Estrogen receptor profiles in human peripheral blood lymphocytes

Immunology Letters 132 (2010) 79–85

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Estrogen receptor profiles in human peripheral blood lymphocytes Marina Pierdominici a , Angela Maselli b , Tania Colasanti a,c , Anna Maria Giammarioli b , Federica Delunardo a , Davide Vacirca a , Massimo Sanchez a , Antonello Giovannetti d , Walter Malorni b,c,∗,1 , Elena Ortona a,1 a

Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Rome, Italy San Raffaele Institute Sulmona, L’Aquila, Italy d Department of Clinical Medicine, Division of Clinical Immunology, “Sapienza” University, Rome, Italy b c

a r t i c l e

i n f o

Article history: Received 23 February 2010 Received in revised form 28 May 2010 Accepted 3 June 2010 Available online 11 June 2010 Keywords: 17␤-Estradiol Estrogen receptors Human peripheral lymphocytes

a b s t r a c t Estrogens are well-known regulators of the immune responses. Most of their effects are mediated by two receptors: estrogen receptor (ER)␣ and ER␤. Up to date the presence of intracellular ER in human immune cells represents a controversial issue, while their surface membrane expression has scarcely been explored. In this study we investigated the intracellular and cell surface expression of ER␣ and ER␤ in human peripheral blood lymphocytes (PBL) by flow and static cytometry as well as by Western Blot. To this aim we used five different commercial antibodies recognizing distinct ER epitopes. We observed that CD4+ and CD8+ T lymphocytes, B lymphocytes and NK cells contain intracellular ER␣ and ER␤, being the ER␣46 isoform the most represented ER. However, significant differences could be observed among the antibodies studied in terms of immunoreactivity and specificity. Importantly, we also found a cell surface expression of ER␣46 isoform. We also observed that a membrane-impermeant form of E2 induced a rapid phosphorylation of extracellular signal-regulated kinase (ERK), a significant proliferation of T lymphocytes, and IFN-␥ production by NK cells, thus suggesting the expression of a functional mER␣. In conclusion our data could provide new insights as concerns the estrogen-related mechanisms of immune system modulation. They also suggest the need for a reappraisal of the experimental conditions for the characterization of the ER expression. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Estrogens play important roles in a number of physiological processes and pathologic conditions [1,2]. There is ample evidence that 17␤-estradiol (E2) directly modulates the development and function of immune cells, although the mechanism by which this might occur is not well understood [3–5]. The primary mechanism of E2 action is mediated by transcriptional activity of the intracellular estrogen receptors (ER), ER␣ and ER␤ [6]. Regarding ER␣, two isoforms have been reported, i.e. the full-length 66 kDa isoform (ER␣66) and the short isoform of 46 kDa (ER␣46) lacking of the N-terminal region. Increasing evidence indicates the existence of membrane-localized ER (mER), structurally similar to their intracellular counterparts, which initiate rapid events leading to the activation of various protein kinase cascades [7]. Particularly, E2-induced effects involve a concurrent activation of two MAPK sig-

naling pathways, ERK pathway by ER␣, and p38 pathway by ER␤ [1,8]. Although the mRNA expression of ER␣ and ER␤ has been demonstrated in human peripheral blood lymphocytes (PBL) [9–11], contrasting results have been reported regarding the intracellular presence of these receptors. This is probably due to the different analytical procedures (i.e. differences in antibody specificity) and to the criteria for donor selection (i.e. gender, age, menstrual cycle phases) [12–17]. Conversely, the presence and the functional significance of mER have scarcely been explored [18]. In this study we analyzed the expression of both intracellular and cell surface ER␣ and ER␤ in PBL from male healthy donors by different methodological approaches. We also investigated the ability of the mER to induce a rapid intracellular signaling and to modulate immunological functions. 2. Materials and methods

∗ Corresponding author at: Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Rome, Italy. Tel.: +39 0649902905; fax: +39 0649903691. E-mail addresses: [email protected], [email protected] (W. Malorni). 1 These authors contribute equally to this work. 0165-2478/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2010.06.003

2.1. Cell cultures and functional studies Blood samples were obtained from 10 male healthy donors between the ages of 24 and 70 years with informed consent. Periph-

