Reprints Available Directly from the Publisher Photocopying Permitted by License Only Estrogen-receptor Expression and Function in Thymocytes in Relation to Gender and Age

The expression of estrogen receptor (ER) in thymocytes was studied in young, middle-aged, and old (2, 12, and 24 months, respectively) female and male C57BL/6J mice. Western immunoblots prepared from the thymocytes of females of all age groups showed the presence of a 67-kD protein band, which has been associated with the apparent MW of denatured ER. Flow cytometry analysis o,f cells stained with a monoclonal anti-ER antibody (clone 13H2) disclosed ER expression in both females and males of all age groups. In vivo treatment with estradiol (E2) led to an increase in the specific activity of thymic creatine kinase (CK) in the female mice, whereas the male thymocytes responded with an increase in CK activity only on treatment with dihydrotestosterone (DHT). The data show no differences in ER expression between male and females, but the receptor appears not to be functional in males. Interestingly, when estradiol was applied to co-cultures of lymphoid-depleted fetal thymus (FT) explants and bone-marrow cells, or thymocytes, from young and old females, it resulted in increased cellularity of cultures containing cells of the young, and not those of the old. The proportion of CD4/CD8 phenotypes of the developing cells in these cultures was not affected by E2 treatment. These observations provide a new insight into ER expression and function in T-cell development in relation to gender and age.


e thymus was
ound mainly in the epithelial cells (Luster et al., 1984; Marchetti et  al., 1984), suggesting the involvement of estrogen function in T-lymphocyte development.In previous studies, using a variety of experimental criteria, we demonstrated that ER is expressed in thymocytes of young adult female mice and rats (Amir-Zaltsman et al., 1993).Hence, (1) Western blot analysis of thymocytes showed the presence of a 67-kD protein band; (2) Northern blot analysis of poly(A+)-enriched RNA fraction obtained from the thymocytes showed the presence of a transcript of 6.2 kb, corresponding to the size of ER mRNA; (3) immunofluorescence studies using anti-idiotypic antibody clone 1D5 that interacts with ER (Mor et al., 1992) showed staining of the thymocyte nuclei; (4) the mitogen-induced proliferative response was reduced in the presence of estradiol; (5) administration of estradiol to immature female rats caused a significant increase in the thymic ER mRNA and Creatine kinase B (CKb) mRNA (Amir-Zaltsman et al., 1993).

The function of estrogen in the immune system has been of particular interest, in view of its relevance to pregnancy (Clarke and Kendall 1994) and autoim- munity (Cutolo et al., 1995).In addition, since generation of T lymphocytes continues throughout the lifespan, a decline in ER expression in the aging thymus may play a role in developmental processes that change with age (Globerson 1994, 1995).How- ever, there is hardly any information on the status of ER in the aging thymus.

The present study was desi ned to determine whether the expression and function of ER in thymocytes are age-and gender-specific and to examine its role in lymphoid development in the thymus.


RESULTS


Expression of ER in Thymocytes

The first series of experiments was conducted to examine the expression of ER in thymocytes of female and male mice, as related to age.We used Western immunoblots and flow cytometry methods, as in our previous report (Amir-Zaltzman et al.,  1993).

Western immunoblots prepared from t ymocytes of female mice showed the presence of a 67-kD protein band (lane A in Figure l a), which has been associated with the apparent MW of denatured ER (Greene et al., 1986).This band was

thymocytes of the three age gro
ps (lanes B, C, and D in Figure a), and was absent when the primary, specific anti-E2 antibody was omitted in the control gel (Figure b).The specific anti-E2 antibody stained the 32-kD fragment in the thymocytes of all mice in the group (lanes B, C and D in Figure a) and was absent in the control blot (lanes B, C, D, and E in Figure lb).

Representative flow cytometry profiles of thymocytes from young and old females show specific staining with the anti-ER antibody and PE-rabbit anti- mouse IgG, as related to the control of second antibody alone.Similar results were obtained with FITC-labeled goat anti-mouse IgG as a second antibody (Figure 2) or when the directly labeled FITC-anti-ER antibody was used (data not shown).Analysis of thymocytes from females and males of the different age groups revealed a similar proportion of ER cells in all cases (Tabl I).

