Hum. Reprod.-2006-García-Pérez-880-7

7/27/2019 Hum. Reprod.-2006-Garca-Prez-880-7

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Human Reproduction Vol.21, No.4 pp. 880887, 2006 doi:10.1093/humrep/dei413

Advance Access publication February 3, 2006.

880 The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.For Permissions, please email: [email protected]

Alterations in the phenotype and function of immune cells inovariectomy-induced osteopenic mice

M.A.Garca-Prez

1,7

, I.Noguera

2

, C.Hermenegildo

1,3

, A.Martnez-Romero

4

, J.J.Tarn

5

and A.Cano6

1Research Unit, Hospital Clnico Universitario of Valencia, Av. Blasco Ibez 17, 46010 Valencia, Spain, 2Research Unit, Faculty of

Medicine, University of Valencia, 46010 Valencia, Spain, 3Department of Physiology, Faculty of Medicine, University of Valencia,

46010 Valencia, Spain, 4Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Valencia, 46010

Valencia, Spain, 5Department of Functional Biology and Physical Anthropology, Faculty of Biological Sciences, University of Valencia,

46100 Burjassot, Spain and 6Department of Pediatrics, Obstetrics and Gynecology, Faculty of Medicine, University of Valencia, 46010

Valencia, Spain

7To whom correspondence should be addressed. E-mail: [email protected]

BACKGROUND: Within the last few years, much evidence has been presented on the involvement of the immune

system in certain types of bone loss, such as activated T cells in rheumatoid arthritis and in periodontitis. Estrogen

deficiency induces bone loss; however, how this deficiency affects the immune system has not been sufficiently stud-

ied. METHODS: To evaluate the effects of estrogen withdrawal on the status and functionality of the immune system,

mice were ovariectomized or sham-operated, and 5 weeks after surgery, when osteopenia had developed, several

parameters were analysed in spleen and in bone marrow. We analysed bone turnover, cell phenotype by flow cytom-

etry, cell function by cell proliferation assays, and the expression of several genes related to the process. RESULTS:

Five weeks after ovariectomy, augmented osteoclastogenesis persisted in the bone marrow. In addition, the ovariect-

omized mice had more B-cells and CD3+ T-cells expressing the receptor activator of NF-kB ligand (CD3+/RANKL+).

The ovariectomized mice had lower serum alkaline phosphatase activity, a normal amount of T cells, lower percentages

of CD11b+ and CD51+ cells in the bone marrow, and a lower serum interferon- level compared with sham-operated

controls. CONCLUSIONS: The data suggest that, 5 weeks after ovariectomy, bone turnover remains imbalanced,

with increased osteoclastogenesis and a decreased rate of bone formation. Moreover, there is an increase in B-cell

formation, with normal and decreased percentages of T cells and myelomonocytic cells (CD11b+), respectively, in the

bone marrow. Decreased serum interferon- levels could be involved in the increased osteoclastogenesis found in thepresent work.

Key words: bone metabolism/estrogen deficiency/immune system/mouse/ovariectomy

Introduction

Recent research has uncovered molecules crucial to bone

metabolism, and in particular to the generation, development

and activation of osteoclasts, the cells that resorb bone. These

molecules include the receptor activator of the NF-B ligand(RANKL, also known as TRANCE, ODF and OPGL), its

receptor RANK, and osteoprotegerin, all of which are keymolecules for osteoclastogenesis. This was demonstrated

through the use of mice deficient in these molecules (Theill

et al., 2002). RANKL and RANK were first described in

immune system cells, where they are expressed in activated T

cells, and in dendritic cells, respectively, and are implicated in

cell survival and immunomodulation (Anderson et al., 1997).

In bone, the binding of RANKL on stromal or osteoblastic

cells to RANK on pre-osteoclasts, together with macrophage

colony stimulating factor (M-CSF), is necessary and sufficient

for the generation, differentiation and activation of osteoclasts

(Lacey et al., 1998), although other molecules in the bone mar-

row environment, such as interleukin (IL)-1, IL-6 and tumour

necrosis factor (TNF)-, intervene in the process (Jilka, 1998).These discoveries have led to the creation of a new field of

research, osteoimmunology (Arron and Choi, 2000).

