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Blood, Vol. 95 No. 9 (May 1), 2000:
pp. 2875-2882
IMMUNOBIOLOGY
From the Turku Immunology Centre, Department of Medical
Microbiology, Turku University, Kiinamyllynkatu 13, 20520 Turku,
Finland.
Dendritic cells (DC) are professional antigen-presenting cells with
a unique capacity to initiate and regulate immune responses. Immature
CD1a+ DC can be cultured from CD14+
monocytes in the presence of interleukin (IL)-4 and granulocyte macrophage colony-stimulating factor in vitro. Results of this study
show that the nonsteroidal anti-estrogens toremifene and tamoxifen
inhibit this differentiation. In the presence of anti-estrogens the
cells lose CD14 expression, but remain CD1a
Dendritic cells (DC) are antigen-presenting cells (APC)
that differentiate from bone marrow-derived precursors.1,2
Because of their capacity to stimulate naive T cells, DC have a central role in the initiation3 of primary immune responses and
regulation of self-tolerance and autoimmunity.1,2 Immature
DC reside in peripheral nonlymphoid tissues, where they efficiently
capture and process antigens. Bacterial products, such as
lipopolysaccharide (LPS), or inflammatory cytokines drive the
maturation of DC, which is characterized by the up-regulation of the
major histocompatibility complex (MHC) class II and costimulatory
molecules CD80 (B7-1) and CD86 (B7-2). This results in an increased
capacity to stimulate T cells.1,2 DC migrate into T-cell
areas of secondary lymphoid tissues where they receive terminal
maturation signals via CD40 on the interaction with CD154 (CD40L) on
antigen-specific T cells.4,5 In response to CD40 ligation,
DC produce high levels of interleukin (IL)-12,4,5 a key
cytokine in the development of interferon (IFN)- Nonsteroidal anti-estrogens such as toremifene (TOR) and tamoxifen
(TAM) have a variety of tissue-specific effects. Depending on the
target cells, they can act as estrogen agonists or antagonists and are
therefore also called selective estrogen receptor modulators. In breast
tissue, anti-estrogens antagonize the effect of estrogen and are widely
used in the treatment of breast cancer. On the other hand, they seem to
mimic the beneficial effects of estrogen in cardiovascular tissue and
in bone.12,13 TAM has also been reported to be beneficial
in experimental models of autoimmune diseases such as collagen-induced
arthritis,14 adjuvant-induced arthritis,15 and
systemic lupus erythematosus (SLE).16,17 However, these
studies have provided little insight into the immunomodulatory mechanisms of TAM. TAM has been shown to enhance the production of the
anti-inflammatory cytokine transforming growth factor
(TGF)- The present study characterized the effect of nonsteroidal
anti-estrogens on the functional differentiation of DC and demonstrated that anti-estrogens inhibit DC differentiation by mechanisms
independent of ER or enhanced production of TGF- Reagents and antibodies
Cell isolation and culture
Flow cytometry, cytologic analysis, and immunofluorescence confocal microscopy (ICM) For immunofluorescence staining, the cells were incubated with PE-conjugated monoclonal antibodies (mAbs) or with nonconjugated mAbs for 30 minutes on ice, followed by incubation with isotype-specific secondary mAb. Before and after each incubation, the cells were washed twice with phosphate-buffered saline ([PBS] + 2% FCS + 0.01% NaN3). The cells were analyzed with a FACScan flow cytometer (Becton Dickinson) using CellQuest (Becton Dickinson) software. The data are expressed as mean fluorescence intensity ratios (MFIRs) (mean fluorescence intensity with mAb of interest/mean fluorescence intensity with control mAb). For cytologic examination the cells were cytocentrifuged and stained using a Dade Diff-Quik staining set (DADE, Düdingen, Switzerland). For ICM, the cultured cells were washed with PBS, allowed to settle onto glass slides, and fixed with 4% paraformaldehyde and methanol. The cells were then stained with anti-CD86 mAb or an isotype control Ab and the secondary fluorescein isothiocyanate-conjugated goat-antimouse Ab (Southern Biotechnology Associated). Confocal microscopy was performed using a Leica TCS SP Spectral Confocal Microscope and analyzed using the Leica Lasertechnik GmbH software (Heidelberg, Germany).Mixed lymphocyte reaction Adherent PBMC were first cultured in the presence of IL-4 + GM-CSF with or without anti-estrogens or IL-10 (100 U/mL) for 7 days. Alternatively, immature DC were activated with LPS (1 µg/mL) in the presence or absence of TOR or TAM for 36 hours. The cells were then irradiated (30 Gy) and counted, and 5 to 20 × 103 cells were cocultured with 2 × 105 Ficoll-Paque-isolated allogeneic PBMC for 5 days in U-bottom 96-well plates (Nunclon, Roskilde, Denmark). To determine the proliferative activity, 1 mCi 3H-thymidine (DuPont, Boston, MA) was added to each well 16 to 18 hours before terminating the culture. The cells were harvested to glass fiber filters (Wallac, Turku, Finland) with a 96-well harvester (Tomtec, Orange, CT). The radioactivity was measured with a 1450 Microbeta Plus liquid scintillation counter (Wallac).Detection of estrogen receptor-  -actin was amplified from the same
pool of cDNA. RT was omitted from the control RT reactions. PCR
amplification conditions were 95°C for 1 minute, 55°C for 50 seconds, 72°C for 1 minute for a total of 35 cycles. The second PCR
was performed by adding 1 µL of the primary PCR product in 50 µL
reaction mixtures containing 15 pmol/µL of nested ER- and ER-
primers, 0.2 mmol/L of each dNTP, 1 U of DynaZyme DNA polymerase, and
10× reaction buffer. Thirty-five cycles were performed for ER-
(95°C for 1 minute, 55°C for 50 seconds, 72°C for 1 minute)
and for ER- (95°C for 1 minute, 65°C for 50 seconds,
72°C for 1 minute). The PCR products were analyzed by
electrophoretic separations on a SeaKerm 1.5% agarose gel (FMC
Bioproducts. Rockland, ME) and transferred to Hybond-N+
Nylon membrane (Amersham International, Amersham, United Kingdom) for
hybridization with internal oligonucleotide probe. All the primer sets
for ER- and ER-, including the internal probes, were from
MedProbe (Oslo, Norway) and have been previously
described.25 Primers for human -actin were sense:
5'-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-3' and
antisense: 5'-GGG AGG TAG CAG GTG GCG TTT ACG CCG ATC-3'.
Cytokine measurements Levels of TGF-1 were measured using Quantikine immunoassay
kits obtained from R&D Systems and IL-10 levels using enzyme-linked immunosorbent assay (ELISA) kits obtained from CLB (Amsterdam, The
Netherlands). To detect latent TGF-1, the culture supernatant samples were activated with 1 N HCl for 10 minutes and neutralized by
adding 1.2 N NaOH + 0.5 HEPES. The antibodies for 2-site sandwich ELISA detecting the biologically active IL-12 heterodimer p70 were from
R&D Systems. All ELISAs were performed in duplicate according to the
instructions of the manufacturer.
Statistical analysis The paired Student t test was used for statistical analysis.
The effect of anti-estrogens on the functional differentiation of monocyte-derived DC Adherent PBMC were cultured in the presence of IL-4 + GM-CSF, which resulted in a typical phenotype of immature DC. After 7 days of culture, the cells were CD14 CD1a+
(Figure 1 and Table
1) and at this stage of differentiation CD83low/ (not shown). In the presence of TOR the
cultured cells lost CD14 expression, but remained mostly
CD1a, indicating that phenotypically the cells had
not differentiated into immature DC (Figure 1 and Table 1). Both TOR
and IL-10 inhibited the up-regulation of CD40 and CD80 during the DC
differentiation. Interestingly, the expression of CD86 increased in the
presence of TOR, whereas IL-10 had an opposite effect. The decrease in HLA-DR expression by IL-4 + GM-CSF was inhibited by 10 µmol/L TOR
and, to some extent, by IL-10 (Figure 1 and Table 1). Morphologically, cells treated with TOR had clearly fewer dendritic processes than the
cells cultured in IL-4 + GM-CSF alone, but they had lost the macrophage-like appearance that was completely retained in the presence
of IL-10 (Figure 2). The effect of TOR on
DC differentiation was also functionally significant, because
TOR-treated cells were less potent than immature DC in inducing the
proliferation of allogeneic T cells (Figure
3). To reveal whether there are any differences between TOR and TAM, we compared their effects on the
phenotype of monocyte-derived DC. As shown in Figure
4, the effect of TOR and TAM on DC
phenotype was identical.
