Blaylock, B. L., Comment, C. E., Heindel, J. J., Fox, W. M., Korach, K. S., and Luster, M. I. (1993). Selective prothymocyte targeting by prenatal diethylstilbestrol exposure. Cell. Immunol. 152, 131–142.[ISI][Medline]

Hughes, D. E., Dai, A., Tiffee, J. C., Li, H. H., Mundy, G. R., and Boyce, B. F. (1996). Estrogen promotes apoptosis of murine osteoclasts mediated by TGF-beta. Nat. Med. 2, 1132–1136.[ISI][Medline]

Ju, S. T., Matsui, K., and Ozdemirli, M. (1999). Molecular and cellular mechanisms regulating T and B cell apoptosis through Fas/FasL interaction. Int. Rev. Immunol. 18, 485–513.[Medline]

Kamath, A. B., Camacho, I., Nagarkatti, P. S., and Nagarkatti, M. (1999). Role of Fas-Fas ligand interactions in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced immunotoxicity: Increased resistance of thymocytes from Fas-deficient (lpr) and Fas ligand-defective (gld) mice to TCDD-induced toxicity. Toxicol. Appl. Pharmacol. 160, 141–155.[ISI][Medline]

Kamath, A. B., Nagarkatti, P. S., and Nagarkatti, M. (1998). Characterization of phenotypic alterations induced by 2,3,7,8- tetrachlorodibenzo-p-dioxin on thymocytes in vivo and its effect on apoptosis. Toxicol. Appl. Pharmacol. 150, 117–124.[ISI][Medline]

Kamath, A. B., Xu, H., Nagarkatti, P. S., and Nagarkatti, M. (1997). Evidence for the induction of apoptosis in thymocytes by 2,3,7,8- tetrachlorodibenzo-p-dioxin in vivo. Toxicol. Appl. Pharmacol. 142, 367–377.[ISI][Medline]

Lai, Z. W., Fiore, N. C., Hahn, P. J., Gasiewicz, T. A., and Silverstone, A. E. (2000). Differential effects of diethylstilbestrol and 2,3,7,8- tetrachlorodibenzo-p-dioxin on thymocyte differentiation, proliferation, and apoptosis in bcl-2 transgenic mouse fetal thymus organ culture. Toxicol. Appl. Pharmacol. 168, 15–24.[ISI][Medline]

Luster, M. I., Hayes, H. T., Korach, K., Tucker, A. N., Dean, J. H., Greenlee, W. F., and Boorman, G. A. (1984). Estrogen immunosuppression is regulated through estrogenic responses in the thymus. J. Immunol. 133, 110–116.[Abstract/Free Full Text]

Melnick, S., Cole, P., Anderson, D., and Herbst, A. (1987). Rates and risks of diethylstilbestrol-related clear-cell adenocarcinoma of the vagina and cervix. An update. N. Engl. J. Med. 316, 514–516.[Abstract]

Mor, G., Munoz, A., Redlinger, R., Jr., Silva, I., Song, J., Lim, C., and Kohen, F. (2001). The role of the Fas/Fas ligand system in estrogen-induced thymic alteration. Am. J. Reprod. Immunol. 46, 298–307.[ISI][Medline]

Okasha, S. A., Ryu, S., Do, Y., McKallip, R. J., Nagarkatti, M., and Nagarkatti, P. S. (2001). Evidence for estradiol-induced apoptosis and dysregulated T cell maturation in the thymus. Toxicology. 163, 49–62.[ISI][Medline]

Okuyama, R., Abo, T., Seki, S., Ohteki, T., Sugiura, K., Kusumi, A., and Kumagai, K. (1992). Estrogen administration activates extrathymic T cell differentiation in the liver. J. Exp. Med. 175, 661–669.[Abstract]

Rijhsinghani, A. G., Thompson, K., Bhatia, S. K., and Waldschmidt, T. J. (1996). Estrogen blocks early T cell development in the thymus. Am. J. Reprod. Immunol. 36, 269–277.[ISI][Medline]

