Relationship between soluble markers of immune activation and bone turnover in post-menopausal women with rheumatoid arthritis

P. Oelzner, S. Franke, A. Müller, G. Hein and G. Stein

Department of Internal Medicine IV, Friedrich-Schiller University of Jena, Germany

Correspondence to: P. Oelzner, Department of Internal Medicine IV, Friedrich-Schiller University of Jena, Erlanger Allee 101, 07740 Jena, Germany.


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. Regarding interactions between pro-inflammatory cytokines and bone metabolism in rheumatoid arthritis (RA), we investigated the relationship between the serum levels of interleukin (IL)-1ß, IL-6, soluble interleukin-2 receptor (sIL-2r), C-reactive protein (CRP), the vitamin D metabolites 25-hydroxyvitamin D3 (25OHD3 ) and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3 ], intact parathyroid hormone (iPTH) as well as serum and urinary parameters of bone turnover in 74 post-menopausal women with RA.

Results. SIL-2r correlated negatively with 1,25(OH)2D3 (P<0.01), whereas IL-6 showed a positive correlation with urinary excretion of deoxypyridinoline–collagen cross-links (P<0.01). 1,25(OH)2D3 (P<0.01) and iPTH (P<0.01) were negatively related to CRP, whereas the urinary excretion of pyridinoline (P<0.01) and deoxypyridinoline (P<0.01)–collagen cross-links showed a positive correlation with CRP. 1,25(OH)2D3 (P<0.01) and iPTH (P<0.05) were positively related to bone alkaline phosphatase as a marker of osteoblast function.

Conclusion. Our data indicate that IL-6 is a critical determinant of increased bone resorption in post-menopausal RA women with high disease activity and that serum levels of 1,25(OH)2D3 are inversely related to T-cell activation.

KEY WORDS: Rheumatoid arthritis, Osteoporosis, Pro-inflammatory cytokines, Vitamin D, PTH.


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Systemic osteoporosis is a frequent and serious complication of rheumatoid arthritis (RA). Various pathogenetic mechanisms, such as chronic glucocorticoid treatment and immobilization, contribute to systemic bone loss in this disease [1]. On the other hand, the chronic inflammation process itself has been shown to be an important risk factor in the development of systemic osteoporosis in RA [24]. However, the mediator mechanisms and the link between the local arthritic process and systemic bone loss are still unclear.

In a previous study, we observed a decrease in serum levels of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3 ] and an increase in parameters of bone resorption (pyridinoline and deoxypyridinoline–collagen cross-links) with increasing disease activity in an unselected group of RA patients [5]. Therefore, we suggested that a disease activity-dependent decrease in 1,25(OH)2D3 may be an important mediator mechanism between local inflammation and systemic bone loss in RA. 1,25(OH)2D3 has immunomodulatory functions, including the suppression of activated T cells and the modulation of the secretion of pro-inflammatory cytokines [6, 7]. On the other hand, the pro-inflammatory cytokines interleukin-1 (IL-1), tumour necrosis factor alpha (TNF-{alpha}) and IL-6 have been shown to act on osteoblasts and osteoclasts in vitro and in vivo, resulting in an increase of bone resorption [814]. This bone-resorptive effect of pro-inflammatory cytokines may influence parathyroid hormone (PTH) secretion and 1,25(OH)2D3 synthesis. Furthermore, because the vitamin D receptor is expressed on activated T cells [6, 7], the activation state of T cells may be a critical determinant of the 1,25(OH)2D3 serum level. Thus, interactions between secretion and metabolism of calcium-regulating hormones and pro-inflammatory cytokines certainly have a role in the regulation of bone turnover in chronic inflammatory diseases in general and especially in RA.

