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ARD Online First, published on April 29, 2015 as 10.1136/annrheumdis-2014-207219
Autoantibodies to osteoprotegerin are associated with increased bone resorption in rheumatoid arthritis Periarticular and systemic bone loss are important complications of rheumatoid arthritis (RA).1 The pathogenesis is complex involving local and systemic release of cytokines which promote osteoclastic bone resorption2; production of receptor activator of nuclear factor κB (NFκB) ligand (RANKL) by activated T cells3 and immobility, corticosteroid use4 and the direct osteoclast activating effects of antibodies directed against citrullinated proteins (ACPA).5 6 We previously reported the occurrence of severe osteoporosis in a patient with autoimmune disease who had developed autoantibodies to the bone protective protein osteoprotegerin (OPG).7 In this study, we screened for the presence of similar autoantibodies in RA and evaluated their clinical significance. We developed an ELISA for autoantibodies to OPG, by coating 96-well plates with recombinant human OPG as the capture antigen. Following the addition of patient samples to each well and multiple wash steps, bound OPG antibodies were detected by a peroxidase conjugated, anti-human IgG antibody ( Jackson Immuno Research Labs , 109-035-127). Concentrations of OPG antibody were then quantified using a standard curve generated with a human anti-OPG antibody (Abcam, ab9986). A plot of the antibody concentration versus absorbance calculated by a four parameter logistical equation gave a r2 value of 0.999. The intra-assay coefficient of variation was 11.1% and the reference range (defined as the mean±3 SD) in 199 healthy controls was 7.0–80.5 ng/mL. According to this definition, raised concentrations of OPG antibodies were found in two controls (1%) with values of 93 and 96 ng/mL, respectively, as compared with 7/75 patients with RA (9.3%), a difference that was significant ( p=0.001, Fisher’s exact test). Antibody concentrations in the seven patients with RA ranged
Table 1
Letter
between 106 and 430 ng/mL with a median of 129 ng/mL. Clinical characteristics of the patients with RA with raised OPG antibody concentrations are shown in table 1 as compared with patients with RA and antibody concentrations within the reference range. The OPG antibody-positive patients had longer disease duration, a higher the disease activity score in 28 joints (DAS28) score and higher levels of the bone resorption marker serum cross-linked C-telopeptide of type I collagen (CTX) (measured by ELISA; IDS systems) but bone mineral density (BMD) values did not differ between the groups. There was a positive correlation (Spearman’s r) between OPG antibody levels and DAS28 score (r=0.31, p=0.009) and with CTX values in patients who were not receiving bisphosphonates (r=0.28, p=0.038). We further investigated the functional effects of the OPG antibodies detected by the ELISA in a RANKL-induced NFκB reporter assay in human embryonic kidney 293 (HEK293) cells as previously described.7 Immunoglobulin from four of six OPG antibody-positive patients partially (n=1) or significantly (n=3) reversed the inhibitory effect of OPG on RANKL-induced NFκB activation compared with zero of seven negative patients (p=0.02, Fisher’s exact test). A representative experiment is shown in figure 1. Here, we have shown that a subset of patients with RA develop functional antibodies to OPG and that the presence of these antibodies as detected by ELISA is associated with disease activity and increased bone resorption. Although we did not find an association between the presence of OPG antibodies and BMD, the study had limited power to detect such an effect given the large number of variables that influence BMD.8 We also found a correlation between OPG antibodies and disease activity, but acknowledge that this may be an epiphenomenon given that ACPA-positive patients have more severe disease and may have a greater propensity to produce autoantibodies to several proteins including OPG. While these findings raise the
Clinical characteristics of OPG antibody positive and negative patients with RA
Characteristics
OPG antibody positive (n=7)
OPG antibody negative (n=68)
p Value
Age Female gender BMI (kg/m2)* Disease duration† ACPA or rheumatoid factor positive† DAS28‡ Serum CTX (ng/mL)§ Hip BMD (g/cm2)* Spine BMD (g/cm2)* Medications Treatment naive Current synthetic DMARD Current anti-TNF Ever anti-TNF Current glucocorticoid Current glucocorticoid dose (mg/day)† Bisphosphonate treatment
63.3±11.8 7/7 (100%) 26.62±6.56 13.69±12.72 7/7 (100%) 6.1±0.6 0.30±0.08 0.86±0.26 0.94±0.23
61.5±13.2 47/68 (70%) 26.43±4.52 6.47±7.80 53/66 (80%) 5.4±1.4 0.16±0.10 0.84±0.16 0.91±0.16
