Estrogens in rheumatoid arthritis; the immune system and bone

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Molecular and Cellular Endocrinology 335 (2011) 14–29

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Molecular and Cellular Endocrinology

journa l homepage: www.e lsev ier .com/ locate /mce

strogens in rheumatoid arthritis; the immune system and bone

lrika Islander ∗,1, Caroline Jochems1, Marie K. Lagerquist, Helena Forsblad-d’Elia, Hans Carlstenenter for Bone and Arthritis Research (CBAR), Department of Rheumatology and Inflammation Research, The Sahlgrenska Academy, University of Gothenburg, Sweden

r t i c l e i n f o

rticle history:eceived 6 November 2009eceived in revised form 28 May 2010

a b s t r a c t

Rheumatoid arthritis (RA) is an autoimmune disease that is more common in women than in men. Thepeak incidence in females coincides with menopause when the ovarian production of sex hormonesdrops markedly. RA is characterized by skeletal manifestations where production of pro-inflammatory

ccepted 29 May 2010

eywords:heumatoid arthritisstrogen

mediators, connected to the inflammation in the joint, leads to bone loss. Animal studies have revealeddistinct beneficial effects of estrogens on arthritis, and a positive effect of hormone replacement therapyhas been reported in women with postmenopausal RA. This review will focus on the influence of femalesex hormones in the pathogenesis and progression of RA.

elective estrogen receptor modulatorsmmune systemone

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152. Pathogenesis of rheumatoid arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2.1. CD4+ and CD8+ T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152.2. IL-17 producing T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152.3. Regulatory T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162.4. B cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

3. Osteoimmunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163.1. OPG/RANK/RANKL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163.2. Cytokines that influence bone metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173.3. Skeletal manifestations in RA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

4. Estrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174.1. Estrogen receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174.2. Hormone replacement therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184.3. Selective estrogen receptor modulators (SERM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

5. Estrogens and the immune system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195.1. The innate and adaptive immune system. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

6. Estrogens and bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196.1. Menopause . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196.2. Treatment with sex steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

7. Sex steroids in RA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.1. Sex steroids in animal models of arthritis . . . . . . . . . . . . . . . . . . . . . . . . . .7.2. Effects of sex steroids in human RA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.3. Clinical studies on HRT in RA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8. Treatment of osteoporosis in RA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: BMD, bone mineral density; CII, collagen type II; CIA, collagen-induceesponse element; HRT, hormone replacement therapy; OPG, osteoprotegerin; OPGL, ostectivator of NF�B ligand; SERM, selective estrogen receptor modulator; SLE, systemic lup∗ Corresponding author at: Center for Bone and Arthritis Research (CBAR), Departmentniversity of Gothenburg, Box 480, 405 30 Göteborg, Sweden. Tel.: +46 31 342 64 11; fax

E-mail address: ulrika.islander@rheuma.gu.se (U. Islander).1 Both authors contributed equally to this work.

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d arthritis; DHEA, dihydroepiandrosterone; ER, estrogen receptor; ERE, estrogenoprotegerin-ligand; OVX, ovariectomy; RA, rheumatoid arthritis; RANKL, receptorus erythematosus; TRANCE, TNF-related activation induced cytokine.of Rheumatology and Inflammation Research, The Sahlgrenska Academy,

: +46 31 823925.

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. Introduction

Rheumatoid arthritis (RA) is a progressive systemic autoim-une disease with a prevalence of ∼0.5–1% (Kvien et al., 1997) and

s characterized by symmetrical polyarthritis. Macrophages, T cellsnd B cells infiltrate the synovium that lines the joints, while theynovial fluid is dominated by neutrophils. Chronic inflammationeads to destruction of joint cartilage and bone. Several findingsndicate the involvement of sex hormones in RA. For example, theemale to male incidence ratio is 4–5:1 before 50 years of age, and:1 for patients with a later onset (Kvien et al., 1997, 2006), and theeak incidence in women coincides with menopause (Goemaere etl., 1990). It has been shown that estrogens can affect the diseaseourse of RA in humans (Forsblad-D’Elia et al., 2003a; Ostensen etl., 1983), and in animal models (Holmdahl et al., 1986; Janssonnd Holmdahl, 1989; Yamasaki et al., 2001). Mice subjected tovariectomy (OVX) have decreased levels of estrogens and dis-lay higher frequency and increased severity of collagen-inducedrthritis (CIA), as compared to OVX mice treated with estrogen orham-operated mice with intact levels of estrogen (Holmdahl etl., 1986, 1987; Jochems et al., 2005). In many women with RA theisease activity diminishes during pregnancy when the levels ofemale sex hormones are high (Ostensen et al., 1983; Barrett et al.,999). In contrast, the disease is often aggravated after deliveryOstensen et al., 1983; Barrett et al., 1999, 2000). The complex rolef estrogens in different inflammatory diseases has previously beenescribed in an extensive review by Straub (2007).

Estrogens are also main regulators of skeletal growth and main-enance, as demonstrated in both experimental and human studiesForsblad-D’Elia et al., 2003b; Riggs et al., 2002; Sinigaglia et al.,000; Vanderschueren et al., 2004; Vidal et al., 2000). Previous stud-

es have demonstrated that estrogen deprivation, such as after OVXn animal models and after menopause in women, reduces trabec-lar bone mineral density (BMD) as well as cortical BMD, whilestrogen substitution restores both bone compartments (Reckert al., 1999; Turner, 1999; Windahl et al., 1999). There are sev-ral skeletal manifestations in RA, including joint erosions andoth periarticular and generalized bone loss, due to excess boneesorption by osteoclasts (Hayward and Fiedler-Nagy, 1987). Therequency of generalized osteoporosis in postmenopausal patientsith RA has been reported to be approximately 50% (Forsblad-’Elia et al., 2003a).

This review will start by giving a background to the pathogenesisf RA and the field of osteoimmunology. Then the effects and mech-nisms of estrogen will be described, followed by the influence ofstrogen on the immune system and bone, and finally the influencef female sex hormones will be described in the pathogenesis androgression of RA.

. Pathogenesis of rheumatoid arthritis

The pathogenesis of rheumatoid arthritis (RA) is largelynknown, with genetic and environmental factors influencing dis-ase development and progression (Imboden, 2009). From studiesf animal models resembling RA it has been shown that micexpressing the MHC class II haplotype H2q can develop arthritispon immunization with collagen type II (CII). This well estab-

ished experimental model is called collagen-induced arthritis (CIA)Trentham et al., 1977). In humans it has been proposed that cer-ain HLA-DR4 molecules present peptides of CII, present in jointartilage, which results in susceptibility to develop RA. It has been

his section, lymphocytes involved in the pathogenesis of RA wille described.