Much evidence has linked the immune system to bone loss(Theill et al., 2002). Some of this evidence comes from the study

of animal models of rheumatoid arthritis and periodontitis. It has

been demonstrated that activated T cells express RANKL,

which, in turn, is able to stimulate osteoclastogenesis and bone

loss (Kong et al., 1999; Teng et al., 2000). Moreover, estrogen

regulates T-cell functions through an estrogen receptor-mediated

pathway, and T-cell subset alterations were found in postmeno-

pausal women with osteoporosis (Olsen and Kovacs, 1996). Fur-

thermore, it has been shown that ovariectomy induces B-cell


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Immune system status in osteopenic m

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lymphopoiesis, and that these cells can be a source of osteoclasts

(Masuzawa et al., 1994; Sato et al., 2001). Therefore, immune

system cells may induce bone loss not only during inflammation

but also under conditions of estrogen deficiency.

Estrogen deficiency induces bone loss and osteoporosis

(Jilka, 1998; Riggs et al., 1998), and since both bone and

immune cells have active receptors for estrogens (Bellido

et al., 1993; Komi and Lassila, 2000; Igarashi et al., 2001) it is

conceivable that estrogen deficiency induces changes not only

in bone but also in the immune system, and that these changes

could be associated with increased osteoclastogenesis and bone

loss. The status of the immune system after estrogen deficiency

is established; however, it needs further study.

In this work, ovariectomy was carried out in the mouse as a

model of estrogen deficiency. Ovariectomy induces a rapid aug-

mentation in bone turnover, causing an increase in both bone

resorption and bone formation (Jilka et al., 1998; Tanizawa et

al., 2000). In the mouse, indices of bone remodelling peak at the

first or second week after ovariectomy and then fall progres-

sively. Five weeks after ovariectomy, bone turnover has been

normalized almost completely, and the mouse has developed

osteopenia due to the large amount of trabecular bone lost (Jilkaet al., 1998; Tanizawa et al., 2000; Miyazaki et al., 2004).

The purpose of the present study was to analyse several

aspects of the immune system in the spleen and bone marrow

in mice that had developed osteopenia as a consequence of

estrogen deficiency induced by ovariectomy. We studied the

phenotype and function of both cellular types, several parame-

ters in serum, such as calcium, alkaline phosphatase (ALP)

activity, TNF-, IL-6 and interferon- levels, and the expres-sion of several genes 5 weeks after ovariectomy, when bone

turnover normalized after an initial imbalance provoked by the

abrupt interruption in the supply of estrogens.

Materials and methods

Media and reagents

Recombinant mouse soluble receptor activator of NF-B ligand

(sRANKL) and recombinant murine M-CSF were obtained from

PeproTech (London, UK). Monoclonal CD3 (clone 1452C11), CD28

(clone 37.51), CD3-fluorescein isothiocyanate (FITC) (clone 145

2C11), CD11b-FITC (clone M1/70), CD4-phycoerythrin (PE) (clone

GK1.5), CD8-PE (clone 536.7), CD51-PE (clone RMV-7), RANKL-

PE (clone IK22/5) and rat PE-negative control (clone KLH/G2a-1-1)

were from e-Bioscience (San Diego, CA). Monoclonal CD19-FITC

(clone 6D5), CD25-RPE (clone PC61.5.3), rat anti-mouse RANK

(clone LOB14-8), rat FITC-negative control (clone LO-DNP-16) and

F(ab)2 goat anti-rat immunoglobulin G-FITC were from Serotec(Oxford, UK).

Cells were obtained in calcium- and magnesium-free phosphate-

buffered saline (PBS; Invitrogen, Carlsbad, CA, USA), and incubated

in phenol red-free RPMI-1640 (spleen cells) or in phenol red-free

Minimum Essential Medium Modified (-MEM) (bone marrow

cells). Media was supplemented with 10% charcoal-stripped, heat-

inactivated fetal bovine serum (FBS), 2 mmol/l glutamine, 100 IU/ml

penicillin G and 100 g/ml streptomycin (all from Gibco, Invitrogen).

Calcitriol (1,25-dihydroxy vitamin D3) was from Alexis (San Diego,CA, USA). PCR primers and enzymes for reverse transcription were

from Invitrogen. All other chemical reagents were from Sigma

(Sigma-Aldrich, St Louis, MO, USA).

