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Estrogen Receptor (ER) Deficiency in Macrophages Results in Increased Stimulation of CD4⁺ T Cells while 17-Estradiol Acts through ER to Increase IL-4 and GATA-3 Expression in CD4⁺ T Cells Independent of Antigen Presentation¹

K. Chad Lambert^*,, Edward M. Curran^,, Barbara M. Judy^,, Gregg N. Milligan, Dennis B. Lubahn^, and D. Mark Estes^2,*,,

^* Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 65211; Department of Pediatrics, University of Texas Medical Branch, Sealy Center for Vaccine Development, Galveston, TX 77555; Department of Biochemistry, University of Missouri, Columbia, MO 65211; and University of Missouri Center for Phytonutrient and Phytochemical Research, Columbia, MO 65211

	Abstract

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The effects of 17-estradiol (E₂) on immune function have been extensively reported. The effects are dependent on concentration and duration of exposure and potential differences in signaling between the known E₂ receptors, estrogen receptors (ER) and ER. Through the use of ER-deficient mice, we and others have begun to demonstrate the role of the two known receptors in modulating immune functional activities. Previous studies have shown that cells of the innate immune system have altered function (bactericidal capacity) and patterns of cytokine expression (increased proinflammatory cytokine expression) through amelioration of ER signaling. In this study, we extend these studies to analysis of T cell differentiation and proliferation in APC-dependent and APC-independent in vitro assay systems. Our results demonstrate that ER deficiency in splenic macrophages, but not CD11c⁺ splenic dendritic cells pulsed with OVA significantly enhances proliferative responses and IFN- production by transgenic OVA peptide-specific (OT-II) CD4⁺ T cells when compared with Ag-pulsed APC from wild-type littermates. The addition of E₂ in this culture system did not significantly affect the production of IFN-. In addition, when purified CD4⁺ T cells from ER-deficient and wild-type littermates were stimulated with anti-CD3/CD28 Ab in the absence of E₂, there were no significant differences in IFN- or IL-4 production. However, the addition of E₂ significantly increased IL-4 secretion, as well as increased GATA-3 mRNA levels from ER-replete CD4⁺ T cells, while this effect was abrogated in ER-deficient CD4⁺ T cells.

	Introduction

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Estradiol (17-estradiol (E₂)³) acts through two known hormone receptors, estrogen receptors (ER) and ER, which are tissue specific and expressed to varying degrees by immune cells. E₂ has significant impact on immune function, accounting at least in part for the sexual dimorphism documented to occur between males and females (1, 2, 3, 4, 5, 6). The effects of E₂ on immunity are broad ranging and are applicable to pregnancy, pre- and postmenopause, alterations in immune function during the estrous cycle, autoimmunity, cancer, and infection. Recent studies suggest gender may impact vaccine efficacy (7). Combined with the reported effects of menstrual cycle stage and contraceptive use on HPV-16 candidate vaccine efficacy, these data illustrate the importance of elucidating the mechanisms by which estrogens and their cognate receptors exert their effects on immune function (7, 8).

Given the trend in females to exhibit more vigorous Ab responses to treatment when compared with males, the linkage between Th cell function and E₂ was investigated. The production of IFN- or IL-4 by CD4⁺ T cells is central for an effective cell-mediated (Th1) or humoral (Th2) immune response (9, 10). APC such as macrophages and dendritic cells (DC) present Ag and provide key cytokines and costimulatory molecules to CD4⁺ T cells to drive expansion and differentiation into cytokine-secreting effector cells (10, 11). Administration of E₂ in vivo and ER signaling are critical modulators of immune function and have been shown to significantly affect Th1/Th2-type cytokine production in infection and autoimmune models (12, 13, 14, 15, 16, 17, 18). However, it remains unclear whether E₂ modulates Th1/Th2-type cytokine production by signaling directly through ER(s) expressed by CD4⁺ T cells or indirectly through APCs.

In vitro administration of E₂ to CD4⁺ T cell cultures has been demonstrated to suppress IL-2 production, while CD4⁺ T cells from E₂-treated mice have reduced ability to produce IL-2 in ex vivo culture (19, 20). In addition, IL-4 production by CD4⁺ T cells was demonstrated to be affected by the cyclical variations of circulating E₂ levels (21). However, the specific pathways by which E₂ exerts these effects on CD4⁺ T cells remain unclear. Human CD4⁺ T cells have been reported to express both ER and ER mRNA (22, 23); however, in a different study, only ER protein was detected in stimulated or resting human CD4⁺ T cells (24). Phiel et al. (25) demonstrated that human CD4⁺ T cells had significantly increased expression of ER as compared with ER. The full ER repertoire of murine T cells has yet to be fully elucidated. Aside from the direct effect of E₂ on CD4⁺ T cells, Liu et al. (26) demonstrated that E₂ pretreatment of DCs reduced proliferation and IFN- secretion from Ag-specific CD4⁺ T cells. In, addition, E₂ has also been shown to suppress specific CD4⁺ T cell responses by modulating MHC II expression on bone marrow-derived macrophages (27).

