|Year : 2018 | Volume
| Issue : 2 | Page : 90-94
Circulating levels of osteoprotegerin and sRANKL and the effect of methotrexate in patients with rheumatoid arthritis
Aadhaar Dhooria1, Narendhiran Pandurangan1, Karthik Vinay Mahesh1, Suchet Sachdev2, Aman Sharma1, Shefali Sharma1, Nidhi Gupta1, Varun Dhir1
1 Department of Internal Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Transfusion Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India
|Date of Web Publication||24-May-2018|
Dr. Varun Dhir
Department of Internal Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh - 160 012
Source of Support: None, Conflict of Interest: None
Clinical trial registration CTRI/2012/05/002660
Background: Receptor activator of nuclear factor κβ ligand (RANKL) and osteoprotegerin (OPG) are pivotal molecules involved in osteoclast activation and resorption. In RA, their alterations mediated through inflammatory cytokines are responsible for erosions and systemic bone loss. This study planned to look at the effect of methotrexate on circulating levels of OPG and RANKL in RA.
Methods: Methotrexate-naive patients with active rheumatoid arthritis (RA) between 18 and 65 years of age were included. Controls were derived from voluntary healthy blood donors after written consent. All patients were started on methotrexate at 15 mg/week, increased by 5 mg every 4 weeks till maximum tolerated dose or 25 mg/week whichever was lower. Circulating plasma levels of OPG and RANKL were measured for cases (at baseline and 24 weeks) and controls (at baseline).
Results: Fifty-one consecutive patients with RA and 57 controls were recruited. Circulating OPG (mean ± standard deviation) levels were higher in RA patients as compared to controls, 2879.6 ± 1037.9 and 2214.1 ± 705.3 pg/ml, respectively (P < 0.001). RANKL levels did not differ significantly between cases and controls. After treatment, circulating OPG levels fell significantly, from 2879.6 ± 1037.9 to 2059.8 ± 532.1 pg/ml (P < 0.001), however, no significant change in circulating RANKL levels. No difference was found in OPG and RANKL levels between patients with erosive and nonerosive disease.
Conclusions: OPG levels are higher in RA patients and normalize in response to treatment with methotrexate. The initial higher levels of OPG may represent a compensatory mechanism to osteoclast activation; they normalize on reduction of disease activity.
Keywords: Methotrexate, osteoprotegerin, receptor activator of nuclear factor κβ ligand, rheumatoid arthritis
|How to cite this article:|
Dhooria A, Pandurangan N, Mahesh KV, Sachdev S, Sharma A, Sharma S, Gupta N, Dhir V. Circulating levels of osteoprotegerin and sRANKL and the effect of methotrexate in patients with rheumatoid arthritis. Indian J Rheumatol 2018;13:90-4
|How to cite this URL:|
Dhooria A, Pandurangan N, Mahesh KV, Sachdev S, Sharma A, Sharma S, Gupta N, Dhir V. Circulating levels of osteoprotegerin and sRANKL and the effect of methotrexate in patients with rheumatoid arthritis. Indian J Rheumatol [serial online] 2018 [cited 2019 Jan 23];13:90-4. Available from: http://www.indianjrheumatol.com/text.asp?2018/13/2/90/228284
| Introduction|| |
Rheumatoid arthritis (RA) is a prototype inflammatory disorder with prevalence nearing 1%. Although predominantly a synovial disease, it has significant effects on the bone – both local and systemic. The characteristic local features include erosions and periarticular osteopenia on plain radiographs, whereas systemically, there is osteopenia. Factors responsible for the prevention of erosions along with preventing systemic osteopenia is a key therapeutic target of RA treatment.
Osteoclasts are multinucleated cells derived from monocyte-macrophage lineage and play a key role in bone resorption. Key molecules that regulate its activity and hence bone resorption and formation were discovered at the end of the 20th century. Receptor activator of nuclear factor κβ ligand (RANKL) acts by binding to its receptor RANK and promotes osteoclast differentiation and activation. Osteoprotegerin (OPG) acts as a decoy receptor for RANKL and inhibits its function. Disruption of the RANKL gene leads to osteopetrosis, and OPG knockout leads to severe osteoporosis., These mechanisms regulating bone loss have been implicated in glucocorticoid-induced osteoporosis, metastatic bone loss, and RA.
