Estrogen Preferentially Promotes Differentiation of CD11c + CD11b intermediate Dendritic Cells from Bone Marrow Precursors This information is current as of October 3, 2021. Vladislava Paharkova-Vatchkova, Ruben Maldonado and Susan Kovats J Immunol 2004; 172:1426-1436; ; doi: 10.4049/jimmunol.172.3.1426

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Estrogen Preferentially Promotes the Differentiation of CD11c؉ CD11bintermediate Dendritic Cells from Bone Marrow Precursors1

Vladislava Paharkova-Vatchkova, Ruben Maldonado, and Susan Kovats2

Sex biases in autoimmunity and infection suggest that steroid sex hormones directly modulate immune cells. show in this study that 17-␤-estradiol (E2) promotes the differentiation of functional dendritic cells (DC) from murine bone marrow precursor cells. Remarkably, ex vivo DC differentiation was inhibited in steroid hormone-deficient medium, and was restored by addition of physiological amounts of E2, but not dihydrotestosterone. DC differentiation was inhibited by the estrogen receptor () antag- onists ICI 182,780 and tamoxifen, and from ER␣؊/؊ bone marrow cells, indicating that E2 acted via ERs. E2 addition was most effective in promoting DC differentiation immediately ex vivo, but did not increase DC proliferation. E2 treatment specifically ؉ int promoted differentiation of CD11c CD11b DC population that displayed high levels of cell surface MHC class II and CD86, Downloaded from suggesting that E2 could augment numbers of potent APC. DC that differentiated in E2-supplemented medium were fully func- tional in their capability to mediate presentation of self and foreign Ags and stimulate the proliferation of naive CD4؉ T cells. The requirement for estrogen during DC differentiation suggests a mechanism by which E2 levels in peripheral tissues might modulate both the number and functional capabilities of DC in vivo, thereby influencing immune responses. The Journal of Immunology, 2004, 172: 1426Ð1436. http://www.jimmunol.org/ ex-based differences in immunity are suggested by gender anism(s) by which sex hormones modulate immunity is not com- differences in the incidence of autoimmune diseases (1). pletely understood, estrogen levels currently are being manipulated S Lymphocyte-mediated autoimmune diseases in humans, during therapy for breast cancer and autoimmune disease (multiple .g., multiple sclerosis, rheumatoid arthritis, Graves’ disease, sys- sclerosis), and in postmenopausal women. temic lupus erythematosus, and Hashimoto’s thyroiditis, and mu- The 17-␤-estradiol (E2) is a regulator of growth, differentiation, rine models of these diseases, such as experimental autoimmune survival, or function in many target tissues, including the female encephalomyelitis and collagen-induced arthritis, are more preva- and male reproductive systems, cardiovascular system, and bone lent in females than males. This sexual dimorphism also has been (12). ER are ligand-dependent transcription factors that modulate found in infectious disease pathogenesis (2–4). Sex hormone treat- gene transcription by binding coactivators or corepressors that by guest on October 3, 2021 ment of T cells or whole organisms has been shown to modulate vary in target tissues (13). In addition, rapid biological effects of autoimmunity and immune responses to pathogens in mouse mod- E2 in some cell types suggest that E2 elicits nongenomic effects els (5–9). Together with the finding that lymphocytes and myeloid via cell surface ER linked to signal transduction pathways, e.g., cells express estrogen receptors (ER)3 and androgen receptors (AR) mitogen-activated protein kinase (13). ER␣ and ER␤ are members (6, 10), these observations suggest that the steroid sex hormones, - of the steroid/thyroid hormone superfamily of nuclear receptors; trogen and testosterone, could directly influence the function of cells although both ER bind ligands with similar affinities, the confor- involved in the immune response. Reports to date indicate diverse mationally distinct ligand/receptor complexes mediate different effects of sex hormones or their receptor antagonists on cellular dif- transcriptional effects (12). Both ER␣ and ER␤ are expressed in ferentiation, cytokine production, Th1/2 cell polarization, NO produc- the spleen and thymus (14). Disruption of the murine ER␣ or ER␤ tion, MHC class II (MHCII) expression, and APC recruitment or genes has revealed distinct roles for the two ER in reproductive, function (reviewed in Refs. 1 and 11). Although the underlying mech- skeletal, and cardiovascular tissue (15). Endogenous ER ligands include estrone (E1), E2, and estriol (E3); E2 is the predominant form of estrogen present in males and nonpregnant females. The Division of Immunology, Beckman Research Institute, City of Hope National Med- steroidal compound ICI 182,780 is a pure ER antagonist in all cell ical Center, Duarte, CA 91010 types (16). In contrast, selective ER modulators (SERM), such as Received for publication August 13, 2003. Accepted for publication November 14, 2003. tamoxifen, bind ER and differentially modulate ER-mediated tran- The costs of publication of this article were defrayed in part by the payment of page scription via the recruitment of cell type-specific coactivators or charges. This article must therefore hereby marked advertisement in accordance corepressors (17, 18). with 18 .S.C. Section 1734 solely to indicate this fact. Multiple studies have provided some evidence that APC num- 1 S.K. was supported by grants from the National Institutes of Health, National In- bers and functional capabilities are altered by normal or manipu- stitute of Allergy and Infectious Diseases (AI-44922), and the Arthritis Foundation. lated systemic levels of estrogen in vivo (19–24). Notably, num- 2 Address correspondence and reprint requests to Dr. Susan Kovats, Division of Im- munology, City of Hope Medical Center, 1450 East Duarte Road, Duarte, CA 91010. bers and function of both professional and nonprofessional APC in E-mail address: [email protected] the female rat reproductive tract vary with different estrogen levels 3 Abbreviations used in this paper: ER, estrogen receptor; AR, androgen receptor; during estrus and diestrus (25). A recent study showed that during BM, bone marrow; CDFCS, charcoal-dextran-treated FCS; CLIP, invariant chain deg- development of experimental autoimmune encephalomyelitis, sys- radation intermediate; DC, dendritic cell; DHT, dihydrotestosterone; DIC, differential interference contrast; E2, 17-␤-estradiol; GM, growth medium; HEL, hen egg temic E2 treatment suppressed disease symptoms and led to - lysozyme; int, intermediate; RegFCS, regular FCS; SERM, selective ER modulator. creased numbers of dendritic cells (DC) migrating into the CNS at

