Molecular Human Reproduction Vol.8, No.9 pp.

849854, 2002
Dioxin stimulates RANTES expression in an in-vitro model
of endometriosis
Dong Zhao
1
, Elizabeth A.Pritts
1
, Victor A.Chao
1
, Jean-Francois Savouret
2
and
Robert N.Taylor
1,3
1
Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California,
San Francisco, HSE 1689, CA 941430556, USA and
2
INSERM U 135, Hormones, Ge`nes et Reproduction,
94270 Le Kremlin-Bicetre, France
3
To whom correspondence should be addressed. E-mail: rtaylor@socrates.ucsf.edu
The industrial contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is associated with inammatory disorders in women
and other mammals. The current studies were performed to investigate the effect of TCDD on RANTES expression in an
in-vitro model of endometriosis. The biochemical effects of dioxins are mediated by binding to aryl hydrocarbon receptors
(AhR). This study showed that both normal and endometriotic endometrial stromal cells express AhR protein, which was
observed to be down-regulated by 4060% after exposure to TCDD. Treatment with TCDD for 24 h increased the luciferase
activity of the RANTES promoter by 2.5 1.0-fold in stromal cells derived from normal endometrium and endometriotic
implants. When AhR were over-expressed in these cells, luciferase activity increased 6.1 1.4-fold, and RANTES protein
secretion increased from undetectable to 31 10 pg/100 000 cells. TCDD failed to activate a RANTES construct with a mutated
dioxin response element. Other AhR ligands had similar effects to TCDD on RANTES transcription and secretion. Control
transfections using tumour necrosis factor (TNF)- and nuclear factor (NF)-B response element reporters indicated that these
pathways are not activated by TCDD in endometrial stromal cells. This study has demonstrated that functional AhR are present
in endometrial and endometriotic stromal cells and that TCDD up-regulates the expression of RANTES, providing a possible
mechanistic link between dioxin exposure and chemokine expression in endometriosis.
Key words: AhR/dioxin/endometrial stromal cells/endometriosis/RANTES
Introduction
Endometriosis is a common gynaecological disorder, affecting
515% of women of reproductive age (Goldman and Cramer,
1990; Haney, 1990). Among theories proposed to explain this
anomaly, Sampsons hypothesis of retrograde menstruation is most
widely accepted (Sampson, 1927). This theory, however, falls short
in completely explaining the pathogenesis of this disease. Retrograde
menstruation occurs in 90% of women (Halme et al., 1984), and
the vast majority manifest no pathology. Therefore, other factors
must be involved in the development of endometriosis.
The aryl hydrocarbon receptor (AhR) is a nuclear receptor
whose natural ligand has not yet been identied. Similar to steroid
receptors, AhR acts as a ligand-dependent transcription factor that
mediates a broad range of responses induced by xenobiotic
toxicants including arylhydrocarbons, dioxins, furans and coplanar
polychlorobiphenyls (Fernandez-Salguero, 1996).
The AhR has multiple domains including a basic helixloop
helix (HLH) domain near the N terminus; the basic region
contributes to DNA binding and the HLH region to protein
protein dimerization. The C terminal segment contains a complex
transactivation domain, consisting of multiple stimulatory and
inhibitory subdomains (Whitlock, 1999). The toxic biochemical
effects of dioxins are mediated by AhR binding. The liganded
AhR associates with its arylhydrocarbon receptor nuclear translocator
European Society of Human Reproduction and Embryology 849
(ARNT) and moves from the cytoplasm to the nucleus. The
heteromeric complex acts as a signal transducer and transcription
factor for target genes, including cytochromes P450 (1A1, 1A2,
1B1), detoxifying phase II enzymes, and growth regulatory genes
involved in cell proliferation, differentiation and inammation
(Whitlock, 1990; Sutter et al., 1991). AhR/ARNT complexes
function as transcription factors by binding to dioxin response
elements (DRE) within target genes (Whitlock, 1993). The most
potent AhR ligand is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),
a well known environmental pollutant. TCDD is an established
carcinogen in experimental animals (Chahoud et al., 1989; Huff
et al., 1991), but its effects on humans are less well studied
(Johnson, 1993; Bertazzi et al., 2001).
In monkeys, exposure to dioxin correlates directly with an
increased prevalence and severity of endometriosis (Rier et al.,
1993; Yang et al., 2000). However, the true effect of TCDD on
human endometriosis remains a matter of debate (Koninckx et al.,
1994; Mayani et al., 1997; Scialli, 2001).