Endocrinology 335 (2011) 14–29 15

2.1. CD4+ and CD8+ T cells

In RA, T cells represent a large proportion of the inflamma-tory cells invading the synovial tissue, and T cell activation andmigration into the synovium occurs as an early consequence of thedisease. These cells adopt a pro-inflammatory phenotype and canactivate for example B cells and synovial macrophages via cytokineproduction. In one study using B10q mice, which are highly suscep-tible to CIA, lack of CD4+ T cells resulted in decreased susceptibilityto disease and lower levels of CII antibodies, whereas lack of CD8+T cells did not significantly affect the disease (Ehinger et al., 2001).In contrast, another study in DBA/1 mice revealed that CD8+ cellswere necessary for disease development, while lack of CD4+ cellsdid not decrease the susceptibility to CIA (Tada et al., 1996). Thesedata suggest that CD4+ and CD8+ T lymphocytes may play differ-ential roles in CIA depending on the genetic background of mousestrains.

The balance of CD4+ Th1 and Th2 cells has been suggested to playa role in the pathogenesis of RA (Dolhain et al., 1996; Morita et al.,1998; Yudoh et al., 2000). The Th1 subset is defined by specific pro-duction of IFN-� and IL-2, and by the stimulation of cell-mediatedimmunity, whereas the Th2 subset specifically produces IL-4 andstimulates humoral immunity (Abbas et al., 1996; Mosmann andSad, 1996). Based on analysis of IFN-� and IL-4 production, a dom-inance of Th1 cell activity over Th2 cell activity has been shownin the inflamed joints of RA patients (Dolhain et al., 1996; Moritaet al., 1998). This imbalance of Th1/Th2 cells was shown to corre-late with disease activity scores (Yudoh et al., 2000). Although IL-4production by T cells from the peripheral blood of RA patients isincreased compared with that of healthy controls, this Th2 activityseems to be insufficient to control Th1-associated inflammation inRA (al-Janadi et al., 1996; Haddad et al., 1998; van Roon et al., 1997).

2.2. IL-17 producing T cells

A large proportion of the T cells infiltrating the joints in RAproduce the pro-inflammatory cytokine IL-17. IL-17 promotes theproduction of pro-inflammatory mediators from cells present inthe joint including synovial fibroblasts, monocytes, macrophagesand chondrocytes, which result in cartilage damage and bone ero-sion (Chabaud et al., 1999; Miossec, 2007). It has been shown thatIL-17 enhances the development of CIA, and IL-17 deficiency pro-tects against development of CIA (Chu et al., 2007; Lubberts et al.,2002a; Nakae et al., 2003). Recently, the first clinical study of RApatients treated with a monoclonal antibody against IL-17 was pub-lished, and the results showed improved signs and symptoms ofRA with no strong adverse safety signals noted (Genovese et al.,2010). IL-17 producing CD4+ T helper cells (Th17 cells) are stronglypro-inflammatory and have an important role in both systemic andorgan-specific autoimmunity (Bettelli et al., 2007). The Th17 cellpopulation was recently shown to develop in the thymus (Markset al., 2009), and the factors required for driving the differentiationof peripheral naïve cells to Th17 cells were first shown in mice tobe TGF� and IL-6, whereas in humans IL-1� and IL-23 are neededin addition to TGF� and IL-6 (Manel et al., 2008; Veldhoen et al.,2006; Volpe et al., 2008). In an experimental model of arthritis itwas shown that Th17 cell differentiation is initiated in draininglymph nodes, while the IL-17 producing cells are restricted to theinflamed synovium (Egan et al., 2008). Blockade of TNF� in CIAresults in increased numbers of Th1 and Th17 cells in lymph nodes,but inhibits their accumulation in the joints, providing an expla-

��T cells are an innate source of IL-17, which have been shownto play an important role in CIA (Roark et al., 2007). ��T cells are

1 ellular Endocrinology 335 (2011) 14–29

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Fig. 1. “Osteoimmunology”, represents the interplay between the immune systemand bone. RANKL is either soluble or bound to the cell membrane, and is pro-duced by many different cells including activated T cells, B cells and osteoblasts.It promotes osteoclast differentiation and activation by binding to RANK, its recep-tor on pre-osteoclasts and osteoclasts. Signalling through RANK stimulates matureosteoclasts to resorb bone and also inhibits osteoclast apoptosis. In addition toRANKL, osteoblasts also produce osteoprotegerin (OPG). OPG acts as a decoy recep-tor, binding and neutralizing soluble or membrane-bound RANKL, thus preventing

6 U. Islander et al. / Molecular and C

he predominant population of IL-17 producing cells in the swollenoints of mice with CIA (Ito et al., 2009), while in draining lymphodes the number of ��T cells is equivalent to that of Th17 cellsRoark et al., 2007; Ito et al., 2009).

.3. Regulatory T cells

Regulatory T cells (Treg) are a T cell subset involved in pre-enting both autoimmune and other inflammatory reactions, byuppression of proliferation and cytokine production from bothh1 and Th2 cells (Sakaguchi et al., 2006). Mice depleted of Treghow an accelerated and more severe form of CIA (Kelchtermanst al., 2005; Morgan et al., 2003), and activated Treg cells improvelinical symptoms of CIA (Kelchtermans et al., 2009). Treg are bothonstitutively produced as a specific cell-line in the thymus, andnduced peripherally from naïve T cells (Sakaguchi et al., 2006;

ing et al., 2005). These cells are characterized by surface expres-ion of CD25, low levels of CD127, and by intracellular expression ofOXP3 and CTLA-4 (Sakaguchi et al., 2006; Hartigan-O’Connor et al.,007). Treg produce TGF� (Oida et al., 2006), and the induction ofreg has been shown to be dependent on TGF�, while the suppres-ive function of the cells may be inhibited by pro-inflammatoryytokines such as IL-6 and TNF� (Afzali et al., 2007; Pasare andedzhitov, 2003). In RA patients, the frequency of Treg is increased

n synovial fluid compared to peripheral blood (Cao et al., 2003; vanmelsfort et al., 2004). However, the interaction of Treg cells withctivated monocytes in the joint could diminish their suppressivectivity, thus contributing to the chronic inflammation in RA (vanmelsfort et al., 2007). Interestingly, it has been shown that theevelopment of Treg cells is linked to that of Th17 cells (Bettelli etl., 2006). Naïve T cells that differentiate under influence of TGF�an develop into either Treg or Th17 cells depending on the levels ofL-6 present (Veldhoen et al., 2006; Bettelli et al., 2006). In addition,reg cells have been shown to differentiate into IL-17 producingells in both human and murine cultures (Koenen et al., 2008; Xut al., 2007).

