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(The FASEB Journal. 1999;13:793-803.)
© 1999 FASEB

Research Communications

Estrogen and progesterone induction of survival of monoblastoid cells undergoing TNF-{alpha}-induced apoptosis

ELISABETTA VEGETO*, GIUSEPPE POLLIO*, CARLO PELLICCIARI{dagger} and ADRIANA MAGGI*1

* Molecular Pharmacology Lab, University of Milan, 20133-Milan, Italy; and
{dagger} Department Animal Biology and CNR Center for Study on Histochemistry, University of Pavia, 27100-Pavia, Italy

1Correspondence: MPL, Institute of Pharmacological Sciences, via Balzaretti, 9 Milan-20133 Italy. E-mail: adriana.maggi{at}unimi.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Induction of apoptosis of mononucleated cells is a physiological process for regulating the intensity of the immune response. The female steroid hormones estrogen (E2) and progesterone (Prog) are known to modulate the reactivity of the immune system; recently it has been demonstrated that they can regulate induction of apoptosis of endothelial cells and osteoblasts. TNF-{alpha}-mediated induction of apoptosis has been well characterized in myeloid cells. We investigated whether E2 and Prog could interfere with TNF-{alpha}-induced apoptosis of the monoblastoid U937 cell line. Treatment with E2 or Prog increased survival and prevented apoptosis induced by TNF-{alpha} in both undifferentiated and macrophage-like PMA-differentiated U937 cells, as assessed by trypan blue exclusion cell counting, thymidine incorporation, AnnexinV labeling, followed by flow cytometry and DNA fragmentation studies. This effect can be associated with the activation of specific hormone receptors, since we observed the expression of the estrogen receptor {alpha} (ER-{alpha}), ER-ß, and progesterone receptor (PR) mRNAs; the ER-{alpha} protein expression was confirmed by immunocytochemical analysis. In addition, hormone-mediated survival against apoptosis was concentration dependent, reaching the half-maximal effect at 10 nM and blocked by the ER antagonist ICI 182,780 in undifferentiated cells, further supporting a receptor-mediated mechanism of cell survival. Other steroid receptor drugs such as Raloxifene, RU486, or the ICI 182,780 in PMA-differentiated cells displayed agonist activity by preventing TNF-{alpha}-induced apoptosis as efficiently as the hormones alone, providing further evidence to the notion that steroid receptor drugs may manifest agonist or antagonist activities depending on the cellular context in which they are studied. Treatment with E2 was also associated with a time-dependent decrease in the mRNA level of the proapoptotic Nip-2 protein, supporting the hypothesis that hormone responsiveness of U937 cells is mediated by target gene transcription. Together, these results demonstrate that ER and PR can be activated by endogenous or exogenous ligands to induce a genetic response that impairs TNF-{alpha}-induced apoptosis in U937 cells. The data presented here suggest that the female steroid receptors play a role in regulation of the immune response by preventing apoptosis of monoblastoid cells; this effect might have important consequences in the clinical use of steroid receptor drugs.—Vegeto, E., Pollio, G., Pellicciari, C., Maggi, A. Estrogen and progesterone induction of survival of monoblastoid cells undergoing TNF-{alpha}-inuced apoptosis.


Key Words: estrogen receptor • progesterone receptor • U937 cells • TNF-{alpha} • apoptosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
NUMEROUS EXPERIMENTAL AND clinical lines of evidence have suggested that sex steroid hormones can control immunological functions. A sexual dimorphism in the reactivity of the immune systems and in the immune-mediated diseases of females and males is well established; in addition, expression of the female steroid hormone receptors has been demonstrated in immunocompetent cell types (1, 2) . On the other hand, it has been suggested that disorders frequently affecting women after menopause, such as cardiovascular disease, osteoporosis, or neurodegenerative disorders, can be ascribed to the loss of sex hormone-dependent regulation of physiological functions, as well as to a modification of the immunological functions of resident cells (3 4 5 6) . Indeed, estrogens have been shown to have various effects on monocytes and macrophages (7 8 9 10 11 12 13 14) ; molecular biology studies have provided clear evidence for a direct activity of female sex hormones on immune cells, showing that the transcription of some of the genes that regulate leukocyte adhesion (15) , tissue remodeling (16 17 18) , or inflammation (19 20 21 22 23 24 25 26) can be modulated by the activation of steroid receptors.