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 1427 disease onset, and decreased DC production of TNF-␣, IL-12, and Generation of DC from BM cells ␥ ␮ IFN- (23). In vitro, high concentrations (5–10 M) of the non- The 129S6, C57BL/6, and ER␣Ϫ/Ϫ (B6.129-Estratm1 N10) mice (Taconic steroidal antiestrogens toremifene and tamoxifen inhibited the Farms, Germantown, NY) were housed at the City of Hope Animal Re- ϩ GM-CSF- and IL-4-mediated differentiation of human ER source Center in accordance with institutional and federal guidelines. BM CD14ϩ monocytes into CD1aϩ CD14Ϫ DC (10). These antiestro- cells were isolated from the femurs and tibiae of 8- to 12-wk-old female gens acted independently of the ER, because neither E2 nor the ER and male mice, and cultured with GM-CSF according to published proto- cols (35). GM-CSF containing conditioned medium from J558L cells trans- antagonist ICI 182,780 had an effect in this cell system. Con- fected with the murine GM-CSF gene (36) was used; the J558L cells were versely, a separate study reported that E2 or testosterone addition kindly provided by R. Steinman (The Rockefeller University, New York, to human monocyte cultures increased the number of monocyte- NY). Briefly, on day 0, 2 ϫ 106 total BM cells (including erythrocytes) derived DC (26). A consistent pattern of estrogen effects on APC were put in 10 ml medium containing GM-CSF (3.3% J558L conditioned medium v/v) in 100 mm bacteriological petri dishes. On day 3, 10 ml of does not emerge from these diverse studies; it is possible that dif- medium containing GM-CSF was added to the plates. On day 6, half of the ferences between in vitro and in vivo systems may be explained culture supernatant was removed, and cells were spun and resuspended in partially by differential effects of low vs high doses of E2, or - an equal volume of fresh medium containing GM-CSF. In some of the fects of systemic E2 treatment on the levels of other steroid hor- cultures, LPS (2 ␮g/ml) (from Escherichia coli serotype 055:B5) (Sigma- mones, e.g., progesterone, known to influence immune cell func- Aldrich) was added from day 6–7 or day 8–9 to mature the DC. Immature and mature DC were harvested on day 7–9. E2, DHT, or ER antagonists tion (27–29). Thus, we chose to investigate a potential role for E2 were added on days 0, 3, and 6, or as indicated in the figures. in DC biology using a well-defined ex vivo culture system for the development of murine DC. mAbs and flow cytometry DC are potent APC involved in the initiation of immune re- sponses by naive T cells and contribute to polarization of Th1 or Fluorochrome- or biotin-labeled mAbs specific for CD11c, CD11b, CD86, Downloaded from CD80, CD40, Gr-1, B220, CD4, and CD8␣ were obtained from BD Th2 cell responses (30) and maintenance of peripheral self-toler- PharMingen (San Diego, CA). anti-DEC205 was obtained from Cedar- ance (31, 32). In peripheral tissues, immature DC acquire Ags, and lane Laboratories (Hornby, Ontario, Canada). mAbs specific for MHCII upon an inflammatory and/or pathogen stimulus, migrate to the (Y3P) and macrophages (F4/80) were purified from hybridoma (American lymph nodes, whereupon the now mature activated DC express Type Culture Collection, Manassas, VA) supernatant and biotinylated. Pu- rified and labeled mAbs specific for -Ab/E␣52–68 complexes (YAe)or high levels of surface MHCII and CD86 and are capable of acti- I-Ab/invariant chain degradation intermediate (CLIP) complexes (15G4) http://www.jimmunol.org/ vating naive T cells (31). DC arise from both myeloid and lym- were kindly provided by A. Rudensky (University of Washington, Seattle, phoid progenitors present in bone marrow (BM) (32, 33). WA). Biotinylated mAbs were detected with streptavidin-CyChrome or To determine how sex hormones might modulate DC differen- streptavidin-APC (BD Pharmingen). Appropriate fluorochrome or biotin- tiation or Ag-presenting function, we used an ex vivo BM culture ylated isotype control mAbs of each species were used as negative controls. Cells were preincubated with mAb 2.4G2 (anti-CD16/32) to block Fc re- system in which GM-CSF promotes DC differentiation from my- ceptors, and labeled with mAbs in PBS, 5% heat-inactivated newborn calf eloid precursors (34). These BM cultures typically yield two popu- serum, and 0.1% sodium azide. Labeled cells were run on a FACSCalibur lations of CD11cϩ DC that display either high or intermediate surface (BD Biosciences, San Jose, CA), and data were analyzed using CellQuest b expression of CD11b. To define the effects of estrogen and testoster- software. To detect I-A /E␣52–68 complexes, DC were incubated with 30 ␮ME␣52–68 peptide for3hat37oC before labeling with mAb YAe and one, BM cells were cultured for 7 days in steroid hormone-deficient Cy5-labeled donkey anti-mouse IgG (Jackson ImmunoResearch Laborato- by guest on October 3, 2021 medium supplemented with defined amounts of E2 or dihydrotest- ries, West Grove, PA). Cell sorting was performed on a Mo-Flo cytometer ϩ osterone (DHT). Remarkably, CD11c CD11bint DC differentia- (Cytomation, Fort Collins, CO) in the City of Hope National Medical Cen- tion in steroid hormone-deficient medium was significantly inhib- ter Analytical Cytometry core facility. ited, and could be restored by addition of physiological amounts of E2 to the cultures. A threshold amount of E2 acting via ERs was T cell hybrid assays required during ex vivo DC differentiation from BM precursor T cell hybrids used to assess Ag presentation were specific for epitopes ϩ cells isolated from either male or female mice. The CD11c derived from exogenous Ags, IgM 377–392 (77.1) (37) and hen egg ly- int sozyme (HEL) 74–88 (B04) (38), or the endogenous Ag rab5 86–101 (2.3) CD11b DC produced by these culture conditions displayed ϩ higher intrinsic levels of MHCII and costimulatory molecules than (37). Immature CD11c DC from day 7 cultures (unfractionated or purified ϩ high using anti-CD11c microbeads and the Miltenyi Biotec (Auburn, CA) mag- CD11c CD11b DC, and were capable of presentation of self netic column separation system) were incubated for 20 h with the native and foreign Ags by MHCII molecules as well as stimulation of Ags IgM (2–5 ␮g/ml) or HEL (100–400 ␮g/ml). Subsequently, the cells naive CD4ϩ T cell proliferation. were matured with LPS for 20 h, washed, and titrated (between 5 ϫ 104 and 3 ϫ 102/well) in wells before addition of T cells (105/well). No T cell responses were detected in the absence of Ag. For rab5-specific responses, Materials and Methods T cells were incubated with variable numbers of DC for 20 h. For all three Cell culture medium and reagents T cell hybrids, antigenic peptides were added to non-Ag-pulsed DC as a positive control for T cell reactivity. IL-2 production by the T cell hybrids Regular culture medium (regular FCS (RegFCS)) was RPMI 1640 supple- was assessed by proliferation of HT-2 cells (39) using an Alamar blue mented with 10% FBS ( Scientific, Tarzana, CA), 2 mM glutamine, colorimetric assay; results are expressed as arbitrary units of OD at 570 vs 100 U penicillin/0.1 mg streptomycin/ml, 10 mM HEPES buffer, 50 ␮M 600 nm, average of duplicate wells. 2-ME, and 1 mM sodium pyruvate. Hormone-deficient medium (charcoal- dextran-treated FCS (CDFCS)) was RPMI 1640 lacking phenol red, sup- Allogeneic T cell assays plemented with 10% charcoal-dextran-treated FBS (Omega Scientific). The DC-GM serum-free medium (Stemgenix, Amherst, NY) was supplemented Immature CD11cϩ DC from day 7 cultures of 129S6 BM cells, containing with 50 ␮M 2-ME. Stock solutions (0.01 M) in ethanol of E2 and DHT either RegFCS or CDFCS without or with supplemented E2, were used as (both from Sigma-Aldrich, St. Louis, MO) were diluted into BM cultures an unfractionated population, or purified using anti-CD11c microbeads. at varying concentrations on days indicated in the figures. DHT was used DC were activated by incubation for 20 h with LPS and ␥ irradiated (3000 because it is not a substrate for aromatase conversion to estrogen. Stock rad), or were incubated with LPS for 3 h and paraformaldehyde fixed. solutions of ICI 182,780 (1 mM) (Faslodex; AstraZeneca, Cheshire, U.K.) Prepared APC were titrated in wells and incubated with 2–2.5 ϫ 105 per and tamoxifen (10 mM) (Sigma-Aldrich) in DMSO were prepared and well naive allogeneic CD4ϩ T cells that were isolated from a B10.M (H-2f) diluted into BM cultures at varying concentrations in the presence or ab- mouse by cell sorting. Proliferation of T cells was assessed by addition of sence of exogenous E2. Addition of ethanol or DMSO alone to BM cul- 0.5 ␮Ci/well of [3H]Tdr during the last 16 h of an 86-h culture period. tures did not change DC differentiation or surface marker expression rel- Stimulation index was calculated as: (cpm responder ϩ stimulator)/(cpm ative to completely untreated cultures. responder alone). 1428 ESTROGEN PROMOTES DC DIFFERENTIATION

