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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 12 6097-6099
Copyright © 2004 by The Endocrine Society

COMMENT

Raloxifene Modulates Interleukin-6 and Tumor Necrosis Factor-{alpha} Synthesis in Vivo: Results from a Pilot Clinical Study

Walter Gianni1, Andrea Ricci1, Paola Gazzaniga, Marina Brama, Maria Pietropaolo, Sergio Votano, Francesco Patanè2, Anna Maria Aglianò, Giovanni Spera, Vincenzo Marigliano, Sergio Ammendola, Donato Agnusdei, Silvia Migliaccio3 and Roberto Scandurra3

Unità Operativa Geriatria Oncologica, INRCA (W.G.), Rome, Italy; Departments of Biochemical Sciences (A.R., M.B., S.A., R.S.), Experimental Medicine (P.G., F.P., A.M.A.), Geriatrics (M.P., V.M.), Medical Physiopathology (S.M., G.S.), University of "La Sapienza," 00161 Rome, Italy; Hospital San Carlo-IDI Sanità (S.V.), 00100 Rome, Italy; and University of Siena, Siena, Italy, and Eli Lilly & Co. (D.A.), 50019 Florence, Italy

Address all correspondence and requests for reprints to: Dr. Silvia Migliaccio, Dipartimento di Fisiopatologia Medica, Università "La Sapienza," Policlinico Umberto I, Viale del Policlinico 155, 00161 Rome, Italy. E-mail: silvia.migliaccio{at}uniroma1.it.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Raloxifene (RAL), a selective estrogen receptor modulator, is indicated for the prevention and treatment of postmenopausal osteoporosis. RAL, by decreasing bone turnover, prevents bone loss and microarchitecture damage, reducing the incidence of osteoporotic fractures. Our previous in vitro data demonstrated that RAL modulates osteoclast activity by, at least in part, an IL-6- and TNF-{alpha}-dependent mechanism. In this study we evaluated the effects of RAL treatment (60 mg/d) on circulating levels of these cytokines in 14 postmenopausal women with osteoporosis. Lumbar bone density (determined by dual energy x-ray absorptiometry) and IL-6 and TNF-{alpha} levels were measured before and after 6 and 24 months of therapy. After 24 months, RAL increased bone density. IL-6 and TNF-{alpha} expression, elevated before treatment, significantly decreased (50% and 30%, respectively) after 6 months. This effect was sustained up to the end of the treatment (75% and 35%, respectively). Thus, our data show that RAL can modulate circulating levels of cytokines involved in osteoclastogenesis and bone resorption, suggesting that modulation of soluble factors could play a pivotal role in the mechanisms of the osteoprotective effect of RAL.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
POSTMENOPAUSAL OSTEOPOROSIS IS a metabolic bone disease characterized by decreased skeletal strength, which leads to increased fracture risk (1). The decreased bone mass and the alteration of bone turnover caused by ovarian failure play pivotal roles in the pathogenesis of osteoporosis. Estrogen therapy prevents bone loss (2). The selective estrogen receptor modulators (SERMs) protect skeletal health by a direct estrogen receptor-mediated mechanism. Raloxifene (RAL) is the only SERM currently available for the treatment and prevention of postmenopausal osteoporosis (3). This compound prevents bone loss by modulating bone turnover (4), because it inhibits osteoclast (OCS) activity both directly and by an osteoblast (OBS)-mediated effect (4). OCS are highly specialized multinucleated cells, developing from hemopoietic precursors of the bone marrow, which differentiate in mature cells at the bone surface (5). OCS differentiation and activity require the presence of factors produced in the bone microenvironment, among which are the proinflammatory cytokines IL-6 and TNF-{alpha} (5, 6, 7). Additionally, it involves the interaction between the receptor activator for nuclear factor-{kappa}B (RANK) and RANK ligand (RANKL) (5, 6, 7). Several reports show that IL-6 and TNF-{alpha} play roles in mediating bone loss induced by estrogen deficiency (8, 9, 10); indeed, high circulating cytokine levels are found in postmenopausal women (11). Recent data reported by Pacifici’s group (12) also stress the roles of these cytokines among the mechanisms involved in postmenopausal bone loss. We demonstrated that RAL inhibits both IL-6 and TNF-{alpha} expression and activity in vitro (4), suggesting that it may modulate bone cell activity through, at least in part, the expression of these cytokines. However, no data are as yet available in vivo on modulation of these cytokines by RAL. Thus, the aim of the present study was to evaluate whether RAL treatment could modulate IL-6 and TNF-{alpha} in osteoporotic postmenopausal women.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patients

