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Suppression of type II collagen-induced arthritis by the endogenous estrogen metabolite 2-methoxyestradiol.

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Vol. 40, No. 1, January 1997, pp 154-163
0 1997, American College of Rheumatology
Objective. To evaluate the antiarthritic properties
of 2-methoxyestradiol, an endogenous metabolite of
estradiol, on type I1 collagen-induced arthritis (CIA) in
DBN1 mice.
Methods. The effects of treatment with 2methoxyestradiol on the development of CIA were evaluated clinically and histologically. The in vitro effects of
2-methoxyestradiol on lymphocyte and endothelial cell
proliferation and differentiation were analyzed by standard methods.
Results. The development of CIA was significantly
suppressed by 2-methoxyestradiol. Incubation with
2-methoxyestradiol suppressed the in vitro proliferation of endothelial cells, indicating that this compound down-regulates angiogenesis. Endothelial cell
production of nitric oxide (NO) was also down-regulated
by 2-methoxyestradiol. In contrast to estradiol, 2methoxyestradiol exerted neither detectable feminizing
effects on the sex organs nor inhibition of leukocyte
development in hematopoietic organs.
Conclusion. The development of CIA is suppressed by 2-methoxyestradiol, possibly via inhibition of
angiogenesis. Diminished NO production could be of
importance in vivo because it is a potent proinflammatory mediator. Since 2-methoxyestradiol exerts only
mild side effects compared with estradiol, it is a n
interesting candidate for therapeutic use in inflammatory diseases.
Supported by grants from the Swedish Medical Research
Council, the Goteborg Medical Society, the Swedish Association
Against Rheumatism, the University of Goteborg, the Sahlgrenska
Foundation, King Gustaf V’s 80-Year Fund, and by the N. Svartz, B.
Dahlin, Tornspiran, A-G Crafoord, F. and I. Thuring, and W. and M.
Lundgren Foundations.
Elisabet Josefsson, PhD, Andrej Tarkowski, MD, PhD: University of Goteborg, Goteborg, Sweden.
Address reprint requests to Elisabet Josefsson, PhD, Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity
College, Dublin 2, Ireland.
Submitted for publication February 12, 1996; accepted in
revised form July 30, 1996.
Much interest has been focused on the ameliorating effects of estrogen on rheumatoid arthritis (RA).
For example, estrogen-containing contraceptives have
been shown to decrease the prevalence of RA (l),and
the severity of the manifestations of RA is ameliorated
during pregnancy (2). Since RA is a T cell-dependent
disease, one possible mode of action of estrogen in
ameliorating arthritis could be the inhibition of T lymphocyte responses. This assumption is supported by the
efficient suppression of collagen-induced arthritis
(CIA), a T cell-dependent disease, by treatment with
17p-estradiol (3). Estrogen exerts numerous effects on
different organs in the body. However, when administered in vivo, the metabolites of estradiol must also be
considered as potential mediators of the immunomodulatory properties. The endogenous estrogen metabolite,
2-methoxyestradiol, was recently shown to be the most
potent inhibitor of in vitro proliferation of bovine brain
capillary endothelial cells when comparing estrogens
and estrogen metabolites (4). The 2-methoxyestradiol
metabolite is derived after hydroxylation of 17pestradiol to 2-hydroxyestradiol and subsequent methylation to 2-methoxyestradiol. Since angiogenesis plays a
central role in the development of arthritis ( 5 ) , we
assessed the antiinflammatory role of 2-methoxyestradiol in murine CIA, a model of human RA (6).
Estrogen was used as a control treatment. Our results
indicate that 2-methoxyestradiol exerts antiinflammatory properties by a concerted inhibitory action on
endothelial cell proliferation and nitric oxide (NO)
Mice. Male and female DBN1 mice, 7-18 weeks old,
and female Swiss mice, 15-17 weeks old, were originally
purchased from Harlan Olac farm (Bicester, UK) and ALAB
(Stockholm, Sweden), respectively. The mice were bred in the
animal facility of the Department of Clinical Immunology
(University of Goteborg). The mice were housed 10 in each
cage and were fed standard laboratory chow and water ad
libitum, under standard conditions of temperature and light.
Ovariectomy and hormone treatment. Female mice
were subjected to ovariectomy at ages 6-13 weeks. The ovaries
were removed via flank incision, which was closed with metallic
clips. The operations were carried out under pentobarbital
Male and female mice were treated with high or low
doses of estradiol or 2-methoxyestradiol. High-dose estrogen
was administered by subcutaneous (SC) implantation of 5-mmlong silastic tubes containing 2.5 mg of 17P-estradiol 3-benzoate
(estradiol; Sigma, St. Louis, MO) or 2-methoxyestradiol (Sigma),
which was performed on day 1 after ovariectomy. Low-dose
estrogen was administered by SC injections (twice a week) of 3.2
p g of estradiol or an equimolar amount (2.6 pg) of
2-methoxyestradioldissolved in 0.1 ml of olive oil, starting 4 days
after ovariectomy.While the high dose of estrogen clearly exceeds
the physiologic spectrum, the low dose results in serum levels of
17P-estradiol that are similar to those in pregnant mice (7). The
low dose gives rise to a transient serum peak of 17P-estradiolthat
disappears within 48 hours (8).