Labeling of cells with CFSE Light microscopy Upon isolation, BM cells (2 ϫ 107/ml) were labeled with 10 ␮M CFSE Differential interference contrast (DIC) images of live cells were taken on (Molecular Probes, Eugene, OR) from a 10 mM stock solution (in DMSO) an Olympus AX70 microscope, original magnification ϫ400. Images were for 10 min at 37oC. Cells were washed and put into culture, and intensity analyzed with Image Pro software. of CFSE signal was monitored every 2 days by flow cytometry. CFSE- labeled cells were labeled with biotin anti-CD11c and streptavidin-APC, as Results described above. Estradiol promotes DC differentiation from BM precursors Detection of sex hormone receptors by immunoblotting To determine an effect of E2 on DC differentiation or function, we Cells from day 7 BM cultures or all cells from BM directly ex vivo were used a well-defined ex vivo culture system for GM-CSF-mediated solubilized in Nonidet P-40 lysis buffer, and variable amounts (10–60 ␮g) generation of DC from BM precursors (34). Unfractionated BM of cell lysate/lane were electrophoresed on 8% SDS-polyacrylamide gels cells from 129S6 mice were incubated in GM-CSF, as described and transferred to nitrocellulose, as described (40). To detect sex hormone receptors, the nitrocellulose was probed with polyclonal anti-ER␣ (MC- (35), and on day 7, cells were assessed for the surface molecules 20), anti-ER␤ (H-150), or anti-AR (N-20) from Santa Cruz Biotechnology characteristic of murine DC by flow cytometry (Fig. 1A). Approx- (Santa Cruz, CA), and HRP-labeled goat anti-rabbit IgG (Jackson Immu- imately 40–60% of cells in these cultures were CD11bϩ CD11cϩ noResearch Laboratories). Breast (MCF-7) and prostate (LNCaP) cancer DC that were F4/80ϩ Gr-1Ϫ CD4Ϫ CD8␣Ϫ B220Ϫ DEC-205Ϫ,as cell lines known to express ER and AR were used as controls, and were ϩ is typical for some splenic DC (41). CD11c DC could be subdi- kindly provided by S. Chen (City of Hope National Medical Center). Blots high were developed using an ECL substrate (Amersham Biosciences, Piscat- vided based on levels of CD11b surface expression, CD11b and away, NJ) and exposure to film. CD11bint. The DC displayed the low levels of surface MHCII and Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 1. Estradiol promotes DC differentiation from BM precursors. A, E2 restores DC differentiation in CDFCS. Female 129S6 BM was cultured in RegFCS (RPMI, 10% FCS) or CDFCS (phenol red-free RPMI, 10% charcoal-dextran-treated FCS) Ϯ 1 nM E2 for 7 days before assessment of CD11b and CD11c expression by flow cytometry. Shown are dot plots of anti-CD11b PE vs anti-CD11c FITC (left panels) and the corresponding forward light scatter vs side scatter plots (right panels). The percentages of CD11bhigh or CD11bint CD11cϩ DC are indicated. Isotype control mAbs (hamster IgG FITC or rat IgG PE) did not bind to any cells in the BM cultures (data not shown). Data are representative of nine independent experiments. B, Amounts of E2 normally present in FCS restore DC differentiation in CDFCS. BM was cultured in CDFCS with variable amounts of E2 for 7 days. Numbers of CD11bhigh or CD11bint CD11cϩ DC that differentiated at each E2 are shown from two representative experiments. C, DHT does not restore DC differentiation in CDFCS. BM cells were incubated in CDFCS Ϯ DHT (100 nM) before determination of CD11c and CD11b expression. Results are representative of two independent experiments. D, The inhibition of DC differentiation in serum-free medium is restored by addition of E2. BM was cultured in DC-GEM serum-free medium Ϯ variable doses (0.01–0.5 nM) of E2 for 7 days, followed by assessment of CD11b and CD11c expression. The number of CD11cϩ DC that differentiated at each E2 is indicated (right panel). Data are representative of two independent experiments. E, DC in E2-supplemented CDFCS display typical DC surface markers. Surface expression of MHCII or CD86 on resting (top panels) or LPS-activated (bottom panels) CD11cϩ DC present in cultures (shown in A) containing RegFCS (shaded histograms), CDFCS (thin lines), or E2-supplemented CDFCS (thick lines) was assessed by flow cytometry. The Journal of Immunology 1429

CD86 characteristic of immature, resting DC, and activation by bacterial LPS (2 ␮g/ml) for 18 h to affect DC maturation resulted in increased levels of surface MHCII and CD86 (Fig. 1E). The CD11cϪ (CD11bhigh or CD11blow) cells in the culture displayed the myeloid differentiation marker Gr-1, but not MHCII nor CD86 either before or after LPS activation, suggesting that they are DC precursors (data not shown). We reasoned that the influence of E2 on DC differentiation would be determined most accurately if cells were cultured in ste- roid hormone-deficient medium that is composed of CDFCS in phenol red-free RPMI. Passage of RegFCS over charcoal-dextran reduces the level of steroid hormones (including E2, estrone, and testosterone) (42). Levels of E2 in RegFCS are typically ϳ8–11 pg/ml (0.03–0.05 nM), and charcoal-dextran treatment reduces E2 to levels below the sensitivity of standard E2 RIA (Ͻ5 pg/ml). Phenol red has weak estrogenic activity at 15–45 ␮M, the con-

centration at which it is present in RPMI medium; the KD of phe- nol red for the ER has been estimated to be 2 ϫ 10Ϫ5 M, 0.001% of the affinity of E2 (43). In contrast to BM cells cultured in RegFCS, steroid hormone- Downloaded from deficient medium (CDFCS) does not support efficient DC differ- entiation, as indicated by the significant reduction of CD11bϩ CD11cϩ immature DC on day 7; the majority of cells remain CD11bϩ (Fig. 1A). Remarkably, addition of 0.01–1 nM E2 to cells in hormone-deficient medium on days 0, 3, and 6 of the BM cul- ture, during medium and GM-CSF renewal, led to the recovery of http://www.jimmunol.org/ CD11cϩ DC on day 7 (Fig. 1, A and B). The paucity of DC in CDFCS may be visualized on forward light scatter vs side scatter plots, in which the majority of cells present in the absence of E2 have lesser forward and side scatter than is typical for DC (Fig. 1A). The number of differentiated CD11cϩ DC was markedly aug- mented by addition of amounts of E2 (0.01–0.1 nM) comparable to that found in RegFCS (0.03–0.05 nM) (Fig. 1B). The CD11cϩ CD11bint DC population was most significantly affected by E2 by guest on October 3, 2021 levels in the culture medium; this population was most clearly reduced in CDFCS and increased ϳ5-fold upon addition of E2 to CDFCS (Fig. 1B). The CD11cϩ CD11bhigh population was less dependent upon E2, although an E2-mediated increase in numbers of this population was observed in some experiments; an example