Mice

The ethics committee of our institution approved all animal proc

dures. Ten-week-old female C57BL/6 mice (Harlan Interfau

Ibrica, Barcelona, Spain) were subjected to either dorsal ovarie

tomy or sham operation under general anaesthesia by using 150 mg/

of ketamine (Merial, Lyon, France) and 5 mg/kg acepromazi

(Calmo Neosan, Pfizer, NY, USA). Animals were kept at 21C with

12-light:12-h dark cycle and were allowed free access to a pellete

standard mouse laboratory diet containing 0.88% calcium and 0.59

phosphorus (Panlab, Barcelona, Spain) and tap water. The mice wekilled 5 weeks after surgery under halothane anaesthesia (Fluothan

Zeneca, Macclesfield, UK) by cardiac puncture, and the bloo

spleen and femora were removed aseptically. The uterus of ea

mouse was also removed and weighed to confirm the success of ov

riectomy surgery. Blood was allowed to clot and the serum was sep

rated and frozen at 80C until analysis. The spleen and femora weprocessed immediately. None of the mice exhibited evidence

infectious disease, impaired growth (see Results), immunosuppre

sion or other side-effects.

Cell culture

The spleen cells were isolated by careful disintegration of spleen with

scalpel in PBS or by passing the tissue through a sterile 90-m nyl

mesh in PBS. The bone marrow cells were isolated from the femusing centrifugation (Cenci et al., 2000). Briefly, after removal of bon

adherent soft tissues, femur ends were cut off with scissors and insert

into a 0.6-ml microcentrifuge tube with a hole in the bottom and plac

in a 1.5-ml carrier microcentrifuge tube. Cells were extracted from t

bones by centrifugation at 4C and resuspended immediately in PBS.Spleen cells were carefully layered over Histopaque 1077 (Sigm

and centrifuged for 30 min at 400 g at room temperature. Monon

clear cells were collected, washed twice in PBS, resuspended in RPM

containing FBS and then incubated. The bone marrow suspensi

cells were treated similarly, except that they were allowed to adhe

overnight in six-well plastic plates to deplete mature stromal ce

(Dao et al., 1997; Cenci et al., 2000), with the exception of flo

cytometry analysis and RNA extraction, in which this step of adhesi

was omitted. After incubation, non-adherent cells were harvested

pipetting, and mononuclear cells were obtained as described f

spleen cells. Mononuclear non-adherent bone marrow cells we

resuspended in -MEM containing FBS. Spleen and bone marrocells were counted using a haemocytometer, and aliquots of the

cells were used for RNA isolation.

Serum biochemistry

Serum calcium, measured by reaction with o-cresolphthalein, and AL

were assayed using commercial kits (Sigma). Serum levels of TNF-

IL-6, interferon-and IL-2 were measured by an enzyme-linked immnosorbent assay according to the manufacturers instructions (Diaclon

Besanon, France). Total tartrate-resistant acid phosphatase (TRA

was determined by a rapid microplate colorimetric assay with modifictions (Lau et al., 1987).p-Nitrophenyl phosphate (10 mmol/l) was us

as substrate in a buffer containing 100 mmol/l sodium acetate, pH 5

20 mmol/l sodium tartrate and 0.1% (v/v) Triton X-100. Serum sampl

(10 l) were added to 200 l of substrate and incubated at 37C for min. The reaction was stopped by the addition of 100 l 0.5 moNaOH, and absorbance was read at 405 nm with a microplate read

(Model 550, Biop-Rad, Richmond, CA, USA).

Resorption pit assay

Bone marrow preosteoclasts were characterized by assessing the

ability to form resorption pits on calcium phosphate-coated cultu


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882

wells. For this, 5 104 non-adherent mononuclear bone marrow cellsin -MEM containing 10% FBS were seeded onto calcium phosphate-coated osteological discs (Millenium Biologix, Kingston, Ontario,

Canada) and incubated for 12 days in a humidified atmosphere of 5%

CO2 in air in the presence of vehicle, M-CSF (20 ng/ml) or M-CSF

(20 ng/ml) plus murine sRANKL (20 ng/ml). The medium was

changed three times per week, eliminating half of the medium and

replacing it with fresh medium plus stimuli. After 12 days, adherent

cells were removed with bleach solution (6% NaOCl and 5.2% NaCl).

The discs were examined for the presence of resorption lacunae bylight microscopy after von Kossa staining, according to the manufac-

turers instructions.