Normal and malignant myeloid cells have been demonstrated to undergo apoptosis when challenged with endogenous molecules, such as cytokines or other mediators of inflammation, or with exogenous cytotoxic chemicals used in the treatment of myeloid cancers (27 28 29 30) . It is now widely accepted that induction of immune cell apoptosis plays an important role in inflammation and immunity, and defective apoptosis has been proposed as the underlying event in the onset or progression of several diseases associated with the immune system. In the cardiovascular system, for instance, it has been hypothesized that macrophage apoptosis is critical for atherosclerotic plaque stability (31) . Recently, it has been shown that estrogen interferes with the apoptotic program of diverse cell systems (32 33 34) . Considering the wide use of natural or synthetic ligands of steroid receptors in fertility control, cancer endocrine therapy, or the prevention of menopause-related disorders, we investigated whether estrogen and progesterone could hinder the induction of the apoptotic program of cells of the monocyte-macrophage lineage. The model system selected for this study was the monoblastoid cell line U937, which undergoes tumor necrosis factor {alpha} (TNF-{alpha})2-dependent apoptosis, thus mimicking, in vitro, the apoptotic process of mononucleated blood cells (35) . Estrogen and progesterone could interfere with TNF-{alpha}-induced apoptotic program. The observed effects were mediated by hormone receptors, which we found expressed in this cell system; a possible mechanism for the described antiapoptotic effect of estrogen is suggested.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
U937 cells were grown at 37°C in RPMI 1640 (Sigma, Milan, Italy) supplemented with 10% fetal bovine serum (Oxoid, Milan, Italy), 1% nonessential amino acids, 5 mM L-glutamine, 100 µg/ml streptomycin, and 100 IU/ml penicillin (Life Technologies, Inc., Milan, Italy), at 37°C under a humidified 5% CO2/95% air atmosphere. Cells were subcultured every week in 25 cm2 flasks (Corning Glassworks, Corning, N.Y.) at a density of 1 x 105 cells/ml and diluted 1:2 on the fourth day. Two days before the experiment, medium was replaced with RPMI 1640 without phenol red (Sigma), supplemented with 10% charcoal-treated (DCC) fetal bovine serum (DCC-wRPMI). On the day of the experiment, cells were harvested, resuspended in 10% DCC-wRPMI at a density of 3 x 105 cells/ml, and grown for the times indicated in the absence or presence of 20 ng/ml hrTNF-{alpha} (Sigma) 17ß-estradiol, 17{alpha}-estradiol, and progesterone (Sigma); ICI 182,780 (Zeneca Pharm., Milan, Italy), Raloxifene (Ely Lilly, Indianapolis, Ind.), and RU486 (Roussel Uclaf, France) were added as specified in the figure legends. To induce cell differentiation, U937 cells were grown for 3 days in 10% DCC-wRPMI supplemented with 320 nM phorbol 12-myristate,13-acetate (PMA) purchased from Sigma.

Cell proliferation and [3H]thymidine incorporation
U937 cells were seeded in 24-well culture plates (Corning) at a density of 3 x 105 cells/ml (500 µl/well) in 10% DCC-wRPMI. For proliferation and survival studies, triplicate samples for each treatment were harvested and counted in a Burker chamber using 0.4% trypan blue vital staining. Each assay was performed at least three times independently. For thymidine incorporation, 1 µCi/well [3H]thymidine (Amersham, Milan, Italy) was added for 4 h at 37°C to U937 cells grown for 24 and 48 h, as specified in the legend to Fig. 1 ; cell suspensions were centrifuged, washed twice in phosphate-buffered saline (PBS) containing 2 mM cold thymidine, and resuspended in 300 µl 0.1 M NaOH. Protein concentration was determined on 6 µl aliquots using the Bio-Rad Protein Assay Reagent (Bio-Rad, Milan, Italy), while the remaining cell lysates were incubated for 30 min at 4°C with an equal volume of 20% trichloroacetic acid. DNA precipitates were filtered on GF/C filters (Watmann, Maidstone, U.K.) and counted in a scintillation counter. Each experimental treatment was analyzed in triplicate.



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  		Figure 1. [3H]Thymidine incorporation of U937 cells. Cells were grown in the absence (-, open bars) or presence of 10 nM estradiol (E, light gray bars) or 10 nM progesterone (P, black bars) for 24 and 48 h; labeling of viable cells was performed by a 4 h incubation with 6.6 µCi [3H]thymidine/1 x 106 cells. Cell lysates and DNA extraction was performed as specified in Materials and Methods. A) Effect of the hormones alone; B) effect of 20 ng/ml TNF-{alpha} alone (-) or in the presence of the hormones (E or P). Data are the mean ± SD of three separate determinations and represent three different experiments. ## = P<0.01, as calculated by ANOVA test, followed by a Bonferroni analysis.

Labeling with FITC-conjugated AnnexinV, counterstaining with propidium iodide, and flow cytometry
About 106 cells were labeled for 15 min with FITC-conjugated Annexin V (Bender Med-System, Prodotti Gianni, Milano, Italy) in culture medium (final concentration 0.1–1 µg/ml). Cells were then counterstained with 2 µg propidium iodide (PI, purchased from Sigma)/ml culture medium; under these conditions, PI stains necrotic and late apoptotic cells only, while being excluded by intact (normal and early apoptotic) cells. Bivariate measurements of green fluorescence (identifying Annexin-labeled cells) vs. red fluorescence (identifying cells with damaged membranes) were made with a Becton Dickinson (San Jose, Calif.) FACStar flow cytometer, under the following conditions: argon-ion laser excitation power 200 mW at 488 nm, 560 nm beam splitter, 510 to 540-nm band pass filter for the green fluorescence signals, and 610 nm-long pass filter for the red fluorescence. Dual parameter cytometric data were evaluated with rectangular region analysis. At least 20,000 cells were considered in the gated region used for calculations.

DNA fragmentation assay
Five x 106 cells were seeded in 25 cm2 flasks in 10% DCC-wRPMI with 20 ng/ml of TNF-{alpha} in the absence or presence of 10-8 M estradiol or 10-8 M progesterone. After 6 h, cells were lysed in 600 µl of 0.5% Triton X-100, 10 mM Tris-base, pH 7.4, and 1 mM EDTA (ethylene diaminetetra-acetic acid) for 30 min on ice and centrifuged at 4°C for 30 min at 8000 g. The supernatants were extracted twice with phenol/chloroform and precipitated with 0.1 M NaCl in 99% ethanol o/n at -20°C. DNA was centrifuged at 8000 g for 30 min, washed with 70% ethanol, and resuspended in 20 µl 10 mM Tris, 1 mM sodium-EDTA, and 0.5 mg/ml DNase-free RNase A (Boehringer Mannheim, Milan, Italy). After incubation at 37°C for 30 min, 10 µg DNA, as calculated by spectrophotometry, was loaded onto a 1.5% agarose gel in Tris borate/EDTA without ethidium bromide. Electrophoresis was conducted at 50 V for 5 h. Staining/destaining in 2 mg/ml ethidium bromide and H2O allowed us to visualize the DNA fragments under the UV light.

Reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot
RNA preparation
Cells were harvested by centrifugation, washed twice in PBS, and resuspended in Bio/RNA-X Cell (Bio/Gene, Kimbolton Cambs, U.K.) (1 x 106 cells/ml). RNA was isolated according to the manufacturer's instructions.

cDNA preparation
RNA was denatured at 68°C and digested at room temperature for 40 min with Dnase I (Boehringer Mannheim) at the concentration of 1 U/µg RNA. After phenol-chloroform extraction and ethanol precipitation, 1 µg RNA was denatured at 68°C with 10 pmol oligo-dT(12 13 14 15 16 17 18) (Perkin Elmer, Milan, Italy) for detection of estrogen receptor {alpha} (ER-{alpha}) and ER-ß mRNA; hPRb-400 and GAPDH-b824 were used for PR (progesterone receptor; see below for primer sequences) in 10 µl final volume. Primer RNA mixes were cooled at room temperature for 15 min, dNTPs (Pharmacia, Milan, Italy) and MuMLV RT (Vios Instrument, Milan, Italy) were added at 200 µM and 1 U/µl final concentrations, respectively, in a final volume of 20 µl. The RT reaction was performed at 37°C for 1 h, the enzyme was inactivated at 75°C for 5 min, and cDNA mixes were stored in 100 µl final volume at -20°C. Control reactions without addition of the RT enzyme were performed for each sample; no bands were detected in the subsequent hybridizations (data not shown).

PCR
Three µl cDNA were incubated with 400 nM dNTPs, 200 nM each primer and 2 Units of DynaZyme DNA polymerase (Finezyme OY, Espoo, Finland) in 100 µl final volume. For both cDNA amplification and probe generation for Southern blot hybridization, the following primers (from MWG Biotech, Ebersberg, Germany) were used: human ER-{alpha}, h{alpha}6a-1539 (5'-AATGTGTAGAGGGGCATGG-3') and h{alpha}7b-1835 (5'-TGATGTGGGAGAGGATAGG-3'); human ER-ß, hßa-33 (5'-TCCCAGCAATGTCACTAAC-3') and hßb-252 (5'-TCCCCACTAACCTTCCTTT-3'); hPR, hPRa-76 (5'-ACTGCTGTGTCGCCCAGC-3') and hPRb-400 (5'- AAGAGCTGGTGACCTCGC-3'). Amplification products were 296, 219, and 324 base pairs long, respectively. In the case of hER-{alpha}, the primers used spanned from the 3'-end of exon 6 to the 5'-end of exon 7 in order to elude amplification of residual genomic DNA. Amplification of the constitutively-expressed enzyme glyceraldehyde phosphodehydrogenase (GAPDH) was performed on cDNA preparation to assess the reaction efficiency using the primers GAPDH-a134 (5'-ATGACCCTTCATTGACC-3') and GAPDH-b824 (5'-TGCTTCACCACCTTCTTG-3'). The PCR reactions were performed as follows: 1) for ER-{alpha}, ER-ß, and GAPDH, 95°C for 5 min, followed by 40 cycles at 92°C for 1 min, 50°C for 1 min, and 72°C for 1 min; 2) for PR, 95°C for 5 min, then 40 cycles at 92°C for 1 min, 55°C for 1 min, and 72°C for 1 min. All PCR reactions were performed on a Perkin Elmer Thermal Cycler 480.

Probe preparation
pSVwt-hER, phPR-B (a gift from Jeoffrey Greene) and pCMV5-hER-ß (a gift from Jan-Ake Gustaffson) plasmids were used as templates to prepare nonradioactively labeled DNA probes by means of PCR; probes corresponded from nucleotide 1539 to 1835 of the human ER-{alpha}, from nucleotide 33 to 252 of the human ER-ß, and from nucleotide 77 to 417 for the hPR, respectively. PCR reaction mixes contained 15 ng plasmid DNA, 0.8 µM primer set, 0.35 mM Dig-11 dUTP (Boehringer Mannheim), 0.65 mM dTTP, and 1 mM each of dATP, dCTP, and dGTP, and 2 U Taq DNA polymerase (Perkin Elmer) in 20 µl final volume. PCR reaction profile was as follows: 95°C for 5 min, then 30 cycles at 92°C for 1 min, 50°C for 1 min, and 72°C for 1 min. Amplification products were purified on a 1% agarose gel in Tris acetate EDTA.

