Abstract

Objective. To assess bone mineral density (BMD), serum levels of soluble receptor activator of nuclear factors-κB ligand (sRANKL) and osteoprotegerin (OPG) in patients with ankylosing spondylitis (AS), and to determine their relationships with disease activities.

Methods. Serum levels of sRANKL and OPG in AS were measured by sandwich enzyme-linked immunosorbent assay. The disease activities were determined using Bath Ankylosing Spondylitis Disease Activity Score Index, Bath Ankylosing Spondylitis Functional Index , Bath Ankylosing Spondylitis Metrology Index and Bath Ankylosing Spondylitis Patient Global Score. BMD of femur and lumbar spine was measured by dual energy X-ray absorptiometry. Radiological grading was determined by New York criteria for sacroiliitis and modified Stoke Ankylosing Spondylitis Spine Score.

Results. Osteoporosis and osteopaenia of femoral neck were found in 33 and 41% of patients, respectively. BMD of femoral neck showed negative correlation with disease activity indexes, erythrocyte sedimentation rate and C-reactive protein. The serum sRANKL levels and the ratio of sRANKL to OPG were significantly higher in patients with AS than those of controls. The sRANKL/OPG ratio tended to increase in patients with reduced BMD and radiological findings of active inflammation.

Conclusion . About 74% of AS patients have reduced BMD and this change reflects disease activity. Serum sRANKL levels and sRANKL/OPG ratios are up-regulated in patients with AS and have relationship with BMD and radiological changes. These results suggest that the imbalance between RANKL and OPG might be involved in the pathogenesis and clinical courses of osteoporosis in AS.

Introduction

Ankylosing spondylitis (AS) is a chronic inflammatory disease, which is characterized by main involvement of axial joints and bilateral sacroiliitis, but sometimes peripheral joints and extra-articular organs are involved. Decreased bone mineral density (BMD) is typical of extra-articular symptoms and many patients with AS have osteoporosis and consequent non-traumatic fractures in spite of their young age and maleness. However, the exact mechanisms and causes of the bone loss in AS are not fully identified yet [ 1 ]. BMD of femoral neck seems to be related with disease activity based upon Schober index [ 2 ], and bone turnover markers have positive correlation with C-reactive protein (CRP) and Larsen radiological hip scores [ 3 ]. Putting together, lower BMD seems to be related with the activity and severity of AS.

Receptor activator of nuclear factors-κB ligand (RANKL) is a potent stimulator of bone resorption by binding receptor activator of nuclear factors-κB (RANK) in the cell membrane of osteoclasts. In contrast, osteoprotegerin (OPG) is a soluble decoy receptor for RANKL by interfering the RANKL/RANK binding, and it inhibits the maturation and activation of osteoclasts and their precursors. Because the balance of RANKL/RANK and OPG has a central role in the regulation of bone remodelling and loss, this balance participates directly in the bone loss and destruction of some diseases, such as osteoporosis, chronic inflammatory arthritis and the osteolytic bony metastasis of malignancies.

In the investigation of the expression of RANKL and OPG in inflammatory arthritis, such as rheumatoid arthritis (RA) and AS, the expression of RANKL is much higher in the tissues of RA and AS with active synovitis than normal tissues [ 4 ]. The expression of OPG is decreased in active RA tissues, but such reduced expression is not seen in AS synovial tissues. From these results, we could presume that there is some different mechanisms of the interplay and balance of RANKL and OPG in the two different diseases [ 5 ]. Until now, the role of OPG and RANKL in AS is open to dispute because there is no agreement of serum OPG levels or data to examine its balanced relationship with RANKL. More considerable works are needed to understand the pathogenesis of osteoporosis in AS [ 6–8 ].

The aims of this article are an analysis of the frequency and clinical characteristics of osteoporosis in AS patients, a measurement of serum concentrations of soluble RANKL (sRANKL) and OPG and a determination of their relationship with disease activity.