Endometriosis is associated with abnormal immune responses in
women affected by this disease (Steele et al., 1984; Braun and
Dmowski, 1998). The peritoneal environment in these women
reveals a predominant monocytic inltrate (Halme et al., 1987;
Oosterlynck et al., 1994). RANTES is a key chemokine expressed
by normal endometrium (NE) and endometriotic implant (EI)
D.Zhao et al.
stromal cells (Hornung et al., 1997). The concentration (Khorram
et al., 1993) and bioactivity (Hornung et al., 2001a) of RANTES
are elevated in the peritoneal uid of women with endometriosis.
We postulated that xenobiotic agents, such as TCDD, might
inuence the immune response in women with endometriosis. An
in-vitro model of endometriosis was therefore used to investigate
the effect of TCDD on RANTES gene expression.
Materials and methods
Materials
A total of 10 nmol/l TCDD (Radian Corp., CA, USA) was added in 0.1%
ethanol carrier directly to the cells. A total of 1 mol/l 9,10-dimethyl-1,2-
benzanthracene (DMBA) and benzo(a)pyrene (BaP) (Sigma Chemicals, St
Louis, MO, USA) were suspended in 0.1% ethanol and mixed before
treatment (Casper et al., 1999). Vehicle controls (0.1% ethanol) were used
as a comparison.
Subjects
Healthy women with ovulatory menstrual cycles who had not received
hormones or GnRH agonist therapy for at least 6 months before surgery
were enrolled in this study. Subjects undergoing elective surgery for
leiomyomata uteri or other benign uterine conditions were recruited for
NE biopsies. Women with laparoscopically conrmed endometriomas were
recruited for EI biopsies. Biopsies were obtained after the patients provided
written informed consent, under a study protocol approved by the Committee
on Human Research at the University of California, San Francisco (UCSF).
Endometrial tissue was obtained by Pipelle

(Cooper Surgical, Shelton,

CT, USA) aspiration biopsies. All samples were obtained in the proliferative
phase of the cycle and this was conrmed histologically according to
established criteria (Noyes et al., 1950).
Cell culture
In brief, dispersed stromal cells were separated from glandular cells and
debris by ltration through narrow gauge sieves with apertures of 40 m.
Stromal cells were plated and subcultured to eliminate contamination by
macrophages or other leukocytes in Modied Eagles Medium (MEM)-
supplemented with 10% fetal calf serum (FCS), antibiotics and nucleosides.
All experiments were initiated after 24 h incubation in 2.5% charcoal-
stripped FCS. Extensive characterization of cell cultures prepared using
this protocol previously conrmed 95% purity with cells retaining
functional steroid receptors and cytoskeletal markers of their endometrial
stromal origin (Ryan et al., 1994).
Immunocytochemistry
NE and EI stromal cells were plated onto Lab-Tek (Miles Laboratories,
Naperville, IL, USA) 8-chamber slides and stained using the Vectastain Elite
ABC Kit (Vector Laboratories, Burlingame, CA, USA). Immunoperoxidase
staining was performed using mouse monoclonal IgG antibodies against
human vimentin (1:100 dilution; Sigma), rabbit polyclonal IgG antibodies
against human cytokeratin (1:200 dilution; Sigma) and AhR (2 g/ml;
Santa Cruz Biotechnology, CA, USA). Non-immune, species-specic
antibodies at the same concentrations were used as controls.
Western blots
Media were removed by aspiration, the cells were washed with 10 ml
cold Tris-buffered saline (TBS), and scraped into 500 l of extraction
buffer (20 mmol/l Tris, pH 8.0, 137 mmol/l NaCl, 10% glycerol, 1%
Triton X-100, 2 mmol/l EDTA, 1 mmol/l Pefablock, 2 mol/l leupeptin,
0.14 IU/ml aprotinin, 1 mmol/l vanadate). Cells were lysed by repeated
freezethaw cycles and centrifuged at 15 000 g for 15 min at 4C. Super-
natants of extracts were denatured in sample buffer and 100 g total
protein sample was electrophoresed on 8% polyacrylamide sodium dodecyl
sulphate (SDS) gels at 30 mA for 2 h. Proteins were transferred to
Immobilon PVDF membranes (Millipore) for 2 h in a semi-dry transfer
system (Hoefer Scientic Instruments, San Francisco, CA, USA). Membranes
were blocked for 1 h in TBS-Tween plus 5% instant non-fat dry milk,
850
then probed with 1.0 g/ml anti-AhR polyclonal antibody H-211 (Santa
Cruz Biotechnology) at 4C overnight. The membranes were washed in
the same solution before addition of 1:5000 dilution of horseradish
peroxidase-conjugated anti-rabbit IgG, and specic bands were visualized
by the ECL (Amersham) method on Kodak X-omat lm. Bands were
quantied by transmission scanning using NIH Image 1.60 software.