Studies using ER-deficient mice have demonstrated an essential role for this receptor in immune function (28, 29). Previous studies from our laboratory have suggested that nonliganded ER can suppress mycobacterial killing and TNF- production by peritoneal macrophages in a mouse model of infection (13). Furthermore, significant increases in IFN- production were observed in ER knockout (KO) mice chronically infected with Mycobacterium avium, a facultative intracellular pathogen (12). The studies described in this work delineate the specific ER repertoire of CD4⁺ T cells and their role in E₂ modulation of Th1/Th2-type cytokine secretion. We demonstrate that E₂ treatment of CD4⁺ T cells resulted in increased IL-4 secretion after anti-CD3/CD28 stimulation, which was directly correlated with increased expression of GATA-3 transcripts, an essential Th2 transcription factor (30, 31). E₂-induced IL-4 production was abrogated in CD4⁺ T cells from ER KO mice, suggesting E₂ acts specifically through this receptor and not ER in CD4⁺ T cells. Finally, we observed significant increases in proliferation and IFN- production by OT-II CD4⁺ T cells when stimulated by ER-deficient APCs relative to their wild-type (WT) littermates. Our data demonstrate this effect on CD4⁺ T cells is due to ER deficiency in splenic macrophages and not splenic CD11c⁺ DCs.

	Materials and Methods

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Mice

Homozygous ER-deficient mice and their WT littermates were generated from C57BL/6 SCID and/or immunocompetent mice heterozygous for the disrupted ER sequence and rederived by Caesarean section (13, 17, 32). C57BL/6 female mice were purchased from The Jackson Laboratory. OVA_323–339-specific, MHC II-restricted, TCR transgenic OT-II mice were supplied by G. N. Milligan. OT-II transgenic mice on a Rag^–/– background were originally purchased from Taconic Farms. All mice were housed under germfree conditions in a 12-h light/dark cycle on water and standard rodent chow ad libitum. Experiments were conducted according to animal care and use guidelines, as recommended by the National Institutes of Health. Female mice 6–8 wk old were either ovariectomized or had sham operations, as previously described (17). Briefly, mice were anesthetized with isoflourane; then bilateral incisions were made in the abdominal cavity. Ovaries were removed and incisions were closed with surgical staples. A recovery time of 2 wk was allowed for all mice before experiments began.

CD4⁺ T cell isolation and activation

Spleens were removed (n = 3–5) and macerated, and the resulting cell suspension was filtered through a 70-µm nylon mesh filter. The filtered suspension (2 spleens per 5 ml) was layered over Lympholyte-M (Accurate Chemical & Scientific). Gradients were centrifuged at room temperature at 800 x g for 20 min. The cellular interface was removed and washed twice with RPMI 1640, and cell suspensions were then placed in a 37°C, 5% CO₂ incubator for 1 h to remove adherent cells. Nonadherent cell suspension was removed, washed once, and resuspended in running buffer (1x PBS, 0.5% BSA, 2 mM EDTA (pH 7.4)). CD4⁺ and CD8⁺ T cells were isolated using CD4 or CD8 microbeads and AutoMacs Cell Sorter, according to manufacturer’s protocol (Miltenyi Biotec). Flow cytometric analysis demonstrated >95% purity for both CD4⁺ and CD8⁺ T cell preparations.

CD4⁺ or CD8⁺ T cells were seeded in 96-well plates at a concentration of 1 x 10⁵ cells/well in 200 µl of complete RPMI 1640 (phenol red-free RPMI 1640 supplemented with 2 mM L-glutamine, 5 x 10^–5 M 2-ME, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, and 5% charcoal/dextran-treated heat-inactivated FBS (HyClone)). Complete RPMI 1640 was shown to be free of estrogenic activity using a MCF-7 bioassay and specific inhibitors of E₂, as previously described (13). T cell cultures were treated with 10^–9, 10^–10, and 10^–11 M concentrations of 17-estradiol (catalogue E-2257; Sigma-Aldrich) for 12–16 h. Control wells not treated with E₂ contained 0.1% methanol, as E₂ stock solutions were dissolved in HPLC grade methanol. In some experiments, ICI 182,780 (Tocris Cookson) was used as a specific inhibitor of E₂, as indicated. Mouse rIL-2 (R&D Systems) at a concentration of 1 ng/ml was added to all wells. CD4⁺ and CD8⁺ T cells were stimulated by the method of Haas et al. (33) with modification. Briefly, avidin-D agarose beads (Vector Laboratories) were incubated with biotinylated anti-CD3 (clone 145-2C11; BD Pharmingen) and biotinylated anti-CD28 (clone 37.51) (BD Pharmingen) at room temperature for 15 min with mixing at a concentration of 2 mg of total Ab/1 mg of agarose bead. The Ab/bead conjugates (immobilized anti-CD3/CD28) were washed three times with a 10 mM HEPES, 15 mM NaCl (pH 7.5) solution. Ab conjugates were added to CD4⁺ or CD8⁺ T cells at a final concentration of 10 µg/ml anti-CD3 (clone 145-2C11) and 20 µg/ml anti-CD28 (clone 37.51).