Materials and methods

Patients

We studied 60 patients with AS who fulfilled the modified New York criteria for AS. Informed consents were obtained from all patients and healthy volunteers enrolled, and this study was approved by the Ethical Committees in Kang-Nam St Mary's Hospital. X-rays of spines and sacroiliac joints were performed, and CRP, erythrocyte sedimentation rate (ESR) and human leukocyte antigen (HLA) B27 were measured. Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) [ 9 ], Bath Ankylosing Spondylitis Functional Index (BASFI) [ 10 ], Bath Ankylosing Spondylitis Metrology Index (BASMI) [ 11 ] and Bath Ankylosing Spondylitis Patient Global Score (BAS-G) [ 12 ] were determined at the same moment.

Radiological studies

For radiological scoring of spine, M-SASSS (modified Stoke Ankylosing Spondylitis Spine Score) was applied and transformed, in that the patients were divided into those with syndesmophytes and those without. From cervical to lumbar spines, the scores were lay by 0 = normal, 1 = bony erosion, sclerosis or squaring, 2 = syndesmophyte and 3 = bridging syndesmophyte, and the score of a spine which had a highest M-SASSS score became a final score of the patient [ 13 ]. For radiological grading of sacroiliitis, the New York criteria for sacroiliitis was applied.

Assessment of bone mineral density (BMD)

BMD of the lumbar spine and right femoral neck was measured by dual energy X-ray absorptiometry (Hologic QDR-4500A, Hologic Inc., Waltharn, USA). Lumbar spine BMD was measured in L1 through L4 with anterior–posterior view. BMD was expressed by gram per square centimetre and T score, and the coefficient of variation was 1% for the lumbar spine and 1.2% for the femur. Osteoporosis and osteopaenia were defined according to the criteria of the World Health Organization (WHO).

Measurement of serum sRANKL, OPG, TNF-α and IL-17

Sera were obtained from AS patients and age- and sex-matched controls, and stored at −20°C. Serum levels of sRANKL, OPG, tumour necrosis factor-α (TNF-α) and interleukin-17 (IL-17) were measured using sandwich enzyme-linked immunosorbent assay (ELISA), as previously described [ 14 , 15 ].

Statistical analysis

Statistical analysis was performed with SPSS 11.5 software for Windows (SPSS, Chicago, IL, USA). Data were expressed by mean±SEM. The difference of data between patients and controls were analysed using Student's t -test and the correlations were presented using Spearman's correlation. P -values<0.05 were considered statistically significant.

Results

Table 1 shows the clinical data of the patients with AS included in the study. The patients with AS presented reduced BMD, with mean BMD of spine 0.97±0.03 g/cm 2 , and mean T score of spine −0.93±0.25. Mean BMD was 0.77±0.03 g/cm 2 , and T score was −1.78±0.24 in femoral neck. The osteoporosis and osteopaenia of lumbar spines were observed in 19 and 37% of the patients, respectively, and in femoral neck, 33 and 41% of the patients had osteoporosis and osteopaenia, respectively. The lesser the BMD of femoral neck, higher the AS disease activity indexes the patients had, with good correlation between BMD and clinical data, such as BASDAI ( R = −0.43, P = 0.006), BASFI ( R = −0.41, P = 0.008), BASMI ( R = −0.52, P = 0.001), BAS-G ( R = −0.44, P = 0.004), ESR ( R = −0.45, P = 0.003) and CRP levels ( R = −0.39, P = 0.01). But, there was poor correlation between BMD of lumbar spines and disease activity indexes or radiological scores. The patients who had scores 3–4 of New York criteria for sacroiliitis had lower BMD compared with the patients having scores 0–2 (T score, −1.73±0.19 vs −0.95±0.34, P = 0.04).

T able 1.

Clinical characteristics of 60 patients with ankylosing spondylitis

Age 32.1 ± 1.2 (range 17–58 yrs) 
Sex 
    Male 51 (85%) 
    Female 9 (15%) 
HLA-B27 (+) 50 (83%) 
Disease duration (yr) 5.5 ± 0.9 (range 0–20 yrs) 
BASDAI 3.7 ± 0.3 (range 0.4–10.0) 
BASFI 1.8 ± 0.3 (range 0–8.0) 
BASMI 1.5 ± 0.3 (range 0–8) 
BAS-G 4.2 ± 0.4 (range 0–10) 
ESR (mm/h) 17.7 ± 2.2 (range 1–68 mm/h) 
CRP (mg/dl) 1.1 ± 0.2 (range 0.1–6.0 mg/dl) 
Age 32.1 ± 1.2 (range 17–58 yrs) 
Sex 
    Male 51 (85%) 
    Female 9 (15%) 
HLA-B27 (+) 50 (83%) 
Disease duration (yr) 5.5 ± 0.9 (range 0–20 yrs) 
BASDAI 3.7 ± 0.3 (range 0.4–10.0) 
BASFI 1.8 ± 0.3 (range 0–8.0) 
BASMI 1.5 ± 0.3 (range 0–8) 
BAS-G 4.2 ± 0.4 (range 0–10) 
ESR (mm/h) 17.7 ± 2.2 (range 1–68 mm/h) 
CRP (mg/dl) 1.1 ± 0.2 (range 0.1–6.0 mg/dl) 

