Research Update: Osteoimmunology

Linking autoantibody production to bone loss

in rheumatoid arthritis

Autoantibodies against citrullinated proteins (ACPA) are found in people with rheumatoid arthritis and are one of the strongest risk factors for bone destruction in this disease. A recent study now directly links the formation of antibodies binding to mutated citrullinated vimentin (anti-MCV) to bone loss in rheumatoid arthritis, indicating that these autoantibodies act on osteoclasts, the bone cells responsible for bone resorption.

Harre U, Georgess D, Bang H, Bozec A, Axmann R, Ossipova E et al. Induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin. J Clin Invest 2012; 122(5):1791-802. (1)

The research of U. Harre, G. Schett and their coworkers provides fundamental new insights into the interaction between bone and the immune system in the inflammatory process leading to the development of rheumatoid arthritis.

There are two major aspects that determine the clinical picture of rheumatic diseases: First, chronic immune activation, production of autoantibodies and accumulation of cells of the adaptive immune system at the sites of inflammation, and second, progressive damage of musculoskeletal tissue, which leads to functional impairment, pain and disability. (2) Thus, deeper insight into the mutual interactions between the immune system and the bone will provide the basis for understanding the pathophysiological processes leading to the development of rheumatic diseases.

Presence of autoantibodies against citrullinated proteins (ACPA) is a strong predictor for bone erosive RA, suggesting a direct association of the autoantibody response and structural bone damage. In particular, antibodies against mutated citrullinated vimentin (anti-MCV) are highly specific for RA and the protein antigen mutated citrullinated vimentin is expressed in mononuclear cells and osteoclasts. (3-6)

ACPA are associated with increased bone resorption

First, U. Harre et al analyzed ACPA-positive serum samples from RA patients for biomarkers of bone resorption and bone formation and compared these results to samples of ACPA-negative RA patients and healthy controls. They observed that presence and levels of ACPA correlated with increased amounts of different serum markers for bone resorption in patients with rheumatoid arthritis. In contrast, markers for bone formation showed no difference between RA patients with or without ACPA. These data document the direct link from ACPA presence to bone destruction and support the hypothesis that ACPA can provoke bone loss by interfering with the bone metabolism.

Mutated citrullinated vimentin is involved in RA pathogenesis

Autoantibodies to mutated citrullinated vimentin are established diagnostic markers for RA (3;7;8) and the production of these antibodies is highly specific for the disease. Moreover, it has been demonstrated, that anti-MCV levels are elevated in synovial fluid of RA patients suggesting that there is local antibody production or retention of these autoantibodies at the site of inflammation governed by RA-predisposing genes. (9)

In recent years, several studies have demonstrated the role of vimentin as a key autoantigen in RA pathogenesis:
Feitsma et al. (10) identified two naturally processed peptides from vimentin that bind to HLA-DRB1*0401-restricted T cells in a citrulline-specific manner. These peptides were recognized by T cells in ACPA-positive RA patients with HLA-DR4 genotype. Similarly, Snir et al. (11) found T cells that were reactive to citrullinated vimentin in HLA-DRB1*0401-positive rheumatoid arthritis patients and healthy controls. A proinflammatory response was detectable in patients with RA exclusivly.
Tabushi et al. (12) performed a proteomics-based analysis of synovial fluids from RA patients and detected three citrullinated proteins – citrullinated fibrinogen, citrullinated fibronectin and citrullinated vimentin. Likewise, with Western blots, 2D gel images of cytosolic synovial tissue extracts and by mass spectrometry, Tilleman et al. (13) identified citrullinated isoforms of vimentin in patients with inflammatory arthritides. They found presence of autoantibodies against these citrullinated processed forms of vimentin to be predominantly associated with RA patients, suggesting a possible origin of the ACPA immune response in RA.
Van Steendam et al. (14) analysed the antigens in immune complexes from sera of healthy persons, RA patients and from synovial fluid of RA patients and patients with spondyloarthropathy (SpA). They detected citrullinated fibrinogen-beta and citrullinated vimentin only in synovial fluids of ACPA-positive RA patients. No citrullinated antigens were present in immuncomplexes from synovial fluid of ACPA-negative RA patients or SpA patients, or in the immuncomplexes from serum of RA patients and healthy volunteers. These findings all strengthen the hypothesis that citrullinated vimentin plays an important role in the pathogenesis of RA.