FIGURE 2. E2 preferentially promotes the differentiation of CD11cϩ CD11bint DC. A, Excess E2 doubles the percentage of CD11bint DC that develops in GM-CSF-treated BM cultures in RegFCS. Female 129S6 BM cells were incubated Ϯ 10 nM E2 for 7 days before assessment of CD11b and CD11c surface expression. The percentage of cells in each boxed region is indicated. Results are representative of 13 independent experiments. B, The number of CD11cϩ CD11bhigh or CD11cϩ CD11bint DC that differentiated in cultures containing RegFCS with variable amounts (0.01–10 nM) of E2 is indicated. C, CD11bint DC display the highest levels of MHCII and CD86 before and after LPS activation. MHCII or CD86 surface expression on CD11cϩ DC present in the day 7 BM cultures (shown in A) without (shaded histograms) or with (thick line histograms) excess 10 nM E2 was assessed by flow cytometry. MHCII or CD86 expression on CD11bhigh or CD11bint DC from either immature or LPS-activated cultures is shown. Biotinylated irrele- vant mouse or rat IgG mAbs were used as isotype controls for biotinylated forms of the murine anti-MHCII mAb Y3P or the rat anti-CD86 mAb, respectively (top panels); mAb binding was detected with streptavidin- CyChrome. Results are representative of 13 independent experiments. D,E2 most effectively promotes DC differentiation from BM cells in RegFCS when added on day 0 of culture. E2 (10 nM) was added to BM cells on either day 0, 1, 3, or 5, and the percentage of CD11cϩ CD11bint DC (top panel) and CD11cϩ CD11bhigh DC (bottom panel) on day 7 in each culture was deter- mined by flow cytometry (F). The percentage of differentiated DC on day 7 in the absence of supplemental E2 is indicated (E). Results are representative of two independent experiments. 1430 ESTROGEN PROMOTES DC DIFFERENTIATION of each result is shown in Fig. 1B. Addition of 1 nM E2 only on of CD11bϩ CD11cϪ cells (Fig. 2, A and B). In 13 independent day 6–7 during the period of LPS activation did not restore DC in experiments in which BM cultures in RegFCS from individual the cultures (data not shown). Similar E2 dose responses were male or female mice were supplemented with 10 nM E2, a 10– observed with both female and male BM cells. Addition of DHT 50% increase in numbers of CD11cϩ DC was observed (geometric or testosterone (Յ100 nM) from day 0 of BM culture did not mean ratio E2/control 1.26). E2 markedly induced the accumula- restore the differentiation of DC (Fig. 1C and our unpublished tion of CD11bint DC, while the number of CD11bhigh DC was only data). slightly increased by E2 addition. Compared with CD11bhigh DC, Because the low amount of E2 remaining in CDFCS cannot be CD11bint DC display higher cell surface MHCII and CD86 before measured accurately using typical E2 RIA (HyClone Laboratories, and after LPS activation (Fig. 2C). In addition, differentiation in unpublished observations), we assessed GM-CSF-induced DC dif- excess E2 augmented the MHCII and CD86 on immature ferentiation in a defined serum-free medium (DC-GM) that lacks CD11bhigh and CD11bint DC (Fig. 2C). steroid hormones. Very few DC developed in DC-GM, and the To determine whether the presence of E2 was required during viability of the cultures was poor. Addition of 0.01–0.1 nM E2 the entire 7-day culture period to promote DC differentiation, E2 increased the number of differentiated DC ϳ5-fold, indicating that was added to BM cells on different days after isolation (day 0, 1, amounts of E2 normally present in FCS (0.03–0.05 nM) are suf- 3, or 5) and retained in the culture from the day of addition until ficient to promote DC differentiation (Fig. 1D). Amounts of E2 the cells were harvested on day 7. Exogenous E2 was most effec- above 0.1 nM did not further augment DC recovery. However, tive in augmenting CD11bint DC differentiation when added from viability of the cultures with added E2 remained poor, and the total day 0 of culture; the differentiation-promoting effects of E2 de- number of cells recovered was less than numbers recovered in creased daily as the culture progressed; numbers of CD11bhigh CDFCS cultures, suggesting that serum-free medium lacks other cells remained relatively constant (Fig. 2D). Thus, it is likely that Downloaded from factors needed for viability of all cell types in BM cultures. E2 acts on cells present early in the culture period; as the culture The DC in the E2-supplemented CDFCS displayed higher sur- progresses, such precursor cells appear to be depleted or have in- face CD86 expression than observed on DC differentiated in reg- sufficient time to complete differentiation. ular medium, while expression of MHCII and CD86 after LPS To confirm that the two distinct CD11cϩ populations distin- activation was similar in both cultures (Fig. 1E). The small num- guishable by differential CD11b expression in these BM cultures bers of CD11bϩ CD11cϩ cells that did develop in CDFCS without are DC, DIC microscopy was used to assess cell morphology. http://www.jimmunol.org/ added E2 (Fig. 1A) appear to be DC because they also expressed CD11cϩ CD11bhigh and CD11cϩ CD11bint populations were MHCII and CD86 (Fig. 1E); these cells may develop in response sorted with a flow cytometer and plated in dishes for several hours to the low amounts of E2 remaining in the hormone-deficient me- before microscopy. Cells from both CD11cϩ CD11bhigh and dium, or their differentiation may be E2 independent. These data CD11cϩ CD11bint populations that were present in BM cultures obtained from culture of BM in CDFCS or serum-free medium containing RegFCS or CDFCS ϩ E2 (0.1 nM) exhibit the den- indicate that E2 promotes DC differentiation by acting on DC pre- drites characteristic of DC (Fig. 3). These morphological data cor- cursors present in freshly isolated BM, and suggest that a threshold roborate our cell surface marker and functional data (see below) to amount of E2 is required to effect differentiation. indicate that the cells that differentiate in E2-supplemented CD- by guest on October 3, 2021 FCS are DC. E2 preferentially promotes the differentiation of CD11cϩ CD11bint DC BM-derived DC express ER, but not AR The DC-promoting effect of E2 also was observed in RegFCS. The E2 was effective at promoting DC differentiation in CDFCS at presence of E2 (0.01–10 nM) significantly increased the number of 0.1–1nM(27–272 pg/ml), at the KD of the ER, consistent with the differentiated CD11cϩ CD11bint DC, concomitant with a reduction concentration of E2 in adult mouse serum during female diestrus