.4. B cells

B lymphocytes are important in the pathogenesis of RA, by pro-uction of antibodies to CII, and for T cell activation (Takemurat al., 2001). Indeed, B lymphocyte deficient mice are resistant toIA (Svensson et al., 1998). Anti-CII antibodies bind to the artic-lar cartilage and initiate complement activation, which recruits

nflammatory cells to the site (Terato et al., 1992). First, neutrophilsre recruited, and then monocytes and lymphocytes. Antibodieso CII have been detected in serum and synovial fluid of patientsith RA (Morgan et al., 1987; Terato et al., 1990), and CII antibody-roducing B cells have been found in synovial fluid and synovialissue (Rudolphi et al., 1997; Tarkowski et al., 1989). Transfer ofII-antibodies can induce arthritis in mice (Terato et al., 1992).dministration of B cell-depleting anti-CD20 antibodies has beenhown to be a successful treatment for some RA patients (Emery etl., 2006; Mease et al., 2008).

. Osteoimmunology

Bone loss is common in various inflammatory conditionsGinaldi et al., 2005). In inflammatory states, infiltrating cells ofhe immune system provide cytokines that affect bone metabolism

nteraction between immune cells, bone cells and their products.hus, the bone and the immune system are connected and recentlyhe fields of bone biology and immunology merged into a niche

osteoclastogenesis and bone resorption, and increasing apoptosis of osteoclasts. Sev-eral different cytokines produced by cells in the immune system can also influenceosteoblasts and osteoclasts and thereby provide a link between activation of theimmune system and bone metabolism.

field called “osteoimmunology” (Arron and Choi, 2000; Takayanagi,2007) (Fig. 1).

3.1. OPG/RANK/RANKL

The first interactive molecule to be recognized was recep-tor activator of NF�B ligand (RANKL), also called TNF-relatedactivation induced cytokine (TRANCE), or osteoprotegerin-ligand(OPGL) (Lacey et al., 1998; Wong et al., 1997). RANKL is eithersoluble or bound to the cell membrane, and is produced byactivated T cells (Horwood et al., 1999), B cells (Manabe et al.,2001), osteoblasts (Yasuda et al., 1998), bone-lining cells (Zhanget al., 2001), macrophages (Crotti et al., 2002), synovial fibrob-lasts (Shigeyama et al., 2000), chondrocytes (Komuro et al., 2001),endothelium (Collin-Osdoby et al., 2001) and neutrophils (Poubelleet al., 2007). RANKL regulates the communication between T cellsand dendritic cells, dendritic cell survival, lymph node forma-tion and formation of lactating mammary glands (Anderson et al.,

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ng osteoclastogenesis by RANKL expression, B lymphocyte lineageells can also serve as osteoclast precursors (Manabe et al., 2001).oth RANK- and RANKL-knock out mice develop grave osteopetro-is, since they have no osteoclasts (Kong et al., 1999a; Li et al., 2000),nd RANKL knock out mice can develop severe serum transfer-nduced arthritis without any bone destruction (Pettit et al., 2001).t has been shown that vitamin D3, parathyroid hormone and IL-1an induce RANKL expression on osteoblasts (Horwood et al., 1998).

In addition to RANKL, osteoblasts and bone marrow stromalells also produce osteoprotegerin (OPG) (Simonet et al., 1997).PG acts as a decoy receptor, binding and neutralizing soluble orembrane-bound RANKL, thus preventing osteoclastogenesis and

one resorption, and increasing apoptosis of osteoclasts. The pro-iferation and differentiation of B cells are inhibited by OPG (Yunt al., 2001). OPG-deficient mice develop early osteoporosis (Bucayt al., 1998). The OPG/RANKL ratio determines the net degree ofsteoclast activation (Fig. 1).

.2. Cytokines that influence bone metabolism

Several different cytokines produced by cells in the immune sys-em can influence bone cells and thereby provide a link betweenctivation of the immune system and bone metabolism (Fig. 1).

TNF� stimulates osteoporosis development by increasingANKL production in bone-lining cells leading to an increasedumber of osteoclasts (Zhang et al., 2001; Lam et al., 2000), by stim-lating osteoclast activity (Fuller et al., 2002), and by increasing thepoptosis of osteoblasts (Tsuboi et al., 1999).

IL-1� stimulates pre-osteoclast fusion (Jimi et al., 1999), osteo-last activation and survival (Jimi et al., 1998), and increasessteoblast apoptosis (Tsuboi et al., 1999), thus contributing to boneoss. IL-1 receptor antagonist is used to hamper inflammation, andlso inhibits osteoclast differentiation and bone resorption (Ljungt al., 2007).

IL-7 induces TNF� and RANKL secretion from T cells, increases Bymphopoiesis and induces bone loss (Miyaura et al., 1997; Toraldot al., 2003). IL-7 knock out mice have increased bone volume andecreased B lymphopoiesis (Miyaura et al., 1997).

IL-17 stimulates differentiation of osteoblasts (Rifas, 2006), andncreases the RANKL/OPG ratio (Nakashima et al., 2000). Th17 cellstimulate osteoclastogenesis (Sato et al., 2006).

TGF� is stored in an inactive form in the bone matrix (Bonewaldnd Dallas, 1994). Its effects are anti-osteoporotic, inhibiting boneesorption and fusion and proliferation of pre-osteoclasts, andncreasing osteoclast apoptosis (Chenu et al., 1988; Hughes et al.,996). It also stimulates osteoblast proliferation and differentationBonewald and Dallas, 1994). In addition, regulatory T cells, whichroduce TGF�, have been demonstrated to suppress osteoclastormation in vitro via direct cell–cell contact (Zaiss et al., 2007), andlso to inhibit osteoclastogenesis in vitro and in vivo (Kelchtermanst al., 2009).