Electrophoresis, blotting, and hybridization
10 µl of cDNA were loaded on 2% agarose gel in Tris borate EDTA and subjected to electrophoresis performed at 100 V. The gel was denatured at room temperature for 30 min in 0.5 M NaOH and 1 M NaCl and neutralized in 0.5 M Tris pH 7.7, 1.5 M NaCl and 1 mM EDTA for 15 min at room temperature. DNA was transferred onto nylon membrane (Hybond-N, Amersham) by capillarity blotting o/n in 20X SSC (sodium chloride and citrate buffer) and then fixed to the membrane by UV irradiation and baking at 80°C for 24 h. After two subsequent prehybridizations of 1 h at 65°C in `pre 1' solution [250 mM Na-phosphate pH 7.2, 7% sodium dodecyl sulfate (SDS), 1% bovine serum albumin, and 1 mM EDTA] and `pre2' solution [5X SSC, 50% formamide, 0.2% SDS, 1% Sarcosyl and blocking reagent (Boehringer Mannheim) dissolved in 0.01 M maleic acid, 15 mM NaCl], heat-denatured Dig-11dUTP-labeled probes were annealed at 42°C o/n and washed at room temperature twice in 5X SSC for 30 min, once in 1X SSC containing 0.1% SDS for 30 min, and twice in 0.1X SSC with 0.1% SDS for 15 min. The subsequent enhanced chemiluminescent reaction was performed as specified by the manufacturer (Boehringer Mannheim).

Immunocytochemistry
PMA-treated cells were grown in 24-well plate on 5% gelatin-coated coverslips for 3 days, then fixed for 10 min in 4% paraformaldehyde in 0.1 M PBS (pH 7.5). Cells were washed three times with PBS and incubated for 20 min at room temperature with blocking solution (5% horse serum, 0.1% Triton-X100 in PBS). After three washes in PBS, cells were incubated with 100 µl of 1:500 PBS dilution of the antihuman ER-{alpha} monoclonal antibody (C542) (Stressgen Biothechnology Corp., Milan, Italy) o/n at 4°C. Cells were washed three times prior to incubation with the secondary biotinylated horse antimouse antibody for 90 min. The staining was visualized after incubation with avidin-horseradish peroxidase and diaminobenzidine (Vector Laboratories, Milan, Italy). ER-{alpha} immunoreactivity was observed with a Zeiss Axioskop Microscope (Zeiss, Milan, Italy). Cells were photographed using a Kodak 200 ASA film.

Northern blot analysis
Probe preparation
The pcDNA 3-HA-NIP2 plasmid (a gift from G. Chinnadurai) was digested with HindIII and XhoI to excise the cDNA of Nip-2, which was labeled with the DNA Megaprime Labeling System purchased from Amersham, using 32P-dCTP (Amersham) to a specific activity of 5 x 108 dpm/mg.

Electrophoresis, blotting and hybridization
Twenty micrograms of total RNA, isolated as described earlier, were loaded on a 1% denaturing agarose gel containing 2.2 M formaldehyde. Electrophoresis was conducted at 100 V for 2 h. Equal loading was confirmed by densitometric scanning of the 18S RNA bands obtained from a photography of the gel. RNA was transferred to a positively charged nylon membrane (Nylon-N+, Amersham) by capillarity blotting. After 1 h prehybridization at 68°C in Quick Hyb solution (Stratagene, La Jolla), heat-denatured 32P-labeled Nip2 cDNA was added for 1 h at the same temperature and washed at 42°C, first in 5X SSPE (sodium chloride and phosphate EDTA buffer) for 30 min and then in 1X SSPE containing 0.1% SDS for 20 min. The membranes were exposed to autoradiographic film (Hyperfilm, Amersham) with intensifying screens at -80°C for 7 days.

Data analysis
Data are presented as mean ±SD of triplicate samples and are representative of at least three independently performed experiments. Analysis of variance (ANOVA) was performed to evaluate the statistical significance of differences between experimental groups with the Bonferroni test. Statistical significance was assigned to the level of P<0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Interference between TNF-{alpha} and female steroid hormones on U937 cell apoptosis
Cell counting and [3H]thymidine incorporation studies reveal reduced TNF-{alpha}-induced blockage of proliferation of U937 cells treated with estrogen or progesterone
To investigate female steroid hormone activity on U937 cell proliferation and survival, we first conducted a viability assay by counting the number of trypan blue stained cells after 24, 48, and 72 h incubation with 10 nM 17ß-estradiol (E2) or 10 nM progesterone (Prog). Table 1 shows that E2 and Prog did not alter the proliferation rate of U937 cells at each incubation time: the number (and percentage, data not shown) of viable cells counted after hormonal treatment did not significantly change when compared with controls; similarly, if cells were grown for 8 days in the absence or presence of 10 nM E2 or 10 nM Prog, with medium replaced every second day, the cell number obtained was reproducible (data not shown). When U937 cells were treated with TNF-{alpha}, the number and percentage of viable cells were significantly decreased with the progression of time. In fact, after 24 h incubation, viable cells in TNF-{alpha}-treated samples were 80% when compared with the number of viable cells in untreated samples; after 48 and 72 h treatment, TNF-{alpha} induced a further decrease in cell viability, reaching 23% and 21%, respectively, of the values obtained in control samples. The percentage of viable cells calculated by trypan blue staining decreased consistently with time from 83% after 24 h to 41% after 72 h (data not shown). In TNF-{alpha} treated cells, the presence of 10 nM E2 or 10 nM Prog lead to an increase in the number of viable cells and in the percentage of viable/total cells. In particular, the number of viable cells with respect to that of control cells was E2 = 123% and Prog = 108% at 24 h; E2 = 38% and Prog = 60% at 48 h; E2 = 40% and Prog = 35% at 72 h; these values are significantly higher than those obtained with TNF-{alpha} alone, suggesting that hormone treatment decreased the number of cells responsive to TNF-{alpha}. Induction of cell survival was also observed by comparing the percentages of viable cells treated with TNF-{alpha} in the absence or presence of the hormones: these were particularly higher after 72 h, when incubation with E2 and Prog resulted in 81% and 89% viability, respectively (data not shown).