Cell proliferation and viability analysis

Cell proliferation and viability were evaluated with an XTT (sodium 3-

[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro)

benzene sulfonic acid hydrate)-based colorimetric assay (Roche Diag-

nostics, Mannheim, Germany) according to the manufacturers instruc-

tions. Briefly, 1.5 105 spleen or 4 104 non-adherent bone marrowmononuclear cells in 100 l were placed in 96-well microtitre plates(Orange Scientific, Braine-lAlleud, Belgium) supplemented with

glutamine, antibiotics and 10% FBS at 37C in a humidified atmosphereof 5% CO2 in air for 4 days. The stimuli and their combinations used are

indicated in Table II, and were added in triplicate. After incubation, 50 lof XTT substrate was added to each well and incubated in the conditions

described above. Absorbance of each well was determined at 450 nm

using a microplate reader. Proliferation data in Table II represent the

quotient of proliferation for each stimulus (in triplicate) and proliferation

of the same cells in the same culture plate incubated with vehicle.

Flow cytometry and DNA staining

Cells were labelled with antibodies at the manufacturers recom-

mended dilutions and conditions. Mononuclear spleen and bone mar-

row cells (1 106) were incubated for 30 min on ice with the indicatedantibodies and then washed and resuspended in PBS containing 1%

bovine serum albumin and 0.1% sodium azide. Non-specific signal

was estimated by incubation with rat FITC- and PE-conjugated IgGisotype controls. For analysis of the cell cycle, cells were labelled with

Coulter DNA-Prep (Beckman Coulter, Fullerton, CA, USA) accord-

ing to the manufacturers instructions. Labelled cells were analysed

with an EPICS-XL flow cytometry system (Beckman Coulter). Data

were expressed as the percentage of positive cells since, although ova-

riectomy induced approximately 20% increment in spleen and bone

marrow total cell content, there were no significant differences

between ovariectomized and sham-operated mice.

RNA isolation and semiquantitative RT-PCR

Total RNA was extracted from mononuclear bone or spleen cells with

Trizol reagent (Invitrogen) according to the manufacturers protocol,

and the integrity of the RNA preparations was examined by agarose gel

electrophoresis. One microgram of total RNA was reverse-transcribedin a 20-l reaction volume into single-stranded cDNA with a first-strand cDNA synthesis kit using an oligo-dT primer (Invitrogen). The

subsequent PCR was performed with specific primers for each gene

with Taq polymerase from Sigma. To ensure equal starting quantities

of cDNA for the experiment and to allow the semiquantification of the

PCR products, reverse-transcribed RNA samples were amplified with

primers specific for glyceraldehyde phosphate dehydrogenase (GAPDH).

Amplifications were done using a GeneAmp 9600 thermal cycler (Per-

kin-Elmer), with the temperature cycling set according to the primer

length and Tm value. The number of cycles for each primer pair was

determined according to a linear amplification curve established from

primary experiments. For each PCR reaction, several samples under-

went 1215 extra cycles of amplification to guarantee the semiquantifi-

cation of the experiment. Amplified PCR products were separated on

2% agarose gel and stained with ethidium bromide for visualization.

The intensity of ethidium bromide-stained bands was quantified using

the histogram function in Adobe Photoshop (version 7.0) and was nor-

malized with the GAPDH housekeeping gene.

The sequence of primers used were the following: GAPDH, sense

5-ACC ACA GTC CAT GCC ATC AC-3 and antisense 5-TCC

ACC ACC CTG TTG CTG TA-3; CD25, sense 5-CTC TCC TACAAG AAC GGC AC-3 and antisense 5-TCA CTA GCC AGA AATCGG TGG-3; core binding factor a1 (Cbfa1), sense 5-CCG CACGAC AAC CGC ACC AT-3 and antisense 5-CGC TCC GGC CCACAA ATC TC-3; RANKL, sense 5-CAT TTG CAC ACC TCACCA TC-3 and antisense 5-AAG GGT TGG ACA CCT GAA TG-3; IL-6, sense 5-ATG AAG TTC CTC TCT GCA AGA GAC T-3 and antisense 5-CAC TAG GTT TGC CGA GTA GAT CTC-3;TRAP, sense 5-ACT TCC CCA GCC CTT ACT ACC-3 and anti-sense 5-TCA GCA CAT AGC CCA CAC CG-3; TNF-, sense 5-TCT TCT GTC TAC TGA ACT TCG G-3 and antisense 5-GTAGAG AAT GGA TGA ACA CCC-3; interferon-, sense 5-TCTTGG CTT TGC AGC TCT TCC-3 and antisense 5-CGA ATC AGC

AGC GAC TCC TTT TC-3.