RTPCR
Total RNA was extracted from stromal cell cultures (NE and EI) using
the TRIzol reagent kit (Gibco BRL, Gaithersburg, MD, USA). The
conditions for preparing the internal standards, the RTPCR reaction and
the primers used for semi-quantitative RTPCR have been described
previously (Hornung et al., 1997). Briey, specic oligonucleotide primers
were designed to amplify sequences from human RANTES mRNA (193
bp) and human GAPDH (240 bp) as a positive control (Ercolani et al.,
1988) and reaction products were separated on 2% agarose gels.
ELISA
A specic sandwich ELISA was used to quantify RANTES in conditioned
media (Quantikine; R&D Systems, MN, USA). Aliquots of culture
supernatants were each tested in duplicate at several dilutions to ensure
linearity and compared with reference standards of recombinant human
RANTES. In our laboratory, the assay is sensitive to 8 pg/ml, with intra-
and inter-assay coefcients of variation of 3 and 7% respectively. The
assay is specic for human RANTES, with no known cross-reactivity with
other cytokines or chemokines (R&D Systems Catalogue, 2001).
Gene promoters and luciferase assays
A full length (961 bp) fragment of the human RANTES promoter (Nelson
et al., 1993) was subcloned into the pGL2 vector (Promega, Madison, WI,
USA). The DRE of the wild-type RANTES promoter CACGCG at nucleotide
positions 821/816 was mutated to CTCGAG with QuickChange

site-
directed mutagenesis kits (Stratagene) using oligonucleotides containing
the desired mutation. Nuclear factor (NF)-B and tumour necrosis factor
(TNF)- response element reporter constructs were gifts from Dr Dale
Leitman (UCSF). The recombinant human (r)AhR vector [psG5-(hu)AhR]
was engineered in the laboratory of Dr Jean-Francois Savouret (INSERM
Unite 135, Bicetre, France) from a sequence generously provided by Dr
Chris Bradeld (McArdle Cancer Research Center, University of Wisconsin,
USA). All constructs and mutants were sequenced by the UCSF Biomolecular
Resource Center to verify that the correct sequences and mutations
were present.
Transient transfections were performed in human NE and EI stromal
cells grown in MEM- with 2.5% FCS and antibiotics in 12-well plates
at ~50% conuence. A total of 0.45 g of pGL2-RANTES promoter
(rey luciferase, experimental reporter) was added to each well using
QIAGEN Effectene

reagent (Valencia, CA, USA). The RANTES promoter

transfection efciencies were normalized to an independent control plasmid
(0.1 g renilla luciferase reporter) co-transfected simultaneously. In some
experiments, AhR was over-expressed by co-transfection of an expression
vector (0.25 g/well). Salmon sperm DNA was added to normalize total
transfected DNA in each well. The results are presented as a percentage
of luciferase activity between treated and untreated cells after correcting
for transfection efciency. Each reporter vector was assayed in at least
three independent cultures. The same amount of empty pGL2 vector was
analysed in pilot experiments as a control and revealed low basal activity.
Data are presented as mean SD. Comparisons between treatments
were evaluated using Wilcoxon non-parametric tests with StatView software.
Two-tailed tests with P 0.05 were considered signicant.
Results
Immunocytochemistry revealed that NE and EI stromal cells
express AhR. The receptor protein was localized in both the
cytoplasm and nuclei of endometrial stromal cells (Figure 1).
Vimentin and cytokeratin served as positive and negative controls
respectively for the immunocytochemistry analyses.
The specicity of the AhR immunolocalization was conrmed
AhR activation induces RANTES expression
Figure 1. Immunocytochemisty of endometrial stromal cells. (A,B) Normal endometrium (NE) and endometriotic implant (EI) stromal cells stained with
anti-vimentin antibody as a positive control. (C,D) NE and EI stromal cells stained with anti-cytokeratin antibody as a negative control. (E,F) NE and
EI stromal cells stained with anti-AhR antibody; AhR is present in both the cytoplasm and nucleus.
Figure 2. TCDD exposure decreases the amount of AhR protein.