Control mice were subjected to ovariectomy and SC
implantation of empty silastic tubes or SC injections of vehicle
twice a week. The morphology of cells from vaginal smears
was determined before and during the estradiol and
2-methoxyestradiol treatment.
Induction of CIA. Male DBA/1 mice were immunized
intradermally at the tail base with 50 pg of native type I1
collagen (a kind gift from Dr. R. Holmdahl, University of
Lund, Lund, Sweden) emulsified in Freund’s complete adjuvant. The degree of swelling, redness, and movement in each
limb of the immunized mice was evaluated macroscopically.
The intensity of arthritis was quantified using a clinical scoring
system (0-3-point scale), where 1 = mild swelling and/or
erythema, 2 = moderate swelling and erythema, and 3 =
marked swelling, erythema, and occasionally ankylosis. An
arthritis index was calculated for each animal by adding the
scores for all 4 limbs, as previously described (9).
Seven weeks after type 11 collagen immunization, the
mice were killed and the arthritis was evaluated histologically
after routine fixation, decalcification, paraffin embedding,
cutting, and staining with hematoxylin and eosin. All the slides
were coded and evaluated by a blinded observer. The degree of
synovitis in the hind limbs was evaluated by scoring the
changes in the knee, ankle, and tarsal joints (0-3-point scale)
as follows: 1 = proliferation of the synovial lining layer; 2 =
proliferation of the synovial lining layer plus moderate leukocyte infiltration within the synovium; and 3 = synovial hypertrophy and proliferation and extensive leukocyte infiltration in
the synovium. The presence of bone and cartilage destruction
was scored 1 point, and no detectable joint destruction was
scored 0 points. A mean of the synovitis or joint destruction
score was calculated for the hind limbs for each type of joint.
Estimation of serum levels of type I1 collagen antibodies. Mice were bled 34 and 49 days after collagen immunization. All sera were collected individually and stored at -20°C
until used. Serum antibody levels were measured by an
enzyme-linked immunosorbent assay. Ninety-six-well microplates (Greiner, Dursley, UK) were coated overnight at 4°C
with 10 pg/ml of type I1 collagen, the concentration found in
checkerboard titration to give optimal coating. After 3 washes
with phosphate buffered saline (PBS), the plates were blocked
with 0.5% bovine serum albumin (BSA) for 1 hour at 20°C.
The sera were serially diluted in PBS-0.5% BSA and incubated
overnight at 4°C. The plates were washed, and affinity-purified
biotinylated F(ab’), fragments of goat anti-mouse IgG antibodies (Jackson, Baltimore, MD), diluted in PBS-Tween to a
final concentration of 0.3 pg/ml, were incubated for 2 hours at
20°C. Washing was followed by incubation of peroxidaseconjugated avidin (Sigma), 0.5 pdml in PBS-Tween, for 1 hour
at 20°C.
After washing, the antigen-antibody reaction was
quantified by addition of the enzyme substrate ABTS (Sigma)
at a concentration of 0.25% (weight/volume) in a buffer of pH
4.2 and containing 24 mM sodium acetate, 50 mM sodium
dihydrogenphosphate, 0.425% (v/v) concentrated acetic acid,
and 0.0075% (w/v) H,O,. After 10 minutes, the absorbance at
405 nm was recorded in a Labsystems Multiscan (Labsystems,
Helsinki, Finland). The calibration curve was constructed using
standards with known concentrations of IgG anti-type I1
collagen antibodies and a computer program based on
weighted logit-log models (10).
Interleukin-6 (IL-6) assay. For IL-6 determinations,
the B9 cell line was used (11,12). B9 cells were harvested from
tissue culture flasks, seeded into microtiter plates (Nunc,
Roskilde, Denmark) at a concentration of 5,000 cells per well,
and cultured in complete Iscove’s medium with sera from CIA
mice or H5V cell culture supernatants. After 68 hours of
culture, 3H-thymidine was added. and the cells were harvested
4 hours later. Mice were bled 34 and 49 days after type I1
collagen immunization, and the sera were tested in 2-fold
dilutions and compared with a recombinant mouse IL-6 standard (Genzyme, Cambridge, MA) (13).
Measurement of serum immunoglobulins. Serum levels of total IgG1, IgG2a, IgG3, and IgM were measured 34 and
49 days after collagen immunization by the radial immunodiffusion technique, as previously described (14). Sera were
diluted 200 and 50 times for the IgGl and IgG2a assays,
respectively, and 20 times for the total IgG3 and IgM assays.