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eral blood mononuclear cells were isolated by Ficoll-Hypaque density-gradient centrifugation (Lympholyte-H; Cedarlane Laboratories, Hornby, Ontario, Canada) and depleted of monocytes by adherence to plastic. PBL were cultured in RPMI-1640 medium without phenol red (GIBCO BRL, Grand Island, NY) supplemented with 10% charcoal-stripped fetal bovine serum (Euroclone, Pero, Milan, Italy), 2 mM glutamine (Sigma, St. Louis, MO), 50 ␮g/ml gentamycin (Sigma), and treated with membrane-impermeant E2albumin construct (E2–BSA, molar ratio E2:BSA = 30:1; Sigma) at concentration of 1 nM, 10 nM, 100 nM and 1 ␮M for 15 min. To quantify lymphocyte proliferation, PBL were labeled with 10 ␮M carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes, Eugene, OR) [19] according to the manufacturer’s instructions and then seeded in 24-well or 96-well culture plates (2.0 × 106 cells/ml) in the presence of different stimuli and subjected to different treatments. Cells were incubated for 1 h with 1 ␮M E2–BSA or with an equal amount of BSA alone, washed and stimulated with optimal and suboptimal concentrations of: (i) precoated anti-CD3 monoclonal antibody (mAb) (4 ␮g/ml or 0.4 ␮g/ml; OKT3, Immunotech, Marseilles, France) for 3 days and (ii) CpG oligodeoxynucleotide (ODN)-2006 (2.5 ␮g/ml or 0.25 ␮g/ml, MWG Biotech, M-Medical, Milan, Italy), interleukin-2 (IL-2) (50 UI/ml, PeproTech Inc., Rocky Hill, NJ) and anti-Ig mAb (2 ␮g/ml, Jackson ImmunoResearch Laboratories, Suffolk, United Kingdom) for 5 days. Each assay included untreated control cells. After 3 or 5 days of culture, cells were washed and stained with appropriate mAb (see below). To evaluate IFN-␥-producing CD3− CD56+ NK cells, PBL were stimulated for 16 h with optimal and suboptimal concentrations of ionomycin (1 ␮g/ml or 0.1 ␮g/ml, Sigma) and phorbol myristate acetate (PMA) (25 ng/ml or 2.5 ng/ml, Sigma) in the presence of 10 ␮g/ml brefeldin A to inhibit cytokine secretion. Cells were then washed and stained with appropriate mAb (see below). Optimal and suboptimal concentrations of the different cell activators were determined in preliminary dose–effect experiments. 2.2. Flow cytometry Cell surface and intracellular phenotyping of freshly isolated PBL was performed using direct and indirect immunofluorescence assays as described before with minor modifications [20]. Staining of ER required extensive blocking with the use of FcR blocking reagent (Miltenyi Biotec, Bergisch-Gladbach, Germany) for 10 min in the refrigerator or appropriate serum for 30 min on ice. After blocking, for cell-surface phenotyping PBL were stained for 30 min at 4 ◦ C with the following polyclonal antibodies (pAb) or mAb: rabbit anti-human ER␣ MC-20 pAb directed to the C-terminus of the ER␣; mouse anti-human ER␣ F-10 and D-12 mAb directed to the C-terminus and N-terminus of the ER␣, respectively; goat anti-human ER␤ L-20 pAb and mouse anti-human ER␤ 1531 mAb, both directed to the C-terminus of the ER␤. All these antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, and 1 ␮g per sample (1 × 106 cells) was used. Equal amounts of appropriate isotype controls (Santa Cruz) were used as negative controls. The primary antibody was visualized by fluorescein isothiocyanate (FITC)-conjugated or phycoerythrin (PE)-conjugated F(ab )2 fragment secondary antibody (Abcam, Cambridge, UK). Allophycocyanin (APC)-conjugated antiCD3, APC- or PE-conjugated anti-CD4, peridinin chlorophyll protein (PerCP)-conjugated anti-CD8, PE-conjugated anti-CD56 and PerCPconjugated anti-CD19 (all from BD Immunocytometry Systems, San Jose, CA) were also used to identify ER␣ F-10 and ER␤ 1531 expression in lymphocyte subsets. In this case, directly conjugated mAb were added after blocking the free binding site of goat anti-mouse with normal mouse serum. For intracellular phenotyping, cells were fixed with 4% paraformaldehyde (PFA) by incubation for 5 min at room temperature (RT), permeabilized with FACS permeabiliz-