Stimulation of OT-II CD4⁺ T cells

DCs and macrophages were purified from ER-deficient SCID (n = 5) and WT littermate mice (n = 5), as indicated and described previously (13). Briefly, spleens were treated with 400 U/ml collagenase (Invitrogen Life Technologies) in a 1x balanced salt solution, and the resulting cell suspension was layered over a BSA gradient and centrifuged at 10,000 x g for 20 min at 4°C. Interface cells were removed and rinsed with RPMI 1640, followed by a 90-min incubation to allow adherent cells to attach. Adherent cells (macrophages and DC) were washed three times to remove nonadherent cells and then incubated with 500 µg/ml OVA (Sigma-Aldrich) or 0.1 µm of OVA 323–339 peptide (a gift from G. N. Milligan) with no further stimulation to induce expression of MHC II or costimulatory molecules. In some experiments, adherent cells were incubated for an additional 18 h, followed by removal of nonadherent cells (DCs). DCs were incubated with CD11c microbeads and sorted with an AutoMacs cell sorter (Miltenyi Biotec). Flow cytometric analysis demonstrated a 95% purity of CD11c⁺ cells. DCs or adherent macrophages were then incubated overnight with 500 µg/ml OVA or peptide (1 µm). The next day, OT-II CD4⁺ T cells were purified, as described above, and 1 x 10⁵ OT-II CD4⁺ T cells were added to APC wells at ratios of 10:1, 5:1, 2:1, and 1:1 (T cell:APC), as indicated.

RNA preparation and RT-PCR for ER repertoire of murine T cells

Total RNA was extracted from ovaries in addition to purified CD4⁺ and CD8⁺ T cells from C57BL/6 female mice using the Qiagen RNeasy minikit, according to the manufacturer’s protocol. Total RNA was treated with a DNase Treatment and Removal kit (Ambion), and 1 µg of total RNA was analyzed by RT-PCR for expression of ER and ER using the Titan One Tube RT-PCR system (Roche). Mouse ovary total RNA served as a positive control for ER and ER mRNA expression. The protocols and specific primers used for ER coding sequence and each of the eight exons of ER spanned intron/exon junctions, as described previously (13). Briefly, RT-PCR conditions for ER were as follows: reverse transcription at 48°C for 45 min, heat inactivation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 40 s, annealing 60°C for 40 s, and an elongation time of 2 min at 68°C (PerkinElmer Gene AMP 2400 Thermocycler). RT-PCR conditions for the ER reactions were the same, except for annealing at 51°C for 40 s. Products were separated by gel electrophoresis on a 2% agarose gel, and bands were visualized by ethidium bromide staining under UV light.

Real-time PCR detection of GATA3 and T-bet mRNA

RNA was extracted from immobilized anti-CD3/CD28-stimulated CD4⁺ T cells 72 h poststimulation using the Qiagen RNeasy minikit, according to the manufacturer’s protocol. DNA was removed from total RNA using the DNase Treatment and Removal kit (Ambion). Total RNA was reverse transcribed to cDNA and amplified using random hexamers. PCR probes and primers were generated using the Applied Biosystems assays on demand 20x assay mix of primers and TaqMan MGB probe (FAM dye labeled) and 18S rRNA VIC-dye labeled probe as an endogenous control. GATA-3 NM_008091, X55123, AK090089, BC062915 GATA-3 probe CTCCGACCCACCACGGGAGCCAGGT. T-bet NM_019507, AF093099, AF241242, AK054495; T-bet probe-AGCAAGGACGGCGAATGTTCCCATT. To determine the relative quantity of gene expression separate tubes (Singleplex), real-time PCR was done with 40 ng of cDNA for both target gene and endogenous control and the universal PCR master mix reagent kit (Applied Biosystems). The parameters for real-time PCR were 40 cycles of: uracil-N-glycosylase activation at 50°C for 2 min, AmpliTaq activation at 95°C for 10 min, denaturation at 95°C for 15 s, and annealing/extension at 60°C for 1 min on the ABI 7000 Sequence Detection System. Duplicate threshold cycle (C_T) values were analyzed in Microsoft Excel using the comparative C_T ( C_T) method, as described by the manufacturer (Applied Biosystems). The amount of target (2^{– CT}) was normalized to endogenous reference 18S rRNA relative to a calibrator (a nonestrogen-treated experimental sample).