Response to Gonadal-Hormone Treatment In Vivo Functional manifestation of ER in thymocytes was investigated in hormone-treated mice, by measuring the CK-response studies.Female and male mice of the Mouse thymocyles (2 105 cells/lane), cDNA estrogen-receptor (ER) protein expressed in yeast and the 32-kD purified ER fragment from porcine uteri were subjected to 10% SDS-PAGE and transferred to nitrocellulase and probed first with anti-ER antibody (clone 13H2), followed with rabbit anti-mouse IgG1 peroxidase conjugate as described (Amiz-Zaltsman, et al, 1993).The blots were then visualized using enhanced chemiluminescence, and the reagents provided by Amersham.Mol wt markers are shown on the right. (a) Lane A: cDNA ER protein expressed in yeast.Lane B: Mouse thymocytes derived from a 2-month-old C57BL female mouse.Lane C: Mouse thymocytes derived from a 1-year-old C57BL female mouse.Lane D: Mouse thymocytes derived from a 2-year-old C57BL female mouse.Lane E: The 34-kD ER fragment derived from porcine uteri.(b) Controls of lanes A through D probed only with the second antibody rat anti-mouse IgG peroxidase.

different age groups were injected with either E2, DHT, or PBS, respectively.Analysis of CK response to hormonal treatment was performed on the intact thymus tissue, to correlate with previous studies (Marchetti et al., 1984), as well as on separated thymocytes, leading to similar results.Figure 3 shows representative results obtained on intact thymus tissue.Hence, CK levels were increased in females treated with E2 and not with DHT, whereas the males showed a response to DHT and not to E2.The sex- specific-induced increase in CK response in the thymus (Figure 3) and in isolated thymocytes (data not shown) was observed in all age groups.


Effects of E2 on Thymocytopoiesis In Vitro

To find out if ER expression plays any role in thymocytopoiesis, we seeded thymocytes and bone- marrow cells, from young and old female mice, onto individual lymphoid-depleted FT lobes.E2 was applied for the first 3 days of cell seeding onto the FT lobes (under the "hanging-drop" condit

ns), or during the subsequent organ culture
period, or throughout the entire in vitro period.Control cultures were set up in parallel, without E2.The cultures were sacrificed after 7 days, and cell numbers were counted.In addition, cells were analyzed for CD4/ CD8 phenotypes, using a direct double-staining procedure.The results showed a significant increase in numbers of cells originating from young donors, following incubation with E2 during the seeding phase of the culture.No effect was observed in any of the cultures of old donor cells (Table II).On the other hand, the proportions of CD4/CD8 phenotypes were not affected by E 2 treatment (Table III).


DISCUSSION

Expression of ER in the thymus has been demon- strated in the past; however, there has been little information on its expression and function in aging.

Our results provide new information regarding ER in relation to gender and age.Whereas ER is expressed in both females and males, only female cells respond to estrogen.Reg

ding aging,
we observed no signifi-cant age-related decline in ER expression, as revealed from Western immunoblots, flow cytometry, and CK response.It thus appear d that ER continues to function in advanced age.However, experiments designed to determine if ER expression in aging plays a role in thymocytopoiesis showed that E 2 treatment resulted in cellular expansion in the young, but not in the old donor-derived thymocytes.

The finding that thymocytes of male mice express the ER, with no increase in CK activity in response to treatment with E2, suggests that the ER in males is not functional.On the other hand, the male thymocytes responded to DHT, indicating the presence of func- tional androgen receptors in this organ.These findings are in accordance with he previous results on skeletal cells, where E2 caused specific stimulation of CK activity only in female-and not in male-derived skeletal cells, whereas the male cells responded to YOUNG A OLD B A Fluorescence FIGURE 2 Flow cytometry profiles of ER thymocytes of young (2 months) and old (24 months) C57BL/6J females.(Curve A) Background control cells stained with the second antibody only.(Curve B) Thymocytes stained in a two-step procedure, using monoclonal anti-ER antibodies (13H2 clone) and PE-labeled rabbit anti-mouse IgG.Y axis: cell number (arbitrary units).aValues represent mean SD of percent ER+ thymocytes from six individual mice per group, calculated from t o-step staining with an anti- ER monoclonal antibody (13H2) and subsequently with a second step of either FITC-goat anti-mouse or RPE-Rabbit anti-mouse IgG.Mice of all the age groups were examined in parallel in each of three independent experiments.

DHT (S6mjen et al., 1995).Interestingly, it was recently shown that estrogen resistance caused by a mutation in the ER gene in a 28-year-old man resulted in os eoporosis, implicating a role of ER in males (Smith et al., 1994).Our finding that ER is expressed in males, yet, it is not functional in response to E2, as manifested in the CK assay, raises the question of whether it has other possible functions.