.3. Skeletal manifestations in RA

RA is characterized by different skeletal manifestations includ-ng bone erosions (Scott, 2000), periarticular osteopenia (Stewartt al., 2004) and generalized osteoporosis (Forsblad-D’Elia et al.,003b; Sinigaglia et al., 2000; Haugeberg et al., 2002, 2000;ambrook et al., 1995). Joint inflammation causes production ofro-inflammatory cytokines that induce osteoclast developmentnd activation, leading to focal bone loss. In addition, the inflamed

Endocrinology 335 (2011) 14–29 17

osteoclasts, expressing TRAP, cathepsin K and calcitonin receptor(Gravallese et al., 1998, 2000). They are also found in bone erosionsof mice with CIA (Suzuki et al., 1998).

There are several factors affecting bone cells in RA. RANKL isfound at sites of bone erosion and in synovial tissue from RApatients (Pettit et al., 2006). The RANKL/OPG ratio is increased inactive RA, and correlates with increased bone resorption (Hayneset al., 2001). Increased levels of RANKL have been found in mouseand rat CIA (Lubberts et al., 2002b; Mori et al., 2002; Stolina etal., 2005), and RANKL knock out mice are protected from boneerosions in serum transfer-induced arthritis (Pettit et al., 2001).Neutrophils are abundant in joints of RA patients, and expressmembrane-bound RANKL, RANK and OPG (Poubelle et al., 2007).Treatment with OPG has been found to reduce bone loss in experi-mental arthritis (Kong et al., 1999b; Redlich et al., 2002; Romas etal., 2002; Schett et al., 2003), as well as in postmenopausal arthritisin women (Bekker et al., 2001). In RA patients, IL-17 induces RANKLexpression and decreases OPG expression in osteoblasts, as well asincreases RANKL, IL-1, IL-6 and TNF� expression in synoviocytes(Kehlen et al., 2003; Kotake et al., 1999). IL-1�, IL-6, the solubleIL-6 receptor and IL-7 are elevated in patients with RA (Forsblad-D’Elia et al., 2003b; Kotake et al., 1996; Okamoto et al., 1997; vanRoon et al., 2005). TNF� is elevated during inflammatory diseases,leading to increased bone resorption (Cenci et al., 2000). Treat-ment with monoclonal anti-TNF� antibodies has been shown topreserve the BMD in patients with RA (Lange et al., 2005; Marotteet al., 2007; Seriolo et al., 2006; Vis et al., 2006). Osteoblasts arealso affected by the inflammatory process e.g. IL-1� and TNF� bothinduce osteoblast apoptosis, and other molecules influence theirsurvival and function by inhibiting bone morphogenetic proteins(Tsuboi et al., 1999; Chen et al., 2004; Groppe et al., 2002).

4. Estrogen

The female sex hormone estrogen has many physiologicaleffects, for example the development and maturation of thereproductive system, skeleton, and immune-, nervous-, and car-diovascular systems. Estradiol is produced by the granulosa cells ofthe ovary, and to some degree by the adrenal cortex, adipose tissueand testicles, by aromatization of testosterone (Gruber et al., 2002).At menopause, most of the ovarian production of sex hormonesceases, although some production of testosterone, androstendione,dihydroepiandrosterone (DHEA), estrone and estradiol has beenshown 10 years after menopause. Ovariectomy of postmenopausalwomen significantly decreases the serum levels of estrone andtestosterone, revealing some remaining ovarian sex hormone pro-duction even after menopause (Fogle et al., 2007).

4.1. Estrogen receptors

The classical estrogen receptors (ER) ER� and ER� were clonedin 1986 and 1996, respectively (Green et al., 1986; Kuiper et al.,1996). The distribution of ER� and ER� varies in different tissues.In the classical transcription pathway the ERs bind to estrogen, forma receptor dimer and translocate into the cell nucleus (Petterssonet al., 1997). There, they form a complex with co-regulatory pro-teins and bind to the estrogen response element (ERE) to initiatetranscription (Levy et al., 2007). The EREs are located in the pro-moter regions of different genes that are regulated by estrogens(Gruber et al., 2004) (Fig. 2, pathway 1). In the non-classical tran-

18 U. Islander et al. / Molecular and Cellular Endocrinology 335 (2011) 14–29

Fig. 2. Estrogen signalling. Pathway 1: In the classical transcription pathway theestrogen receptors (ER) bind to estrogen, form a receptor dimer and translocateinto the cell nucleus. There, they form a complex with co-regulatory proteins andbind to the estrogen response element (ERE) to initiate transcription. Pathway 2:In the non-classical transcription pathway, the estrogen/ER-complex starts tran-scription by binding to alternative transcription factors e.g. AP-1, SP-1 and NF�B,which bind non-ERE sites. Pathways 3 and 4: There are also membrane associatedestrogen receptors e.g. ER or GPR30. Binding of estrogen to a membrane associatedr(((

GsswFeaPvmstrom11

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eceptor leads to rapid activation or repression of intracellular signalling pathwayscalcium mobilization and PI3K activation), leading either to non-genomic signallingpathway 3) or altered transcriptional activity via other transcription factors (TF)pathway 4).

-protein-coupled receptor, and this receptor has recently beenuggested to be tightly coupled to estrogen membrane receptorignalling and may thereby contribute to normal physiological asell as pathophysiological estrogenic effects (Benten et al., 2001;

ilardo et al., 2007; Levin, 2009; Pedram et al., 2006; Revankart al., 2005, 2007). Some studies have indicated that ER� maylso be membrane associated in some cells (Pedram et al., 2006;appas et al., 1995). Binding of these receptors leads to rapid acti-ation or repression of intracellular signalling pathways (calciumobilization and PI3K activation), leading either to non-genomic

ignalling or altered transcriptional activity via other transcrip-ion factors (TF) (Fig. 2, pathways 3 and 4). In addition, estrogeneceptors can be activated through phosphorylation in the absencef estrogen by dopamine, insulin-like growth factor-1, epider-al growth factor and cyclic AMP (Aronica and Katzenellenbogen,

991; Curtis et al., 1996; Newton et al., 1994; Smith et al.,993).

.2. Hormone replacement therapy

Hormone replacement therapy (HRT) with estradiol afterenopause was first started in 1941, and was successful since

he clinical symptoms from loss of estrogen could be abated. These of estrogens further increased during the 60’s and 70’s, but

n 1975 a study showed the relationship between estrogen treat-ent and endometrial cancer, which led to decreased use. The

nding that addition of progesterone protects from endometrial

n 2002 the Women’s Health Initiative study, which was the biggesttudy ever of the long-term effects of HRT, was prematurely inter-

Fig. 3. Molecular structures of 17�-estradiol, ICI 182,780, Tamoxifene and Ralox-ifene.

rupted due to severe side effects. The combination of conjugatedequine estrogen and progesterone was shown to increase the riskof coronary heart disease and stroke, in addition to the previ-ously known risk of venous thrombo-embolic disease and invasivebreast cancer (Rossouw et al., 2002). One and a half years later,the group taking only conjugated equine estrogen was also termi-nated due to increased risk of stroke and no evidence for benefitson coronary heart disease (Anderson et al., 2004). No increasedrisk of breast cancer was shown after taking estrogen alone(Anderson et al., 2004). Since then, the use of HRT has decreasedworldwide (Stefanick, 2005), and the search for other drugs withthe beneficial effects of estrogen, but without the side effects,continues.