CD4⁺ T cell proliferation

For [³H]TdR (MP Biomedicals) incorporation assays, triplicate T cell cultures were pulsed with 1 µCi of ³H/well for 18 h and then harvested 72 h poststimulation. [³H]TdR incorporation was determined using a 1450 Microbeta liquid and luminescence counter (PerkinElmer). Purified CD4⁺ T cells with only avidin-agarose beads or APCs without OVA added and cocultured with purified OT-II CD4⁺ T cells served as negative controls. All assay conditions were repeated in at least three independent experiments.

Flow cytometric assessment of MHC II

Adherent splenic macrophages, which demonstrated typical macrophage morphology, were either treated with IFN- (20 U/ml) (R&D Systems) for 24 h, or loaded with OVA overnight and then treated with dispase (1 mg/ml) (Sigma-Aldrich) for 5 min at 37°C and lightly scraped with a rubber policeman. PE-labeled Ab specific for MHC II I-A^b (clone AF6–120.1) (BD Pharmingen) or an isotype-matched control was used to analyze the expression of MHC II. Unlabeled Abs specific for CD80 (clone 1G10) and CD86 (clone GL1), or an isotype control (keyhole limpet hemocyanin/G2a-1-1; Southern Biotechnology Associates) were incubated with APCs for 20 min on ice and then washed twice, followed by incubation with PE-labeled Abs specific for rat IgG2a (clone RG711.30), followed by two washes and fixation in 4% paraformaldehyde.

ELISA and MultiPlex cytokine assay

To determine the level of cytokine secretion, culture supernatants were harvested 72 h poststimulation from 96-well plates after centrifugation at 500 x g for 8 min. The supernatants were assayed by ELISA, according to the manufacturer’s instructions, for IFN-, IL-4 (BD Pharmingen), and IL-10 (R&D Systems). Absorbance was determined at 450 nm using a SoftmaxPro 4.0 plate reader (Molecular Devices), and data were analyzed with accompanying software. Cytokine concentrations were estimated by linear regression relative to the manufacturer’s standard. A Bio-Plex Mouse 18-Plex Assay (Bio-Rad) was used to initially screen samples for differences in IL-2, IL-12 p40, IL-12 p70, IL-4, IL-10, and IFN- cytokine production. Briefly, triplicate wells were pooled and assayed in duplicate. Samples were incubated with Ab-coupled beads for 30 min, and then washed and incubated with biotinylated detection Ab, as indicated, for 30 min. This was followed by washing and incubation with streptavidin-PE for 10 min. Samples were then analyzed on the Bio-Plex suspension array system.

Data analysis

Sigma Stat 3.0 was used to estimate significance of treatment differences. The means and SE were obtained from triplicate wells for each treatment assayed in duplicate. One-way ANOVA was used to analyze data when multiple treatments were compared within a single genotype, and two-way ANOVA was used for treatments on two different genotypes. A Student’s t test was used when comparing single E₂ dose with control within a genotype. Significance was established at p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
CD4⁺ and CD8⁺ T cells express detectable ER mRNA, but not ER mRNA

There are two known intracellular ERs, ER and ER, which are expressed in a tissue-specific manner. Previously, we reported that murine macrophages and CD11c⁺ splenic DCs express ER transcripts, but no detectable ER transcripts (13). Both CD4⁺ and CD8⁺ T cells expressed ER transcripts (Fig. 1, upper panel), but we were unable to detect ER transcripts using RT-PCR. Transcripts for both ERs were readily detected from mRNA extracted from ovaries. To rule out the possibility of alternatively spliced isoforms of ER mRNA, we used exon-specific primer sets spanning all seven exon-intron junctions of the mature ER transcript to detect alternative splice variants of ER (13). These results demonstrate that the ovary (positive control), but not CD4⁺ and CD8⁺ T cells, express ER mRNA (exons 2–3; Fig. 1, lower panel). We were unable to detect any ER transcripts in murine CD4⁺ or CD8⁺ T cells using these seven different primer sets (data not shown).

View larger version (64K): [in this window] [in a new window]   FIGURE 1. Purified CD4+ and CD8+ T cells express mRNA for ER, but not ER. RT-PCR with specific primers for ER (upper panel) and exon-specific primers spanning the seven intron junctions of ER (only exon 2–3 is shown in the lower panel) were used to determine whether CD4+ or CD8+ T cells expressed detectable ER transcripts. None of the other primer sets for ER showed expression in these cell types (data not shown). Mouse ovary serves as a positive control for ER transcript expression. The J774 macrophage cell line served as an additional negative for ER, in addition to H2O. A total of 1.0 ng of total RNA was used in all ER reactions, and 0.2 ng was used in G3PDH control reactions.