FIGURE 3. CD11bhigh and CD11bint DC ex- hibit characteristic dendritic morphology. DC pop- ulations were sorted from day 7 BM cultures, and DIC images of live cells were obtained with an Olympus AX70 microscope, original magnifica- tion ϫ400. Shown are A, CD11cϩ CD11bhigh DC or B, CD11cϩ CD11bint DC from BM cultures containing CDFCS ϩ 0.1 nM E2. Shown are C, CD11cϩ CD11bhigh DC or D, CD11cϩ CD11bint DC from BM cultures containing RegFCS. The Journal of Immunology 1431

amount of ICI 182,780 increased between 1 and 100 nM, the num- ber of DC developing in BM cultures supplemented with both E2 and ICI 182,780 decreased 3- to 4-fold, indicating that E2 was competitively inhibited by ICI 182,780 and thus was acting via the ER to promote DC differentiation. The numbers of DC that dif- ferentiate in CDFCS alone also were reduced by addition of ICI 182,780, suggesting that this differentiation is E2 dependent. To confirm that differentiating DC require the E2 present in RegFCS, titrated amounts (1–100 nM) of ICI 182,780 were added to BM cultures containing RegFCS (Fig. 5B). Treatment of BM cells from day 0 with ICI 182,780 results in a 3- to 4-fold reduction in the number of CD11bint DC on day 7. The CD11bhigh DC were not significantly affected by ICI 182,780. These data indicate that ␣ ␤ FIGURE 4. BM cells and DC express ER and ER . AÐC, Immuno- ICI 182,780 preferentially inhibits the differentiation of the blots containing variable amounts (10–60 ␮g) of cell lysate isolated from CD11bint DC population and that these cells respond to E2 present all cells in DC cultures on day 7 were probed with rabbit antisera specific for ER␣,ER␤, or AR, as indicated. For ER blots, control lysate was from in RegFCS via ER. Culture of BM cells with both ICI 182,780 the breast cancer cell line MCF-7 that expresses ER␣ isoforms ranging (1–100 nM) and E2 (10 nM) showed that a 2- to 10-fold excess of between 63 and 78 kDa and ER␤ isoforms ranging between 56 and 62 kDa; ICI 182,780 was required to inhibit the DC-promoting effect of E2

for the AR blot, control lysate was from the prostate cancer cell line LN- (data not shown). Downloaded from CaP, which expresses the 110-kDa AR. D, Female (BM-F) or male The SERM tamoxifen may act as an agonist or an antagonist (BM-M) BM cells were lysed immediately upon isolation, and 20 ␮gof depending on the cell type (17). Because long-term systemic treat- cell lysate was electrophoresed, transferred to nitrocellulose, and probed ment of tamoxifen is used for breast cancer prevention, it was of with a rabbit antiserum specific for ER␣. interest to determine the effects of tamoxifen on DC differentiation. ϳ The KD of tamoxifen for the ER is 6 nM, or 3% of E2 (43); ϳ (25–35 pg/ml) and estrus (100–200 pg/ml) and in males (8–15 plasma levels typically achieved in human patients are 200 nM. http://www.jimmunol.org/ pg/ml) (15, 44). Conversely, DHT did not promote DC differen- When present during the 7-day culture period in medium contain- tiation at doses between 10 and 1000 nM. The KD of the AR also ing RegFCS, tamoxifen (10–500 nM) reduced the number of dif- is 0.1–1nM(29–290 pg/ml), and normal serum levels of testos- ferentiated DC, with a greater reduction of CD11bint cells at the terone are 0.1–0.8 ng/ml in females and 5–15 ng/ml in males (15, lower doses (Fig. 5C). Tamoxifen competed with E2 for effect, 44). Female and male cells in GM-CSF-supplemented BM cultures indicating its action via the ER, and has antagonist activity in BM on day 7 were shown to contain known ER, ER␣, and ER␤, but not cells, consistent with our observation that tamoxifen does not re- the AR, by immunoblotting with specific antisera (Fig. 4, AÐC). store DC differentiation in CDFCS (data not shown). Unfractionated BM cells analyzed directly ex vivo also expressed Treatment of cultures with ICI 182,780 or tamoxifen led to a ER (Fig. 4D). decrease in MHCII and CD86 on CD11cϩ CD11bhigh cells that by guest on October 3, 2021 remained in the cultures (Fig. 5D). Tamoxifen disrupts the pH of ER antagonists inhibit DC differentiation endocytic vesicles independently of the ER (45), which could in- To demonstrate that E2 is acting via the ER to promote DC dif- hibit intracellular trafficking of MHCII; however, reduction of ferentiation in CDFCS, the steroidal ER antagonist ICI 182,780 MHCII and CD86 was similar in ICI 182,780 and tamoxifen- was titrated into cultures containing 1 nM E2 (Fig. 5A). As the treated cultures, suggesting that it occurred as a consequence of ER

FIGURE 5. The ER antagonists ICI 182,780 and tamoxifen inhibit DC differentiation. A, The ability of E2 to restore DC differentiation in CDFCS is competitively inhibited by the ER antagonist ICI 182,780. BM cells were incubated in CDFCS Ϯ E2 (1 nM), and variable amounts of ICI 182,780 (1–100 nM) were titrated into the cultures, as indicated. Shown is the number of CD11cϩ DC that developed in each of these cultures in the absence (E) or presence (F) of supplemental E2. Results are representative of four independent experiments. B, ICI 182,780 competitively inhibits the ability of the E2 present in RegFCS to promote DC differentiation. BM cells cultured in RegFCS were treated with variable amounts (1–100 nM) of ICI 182,780 or DMSO alone for 7 days before assessment of CD11b and CD11c expression on resting DC by flow cytometry. The numbers of CD11cϩ CD11bhigh DC (E) or CD11cϩ CD11bint DC (F) at each ICI 182,780 are plotted. Results are representative of three independent experiments. C, Tamoxifen competitively inhibits the ability of the E2 present in RegFCS to promote DC differentiation. BM cells in RegFCS were cultured for 7 days with variable amounts of tamoxifen (1–500 nM). The numbers of CD11cϩ CD11bhigh DC (E) or CD11cϩ CD11bint DC (F) at each tamoxifen are plotted. Results are representative of four independent experiments. D, ER antagonists decrease expression of MHCII and CD86 on immature DC. Expression of MHCII (left panels) and CD86 (right panels) on control (shaded histogram), 10 nM ICI 182,780-treated (top panels, thick line), or 50 nM tamoxifen-treated (bottom panels, thick line) CD11cϩ DC was determined. Results are representative of four independent experiments. 1432 ESTROGEN PROMOTES DC DIFFERENTIATION ligation. Taken together, our observations show that levels of cell Estradiol-treated DC retain the ability to degrade and present surface MHCII and CD86 on DC are modulated by both ER ago- self and foreign Ags via MHCII nists and antagonists (Figs. 1E,2C, and 5D). To determine whether E2 altered the function of DC, the intrinsic ability of DC to process and present Ags via MHCII was moni- ␣ DC differentiation from BM cells lacking ER is inhibited tored using T cell hybrids as specific probes for peptide/MHC BM cells express both ER␣ and ER␤. To determine a role for ER␣ complexes. Because addition of E2 increases the number of DC, in DC differentiation, BM was isolated from female ER␣Ϫ/Ϫ CD11cϩ DC were purified from BM cultures (Ϯ10 nM E2) in (B6.129-Estratm1 N10) or control C57BL/6 mice and incubated RegFCS on day 7 using anti-CD11c magnetic beads. Purified DC without or with E2 (10 nM) in RegFCS (Fig. 6). DC differentiation from control and E2-treated cultures displayed equivalent levels of from the ER␣Ϫ/Ϫ BM (22% CD11cϩ cells) was reduced signifi- surface MHCII and CD86 after LPS activation; therefore, our T cantly compared with C57BL/6 BM (52% CD11cϩ cells), with the cell hybrid assay directly measured the presentation of specific greatest reduction in CD11bint cells (Fig. 6, A and D). The majority peptide/MHCII complexes. DC were incubated with the exoge- of DC that did develop from the ER␣Ϫ/Ϫ BM were CD11bhigh,a nous Ags HEL and IgM, activated by LPS, titrated in wells, and phenotype remarkably similar to control BM treated with ICI incubated with T cell hybrids specific for HEL 74–88 and IgM 182,780 (Fig. 6C). Addition of 10 nM E2 to the ER␣Ϫ/Ϫ BM cells 377–392 epitopes. No significant differences were observed in the resulted in differentiation of near normal numbers of both DC pop- ability of control or E2-treated DC to present these Ags, indicating ulations, comparable to DC numbers derived from untreated that E2-treated DC are fully functional (Fig. 8, A and B). Presen- C57BL/6 BM, but less than from E2-treated C57BL/6 BM (Fig. 6, tation of an epitope derived from the endogenous self Ag rab5 also