Anti-estrogens inhibit the differentiation of DC by mechanisms not involving the ER Next we wanted to detect whether ER transcripts are expressed in cells representing various stages of DC differentiation. Therefore, resting CD14+ monocytes, cultured immature CD1a+, and mature CD83+ DC were sorted by MACS. In RT-PCR analysis both ER- and ER- mRNA were expressed, although
at low levels, in CD14+, CD1a+, and
CD83+ cells (Figure 5). These
results indicated that ER- and ER- are expressed at all stages of
DC differentiation and that the effect of anti-estrogens could be
mediated via the ER. TAM and TOR bind to the ER at similar affinity,
which is about 3% of the binding affinity of the natural ER ligand
17-estradiol (E2),26 whereas the binding affinity of a
pure antagonist ICI 182780 is about 90% of that of E2.27
However, E2 or ICI 182780 did not modulate the phenotype of immature
DC, when administered at concentrations up to 1 µmol/L (Figure
6). Furthermore, 1 µmol/L of E2 or ICI 182780 could not counteract the effect of 5 µmol/L TOR or TAM on the
expression of CD1a (Figure 7), suggesting
that TOR and TAM inhibit DC differentiation by ER-independent
mechanisms. To exclude the effect of endogenous hormones in the culture
medium, we also studied the effect of TOR, TAM, E2, and ICI 182780 on DC differentiation using dextran-charcoal stripped FCS and medium without phenol red, which has weak estrogen-like effects.28 Under these culture conditions, 10% to 50% of the cells expressed CD1a and about 50% of the cells remained CD14+ after 7 days of culture with IL-4 + GM-CSF. However, only TOR and TAM, but
not E2 or ICI 182780, had a significant effect on the expression of the
phenotypic markers of DC (data not shown).
TOR-induced changes in the DC phenotype are not due to the enhanced
production of TGF- 1 has been suggested to be a
potential mechanism by which anti-estrogens have therapeutic effects in
autoimmune diseases.14 When TGF-1 is added into culture
with IL-4 + GM-CSF, the monocyte-derived cells differentiate into
Langerhans cells, which express E-cadherin.29 We observed that addition of TOR into culture with IL-4 + GM-CSF enhanced the
expression of E-cadherin in monocyte-derived cells (unpublished data).
Therefore, we studied the effect of TOR on the production of TGF-1
in adherent PBMC cultured together with IL-4 + GM-CSF. After 1, 3, and 7 days, no detectable levels of the biologically active form of
TGF-1 were found in the culture supernatants. In contrast to active
TGF-1, high levels of latent TGF-1 were observed in all cultures.
Only a minority of the latent TGF-1 was produced by
monocytes/immature DC, because the FCS in the culture medium contained
high amounts of latent TGF-1 (Table 2).
Addition of TOR to the culture with IL-4 + GM-CSF did not increase
the levels of active or latent TGF-1 (Table 2), indicating that TOR
does not inhibit DC differentiation by enhancing the production of
TGF-1. To exclude the possibility that TOR inhibits DC
differentiation by enhancing the production of IL-10, we also measured
IL-10 levels after 1, 3, and 7 days in culture. However, IL-10
levels were undetectable or very low and were not found increased in
the presence of TOR (data not shown).
Anti-estrogens exert differential effects on the expression of costimulatory molecules CD80 and CD86 The expression of CD80 and CD86 on monocyte-derived cells seemed to be differentially regulated by anti-estrogens (Table 1 and Figures 1, 4, and 6). A similar effect was also observed in macrophages from the synovial fluid of patients with rheumatoid arthritis (unpublished data). To characterize these findings in more detail, we studied the kinetics of CD80 and CD86 expression during DC differentiation. CD80 was not expressed on freshly isolated monocytes but it was up-regulated in the presence of IL-4 + GM-CSF. This up-regulation was inhibited by both TOR and IL-10 (Figure 8A). On the other hand, CD86 was already expressed on freshly isolated monocytes. In the presence of IL-4 + GM-CSF, CD86 was strongly up-regulated after 24 hours of culture and its expression decreased thereafter. IL-10 inhibited the up-regulation of CD86, whereas TOR clearly enhanced and prolonged its expression (Figure 8B). E2 or ICI 182780 did not enhance CD86 expression or modulate the TOR-induced up-regulation of CD86, suggesting that anti-estrogens regulate CD86 expression by ER-independent mechanisms (Figure 9). To reveal whether TOR affects the distribution of CD86 protein between the cell surface and the intracellular reservoir recently described in monocytes,30 we performed ICM. When monocytes were cultured with IL-4 + GM-CSF for 48 hours, CD86 protein was found to be localized both on the cell surface and in the intracellular compartment, whereas in the presence of TOR, CD86 protein was concentrated on the cell surface (Figure 10).