In RA, inflammatory cytokines such as tumor necrosis factor α (TNFα), interleukin-6, and interleukin-17 have been shown to trigger bony resorption both locally and systemically. This has been shown to occur (in part) through the secretion of RANKL by synovial lymphocytes and osteoblasts leading to osteoclast activation. Increased expression of RANKL has been shown at the pannus-bone interface at sites of erosions in patients with RA. Thus, modulation of the RANKL-OPG axis has gained prominence to discover a way to stop erosions (through osteoclast inhibition) that may be independent of disease activity (delinking the two processes). Indeed, in animal models, OPG-treated mice exhibit minimal loss of bone and trabecular bone by preventing osteoclast accumulation in contrast to untreated mice where erosions develop. Further, OPG has been linked to cardiovascular risk (CVD) in RA, and thus, its modulation may be the mechanism CVD improves in RA.
Methotrexate forms the cornerstone of RA treatment and acts as a disease-modifying antirheumatic drug (DMARD), i.e., modifies the natural history to prevent erosions. It is unclear whether it modulates the RANKL/OPG axis. Thus, we wanted to look at the effect of methotrexate on the circulating levels of RANKL and OPG.
| Methods|| |
This study had a cross-sectional component where a comparison of levels of circulating bone markers was made between cases of RA (not on treatment with methotrexate) and controls. In addition, it had a longitudinal component where the levels of markers were again measured in patients after 24 weeks of treatment with methotrexate.
Patients and controls
Patients with active RA between 18 and 65 years of age who were not on methotrexate were included. All patients who fulfilled the 1987 American College of Rheumatology (formerly American Rheumatism Association) criteria for RA  had a modified disease activity score using 3 variables (DAS28-3) ≥3.2 and were not on methotrexate for at least the prior 2 months. Stable doses (at least 4 months) of prednisolone, leflunomide, and/or sulfasalazine were permitted. Patients receiving bisphosphonates were not included. Patients with chronic conditions such as chronic kidney disease, liver disease, chronic obstructive pulmonary disease, inflammatory bowel disease, or malabsorption syndromes were also excluded. Patients who had any contraindication to the use of methotrexate including pregnancy/breastfeeding were not a part of the study. Controls were derived from voluntary healthy blood donors.
Assessment of clinical parameters
Disease activity was measured using the modified DAS using 3 variables (DAS28-3); a validated variation of modified DAS that excludes patient global assessment on visual analog scale but displays similar characteristics as the 4 variable DAS28. It is calculated using the formula DAS28-3 = (0.56* √(TJC28) + 0.28* √(SJC28) + 0.70* ln [erythrocyte sedimentation rate (ESR)]) 1.08 + 0.16 where TJC28: 28 tender joint count; SJC28: 28 swollen joint count; ESR: erythrocyte sedimentation rate. Patient's global assessment was excluded as it has been found to be difficult to perform in our part of the world. This was measured at baseline and again at 24 weeks after methotrexate treatment. EULAR criteria were used to determine response status, with good and moderate responders clubbed as “responders” and patients with no response as “nonresponders.” Indian Health assessment questionnaire (HAQ) was used to measure functional disability.
All patients were started on methotrexate at a dose of 15 mg/week that was increased by 5 mg every 4 weeks till the maximum tolerated dose or 25 mg/week whichever was lower. In addition, the dose escalation was only done if the DAS28-3 ≥2.6 (patient was not in remission); there was no transaminitis (defined as SGOT or SGPT >2 times upper limit of normal) and there were no cytopenias (white blood count <4000 or platelet count <1,00,000).
Measurement of rheumatoid factor and anti-cyclic citrullinated peptide
For the purpose of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP), blood was collected in plain vacutainers – allowed to clot over 1 h, centrifuged at 2000 RPM for 10 min, and serum separated. RF was measured by latex agglutination (Omega diagnostics, Scotland, UK) and reported as positive or negative (cutoff 16 IU/L). Measurement of anti-CCP levels was assayed using a commercial ELISA Immunoscan CCPlus test kit (Euro-Diagnostica, Arnhem, The Netherlands), and a cutoff of 25 U/L was taken as positive.