0.64 0.18 0.93 0.03 0.34 0.02 0.01 0.82 0.67
1/7 (14.3%) 2/7(28.6%) 1/7 (14.3%) 4/7(57.1%) 3/7 (42.9%) 3.33±2.31 2/7 (28.6%)
12/68 (17.6%) 33/68 (48.5%) 7/68 (10.3%) 15/68 (22.1%) 20/68 (29.4%) 5.33±5.25 13/68 (19.1%)
0.82 0.44 0.75 0.06 0.67 0.53 0.55
Data are shown as mean±SD or number (%). Differences between groups were analysed by Student’s t test for continuous data or by Mann–Whitney U test if the variables were not normally distributed. The χ2 test was used to test for differences between categorical variables. *Spine BMD measurements were available in 61 patients with RA, hip BMD on 60 patients and BMI measurements in 62 patients with RA. †Data on ACPA and rheumatoid factor status, disease duration and current glucocorticoid dose were unavailable for two patients. ‡DAS28 was not recorded in five patients. §Comparison of CTX levels was performed after exclusion of patients on bisphosphonate therapy. ACPA, anti-citrullinated protein antibodies; BMD, bone mineral density; BMI, body mass index; DMARD, disease modifying anti rheumatic drug; OPG, osteoprotegerin; RA, rheumatoid arthritis; TNF, tumour necrosis factor.
Ann Rheum Dis Month 2015 Vol 0 No 0
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Letter 1
Rheumatic Diseases Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK 2 Centre for Population Health Sciences, University of Edinburgh, Edinburgh, UK Correspondence to Professor Stuart H Ralston, Rheumatic Diseases Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK; [email protected] Contributors Study concept and design: BH, PLR and SHR. Acquisition of data: BH, JFW and SHR. Analysis and interpretation of data: BH, TG, MRV, PLR and SHR. Obtained funding: BH, JFW and SHR. Study supervision: PLR and SHR. Funding Arthritis Research UK (19299). Competing interests SHR and PLR are inventors on a patent application on the use of OPG antibodies as a diagnostic marker for bone disease and vascular calcification. Ethics approval Lothian Research Ethics Committee. Provenance and peer review Not commissioned; externally peer reviewed. ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/annrheumdis-2014-207219). To cite Hauser B, Riches PL, Gilchrist T, et al. Ann Rheum Dis Published Online First: [ please include Day Month Year] doi:10.1136/annrheumdis-2014-207219
Figure 1 Effect of osteoprotegerin (OPG) antibody positive and negative samples on receptor activator of nuclear factor κB (NFκB) ligand (RANKL) induced NFκB signalling. The vertical axis shows levels of NFκB activation in HEK293 cells in response to the effects of RANKL, OPG and purified IgG from patient samples. The cells were stimulated with 50 mg/mL RANKL and 100 mg/mL OPG, in the presence or absence of purified IgG from patients with RA that tested positive (samples #1 and #2) or negative for OPG antibodies (samples #3 and #4) as detected by ELISA in this study. The data shown are mean and SEM from three experiments, corrected to vehicle-treated control wells adjusted for cell number. Differences between groups are indicated by ***p=0.001 and +++p=0.004, not significantly (ns) different from RANKL and OPG-treated wells. Patient samples #3 and #4 were not significantly different from RANKL and OPG-treated cells. possibility that autoantibodies to OPG may contribute to the bone loss that accompanies RA, further studies in larger cohorts will be required to fully evaluate their clinical significance. Barbara Hauser,1 Philip L Riches,1 Tamara Gilchrist,1 Micaela R Visconti,1 James F Wilson,2 Stuart H Ralston1
2
Received 23 December 2014 Revised 9 April 2015 Accepted 12 April 2015 Ann Rheum Dis 2015;0:1–2. doi:10.1136/annrheumdis-2014-207219
REFERENCES 1 2 3
4 5
6
7 8
Haugeberg G. Focal and generalized bone loss in rheumatoid arthritis: separate or similar concepts? Nat Clin Pract Rheumatol 2008;4:402–3. Schett G, Gravallese E. Bone erosion in rheumatoid arthritis: mechanisms, diagnosis and treatment. Nat Rev Rheumatol 2012;8:656–64. Kong YY, Feige U, Sarosi I, et al. Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand. Nature 1999;402:304–9. van Staa TP. The pathogenesis, epidemiology and management of glucocorticoid-induced osteoporosis. Calcif Tissue Int 2006;79:129–37. Kleyer A, Finzel S, Rech J, et al. Bone loss before the clinical onset of rheumatoid arthritis in subjects with anticitrullinated protein antibodies. Ann Rheum Dis 2014;73:854–60. Harre U, Georgess D, Bang H, et al. Induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin. J Clin Invest 2012;122:1791–802. Riches PL, McRorie E, Fraser WD, et al. Osteoporosis associated with neutralizing autoantibodies against osteoprotegerin. N Engl J Med 2009;361:1459–65. Sambrook P, Cooper C. Osteoporosis. Lancet 2006;367:2010–18.