B and E). Similar data were obtained upon analyses of multiple was identical in control and E2-treated DC (Fig. 8C). These data Downloaded from ER␣Ϫ/Ϫ and C57BL/6 mice (2 females and 2 males of each strain). indicate that while E2 promotes the differentiation of DC, resulting These data indicate that ER␣ has an important role in mediating E2 in increased DC numbers and increased Ag-presenting function on responsiveness in DC precursors; however, if the ER␣Ϫ/Ϫ DC pre- a population level (data not shown), individual mature DC that had cursors are exposed to sufficient amounts of E2, then ER␤ is ca- differentiated in the presence of E2 do not differ in intrinsic ability pable of mediating those E2 responses, resulting in DC to present model Ags to T cells. differentiation. http://www.jimmunol.org/ DC that differentiate in E2-supplemented CDFCS stimulate Estradiol promotes DC differentiation without increasing DC proliferation of allogeneic naive T cells proliferation To determine whether the DC that had differentiated in E2-sup- Our data with E2-supplemented regular and hormone-deficient plemented (0.1 nM) CDFCS were functional, we assessed the abil- medium suggest that E2 promotes DC differentiation from precur- ity of purified, LPS-activated CD11cϩ DC (129S6, H-2b) to stim- sor cells, or the survival of DC or DC precursors. Alternately, E2 ulate proliferation of naive allogeneic (B10.M, H-2f) CD4ϩ T cells could induce more rapid proliferation of already differentiated DC. (Fig. 9). Compared with CD11cϩ DC present in BM cultures con- To monitor proliferation in BM cultures, cytosolic proteins in BM taining RegFCS, CD11cϩ DC from E2-supplemented CDFCS ex- cells were labeled on day 0 with the dye CFSE. BM cells incubated hibited superior ability to induce the proliferation of naive CD4ϩ by guest on October 3, 2021 in the absence or presence of E2 were analyzed on days 1, 3, 5, and T cells (Fig. 9A). Because the CD11cϩ CD11bint DC display 7 for intensity of CFSE and CD11c expression by flow cytometry higher levels of cell surface CD86, this observation may be ex- (Fig. 7, AÐC). Although E2-treated cultures had more CD11cϩ plained in part by the greater number of CD11cϩ CD11bint DC in cells by day 3, the intensity of the CFSE signal decreased in the the cultures containing E2-supplemented CDFCS (50% CD11bint) two populations equally between days 1 and 7, indicating that cells than in cultures containing RegFCS (38% CD11bint). The geomet- were dividing in the culture at similar rates. These data suggest that ric means of CD86 expression on the combined CD11cϩ DC pop- E2 promotes DC differentiation or survival, rather than increasing ulations were 144 in CDFCS and 71 in RegFCS. To determine proliferation of already existing DC. whether the small numbers of DC that differentiated in CDFCS

FIGURE 6. DC differentiation from ER␣Ϫ/Ϫ BM cells is inhibited. DC differen- tiation from ER␣Ϫ/Ϫ BM cells is signifi- cantly reduced, but may be restored by addi- tion of E2. BM cells from female C57BL/6 (AÐC) and ER␣Ϫ/Ϫ (D and E) mice were in- cubated in RegFCS without additives or with E2 (10 nM) or ICI 182,780 (10 nM) for 7 days before determination of CD11c and CD11b expression by flow cytometry. The percentages of CD11cϩ CD11bhigh and CD11cϩ CD11bint cells in each culture are indicated. Data are representative of four in- dependent experiments. The Journal of Immunology 1433

FIGURE 8. Estradiol-treated DC function to present Ags via MHCII. LPS-activated DC differentiated in RegFCS in the presence of excess E2 retain the intrinsic ability to present Ag via MHCII. Sorted CD11cϩ DC from control or E2-treated cultures, displaying comparable amounts of cell surface MHCII and CD86, were incubated overnight with A, soluble IgM (2 ␮g/ml) or B, HEL (400 ␮g/ml), followed by LPS activation, titration in wells, and incubation with Ag-specific T cell hybrids. C, LPS-activated DC also were incubated with T cells specific for the self Ag rab5. IL-2 production of T cells was assessed by HT-2 proliferation using an Alamar blue colorimetric assay; results are expressed as arbitrary units of OD at 570–600, average of duplicate wells.

Ͻ Downloaded from Responses of the T cells to APC in the absence of Ag were 0.01 OD570–600. Cognate peptide (1 ␮g/ml) was added to LPS-activated DC (400 cells) at the Ͼ time of the T cell assay and resulted in 0.5 OD570–600. Results are repre- sentative of two independent experiments.

