Identification of anti-citrullinated osteopontin antibodies and increased inflammatory response by enhancement of osteopontin binding to fibroblast-like synoviocytes in rheumatoid arthritis

Abstract

Background: Anti-citrullinated protein/peptide antibodies (ACPAs) are present in patients at onset and have important pathogenic roles during the course of rheumatoid arthritis (RA). The characteristics of several molecules recognized by ACPA have been studied in RA, but the positivity rate of autoantibodies against each antigen is not high, and the pathogenic mechanism of each antibody is not fully understood. We investigated the role of anti-citrullinated osteopontin (anti-cit-OPN) antibodies in RA pathogenesis.

Methods: Enzyme-linked immunosorbent assays on RA patients' sera were used to detect autoantibodies against OPN. Fibroblast-like synoviocytes (FLS) isolated from RA patients were used to test the binding activity and inflammatory response of OPN mediated by anti-cit-OPN antibodies, and their effect was tested using an inflammatory arthritis mouse model immunized with cit-OPN. Anti-cit-OPN antibody positivity and clinical characteristics were investigated in the patients as well.

Results: Using sera from 224 RA patients, anti-cit-OPN antibodies were positive in approximately 44% of RA patients, while approximately 78% of patients were positive for the cyclic citrullinated peptide (CCP2) assay. IgG from patients with anti-cit-OPN antibody increased the binding activity of OPN to FLSs, which further increased matrix metalloproteinase and interleukin-6 production in TNF-stimulated FLSs. Mice immunized with cit-OPN antibodies experienced severe arthritis. Anti-cit-OPN antibodies in RA patients decreased the drug survival rate of tumor necrosis factor (TNF) inhibitors, while it did not decrease that of CTLA4-Ig.

Conclusions: Anti-cit-OPN antibodies were detected in patients with RA. IgG from patients with anti-cit-OPN antibodies aggravated RA, and anti-cit-OPN antibody was a marker of reduced the survival rate of TNF inhibitors in RA patients.

Keywords: Anti-citrullinated protein antibody; Citrullination; Integrin; Osteopontin; Rheumatoid arthritis.

Fig. 1

Positivity of autoantigen in sera of patients with rheumatoid arthritis (RA). A Serum IgG antibodies against osteopontin (OPN), fibronectin (FN), tenascin-C (TNC), α-enolase (α-ENO), bone sialoprotein (BSP), type 2 collagen (Col II), vimentin (Vim), and fibrinogen (Fgn) were quantified by enzyme-linked immunosorbent assay (ELISA). B Antibodies against citrullinated osteopontin (cit-OPN), citrullinated fibronectin (cit-FN), citrullinated tenascin-C (cit-TNC), citrullinated α-enolase (cit-α-ENO), citrullinated bone sialoprotein (cit-BSP), citrullinated type 2 collagen (cit-Col II), citrullinated vimentin (cit-Vim), and citrullinated fibrinogen (cit-Fgn) were tested. Serum samples from 30 RA patients and 7 healthy donors were used. The dashed line indicates the cutoff, defined as the mean plus two standard deviations (SDs), and the positivity of each antibody in RA patients is shown

Fig. 2

IgG from RA patients’ sera with anti-cit-OPN autoantibodies enhanced binding of fibroblast-like synoviocyte (FLSs) with OPN. A The 96-well plates were coated with OPN. FLSs were transferred to protein-coated plates and incubated at 37 °C for 120 min with pooled IgG from anti-cit-OPN positive RA patients or anti-cit-OPN negative RA patients. After washing, the number of cells was estimated using a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. B Quantification of the five different donors. Data are shown as mean ± SEM from the aggregate data. * p < 0.05 and ** p < 0.01 by Tukey–Kramer test

Fig. 3

OPN induced IL-6 and MMP production in TNF-stimulated FLSs and IgG from patients with anti-OPN antibody increased their production. A RT-qPCR analysis of inflammatory genes (normalized relative to GAPDH mRNA). FLSs were stimulated with TNF (5 ng/ml) and OPN, cit-OPN, Col II, α-ENO, or Vim for 24 h. Data represent the mean ± SEM of triplicates from one representative experiment of three independent donors. B Proliferation of FLSs. The FLSs were incubated with OPN or cit-OPN for 1 week. The number of cells was quantified using the MTT assay. Results are presented as the mean ± SD from three independent donors. C FLSs were stimulated with TNF (5 ng/ml) and OPN for 24 h. IgG from anti-cit-OPN-positive RA patients or anti-cit-OPN-negative RA patients were added simultaneously. Data represent the mean ± SEM of 10 independent donors. Data are shown as the mean ± SEM. * p < 0.05 and ** p < 0.01 by Tukey–Kramer test (B) or Mann–Whitney’s U test (C)

Fig. 4

Anti-cit-OPN antibody aggravates inflammatory arthritis. A Arthritis in DBA/1 mice immunized with ovalbumin or citrullinated osteopontin was induced by intraperitoneal injection of KBxN serum. B Time course of changes in the arthritis severity score and joint swelling. C, D Histologic sections from the ankle stained with hematoxylin and eosin staining (C) and assessed for the histologic synovitis scores (D) (n = 4–7 from two independent experiments). E Arthritis in SKG mice immunized with ovalbumin or citrullinated osteopontin was induced by mannan. F Time course of changes in the arthritis severity score and joint swelling. G, H Histological sections from the ankle stained with hematoxylin and eosin (G) and assessed for histological synovitis scores (H) (n = 6, from two independent experiments). All data are shown as the mean ± SEM. * p < 0.05 and *** p < 0.001 by Holm–Sidak test (B, F) or Mann–Whitney’s U test (D, H)

Fig. 5

Survival rate of anti-rheumatic drugs between RA patients with and without anti-cit-OPN antibodies. A Positivity of anti-cit-OPN antibody from RA sera collected over 3 consecutive years, measured by ELISA. B Relationship between serum anti-CCP antibody and anti-cit-OPN antibody using stored serum in the 2016 cohort. C Survival rate of TNF inhibitors due to lack of efficacy between RA patients with and without anti-cit-OPN antibodies in the 2016 cohort. Patients in whom TNF inhibitors were discontinued within 90 days were excluded. D Survival rate of CTLA4-Ig due to lack of efficacy between RA patients with and without anti-cit-OPN antibodies. Patients in whom the drug was discontinued within 90 days were excluded