Antisera and immunoglobulin standards specific for IgG1,
IgG2a, IgG3, and IgM were purchased from Sigma.
Olive oil-induced inflammation. After 2 weeks of
low-dose or high-dose 2-methoxyestradiol or estradiol treatment of female D B N 1 mice, granulocyte-mediated and T
cell-independent inflammation was induced by SC injection of
30 p1 of olive oil (Apoteksbolaget, Goteborg, Sweden) into a
hind footpad as previously described (15J6). Before and 24
hours after the olive oil injection, the thickness of the footpad
was measured with an Oditest spring calipers (Kroplin,
Schliichtern, Germany). The inflammatory response was expressed as the increase in footpad thickness (in mm).
Immunization procedure and recording of delayedtype hypersensitivity (DTH). T cell-dependent inflammation
was induced in female DBN1 mice after 24 days of high-dose
treatment with 2-methoxyestradiol or estradiol by elicitation of
a DTH reaction. Mice were sensitized by epicutaneous application of 150 pl of a mixture of absolute ethanol and acetone
(2:l) containing 3% 4-ethoxymethylene-2-phenyloxazolin-5one (oxazolone; Sigma) on the skin of the shaved abdomen and
thorax. Seven days later, the mice were booster immunized by
topical application of 30 pl of 3% oxazolone dissolved in olive
oil on each side of one ear. The ear thickness was registered
before and 24 hours after the challenge, using Oditest spring
calipers as described elsewhere (17,18). Challenge and measurement procedures were performed under light anesthesia.
The intensity of the DTH reaction was expressed as the
thickness of the ear after the booster immunization minus the
thickness of the ear before the booster immunization (in mm).
Quantitation of peripheral blood and bone marrow
leukocytes. Peripheral blood cells and bone marrow leukocytes
were investigated in female D B N l mice after 5 weeks of
treatment with high doses of 2-methoxyestradiol or estradiol.
Peripheral blood was obtained by venesection and collected
into tubes containing heparin. Bone marrow cells were obtained by resecting the proximal and distal ends of both
femurs, followed by flushing of the bone marrow cavity with
physiologic saline. The bone marrow cells were centrifuged
and reconstituted with saline. Peripheral blood and bone
marrow cells were analyzed in an automated cell counter
(Sysmex, Kobe, Japan).
Fluorescence-activated cell sorter (FACS) analysis.
After 5 weeks of high-dose treatment with 2-methoxyestradiol
or estradiol, splenocytes and thymocytes of female D B N 1
mice were analyzed for expression of CD4 and CD8 antigens
and, in the case of splenocytes, for immunoglobulins by FACS.
Cells suspended in 1% BSA-PBS were incubated at 0°C for 30
minutes. For single-color analysis, cells were stained with a
1:100 dilution of fluorescein-canjugated anti-mouse Ig (Dakopatts, Glostrup, Denmark). For analysis of CD4 and CD8
expression, cells were stained with a combination of
phycoerythrin-conjugated anti-GK1.5 (Becton Dickinson,
Mountain View, CA) and fluorescein-conjugated anti-Lyt2
(Becton Dickinson), both diluted 1:375. All the samples were
analyzed with a FACScan cytometer with Lysis I1 software
(Becton Dickinson).
Culture of H5V endothelial cells. Endothelioma line.
The murine endothelioma cell line H5V (heart endothelial
cells from BALB/c mice transformed by polyoma virus middle
T antigen) was kindly provided by Dr. A. Mantovani (Instituto
di Ricerche Farmacologiche Mario Negri, Milan, Italy). The
endothelial cells were grown in Dulbecco’s modified Eagle’s
medium (DMEM; Sigma) with a high glucose content (4.5
gm/liter) supplemented with 10% fetal calf serum (Biological
Inc., Beit Haemek, Israel), 2 mM L-glutamine, 50 pg/ml
gentarnicin, and 50 pA4 2-mercaptoethanol. The cells were
detached from the plastic surface by incubation with 4%
trypsin in EDTA solution.
Stock solutions of 1.3 X lO-’M 2-methoxyestradiol or
estradiol in ethanol were used for the in vitro assays. The trace
volumes of ethanol in the culture media dilutions had no
significant effect on cell proliferation or differentiation (results
not shown).
Proliferation assays. H5V cells were seeded in 96-well
flat-bottomed microtiter plates (Nunc, Roskilde, Denmark) in
0.1 ml of cuIture medium and grown at 37°C in 5% CO, and
95% humidity until the cells covered about 75% of the wells.
New medium containing 2-methoxyestradiol or estradiol in
concentrations ranging from 0 to 100 +I4 was then added into
the wells. The endothelial cells were cultured with
2-methoxyestradiol or estradiol for 48 hours or .were pretreated for 4 hours, washed with 37°C culture medium, and
kept in culture medium alone for another 18 hours. In both
proliferation assays, 1 pCi of 3H-thymidine (Radiochemical
Centre, Amersham, UK) was included in each well during the
final 18 hours of culture. The endothelial cells were incubated
with trypsin for 90 minutes before being harvested into glassfiber filters. Thymidine uptake was counted in a beta counter.