ferentiation and function, we provided E2 or DHT during ex vivo differentiation of DC from murine BM cells in steroid hormone- http://www.jimmunol.org/ deficient medium. Remarkably, efficient DC differentiation was not supported by CDFCS or serum-free medium, and was restored upon addition of amounts of E2 normally found in FCS and mouse serum, while DHT did not have this effect. The CD11cϩ cells in these BM cultures may be divided into two groups based on the magnitude of CD11b expression (CD11bhigh vs CD11bint); imma- ture CD11bint DC exhibited higher, more uniform MHCII and CD86 expression than immature CD11bhigh DC. Addition of E2 by guest on October 3, 2021 induced selective accumulation of CD11bint DC, while CD11bhigh DC numbers were less affected by E2 levels. Conversely, treatment with ER antagonists or the absence of ER␣ primarily blocked the accumulation of the CD11bint DC. Thus, E2 preferentially pro- motes the development of one of two types of DC, distinguishable by levels of CD11b expression, that differentiate in GM-CSF-sup- FIGURE 7. E2 promotes DC differentiation without increasing DC pro- plemented BM cultures. liferation. A, Control and E2-treated DC divide at similar rates. BM cells Our data support the hypothesis that E2 promotes the differen- were labeled with CFSE on day 0 and cultured ϮE2 (10 nM) in RegFCS tiation of DC from precursors in the BM. During the 7-day culture for 7 days. An aliquot of cells from each culture was taken on day 1, 3, 5, period, as the number of CD11cϩ CD11bint cells increases, or 7 and analyzed for intensity of CFSE and CD11c expression. B, The Ϫ low ϩ CD11c CD11b cells are depleted. Recent work of others has percentages of CD11c DC on each day (ϮE2) are plotted; CD11c levels shown that CD11cϪ cells present on day 7 of GM-CSF-supple- greater than fluorescence intensity of 25 (log scale) were considered pos- ϩ itive for this graph. C, The total number of cells in each culture (ϮE2) did mented BM cultures include direct precursors of CD11c cells not vary significantly on each day. (46). Monitoring of cell division using the cytosolic dye CFSE showed that E2 did not increase proliferation of differentiated CD11cϩ DC, suggesting that E2 promotes the development of without addition of E2 were functional, unfractionated cells from CD11cϩ cells at the expense of CD11blow CD11cϪ precursor ϩ these cultures (containing 15% CD11c CD11bhigh DC) were in- cells. Furthermore, addition of E2 has maximal effect at the be- ϩ cubated with naive CD4 T cells (Fig. 9B). After LPS activation, ginning of the culture, consistent with E2 action on precursor cells the unfractionated cells elicited a minor amount of T cell prolif- rather than already differentiated DC. Alternately, E2 may regulate eration compared with purified DC from E2-supplemented CDFCS the survival of differentiated DC. cultures. However, on a per cell basis, the DC that differentiated in The influence of E2 on hemopoietic cell differentiation is not ϩ CDFCS elicited the proliferation of naive CD4 T cells nearly as unprecedented, because sustained elevated systemic E2 (or DHT) well as DC from cultures in E2-supplemented CDFCS, suggesting selectively depletes lymphoid-restricted progenitors (LinϪ IL- that they are fully functional. 7R␣ϩ c-kitlow TdTϩ) from murine BM and the thymus (47, 48). Conversely, decreased levels of systemic sex steroids resulted in Discussion greater numbers of BM B cell precursors. Although B and T cell Sex biases in lymphocyte-mediated autoimmune diseases and APC precursors were decreased in the LinϪ c-kithigh fraction of BM of expression of ER and AR suggest that sex hormones could directly mice with elevated systemic E2, myeloid precursors were present influence APC. To determine a role for sex hormones in DC dif- in normal numbers, and a common myeloid progenitor population 1434 ESTROGEN PROMOTES DC DIFFERENTIATION

hormone receptors in DC precursors will lead to a greater understand- ing of molecular events underlying DC differentiation. The relationship between the two DC populations observed in these GM-CSF-supplemented BM cultures remains unclear; how- ever, our data suggest that the CD11cϩ CD11bint population con- tains the more potent APC. Immature CD11bint DC exhibited higher, more uniform MHCII and CD86 expression than CD11bhigh DC, and showed a greater increase in cell surface MH- CII and CD86 after activation with bacterial LPS. E2 supplemen- tation of BM cultures increased the overall levels of MHCII and ϩ FIGURE 9. DC that differentiate in E2-supplemented CDFCS stimulate CD86 on CD11c DC in two ways: 1) by increasing the number the proliferation of naive allogeneic CD4ϩ T cells. A, CD11cϩ DC (129S6, of CD11bint DC, and 2) by increasing the level of cell surface H-2b) were purified from cultures containing CDFCS ϩ 0.1 nM E2 (F)or MHCII and CD86 on immature CD11bhigh and CD11bint DC. A RegFCS (f), LPS activated to induce maturation and titrated in wells be- similar increase in CD86 was found on immature DC that devel- ϩ f fore addition of purified CD4 T cells (B10.M, H-2 ). Unfractionated im- oped in E2-supplemented CDFCS. In contrast to effects on MHCII Ϫ mature DC from CDFCS ( E2) cultures were used as a negative control and CD86, E2 did not induce expression of CD40 on immature Ⅺ ϩ ϩ ( ). B, Purified CD11c DC from cultures containing CDFCS 0.1 nM DC, nor change CD40 expression on LPS-activated DC (our un- E2 (F) or unfractionated cells (containing 15% CD11cϩ DC) from CDFCS published data). Addition of E2 to cultures only on day 6–7 during (ϪE2) cultures (E) were LPS activated and titrated in wells before addition ϩ LPS activation did not increase surface expression of MHCII nor

of purified CD4 T cells. Unfractionated immature DC from CDFCS Downloaded from (ϩE2) cultures were used as a negative control (triangles). Proliferation of CD86, suggesting E2 does not directly modulate signals resulting T cells was assessed by addition of 0.5 ␮Ci/well of [3H]Tdr during the last from LPS ligation of Toll-like receptors that ultimately lead to 16 h of an 86-h incubation period. Proliferation of T cells alone or APC CD86 up-regulation (our unpublished data). The combination of alone was Ͻ100 cpm. Numbers next to symbols are the stimulation index 10 nM E2 and very low amounts of LPS (0.2 ng/ml) did not in- at the indicated responder to stimulator ratio. crease CD86 nor MHCII (data not shown), indicating that E2 does not synergize with suboptimal amounts of LPS to affect DC maturation. http://www.jimmunol.org/ (LinϪ c-kithigh Sca-1Ϫ IL-7R␣Ϫ CD34ϩ/Ϫ FcRII/IIIϩ/Ϫ) was un- Mechanisms that account for the increased MHCII expression changed. LinϪ c-kithigh cells with potential for lymphoid or my- on LPS-activated DC may apply to immature DC that develop in eloid differentiation were found to be directly sensitive to E2 in E2-supplemented cultures: increased MHCII synthesis, invariant serum- and stromal cell-free in vitro cultures supplemented with chain degradation, peptide loading, or transit of MHCII molecules stem cell factor, IL-7, and Flt2/Flk3 ligand (49). E2 addition (10 from intracellular vesicles to the cell surface (31). For example, in- nM) to both LinϪ c-kithigh and c-kitlow precursors resulted in in- flammatory stimuli normally lead to fewer MHCII molecules bound hibition of CD19ϩ B cells, but an increase (up to 2.5-fold) in to the invariant chain degradation intermediate CLIP and greater Gr-1ϩ myeloid cells, and elevation of absolute numbers of numbers of MHCII molecules bound to high affinity self or foreign by guest on October 3, 2021 CD45Rϩ CD19Ϫ cells. Taken together with our data, these obser- peptides (31). To determine whether E2 affects the self peptide rep- vations suggest that E2 has opposing effects on BM lymphoid and ertoire of surface MHCII molecules, DC were assessed for binding of myeloid differentiation, and that E2 may promote DC differentia- the anti-CLIP/I-Ab mAb 15G4 (56). Although E2-treated immature tion from a LinϪ c-kithigh myeloid progenitor (50). DC display higher surface MHCII than control DC, the amount of E2 acted via the ER on DC precursors because the ER antago- mAb 15G4 staining was identical on the two DC populations (our nists ICI 182,780 and tamoxifen blocked the differentiation of the unpublished data), suggesting that E2-treated DC contained a lower E2-responsive DC population and competed with E2 for effect. portion of MHCII molecules bound to CLIP. To determine whether CD11cϩ CD11bint DC differentiation from BM cells isolated from surface MHCII on E2-treated DC contained more tightly bound high ER␣Ϫ/Ϫ mice also was inhibited, but could be restored partially by affinity peptides, the ability of surface MHCII to bind exogenously exogenous E2. Thus, our data with BM cultures from ER␣Ϫ/Ϫ supplied peptides was monitored. DC incubated with the E␣52–68 mice suggest roles for both ER␣ and ER␤ in DC differentiation, peptide showed no difference in binding of the E␣52–68/I-Ab com- although ER␤ is unable to efficiently compensate for the lack of plex-specific mAb YAe (57); thus, a dramatic change in the affinity of ER␣ unless the cultures are supplemented with additional E2. surface MHCII-bound peptides did not result from E2 treatment (our These data support a model in which E2-regulated genes required unpublished data). for DC differentiation are more efficiently activated by ER␣ than Our experiments show that E2 promotes DC differentiation, re- ER␤; thus, exposure to higher levels of E2 is required for ER␤- sulting in increased numbers of CD11cϩ CD11bint DC that, upon mediated gene expression. Alternately, the E2-responsive myeloid LPS activation, display higher levels of MHCII and CD86 than the precursors may have lower numbers of ER␤ than ER␣; relative CD11cϩ CD11bhigh DC population. In vivo, the E2 produced in numbers of the two ER vary among cell types (14). ER␣ was extragonadal sites, e.g., bone or adipose tissue, is thought to exert shown previously to have a role in murine hemopoietic stem cell its biological influence locally (58), and therefore the local con- development and B cell maturation (51). centrations of E2 in peripheral tissues may increase numbers of Other steroid hormones have been shown to modulate human CD11cϩ CD11bint DC that display augmented levels of MHCII monocyte to DC differentiation or murine DC differentiation from and costimulatory molecules, leading to their greater presentation