Data were expressed by mean±SEM. BASDAI, Bath Ankylosing Spondylitis Disease Activity Index; BASFI, Bath Ankylosing Spondylitis Functional Index; BASMI, Bath Ankylosing Spondylitis Metrology Index; BAS-G, Bath Ankylosing Spondylitis Patient Global Score; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein.

Serum levels of sRANKL in AS patients were significantly higher than those in normal controls (1187.4±419.0 pg/ml vs 301.0±110.3 pg/ml, P = 0.045). However, serum levels of OPG in AS patients were not different from those in normal controls (928.4±61.7 pg/ml vs 1158.2±266.1 pg/ml, P >0.05). In particular, the ratio sRANKL to OPG was clearly higher in AS than in controls (1.49±0.5 vs 0.34±0.1, P = 0.033, Fig. 1 ). In spine, patients who had syndesmophytes had lower sRANKL:OPG ratio than the patients without syndesmophytes (2.0±0.7 vs 0.2±0.03, P = 0.01). In sacroiliac joints, patients who had the scores 3–4 of New York criteria for sacroiliitis tended to have higher sRANKL:OPG ratio than those who had the score 0–2, but the difference was not statistically significant (2.7±1.5 vs 1.0±0.4, P >0.05). However, there was no correlation between sRANKL:OPG ratio and clinical data, such as CRP, ESR, HLA-B27 and AS disease activity indexes, disease duration or BMD. Patients with osteoporosis or osteopaenia tended to have higher sRANKL:OPG ratio than those without, but the difference was not statistically significant (2.09±0.94 vs 1.21±0.71, P >0.05).

F ig . 1.

( A ) Serum levels of soluble receptor activator of nuclear factors-κB ligand (sRANKL) and osteoprotegerin (OPG) in ankylosing spondylitis (AS) and healthy controls (HC) are measured by sandwich ELISA. Open bars indicate the serum levels of AS patients and solid bars indicate the serum levels of controls. Serum levels of sRANKL are significantly higher in AS patients than in the controls. ( B ) sRANKL:OPG ratios are also higher in AS patients than in the controls. * P <0.05.

F ig . 1.

( A ) Serum levels of soluble receptor activator of nuclear factors-κB ligand (sRANKL) and osteoprotegerin (OPG) in ankylosing spondylitis (AS) and healthy controls (HC) are measured by sandwich ELISA. Open bars indicate the serum levels of AS patients and solid bars indicate the serum levels of controls. Serum levels of sRANKL are significantly higher in AS patients than in the controls. ( B ) sRANKL:OPG ratios are also higher in AS patients than in the controls. * P <0.05.

Finally, we studied the relationship between TNF-α, IL-17 and RANKL. The serum levels of both TNF-α and IL-17 were also elevated in AS patients (data not shown) and had positive correlation with serum sRANKL levels ( R = 0.49, P <0.001, and R = 0.30, P = 0.02, respectively).

Discussion

AS is a chronic inflammatory disease characterized by an axial joint disease starting from sacroiliitis and enthesopathies. In addition to the bone loss at the sites of chronic inflammation, a new bone formation is a distinctive feature in this disease. Generalized osteoporosis as well as regional osteopaenia is common in AS with high incidence of osteoporosis or osteopaenia, 41–62% in spine and 46–86% in femur. The incidence of consequent non-traumatic fractures is also high as 10–40% [ 2 , 16–18 ]. In long-standing diseases, BMD tends to reduce with disease duration. The exact mechanisms of osteoporosis in the inflammatory arthritis are not fully uncovered, but some possible mechanisms are presumed, such as the activation of osteoclasts induced by inflammatory responses, immobility, adverse effects of corticosteroid, genetic factors and hormonal abnormalities [ 19 ]. In this study, 74% of AS patients presented a reduced BMD in femoral neck. But in lumbar spines, the incidence of osteoporosis and osteopaenia was lower than in femur, and BMD was not related with disease activity indexes and radiological findings. It could be explained that the formation of syndesmophytes and the ossification of ligaments led to the overestimation of spinal BMD.