The cultures were set up in triplicate, and the results were
expressed as the mean counts per minute.
Differentiation assay for NO production. H5V cells
were seeded in 24-well flat-bottomed culture plates (Nunc) in
1 ml of culture medium and grown to confluence (4 days). The
cells were then either continuously incubated with
2-methoxyestradiol or estradiol (0-100 CLM) in fresh medium
for 48 hours or were pretreated with 2-methoxyestradiol or
estradiol for 24 hours, washed with warm culture medium, and
incubated with culture medium alone for another 24 hours.
The supernatants were collected and stored at -20°C until the
nitrite content was analyzed. The culture medium contained
0.4 mM L-arginine, but to all the cell cultures 1 mM L-arginine
(Sigma) was added 24 hours before the harvest.
Differentiation assay for IL-6 production. H5V cells
were grown to confluence, incubated with 2-rnethoxyestradiol
or estradiol(0-100 I.M) for 4 or 24 hours and stimulated with
0-50 units/ml of recombinant murine interferon-? (IFNy;
Genzyme) and 0-10 pg/rnl of tumor necrosis factor a (TNFa;
Genzyme) for 24 hours. The supernatants were stored in
-20°C until analyzed for IL-6.
Nitrite detection. Nitrite was detected by mixing 0.1 ml
of cell culture supernatant with 0.1 ml of Griess reagent (19).
After incubation for 30 minutes in the dark at room temperature, the absorbance was measured at 540 nm in a Labsystems
Multiscan. To avoid measurement bias by the pH indicator,
phenol red-free medium was used in all the experiments
performed for nitrite production.
Immunohistochemistry. Endothelial cells were grown
to confluence on a glass slide chamber (Lab-Tek Nunc,
Naperville, IL) and treated for 24 hours with 0-100 p k l
estradiol or 2-methoxyestradiol during stimulation with 50
units/ml of recombinant murine IFNy and 10 pg/ml of TNFa.
The cells were then fixed in situ with acetone for 5 minutes,
washed in PBS, blocked with 2% BSA in PBS-Tween for 30
minutes, and incubated overnight at 4°C with antibodies to
intercellular adhesion molecule 1 (ICAM-1; CD54), MEL-14
(CD62L), Mac-1 ( C D l l b ) , and lymphocyte functionassociated antigen 1 (LFA-1; CDlla) from YN1/1.7.4, MEL14, M1/, and FD441.8 hybridoma lines, respectively
(American Type Culture Collection, Rockville, MD). The cells
were depleted of endogenous peroxidase by treatment with
0.3% H202for 5 minutes, washed, and incubated for 1 hour
with a biotin-labeled rabbit anti-rat Ig secondary antibody
(Vector, Burlingame, CA) diluted in appropriate concentration with PBS containing 1% BSA. The slides were washed and
incubated stepwise with streptavidin-biotin-peroxidase complexes (Dakopatts) (20) and 3-amino-9-ethylcarbazole (Sigma)
containing H,O, (21).
In vitro stimulation of lymphocytes. Spleens from
female Swiss mice were cut and passed through a nylon sieve.
The cells were suspended in PBS and centrifuged at 515g for 5
minutes. The pelleted cells were resuspended for 10 minutes in
Tris-buffered 0.83% ammonium chloride to lyse erythrocytes.
After 2 washes in PBS, the mononuclear cells were counted.
Table 1. Delayed onset of type I1 collagen-induced arthritis in mice
treated with 2-methoxyestradiol*
- Control
Prevalence of arthritis (%)
Day of
(n = 11-12)
(n = 12)
(n = 8-10)
* 2-methoxyestradiol and estradiol treatment started 20 days after
collagen I1 immunization.
* *** 1 0
*** * *
" . ' . " . . " . I
Days after start
of treatment
Figure 1. Development of type I1 collagen-induced arthritis, expressed as an arthritis index in mice treated with either
2-methoxyestradiol or estradiol beginning at the initiation of the
arthritis (i.e., day 20 after collagen immunization). * = P 5 0.05, ** =
P 5 0.01, and *** = P 5 0.001 versus controls.
Iscove's medium (Gibco, Paisley, UK) with the same supplements as for the DMEM was used as culture medium for the
In proliferation assays, spleen cells were incubated at a
concentration of 1 X lo6 mononuclear cells/ml in 96-well
flat-bottomed microtiter plates in 0.1 ml of culture medium at
37°C in 5% CO, and 95% humidity. Either 2-methoxyestradiol
or estradiol, in concentrations ranging from 0 to 100 pA4, and
2.5 pgjml of concanavalin A (Con A) (Miles Yeda, Rehovot,
Israel) or 10 pgjml of lipopolysaccharide (LPS; Sigma) were
included in the medium. The concentrations of the mitogens
were optimized in preliminary experiments. The cells were
cultured for 72 hours. During the final 18 hours of culture, 1
pCi of 3H-thymidine was included in each well. The cultures
were harvested into glass-fiber filters, processed, and counted
in a beta counter. The cultures were set up in triplicate, and the
results were expressed as the mean cpm.