Fourteen osteoporotic women, according to the WHO definition, were given RAL (60 mg) once a day for 24 months. Characteristics at baseline were as follows: mean age, 61.7 ± 1.0 yr; nonsmokers; body mass index, 25.5 ± 0.6; mean age at menopause, 50.0 ± 0.9 yr; mean T-score, –2.8 ± 0.1, normal concentrations of cholesterol and triglycerides, and free of any therapy for at least 6 months. No side effects or drop-outs were reported. Cytokine measurements were performed at baseline (time zero) and after 6 and 24 months of treatment. Lumbar bone density measurements were performed at baseline and after 6 and 24 months using dual energy x-ray absorptiometry (QDR 2000; Hologic, Inc., San Francisco, CA). The study was performed according to the guidelines of the Helsinki Declaration on human experimentation and was approved by the local ethics committee of University "La Sapienza" (Rome, Italy). All patients gave their written informed consent before the study.

RNA extraction from blood

Two milliliters of peripheral blood were collected by forearm venipuncture using EDTA tubes in the morning after an overnight fasting period. Serum samples for determinations of IL-6 and TNF-{alpha} were stored at –80 C in aliquots until thawed for analysis. RNA extraction was performed using a modified TRIzol LS-based procedure according to the manufacturer’s instructions. Briefly, the blood samples were mixed into a succinyl-linked gelatin (1:1), and the red cells were allowed to settle by gravity to separate all nucleated cells. Supernatant was then centrifuged, and the pellet underwent subsequent RNA extraction. The quantity and quality of RNA preparations were determined by absorbance at 260 and 280 nm.

ELISA

IL-6 was measured in previously unthawed serum samples using a highly sensitive commercial kit according to manufacturer’s instructions (kit DKH035; Euroclone, Milan, Italy). Results are expressed as picograms per milliliter and were converted to international units per milliliter using the National Institute of Biological Standard and Control/WHO IL-6 International Reference Standard 89/548 (conversion factor = 0.131).

RT-PCR

Total RNA (1 µg) from blood samples was reverse transcribed in a final volume of 20 µl with 100 pmol random primers and 50 U Moloney murine leukemia virus reverse transcriptase. Aliquots from blood samples corresponding to 100 ng RNA were amplified in PCR buffer containing 25 pmol of each primer and 1.25 U Taq polymerase in a final volume of 50 µl. Aliquots of cDNA were amplified with human IL-6, human TNF-{alpha} and 3' human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers. PCR-amplified fragments were resolved by electrophoresis with 1.0% agarose/ethidium bromide gel. Bands were quantified by scanning densitometry using Molecular Analyst software (model 670; Bio-Rad Laboratories, Hercules, CA) and were normalized against constitutive GAPDH. Values were reported as a percentage of the GAPDH values. Statistical analysis was performed by ANOVA. Differences were considered significant at P < 0.01.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
In this small group of postmenopausal women, RAL significantly increased lumbar bone mineral density compared with baseline (T-score: baseline, –2.8 ± 0.1; 6 months, –2.57 ± 0.1; 24 months, –2.0 ± 0.1), confirming previous results obtained in a large cohort of osteoporotic women (3, 13).

Because we showed that RAL decreased IL-6 and TNF{alpha} expression in vitro (5), we evaluated potential similar changes in women taking RAL. As expected, IL-6 was highly expressed in postmenopausal women (Fig. 1Go). RAL significantly decreased (50%) circulating IL-6 expression levels after 6 months. At the end of the 24-month treatment, circulating IL-6 levels were further (70%) decreased (Fig. 1Go).


Figure 1
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  		FIG. 1. Left panel, Data are presented as the mean ± SE of 14 patients before ({blacksquare}) and after 6 (Figure 1) and 24 ({square}) months of therapy. *, P < 0.01 vs. month 0. Right panel, Representative PCR analysis in one of the patients. Right top panel, IL-6 gene; right bottom panel, GAPDH gene; left top panel, TNF-{alpha}; left bottom panel, GAPDH.

 
Because the above-described IL-6 decrease was at the level of gene expression, our aim was to confirm modulation at protein levels. Thus, IL-6 protein levels were measured by ELISA. Serum bioactive IL-6 levels among postmenopausal women were high, as reported in other clinical studies (11.7 ± 1.3 pg/ml; equal to Systeme Internationale units, 1.53 ± 0.17 IU/ml) (11). Treatment with RAL determined a reduction of levels of this cytokine after 6 months (6.1 ± 0.6 pg/ml; equal to Systeme Internationale units, 0.79 ± 0.07 IU/ml; P < 0.001). This effect was sustained up to the end of treatment (4.0 ± 0.4 pg/ml; equal to 0.52 ± 0.052 IU/ml; P < 0.001 compared with baseline).

Additionally, TNF-{alpha} expression levels were high in postmenopausal women (Fig. 1Go). Six-month treatment with RAL significantly reduced expression levels of this TNF-{alpha} (30%), albeit to a lesser extent than IL-6. The decrease in TNF-{alpha} expression was maintained after 24 months of treatment (35%; Fig. 1Go) as well.