Ann Rheum Dis Month 2015 Vol 0 No 0
Downloaded from http://ard.bmj.com/ on May 5, 2015 - Published by group.bmj.com
Autoantibodies to osteoprotegerin are associated with increased bone resorption in rheumatoid arthritis Barbara Hauser, Philip L Riches, Tamara Gilchrist, Micaela R Visconti, James F Wilson and Stuart H Ralston Ann Rheum Dis published online April 29, 2015
Updated information and services can be found at: http://ard.bmj.com/content/early/2015/04/29/annrheumdis-2014-2072 19
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References Email alerting service
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ARD Online First, published on April 29, 2015 as 10.1136/annrheumdis-2014-207219
Autoantibodies to osteoprotegerin are associated with increased bone resorption in rheumatoid arthritis Periarticular and systemic bone loss are important complications of rheumatoid arthritis (RA).1 The pathogenesis is complex involving local and systemic release of cytokines which promote osteoclastic bone resorption2; production of receptor activator of nuclear factor κB (NFκB) ligand (RANKL) by activated T cells3 and immobility, corticosteroid use4 and the direct osteoclast activating effects of antibodies directed against citrullinated proteins (ACPA).5 6 We previously reported the occurrence of severe osteoporosis in a patient with autoimmune disease who had developed autoantibodies to the bone protective protein osteoprotegerin (OPG).7 In this study, we screened for the presence of similar autoantibodies in RA and evaluated their clinical significance. We developed an ELISA for autoantibodies to OPG, by coating 96-well plates with recombinant human OPG as the capture antigen. Following the addition of patient samples to each well and multiple wash steps, bound OPG antibodies were detected by a peroxidase conjugated, anti-human IgG antibody ( Jackson Immuno Research Labs , 109-035-127). Concentrations of OPG antibody were then quantified using a standard curve generated with a human anti-OPG antibody (Abcam, ab9986). A plot of the antibody concentration versus absorbance calculated by a four parameter logistical equation gave a r2 value of 0.999. The intra-assay coefficient of variation was 11.1% and the reference range (defined as the mean±3 SD) in 199 healthy controls was 7.0–80.5 ng/mL. According to this definition, raised concentrations of OPG antibodies were found in two controls (1%) with values of 93 and 96 ng/mL, respectively, as compared with 7/75 patients with RA (9.3%), a difference that was significant ( p=0.001, Fisher’s exact test). Antibody concentrations in the seven patients with RA ranged
Table 1
Letter
between 106 and 430 ng/mL with a median of 129 ng/mL. Clinical characteristics of the patients with RA with raised OPG antibody concentrations are shown in table 1 as compared with patients with RA and antibody concentrations within the reference range. The OPG antibody-positive patients had longer disease duration, a higher the disease activity score in 28 joints (DAS28) score and higher levels of the bone resorption marker serum cross-linked C-telopeptide of type I collagen (CTX) (measured by ELISA; IDS systems) but bone mineral density (BMD) values did not differ between the groups. There was a positive correlation (Spearman’s