Statistical analysis. The level of significance of differences between groups was calculated using Student's 2-tailed
t-test, except for the arthritis index (ordinal data), for which
Wilcoxon's rank sum test was used. Data are presented as the
mean plus or minus the standard error of the mean or the
standard deviation, as indicated.
Ameliorating effects of 2-methoxyestradiolon the
course of CIA. At the onset of CIA (20 days after the
injection of type 11 collagen), the mice were implanted
SC with control tubes or with tubes containing
2-methoxyestradiol or estradiol. After 8 days of treatment with 2-methoxyestradiol, there was already a
significant decrease in the arthritis index compared
with that in the control mice (P < 0.05) (Figure 1).
The course of arthritis was ameliorated by
2-methoxyestradiol during the entire 28-day period of
treatment. Estradiol treatment almost totally inhibited
the development of arthritis. Also, the onset of arthritis
was strikingly different between the 2-methoxyestradioltreated mice and the estradiol-treated mice compared
with the controls (Table 1).
All mice, including the controls, experienced a
decrease in body weight during the development of
CIA. Administration of 2-methoxyestradiol significantly
reduced the amount of the body weight decrease
compared with the control group (mean % SD -0.5 ?
0.7 gm versus -1.3 ? 0.6 gm; P = 0.008). In contrast,
estradiol treatment gave rise to a significantly greater
loss of body weight (-3.7 t 1.8 gm; P 5 0.001)
compared with the control mice and with the 2methoxyestradiol-treated mice.
Table 2. Histologic evaluation of the degree of synovitis in the knee,
ankle, and tarsal joints of type I1 collagen-immunized mice treated
with 2-methoxyestradiol or estradiol*
(mean -+
(mean f
(mean k
516 2.0 t 1.1 819 1.9 2 0.9 8/10 2.1 ? 0.8
2-methoxyestradiol 617 1.7 -C 0.7 618 0.9 -C 0.6$ 9/11 1.4 -+ 0.5
2/7 0.5 -t 0
2/8 0.8 % 0.4 0/8
* The knee, ankle, and tarsal joints of both hind legs of each mouse
were evaluated and a mean synovitis score was calculated for each
joint. Values are the mean synovitis score in only the mice with
t Values are the number of mice with arthritisitotal number of mice.
$ P < 0.05 versus controls.
Table 3. Levels of anti-type I1 collagen antibodies and interleukin-6 (IL-6) in the sera of
2-methoxyestradiol-treated or estradiol-treated mice 5 and 7 weeks after collagen immunization*
IgG anticollagen antibodies
5 weeks
7 weeks
5 weeks
7 weeks
887 i 432 (11)
699 2 461 (12)
647 2 415 (11)
652 i 314 (11)
718 ? 571 (12)
148 t 93 (S)?
0.26 t 0.21 (11)
0.39 2 0.23 (11)
0.34 i 0.25 (11)
0.25 i 0.20 (11)
0.18 t 0.15 (12)
0.56 2 0.47 (8)
* Values are the mean
i SD (number of mice).
7 P < 0.001 versus controls.
Seven weeks after collagen immunization, the
knee, ankle, and tarsal joints were examined histologically for the degree of synovitis. In animals that had been
treated with 2-methoxyestradiol since day 20 after
collagen immunization, the synovitis was significantly
decreased in the ankle joints (I' < 0.05) (Table 2).
In nearly all cases of synovitis, bone and cartilage destruction was also detected. Joint destruction was
present in 2-methoxyestradiol-treated mice to the same
extent as in control mice (data not shown). Thus, 7
weeks after collagen immunization, administration of
2-methoxyestradiol diminished the degree of synovitis
but did not affect the degree of joint destruction.
Specific anti-type I1 collagen antibody responses
and IL-6 production were measured in 2methoxyestradiol-treated and estradiol-treated mice 5
and 7 weeks after type I1 collagen immunization, which
is equivalent to 2 and 4 weeks of steroid treatment,
respectively. Anti-type I1 collagen antibody titers and
IL-6 levels were not significantly affected by the
2-methoxyestradiol treatment, whereas estradiol-treated
animals had significantly lower levels of anti-type I1
collagen antibodies and high, but not significantly higher,
levels of IL-6 after 29 days of treatment (Table 3).
Levels of IgG1, IgG2a, IgG3, and IgM were
measured at the same times after collagen administration. There were no significant changes in any of these
levels in any treatment group (data not shown).