Estrogens influence many developmental and physiological responses in target cells by regulating specific gene activity (Parker, 1993).Expression of the receptor in thymic epithelial cells may play a role in processes of stromal-cell induction of lymphocyte development, whereas expression in the thymic lymphoid cells also sug, gests direct hormonal effects on these cells.Estrogen may thus affect various types of processes in the thymus, including cell division, differentiation, and apoptosis, either directly or via stimulation of cytokine function.Our present study reveals a role of ER on cell division, and no effect on thymocyte subset differentiation, as indicated from the results on young donor cells.The observation of no effect on cell division in case of the old may be attributed to downstream pr

ging
Globerson, 1995).The mechanisms underlying the functional manifestation and the immunological relevance of the receptor in thymocytes, as well as the nature of the ER in males, will need to be further elucidated.


MATERIALS AND METHODS


Mice

Young (2 to 3 months), middle-aged (12 months), and old (24 months) female and male C57BL/6J (Jackson Laboratories, Bar Harbor, ME) and BALB/c mice (OLAC, UK) were used throughout the study.The mice were virus-and pathogen-free (SPF), bred in isolators, and maintained in Millipore-top cages, on sterile bedding, with food and water ad libitum.

Cleaning and handling

ere performed under la
inar flow hoods.The mice were routinely monitored for possible viral, bacterial, or parasitic contaminations.

Only mice with no overt malignancy or any other gross pathological manifestation were included in the study.


Western Blot Analysis

Mouse thymocytes (2 10 5 cells/lane), the 32-kD ER fragment purified from porcine uteri (Thole et al.,  1991) and the cDNA ER protein expressed in yeast (Greene et al., 1986) wer

solubilized
in buffer containing 0.07 M TrisHC1, pH 6.8, 10% glycerol, 1%

SDS, /3-mercaptoethanol, and bromophenol blue by heating at 100C for 5 min, and processed for Western blot analysis as described previously (Amir-Zaltsman et al., 1993).


Antibodies

Mouse monoclonal antibody, clone 13H2, raised against porcine 32-kD hormone-binding estrogen receptor (ER) fragment (Thole et al., 1991; Thole and Jakob, 1993) was prepared as described previously.This antibody was conjugated with FITC for direct staining of thymocytes in flow cytometry, using conditions previously described (Mot et al., 1992).Rabbit anti-mouse IgG1 peroxidase and FITC-labeled goat anti-mouse IgG were from Zymed Lab.(South San Francisco) and rabbit anti-mouse

E, affinity- DHT FIGURE 3 Ho
monal stimulation of CK-specific activity in mouse thymus.Young and old, female and male mice were injected with E2, DTH, or PBS, as described in Materials and Methods.The thymus of each mouse was then assayed for CK-specific activity.Results are expressed as mean S.E.M.; n 5; *P --< 0.05.Note: BM cells were seeded onto irradiated FT explants hr after exposure.E2 was applied during the first 2 days in hanging-drop cultures.

Cells were harvested for analysis after 7 days in organ cultures.

isolated F(ab-)2 conjugate was from Dako (Den- mark).Anti-mouse CD4 conjugated with PE and FITC-anti-mouse CD8 (Serotec, UK) were used in direct double staining.


Hormonal Treatment In Vivo

The changes in creatine kinase (CK) specific activity in the thymus induced by the short treatment of estradiol (E2), dihydrotestosterone (DHT), or p

s- phate-buffered saline
(PBS, control) were studied in male and female mice of the three age categories.The animals (five mice/group) were sacrificed 24 hr after i.p. injection of E2 (5 /zg/animal), DHT (10 #g/ animal), or PBS.

Hormonal Treatment In Vitro Organ cultures were prepared as originally described (Eren et al., 1988).Briefly, fetal (day 14 of gestation) thymus (FT) lobes were depleted of lymphoid cells by treatment with 2-deoxyguanosine (1.35 mM) for 5 days at 37C (Jenkinson et al., 1982), or by exposure to irradiation (20 Gy; Fridkis-Hareli et al., 1991), as specified.The FT lobes were subsequently incubated with the donor cells (60 103 cells/lobe), in hanging drops, in Terasaki plates (Nunclon; Denmark), for 3 days; then rinsed and cultivated in organ cultures for 7 days.Donor cells included bon

marrow, or immature (PNA
) thymocytes, prepared in accord- ance to standard procedures (Reisner et al., 1976).Hormonal treatment (10 -7 M E2) to the cultures was applied either in the hanging drops, or in the

rgan cultures,
or throughout the in vitro incubation period.


Flow Cytometry Analysis

Thymocytes, prepared from each mouse separately (2 X 106 cells/pellet), were fixed in 50% ethanol at -20