r) between OPG antibody levels and DAS28 score (r=0.31, p=0.009) and with CTX values in patients who were not receiving bisphosphonates (r=0.28, p=0.038). We further investigated the functional effects of the OPG antibodies detected by the ELISA in a RANKL-induced NFκB reporter assay in human embryonic kidney 293 (HEK293) cells as previously described.7 Immunoglobulin from four of six OPG antibody-positive patients partially (n=1) or significantly (n=3) reversed the inhibitory effect of OPG on RANKL-induced NFκB activation compared with zero of seven negative patients (p=0.02, Fisher’s exact test). A representative experiment is shown in figure 1. Here, we have shown that a subset of patients with RA develop functional antibodies to OPG and that the presence of these antibodies as detected by ELISA is associated with disease activity and increased bone resorption. Although we did not find an association between the presence of OPG antibodies and BMD, the study had limited power to detect such an effect given the large number of variables that influence BMD.8 We also found a correlation between OPG antibodies and disease activity, but acknowledge that this may be an epiphenomenon given that ACPA-positive patients have more severe disease and may have a greater propensity to produce autoantibodies to several proteins including OPG. While these findings raise the
Clinical characteristics of OPG antibody positive and negative patients with RA
Characteristics
OPG antibody positive (n=7)
OPG antibody negative (n=68)
p Value
Age Female gender BMI (kg/m2)* Disease duration† ACPA or rheumatoid factor positive† DAS28‡ Serum CTX (ng/mL)§ Hip BMD (g/cm2)* Spine BMD (g/cm2)* Medications Treatment naive Current synthetic DMARD Current anti-TNF Ever anti-TNF Current glucocorticoid Current glucocorticoid dose (mg/day)† Bisphosphonate treatment
63.3±11.8 7/7 (100%) 26.62±6.56 13.69±12.72 7/7 (100%) 6.1±0.6 0.30±0.08 0.86±0.26 0.94±0.23
61.5±13.2 47/68 (70%) 26.43±4.52 6.47±7.80 53/66 (80%) 5.4±1.4 0.16±0.10 0.84±0.16 0.91±0.16
0.64 0.18 0.93 0.03 0.34 0.02 0.01 0.82 0.67
1/7 (14.3%) 2/7(28.6%) 1/7 (14.3%) 4/7(57.1%) 3/7 (42.9%) 3.33±2.31 2/7 (28.6%)
12/68 (17.6%) 33/68 (48.5%) 7/68 (10.3%) 15/68 (22.1%) 20/68 (29.4%) 5.33±5.25 13/68 (19.1%)
0.82 0.44 0.75 0.06 0.67 0.53 0.55
Data are shown as mean±SD or number (%). Differences between groups were analysed by Student’s t test for continuous data or by Mann–Whitney U test if the variables were not normally distributed. The χ2 test was used to test for differences between categorical variables. *Spine BMD measurements were available in 61 patients with RA, hip BMD on 60 patients and BMI measurements in 62 patients with RA. †Data on ACPA and rheumatoid factor status, disease duration and current glucocorticoid dose were unavailable for two patients. ‡DAS28 was not recorded in five patients. §Comparison of CTX levels was performed after exclusion of patients on bisphosphonate therapy. ACPA, anti-citrullinated protein antibodies; BMD, bone mineral density; BMI, body mass index; DMARD, disease modifying anti rheumatic drug; OPG, osteoprotegerin; RA, rheumatoid arthritis; TNF, tumour necrosis factor.
Ann Rheum Dis Month 2015 Vol 0 No 0
1
Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd (& EULAR) under licence.
