Anti-citrullinated protein antibodies promote apoptosis of mature human Saos-2 osteoblasts via cell-surface binding to citrullinated heat shock protein 60.
We hypothesized that anti-citrullinated protein antibodies (ACPAs) react with osteoblast surface citrullinated proteins and affect cell function, leading to joint damage in patients with rheumatoid arthritis (RA). First, we purified ACPAs by cyclic citrullinated peptide (CCP)-conjugated affinity column chromatography. The cognate antigens of ACPAs on Saos-2 cells, a sarcoma osteogenic cell line generated from human osteoblasts, were probed by ACPAs, and the reactive bands were analyzed using proteomic analyses. We found that ACPAs bind to Saos-2 cell membrane, and several protein candidates, including HSP60, were identified. We then cloned and purified recombinant heat shock protein 60 (HSP60) and citrullinated HSP60 (citHSP60) and investigated the effect of ACPAs on Saos-2 cell. We confirmed that HSP60 obtained from Saos-2 cell membrane were citrullinated and reacted with ACPAs, which induces Saos-2 cells apoptosis via binding to surface-expressed citHSP60 through Toll-like receptor 4 signaling. ACPAs promoted interleukin (IL)-6 and IL-8 expression in Saos-2 cells. Finally, sera from patients with RA and healthy controls were examined for their titers of anti-HSP60 and anti-citHSP60 antibodies using an enzyme-linked immunosorbent assay. The radiographic change in patients with RA was evaluated using the Genant-modified Sharp scoring system. Patients with RA showed higher sera titers of anti-citHSP60, but not anti-HSP60, antibodies when compared with controls. In addition, the anti-citHSP60 level was positively associated with increased joint damage in patients with RA. In conclusion, Saos-2 cell apoptosis was mediated by ACPAs via binding to cell surface-expressed citHSP60 and the titer of anti-citHSP60 in patients with RA positively associated with joint damage.
Division of Allergy, Immunology and Rheumatology, Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Chiayi, Taiwan; School of Medicine, Tzu Chi University, Hualien, Taiwan.
Ming-Chi
MC
Lu
Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan.
Chia-Li
CL
Yu
Division of Allergy, Immunology and Rheumatology, Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Chiayi, Taiwan.
Hui-Chun
HC
Yu
Department of Life Science and Institute of Molecular Biology, National Chung Cheng University, Chiayi, Taiwan.
Hsien-Bin
HB
Huang
Department of Medical Research, Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Chiayi, Taiwan; Dalla Lana School of Public Health, University of Toronto, Ontario, Canada.
Malcolm
M
Koo
Division of Allergy, Immunology and Rheumatology, Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Chiayi, Taiwan; School of Medicine, Tzu Chi University, Hualien, Taiwan. Electronic address: tzuchilai@gmail.com.
Ning-Sheng
NS
Lai
D000328:Adult; D000368:Aged; D017209:Apoptosis / Q000187:drug effects; D001172:Arthritis, Rheumatoid / Q000235:genetics / Q000276:immunology* / Q000473:pathology; D001323:Autoantibodies / Q000096:biosynthesis / Q000494:pharmacology*; D001324:Autoantigens / Q000235:genetics / Q000276:immunology; D016022:Case-Control Studies; D002460:Cell Line; D018834:Chaperonin 60 / Q000235:genetics / Q000276:immunology*; D002956:Citrulline / Q000276:immunology / Q000378:metabolism*; D003001:Cloning, Molecular; D005260:Female; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics / Q000276:immunology; D016209:Interleukin-8 / Q000235:genetics / Q000276:immunology; D007596:Joints / Q000187:drug effects / Q000276:immunology* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D024101:Mitochondrial Proteins / Q000235:genetics / Q000276:immunology*; D010006:Osteoblasts / Q000187:drug effects / Q000276:immunology / Q000473:pathology; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000276:immunology
D001323:Autoantibodies; D001324:Autoantigens; D018834:Chaperonin 60; C060293:HSPD1 protein, human; C508600:IL6 protein, human; D015850:Interleukin-6; D016209:Interleukin-8; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D002956:Citrulline
Anti-citrullinated protein antibodies; Apoptosis; Dheumatoid arthritis; Heat shock protein 60; Osteoblast