ing solution (BD Immunocytometry Systems) for 10 min, washed and stained as described above. Intracellular IFN-␥ expression in CD3− CD56+ NK cells was detected using FITC-conjugated anti-IFN␥, APC-conjugated anti-CD3, and PE-conjugated anti-CD56 mAb (BD Immunocytometry Systems). Data were expressed as the percentage of IFN-␥ within the NK cell population considered as 100%. The amounts of cell proliferation were quantified by monitoring the sequential loss of green fluorescence intensity of the CFSE-labeled cells in lymphocyte subsets using PE-conjugated antiCD3, APC-conjugated anti-CD4, PerCP-conjugated anti-CD8, and APC-conjugated anti-CD19 mAb (all from BD Immunocytometry Systems). Dead cells were excluded with Sytox Blue (Invitrogen, Carlsbad, CA) staining. Acquisition was performed on FACSCalibur and FACSAria flow cytometers (BD Immunocytometry Systems) and 50,000 events per sample were run. Data were analyzed using the Cell Quest Pro (BD Immunocytometry Systems) and the FlowJo version 7.2.5 (Tree Star, Ashland, OR) softwares. Untreated cell cultures at day 3 or 5 were considered as the starting point for the proliferative profile analyses. A cell division index (DI), i.e. the average number of cell divisions of the responding cells, was determined for each stimulated sample through comparison with results from an unstimulated sample [21]. The resolution index (RI) between two fluorescence distributions was calculated according to this general formula [22]: RI =



Xi − Xo SDi 2 + SDo 2

where Xi and SDi = mean intensity and standard deviation of the population with the higher mean intensity (specific antibody), respectively; Xo and SDo = mean intensity and standard deviation of the population with the lesser mean intensity (isotype antibody), respectively. Purification of lymphocyte subsets (i.e. CD3+ CD4+ , CD3+ CD8+ , CD19+ , and CD3− CD56+ lymphocytes) was performed by immunomagnetic-based pre-enrichment using CD3 microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany), followed by multicolor flow cytometric sorting using a FACSAria flow cytometer (BD Immunocytometry Systems). For cell sorting, enriched CD3+ lymphocytes were stained with FITC-conjugated anti-CD3, APCconjugated anti-CD4 and PerCP-conjugated anti-CD8 mAb whereas enriched CD3− lymphocytes were stained with FITC-conjugated anti-CD3, PE-conjugated anti-CD56 and PerCP-conjugated antiCD19 mAb. Purity of sorted cells, assessed by flow cytometer, was ≥99%.

2.3. Static cytometry To visualize intracellular distribution of ER static cytometry analysis was also performed. Briefly, cells were fixed with 4% PFA for 30 min at RT and then permeabilized with 0.5% Triton X-100 in PBS for 5 min at RT. Extensive blocking was performed with human serum before staining. Cells were stained with the pAb or mAb described above, for 1 h at 37 ◦ C and then incubated for 30 min at 37 ◦ C with appropriate Alexa Fluor 488-coniugated secondary antibodies (Molecular Probes). Cells were then collected by centrifugation, attached to glass coverslips, mounted with glycerol/PBS (1/1), and observed by intensified video microscopy using a Nikon Microphot fluorescence microscope (Nikon, Tokyo, Japan) equipped with a color chilled 3CCD camera (Zeiss, Germany). Normalization and background subtraction were performed for each captured image. Figures were obtained by the OPTILAB (Graftek, France) software for image analysis.