RANKL is over-expressed and OPG is under-expressed in the tissues of active synovitis in RA, and there is a general agreement about the fact that the imbalance of these two molecules has an important role in the bone loss and destruction in RA [ 4 , 5 ]. But, unlike RA, which results in bone loss and erosions mainly, AS which has another characteristic such as a new bone formation may have some different mechanism in the interplay of RANKL and OPG. This difference may lead to the different characteristics and pathogenesis in two types of inflammatory arthritis. So far, little is known of the correlation of RANKL/OPG system and AS. In immunohistochemical staining, RANKL is over-expressed in the tissue of active synovitis of AS, but the expression of OPG is not decreased, unlike RA [ 5 ]. There is no consensus to the serum levels of OPG in AS; some studies showed the results of increased OPG levels, but others showed the results of decreased levels [ 6–8 ]. The serum or synovial fluid levels of RANKL in AS are not yet determined. This study aimed to determine the serum levels of OPG and RANKL, and we found out that serum OPG levels in AS was not so much lower, but the sRANKL levels and RANKL:OPG ratio are much higher in AS than in controls. These results are first described in the present study. These findings mean that the imbalance and the disturbed interaction of RANKL and OPG, rather than each serum level, may be an important cause and pathogenesis in reduced BMD in AS.

Of special interest of this study is the detailed analysis of the correlation between BMD and various disease activity indexes. Until now, there are only simple and separate data that assesses the correlation between BMD and spinal mobility, CRP or radiological changes using BASRI scores [ 6 ], but we assessed the relationships using both subjective and objective indexes. The objective measurement consists of BASMI, CRP and ESR, and the subjective measurement includes BASDAI, BASFI and BAS-G. The BMD is found to have significant correlation with subjective indexes as well as objective indexes. This result implies that AS patients who have more active and severe disease tend to have lower BMD. By analogy with these facts, BMD could be assessed as indirect illumination of the AS disease activity.

Finally, we assessed the correlation of the key inflammatory cytokines contributory to osteoclastogenesis in inflammatory arthritis and RANKL expression in AS. TNF-α has an important role in AS because it is up-regulated in AS and the inhibition of this cytokine is effective in the treatment of AS. Until now, there is no study investigating IL-17 in patients with AS. IL-17 is a potent stimulator of the osteoclast activation in bone remodelling and it is up-regulated in synovial fluids of RA [ 20 ]. In this study, the serum levels of these two cytokines were higher in AS than controls (data not shown), and had significant correlation with serum sRANKL levels. We found serum levels of IL-17 are up-regulated in AS at the first time, but the levels had no correlation with disease activity indexes or BMD.

In summary, we attempted to determine the clinical characteristics of osteoporosis, to measure the serum levels of RANKL and OPG, and to find the correlation between BMD, RANKL/OPG levels and disease activities in AS. About 74% of AS patients had reduced BMD and this change reflected disease activity. Serum levels of RANKL and sRANKL/OPG ratios were up-regulated in patients with AS and tended to be related to BMD of femoral neck and radiological changes. Our data suggest that the imbalance of sRANKL and OPG might be one of the mechanisms leading to osteoporosis in AS. When managing patients with AS, clinicians always should pay attention as osteoporosis is quite common in AS despite young age and it could reflect disease activity indirectly. Future therapies should target against the restoration of unbalanced RANKL/OPG ratio as well as blocking of the key inflammatory cytokines with regard to this respect.

graphic

Acknowledgements

This work was supported by grant (R11-2002-098-05001-0) from Korea Science & Engineering Foundation through the RhRC (Rheumatism Research Center) at the Catholic University of Korea.

The authors have declared no conflict of interest.

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