In a second set of experiments, 2-methoxyestradiol high-dose treatment was given 2 weeks after the
onset of arthritis. This treatment significantly delayed
the progression of arthritis for 6 days as compared with
the control mice. However, the mean arthritis index was
not significantly decreased by 2-methoxyestradiol treatment in this setting (data not shown).
Differential outcome of polymorphonuclear cellmediated and T cell-mediated inflammatory responses
in mice treated with 2-methoxyestradiol and estradiol.
Polymorphonuclear leukocyte-mediated inflammation
was induced in female DBNl mice by SC injection of
olive oil. Neither low-dose nor high-dose treatment with
2-methoxyestradiol significantly influenced the inflammatory response to olive oil (Table 4). This contrasted
with the suppressive effects of both low-dose and high-
Table 4. Effects of estradiol and 2-methoxyestradiol treatment on olive oil-induced, granulocytemediated inflammation and delayed-type hypersensitivity (DTH) responses to ozazolone*
Olive oil-induced inflammation,
increase in footpad thickness (mm)
DTH response
to oxazolone,
increase in ear
thickness (mm)
0.74 -+ 0.17 (9)
0.66 -+ 0.14 (7)
0.48 i 0.22 (ll)?
0.38 i 0.09 (10)
0.33 t 0.06 (10)
0.21 i 0.03 (7)$
0.29 i 0.05 (8)
0.37 i 0.07 (9)
0.18 2 0.04 (8)t
* The mice were treated with the steroids for 2 weeks before olive oil injection or sensitization. Values are
the mean 2 SD (number of mice).
t P < 0.05 versus controls.
i P < 0.001 versus controls.
Table 5. Effects of high-dose administration of 2-methoxyestradiol and estradiol on leukocyte numbers
in different body compartments*
Total leukocytes ( X lo6)
Bone marrow
Peripheral blood
(Per m 9
15.8 2 4.7 (10)
15.8 2 4.0 (10)
7.7 2 1.4 (10)
7.0 2 3.0 (10)
4.2 2 1.3 (8)$
93 2 21 (5)
84 2 8 ( 5 )
17 t 9 (5)$
167 2 13 (5)
131 2 21 (5)t
123 ir 22 ( 5 ) s
* Values are the mean 2 SD (number of mice). Bone marrow was totally depleted in estradiol-treated
t P < 0.05 versus controls.
$ P 5 0.001 versus controls.
0 P < 0.01 versus controls.
dose estradiol on the polymorphonuclear cell inflammatory response.
The T cell-mediated DTH response to oxazolone
in female D B N 1 mice was not significantly affected by
2-methoxyestradiol treatment (Table 4). However, estradiol suppressed the DTH response significantly.
Effects of in vivo treatment with 2-methoxyestradiol
and estradiol on body weight, leukopoiesis, lymphocyte
populations, and sex organ differentiation. Mouse body
weight was not significantly affected by high-dose treatment with either 2-rnethoxyestradiol or estradiol (n = 2
experiments) (data not shown).
The numbers of leukocytes in the bone marrow,
peripheral blood, and thymus were not significantly
affected by 2-methoxyestradiol treatment (Table 5).
However, the number of splenocytes was slightly but
significantly reduced in 2-methoxyestradiol-treated
mice. In contrast, estradiol treatment strikingly reduced
the leukocyte numbers in peripheral blood, thymus, and
spleen, and totally eliminated leukocytes from the bone
Spleen and thymus lymphocyte populations in
control, 2-methoxye~tradiol-treated~and estradioltreated animals were investigated. CD4+ and CD8+ T
cells and Ig+ B cells were stained and analyzed by
FACS. Treatment with 2-methoxyestradiol significantly
diminished the number of Ig+ splenocytes, but did not
significantly affect the numbers of CD4+ or CD8+ cells
either in the spleen or in the thymus (Table 6). The
numbers of CD4+, CD8+, and Ig+ lymphocytes were
significantly diminished in the spleens of the estradioltreated animals. This treatment also significantly suppressed the numbers of CD4+, CD8+, and CD4+:
CD8+ lymphocytes in the thymus (Table 6).
Treatment with 2-methoxyestradiol did not produce any sex hormone effects. Ovariectomized females
treated with 2.6 pg of 2-methoxyestradiol twice a week
remained at the diestrus stage, according to the morphology of the cells on vaginal smears, whereas treatment with a similar dose of estradiol precipitated the
estrus phase in all mice within 6 days (data not shown).
The weight of the seminal vesicles in males did
not change significantly in those implanted with
2-methoxyestradiol (mean ? SD 74 2 17 mg versus 73 2
14 mg in controls). In contrast, estradiol treatment
significantly reduced the seminal vesicle weight (28 ? 5
mg; P < 0.001). The weight of the testes was not affected
Table 6. Analysis of spleen and thymus lymphocytes in 2-methoxyestradiol-treated and estradiol-treated
Splenocytes ( x lo6)
CD4 +
18.8 t 2.4
2-methoxyestradiol 16.4 t 2.8
8.5 t 2.01
* Values are the mean
4.9 + 0.7
4.0 t 0.7
2.2 t 0.51
8.1 t 2.1
89.9 t 10.4
7.8 2 0.9
64.6 t 9.81
44.8 t 12.7% 1.7 t 0.9$
2 SD (n = 5 mice per group).
t P < 0.01 versus controls.