4.3. Selective estrogen receptor modulators (SERM)

Selective estrogen receptor modulators (SERM) are non-steroidal molecules, which bind to the estrogen receptors anddisplay estrogen-like effects in some tissues, but antagonisticeffects in other tissues. The tissue selectivity of a SERM depends onthe relative amount of ER� and ER� in that tissue, the affinity of theSERM, and upon the availability of co-activators and co-repressors.To this date there are three SERMs used in clinical practise, Tamox-ifene, Raloxifene and ICI 182,780 (Fulvestrant) (Fig. 3). Tamoxifeneis approved for treatment of ER-positive breast cancer. It acts asan estrogen antagonist in breast tissue, but has agonistic effectson endometrium (Rutqvist et al., 1995). Raloxifene is approved asprophylaxis of invasive breast cancer (Temin, 2009) and for treat-ment of postmenopausal osteoporosis (Stefanick, 2005). It bindswith high affinity to ER� and functions as an estrogen agonist inbone and on serum lipids, but acts as an antagonist in uterus andbreast tissue (Cummings et al., 1999; Ettinger et al., 1999; Li et al.,1998; Sato et al., 1995, 1996). ICI 182,780 (Fulvestrant) is a pure ER�and ER� antagonist without any known agonistic properties, and isused as adjuvant treatment for ER-positive breast cancer (Wakelinget al., 1991).

Numerous new SERMs are currently undergoing clinical devel-opment for the prevention and/or treatment of postmenopausalosteoporosis (Miller and Derman, 2010). For example, Lasofoxifenewas recently described to be associated with reduced risks of frac-tures, ER-positive breast cancer, coronary heart disease and stroke

ellular

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b((cettwc(c(a(b(as

. Estrogens and the immune system

Estrogen affects the immune system in multiple ways. Thestrogen receptors ER� and ER� are found in cells of both thennate and the adaptive immune system in both sexes (Stygart al., 2006). Women have stronger humoral and cell-mediatedmmune responses to infections than men (Halme et al., 1998).n contrast, women have less potent innate immune responses,s measured in vitro by 30% lower TNF� secretion after stimu-ation of whole blood with LPS (Moxley et al., 2002). Because ofhese and many other dual effects on the immune system, estrogen

ay have an ameliorating or an enhancing influence in differentutoimmune diseases (Straub, 2007). We have previously shownhat estrogen inhibits T cell-dependent immune reactions whilentibody production from B cells is enhanced (Carlsten et al., 1989;rlandsson et al., 2003). Estrogen has been shown to aggravateystemic lupus erythematosus (SLE) in murine models, and tonduce flares and increased antibody production in patients withLE (Carlsten and Tarkowski, 1993; Carlsten et al., 1990; Gompelnd Piette, 2007; Grimaldi et al., 2006; Kanda et al., 1999). In con-rast, RA and multiple sclerosis as well as their murine equivalentsIA and experimental autoimmune encephalitis, are amelioratedy endogenous and exogenous estrogen (Forsblad-D’Elia et al.,003a; Holmdahl et al., 1986; Offner et al., 2000; Voskuhl, 2003).

nterestingly, the lupus-like disease is aggravated and the arthriticisease is ameliorated by estradiol in MRL/lpr mice that sponta-eously develop SLE (Carlsten et al., 1992; Ratkay et al., 1994).resently, autoimmune diseases are not classified according tohe immunological mechanisms involved, however it has beenuggested that autoimmune diseases could be divided into tworoups, one in which estrogen accelerates the disease progres-ion and another in which estrogen is beneficial (Holmdahl et al.,989).

.1. The innate and adaptive immune system

Estrogen affects cells of both the innate and adaptive immuneystem (Straub, 2007). For example it inhibits neutrophil functionnd adhesion to endothelium, and the number of neutrophils ineripheral blood (Bekesi et al., 2007; Buyon et al., 1984; Geraldest al., 2006; Josefsson et al., 1992). NK cell activity is decreasedNilsson and Carlsten, 1994). Estrogen induces apoptosis in human

onocytes, and also modulates the pro-inflammatory cytokineelease from activated monocytes and macrophages (Kramer et al.,004; Mor et al., 2003). Serum levels of IL-1, IL-6 and TNF� are

ncreased after menopause, and decreased by HRT (Pfeilschifter etl., 2002; Rachon et al., 2002).

The adaptive immune system is affected in differential waysy estrogen. In mice, the delayed type hypersensitivity reactionDTH), mediated by macrophages and T cells, is reduced by estrogenCarlsten et al., 1989; Taube et al., 1998). Treatment with estradiolauses thymic involution, and reduces T lymphopoiesis (Erlandssont al., 2001; Marotti et al., 1984; Rijhsinghani et al., 1996). Con-ribution of the GPR30 membrane receptor to estrogen-inducedhymocyte apoptosis was recently shown (Wang et al., 2008). Itas found that the levels of estrogen present during pregnancy

an stimulate proliferation and differentiation of regulatory T cellsPrieto and Rosenstein, 2006; Tai et al., 2008), and that these effectsan be inhibited by ICI 182,780, the specific inhibitor of ER� and ER�Tai et al., 2008). B lymphopoiesis is down-regulated by estrogen,nd both B and T lymphopoiesis are increased after ovariectomy

Endocrinology 335 (2011) 14–29 19

late immunoglobulin production in spleen cells (Erlandsson et al.,2002).

By using ER knock out mice, the impact of estrogen signallingon the immune system has been studied. It has been shown thatmice lacking ER� have smaller thymi, and display less thymic atro-phy after exposure to estrogen compared to WT mice (Staples et al.,1999). These results were confirmed and expanded in a study show-ing that deletion of ER� alone, or ER� + ER�, results in hypoplasiaof both thymus and spleen (Erlandsson et al., 2001). Furthermore, ahigher frequency of double positive (CD4+ CD8+) T cells, but a lowerfrequency of single positive T cells, was found in thymus of micelacking ER�. Estrogen treatment of mice lacking ER� results in asimilar degree of thymic atrophy compared with WT mice, but dis-plays no alteration in the frequency of double positive thymocytes(Erlandsson et al., 2001).