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
In the present study we show that RAL treatment inhibits the production of IL-6 and TNF-{alpha} in postmenopausal women and that this effect is sustained over a 24-month treatment period. The above-described effect of RAL in lowering levels of IL-6 and TNF-{alpha} is consistent with our previous data showing that this molecule inhibits the expression and activity of these cytokines in OBS (4).

RAL, the first SERM used for the treatment of postmenopausal osteoporosis, prevents postmenopausal bone loss and skeletal fracture by preservation of both bone density and bone quality (3, 13). Modulation of bone turnover by RAL might play a role in fracture prevention. High bone turnover has, in fact, been claimed to be a risk factor involved in fracture development (14). The equilibrium in bone turnover is maintained by the concerted actions of OBS and OCS (15, 16, 17). In vitro and in vivo preclinical data show that OCS differentiation and activity require factors either present in the hematic compartment or produced in the bone microenvironment (5, 6, 7). Among these, IL-6 and TNF-{alpha} seem to control OCS development and activity. TNF-{alpha} increases RANKL-induced osteoclastogenesis (17, 18, 19). Estrogens are able to inhibit the production of both IL-6 and TNF-{alpha} (10, 19), and it appears that inhibition of TNF-{alpha} production in an experimental animal model accounts for the ability of estrogen to block OCS formation, whereas other cytokines, such as IL-6, are important in the modulation of OCS activity (19). The data also suggest that OCS formation and activity stimulation supported by IL-6 can occur by, at least in part, a RANKL-independent mechanism (5).

Interestingly, levels of TNF-{alpha} and IL-6 increase after menopause (11), when estrogen levels decrease, and during the aging processes (20), suggesting a role for these factors in the pathogenesis of postmenopausal osteoporosis as well as other aged-related disorders (20). Estrogen replacement therapy can block this increase (8, 9, 10, 11, 12). In agreement with these hypotheses, we demonstrated that RAL inhibited IL-6 and TNF-{alpha} activity in an in vitro cellular model system (4), and that this inhibition was related to the modulation of osteoclastogenesis and OCS activity (4). Our present data indicate for the first time that RAL in vivo, as well as estrogens (8, 10, 15), can modulate OCS activity and, thus, bone turnover in postmenopausal women by, at least in part, an IL-6- and TNF-{alpha}-dependent mechanism, as demonstrated in vitro.

In conclusion, these results support the hypothesis that RAL, modulating cytokine levels, might preserve bone mass by reducing increased bone turnover in postmenopausal women by an estrogenic-like mechanism(s) similar to those operative in women receiving estrogen replacement therapy. Additional clinical trials are needed to fully clarify the mechanism of this SERM in bone homeostasis modulation in postmenopausal women and confirm the modulation of proinflammatory cytokines in vivo.


   Footnotes
 
This work was supported by grants from the Agenzia Spaziale Italiana (Contract I/R/110/00; to S.M.) and European Union Project IGOID (Contract G5RD-CT-2000-00377; to R.S.).

1 W.G. and A.R contributed equally to this study. Back

2 F.P. died on May 19, 2004. Back

3 S.M. and R.S. contributed equally to this study. Back

Abbreviations: GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; OBS, osteoblast; OCS, osteoclast; RAL, raloxifene; RANK, receptor activator for nuclear factor-{kappa}B; RANKL, receptor activator for nuclear factor-{kappa}B ligand; SERM, selective estrogen receptor modulator.

Received May 26, 2004.

Accepted August 13, 2004.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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  5. Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Martin TJ 1999 Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev 20:345–357[Abstract/Free Full Text]
  6. Kudo O, Sabokbar A, Pocock A, Itonaga I, Fujikawa Y, Athanasou NA 2003 Interleukin-6 and interleukin-11 support human osteoclast formation by a RANKL-independent mechanism. Bone 32:1–7[Medline]
  7. Takahashi N, Udagawa N, Suda T 1999 A new member of tumor necrosis factor ligand family, ODF/OPGL/TRANCE/RANKL, regulates osteoclast differentiation and function. Biochem Biophys Res Commun 256:449–455[CrossRef][Medline]
  8. Jilka R, Hangoc G, Girasole G, Passeri G, Williams D, Abrams J, Boyce B, Broxmeyer H, Manolagas S 1992 Increased osteoclast development after estrogen loss: mediation by IL-6. Science 257:88–91[Abstract/Free Full Text]
  9. Poli V, Balena R, Fattori E, Markatos A, Yamamoto A, Tanaka H, Ciliberto G, Rodan G, Costantini F 1994 Interleukin-6 deficient mice are protected from bone loss caused by estrogen depletion. EMBO J 13:1189–1196[Medline]
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