Statistical analysis

Analysis of variance test was used for statistical comparisons between

means of different groups. Results are presented as mean SD. Thenumbers of mice used in each experiment appear in the tables or in the

text of the figures; they correspond to three surgical interventions in

different weeks. A P value < 0.05 of was considered statistically

significant. The entire statistical analysis was carried out using the

Statistical Package for Social Sciences (SPSS, Chicago, IL, USA), v.

11.0 for Windows.

Results

Serum parameters in ovariectomized mice

The success of ovariectomy was determined from the uterine

hypoplasia caused by estrogen deficiency (Table I and Figure 3A).

Ovariectomy induced a dramatic decrease in uterine weight but

not body weight. Ovariectomy increased bone resorption, as

shown by increased values of serum calcium (P < 0.01; Table I).

Serum levels of ALP, a formation marker of bone remodelling,

Table I. Physical parameters, biochemical serum values and number ofresorption pits of sham-operated and ovariectomized mice

Data are mean SD corresponding to 1628 mice from two experiments,except for cytokines (1012 mice) and for resorption pit assay (four mice).ALP, alkaline phosphatase; TRAP, tartrate-resistant alkaline phosphatase.

Sham-operated Ovariectomized P

Body weight (g) 22.73 1.41 23.94 2.68 NSUterus weight (mg) 102.06 23.71 21.66 6.41


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were lower in ovariectomized mice (P < 0.01). Furthermore,

ovariectomy caused a significant decrease in serum levels ofinterferon-(P < 0.01), a T-cell produced cytokine that inhibitsosteoclastogenesis in vitro. Other markers (TNF-, IL-6, IL-2and TRAP) remained unchanged. The number of resorption

pits generated by mononuclear non-adherent ovariectomy bone

marrow cells after incubation with RANKL and M-CSF

was more than twice that found in sham-operated operated

mice (P < 0.05; Table I and Figure 1). The number of pits

formed when cells were incubated with vehicle or M-CSF

alone was very small (


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This agrees with previous work in which an increased number

of osteoclastic cells 5 weeks after ovariectomy was described,

although osteoclastic cell numbers declined over time (Jilka

et al., 1998). Results of the present work show no increases in

serum levels of IL-6 and TFN-, although a trend can beobserved (Table I). Nevertheless, inflammatory cytokines are

involved in the process since it has been reported that blocking

IL-1 and TNF- by antibodies prevents bone loss after ovariec-tomy (Kimble et al., 1995).

Two unexpected results can be seen in Table I: the small

amount of serum ALP-activity and the low level of interferon-after ovariectomy. ALP activity is considered a marker of bone

remodelling (Weaver et al., 1997; Minisola et al., 1998), and a

high correlation exists between bone-specific and total ALP(Takahashi et al., 1997). After ovariectomy, levels of total and

bone-specific ALP increase, peak between the first and second

weeks, and then decline progressively (Tanizawa et al., 2000;

Miyazaki et al., 2004). Although total ALP activity is not

bone-specific, it can be assumed that, if there is no hepatic dis-

ease (Minisola et al., 1998), it should reflect changes caused by

bone marrow cells (Weaver et al., 1997). This decreased level

of ALP activity 5 weeks after ovariectomy implies that bone

formation is impaired, and together with the increase in resorp-

tion pit-generating cells in bone marrow, could explain the

increased calcaemia detected, thus linking estrogen deficiency

with osteoblastic function. We cannot discard the possibility

that the decreased levels of ALP after ovariectomy, however,are related to the low bone density reached by our strain of

mouse (C57BL/6) compared with others, as a consequence of a

lower rate of bone formation (Richman et al., 2001). In accord-

ance with our data, after ovariectomy, decreases have been

described in the percentage of preosteoblastic marrow stromal

cells in early postmenopausal women (Eghbali-Fatourechi

et al., 2003), in serum ALP activity (Sakakura et al., 2001), in

the number of ALP-positive colony-forming units (Pei et al.,

2003) and in ALP activity in individual osteoblasts (Gevers

et al., 2002).