Natural hormonal fluctuations in females have been shown to affect ex vivo cytokine secretion of CD4⁺ T cells (18, 21). To minimize this variable, WT mice were ovariectomized 2 wk before obtaining splenic CD4⁺ T cells to control for the effect of prior exposure to endogenous E₂ on ex vivo CD4⁺ T cell cytokine secretion. E₂ and ER have been shown to alter IL-2 production and CD4⁺ T proliferation (19, 34); therefore, supplemental IL-2 was added to all CD4⁺ T cell cultures to eliminate E₂ modulation of IL-2 production as a potential confounding parameter. Purified CD4⁺ T cells were treated with E₂ and stimulated with immobilized anti-CD3/CD28 and IL-2 for 72 h, followed by analysis of cytokine production. Our previous in vivo studies have demonstrated that the effects of E₂ are both dose and duration of exposure dependent, and thus given this precedent, we sought to mimic as closely as possible the in vivo response in our tissue culture model system by pretreating CD4⁺ T cells with E₂ (12). The addition of E₂^–9 M for 16 h prestimulation significantly increased IL-4 secretion (p < 0.05) from CD4⁺ T cells relative to no supplemental E₂ (Fig. 2A). In addition, the E₂-treated CD4⁺ T cell cultures expressed increased amounts of GATA-3 mRNA and decreased T-bet transcripts compared with untreated controls (Fig. 2B). However, there was no significant impact on IFN-, IL-10, or IL-2 secretion (Fig. 2, C–E, respectively). The significant increase in IL-4 production due to E₂ was not due to changes in proliferation of these cells, as E₂ had no effect on CD4⁺ T cell proliferation in our system (data not shown).

View larger version (26K): [in this window] [in a new window]   FIGURE 2. Cytokine production by CD4+ T cells cultured with E2 and anti-CD3/CD28 beads following overnight culture. Purified CD4+ T cells from ovariectomized mice (n = 3–5) were stimulated with biotin-labeled anti-CD3 (10 µg/ml) and anti-CD28 (20 µg/ml) Ab bound to agarose beads in 96-well plates in the presence of 1 ng/ml IL-2. E2 was added to a concentration of 10–9 M (272.4 pg/ml). T cells were treated with E2 in triplicate wells 12–16 h before stimulation at the concentrations shown. Supernatants were harvested 72 h poststimulation and assayed by ELISA or RT-PCR for secreted IL-4 (A), T-bet mRNA (B), IFN- (C), IL-10 (D), or IL-2 (E). Total RNA was extracted from CD4+ T cells, and relative expression of transcripts for GATA-3 and T-bet was measured by real-time PCR, as described in Materials and Methods. Results are shown as ±SEM of at least triplicate pooled cultures assay in duplicate. The results are representative of three independent experiments. Significance was determined using a Student’s t test comparing E2 10–9 M treatments with nontreated controls (*, p < 0.05).

ER expression by CD4⁺ T cells is necessary for E₂-induced IL-4 production

To assess whether the production of IL-4 and IFN- from CD4⁺ T cells ex vivo is dependent upon ER expression, purified splenic CD4⁺ T cells from ER-deficient mice and WT littermates (n = 3–5) were stimulated ex vivo with immobilized anti-CD3/CD28 and different physiologically relevant concentrations (10^–9-10^–11 M) of E₂. IL-4 (Fig. 3, A and B) or IFN- (Fig. 3, C and D) production by ER KO CD4⁺ T cells was not significantly different from WT counterparts when cultures were not treated with E₂. These data suggest that ER deficiency in CD4⁺ T cells does not have a direct effect on secretion of IFN- or IL-4. However, E₂ (10^–9 M) increased secretion of IL-4 by ER-replete CD4⁺ T cells (Fig. 3B) (p < 0.05), which was completely abrogated in ER KO CD4⁺ T cells (Fig. 3A). These results suggest that E₂ acts through ER to modulate the production of IL-4 by CD4⁺ T cells independent of IL-2. We observed a moderate decrease in IFN- secretion in WT CD4⁺ T cells treated with E₂ (10^–9 M). Although this suppression was not significant, the modest decrease in IFN- secretion due to E₂ (Fig. 3D) was also abrogated in ER KO CD4⁺ T cells (Fig. 3C).