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2.4. Protein membrane purification Cell surface membrane proteins were purified from PBL using the Pierce Cell Surface Protein Isolation Kit, according to the manufacturer’s instructions with slight modifications (Pierce, Rockford, IL). Briefly, 1 × 107 cells were incubated in 1 ml biotin solution. After the biotinylation step, we washed the cells twice with Tris Buffered Saline. The cells were subjected to sonication and the biotinylated proteins were incubated with Immobilized NeutrAvidin Gel (Pierce). After extensive lavage of the gel (nine times), the proteins were eluted according to the protocol. 2.5. SDS-PAGE and Western Blot Cells were lysed in RIPA buffer (100 mM Tris–HCL, pH 8, 150 mM NaCl, 1% Triton X-100, 1 mM MgCl) in presence of complete protease–inhibitor mixture (Sigma). Protein content was determined by the Bradford assay (BioRad, Richmond, CA). Cell lysate, cell surface proteins and recombinant ER␣ and ER␤ (Sigma) were run in 10% SDS-PAGE. Cell protein extract (60 ␮g) and recombinant proteins (1 ␮g) were loaded in each lane. After protein transfer, nitrocellulose membranes were incubated with specific antisera diluted 1:50 in phosphate buffer (Santa Cruz). Peroxidase-conjugated goat anti-mouse IgG, goat anti-rabbit IgG and rabbit anti-goat IgG (Biorad) were used as second antibodies and the reactions were developed using the SuperSignal West Pico Chemiluminescent Substrate (Pierce). To ensure the presence of equal amounts of protein, the membranes were re-probed with a mouse anti-␤-tubulin mAb (Amersham, Gent, Belgium). The levels of phosphorylated ERK (pERK) were analyzed by Western Blot analysis essentially as previously described [23]. PBL lysate (25 ␮g) was loaded in SDS-PAGE and Western Blot was performed using anti-pERK-1/2 mAb (Cell Signaling Technology, Beverly, MA). Quantification of protein expression was performed by densitometry analysis of the autoradiograms (GS-700 Imaging Densitometer, BioRad). 2.6. Statistical analysis Student’s t-test was used to establish significant differences between samples. Results were considered statistically significant when p < 0.05. Flow cytometry data were statistically evaluated using the Kolmogorov–Smirnov test [24] according to the Cell Quest Pro software guide (BD Immunocytometry Systems) and a D/s(n) ratio ≥ 15 was accepted as significant in the experimental condition used. 3. Results 3.1. Intracellular expression of ER in total PBL First, we examined by flow and static cytometry and Western Blot the intracellular expression of ER in PBL from healthy donors using commercially available antibodies recognizing different ER epitopes. Male subjects were selected in order to exclude differences of E2 concentration in serum, which may influence ER expression in the studied cell populations. Regarding flow cytometry analysis, as shown in Fig. 1A, positive ER␣ signals were observed using the F-10 and MC-20 antibodies, directed to the C-terminus of this receptor, and the D-12 antibody directed to the N-terminus of ER␣. Similarly, positive ER␤ signals were detected either using the 1531 or the L-20 antibodies, directed to distinct epitopes of the C-terminus of ER␤. All the antibodies under study showed a unimodal ER distribution with overlapping curves (except for MC-20), as result of isotype or specific staining, and positivity for ER expression was valuated using

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Table 1 Immunoreactivity and specificity of different anti-ER␣ and -ER␤ antibodies. Antibody

Ligand

Flow cytometry (RI)

F-10 MC-20 D-12 1531 L-20

ER␣ ER␣ ER␣ ER␤ ER␤

1.5 28 1.2 2.5 0.7

± ± ± ± ±

0.2 1 0.3 0.1 0.1

Western Blot (specificity) 46 kDa (strong); 66 kDa (weak) Multiple bands Multiple bands 56 kDa Multiple bands

RI, resolution index.

the Kolmogorov–Smirnov analysis. RI for all the antibodies under study were shown in Table 1. Regarding anti-ER␣ antibodies note that although the MC-20 showed a good RI, a high level of background was detected with this antibody and an extensive blocking was required using 60% human serum in PBS (data not shown). Immunofluorescence analysis confirmed the expression of ER␣ and ER␤ in total PBL (Fig. 1B). However, a more marked positivity of ER␣ and ER␤ signals was detected with MC-20 and L-20 pAb in comparison with the other antibodies tested. The reactivity of the above mentioned antibodies specific to ER␣ and ER␤ was tested in Western Blot using PBL lysate as antigen under carefully controlled conditions to eliminate proteolytic cleavage [25] (Fig. 1C). The F-10 mAb showed a strong band at 46 kDa and a very weak band at 66 kDa whereas the MC-20 pAb revealed a series of bands of different molecular weight. The D-12 mAb did not recognize the truncated ER␣46 isoform lacking of the N-terminal region, but reacted with ER␣66 isoform. Non-specific bands at higher and lower molecular weights were also detected with this antibody. All the three commercial antibodies strongly recognized the 66 kDa recombinant ER␣. Analyzing the expression of ER␤ in PBL, we observed a specific band of 56 kDa using the 1531 mAb, whereas the L-20 pAb revealed a series of bands at different molecular weight. Both antibodies reacted with the recombinant ER␤. In all cases, the immunoenzymatic reaction without the incubation with the primary antibody resulted completely negative excluding a non-specific binding of secondary antibodies. The multiple bands recognized by the MC-20 and L-20 pAb could be due to the binding with non-selective proteins. These results are summarized in Table 1 where antibody immunoreactivity (flow cytometry) and specificity (Western Blot) were shown. 3.2. Intracellular expression of ER in lymphocyte subsets On the basis of the above results, we decided to use the F-10 and 1531 mAb to analyze the intracellular expression of ER␣ and ER␤ in lymphocyte subsets, namely CD4+ and CD8+ T lymphocytes, CD19+ B lymphocytes and CD3− CD56+ NK cells. As shown by flow (Fig. 2A) and static (Fig. 2B) cytometry analyses, positive ER␣ signals were detected in all lymphocyte subsets without significant differences among them; similarly, positive ER␤ signals were observed in all studied cell populations. Regarding ER␣, we detected by Western Blot a very strong signal for the ER␣46 isoform in all lymphocyte subsets, and no signal for the ER␣66 isoform, with the exception of CD19+ B lymphocytes in which a weak band was detected (Fig. 2C). ER␤ was expressed in all the lymphocyte subsets although our data suggest a lower expression level of this receptor with respect to ER␣46. 3.3. Cell surface expression and function of ER Cytometry analysis showed that both mER␣ and mER␤ were not detectable at the surface of PBL (data not shown). Membrane expression of ER␣ and ER␤ was further analyzed by Western Blot after purification of proteins expressed on the PBL surface (Fig. 3A).