Therefore, the purpose of our study was to investigate whether there are relationships between serum levels of pro-inflammatory cytokines, soluble markers of T-cell activation, calcium-regulating hormones and bone-turnover parameters in RA patients which may be of particular importance regarding the pathogenesis of osteoporosis in RA.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients
Because of the well-known influences of sex and menopausal state on bone metabolism and secretion of pro-inflammatory cytokines, only post-menopausal RA women without hormone replacement therapy were included in our study. We investigated 74 post-menopausal women with RA aged between 49 and 80 yr (mean 64.8 yr), and with a disease duration ranging from 6 months to 44 yr (mean 8.6 yr). All patients fulfilled at least four of the 1987 revised ACR criteria for RA [15]. With respect to the renal synthesis of 1,25(OH)2D3 , only patients with normal serum creatinine levels were included in the study.

Fifty-one patients aged between 50 and 79 yr (mean 64.8 yr), and with a disease duration between 6 months and 44 yr (mean 9.1 yr), were receiving glucocorticoid treatment (5–15 mg prednisolone/day), whereas 23 patients aged between 49 and 80 yr (mean 64.9 yr), and with a disease duration between 6 months and 39 yr (mean 7.4 yr), were not. None of the patients were administered i.m., intra-articular or i.v. steroids during the last 3 months before investigation. Twenty women were receiving disease-modifying anti-rheumatic drugs (DMARDs) (11 methotrexate, four azathioprine, four sulphasalazine and one hydroxychloroquine).

Laboratory tests and analytical techniques
The following parameters were investigated in the sera of patients: IL-1ß, IL-6, soluble interleukin-2 receptor (sIL-2r), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), albumin, the vitamin D3 metabolites 25-hydroxyvitamin D3 (25OHD3 ) and 1,25(OH)2D3 , intact PTH (iPTH), bone alkaline phosphatase (bALP), osteocalcin (OC), tartrate-resistant acid phosphatase (TRAP), calcium (Ca), calcium corrected for albumin (Ca-alb.), and phosphate (PO4). Fasting venous blood samples were drawn between 7 and 9 a.m. For the determination of vitamin D metabolites, iPTH and cytokines, serum aliquots were frozen and stored at -70°C until analysed. The measuring procedure, producer and intra-assay/interassay coefficients of variation of calcium-regulating hormones, cytokines and cytokine receptors are listed in Table 1Go.


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TABLE 1.  Measuring procedure, producer and intra-assay/interassay coefficients of variation of calcium-regulating hormones, cytokines and cytokine receptors
 
Furthermore, the excretion of pyridinoline (Pyd) and deoxypyridinoline (Dpyd) cross-links, as well as the calcium/creatinine ratio (Ca/crea) in the morning urine and the daily urinary calcium excretion, were measured. Pyd and Dpyd were determined in urine by a reversed-phase HPLC method, described in detail by Müller et al. [16], and expressed as the Pyd/creatinine ratio (Pyd/crea) and as the Dpyd/creatinine ratio (Dpyd/crea), respectively.

Statistical analysis
The data were analysed statistically using the SPSS for Windows Statistical Program. For correlation analysis, we used the Spearman correlation coefficient. To compare the parameters of the various patient groups, the Mann–Whitney U-test was used. Differences of P<0.05 were considered significant.


    Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Influence of therapy on parameters of disease activity including serum cytokine levels and parameters of bone turnover
According to therapy at the time of investigation, the post-menopausal women with RA were divided into four groups: patients receiving neither glucocorticoids nor DMARDs, patients with glucocorticoid treatment but without DMARDs, patients treated with glucocorticoids and DMARDs and, as a subgroup of these, patients who received methotrexate (MTX) (Table 2Go). The ESR was lower in patients receiving glucocorticoids but no DMARDs (P<0.05), in patients treated with glucocorticoids and DMARDs (P<0.01), and in the subgroup with MTX therapy (P<0.01) compared to patients receiving neither glucocorticoids nor DMARDs. Patients treated with MTX had a lower ESR (P<0.05) and a higher serum albumin level (P<0.05) than those treated with glucocorticoids only. sIL-2r was significantly lower in patients treated with glucocorticoids than in those receiving neither glucocorticoids nor DMARDs (P<0.01). In patients treated with glucocorticoids alone (P<0.05) and in those receiving glucocorticoids and DMARDs (P<0.01), we found a higher bALP activity than in patients receiving neither glucocorticoids nor DMARDs. The serum concentration of PO4 was slightly lower in patients treated with MTX than in those receiving neither glucocorticoids nor DMARDs (P<0.05). The other parameters of bone turnover, especially serum levels of vitamin D metabolites and iPTH, and the markers of bone resorption (Pyd/crea, Dpyd/crea and TRAP), were not different between the four groups.