$ P < 0.001 versus controls.
5 P < 0.1 versus controls.
( X 10")
1.6 t 0.5
1.4 t 0.2
0.6 t 0.59
77.8 t 17.3
69.0 ? 8.4
13.1 t 7.7$
2-methoxyestradiol 4 h
2-methoxyestradiol 2 d
Estradiol 2 d
0.1 pM
1 pM
10 KM
100 pM
Figure 2. Proliferation of endothelial cells, measured as 'H-thymidine
uptake, after 4 hours of pretreatment or after 2 days of treatment with
2-methoxyestradiol or estradiol. The data were collected from 2
independent experiments.
by either 2-methoxyestradiol or high-dose estradiol
treatment (data not shown).
In an experiment with females treated with highdose 2-methoxyestradiolor estradiol, the morphology of
the cells on vaginal smear after 36 days of treatment
demonstrated diestrus phase in all controls and 2methoxyestradiol-treated mice, whereas all estradioltreated mice had estrus phase morphology.
Effects of 2-methoqestradiol on proliferative
responses of endothelial cells. Endothelial cell cultures
were exposed to estradiol or 2-methoxyestradiol for 48
hours. The proliferation of the endothelial cells was
suppressed in a dose-dependent manner by both
2-methoxyestradiol and estradiol (Figure 2). Even pretreatment of the endothelial cells for only 4 hours with
estradiol or 2-methoxyestradiol inhibited the proliferation of the endothelioma cells in a dose-dependent
manner (Figure 2).
Effects of 2-methoqestradiol on IL-6 production
by endothelial cells. IL-6 production by HSV cells was
increased 30-60 times on incubation with TNFa and
IFNy. However, pretreatment of the cell cultures with
up to 100 pM estradiol and 2-methoxyestradiol did not
significantly influence the production of IL-6. The spontaneous production of IL-6 by the endothelial cell line
was not affected by the addition of estradiol or its
metabolite (data not shown).
Inhibitory effects of 2-methoqestradiol on NO
production by endothelial cells. Endothelial cell cultures
were pretreated with 2-methoxyestradiol or estradiol for
24 hours and were incubated with culture medium alone
for the following 24 hours or the cells were treated
continuously with 2-methoxyestradiol or estradiol for
48 hours. The production of NO, as evaluated by the
nitrite concentration, was inhibited in a dose-dependent
manner in both the estradiol-treated and the 2methoxyestradiol-treated cell cultures. However,
2-methoxyestradiol displayed inhibitory properties at a
10 times lower concentration than estradiol (Figure 3).
Similar data were obtained for pretreated cell cultures.
The mean f SEM nitrite concentrations were 8.3 5 0.2
f l in controls, 7.2 2 0.2 pM (P = 0.017 versus controls)
and 6.3 ? 0.1 p M (P < 0.001) in cultures pretreated with
10 pM or 100 pM 2-methoxyestradiol, respectively, and
5.7 2 0.4 f l in cultures pretreated with 100 pA4
estradiol (P < 0.001).
Effects of estrogens on the phenotypic expression
of adhesion molecules. The H5V endothelioma line
expressed ICAM-1 while neither Mac-1, MEL-14, nor
LFA-1 was detected with the monoclonal antibodies
used. ICAM-1 expression was not influenced by incubation with 2-methoxyestradiol or estradiol (data not
Inhibitory effects of 2-methoxyestradiol on
mitogen-inducedproliferationof lymphocytes. Spleen cells
were incubated with estradiol and 2-methoxyestradiol
during mitogenic stimulation. The proliferative responses of splenocytes to Con A and LPS were suppressed in a dose-dependent manner by both estradiol
and 2-methoxyestradiol (Figures 4A and B). However,
2-methoxyestradiol diminished the proliferative response to the T cell mitogen Con A significantly more
than estradiol at 1 f l and 10 pM concentrations (P 5
Concentration (pM)
Figure 3. Nitrite concentration in supernatants of endothelial cell
cultures incubated for 48 hours with 2-methoxyestradiol or estradiol.
*** = P 5 0.001 versus controls.
0.001 0.01
Concentration (BM)
Concentration (pM)
Figure 4. Proliferative responses of spleen cells to A, the T cell mitogen concanavalin A (Con A) and B,the B cell mitogen lipopolysaccharide (LPS)
during concomitant treatment with 2-methoxyestradiol or estradiol. The data were collected from 2 independent experiments. * = P I0.05.
0.05). The effects of 2-methoxyestradiol and estradiol on
the proliferative response in the LPS-driven cultures
were similar.