Western blot of normal endometrium (NE) and endometriotic implant
(EI) stromal cells lysates, probed for human AhR (110 kDa). Cells were
treated with or without TCDD (10 nmol/l) for 2 days. Stromal cells
transfected with rAhR vectors were included as positive controls.
by Western immunoblots. Western blot analysis of samples from
isolated endometrial stromal cells revealed a dominant band
corresponding to 110 kDa as reported for the human AhR (Probst
et al., 1993; Wang et al., 1995). Treatment with TCDD caused a
4060% decrease in AhR protein in NE and EI stromal cells
(Figure 2).
To assess the expression of RANTES mRNA in cultured
endometrial stromal cells treated with and without TCDD for 48 h,
851
Figure 3. TCDD up-regulates RANTES mRNA. Semi-quantitative
RTPCR of normal endometrium (NE) and endometriotic implant (EI)
stromal cells treated with or without TCDD. TCDD treatment increased
RANTES mRNA over the basal amount.
we used semi-quantitative RTPCR assays. TCDD treatment
increased the expression of RANTES mRNA in NE and EI stromal
cells by ~2-fold (Figure 3). Intron-spanning primers used to amplify
transcripts of a constitutive gene, GAPDH, indicated that the RNA
D.Zhao et al.
Figure 4. TCDD treatment increases RANTES protein secretion.
Supernatants of normal endometrium (NE) and endometriotic implant
(EI) stromal cells treated with TCDD were analysed for RANTES
production by ELISA. TCDD treatment increased the secretion of
RANTES protein by stromal cells transfected with rAhR, but not in
non-transfected cells. *P 0.05 compared with basal RANTES
secretion.
Figure 5. TCDD activates RANTES gene expression. Luciferase reporter
gene assay in endometrial stromal cells using the wild-type RANTES
promoter reporter construct (961 bp, 0.3 g/well). Transfection
efciency was normalized by renilla luciferase. In the absence of
rAhR vectors, TCDD increased luciferase activity by 2.5 1.0-fold,
*P 0.05 compared with control RANTES expression. In the presence
of rAhR vectors, TCDD treatment increased luciferase activity by 6.1
1.4-fold, *P 0.05 compared with controls. Even in the presence of
rAhR, TCDD did not increase luciferase activity from a RANTES
construct with a mutated DRE site.
preparations were of good quality and not contaminated by
genomic DNA.
With over-expression of AhR (Figure 2, lanes 3 and 6),
enhanced production of RANTES protein was detected in endo-
metrial stromal cells treated with TCDD for 48 h; RANTES
secretion by NE and EI stromal cells was increased from
undetectable to 31 10 pg/100 000 cells (n 3, P 0.01,
Figure 4).
To explore the role of specic domains within the RANTES
gene promoter, endometrial stromal cells were transiently transfected
with wild-type RANTES promoter constructs cloned upstream of
the rey luciferase reporter gene.
For the full length RANTES construct (961 bp of 5 anking
DNA), containing a single DRE consensus motif, 24 h treatment
with TCDD increased luciferase activity by 2.5 1.0-fold (P
0.05) in stromal cells from both NE (n 3) and EI (n 3)
sources. When rAhR was over-expressed in these cells, the
luciferase activity increased 6.1 1.4-fold (P 0.05, Figure 5).
To further investigate the role of this DRE site, we used site-
directed mutagenesis to disrupt DRE in the RANTES promoter
construct (Figure 5). With the mutated DRE construct (Figure 6),
852
Figure 6. Cartoon of RANTES reporter construct. 961 wild-type (wt)
construct reveals one Dioxin Responsive Element (DRE) at 821 bp.
The mutations introduced into the DRE site of the mutant construct are
shown.
Figure 7. TCDD fails to activate NF-B and TNF response elements.
(A) A total of 0.3 g NF-B responsive element reporter construct were
co-transfected into endometrial stromal cells. TCDD treatment had no
effect on NF-B signalling. (B) A total of 0.3 g TNF-responsive
element reporter constructs were co-transfected into endometrial stromal
cells. TCDD treatment had no effect on TNF-RE activation.
TCDD failed to increase luciferase activity even when rAhR was
over-expressed (n 3, Figure 5).
TCDD exposure had no effect on endometrial stromal cells
transiently transfected with NF-B or TNF- response element
constructs in the presence of rAhR. The relative luciferase activities
were 1.0 0.2-fold (n 5, Figure 7A) and 1.2 0.1-fold (n
5, Figure 7B) respectively. Neither of the results were statistically
signicant. By contrast, TNF- stimulated the TNF- response
element luciferase construct 3-fold (data not shown).