In contrast to estradiol, Raloxifene does not affect the DTH reac-tion and does not induce thymic involution (Erlandsson et al., 2000).Raloxifene decreases the serum levels of IL-6 in arthritic mice, butdoes not affect IL-6 in non-arthritic mice, and Raloxifene, but notestradiol, decreases the expression of TNF� and RANKL mRNA inspleen from arthritic mice (Jochems et al., 2007).

6. Estrogens and bone

The classical estrogen receptors ER� and ER� are present inosteoblasts, osteocytes, osteoclasts and chondrocytes, mediatingestrogen effects on bone and cartilage (Komm et al., 1988; Oursler etal., 1991; Tomkinson et al., 1998; Ushiyama et al., 1999). Indeed, theclassical transcription pathway is activated in osteoblasts, osteo-cytes and chondrocytes exposed to estradiol (Windahl et al., 2007).

6.1. Menopause

The development of postmenopausal osteoporosis is associatedwith estrogen deficiency. At first, there is a phase of rapid boneloss, dominated by an increase in bone resorption and trabecu-lar thinning, leading to loss of connection between trabeculae. Aslower rate of bone loss follows and is sustained, dominated by adecrease in bone formation and trabecular thinning (Riggs et al.,2002; Eriksen et al., 1990). Estrogenic effects on bone are likely tobe mediated by both direct effects on the different cell types, andchanges of the cytokine milieu of the bone compartment (Fig. 4).Estrogen induces OPG expression in human osteoblastic cells invitro (Hofbauer et al., 1999), and OPG-treatment counteracts thedevelopment of osteoporosis after ovariectomy in rats (Simonet etal., 1997). In early menopausal women it has been demonstratedthat the expression of RANKL is up-regulated on T cells, B cellsand pre-osteoblastic bone marrow cells (Eghbali-Fatourechi et al.,2003). The net effects of estrogen deprivation are increased boneresorption due to a higher number of activated osteoclasts (Eriksenet al., 1999), deeper resorption pits due to increased osteoclast sur-vival (Hughes et al., 1996), and increased bone formation that is notsufficient to compensate for the resorption (Jilka et al., 1998). Theresponse in bone to strain is decreased by estrogen deficiency, dueto reduced ER� activity in osteocytes (Zaman et al., 2006).

The production of several cytokines is influenced by menopause.Thus, the serum levels of IL-1, IL-6, TNF� and M-CSF havebeen shown to increase after natural or surgical menopause, anddecrease upon estrogen therapy (Rachon et al., 2002; Pacifici etal., 1991, 1989; Ralston et al., 1990). Ovariectomy in mice leads toincreased serum levels of IL-6, IL-7 and TNF� (Cenci et al., 2000;

20 U. Islander et al. / Molecular and Cellular Endocrinology 335 (2011) 14–29

F ge anda of regf andT

lp2el(btetep

s(FpDir(Rsrac

ig. 4. Actions of estrogen in arthritis and osteoporosis. Estrogen inhibits macrophand inducers of osteoblast apoptosis. Estrogen induces proliferation and maturationrom osteoblasts and bone-lining cells, which inhibits the RANK–RANKL interactionGF� from osteoblasts and osteocytes, thereby increasing osteoclast apoptosis.

oss (Poli et al., 1994; Roggia et al., 2001), and anti-TNF� treatmentreserves BMD in both experimental and human RA (Lange et al.,005; Seriolo et al., 2006; Vis et al., 2006; Saidenberg-Kermanac’ht al., 2004). IL-6 has pro-osteoporotic properties, and serumevels of IL-6 can predict bone loss in postmenopausal womenScheidt-Nave et al., 2001). Soluble IL-6 receptor acts as an agonist,y binding to IL-6, and then interacting with the same signal-ransduction pathways as the membrane-bound receptor (Tamurat al., 1993). Soluble IL-6 receptor increases after menopause, andhis increase can be prevented and reversed with HRT (Abrahamsent al., 2000). This prevention has also been reported in women withostmenopausal RA (Forsblad-D’Elia et al., 2003c).

.2. Treatment with sex steroids

Estrogen also influences the skeleton through the endocrineystem, increasing the production of insulin-like growth factor 1IGF-1), which has anabolic effects on bone (Riggs et al., 2002;orsblad-D’Elia et al., 2003c). Hormone replacement therapy in RAatients for 2 years results in increased levels of IGF-1 (Forsblad-’Elia et al., 2003c). In osteoblasts, estrogen has been shown to

ncrease the expression of OPG, BMP-6, TGF� and IGF-1, whichesults in osteoblast formation and increased osteoclast apoptosisHughes et al., 1996; Hofbauer et al., 1999; Ernst and Rodan, 1991;

T cell secretion of IL-1 and TNF�, which are stimulators of osteoclast developmentulatory T cells, which inhibit osteoclast development. It stimulates OPG productionresults in decreased activation and development of osteoclasts. Estrogen increases

(Zaiss et al., 2007; Prieto and Rosenstein, 2006; Tai et al., 2008).Interestingly, estrogen withdrawal in women is associated withincreased osteocyte apoptosis (Tomkinson et al., 1997). Osteocytesinhibit osteoclast activity through TGF�, and estrogen enhancesthis function (Heino et al., 2002) (Fig. 4).

Using transgenic knock out mouse models, it has been shownthat signalling via ER� protects against OVX-induced trabecularbone loss (Lindberg et al., 2002a,b; Sims et al., 2002). In contrast,ER� or GPR30 are not essential for this bone-protective effect ofestrogen (Lindberg et al., 2002a,b; Sims et al., 2002; Windahl et al.,2009), although the ER� activity can be slightly modulated by ER�in bone (Lindberg et al., 2003; Windahl et al., 2001).

The SERM Raloxifene is approved for the treatment of post-menopausal osteoporosis (Stefanick, 2005). It has been shown toinfluence the serum levels of bone turnover markers in womenwith postmenopausal osteoporosis (Ozmen et al., 2007). In addi-tion, serum OPG levels are higher in postmenopausal women afterRaloxifene treatment (Messalli et al., 2007), and the RANKL/OPGratio is decreased by Raloxifene treatment of osteoblastic cells invitro (Cheung et al., 2003). Raloxifene also decreases osteocyteapoptosis both in vivo and in vitro (Huber et al., 2007; Mann etal., 2007; van Essen et al., 2007), and prevents bone loss in micewith CIA (Jochems et al., 2007). However, the effect of Raloxifenein postmenopausal RA remains to be evaluated.