Downloaded from http://ard.bmj.com/ on May 5, 2015 - Published by group.bmj.com
Letter 1
Rheumatic Diseases Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK 2 Centre for Population Health Sciences, University of Edinburgh, Edinburgh, UK Correspondence to Professor Stuart H Ralston, Rheumatic Diseases Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK; [email protected] Contributors Study concept and design: BH, PLR and SHR. Acquisition of data: BH, JFW and SHR. Analysis and interpretation of data: BH, TG, MRV, PLR and SHR. Obtained funding: BH, JFW and SHR. Study supervision: PLR and SHR. Funding Arthritis Research UK (19299). Competing interests SHR and PLR are inventors on a patent application on the use of OPG antibodies as a diagnostic marker for bone disease and vascular calcification. Ethics approval Lothian Research Ethics Committee. Provenance and peer review Not commissioned; externally peer reviewed. ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/annrheumdis-2014-207219). To cite Hauser B, Riches PL, Gilchrist T, et al. Ann Rheum Dis Published Online First: [ please include Day Month Year] doi:10.1136/annrheumdis-2014-207219
Figure 1 Effect of osteoprotegerin (OPG) antibody positive and negative samples on receptor activator of nuclear factor κB (NFκB) ligand (RANKL) induced NFκB signalling. The vertical axis shows levels of NFκB activation in HEK293 cells in response to the effects of RANKL, OPG and purified IgG from patient samples. The cells were stimulated with 50 mg/mL RANKL and 100 mg/mL OPG, in the presence or absence of purified IgG from patients with RA that tested positive (samples #1 and #2) or negative for OPG antibodies (samples #3 and #4) as detected by ELISA in this study. The data shown are mean and SEM from three experiments, corrected to vehicle-treated control wells adjusted for cell number. Differences between groups are indicated by ***p=0.001 and +++p=0.004, not significantly (ns) different from RANKL and OPG-treated wells. Patient samples #3 and #4 were not significantly different from RANKL and OPG-treated cells. possibility that autoantibodies to OPG may contribute to the bone loss that accompanies RA, further studies in larger cohorts will be required to fully evaluate their clinical significance. Barbara Hauser,1 Philip L Riches,1 Tamara Gilchrist,1 Micaela R Visconti,1 James F Wilson,2 Stuart H Ralston1
2
Received 23 December 2014 Revised 9 April 2015 Accepted 12 April 2015 Ann Rheum Dis 2015;0:1–2. doi:10.1136/annrheumdis-2014-207219
REFERENCES 1 2 3
4 5
6
7 8
Haugeberg G. Focal and generalized bone loss in rheumatoid arthritis: separate or similar concepts? Nat Clin Pract Rheumatol 2008;4:402–3. Schett G, Gravallese E. Bone erosion in rheumatoid arthritis: mechanisms, diagnosis and treatment. Nat Rev Rheumatol 2012;8:656–64. Kong YY, Feige U, Sarosi I, et al. Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand. Nature 1999;402:304–9. van Staa TP. The pathogenesis, epidemiology and management of glucocorticoid-induced osteoporosis. Calcif Tissue Int 2006;79:129–37. Kleyer A, Finzel S, Rech J, et al. Bone loss before the clinical onset of rheumatoid arthritis in subjects with anticitrullinated protein antibodies. Ann Rheum Dis 2014;73:854–60. Harre U, Georgess D, Bang H, et al. Induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin. J Clin Invest 2012;122:1791–802. Riches PL, McRorie E, Fraser WD, et al. Osteoporosis associated with neutralizing autoantibodies against osteoprotegerin. N Engl J Med 2009;361:1459–65. Sambrook P, Cooper C. Osteoporosis. Lancet 2006;367:2010–18.
Ann Rheum Dis Month 2015 Vol 0 No 0
Downloaded from http://ard.bmj.com/ on May 5, 2015 - Published by group.bmj.com
Autoantibodies to osteoprotegerin are associated with increased bone resorption in rheumatoid arthritis Barbara Hauser, Philip L Riches, Tamara Gilchrist, Micaela R Visconti, James F Wilson and Stuart H Ralston Ann Rheum Dis published online April 29, 2015
Updated information and services can be found at: http://ard.bmj.com/content/early/2015/04/29/annrheumdis-2014-2072 19
These include:
References Email alerting service
This article cites 8 articles, 1 of which you can access for free at: http://ard.bmj.com/content/early/2015/04/29/annrheumdis-2014-2072 19#BIBL Receive free email alerts when new articles cite this article. Sign up in the box at the top right corner of the online article.
Notes
To request permissions go to: http://group.bmj.com/group/rights-licensing/permissions To order reprints go to: http://journals.bmj.com/cgi/reprintform To subscribe to BMJ go to: http://group.bmj.com/subscribe/