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  		Table 1. Effect of 17ß-estradiol (E2) and progesterone (Prog) on U937 cell viability and survival

These results suggest that under these experimental conditions, TNF-{alpha} blocks proliferation and decreases cell number in a time-dependent manner, reaching maximal effect after 48 h incubation and remaining equally effective after 72 h; this may not be surprising since the cytokine was added only on the first day of the experiment. What is more striking is that we found that female sex steroid hormones can induce cell survival, leading ~30% of cells escaping TNF-{alpha}-dependent death.

To further examine the activity of E2 and Prog on U937 cell proliferation, we pulse-labeled the cells with [3H]thymidine. The amount of radioactivity incorporated in U937 cell DNA is shown in Fig. 1 . After 24 and 48 h growth, untreated cells incorporated [3H]thymidine equally, and E2 or Prog did not modify this incorporation rate (see Fig. 1A ). The increase in thymidine incorporation observed after 24 h incubation with Prog is unclear, since we did not evaluate any effect of this hormone on cell number at any time assayed; we believe this effect is linked to a modulatory activity of Prog on the cell cycle, as already reported in other cellular systems (36, 37) . Incubation with TNF-{alpha} led to a decrease in [3H]thymidine incorporation by 50% after 24 h and by 95% after 48 h, reflecting the decrease in cell viability observed in the cell counting experiments (see Fig. 1B ). Addition of E2 and Prog together with TNF-{alpha} prevented the decrease in [3H]thymidine incorporation observed with the cytokine alone.

These results suggest that the female steroid hormones modulate the response of undifferentiated U937 cells to TNF-{alpha} and induce cell proliferation. These and probably other mechanisms might account for the induction of cell survival.

DNA fragmentation studies and AnnexinV/propidium iodide double staining analyzed by flow cytometry provide qualitative and quantitative evidences for the protective effect of steroid hormones on TNF-{alpha}-induced apoptosis
To verify whether TNF-{alpha} caused the death of U937 cells by apoptosis and to examine whether steroid hormones were able to counteract this effect, we analyzed the DNA fragmentation pattern, which is a typical feature of cells undergoing apoptosis, after cytokine and hormones addition. As shown in Fig. 2 , after 6 h exposure to TNF-{alpha}, a clear fragmentation of U937 cells DNA was observed; this effect was strongly inhibited if cells were treated with TNF-{alpha} together with 10 nM E2 or 10 nM Prog. A quantitative measure of the apoptotic events resulting from TNF-{alpha} or TNF-{alpha} and estrogen treatment was obtained by labeling U937 cells with FITC-conjugated AnnexinV, counterstaining with PI, and analysis by flow cytometry. As shown in Fig. 3 , AnnexinV-positive cells were ~23% and 18% after TNF-{alpha} and TNF-{alpha} plus E2, respectively. This suggests that after the latter treatment, ~22% of the cells triggered by TNF-{alpha} were able to survive the cytokine-induced apoptotic signal. These results demonstrate that induction of apoptosis of U937 cells could account, at least in part, for the decrease in cell viability induced by TNF-{alpha}. In addition, E2 and Prog were able to attenuate this effect, suggesting that the hormonal induction of cell survival observed with the previous experiments could also be ascribed to a mechanism of inhibition of the early onset of apoptosis.



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  		Figure 2. DNA fragmentation pattern. U937 cells were grown for 6 h in the presence of 20 ng/ml TNF-{alpha} alone (-) or in the presence of 10 nM 17ß-estradiol (E2) or progesterone (Prog). 10 µg DNA were loaded on each lane. DNA molecular weight marker was loaded on the left of the gel.



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  		Figure 3. Dual-parameter flow cytograms of FITC-labeled Annexin V (abscissa) vs. PI staining in isotonic conditions (ordinate) of U937 cells treated in the absence (A) or presence of either TNF-{alpha} alone (B) or TNF-{alpha} plus 10 nM 17ß-estradiol (C). Normal, nonapoptotic cells were negative for both FITC and PI (quadrant 1); relatively early apoptotic cells were labeled by Annexin V, while being negative for PI (quadrant 2); late apoptotic and secondary necrotic cells were labeled for both Annexin V and PI (quadrant 3). The percentages of cells in quadrant 1 were 93.9 ± 1.25, 64 ± 1.1, and 72.8 ± 1.1 in panels A, B, and C, respectively, whereas those for apoptotic cells in quadrant 2 were 1.7 ± 0.5, 23.4 ± 3, and 18.5 ± 2. These values refer to eight independent experiments; differences between untreated (A) and treated (B, C) samples as well as those between panels B and C were always statistically significant (Student t test, P<0.01).

Receptor-dependent mechanism of action of estrogen and progesterone
Detection of E2 and Prog receptors by RT-PCR in undifferentiated and PMA-differentiated U937 Cells
The concentration of hormone sufficient to observe an effect was compatible with the hypothesis of a receptor-mediated mechanism of action. Therefore, to define whether the steroid effects reported above could be ascribed to an interaction with specific receptors, we first investigated the expression of the ER-{alpha} and ER-ß and PR gene by RT-PCR. These experiments demonstrated the expression of the mRNAs for ER-{alpha}, ER-ß (Fig. 4A, B ), and PR (Fig. 4C ) in U937 cells. Cell differentiation obtained after 3 days incubation with PMA did not change the receptor expression pattern, which was also detected in another human monoblastoid cell line, the THP-1 (see Fig. 4A, B ), suggesting a conserved hormonal signaling pathway in the myeloid cell system. cDNA reaction efficiency was controlled by amplification of the mRNA coding for the enzyme GAPDH (data not shown).



