Three different types of skeletal complications occur in rheumatoid arthritis (RA): focal erosions of marginal and subchondral bone, juxta-articular osteoporosis, and generalized bone loss. New evidence points towards common mechanisms underlying the effects on the skeleton in RA, with osteoclasts as the key mediators. Recently the RANKL/OPG/RANK (receptor activator of nuclear factor κB ligand/osteoprotegerin/receptor activator of nuclear factor κB) system was discovered; this system modifies osteoclast precursors and the differentiation and activation of osteoclasts. OPG, which is a decoy receptor, blocks the osteoclastogenesis effects of RANKL. RANKL, OPG, and RANK act in a network of osteoclast-stimulating cytokines and systemic hormones such as estrogen, 1,25(OH)₂D₃, and parathyroid hormone 1.

Estrogen deficiency is known to increase bone remodeling and resorption, which subsequently leads to an increased risk of osteoporosis. Hormone replacement therapy (HRT) is known to restore this imbalance. Receptors for the sex steroids estrogen, androgen, and progesterone have been shown to be expressed in the osteoblasts and osteoclasts 2. Estrogen, besides having direct effects on bone cells, also acts indirectly, by modulating the production of osteoclast-stimulating and -inhibiting factors by paracrine substances from bone marrow cells and by the osteoblasts 2. Estrogen also influences the skeleton through the endocrine system, increasing the production of insulin-like growth factor 1 (IGF-1), which has anabolic effects on bone 34.

The effects of sex hormones on rheumatic diseases are controversial. Some data suggest that estrogens and HRT may be beneficial in RA 567, whereas other findings did not show amelioration of disease activity by HRT 8. The peak incidence of RA in women coincides with the peri-menopausal age, suggesting a connection with hormonal alterations 9. Furthermore, type-II-collagen-induced arthritis in female mice is exacerbated by ovariectomy and is ameliorated by subsequent treatment with estradiol (E₂) 10. In a recent trial exploring the effects of HRT in RA, we found ameliorating effects on clinical measures of disease activity and inflammation, improved bone mineral density (BMD), and also results pointing towards retardation of joint damage 11.

The aim of this study was to assess the effects of HRT on serum levels of the osteoclast-stimulating cytokines, tumor necrosis factor α (TNF-α), IL-Iβ, IL-6, on their modifiers IL-1-receptor antagonist (IL-1Ra) and soluble IL-6 receptor (sIL-6R), on OPG, and on IGF-1, attempting to understand the mechanisms through which HRT exerts its effects in postmenopausal women with RA.

We have recently reported that 2 years of HRT in post-menopausal women with RA showed signs of decreased laboratory measures of inflammation, resulting in a better disease activity score 28 (DAS 28) response, improved BMD, and indicated a protective effect on joint destruction 11. The objective of the present study was to analyze possible mechanisms behind the effects of HRT in post-menopausal patients with long-lasting and active RA.

HRT acts in a complex, not yet completely elucidated way and there are several possible routes whereby HRT could influence the erosive process and juxta-articular and generalized osteoporosis in RA. Expression of estrogen receptor in human osteoblastic cells was shown in 1988 13 and in osteoclastic cells in 1991 14. Besides having the capacity to decrease osteoclast formation and activity and to increase apoptosis of osteoclasts 1516, estrogen also seems to have a stimulatory effect on bone formation by the osteoblasts 1718. Thus, estrogen seems to have both anti-proliferative and proliferative effects. In addition to these direct effects on bone cells, the major action of estrogens in vivo is believed to be mostly by indirect actions, regulation of growth factors and cytokines in osteoblasts, which in turn regulate osteoclast differentiation and activation 2. Among these factors, we analyzed IL-Iβ, IL-6, TNF-α, all inducers of bone resorption 19, and the decoy receptor OPG, inhibiting activation of osteoclasts and its precursors 20, as well as the bio-activity modifiers IL-1Ra and sIL-6R and the endocrine bone-stimulating agent IGF-1.

IL-6 is one of the key mediators of increased bone loss in postmenopausal women. Production of this cytokine by mononuclear blood cells increases with age and menopause 21 and is inhibited in vitro by E₂ 22. The bioactivity of IL-6 is significantly enhanced in the presence of its agonist, sIL-6R, which renders cells expressing the gp 130 signaling protein responsive to IL-6 23. Therefore, it is suggested that sIL-6R may be of more importance than IL-6 in the postmenopausal acceleration of bone turnover 2425. IL-6/sIL-6R are also believed to be involved in joint destruction through osteoclastogenesis in RA 26. We have recently reported that the radiographic scores of the women with RA showed a significant positive correlation with IL-6 and sIL-6R 27. In this study, serum levels of sIL-6R decreased significantly in the HRT group. Furthermore, the change in E₂ in serum was inversely, though modestly, correlated with the change in sIL-6R. The change of sIL-6R was inversely associated with increase in BMD in the lumbar spine after 2 years.

Since the increase of E₂ in serum was significantly correlated with improvement of BMD in the lumbar spine and total hip, we adjusted the correlation of sIL-6R and bone mass in the spine for the effect of changed E₂ levels. After adjustment, the association was no longer significant (P = 0.075). However the partial correlation coefficient changed only slightly, indicating the possibility of a true correlation between sIL-6R and bone mass in the spine.

In sum, we suggest that estrogen-mediated effects of IL-6/sIL-6R functions may be involved in the protection of the skeleton in RA patients.