7. Sex steroids in RA

The fact that the peak incidence of RA in women coincides withthe time of menopause (Goemaere et al., 1990), while in men it

ellular

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ccurs at 60–70 years of age coinciding with the fall of biologi-ally active testosterone (Goemaere et al., 1990; Doran et al., 2002),learly indicates the involvement of endogenous sex hormones inA. This section of the review will focus on the effects of endoge-ous and exogenous sex steroids in experimental and human RA.

.1. Sex steroids in animal models of arthritis

Mice subjected to OVX display a higher frequency and increasedeverity of CIA compared to controls (Holmdahl et al., 1986;ochems et al., 2005), and several studies have shown that estradiol-reatment of CIA in mice suppresses disease progression (Holmdahlt al., 1986; Jansson and Holmdahl, 1989, 1992). In addition,rthritic mice show amelioration of arthritis during pregnancynd aggravation after delivery (Bond et al., 1997; Buzas et al.,993; Gonzalez et al., 2004; Mattsson et al., 1991; Waites andhyte, 1987). Blocking of the ER� and ER� using the anti-estrogen

CI 182,780 enhances the disease (Jansson and Holmdahl, 2001)emonstrating the importance of signalling through the classicaluclear ERs in this regard. In addition, we have recently shownhat the protective effect of estrogen on development of arthritisnd bone loss in CIA is mediated via ER�, and not ER� or GPR30Engdahl et al., 2010). Raloxifene treatment of ovariectomized miceesults in a reduced frequency of CIA, suppressed disease severitynd preserved joint histology (Jochems et al., 2007). These effectsre also seen during long-term treatment, when therapy is startedn established disease (Jochems et al., 2008).

2-methoxyestradiol is an endogenous estradiol metabolite thatisplays inhibitory effects on cell proliferation and neoangiogen-sis (Straub, 2007). Its antitumor potency has previously beenemonstrated in animal models, and 2-methoxyestradiol is cur-ently being investigated in clinical trials (Mueck and Seeger, 2010).-methoxyestradiol has inhibitory effects in different experimen-al models of arthritis (Brahn et al., 2008; Issekutz and Sapru, 2008;osefsson and Tarkowski, 1997; Plum et al., 2009), and our pre-iminary data suggest a protective effect of 2-methoxyestradiol onrthritis and bone loss in OVX mice with CIA (Karlstrom et al., 2010).

.2. Effects of sex steroids in human RA

Menopause is associated with decreased production of estrogen,rogesterone and adrenal androgens such as dehydroepiandros-erone (DHEA) and DHEA-sulphate (DHEAS) (Kanik and Wilder,000), and the peak incidence of RA in women occurs at this timeGoemaere et al., 1990). On the contrary there is an increase in theevels of estrogen and many other steroid hormones during preg-ancy, and it has been shown that this can affect both the incidencend the progression of RA. In 75% of women with RA the diseasectivity diminishes during pregnancy when the levels of femaleex hormones are high (Ostensen et al., 1983; Barrett et al., 1999;anik and Wilder, 2000; Ostensen, 1999). In contrast, the disease isften aggravated after delivery (Barrett et al., 1999, 2000). Breast-eeding has been shown to increase the risk for RA, which maye due to pro-inflammatory effects of prolactin, the lactation hor-one (Brennan and Silman, 1994). The mechanisms behind these

ffects are not fully established. Oral contraceptives often involveoncomitant administration of estrogens and progestins, and expo-ure to oral contraceptives before disease outbreak has in sometudies been shown to reduce the risk of developing RA (Doran etl., 2004; van Zeben et al., 1990). However, no protective effect ofral contraceptives was demonstrated in the majority of the stud-

The peak incidence for RA in men occurs at 60–70 years of ageGoemaere et al., 1990; Doran et al., 2002). Serum levels of estro-en in male RA patients have been found normal in some studies,

Endocrinology 335 (2011) 14–29 21

and increased in others, whereas the levels of testosterone werefound to be lower than in controls (Tengstrand et al., 2003, 2002).Increased levels of estradiol and decreased levels of androgens havebeen found in synovial fluid of both men and women with RA(Castagnetta et al., 2003). This could be due to increased periph-eral conversion of androgens to estrogens, since pro-inflammatorycytokines have been shown to stimulate the peripheral aromataseactivity (Macdiarmid et al., 1994; Nestler, 1993). Treatment withanti-TNF� antibodies in RA does not influence the hormonal home-ostasis, which is stable independently of the inflammatory level(Ernestam et al., 2007; Straub et al., 2005). In contrast, serum levelsof DHEAS are increased in patients treated with anti-TNF� antibod-ies for 2 years, which could be due to improved adrenal function(Ernestam et al., 2007). It has been suggested that the relative abun-dance of different biologically active estrogen metabolites couldbe involved in the dual pro-inflammatory and anti-inflammatoryeffects of estrogen seen in various inflammatory conditions (Straub,2007). Interestingly, a recent publication shows that precursorestrogens can be converted into estrogen metabolites with a pro-inflammatory phenotype, particularly in synovial cells from RApatients (Schmidt et al., 2009).

7.3. Clinical studies on HRT in RA

Early trials on sex hormones in RA in the 1930s were hopeful(Blais and Demers, 1962; Cohen et al., 1940; Hall, 1938), whilesubsequent reports in the 1960s using a progestogen with someestrogenic properties revealed unequivocal results (Gilbert et al.,1964). In the 1980s and 1990s some studies evaluating the effect ofHRT, composed of estrogen alone or estrogens and progestogen,indicated an anti-inflammatory effect (Bijlsma et al., 1987; Hallet al., 1994a; MacDonald et al., 1994). The effect of administra-tion of 12.5 �g ethinylestradiol to 10 female patients with activeRA was investigated in a prospective double-blind crossover 24weeks study. Some improvement during estrogen treatment wasfound in 30 m walking time, haemoglobin (Hb) concentration, andthrombocytosis (Bijlsma et al., 1987). In the largest HRT study asubgroup of patients (58.4%) who had greater increments in serumE2 whilst taking HRT, ‘compliers’, demonstrated improvements insome parameters of disease activity. After 6 months there weresignificant improvements in joint index and pain score comparedwith placebo. Comparisons between HRT ‘compliers’ and ‘poor-compliers’ confirmed significant improvements in articular index,pain score and morning stiffness in the ‘compliers’. It was alsoconcluded that HRT could be prescribed without fear of a diseaseflare (Hall et al., 1994a). In another, 48-week placebo controlleddouble-blind study of 62 women with 22 patients on placebo and40 patients on HRT, there were significant improvements in well-being as assessed by the Nottingham Health Care Profile and in thejoint index. Although no laboratory evidence was found of a diseasemodifying effect, it was concluded that due to the symptomaticbenefits and improvements in BMD, HRT could be a valuable adju-vant to conventional anti-rheumatic therapy in RA (Hunt et al.,1981). In contrast, van den Brink et al. (1993) did not find any ben-eficial effect of oral HRT on disease activity in a 1 year study of 40women with RA.