Table II. Cell proliferation analysis of mononuclear spleen and bone marrow cells

PHA, phytohaemagglutinin; Con A, concanavalin A; PMA, 4-phorbol-12-myristate-13-acetate; M-CSF, macrophage colony-stimulating factor; RANKL, receptoractivator of NF-B ligand.Data are mean SD of triplicate determinations corresponding to six mice.aP < 0.05.

Spleen Bone marrow

Sham-operated Ovariectomized Sham-operated Ovariectomized

PHA (10 g/ml) 144.5 18.5 167.1 44.3 131.3 16.5 115.1 9.8Con A (10 g/ml) 222.8 70.5 240.8 41.6 152.5 17.8 128.4 18.9PMA (10 ng/ml) 152.0 32.6 195.6 6.8a 139.3 10.5 127.5 6.6CD3 (2 g/ml) 150.7 61.5 115.4 29.3 106.1 12.2 109.9 12.7CD3 (2 g/ml) + CD28 (0.2 g/ml) 173.5 77.4 156.4 58.4 114.7 3.6 109.7 2.1a

M-CSF (25 ng/ml) 281.2 151.1 406.1 66.0 484.5 149.7 398.4 102.3Calcitriol (0.01 mol/l) 92.7 5.1 100.0 13.5 95.5 7.1 96.8 6.7RANKL (25 ng/ml) 100.9 7.4 104.0 5.7 108.1 16.6 103.5 6.6

Figure 2. Representative flow cytometry dot plots sorted from mice.Forward and side light-scatter plot of unstained cells after Histopaquecentrifugation from spleen (A) and bone marrow (B) cells. The gatedregion contains the greatest number of lymphocytes/blast cells inspleen and bone marrow.

Table III. Flow cytometry analysis of mononuclear spleen cells

Data indicate the percentage of positive cells and are mean SD, correspondingto 412 mice from two different experiments.aP < 0.05.

Molecule Sham-operated Ovariectomized

CD3 32.6 7.8 28.5 8.4CD3+/CD4+ 19.3 2.8 17.4 4.4CD3+/CD8+ 13.5 5.0 11.4 4.4CD11b 12.8 4.2 12.1 2.2CD19 38.7 9.8 46.1 8.6CD25 43.0 9.6 56.7 8.5a

CD51 32.0 5.7 28.5 6.2

Table IV. Flow cytometry analysis of mononuclear bone marrow cells

Data indicate the percentage of positive cells and are mean SD, correspond-ing to 612 mice from two different experiments.aP < 0.05; bP < 0.01.

Molecule Sham-operated Ovariectomized

CD3 8.2 5.2 6.5 2.3

CD19 34.6 6.9 41.0 6.5a

CD11b 30.9 13.8 20.6 8.1a

CD25 23.2 5.9 23.1 3.6CD51 41.5 6.5 35.0 2.3b

RANK 2.8 0.5 3.0 0.6CD3+/RANKL+ 2.1 0.7 4.25 1.4b


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Interferon- is an in vitro negative regulator of osteoclas-togenesis (Gowen and Mundy, 1986; Takayanagi et al., 2000).

Nonetheless, in vivo interferon-cures osteopetrosis by stimu-lating osteoclast formation and bone resorption (Key et al.,

1995), and interferon- receptor-deficient mice have beenshown to be protected against ovariectomy-induced bone loss

(Cenci et al., 2003). The issue of interferon- is complexbecause this cytokine is likely to have tissue specific effects.

Data in the literature about interferon-production after gona-dectomy are sparse, however, particularly in the bone marrow

microenvironment. Cenci and colleagues have described an

increase in the percentage of CD4+ T cells expressing inter-

feron- and in the interferon- concentration in culture super-

natants of bone marrow CD90+ T cells after ovariectom

concluding that this increase is critical in explaining the bo

effects in estrogen deficiency (Cenci et al., 2003). Howeve

most data indicate that estrogens up-regulate and gonadectom

down-regulates interferon-production, although these studiwere not carried out in bone marrow cells. For instance, it h

been reported that interferon- production is decreased aftmice gonadectomy (Aloisi et al., 2001; Sun et al., 2003), b

up-regulated by estrogen in spleen and lymph node lym

phocytes (Karpuzoglu-Sahin et al., 2001; Maret et al., 2003

Moreover, a decrease in the interferon- production has albeen described in postmenopausal women (Yang et al., 200