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  		Figure 4. RT-PCR and Southern blot analysis. Expression of ER-{alpha} (A), ER-ß (B), and PR (C) mRNAs was evaluated in undifferentiated (PMA: -), and differentiated cells (PMA: +). The length of the amplification product is reported on the left of each panel and the primers used for each PCR reaction are graphically shown on the right. MCF-7, and T47D mRNAs were used as positive controls. Hybridized membranes were exposed to autoradiographic films for 20 min (A) and 1 h (B, C) at room temperature.

Detection of ER-{alpha} by immunocytochemistry
To confirm that the ER protein was expressed in the cells, we performed an immunological assay on PMA-differentiated U937 cells. Figure 5 shows the positive staining (+AbI) for the ER-{alpha} protein in ~80% of the cells; specificity was confirmed when the primary antibody was omitted (-AbI). This experiment also shows that the ER-{alpha} is not uniformly expressed and that the receptor levels may vary among positive cells. Heterogeneous expression of ER-{alpha} could account for the partial activity of the hormone in the induction of cell survival, as we observed its effect on 30% of cells undergoing apoptosis; it also confirms the need of a highly sensitive method for receptor RNA detection, such as RT-PCR, followed by Southern blot, and could also provide explanations for the failure of the northern and Western blot techniques to detect the specific receptor mRNA and proteins (data not shown).



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  		Figure 5. Immunocytochemistry. PMA-differentiated U937 cells were analyzed for the expression of the ER-{alpha} protein in the presence (+AbI) or absence (-AbI) of the receptor specific monoclonal antibody.

We therefore concluded that the cell survival induced by the female steroid hormones occurs by the activation of intracellular receptors. In addition, these results suggest that steroid receptor expression is active and conserved in two different monoblastoid cell lines, both as undifferentiated or as differentiated cells toward a macrophage-like phenotype.

Hormone-mediated reduction of TNF-{alpha} antiproliferative activity involves ligand–receptor interaction
To further prove that the protective effect of E2 and Prog on TNF-{alpha}-induced apoptosis of U937 cells was receptor-mediated, we carried out a concentration-dependent assay. The U937 cells were treated with a constant amount of TNF-{alpha} and increasing concentrations of E2 or Prog (Fig. 6 A). A significant effect is first observed at 10 pM E2 and 100 pM Prog; the activity profile observed with this experiment reveals an ED50 of 10 nM for both hormones. 17{alpha}-Estradiol, a ligand for the ER-ß but not for the ER-{alpha}, displayed a mild agonist activity, providing evidence for a ligand selectivity on the observed effect. Similarly, ligand specificity was confirmed by the analysis of other steroid molecules, such as ICI 182,780, cholesterol, or dexamethasone (0.01 or 1 µM), which did not change the TNF-{alpha}-induced apoptotic program. To substantiate the hypothesis whereby the hormones operated by activation of their receptors in this cellular system, we tested the hormonal activity in the presence of specific receptor antagonists. Estrogen activity was blocked by the ER antagonist ICI 182,780, which had no effect on TNF-{alpha}-induced apoptosis if added alone (see Fig. 6B ) and reached half-maximal antagonist activity at 10 nM when tested in a dose-dependent competition assay together with 1 nM estradiol (data not shown). The ER ligand Raloxifene and the PR antagonist RU486 instead displayed a nonadditive, hormone-like activity, partially protecting U937 cells from entering apoptosis. It has already been reported that these ligands can trigger a hormone-like response depending on the cell type and gene promoter used to investigate their activity (38) .



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  		Figure 6. Ligand–receptor interactions mediate hormonal antiapoptotic activity. U937 cell viability (viable cells/total cells x 100) was calculated by trypan blue exclusion counting. Cells were grown for 48 h in the presence of 20 ng/ml TNF-{alpha}. A) Increasing concentrations of 17ß-estradiol ({blacktriangleup}), 17{alpha}-estradiol ({bullet}), or progesterone ({blacksquare}) were assayed. B, C) TNF-{alpha} was added together with 1 nM estradiol (E2, closed bars) or 10 nM progesterone (Prog, shaded bars); 100 nM Raloxifene (Ral), 100 nM ICI 182,780, or 100 nM RU486 were assayed as indicated. U937 cells grown in suspension (A, B) or after differentiation with PMA (C) were analyzed. Values represent the mean ± SD of triplicate determinations and are representative of four separate experiments. a: P<0.01 as calculated by ANOVA test, followed by a Bonferroni analysis.

These experiments suggest that the female steroid hormones exert their role in preventing TNF-{alpha}-induced apoptosis by interacting either with their intracellular receptors or in concert with membrane and cytosolic receptor isoforms.

PMA-differentiated U937 cells maintain the hormonal signaling pathway
We demonstrated that PMA-differentiated U937 cells express ER and PR; therefore, we analyzed whether in these macrophage-like differentiated cells E2 and Prog could still protect against TNF-{alpha}-induced apoptosis. As shown in Fig. 6C , hormones were still able to partially protect cells from entering the apoptotic program. This result suggests that E2 and Prog can trigger a biological response in macrophage-like differentiated U937 cells. The pharmacological activity of ICI 182,780 is reverted in differentiated U937 cells, since this ligand protects cells against apoptosis induced by the cytokine.

