Serum RANKL levels associate with anti- citrullinated protein antibodies in early untreated rheumatoid arthritis and are modulated following methotrexate

Abstract

Introduction: Receptor activator of nuclear factor kappa B ligand (RANKL) is a key regulator of bone metabolism. Anti-citrullinated protein antibodies (ACPA) have been suggested to cause bone destruction by osteoclast activation. We investigated the relationship between RANKL and ACPA in patients with early untreated rheumatoid arthritis (RA).

Methods: Patients with newly diagnosed untreated RA (n = 183) were analyzed at baseline and 3 months after initiating methotrexate (MTX) treatment. Serum RANKL (total RANKL), ACPA (anti-CCP2) and ACPA specificities (anti-citrullinated (cit)-vimentin, anti-cit-enolase and anti-cit-fibrinogen) were determined by enzyme-linked immunosorbent assay (ELISA). Synovial RANKL expression was evaluated by immunohistochemistry in a small group of patients (n = 15). The relationship between anti-cit-vim antibodies and bone destruction was further validated in 1116 RA patients included in the EIRA cohort. Pearson's chi-square test, Wilcoxon rank sum test, Wilcoxon signed rank test and linear regression models were used.

Results: Serum RANKL concentration was significantly higher (p <0.05) in ACPA-positive (median: 689 pmol/L, IQR 342-1253) compared with ACPA-negative (median: 159 pmol/L, IQR 96-243) patients and this difference was also seen for synovial RANKL expression. Serum RANKL associated with ACPA (p <0.05) and bone erosions in rheumatoid factor (RF)-negative patients (n = 59). Among ACPA specificites, anti-cit-vimentin (amino acids 60-75) was associated with higher RANKL concentration and higher prevalence of bone erosion (p <0.05). Significant reductions in both serum RANKL and ACPA levels were observed after 3 months of MTX treatment (p <0.05).

Conclusions: RANKL was elevated in ACPA-positive and in anti-cit-vimentin-positive patients with early untreated RA and associated with bone erosions. These findings give further support for an early direct pathogenic link between ACPA and bone destruction in RA.

Fig. 1

Serum and synovial RANKL is increased in ACPA-positive as compared to ACPA-negative rheumatoid arthritis (RA). Graphs illustrate the results of ELISA measurement of total serum RANKL concentrations in RA (a) and in RF-negative RA (e) grouped by ACPA status. Immunohistochemistry staining shows expression of synovial RANKL in one ACPA-positive (b) and one ACPA-negative RA patient (c) and the graph illustrate the results of image analysis in 15 patients (d). Horizontal lines represent median values, ^* p <0.05. ACPA anti-citrullinated protein antibodies, ELISA enzyme-linked immunosorbent assay, RANKL receptor activator of nuclear factor kappa B ligand, RF rheumatoid factor

Fig. 2

Serum RANKL and ACPA associate with bone destruction. Graphs show the results of ELISA measurement of total serum RANKL concentrations in RF-negative RA patients grouped by bone erosion status (a). ACPA-positive and anti-cit-vim-positive patients observed higher prevalence of bone destructions than ACPA-negative or anti-cit-vim-negative patients in both early RA cohorts (b). Horizontal lines represent median values, ^* p <0.05; ns: p >0.05. VC denotes validation cohort. ACPA anti-citrullinated protein antibodies, cit citrullinated, ELISA enzyme-linked immunosorbent assay, RA rheumatoid arthritis, RANKL receptor activator of nuclear factor kappa B ligand, RF rheumatoid factor, vim vimentin

Fig. 3

Serum concentration of ACPA and ACPA specificities. Graphs show the results of ELISA measurements of the antibody concentrations for patients being positive at baseline or at 3 months for the corresponding antibody: ACPA n = 120 (a), anti-cit-enolase (amino acids 5–21) n = 71 (b), anti-citrullinated (cit)-vimentin (vim) (amino acids 60–75) n = 67 (c), anti-cit-fibrinogen (fib) (amino acids 563–583) antibodies n = 42 (d). Horizontal lines represent median values, ^* p <0.05. Dotted lines delineate ELISA cutoff values for each antibody. ACPA anti-citrullinated protein antibodies, ELISA enzyme-linked immunosorbent assay