The effect of anti-estrogens on the terminal maturation of DC To evaluate the capacity of anti-estrogens to interfere the maturation of DC, adherent PBMC were cultured with IL-4 + GM-CSF with or without TOR and stimulated with CD40L. As shown in Figure 11A, the TOR-treated cells were clearly impaired in producing the biologically active IL-12 p70 heterodimer when cocultured with CD40L-transfected cells, showing that they could not respond normally to the CD40 ligation. A similar effect was also observed with TAM (Figure 11B). Next, we stimulated immature DC with LPS or TNF-. These cultures resulted in mature CD83+ DC
(Table 3 and Figure 2) with high capacity
to stimulate the proliferation of allogeneic T cells (Figure 11C). TOR
significantly inhibited LPS- and TNF--induced up-regulation of
CD1a, HLA-DR, and LPS-induced up-regulation of CD83, whereas the
up-regulation of other molecules was inhibited to a lesser degree
(Table 3). Similar findings were also observed with TAM (data not
shown). The effect of TOR and TAM on DC maturation was also
functionally significant, because anti-estrogen-treated cells were
less capable in inducing the proliferation of allogeneic T cells than
their fully matured counterparts (Figure 11C). Altogether, these data indicated that terminal maturation of DC can also be partially inhibited by anti-estrogens.
Nonsteroidal anti-estrogens have been suggested to have therapeutic potential in the treatment of autoimmune diseases. In experimental models TAM has been reported to inhibit the development of collagen-induced arthritis14 and adjuvant-induced polyarthritis,15 and to have beneficial effects in murine models of SLE.16,17 In the present study we provide phenotypical, morphologic, and functional evidence that anti-estrogens are able to inhibit the differentiation of monocyte-derived DC in vitro. DC have a central role in the initiation and regulation of immune responses1,2 and a unique ability to produce high levels of IL-12.4-6 IL-12 is the major cytokine responsible for the generation of inflammatory Th1 cells,4-6 which are the predominant cells in various autoimmune diseases.31 We found that cells treated with anti-estrogens were less potent than immature DC in inducing allogeneic T-cell proliferation and clearly impaired in producing the biologically active IL-12 p70 heterodimer after CD40 ligation. This suggests that by inhibiting the functional differentiation of DC, anti-estrogens may inhibit the development of inflammatory Th1 responses. It also implies that the beneficial effects of anti-estrogens in experimental models of autoimmune disease may occur at the level of APCs. We found no differences between the effects of TOR and TAM. Importantly, both anti-estrogens inhibited DC differentiation at concentrations that can be achieved in vivo. Administration of high-dose TAM and TOR can result in 4 to 6 µmol/L and 10 to 15 µmol/L plasma concentrations, respectively.32,33 Because TOR has been shown to be less genotoxic than TAM in animal studies,34-38 it could have some benefits over TAM when administered at high doses.
We thank Ms Marianne Laine for expert technical assistance. We thank Dr P. Lane for providing the CD40L transfected J558L cell line, Dr A. E. Wakeling for providing ICI 182780 and Dr T. F. Tedder for providing the anti-CD83 mAb. We acknowledge Dr J. Uksila, Dr J. Punnonen, Dr M. Möttönen, Dr D. Smith and Dr H. Arvilommi for their critical reading of the manuscript and Dr P. Lakkakorpi for assistance with the confocal microscopy.
Supported by the Academy of Finland, Turku Graduate School of Biomedical Sciences, Turku University Central Hospital special funds, and the South-Western Cancer Society of Finland.
Submitted May 24, 1999; accepted October 22, 1999.
Reprints: Janne Komi, Turku Immunology Centre, Department of Medical Microbiology, Turku University, Kiinamyllynkatu 13, 20520 Turku, Finland; e-mail: janne.komi{at}utu.fi.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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Differential expression of estrogen receptors |
Top
Abstract
Introduction
-producing Th1
cells.
B,
and -