Kumru et al., 2004). These data suggest a difference in the re

ulation of interferon- production for T cells from periphertissues (spleen and lymph node) and bone marrow T cells aft

gonadectomy. Although the rationale for such impairme

remains unclear, it might be due to differences between sy

temic and local interferon- production. Indeed, interferonimplicated in osteoclastogenesis should be produced mainly

the bone marrow, and it remains to be demonstrated wheth

the decrease in interferon- production detected in this an

other studies has an effect on the increased osteoclastogenesthat is established after gonadectomy (Teitelbaum, 2004).

Estrogen deficiency induced important changes in cellul

subtypes of spleen and bone marrow. Ovariectomy induced

increase in levels of B-cells (CD19+) in bone marrow an

CD25+ cells (IL-2 receptor chain) in spleen as well as bone marrow (total cells; results not shown). Both estroge

deficiency (this work and Masuzawa et al., 1994) and IL

administration (Miyaura et al., 1997) induce B-lymphopoies

a process that may be involved in the mechanism of stimulat

bone loss. In this respect, it has been reported that B-cells are

source of osteoclasts (Sato et al., 2001). Moreover, an increa

in CD25+ T cells in bone marrow of ovariectomized mi

(Cenci et al., 2003) and of CD25+

and HLA-DR+

T cells postmenopausal women (Yang et al., 2000) has be

described, although this effect has not been well explaine

particularly if IL-2 levels do not change with ovariectom

(Table I).

Estrogen receptors are detectable in the reticular strom

cells of the thymus (Barr et al., 1982), suggesting that estr

gen can modulate T-cell lymphopoiesis. Nevertheless, it is n

clear whether ovariectomy modulates T-cell levels. In th

present study, ovariectomy did not affect T-cell levels (tot

cells or percentage), using a CD3 monoclonal antibody. Th

result agrees with other work, in which it has been reporte

that T cells (Thy 1.2+) do not change (total cells or percentag

after mouse ovariectomy (Masuzawa et al., 1994). Aincrease in bone marrow T-cell content has been describe

after ovariectomy (Cenci et al., 2000; Roggia et al., 2001), b

it is possible that the authors gated immature B cells as T cel

since the CD90 monoclonal antibody used is specific f

CD90.1 (Thy 1.1+) and CD90.2 (Thy 1.2+), and an increase

Thy 1.1+ cells of B-cell lineage after ovariectomy has be

described (Erben et al., 1998b). Results of the present stud

support previous data from other authors showing that ovarie

tomy either does not affect or diminishes mouse and rat bon

marrow T-cell levels (Masuzawa et al., 1994; Erben et a

Figure 3. Uterine hypoplasia and mRNA expression levels in severalgenes assessed by semiquantitative RT-PCR. (A) Marked uterine

hypoplasia is obvious in ovariectomized (right uterus) but not insham-operated mice (left uterus) 5 weeks after surgery. RT-PCR ofRNA samples extracted 5 weeks after surgery from spleen and femursfrom ovariectomized and sham-operated mice. Semiquantitative RT-PCR was performed using the indicated primers in spleen (B) andbone marrow (C) mononuclear cells. The intensity of agarose gelbands are represented, after semiquantitative PCR, calculated asdescribed in Materials and methods. Data are mean SD of duplicatedeterminations corresponding to 12 mice. Photographs representbands of the different PCRs and band length is given as the number ofbase pairs (bp).


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886

1998a; Safadi et al., 2000), as well as the results of a study in

postmenopausal women (Yang et al., 2000). However, T-cell-

deficient mice do not lose bone after ovariectomy, thus indicating

a determinant role of T-cells in bone loss in estrogen deficiency

(Cenci et al., 2000). Although it is not possible to discard pos-

sible roles of monocytes and NK cells, T cells are responsible

for increased production of pro-resorptive cytokines after ova-

riectomy (Jilka, 1998; Theill et al., 2002), and the decreased

interferon-level found in the present study after ovariectomy.Ovariectomy did not change the percentage of RANK-

expressing cells. RANKL expression in peripheral tissues is

very low (Anderson et al., 1997; Josien et al., 1999). We have

found, however, that levels of bone marrow CD3+/RANKL+

cells are higher after ovariectomy. The increase in RANKL+

T cells could participate in the increased osteoclastogenesis

after ovariectomy, as has been described (Kong et al., 1999).