Anti-MCV antibodies directly stimulate the differentiation of bone-resorbing cells in vitro

To further elucidate how autoantibodies with specificity to mutated citrullinated vimentin may contribute to bone destruction, Schetts team together with scientists from ORGENTEC developed an efficient method for the purification of human ACPA, and characterized their frequency and fine-specificity pattern. The researchers isolated anti-MCV antibodies directly from the blood of RA patients. These purified natural human antibodies showed specificity to peptides from citrullinated vimentin, but not to non-citrullinated vimentin peptides and they did not recognize citrullinated peptides from other proteins. In addition, the purified human anti-MCV antibodies bound to the surface and the cytoplasm of cultivated osteoclasts, indicating the expression of citrullinated vimentin in these cells.
In osteoclast differentiation assays addition of human anti-MCV increased the formation of osteoclast progenitor cells and induced their differentiation into mature bone resorbing cells. When assessing bone resorption in a cell culture assay, the researchers observed a dose dependent relationship of antibody concentration and increased osteoclast-mediated bone resorption.

Anti-MCV antibodies stimulate TNF-alpha production and induce bone loss in vivo

In lymphocyte-deficient mice, injection of anti-MCV antibodies induced bone loss. This seemed to be mediated by a higher number of osteoclast precursors and upregulated osteoclast differentiation in response to anti-MCV binding. Mice exposed to anti-MCV-antibodies showed increased systemic levels of TNF-alpha, a cytokine that plays a central role in RA-pathogenesis. Interaction with anti-MCV antibodies induced production of TNF-alpha by osteoclast precursors. In turn, TNF-alpha promotes the migration of osteoclast progenitors from bone marrow to the lymphoid organs and stimulates their differentiation into mature osteoclasts. Thus, anti-MCV presence affects TNF-alpha mediated osteoclastogenesis and bone resorption.

In summary, these data demonstrate the direct relationship between autoantibody formation and bone destruction in rheumatoid arthritis and extend current knowledge on the interplay of the immune system and the skeleton in autoimmunity.

References:

(1) Harre U, Georgess D, Bang H, Bozec A, Axmann R, Ossipova E et al. Induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin. J Clin Invest 2012; 122(5):1791-802. (Full text article)

(2) Schett G. Osteoimmunology in rheumatic diseases. Arthritis Res Ther 2009; 11(1):210. (Full text article)

(3) Bang H, Egerer K, Gauliard A, Luthke K, Rudolph PE, Fredenhagen G et al. Mutation and citrullination modifies vimentin to a novel autoantigen for rheumatoid arthritis. Arthritis Rheum 2007; 56(8):2503-11. (Full text article)

(4) Raza K, Mathsson L, Buckley CD, Filer A, Ronnelid J. Anti-modified citrullinated vimentin (MCV) antibodies in patients with very early synovitis. Ann Rheum Dis 2009; 69(3):627-8. (Abstract)

(5) Syversen SW, Goll GL, van der Heijde D, Landewe R, Lie BA, Odegard S et al. Prediction of radiographic progression in rheumatoid arthritis and the role of antibodies against mutated citrullinated vimentin: results from a ten-year prospective study. Ann Rheum Dis 2009; 69(2):345-51. (Abstract)

(6) van der Linden MP, van der Woude D, Ioan-Facsinay A, Levarht EW, Stoeken-Rijsbergen G, Huizinga TW et al. Value of anti-modified citrullinated vimentin and third-generation anti-cyclic citrullinated peptide compared with second-generation anti-cyclic citrullinated peptide and rheumatoid factor in predicting disease outcome in undifferentiated arthritis and rheumatoid arthritis. Arthritis Rheum 2009; 60(8):2232-41. (Full text article)

(7) Tesija KA. Mutated citrullinated vimentin antibodies in rheumatoid arthritis. Clin Chim Acta 2012; 413(1-2):66-73. (Abstract)

(8) Van Steendam K., Tilleman K, Deforce D. The relevance of citrullinated vimentin in the production of antibodies against citrullinated proteins and the pathogenesis of rheumatoid arthritis. Rheumatology (Oxford) 2011. (Full text article)

(9) Snir O, Widhe M, Hermansson M, von SC, Lindberg J, Hensen S et al. Antibodies to several citrullinated antigens are enriched in the joints of rheumatoid arthritis patients. Arthritis Rheum 2009; 62(1):44-52. (Full text article)

(10) Feitsma AL, van der Voort EI, Franken KL, El BH, Elferink BG, Drijfhout JW et al. Identification of citrullinated vimentin peptides as T cell epitopes in HLA-DR4-positive patients with rheumatoid arthritis. Arthritis Rheum 2009; 62(1):117-25. (Full text article)

(11) Snir O, Rieck M, Gebe JA, Yue BB, Rawlings CA, Nepom G et al. Identification and functional characterization of T cells reactive to citrullinated vimentin in HLA-DRB1*0401-positive humanized mice and rheumatoid arthritis patients. Arthritis Rheum 2011; 63(10):2873-83. (Abstract)