Robertson, C. N., Roberson, K. M., Padilla, G. M., O‘Brien, E. T., Cook, J. M., Kim, C. S., and Fine, R. L. (1996). Induction of apoptosis by diethylstilbestrol in hormone-insensitive prostate cancer cells. J. Natl. Cancer. Inst. 88, 908–917.[Abstract/Free Full Text]

Ruggiero, R. J., and Likis, F. E. (2002). Estrogen: Physiology, pharmacology, and formulations for replacement therapy. J. Midwifery Womens Health 47, 130–138.[ISI][Medline]

Safe, S. H., Pallaroni, L., Yoon, K., Gaido, K., Ross, S., Saville, B., and McDonnell, D. (2001). Toxicology of environmental estrogens. Reprod. Fertil. Dev. 13, 307–315.[ISI][Medline]

Screpanti, I., Morrone, S., Meco, D., Santoni, A., Gulino, A., Paolini, R., Crisanti, A., Mathieson, B. J., and Frati, L. (1989). Steroid sensitivity of thymocyte subpopulations during intrathymic differentiation. Effects of 17 beta-estradiol and dexamethasone on subsets expressing T cell antigen receptor or IL-2 receptor. J. Immunol. 142, 3378–3383.[Abstract/Free Full Text]

Shevde, N. K., and Pike, J. W. (1996). Estrogen modulates the recruitment of myelopoietic cell progenitors in rat through a stromal cell-independent mechanism involving apoptosis. Blood 87, 2683–2692.[Abstract/Free Full Text]

Silverstone, A. E., Frazier, D. E., Jr., Fiore, N. C., Soults, J. A., and Gasiewicz, T. A. (1994). Dexamethasone, beta-estradiol, and 2,3,7,8-tetrachlorodibenzo-p-dioxin elicit thymic atrophy through different cellular targets. Toxicol. Appl. Pharmacol. 126, 248–259.[ISI][Medline]

Staples, J. E., Fiore, N. C., Frazier, D. E., Jr., Gasiewicz, T. A., and Silverstone, A. E. (1998). Overexpression of the anti-apoptotic oncogene, bcl-2, in the thymus does not prevent thymic atrophy induced by estradiol or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 151, 200–210.[ISI][Medline]

Staples, J. E., Gasiewicz, T. A., Fiore, N. C., Lubahn, D. B., Korach, K. S., and Silverstone, A. E. (1999). Estrogen receptor alpha is necessary in thymic development and estradiol-induced thymic alterations. J. Immunol. 163, 4168–4174.[Abstract/Free Full Text]

Tai, X. G., Toyo-oka, K., Yamamoto, N., Yashiro, Y., Mu, J., Hamaoka, T., and Fujiwara, H. (1997). Expression of an inducible type of nitric oxide (NO) synthase in the thymus and involvement of NO in deletion of TCR-stimulated double-positive thymocytes. J. Immunol. 158, 4696–4703.[Abstract]

Tornwall, J., Carey, A. B., Fox, R. I., and Fox, H. S. (1999). Estrogen in autoimmunity: Expression of estrogen receptors in thymic and autoimmune T cells. J. Gend. Specif. Med. 2, 33–40.[Medline]

Vermes, I., Haanen, C., Steffens-Nakken, H., and Reutelingsperger, C. (1995). A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Methods 184, 39–51.[ISI][Medline]

Welshons, W. V., Murphy, C. S., Koch, R., Calaf, G., and Jordan, V. C. (1987). Stimulation of breast cancer cells in vitro by the environmental estrogen enterolactone and the phytoestrogen equol. Breast Cancer Res. Treat. 10, 169–175.[ISI][Medline]

Yellayi, S., Teuscher, C., Woods, J. A., Welsh, T. H., Jr., Tung, K. S., Nakai, M., Rosenfeld, C. S., Lubahn, D. B., and Cooke, P. S. (2000). Normal development of thymus in male and female mice requires estrogen/estrogen receptor-alpha signaling pathway. Endocrine 12, 207–213.[ISI][Medline]

Zava, D. T., Blen, M., and Duwe, G. (1997). Estrogenic activity of natural and synthetic estrogens in human breast cancer cells in culture. Environ. Health Perspect. 105(Suppl. 3), 637–645.[ISI][Medline]