Growth hormone (GH) contributes also in the regulation of bone metabolism: a deficiency of this hormone causes reduced BMD and replacement therapy has been shown to increase bone mass 3. GH is the primary inducer of IGF-1 synthesis by the liver and a central regulator of the concentration of circulating IGF-1 3. IGF-1 has been found to induce cellular proliferation and has an antiapoptotic effect 28. Estrogen stimulates the secretion of GH from the pituitary gland 29 and also increases the production of IGF-1 by osteoblastic cell lines 30. In the present investigation, HRT resulted in a significant increase in serum levels of IGF-1. We did not find a significant association between the changes in E₂ levels and IGF-1 after 2 years, which might be caused by the limited number of patients examined.

It has also been reported that IGF-1 is reduced in inflammatory diseases such as juvenile chronic arthritis (JCA) and RA 3132, and treating JCA with human growth hormone increased IGF-1, growth velocity, and markers of bone formation 31. Furthermore, De Benedetti and colleagues proposed that IL-6 mediated the decrease in IGF-1 production that was associated with growth defect in transgenic mice and they also showed that circulating IL-6 levels were negatively correlated with IGF-1 levels in JCA 33. We also found in this study an inverse correlation between IL-6 and IGF-1, although the P value (0.032) was relatively weak and serum levels of IGF-1 were only half of the reference values of age-matched healthy post-menopausal women 34. There was also a strong positive correlation between ESR and IL-6 at baseline, which has previously been observed 35, and, interestingly, the change of ESR during the course of the study was highly correlated with the change of IL-6 and to a lesser degree was inversely correlated with the change of IGF-1. However, it is unclear if the increase of IGF-1 in this trial is caused by estrogen-mediated reduction of the IL-6/sIL-6R pathway or by effects on the GH/IGF-1 axis or by a combination of these mechanisms.

Administration of IL-1Ra or anti-TNF treatment has recently been found to reduce radiological progression in RA 3637. These cytokines and their receptors have also been reported to be involved in bone loss after estrogen deficiency: for instance, administration of IL-1Ra and TNF-binding protein (TNFbp), an inhibitor of TNF, to ovariectomized mice decreased osteoclastogenesis and bone resorption 38. It was also reported that estrogen replacement in women induced reduction of IL-1 and TNF-α in peripheral blood mononuclear cells 39, whereas other researchers did not observe any effect of HRT on serum levels of the cytokines 40. In our trial, serum levels of TNF-α were not significantly altered in any group, but we found a weak inverse correlation between the changes in TNF-α and E₂ after 2 years. The change in TNF-α was also associated weakly with the change in sIL-6R and more strongly with the alterations in IL-1Ra and ESR and inversely to a smaller extent with the change in IGF-1.

IL-1Ra increased significantly during the first year in both study groups, but the rise was not sustained until the end of the trial. One hypothesis to explain this finding is that vitamin D₃, which affects the immune system 41 and was given to all the women studied, may have affected the production of IL-1Ra. We could not tell if HRT had any effect on IL-1β, since half of the participants had undetectable levels.

We were also interested in the effects of HRT on OPG, since treatment with OPG in rat adjuvant arthritis blocked bone and cartilage destruction 42 and 17β-estradiol has been shown to increase OPG mRNA and protein levels in human osteoblastic cell lines 43, whereas other workers did not find that OPG was regulated by estrogen 44. In the present study, OPG increased significantly during the first year in the HRT group compared with the controls. There is a need to be cautious in interpreting this finding, since there were significantly more missing OPG samples in the HRT group and no associations between OPG and the other variables were discovered.

The present study has certain limitations. Corrections for multiple comparisons have not been made, since the findings seem biologically reasonable. Yet, one needs to be cautious about significances with P values at the <0.05 level, which theoretically could have occurred by chance, because quite a lot of tests have been performed. In addition, there were unfortunately some missing samples, in particular from the HRT group, a circumstance that renders some of the data incomplete.

Most of the women in the HRT group received combined treatment with E₂ plus norethisterone acetate continuously or sequentially. Consequently, our findings show the effects of both substances. However, the change in E₂ alone correlated strongly with the improvement of BMD in the total hip and lumbar spine. Our discussion relates mostly to previous findings in HRT and estrogen investigations, because less is known about the effects of progestogen on the immune and endocrine systems. However, progestogens, like androgens, are known to have immunosuppressive effects, and during pregnancy, progesterone acts together with estrogen and cortisol to influence alterations in immunological reactivity, such as decreasing cytokines associated with T-helper-1 cells and increasing those associated with T-helper-2 cells, phenomena that are probably connected with the clinical improvement in RA 4546.

In summary, we found in this controlled clinical trial that the increase of E₂ levels in serum was highly correlated with improved BMD. We have tried to elucidate possible ways, in the network of proinflammatory cytokines and IGF-1, by which HRT exerts its effects on the skeleton in long-lasting active RA. We found that HRT reduces serum levels of sIL-6R, whereas IGF-1 levels were observed to be increased. Both of these results – the effects on the IL-6/ sIL-6R pathway and on IGF-1 in the endocrine system – may be involved in the mechanisms mediating the beneficial effects of HRT. There is a need for larger, controlled, long-term studies of combined treatment in RA – estrogen plus progestogen, and estrogen alone – to support our results and to investigate the effects of the various hormones.

None declared.

BMD = bone mineral density; E₂ = estradiol; ELISA = enzyme-linked immunosorbent assay; ESR = erythrocyte sedimentation rate; GH = growth hormone; HRT = hormone replacement therapy; IGF-1 = insulin-like growth factor 1; IL = interleukin; IL-1Ra = IL-1-receptor antagonist; OPG = osteoprotegerin; RA = rheumatoid arthritis; RANK = receptor activator of nuclear factor κB; RANKL = receptor activator of nuclear factor κB ligand; sIL-6R = soluble IL-6 receptor; TNF-α = tumor necrosis factor α.