In this study, the endogenous estradiol metabolite 2-methoxyestradiol was shown to exert an ameliorating effect on the development of type I1 collageninduced arthritis. The onset of arthritis was delayed
and its severity, measured as soft tissue swelling and
degree of synovitis, was significantly diminished by
2-methoxyestradiol treatment.
What is the mechanism of action of 2methoxyestradiol? Since CIA is a T and B celldependent disease (22,23), it was important to assess the
impact of 2-methoxyestradiol on lymphocyte responses
in vivo. However, the T cell-dependent DTH response
was not suppressed by 2-methoxyestradiol treatment,
even when the treatment was started before sensitization, indicating that 2-methoxyestradiol does not act
upon the T cells. It is therefore not likely that treatment
with 2-methoxyestradiol affected T cell activity in the
arthritis process. This is also supported by the intact
ability of 2-methoxyestradiol-treated mice to produce
TL-6. The B cell activities in the arthritic animals,
measured as the production of specific anti-type I1
collagen antibodies and total levels of Ig subclasses, were
also not influenced by 2-methoxyestradiol. The reduction in the number of Ig+ spleen cells in
2-methoxyestradiol-treated mice did not seem to affect
the total Ig production. Interestingly, proliferative responses of T and B cells in vitro were inhibited by
2-methoxyestradiol in a dose-dependent manner. The
pathway whereby these responses are inhibited by
2-methoxyestradiol and whether this antiproliferative
action is of relevance in vivo remain to be analyzed. Our
preliminary results indicate that 2-methoxyestradiol arrests cell proliferation at the G, phase and simultaneously increases the proportion of apoptotic cells (Josefsson et al: unpublished observations).
In a recent study, 2-methoxyestradiol was proposed to suppress tumor growth by inhibition of angiogenesis (4). The amelioration of arthritis could thus be
due to an inhibition of angiogenesis. Indeed, our data
clearly support the possibility that the inhibition of
angiogenesis as observed by Fotsis et a1 (4) may be due
to the antiproliferative action of 2-methoxyestradiol. In
analogy, another steroid, the cortisol metabolite tetrahydrocortisol, is highly angiostatic in vivo (24). Since
angiogenesis is a central pathogenic mechanism in the
induction and progression of arthritis (9,our finding
may at least partly explain the antiinflammatory effects
of 2-methoxyestradiol. Further support for this view is
provided by the inability of 2-methoxyestradiol treatment to suppress polymorphonuclear cell-mediated inflammation or DTH in vivo (see Table 4), since these
inflammatory responses involving the extravasation of
polymorphonuclear cells, the formation of edema, and
in the case of DTH, the infiltration of mononuclear cells,
develop within hours and therefore do not involve the
formation of new vessels.
Treatment with 2-methoxyestradiol suppressed
not only the proliferative responses of endothelial cells,
but also their production of NO. NO is a potent proinflammatory agent that has arthritogenic properties (2527). Interestingly, NO is not only proinflammatory per
se, but it is also capable of enhancing angiogenesis
(28,29), thereby creating a positive feedback. Notably,
2-methoxyestradiol acted as NO inhibitor at a concentration 10 times lower than that of estradiol. The suppression of NO production by the 2-methoxyestradiol
could be due to a general toxic effect of this metabolite.
This, however, is less likely since identical concentrations of 2-methoxyestradiol did not influence the production of IL-6 or the expression of endothelial
ICAM-1. Thus, our results support the notion that
2-methoxyestradiol may suppress CIA by the simultaneous inhibition of angiogenesis and down-regulation of
endothelial NO production.
The beneficial effect of 2-methoxyestradiol on
the development of arthritis was less striking than that of
estradiol. However, in contrast to estradiol, administration of 2-methoxyestradiol was reasonably free of the
side effects commonly seen with estradiol administration. Thus, the feminizing effects of 2-methoxyestradiol
on sex organs, including the vagina and seminal vesicles,
were absent. This is because of a very low binding
affinity of 2-methoxyestradiol for the estrogen receptor
(30). In addition, in contrast to the estradiol treatment,
administration of 2-methoqestradiol did not grossly
affect the physiologic function of hematologic organs,
such as the bone marrow and thymus. Finally, body
weight decreases during the course of arthritis, which are
typically accelerated by the administration of estradiol,
were halted by 2-methoxyestradiol treatment.
Taken together, the endogenous estradiol metabolite 2-methoxyestradiolameliorates the development of
type I1 collagen-induced arthritis, possibly via inhibition
of angiogenesis. Moreover, 2-methoxyestradiolis a more
efficient inhibitor of NO production by endothelial cells
and of lymphocyte proliferation than is estradiol. Thus,
2-methoxyestradiol may be an attractive therapeutic
agent that can efficiently ameliorate arthritis with only
mild side effects compared with estradiol.
We thank Mrs. Ing-Marie Nilsson and Mrs. Margareta
Verdrengh for skillful technical assistance.
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