BM in vitro. Although glucocorticoids and vitamin D3 inhibit DC of self Ags. Our data suggest a mechanism by which E2 could differentiation (52–54), the thyroid hormones triiodothyronine and enhance overall DC Ag-presenting function in vivo in the absence thyroxine were shown to promote DC differentiation from monocytes of inflammation and thus promote autoimmunity. E2 may render via an unknown mechanism (55). However, we did not observe an immature tissue DC more likely to achieve expression levels of effect of these thyroid hormones in the murine BM DC culture system MHCII/peptide complexes and costimulatory molecules necessary (R.M., unpublished data). Future dissection of signal transduction and to overcome naive T cell self-tolerance. Interestingly, in a murine gene regulatory pathways downstream of the ER and other steroid model of SLE, systemic E2 elevation alters thresholds for B cell The Journal of Immunology 1435 apoptosis and activation, resulting in accumulation of autoreactive 17. Dutertre, M., and C. L. Smith. 2000. Molecular mechanisms of selective estrogen B cells that would be deleted in normal mice (59). receptor modulator (SERM) action. J. Pharmacol. Exp. Ther. 295:431. 18. Shang, Y., and M. Brown. 2002. Molecular determinants for the tissue specificity Conversely, decreases in systemic E2 due to SERM treatment of of SERMs. Science 295:2465. hormone-responsive breast cancer may lead to reduced DC num- 19. Kaushic, C., F. Zhou, A. D. Murdin, and C. R. Wira. 2000. Effects of estradiol bers in these patients (60). In view of our data, therapeutic anties- and progesterone on susceptibility and early immune responses to Chlamydia trachomatis infection in the female reproductive tract. Infect. Immun. 68:4207. trogens may decrease numbers of DC capable of Ag presentation, 20. Salem, M. L., G. Matsuzaki, K. Kishihara, G. A. Madkour, and K. Nomoto. 2000. thereby limiting both pathogen and tumor immunity. SERM bind ␤-Estradiol suppresses T cell-mediated delayed-type hypersensitivity through suppression of antigen-presenting cell function and Th1 induction. Int. Arch. ER and differentially modulate ER-mediated transcription via the Allergy Immunol. 121:161. recruitment of cell type-specific coactivators or corepressors (13, 21. Saito, S., M. L. Foegh, N. Motomura, H. Lou, K. Kent, and P. W. Ramwell. 1998. 18). Thus, an ideal SERM for cancer prevention would have ag- Estradiol inhibits allograft-inducible major histocompatibility complex class II antigen expression and transplant arteriosclerosis in the absence of immunosup- onist activity in bone and myeloid cells, but antagonist activity in pression. Transplantation 66:1424. reproductive tissues. 22. Wilcoxen, S. C., E. Kirkman, K. C. Dowdell, and S. A. Stohlman. 2000. Gender- Our observations will lead to a greater understanding of the dependent IL-12 secretion by APC is regulated by IL-10. J. Immunol. 164:6237. 23. Liu, H. Y., A. C. Buenafe, A. Matejuk, A. Ito, A. Zamora, J. Dwyer, molecular requirements for differentiation of DC, as well as how A. A. Vandenbark, and H. Offner. 2002. Estrogen inhibition of EAE involves estrogen might influence immune responses in vivo. Sex hormones effects on dendritic cell function. J. Neurosci. Res. 70:238. currently are being manipulated during therapy for sex hormone- 24. Wira, C. R., M. A. Roche, and R. M. Rossoll. 2002. Antigen presentation by vaginal cells: role of TGF␤ as a mediator of estradiol inhibition of antigen pre- dependent cancers and autoimmune diseases such as multiple scle- sentation. Endocrinology 143:2872. rosis, and in postmenopausal women (60, 61). Such hormone ther- 25. Prabhala, R. H., and C. R. Wira. 1995. Sex hormone and IL-6 regulation of apies may alter DC differentiation and function, leading to either antigen presentation in the female reproductive tract mucosal tissues. J. Immunol.

155:5566. Downloaded from desired or detrimental effects on DC presentation of tumor Ags or 26. Hoek, A., W. Allaerts, P. J. Leenen, J. Schoemaker, and H. A. Drexhage. 1997. autoantigens. Dendritic cells and macrophages in the pituitary and the gonads: evidence for their role in the fine regulation of the reproductive endocrine response. Eur. J. Endocrinol. 136:8. Acknowledgments 27. Liu, J. W., D. D. Dawson, C. E. Peters, M. A. Baker, and A. M. Walker. 1997. We thank Dr. Alexander Rudensky for mAbs 15G4 and YAe; Dr. Ralph Estrogen replacement in ovariectomized rats results in physiologically significant levels of circulating progesterone, and co-administration of progesterone mark- Steinman for the GM-CSF gene-transfected J558L cells; Karlene Campo edly reduces the circulating estrogen. Endocrine 6:125. http://www.jimmunol.org/ for expert technical assistance; Lucy Brown and Claudio Spalla in the City 28. Miller, L., and J. S. Hunt. 1998. 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