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TABLE 2.  Influence of therapy on serum cytokine levels, parameters of disease activity and of bone metabolism in post-menopausal women with RA (mean values and S.D. )
 
Correlation between parameters of disease activity, serum albumin and cytokine levels (Table 3Go)
CRP correlated positively with ESR (P<0.001), IL-6 (P<0.05) and sIL-2r (P<0.01), and was negatively related to serum albumin (P<0.05). ESR showed a positive correlation with IL-6 (P<0.001) and sIL-2r (P<0.01), but a negative correlation with albumin (P<0.01). IL-1ß and IL-6 were positively related (P<0.01), whereas neither IL-1ß nor IL-6 were related to sIL-2r. Furthermore, IL-6 was negatively related to serum albumin (P<0.05).


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TABLE 3.  Correlations between conventional parameters of disease activity, serum albumin and cytokine levels in post-menopausal women with RA
 
Correlation between markers of disease activity and parameters of bone metabolism
Correlations between serum levels of pro-inflammatory cytokines, conventional parameters of disease activity, calcium-regulating hormones as well as bone turnover parameters are shown in Table 4Go. IL-6 correlated negatively with the renal excretion of Dpyd (P<0.01), but showed no correlation with calcium-regulating hormones, serum calcium and phosphate, and markers of bone formation. The serum level of sIL-2r was negatively related to 1,25(OH)2D3 (P<0.01) and iPTH (P<0.05), but positively related to renal Pyd excretion (P<0.05). IL-1ß yielded no significant correlation with calcium-regulating hormones and bone turnover parameters. CRP correlated negatively with 1,25(OH)2D3 (P<0.01), iPTH (P<0.01) and bALP (P<0.01), and positively with serum levels of Ca-alb. (P<0.01) and with the renal excretion of Pyd (P<0.01) and Dpyd (P<0.01). ESR had a negative correlation with 1,25(OH)2D3 (P<0.01) and bALP (P<0.01), but a positive correlation with serum levels of Ca-alb. (P<0.01) and PO4 (P<0.05), and with the renal excretion of Pyd (P<0.01) and Dpyd (P<0.01).


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TABLE 4.  Correlations of calcium-regulating hormones and bone turnover parameters with markers of disease activity and serum levels of pro-inflammatory cytokines in post-menopausal women with RA
 
Relationship between serum levels of calcium-regulating hormones and parameters of bone turnover (Table 5Go)
Serum levels of 25OHD3 were not related to 1,25(OH)2D3 , iPTH and to the parameters of bone formation and bone resorption. 1,25(OH)2D3 levels correlated positively with iPTH (P<0.001) and bALP (P<0.01), but showed no significant correlation with the parameters of bone resorption. Serum levels of iPTH correlated negatively with serum levels of Ca-alb. (P<0.001) and PO4 (P<0.05), and positively with bALP (P<0.05).


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TABLE 5.  Correlations between serum levels of calcium-regulating hormones and parameters of bone turnover in post-menopausal women with RA
 
Influence of age on serum cytokine levels and bone turnover parameters
Neither serum levels of IL-1ß, IL-6, sIL-2r, CRP, ESR and albumin nor parameters of bone turnover, including serum levels of vitamin D metabolites and iPTH, were correlated with the age of patients (data not shown).