Cells treated with a combination of DMBA/BaP (alternative
AhR ligands) showed similar effects on RANTES as determined
by both luciferase activity and protein secretion (data not shown).
Discussion
Dioxins are a family of halogenated heterocyclic hydrocarbon by-
products of industrial processes such as paper bleaching and
herbicide synthesis. Dioxins directly alter the expression of target
AhR activation induces RANTES expression
genes (Whitlock, 1990) or may act as anti-estrogens or weak
estrogens through interference with the estrogen receptor (Safe
et al., 1991). The increased incidence of endometriosis in
populations from industrialized countries has suggested a possible
link with exposure to dioxins and other AhR ligands (Rier et al.,
1993; Yang et al., 2000). However, social as well as environmental
factors could be responsible for this association. Later age at rst
pregnancy, shorter duration of breast-feeding and smaller family
size may contribute to the risk of endometriosis. While a study
by Pauwels et al. showed an increased odds ratio of 4.3 for the
association of serum dioxin equivalents and endometriosis, this
failed to reach statistical signicance (Pauwels et al., 2001).
Whether endometriosis is associated with high exposure to dioxin-
like compounds remains to be proven.
Our study examined the presence and activation of AhR in
cultures of human NE and EI stromal cells. We found AhR protein
to be localized in both cytoplasm and nuclei of endometrial stromal
cells. Western blotting indicated that AhR protein concentrations
were decreased by TCDD treatment, reportedly through the
ubiquitinproteasome pathway (Pollenz, 1996; Ma and Baldwin,
2000). Our ndings are of interest with respect to a recent study,
which reported high levels of AhR mRNA in endometriotic ovarian
cysts (Khorram et al., 2002). From these and our results,
we suggest that the relationship between dioxin exposure and
endometriosis is not direct. The co-expression of other factors,
such as cellular AhR and ARNT are necessary to activate the
transcription of target genes. The potential AhR modulator
7-ketocholesterol (Savouret et al., 2001) might also inuence the
effect of dioxin exposure in this pathway.
At least two phenomena appear to limit RANTES production
in our in-vitro model. One is the down-regulation of AhR by
TCDD (Ma and Baldwin, 2000) and the other is the spontaneous
loss of nuclear receptors in cultured cells (Sadovsky et al., 1992).
To restore TCDD responsiveness in our cells, rAhR expression
vectors were constructed and transfected. Under this condition,
TCDD treatment for 48 h dramatically increased RANTES secretion.
A combination of DMBA and BaP, also ligands for AhR, caused
a similar stimulation in RANTES secretion. This nding supports
a generalized effect of AhR ligands on this inammatory pathway.
To investigate whether the induction of RANTES secretion is
transcriptionally regulated, we established a model system of
transiently transfected human RANTES promoter-luciferase reporter
constructs in primary human NE and EI stromal cells. Treatment
with TCDD increased luciferase activity up to 6-fold. A similar
stimulation was noted using DMBA and BaP. However, when we
mutated the DRE site, TCDD treatment no longer increased
RANTES expression. These data indicate that TCDD up-regulates
RANTES gene expression, and that this effect is both AhR- and
DRE-dependent.
A previous study from our group showed that NF-B activation
is responsible for interleukin-1 stimulation of RANTES gene
expression in endometriotic stromal cells (Lebovic et al., 2001).
It was also reported that TNF- treatment can increase RANTES
mRNA and protein production in endometrial cells (Altman et al.,
1999; Hornung et al. 2001b). To test the hypothesis that TCDD
could directly activate NF-B or TNF- and thereby stimulate
RANTES gene expression, we transiently transfected NF-B or
TNF- response element luciferase constructs into endometrial
stromal cells. Exposure to TCDD failed to activate NF-B or
TNF- response elements in the absence or presence of over-
expressed AhR. These ndings suggest that the increase in
RANTES expression by AhR complexes is not mediated through
NF-B or TNF- pathways.
853
In conclusion, the combination of TCDD and AhR up-regulates
the expression of RANTES via a pathway that appears to be
DRE-dependent and independent of TNF- and NF-B activation.
Other AhR ligands have similar effects. These ndings provide a
mechanistic link between xenobiotic exposure, macrophage recruit-
ment and inammation in endometriosis.
Acknowledgements
The immunocytochemistry expertise of Ms Emily Ricke is gratefully
appreciated. This work was supported by a National Institute of Health
grant from the NICHD, through co-operative agreement U54-HD37321, as
part of the Specialized Cooperative Centers Program in Reproduction
Research.
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Submitted on February 8, 2002; accepted on June 20, 2002