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Fig. 1. Intracellular ER expression in total PBL by flow and static cytometry analyses and Western Blot. (A) Representative flow cytometry histogram plots show the fluorescence intensity of F-10, MC-20 and D-12 antibodies for ER␣ (upper panels) and 1531 and L-20 antibodies for ER␤ (bottom panels) compared with isotype controls. Isotype control staining is represented by the dotted line and anti-ER␣- and anti-ER␤-labeled cells are represented by the solid line. Statistical differences between the peaks of cells were evaluated by the Kolmogorov–Smirnov test (a D/s(n) ratio ≥ 15 was accepted as significant). Values of the respective mean fluorescence intensity (MFI) fold increase are also reported (fluorescence increase is calculated with respect to isotype control). The mean ± SD from 10 independent experiments is shown. (B) Immunofluorescence images for ER␣ (upper panels) and for ER␤ (bottom panels). The bright field is shown on the right of every frame. The micrographs were acquired using intensified video microscopy technology obtained with a CCD camera (60× objective), and then processed for background using image analysis software (OPTILAB, Graftek, France). Final magnification 2200×. Data from one representative experiment out of 10 are shown. (C) Western Blot analysis of PBL lysates (60 ␮g/lane) for ER␣ (upper left panel) and for ER␤ (upper right panel). Bottom panels represent the reactivity of the commercial antibodies with recombinant ER␣ (left) and recombinant ER␤ (right). Data from one representative experiment out of 10 are shown.

A strong band at 46 kDa, detected by F-10 mAb, revealed the expression of mER␣46 isoform, whereas 1531 mAb failed to detect mER␤ expression by this approach. To provide evidence of a signaling function for the mER␣ expressed on PBL, we determined the effect of the membrane-impermeant form of E2 (E2–BSA) on ERK activation by Western Blot. Both physiological (1 nM, 10 nM, and 100 nM)

and pharmacological (1 ␮M) concentrations of E2 were used in a time not compatible with genomic actions (15 min). E2–BSA induced ERK phosphorylation in a dose dependent manner (Fig. 3B and C). In a second step, we tested the effect of E2–BSA (1 ␮M) on immunological functions, i.e. anti-CD3 stimulation of T cells, anti-