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
RA is associated with periarticular and generalized bone loss [1, 2, 17, 18]. The pyridinium compounds Pyd and Dpyd are measured to assess collagen degradation in RA [1921]. Pyd is a marker of type I and II collagen degradation, and is derived from bone, cartilage, synovium and tendons. Dpyd is a specific marker of type I collagen degradation located in bone and dentine only. Therefore, the urinary Dpyd/crea ratio reflects bone resorption more selectively than the Pyd/crea ratio.

In the post-menopausal women with RA investigated in our study, high disease activity was associated with an increase in bone resorption reflected by the positive correlation between parameters of disease activity (CRP and ESR) and the excretion of collagen cross-links (Pyd and Dpyd). Measurement of renal Pyd and Dpyd excretion is not suitable for differentiating between periarticular and systemic bone resorption. However, because cartilage degradation and periarticular bone resorption induced by pro-inflammatory cytokines with bone-resorbing properties, such as IL-1ß, TNF-{alpha} and IL-6, is a main feature of the inflammation process in RA [22], the significant increase in both Pyd and Dpyd excretion with increasing disease activity is likely to be partly due to joint destruction. Increased periarticular bone resorption in RA has been demonstrated by histomorphometric investigation [17, 18], and the excretion of Pyd has been shown to correlate with disease activity, bone loss at the femoral neck and with the degree of joint damage in RA [1921]. On the other hand, various data from patients with RA and from animal models indicate that an augmented state of activation of monocytes/macrophages with an increased secretion of pro-inflammatory cytokines and an increase in osteoclastogenesis in the bone marrow may contribute to both local and systemic bone loss [2329]. A significant correlation between systemic bone loss and urinary Pyd and Dpyd excretion has been shown in a prospective study in early RA [3], indicating that activation of osteoclasts is the dominant process leading to bone loss in this situation.

In our study group of post-menopausal women with RA, the renal excretion of Dpyd correlated positively with the serum level of IL-6, indicating that increased secretion of IL-6 is of pathogenetic importance in the process of bone resorption in RA. This assumption is in agreement with other in vivo and in vitro findings in RA patients. An increase in osteoclastogenesis, and in the secretion of the pro-inflammatory cytokines IL-6 and IL-8, have been shown in the iliac crest bone marrow in patients with RA [26, 29]. Moreover, it has been demonstrated in vitro that monocytes and macrophages from RA patients are capable of differentiating into osteoclasts [27], and that the osteoclastogenesis in the RA synovium is linked to IL-6 and the soluble IL-6 receptor [28]. Therefore, the activation of the IL-6 system by the pro-inflammatory cytokines IL-1 and TNF-{alpha} is thought to play a critical role in periarticular and systemic bone resorption in RA [23, 25, 28]. This assumption is supported by the positive correlation between IL-1ß and IL-6, and between IL-6 and urinary Dpyd excretion in our patients. The positive correlation of both IL-6 and renal Dpyd excretion with CRP and ESR indicates that bone resorption in our post-menopausal women with RA is due to the inflammatory process rather than to oestrogen deficiency.

Contrary to the markers of bone resorption, the serum levels of the two calcium-regulating hormones with bone-resorptive and osteoanabolic properties, i.e. 1,25(OH)2D3 and iPTH, showed a strong negative correlation with disease activity. The decrease in 1,25(OH)2D3 and iPTH with increasing disease activity may at least in part be induced by an increase in bone resorption and in free ionized serum calcium. This hypothesis is supported by the strong negative correlation between iPTH and serum calcium corrected for albumin, and the positive correlation between iPTH and 1,25(OH)2D3 . Ionized calcium was not measured in this study. However, an increase in ionized calcium in patients with high disease activity may be assumed because of the positive correlation between disease activity and serum calcium corrected for albumin. Although we did not find a significant negative correlation between IL-6 and iPTH, the activation of the IL-6 system may have indirect suppressive effects on PTH and 1,25(OH)2D3 serum levels, because IL-6 is a pleiotropic cytokine which stimulates bone resorption and decreases hepatic albumin synthesis [30, 31]. Both processes may contribute to an increase in ionized calcium.