Measurement of osteoprotegerin and Receptor activator of nuclear factor κβ ligand
For measurement of OPG and RANKL levels, blood was collected in heparinized vials, plasma separated by centrifugation over 10 min and stored in multiple aliquots at −80°C. All plasma samples were stored at −80°C until use. Plasma levels of OPG (DY 805 R and D Systems, Minneapolis, Minnesota, USA) and RANKL (DY 626 R and D Systems, Minneapolis, Minnesota, USA) were measured by commercially available ELISA kits. Briefly, 96-well plates were coated overnight at room temperature with monoclonal mouse antihuman capture antibodies (R and D Systems) in phosphate-buffered saline (PBS). The plates were washed with PBS/Tween and blocked with 1% bovine serum albumin in PBS for 1 h at room temperature. Serum samples were added to the plates and incubated for 2 h at room temperature. Goat antihuman detection antibodies (R and D Systems) were added, and the plates were incubated for another 2 h. Streptavidin-horseradish peroxidase was added and incubated for 20 min. After the plates were washed with PBS, the substrate reagent (R and D Systems) was added for another 20 min. The substrate reaction was stopped on addition of 1 mol/L sulphuric acid, and extinction was measured at a 450-nm wavelength using a multiplate ELISA reader. Subsequently, the standard curve was made using the 4 parameter logistic curve fit plotting the optical densities (OD) and standard concentrations. Then, sample OD values were used to determine the relevant concentrations.
Type of distribution of variable (normal or nonnormal) was decided by using Kolmogorov–Smirnov test and confirmed by looking at the histogram. In case of normal distribution, variables in different groups were compared using the independent t-test (normal distribution) and Mann–Whitney U-test (nonnormal). Comparisons of before-after levels were done using the paired t-test (normal) and the Wilcoxon-signed rank test (nonnormal), respectively. P < 0.05 was considered statistically significant.
The study was approved by the ethics committee of the institute. Informed written consent was obtained from all participants prior to their enrollment in this study.
| Results|| |
Fifty-one consecutive treatment-naive patients with RA and 57 controls were recruited for the purpose of the study. The mean age of patients and controls was 42.6 ± 11.4 years and 36.1 ± 9.8 years, respectively. RF positivity was seen in 29 patients (56.9%) and CCP positivity was exhibited by 41 patients (80.4%). Erosions were seen in 6 patients (11.8%). The median disease duration was 1 year (range – 3.5 months–7 years). Twenty patients were on oral steroids (mean prednisolone daily dose 7.2 ± 1.2 mg/day) and 3 patients were on hydroxychloroquine (400 mg/day). The doses of these medications were not changed during the course of the study. Mean body mass index of RA patients was 22.2 ± 4.5 kg/m 2.
Comparison of circulating osteoprotegerin and receptor activator of nuclear factor κβ ligand between cases and controls
Circulating OPG (mean ± standard deviation) levels were higher in RA patients as compared to controls, 2879.6 ± 1037.9 and 2214.1 ± 705.3 pg/ml, respectively (P < 0.001) [Figure 1]. Circulating RANKL levels did not differ between cases and controls (Median [interquartile range] 90.7 (136.2) and 111.5 (123.4), P = 0.392). No differences in OPG levels was seen between erosive and nonerosive disease (P = 0.76), RF status (P = 0.89) or CCP status (P = 0.77) or taking steroids (P = 0.7)
|Figure 1: Box and whiskers plot overlying a scatter plot showing individual values of osteoprotegerin in cases and controls. Whiskers extend from 5th to 95th percentile|
Click here to view
Change in circulating osteoprotegerin and receptor activator of nuclear factor κβ ligand after treatment with methotrexate
Patients received treatment with methotrexate for a total duration of 24 weeks (mean dose of 22.5 ± 4.0 mg/week). The swollen and tender joint counts, ESR as well as the DAS 28 and Indian HAQ improved significantly between week 0 and week 24 after methotrexate therapy [Table 1]. There were 29 patients who responded (4 good and 25 moderate) and 21 nonresponders to methotrexate. Circulating OPG levels fell significantly, from 2879.6 ± 1037.9 to 2059.8 ± 532.1 pg/ml (P < 0.001) [Figure 2]. However, there was no significant difference between the posttreatment OPG levels and healthy controls (P = 0.2). There was also no difference between responders and nonresponders (EULAR criteria) in terms of fall in OPG. Circulating RANKL levels did not change in response to therapy (P = 0.392).
|Figure 2: Box and whiskers plot overlying a scatter plot showing individual values of osteoprotegerin at baseline and after 24 weeks of treatment with methotrexate. Whiskers extend from 5th to 95th percentile|
Click here to view
| Discussion|| |
We found higher circulating OPG levels in RA patients as compared to controls that declined (or normalized) after treatment with methotrexate. Our finding of higher OPG in patients with RA is similar to many studies. Previously, Feuerherm et al., Ziolkowska et al., Skoumal et al., Çakırca et al., and Liu et al. have also reported that OPG levels are elevated in RA compared to controls. However, some studies have found similar levels as controls  or even lower levels. Elevated OPG in RA is probably related to the effect of inflammatory cytokines, such as TNFα and Interleukin-1, acting on fibroblast such as synoviocytes, synovial fluid mononuclear cells, and peripheral blood mononuclear cells. High OPG levels have been shown in the synovial fluid as well. This elevation of OPG may represent a compensatory change to counteract the osteoclast activation in RA. This compensatory elevation has also been found in postmenopausal osteoporosis. Why this compensatory mechanism fails, and local and generalized bone loss results, remains open to speculation.
