* Department of Microbiology and Immunology,
Department of Pharmacology and Toxicology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298
Received May 21, 2002; accepted July 26, 2002
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ABSTRACT |
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Key Words: estrogen; immunotoxicity; Fas; FasL; apoptosis; thymus.
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INTRODUCTION |
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It has been reported that several microbes and plants produce estrogens, such as enterolactone and coumestrol, respectively (Welshons et al., 1987). 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, 2000
). For example, compounds such as bisphenol A and nonylphenol bind ER and induce transactivation of estrogen-responsive genes (Safe et al., 2001
). 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., 1997
). Estrogen is used in various forms for contraception or for pharmacological purposes in the treatment of postmenopausal women to prevent osteoporosis, Alzheimers disease, and cardiovascular diseases (Ruggiero and Likis, 2002
). 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 510 million Americans received DES during pregnancy or were exposed to the drug in utero during this period (Giusti et al., 1995
). 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., 1987
; Melnick et al., 1987
). Pregnant women treated with DES may also be more susceptible to autoimmunity (Cutolo et al., 1995
).
Estrogen induces thymic atrophy (Holladay et al., 1993; Silverstone et al., 1994
; Staples et al., 1999
). 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., 2001
). Similarly, addition of E2 to fetal thymic organ culture induces apoptosis (Barnden et al., 1997
). 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., 1999). 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.
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MATERIALS AND METHODS |
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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., 2001; Vermes et al., 1995
). 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 manufacturers 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 810 µg of RNA using Super Arrays 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 24 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., 1999). 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., 1997). 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 46 mice. Statistical comparisons of dose-dependent responses were made by one-way analysis of variance (ANOVA). Post hoc comparison was accomplished using Tukeys HSD. The level of significance was based on p < 0.05.
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RESULTS |
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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 9 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 10
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.
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DISCUSSION |
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Estrogen has recently been shown to induce apoptosis in nonlymphoid tissues (Hughes et al., 1996; Robertson et al., 1996
; Shevde and Pike, 1996
), but whether estrogen induces thymic involution by triggering apoptosis is not clear. Staples et al. (1998)
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., 2000
). In an earlier study, we could not detect apoptosis in freshly isolated thymocytes following E2 treatment (Okasha et al., 2001
). 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 1224 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., 1997
, 1998
, 1999
; Okasha et al., 2001
). 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, 2000). 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., 1998
). 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., 1994). 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., 2000
; Tai et al., 1997
). 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, 1999). 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
is expressed on all lymphocyte subsets, including T cells (Tornwall et al., 1999
). 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
.
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., 1996; Screpanti et al., 1989
; Staples et al., 1999
). Furthermore, estrogen at 0.11.0 mg/mouse has been considered to be a pharmacological dose and compares well with the doses used in the current study (Okuyama et al., 1992
). 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., 1994
).
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., 1995). Furthermore, pregnant women treated with synthetic estrogens such as DES are more susceptible to autoimmunity and cancer (Cutolo et al., 1995
). 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., 1987
; Melnick et al., 1987
). 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.
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ACKNOWLEDGMENTS |
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NOTES |
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