The mRNA for the apoptotic protein Nip-2 is regulated by E2
From the studies reported above, we concluded that E2 and Prog were able to induce survival of U937 cells. Since this effect was receptor mediated, we hypothesized that transcription of genes regulating cell survival/proliferation could be under hormonal control. Support for this hypothesis was provided by the observation that the hormones lost their antiapoptotic activity when added 8 h before TNF-{alpha} (data not shown) instead of together with or 2 h before the cytokine. One explanation for this temporal efficacy of the hormones against TNF-{alpha} activity could be that overlapping genetic elements were targeted by both molecules or, indirectly, by the products of their initial transcriptional activity.

The Nip-2 protein, recently cloned by means of its interaction with bcl-2, is believed to play an important role in apoptosis (39) . We have demonstrated that in a neural cell system, in which E2 promotes cell differentiation and prolongs cell survival, activation of ER corresponded to a decrease in the levels of Nip-2 mRNA (40) . A Northern blot analysis, shown in Fig. 7 , reveals that 2 h treatment with 10 nM 17ß-estradiol caused a 50% decrease in Nip-2 mRNA also in U937 cells. The ICI 182,780 blocked the effect of estrogen, confirming that ER was mediating the hormonal transcription signal. The time course experiment of estrogen regulation of Nip2 mRNA demonstrated that, as shown in Fig. 7B, C , the effect of E2 can be observed after 2 and 24 h incubation. The late regulation of Nip2 mRNA could be ascribed to both hormone-independent cell growth-related factors (in fact, Nip2 mRNA decreases in control cells after 1 day of culture) and to a hormone-dependent mechanism, probably mediated by hormone-activated intermediary factors.



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  		Figure 7. Nip-2 mRNA Northern blot analysis. A time course experiment of U937 cells grown in the absence or presence of 1 nM estrogen and 100 nM ICI 182,780 is shown. A) Northern blot showing the hybridization of the Nip2 probe with the Nip-2 mRNA band of the expected molecular weight; in the lower panel, a picture of the 18S RNA of the gel before the Northern blotting is shown. The densitometric values evaluated on the autoradiography were normalized by the densitometric values of the ethidium-bromide stained 18S RNA. Normalized numbers are reported in panels B and C, in which an arbitrary value of 1 was given to the 2 h control number. Open bars represent control values (B, C), whereas estrogen-treated samples are reported as closed bars (B, C). a: P < 0.01 as compared with the 2 h control value; b: P < 0.01 compared to the 24 h control value.

These results provide a genetic evidence for the existence of a steroid hormone signaling pathway in U937 cells; in addition, they suggest that the Nip-2 gene promoter could be a direct target of estrogen induction of cell survival.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Regulation of apoptosis is considered an important mechanism for controlling the number of monocytes available and therefore the intensity of the physiological response to infection or wound healing. Failure in the control of programmed cell death may become particularly relevant in pathological conditions such as tumor growth, autoimmune, and chronic inflammatory disorders, where apoptosis has been identified as a key factor in disease progression or remission (26 27 28 29) .

Massive physiological cell death occurs under the control of hormonal signals in reproductive tissues characterized by cyclic functional changes and remodeling. Detailed cellular and biochemical studies on the menstrual breakdown of the endometrial wall have demonstrated that the resident immune cells play a critical role in the regulation of intercellular signaling that triggers apoptosis and that steroid hormones can regulate, in cells of the stromal compartment, the transcription of effector molecules of apoptosis, such as matrix metalloproteinases (16 17 18) . In agreement with these studies, it has been shown that estrogen promotes apoptosis in osteoclasts (33) , suggesting the development of hormone-like therapeutic agents to prevent postmenopausal osteoporosis. This results from the increased generation and activity of osteoclasts associated with estrogen deficiency. On the contrary, it has recently been reported that estrogen receptor inhibits TNF-{alpha}-induced apoptosis of human endothelial cells, providing another mechanism that may account for the atheroprotective effect of estrogen (32) .

In the present study we have used the premonocytic U937 cell line, which can be differentiated in vitro toward a macrophage-like phenotype, as a model system to investigate whether sex steroid hormones played a role in the manifestation of apoptosis. Our results suggest that both estrogen and progesterone, by interacting with their cognate receptor, partially prevent the onset of TNF-{alpha}-induced apoptosis. That ER and PR are expressed in U937 and THP-1 cell lines is not surprising, since other reports have demonstrated that sex steroid hormone receptors are expressed in cells of the immune system and that cell biology can be regulated by steroid hormones, under both physiological and pathological conditions (1, 2, 5 6 7 8 9 10 11 12 13 14 15) . In addition, outside the hypothalamic-pituitary-ovary axis, steroid receptors have been shown to be expressed and, even at very low concentrations, can be activated by specific ligands to induce a significant biological response (41) . With our experiments we have shown that TNF-{alpha} exerts an antiproliferative and apoptotic activity, as already reported by other authors; more important, when physiological concentrations of estradiol or progesterone were added together with TNF-{alpha}, ~30% cells were induced to survive the apoptotic signal and to proliferate. Some distinctive features of the hormonal activity observed in this cellular system suggest that this effect is mediated by intracellular hormone receptors: 1) nanomolar concentrations of hormones are effective in inducing cell survival; 2) the ER antagonist ICI 182,780 is able to block estrogen activity; and 3) incubation of U937 cells with estrogen resulted in a decrease in Nip2 mRNA. The results on expression and function of the sex steroid receptors in U937 cells provide important informations on this cellular system and represent an interesting background for understanding the genetic mechanism underlying the antiapoptotic activity of these receptors and for the investigation on receptor-directed compounds that could modulate or mimic this activity. When we tested some receptor antagonists such as Raloxifene and RU486, we observed an agonist-like effect, both as undifferentiated and PMA-differentiated cells. As has been widely demonstrated, the pharmacological potential of steroid receptor ligands is strictly tissue specific; receptor drugs may manifest agonist or antagonist activities depending on the environment of nuclear proteins and cofactors that are specific for each cell type (42, 43) . In addition, differentiation of U937 cells with PMA reverted the pharmacological activity of ICI 182,780; it is possible that hyperstimulation of protein kinase C by 3 days of PMA treatment leads to a modification of the phosphorylation events that mediate the ER transcriptional activity induced by the ICI 182,780. Altogether, these studies show that myeloid cells are target cells for female steroid hormones and receptor drugs, whose pharmacological activity needs to be assayed specifically. Prevention of apoptosis of monoblastoid cells mediated by these compounds might have important consequences in the clinical use of steroid receptor drugs and in the therapy of malignant myeloid cell growth.