On treatment with methotrexate, there was a significant fall in OPG levels (P < 0.01), which normalized to the level of healthy controls. Previously, two different studies have also demonstrated a fall in OPG values after treatment with anti-TNFα agents , whereas one study reported an increase in OPG/RANKL after methotrexate treatment (did not mention separately the effect on OPG). It seems that methotrexate has a similar effect as anti-TNFα agents on OPG. Indeed, methotrexate has been shown to lower TNF, IL1, and IL6 levels, and as these are the driving forces for OPG elevation ab initio, it should be not be surprising that it also causes a fall or normalization of OPG levels. Interestingly, even rituximab has been shown to reduce the elevated OPG levels. This may occur as a result of B cell depletion that removes the stimulation for T cells (from B cells) or as an indirect effect of subdued inflammation. The lack of difference between responders and nonresponders may limit its use as a biomarker for this purpose but requires confirmation by a larger study.
This study did not find any difference in RANKL levels between patients and controls. This is contrary to many studies that have found a higher RANKL in RA such as studies by Ziokolswka et al., Hein et al., Xu et al., and Çakırca et al. This could relate either to the sensitivity of the kit used for measurement of RANKL or to the fact that almost a third of the patients were on oral steroids at baseline. We did not find any significant change in RANKL on treatment with methotrexate. Similar results have been reported after anti-TNFα treatment  and by a previous study on treatment with methotrexate. However, some studies have found normalization and reduction of elevated RANKL levels after treatment with both anti-TNF  as well as DMARD regimens. Most of the studies showing a fall in RANKL levels has found elevated levels at baseline whereas studies (like ours) that did not demonstrate any significant change often found levels at baseline to be similar to controls.
We did not find any difference in OPG and RANKL levels between patients with erosive and nonerosive disease. In contrast, Hein et al. found higher synovial and serum levels of RANKL in RA patients as compared to patients with nonerosive arthritis. This might be due to the fact that only 6 of our patients had erosive disease at the time of inclusion in the study. Limitations of our study include the modest sample size. An important highlight is that this is one of the few studies to look at change with methotrexate. Importantly, OPG has been implicated in CVD and coronary artery calcification in RA; normalization of levels may be one of the ways methotrexate reduces CV risk.,
This study confirms higher levels of circulating OPG in RA that normalize on treatment with methotrexate. These higher levels may represent a compensatory mechanism to osteoclast activation.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Vis M, Güler-Yüksel M, Lems WF. Can bone loss in rheumatoid arthritis be prevented? Osteoporos Int 2013;24:2541-53.
Takayanagi H. Osteoimmunology and the effects of the immune system on bone. Nat Rev Rheumatol 2009;5:667-76.
Li J, Sarosi I, Yan XQ, Morony S, Capparelli C, Tan HL, et al.
RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. Proc Natl Acad Sci U S A 2000;97:1566-71.
Mizuno A, Amizuka N, Irie K, Murakami A, Fujise N, Kanno T, et al.
Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/osteoprotegerin. Biochem Biophys Res Commun 1998;247:610-5.
Jones DH, Kong YY, Penninger JM. Role of RANKL and RANK in bone loss and arthritis. Ann Rheum Dis 2002;61 Suppl 2:ii32-9.
Pettit AR, Walsh NC, Manning C, Goldring SR, Gravallese EM. RANKL protein is expressed at the pannus-bone interface at sites of articular bone erosion in rheumatoid arthritis. Rheumatology (Oxford) 2006;45:1068-76.
Asanuma Y, Chung CP, Oeser A, Solus JF, Avalos I, Gebretsadik T, et al.
Serum osteoprotegerin is increased and independently associated with coronary-artery atherosclerosis in patients with rheumatoid arthritis. Atherosclerosis 2007;195:e135-41.
Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al.
The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315-24.