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Fig. 2. Intracellular ER expression in peripheral lymphocyte subsets (CD3+ CD4+ and CD3+ CD8+ T lymphocytes, CD19+ B lymphocytes and CD3− CD56+ NK cells) by flow and static cytometry analyses and Western Blot using F-10 and 1531 mAb for ER␣ and ER␤ detection, respectively. (A) Representative flow cytometry histogram plots show the fluorescence intensity of anti-ER␣ (upper panels) and anti-ER␤ (bottom panels) mAb compared with isotype controls. Isotype control staining is represented by the dotted line and anti-ER␣- and anti-ER␤-labeled cells are represented by the solid line. Statistical differences between the peaks of cells were evaluated by the Kolmogorov–Smirnov test (a D/s(n) ratio ≥ 15 was accepted as significant). Values of the respective mean fluorescence intensity (MFI) fold increase are also reported (fluorescence increase is calculated with respect to isotype control). The mean ± SD from 10 independent experiments is shown. (B) Immunofluorescence images of intracellular expression of ER␣ (upper panels) and ER␤ (bottom panels). ER␣ and ER␤ were diffusely distributed in all lymphocyte subsets. (C) Western Blot analysis of lymphocyte subsets (60 ␮g/lane) for ER␣ and ER␤. To ensure the presence of equal amounts of protein, the membranes were re-probed with a mouse anti-␤-tubulin mAb. Data from one representative experiment out of five are shown.

Ig and CpG stimulation of B cells and IFN-␥ production by NK cells upon PMA and ionomycin activation. In order to evaluate lymphocyte proliferation, PBL were stained with CSFE, a cytoplasmic dye whose fluorescence intensity decreases as it is partitioned among daughter cells, and cultured with appropriate stimuli or left untreated. E2–BSA significantly increased CD4+ and CD8+ T lym-

phocyte proliferation in response to a suboptimal concentration of anti-CD3 mAb (Table 2) whereas no significant effect was observed on anti-Ig and CpG stimulation of B cells (data not shown). Additionally, E2–BSA significantly increased IFN-␥ production by CD56bright NK cells activated with suboptimal concentrations of PMA and ionomycin (Table 2). E2–BSA did not exert any effect on lymphocyte

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Fig. 3. Analysis of cell surface ER expression and functionality in PBL. (A) Purified cell membrane proteins analyzed by Western Blot with F-10 mAb for ER␣ and 1531 mAb for ER-␤. (B) Western Blot analysis of pERK-1/2 in untreated and E2–BSA-treated PBL. E2–BSA was added to cells at different concentrations (1 nM, 10 nM, 100 nM and 1 ␮M) for 15 min. To ensure the presence of equal amounts of protein, the membranes were re-probed with a mouse anti-␤-tubulin mAb. Data from one representative experiment out of five are shown. (C) Histograms show the increase of the ratio ERK/␤-tubulin after exposure to E2–BSA. Data are expressed as mean ± SD from five experiments. *p < 0.05 as compared to untreated cells.

proliferation or cytokine production when optimal concentrations of the different stimuli were used, possibly because a plateau level of cell activation was reached (data not shown). 4. Discussion The influence of estrogens on immune responses is well documented [3–5]. Many of their effects are likely mediated through the interaction with ER, thus the analysis of the ER␣ and ER␤ expression in PBL subsets may provide a useful tool in understanding the responsiveness of these cells to estrogens. In this study, to define the distribution of ER in freshly isolated PBL, we evaluated the intracellular and cell surface expression of ER␣ and ER␤, analyzing five commercial antibodies recognizing different epitopes of ER by distinct methodological approaches. Our data showed intracellular expression of both ER␣ and ER␤ in all lymphocyte subsets, including peripheral NK cells for which no data were available up to date. The ER␣46 isoform appeared to be the most represented ER in lymphocytes. On the contrary, Shim et al. [15] identified ER␤, but not ER␣, in human PBL. The different anti-ER␣ antibodies used in this study might account for these discordant data. Particularly, the use of antibodies specific for the N-terminus of ER␣ might fail to reveal ER because of the weak expression of the ER␣66 isoform. Regarding cell surface expression of ER on human lymphocytes, previously reported data, obtained using estradiol covalently bound to BSA-FITC, indicated that an estrogen binding protein exists on the plasma membrane of human lymphoblastoid B cells [18]. In this study, using epitope-binding technologies, we demonstrated for the first time the cell surface expression of a functionally active ER␣46 isoform on lymphocytes, supporting the idea that E2 level fluctuations may be associated with a prompt PBL activation. We observed a different effect of E2–BSA on lymphocyte functions, being T and NK cells apparently more susceptible to its Table 2 Effect of E2–BSA on immunological functions.

DI CD4+ T lymphocytes DI CD8+ T lymphocytes IFN-␥-expressing CD56bright NK cells (%)

BSA

E2–BSA

p-Value

0.46 ± 0.11 0.56 ± 0.11 19 ± 2

1.8 ± 0.15 2.0 ± 0.10 32 ± 3