It would be of particular interest to investigate whether induction of apoptosis of normal circulating monocytes or resident monocytes–macrophages can also be modified by female steroid hormones. In fact, U937 cells are considered a valid model system with which to study the induction of apoptosis (35) ; these cells derive from a human lymphoma and, therefore, cannot fully represent the physiology of circulating cells. However, it is interesting that a gene such as Nip-2, which we have demonstrated as being regulated by estradiol and that encodes for a protein involved in the onset of apoptosis (39) , can be modulated by estrogen in U937 cells; this result suggests that the genetic pathway of response to steroid hormones is conserved among different cell types. As mentioned above, recent studies have demonstrated a hormonal regulation of the apoptotic program; the opposite results, reported by Hughes et al. (33) , Spyridopoulos et al. (32) , and in this report, have been observed in different cell types; steroid hormones have been frequently associated with a differential activity depending on the cell type or on the cell differentiation state. Although the molecular mechanism for this cell type- and gene-specific activity has not yet been fully clarified, one could speculate that the differential tissue distribution of the receptor cofactors, recently identified proteins that interact with steroid receptors to modulate gene transcription (44) , could account for the cell-specific response to steroid hormones.

Characterization of TNF-{alpha} activity has shown two distinct mechanisms of action in induction of apoptosis by this cytokine: an initial, transcription-independent activity, which operates through the activation of proteases, and a subsequent transcription-dependent antiapoptotic activity by activation of the NF-{kappa}B factors (45) . In this paper we report that interference between estradiol and TNF-{alpha} signaling pathways occurs only when E2 is added along with or 2 h before the cytokine. On the contrary, when estradiol is added 8 h before TNF-{alpha}, protection is lost. These results suggest that induction of apoptosis and/or cell survival might be regulated by overlapping genetic targets of TNF-{alpha} and ER transcriptional activity. Negative interference between ER and TNF-{alpha} signaling pathways has been reported for the interleukin-6 promoter, where transcriptional interference has been proposed to occur through direct physical interaction and reciprocal transcriptional silencing between specific members of the NF-{kappa}B family and ER. Interaction between PR and the NF-{kappa}B family has also been demonstrated; in addition, PR and STAT5, a member of the JAK/STAT family of latent transcription factors that are activated by numerous extracellular signals, have been shown to interact in vitro and to reciprocally interfere when artificially coexpressed in cells, providing further evidence for the hypothesis that the steroid receptors and membrane receptor-associated second messengers can communicate and reciprocally modulate transcriptional efficacy of the respective activating signals (46) . However, we cannot rule out the possibility that target gene regulation by estrogen counteracts the transcription-independent TNF-{alpha} induction of apoptosis and that estrogen-induced biological response declines after a few hours of hormonal stimulation. Further studies will shed more light on this important aspect.

Our results have provided the first evidence for receptor-mediated transcriptional and cellular responses of myeloid cells to estrogen and progesterone, and have highlighted the need to further elucidate the genetic mechanisms triggered by the steroid hormones and receptor drugs in this immune cell system.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the Istituto Superiore della Sanità, Multiple Sclerosis Project, by CEE BioMed Project BMH4-CT97–2286, by Telethon, by AIRC, and by CNR Targeted Project Biotechnology. We are grateful to Monica Rebecchi and Clara Meda for their skillful technical assistance, to Michela Pompini, Roberta Bozzella, Tiziana Piepoli, and Claudia Scarsi for their technical support, and to Paola Agrati, Paolo Ciana, Michael Penlington, and Sabrina Santagati for their helpful discussions. We also thank Simonetta Nicosia for providing us with U937 cells, Corrado Galli for providing us with TNF-{alpha}, and John Termine for providing us with Raloxifene. The cytometric measurements were performed by Maria Grazia Bottone at the Centro Grandi Strumenti, University of Pavia.


   FOOTNOTES
 
2 Abbreviations: ANOVA, analysis of variance; E2, 17ß-estradiol; ER-{alpha}, estrogen receptor {alpha}; EDTA, ethylene diaminetetra-acetic acid, GAPDH, glyceraldehyde phosphodehydrogenase; PBS, phosphate-buffered saline, PI, propidium iodide; PMA, phorbol 12-myristate,13-acetate; PR, progesterone receptor; Prog, progesterone; RT-PCR, reverse transcriptase-polymerase chain reaction; SDS, sodium dodecyl sulfate, SSC, sodium chloride and citrate buffer, SSPE, sodium chloride and phosphate buffer; TNF-{alpha}, tumor necrosis factor {alpha}.

Received for publication September 4, 1998. Revision received January 4, 1999.
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