Fransen J, van Riel PL. The disease activity score and the EULAR response criteria. Clin Exp Rheumatol 2005;23:S93-9.
Kumar A, Malaviya AN, Pandhi A, Singh R. Validation of an indian version of the Health Assessment Questionnaire in patients with rheumatoid arthritis. Rheumatology (Oxford) 2002;41:1457-9.
Feuerherm AJ, Borset M, Seidel C, Sundan A, Leistad L, Ostensen M, et al.
Elevated levels of osteoprotegerin (OPG) and hepatocyte growth factor (HGF) in rheumatoid arthritis. Scand J Rheumatol 2001;30:229-34.
Ziolkowska M, Kurowska M, Radzikowska A, Luszczykiewicz G, Wiland P, Dziewczopolski W, et al.
High levels of osteoprotegerin and soluble receptor activator of nuclear factor kappa B ligand in serum of rheumatoid arthritis patients and their normalization after anti-tumor necrosis factor alpha treatment. Arthritis Rheum 2002;46:1744-53.
Skoumal M, Kolarz G, Woloszczuk W, Hawa G, Klingler A. Serum osteoprotegerin but not receptor activator of NF-kappaB ligand correlates with Larsen score in rheumatoid arthritis. Ann Rheum Dis 2004;63:216-7.
Çakırca G, Batmaz İ, Mete N, Sarıyıldız MA, Ulu MA, Yazmalar L, et al
. The relationship between bone mineral density and levels of RANKL, osteoprotegerin and cathepsin-K in patients with rheumatoid arthritis. Dicle Med J 2012;39:479-84.
Liu YY, Long L, Wang SY, Guo JP, Ye H, Cui LF, et al.
Circulating dickkopf-1 and osteoprotegerin in patients with early and longstanding rheumatoid arthritis. Chin Med J (Engl) 2010;123:1407-12.
Świerkot J, Gruszecka K, Matuszewska A, Wiland P. Assessment of the effect of methotrexate therapy on bone metabolism in patients with rheumatoid arthritis. Arch Immunol Ther Exp (Warsz) 2015;63:397-404.
Xu S, Wang Y, Lu J, Xu J. Osteoprotegerin and RANKL in the pathogenesis of rheumatoid arthritis-induced osteoporosis. Rheumatol Int 2012;32:3397-403.
Yano K, Tsuda E, Washida N, Kobayashi F, Goto M, Harada A, et al.
Immunological characterization of circulating osteoprotegerin/osteoclastogenesis inhibitory factor: Increased serum concentrations in postmenopausal women with osteoporosis. J Bone Miner Res 1999;14:518-27.
González-Alvaro I, Ortiz AM, Tomero EG, Balsa A, Orte J, Laffon A, et al.
Baseline serum RANKL levels may serve to predict remission in rheumatoid arthritis patients treated with TNF antagonists. Ann Rheum Dis 2007;66:1675-8.
Vis M, Havaardsholm EA, Haugeberg G, Uhlig T, Voskuyl AE, van de Stadt RJ, et al.
Evaluation of bone mineral density, bone metabolism, osteoprotegerin and receptor activator of the NFkappaB ligand serum levels during treatment with infliximab in patients with rheumatoid arthritis. Ann Rheum Dis 2006;65:1495-9.
Boumans MJ, Thurlings RM, Yeo L, Scheel-Toellner D, Vos K, Gerlag DM, et al.
Rituximab abrogates joint destruction in rheumatoid arthritis by inhibiting osteoclastogenesis. Ann Rheum Dis 2012;71:108-13.
Hein GE, Meister M, Oelzner P, Franke S. SRANKL and OPG in serum and synovial fluid of patients with rheumatoid arthritis in comparison to non-destructive chronic arthritis. Rheumatol Int 2008;28:765-9.
Wechalekar MD, Lester S, Nagpal S, Cole S, Das A, Hissaria P, et al
. THU0070 RANKL, OPG and OSCAR but Not Dkk-1 Predict Radiographic Progression in An Inception Cohort of Seropositive Rheumatoid Arthritis (RA) Treated-To-Target with Combination Conventional DMARD Therapy. Annals of the Rheumatic Diseases. 2016;75:204.
López-Mejias R, Ubilla B, Genre F, Corrales A, Hernández JL, Ferraz-Amaro I, et al.
Osteoprotegerin concentrations relate independently to established cardiovascular disease in rheumatoid arthritis. J Rheumatol 2015;42:39-45.
[Figure 1], [Figure 2]