In a randomized controlled prospective 2-year trial of 88 post-menopausal women with RA, it was shown that HRT improved bothclinical and laboratory signs of disease activity (Forsblad-D’Elia etal., 2003a), although no effect on serum levels of IgM, IgG or IgAwas found (Forsblad-d’Elia and Carlsten, 2008). The disease activ-

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oth the HRT and control groups, roughly 40% of the patients didot progress radiologically at all. However, among patients withrogressive joint damage, the mean Larsen score (Larsen, 1995)

ncreased significantly more in the control group compared to theRT group (p = 0.026). This suggests a protective effect of HRT on

oint destruction in postmenopausal women with RA. In addition,RT was found to decrease markers of both bone and cartilage

urnover (Forsblad-d’Elia et al., 2004). The study proceeded for 24onths in contrast to the previously mentioned trials with dura-

ion of up to 12 months, and it seems likely that HRT requires aonger period to fully display its anti-arthritic qualities. The moreavourable outcome in this study may also be associated with theype of HRT chosen.

In most studies, the HRT treatment is a combination of estra-iol and progesterone and it is therefore not possible to evaluatehe direct effect of the individual substances given. In the trialsescribed above, different combinations of estrogens and proges-erones, as well as different modes of administration, have beensed which makes it difficult to generalize the results to all kindsf HRT.

. Treatment of osteoporosis in RA

The prevalence of generalized osteoporosis in postmenopausalA is more than 50%, resulting in increased risk of fracturesForsblad-D’Elia et al., 2003b; Sinigaglia et al., 2000; Haugebergt al., 2002, 2000; Sambrook et al., 1995; Hooyman et al., 1984;pector et al., 1993). The prevalence of osteoporosis is alsolevated in men with RA, compared to a healthy reference popu-ation (Tengstrand and Hafstrom, 2002). Different therapies haveeen used in the management of osteoporosis in RA. Today,nti-osteoporosis therapies are divided into therapies that pre-ominantly stimulate bone formation (parathyroid hormone andtrontium ranelate), or inhibit bone resorption (bisphosphonates,trontium ranelate, HRT and SERM) (Stepan et al., 2003). In addition,isphosphonates, estrogen and SERM seem to inhibit osteocytepoptosis (Plotkin et al., 2005). Both estrogen deficiency andrthritic disease have deteriorating effects on bone density, andRT was found to ameliorate arthritis in some studies (Forsblad-’Elia et al., 2003a; Hall et al., 1994a; MacDonald et al., 1994), and to

ncrease BMD (Forsblad-D’Elia et al., 2003a; MacDonald et al., 1994;an den Brink et al., 1993; Hall et al., 1994b). However, the use ofRT has decreased over the last years, due to the possibility of seri-us side effects (Rossouw et al., 2002; Anderson et al., 2004; Beral,003). Bisphosphonates are often used in osteoporosis, and inter-ittent treatment with the bisphosphonate etidronate was found

o increase BMD as well as retard the development of erosionsn RA, but in another trial radiological outcome was not influ-nced (Hasegawa et al., 2003; Valleala et al., 2003). Etidronate alsoncreased BMD in steroid treated patients with RA and polymyalgiaheumatica (Adachi et al., 1997). Both alendronate and risedronateave been shown to increase BMD in RA patients on glucocorticos-eroid therapy (Eastell et al., 2000; Saag et al., 1998; Yilmaz et al.,001). Risedronate also reduced the risk of vertebral fractures inlarge study of patients treated with corticosteroids with differ-

nt diagnoses, including RA (Wallach et al., 2000). Treatment withalcium and vitamin D3 increased BMD in the lumbar spine androchanter in RA patients on long-term low-dose glucocorticos-eroids (Buckley et al., 1996). In postmenopausal women on HRTith corticosteroid-induced osteoporosis (∼50% were RA patients)

reatment with the anabolic agent parathyroid hormone (PTH) ana-ogue improved BMD significantly more in the lumbar spine in

omen treated with both HRT and PTH as compared to only HRTLane et al., 1998). New therapeutic compounds preventing botheneralized bone loss and erosions in RA are under development,

Endocrinology 335 (2011) 14–29

such as substances influencing the RANKL/OPG/RANK system,thereby reducing osteoclast differentiation and activation (Rehmanand Lane, 2001). It has been shown that a human monoclonal anti-body to RANKL, Denosumab, increases BMD in postmenopausalwomen (McClung et al., 2006), and also increases BMD, reducesbone turnover and reduces the development of erosions in patientswith RA (Cohen et al., 2008; Dore et al., 2010). Raloxifene, whichhas both anti-arthritic and anti-osteoporotic effects in experimen-tal postmenopausal arthritis in mice, also seems to be a promisingtreatment candidate in postmenopausal RA (Jochems et al., 2007,2008).

9. Concluding remarks

Estrogens influence the immune system in a complex multi-faceted way, not yet completely understood. It has stimulatoryand inhibitory effects on different parts of the immune system,and some functions may not be the same in vivo as in vitro. BothT and B cells are important in the pathogenesis of RA, and morestudies need to be performed in order to elucidate the specificeffects of sex steroids on the inflammatory process. Clinical tri-als of postmenopausal RA patients have shown that HRT decreasesdisease activity, improves BMD, and in addition, an indication ofa joint protective effect has been found. Also, the bone and car-tilage turnover is reduced by HRT. Thus, the treatment seems tohave several important effects in postmenopausal RA. However,HRT is no longer recommended for long-term therapy due to therisk of serious side effects. Based on our studies in mice we sug-gest that selective modulation of estrogen receptors could providea way of obtaining the positive effects of estrogen therapy on RA,while avoiding the serious side effects. This area needs to be furtherstudied.

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Estrogens in rheumatoid arthritis; the immune system and bone

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