Percentages of CD11b+ and of CD51+ cells were smaller in

the bone marrow of ovariectomized mice, although, confirm-

ing previous results (Masuzawa et al., 1994), the total cell con-

tent remained unchanged (results not shown). CD11b (Mac-1)

is a marker of the myeloid lineage, while CD51 is directed

against the V-chain of the vitronectin receptor (CD51/CD61,V3) and is considered an osteoclast marker, although it isexpressed in platelets, T cells and granulocytes. Since osteo-

clasts derive from myelomonocytic lineage cells, one could

expect that ovariectomy would increase the monocyte

macrophage precursors in bone marrow. Confirming our

results, a time-course study showed that percentages of

CD11b+ cells and Gr-1+ cells (myeloid cells and granulocytes,

respectively) decreased but B220+ cells (B cells) were

selectively increased 24 weeks after ovariectomy (Masuzawa

et al., 1994). It is possible, though, that the transitory increase

in myeloid precursors is lost or that the osteoclast precursor

represents only a small fraction of CD11b+ and CD51+ cells. In

addition, it is possible that we need a better specific marker todetect increases in osteoclast lineage cells. In this respect, a

transitory increase in ED1+ cells (myeloid marker of monocyte

and osteoclast lineage) has been described 2 weeks after ova-

riectomy coinciding with the up-regulation of osteoclast num-

bers (Erben et al., 1998b; Jilka et al., 1998), the expression

returning to normal after 4 weeks (Erben et al., 1998b).

In the present study, cell functionality was similar in ova-

riectomized and sham-operated mice. Spleen cells from ova-

riectomized mice showed increased proliferation in response to

PMA, a specific activator of protein kinase C (PKC), suggest-

ing that there is an increase in the amount and/or activity of

PKC. In fact, it has been described that the PKC- gene is

upregulated during osteoclastogenesis (Lee et al., 2003).Mononuclear bone marrow cells from ovariectomized mice

exhibited lessened proliferation in response to specific stimula-

tion of T-cells (CD3 + CD28 monoclonal antibodies), which

could be due to impairment of T-cell functionality and could

explain the low level of interferon-observed in this work.Gene expression analysis by RT-PCR did not offer substan-

tial data. The only significant difference found was higher

CD25 expression in bone marrow. Other authors have found

changes in rat osteoprotegerin and RANKL gene expression in

the first days after ovariectomy, but not in the fourth week

(Bonnelye et al., 2002), suggesting that many of the changes in

gene expression occur in the first days after ovariectomy

(Tanizawa et al., 2000; Miyazaki et al., 2004).

In summary, the main effects of estrogen deficiency on the

immune system seem to involve haematopoiesis. It seems that

estrogen receptors are present only in certain immature

immune cells or in a small population of CD8+ T cells and

macrophages (Igarashi et al., 2001). Therefore, the effects of

estrogen could be due to direct action on mature lymphoid cells

or, more probably, through interaction with other estrogen

receptor-expressing cells that can regulate lymphoid cells, such

as dendritic or bone marrow stromal cells (Bellido et al., 1993;

Komi and Lassila, 2000). In bone marrow, ovariectomy up-

regulated CD3+/RANKL+ cells and B cells, did not affect T-cell

levels, and decreased the percentage of myeloid cells, although

only B cells increased in absolute numbers after ovariectomy.

The decrease in serum interferon- level, which can be in thebase of the incremented osteoclastogenesis found, could reflect

impaired T-cell functionality. Furthermore, ovariectomy signi-

ficantly diminished serum ALP activity, indicating a lower rate

of bone formation 5 weeks after ovariectomy.

Acknowledgements

The authors are indebted to Drs Enrique OConnor and GuadalupeHerrera for their expert assistance with flow cytometry, and toMrs Rosa Aliaga and Mrs Elvira Calap for their excellent technicalassistance. This work was supported by grant 01/3051 from the Fondode Investigaciones Sanitarias (FIS).

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Submitted on August 24, 2005; resubmitted on October 21, 2005; accepted November 3, 2005

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