View larger version (37K): [in this window] [in a new window]   FIGURE 3. The absence of ER abrogates the effect of E2 treatment on IL-4 production. CD4+ T cells were purified from ER KO mice (n = 3–5) and their WT littermates (n = 3–5), treated with various concentrations of E2 in triplicate wells, and stimulated with anti-CD3 (10 µg/ml) and anti-CD28 (20 µg/ml) Ab bound to agarose beads in 96-well plates with 1 ng/ml IL-2 in 96-well plates, as indicated. Supernatants were harvested 72 h poststimulation and assayed by ELISA for secreted IFN- and IL-4. Comparisons of IFN- (C and D) and IL-4 (A and B) secretion by CD4+ T cells from ER KO and littermate (WT) mice without E2 treatment are shown. Treatment groups receiving no stimulation (avidin-agarose beads only) had below detectable levels of cytokine present. Significance was determined using a Student’s t test (*, p > 0.05). Results are representative of the mean + SEM of at least triplicate pooled cultures assay in duplicate.

Modulation of proliferation and IFN- production by ER-deficient APCs

ER deficiency in CD4⁺ or CD8⁺ T cells did not directly affect the ability of these cells to secrete IFN- during ex vivo culture. In contrast, we have previously shown that ER deficiency resulted in 3- to 4-fold increases of IFN- levels in a chronic M. avium infection model (12). A potential explanation for the variation in ex vivo vs in vivo results may relate to the functional role of ER in APCs. To determine whether ER modulates Ag presentation in APCs, we used OVA peptide-specific OT-II CD4⁺ T cells stimulated with APCs (2:1) from WT and ER KO mice pulsed with OVA and measured proliferation and production of IFN- in the context of Ag presentation. The OT-II CD4⁺ T cells showed a significant increase (40%) in proliferation (p < 0.05) when Ag was presented by ER KO APCs vs APCs that possess ER (Fig. 4A). Moreover, Ag presentation by ER KO APCs yielded a 3-fold increase (p < 0.05) in IFN- production by CD4⁺ T cells (Fig. 4C), but no significant difference in IL-4 production (Fig. 4E). This effect was independent of E₂ or treatment with the ER antagonist ICI 182,780 (data not shown). In contrast, we observed no differences in the ability of APCs from ER KO mice to stimulate proliferation or IFN- and IL-4 production by OT-II CD4⁺ T cells as compared with WT APCs (Fig. 4, B, D, and F, respectively). The lack of ER is thus a major contributing factor to the increased proliferation and IFN- production by OT-II T cells when Ag is presented by ER KO APCs.

View larger version (32K): [in this window] [in a new window]   FIGURE 4. ER deficiency in APCs results in increased proliferative responses and IFN- secretion by CD4+ T cells. APCs were purified from ER-deficient SCID mice (n = 3–5) and their WT littermates (n = 3–5) and incubated with OVA protein (500 µg/ml) overnight. Purified CD4+ T cells from OT-II mice (n = 3–5) were cocultured with either ER KO or WT OVA-loaded APCs at a ratio of 1 APC:2 CD4+ T cells. Controls for proliferation studies were OT-II CD4+ T cells incubated with APC that had not been loaded with OVA. APCs not loaded with OVA cocultured with OT-II CD4+ T cells produced undetectable amounts of IFN- or IL-4. Proliferation of OT-II CD4+ T cells with APC derived from ER (A) or ER (B) donors. IFN- and IL-4 production of OT-II CD4+ T cells with APC derived from ER (C and E, respectively) or ER (D and F, respectively) donors. Results are shown as the mean + SEM of at least triplicate pooled cultures assay in duplicate and are representative of three independent experiments comparing ER KO APCs vs WT APC. ER KO APCs vs WT was performed twice. Statistical significance was assessed with Student’s t test, and *, p < 0.05.

Macrophages are responsible for increased IFN- by OT-II CD4⁺ T cells

To delineate the specific APC affected by ER deficiency, we purified CD11c⁺ DCs and macrophages from ER KO or WT mouse spleens and loaded them with OVA overnight. We then cocultured the OVA-loaded, ER KO, or WT DCs and macrophages with WT CD4⁺ T cells purified from OT-II mice. Ag presentation by macrophages from ER KO mice elicited significant increases in IFN- production (p < 0.05) by CD4⁺ T cells when compared with that elicited by WT macrophages (Fig. 5B). In contrast, no differences were observed in the ability of ER KO or WT CD11c⁺ DCs to elicit IFN- production from CD4⁺ T cells (Fig. 5A). In addition, stimulation of purified CD4⁺ T cells with anti-CD3 Ab alone (10 µg/ml) for 72 h resulted in negligible IFN- production (<50 pg/ml) (data not shown), suggesting a relatively naive population.

View larger version (37K): [in this window] [in a new window]   FIGURE 5. ER KO splenic macrophages, but not splenic CD11c+ DCs mediate increased proliferative and IFN- responses by OT-II CD4 T cells in response to OVA. Purified CD11c+ DCs were incubated overnight with OVA (500 µg/ml) and cocultured with OT-II CD4+ T cells at T cell:DC ratios of 2:1 or 10:1 (A). Splenic macrophages were incubated overnight with OVA (500 µg/ml) and cocultured with OT-II CD4+ T cells at T cell:macrophage ratios of 1:1, 2:1, 5:1, and 10:1 (B). The results presented are representative of two independent experiments. The results are reported as the mean + SEM of duplicate assays with triplicate wells in each assay. Statistical significance was assessed with a Student’s t test (*, p < 0.05).