Other additional mechanisms may be of importance for the decrease in 1,25(OH)2D3 with increasing disease activity. Although renal 1,25(OH)2D3 synthesis has been shown to decrease with aging [32], an age-related decrease in 1,25(OH)2D3 serum levels has been excluded in our patients. Interestingly, serum levels of 1,25(OH)2D3 in our RA patients showed a marked negative correlation with sIL-2r. sIL-2r is an accepted marker of T-cell activation. Not silent, however, activated T cells express the vitamin D receptor (VDR), and 1,25(OH)2D3 is known to have suppressive effects on Th1 cytokine production by inducing an inhibition of IL-12 secretion by monocytes/macrophages [33]. sIL-2r showed a significant positive correlation with disease activity in our RA patients. Therefore, an increased binding of 1,25(OH)2D3 to the VDR on activated T cells may contribute to the decreased 1,25(OH)2D3 serum levels in RA patients with high disease activity. In addition to a decreased renal 1{alpha}-hydroxylation and to an increased binding to the VDR, an increased extra-renal metabolism of 1,25(OH)2D3 to inactive metabolites could contribute to the lower 1,25(OH)2D3 serum levels in RA patients with high disease activity [3436]. The extra-renal 1{alpha}-hydroxylation of 25OHD3 to 1,25(OH)2D3 in the synovial tissue of patients with RA has been shown to be followed by a 24-hydroxylation of 1,25(OH)2D3 to 1,24,25(OH)3D3 [36]. Bone ALP, as a marker of osteoblast function, was negatively correlated with disease activity and positively related to both iPTH and 1,25(OH)2D3 . PTH and 1,25(OH)2D3 are hormones with bone-resorptive, but also osteoanabolic effects, and 1,25(OH)2D3 induces the expression of alkaline phosphatase in osteoblasts [37]. Therefore, the decrease in iPTH and 1,25(OH)2D3 may contribute to a suppression of bone formation in RA patients with high disease activity.

Although our study group consists of post-menopausal women without hormone replacement therapy, the group is heterogeneous with respect to age and therapy. Especially glucocorticoid treatment, but also DMARDs, could affect calcium-regulating hormones and bone turnover [1]. However, with the exception of bALP, the serum levels of calcium-regulating hormones and bone-turnover parameters were not influenced by therapy with glucocorticoids and/or with DMARDs. The lower levels of bone ALP in patients receiving neither glucocorticoids nor DMARDs may result from a slightly higher disease activity in these patients in comparison with patients treated with glucocorticoids and/or DMARDs. Pro-inflammatory cytokines inhibited by glucocorticoids and some DMARDs [38, 39] have been shown to suppress the expression of ALP in human osteoblasts [9, 10]. Methotrexate has been shown to have suppressive effects on osteoblasts in vitro and in vivo [40, 41], and stimulates bone resorption in vivo [41], resulting in bone loss in healthy rats [41]. However, in our RA women receiving MTX, the parameters of bone metabolism were not different from those of other patients. Thus, age and therapy may have only modest effects on the results of the study.

In summary, our data indicate that in post-menopausal women suffering from RA with high disease activity, an increased periarticular and systemic bone resorption is associated with an increased secretion of IL-6, and with a downregulation of PTH and 1,25(OH)2D3 serum levels resulting in decreased bone formation. With respect to an interruption of these postulated pathogenetic mechanisms, the suppression of bone resorption by bisphosphonates and the application of active vitamin D metabolites are of particular importance in the prevention of systemic osteoporosis in RA patients with high disease activity.


    Acknowledgments
 
This work was supported by the German Ministry of Research, Science, Education and Technology (FKZ 01 ZZ 9602).


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 28 September 1998; revised version accepted 6 April 1999.



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