MHC II expression is not altered by ER deficiency in macrophages after OVA uptake or IFN- stimulation

We next sought to determine potential mechanisms by which ER KO splenic macrophages increased OT-II CD4⁺ T cell proliferation and IFN- production. Because E₂ had been shown to alter MHC-II expression on primary macrophages, we used flow cytometric analysis to determine whether there were differences in MHC-II expression between ER KO and WT splenic macrophages. We observed no significant differences in levels of MHC-II expression between ER KO and WT splenic macrophages after OVA uptake (median fluorescence-14 vs 14, respectively) or IFN- stimulation (median fluorescence-2017 vs 2288, respectively) (Fig. 6). CD80/86 expression was detectable after OVA uptake, although there were no significant differences between ER KO and WT phenotypes (data not shown). In addition, we assayed 3-day supernatants with a mouse MultiPlex cytokine kit and found no significant differences in Th1/Th2-polarizing cytokines, such as IL-12p70 or IL-4 (Table I).

View larger version (56K): [in this window] [in a new window]   FIGURE 6. Levels of MHC II expression are not altered in ER KO splenic macrophages. Splenic macrophages obtained from ER KO mice (A; median = 14) or WT mice (B; median = 14) were loaded with OVA overnight and then analyzed for levels of MHC II expression. Additionally, ER KO splenic macrophages (C; median = 2017) and WT splenic macrophages (D; median = 2288) were treated with IFN- for 24 h and also analyzed for MHC II expression. Isotype control staining is shown in black, with MHC class II staining shown in gray. The results are representative of two independent experiments.

View this table: [in this window] [in a new window]   Table I. Cytokine production of 3-day OVA-loaded ER KO and WT splenic macrophage/OT-II CD4 T cell coculturesa

	Discussion

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We established that murine CD4⁺ T cells express detectable transcripts for ER; however, we were unable to detect ER transcripts using seven different exon-specific primer sets. It appears there are species-specific differences in the expression of ER by T cells, because several reports indicate that ER is expressed in human T cells, although relative expression of ER by human CD4⁺ T cells was reduced when compared with ER expression (23, 25).

To determine the functional consequences of ER expression in T cells, we measured cytokine secretion from CD4⁺ or CD8⁺ T cells. In agreement with in vivo and ex vivo experiments in experimental autoimmune encephalomyelitis or OVA animal models indicating that ER deficiency in T cells cannot account for disparities in immune responses (38, 39), we saw no differences in cytokine secretion between untreated ER KO and WT CD4⁺ or CD8⁺ T cells after stimulation with anti-CD3/CD28. However, we observed a significant E₂ induction of IL-4 secretion and increased GATA-3 expression by purified CD4⁺ T cells stimulated via CD3/CD28. These effects were abrogated in ER KO CD4⁺ T cells, suggesting that E₂ regulates IL-4 secretion by CD4⁺ T cells through ER in a classical ligand-dependent manner. It has been demonstrated that a significant correlation exists between the expression of ER and GATA-3 in breast cancer cell lines (40, 41). Although this may serve as a potential explanation for the abrogation of E₂-induced IL-4 secretion in ER KO CD4⁺ T cells, the specific mechanisms by which E₂ increases IL-4 production by WT CD4⁺ T cell are yet to be determined. E₂ bound to ER has been shown to alter activities of key immune transcriptional activators, such as AP-1 and NF-B (42, 43). ER has been demonstrated to undergo phosphorylation and activation by signaling pathways important in T cell signaling, such as the PI3K/protein kinase B (PKB/Akt) pathway, as well as MAPK pathways (44, 45). The specific transcriptional factors other than GATA-3, which serve to increase IL-4 in our system, are yet to be determined.

We have previously demonstrated that ER KO mice had significantly increased IFN- production compared with WT littermates in an in vivo M. avium infection model (12), and somewhat paradoxically we detected no significant differences in IFN- production from purified ER KO CD4⁺ or CD8⁺ T cells compared with littermates. To reconcile these findings, we postulated that ER deficiency may affect APC signaling to CD4⁺ or CD8⁺ T cells. In our system, OVA-specific OT-II CD4⁺ T cells cultured ex vivo with APCs from ER KO mice that had been pulsed with OVA or specific peptide exhibited significantly increased proliferation and IFN- production (2- to 3-fold) as compared with OT-II CD4⁺ T cells cultured with WT APCs. Furthermore, Ag presented by ER KO splenic macrophages, but not ER KO splenic CDllc⁺ DCs, increased IFN- production by OT-II CD4⁺ T cells. Thus, it appears that ER deficiency in splenic macrophages enhances Ag presentation and/or costimulation. Paharkova-Vatchkova et al. (46) demonstrated that E₂ acting through ER plays an important role in bone marrow-derived CD11c⁺-CD11b^int DC differentiation. In contrast, we observed no significant differences in total CD11c⁺ numbers between WT and ER KO mice (data not shown), or in their ability to stimulate OVA-specific CD4⁺ T cells. Therefore, we did not further purify DC subsets to stimulate OVA-specific CD4⁺ T cells. However, ER deficiency may affect the ability of certain DC subsets to present Ag. In addition, E₂ signaling has been shown to affect MHC II expression in vivo and in vitro. In vivo, the removal of E₂ increased MHC II expression on primary macrophages in response to IFN- (27). In vitro, Adamski et al. (47, 48) demonstrated suppressive effects of E₂ on MHC II expression in an intrahepatic biliary epithelial brain endothelial cell line and in IFN--stimulated RAW macrophage cells. These data suggest that ER modulates MHC II expression. However, we were unable to detect differences in MHC II expression measured by flow cytometry between ER KO or WT splenic macrophages. We also observed no significant differences in polarizing cytokines (Table I), such as IL-12 or IL-4 in these cultures. Thus, the mechanism(s) by which ER-deficient macrophages increase proliferation and IFN- production is still under investigation. Interestingly, we detected no significant effect of E₂ or ICI 182,780 treatment on the ability of either WT or ER KO APCs to stimulate CD4⁺ T cells as measured by proliferation or IFN- production. Our results combined with previous studies suggest that the presence of nonliganded ER may have suppressive effects on immune function (12, 13). The actions of nonliganded ER have been demonstrated to affect many different transcriptional systems through direct binding of DNA after phosphorylation (49). It has also been postulated that certain phosphorylation events of nonliganded ERs may alter the activity of coactivators that bind ER (50). In addition, nonliganded ER has been reported to suppress NF-B activity in the transfected osteoblastic U2-OS cell line (51). Although our system does not fully establish that a nonliganded form of ER in splenic macrophages is responsible for these effects on CD4⁺ T cells, it does suggest a novel and potentially important signaling pathway that warrants further investigation.

In contrast to our findings that OVA-loaded ER KO macrophages increased IFN- production by OT-II CD4⁺ T cells independent of E₂, Maret et al. (16) reported that in vivo administration of E₂ increased production of IFN- from CD4⁺ T cells and that ER expression in hemopoietic cells was necessary for this increased IFN- production by CD4⁺ T cells taken from lymph node. These differences may be explained by the assay systems used in the respective studies. We administered E₂ in vitro to ER KO or WT littermate OVA-loaded APCs ex vivo to stimulate OVA-specific OT-II CD4⁺ T cells, whereas Maret et al. administered E₂ in vivo to WT or chimeric B6 mice that had been immunized with OVA to increase OVA-specific CD4⁺ T cells in draining lymph nodes, and then lymph node cells were pulsed with OVA. The in vivo effects of E₂ on immune function are complex and may involve both nonhemopoietic and hemopoietic cell populations.

In summary, our data demonstrate that, in vitro, E₂ has the potential to directly modulate IL-4 production by CD4⁺ T cells independent of APC function. The increase in IL-4 production resulting from the addition of E₂ is abrogated in ER KO CD4⁺ T cells, which suggests that E₂ acts through ER in a ligand-dependent manner to modulate IL-4 production. Although we observed no differences in IFN- production between CD4⁺ T cells isolated from female ER KO and their WT littermates in the absence of E₂, we did observe significant differences in OT-II CD4⁺ T cell proliferation and IFN- production when ER KO macrophages were used to present Ag compared with WT macrophages. These data suggest that ER affects Ag-presenting pathways in splenic macrophages independently of E₂ binding.

	Acknowledgments

 
We sincerely thank Dr. Charles Brown and Leslie Newton in helping to coordinate mouse experiments, as well as Ngozi Duru and Marguerite Maurer for their care and handling of animals. We also thank and acknowledge Dr. Huiping Guo for her real-time PCR expertise, and Dr. Victor Reyes and Giovanni Suarez for the analysis of the MultiPlex cytokine assays.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by U.S. Public Health Services Grant NIEHS PO1 ES10535.

² Address correspondence and reprint requests to Dr. D. Mark Estes, Department of Pediatrics, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0372. E-mail address: dmestes{at}utmb.edu

³ Abbreviations used in this paper: E₂, 17-estradiol; ER, estrogen receptor; C_T, threshold cycle; KO, knockout; WT, wild type.

Received for publication April 28, 2005. Accepted for publication August 17, 2005.

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