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Characterization of a Synovial B Cell–Derived Recombinant Monoclonal Antibody Targeting Stromal Calreticulin in the Rheumatoid Joints

Rheumatoid arthritis (RA) is characterized by formation of synovial ectopic lymphoid structures (ELS) supporting B cell autoreactivity toward locally generated citrullinated (cit) antigens, including those contained in neutrophil extracellular traps (NETs). However, only a minority of RA-rmAbs from...

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Detalles Bibliográficos
Autores principales: Corsiero, Elisa, Jagemann, Lucas, Perretti, Mauro, Pitzalis, Costantino, Bombardieri, Michele
Formato: Online Artículo Texto
Lenguaje:English
Publicado: AAI 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6099528/
https://www.ncbi.nlm.nih.gov/pubmed/30045972
http://dx.doi.org/10.4049/jimmunol.1800346
Descripción
Sumario:Rheumatoid arthritis (RA) is characterized by formation of synovial ectopic lymphoid structures (ELS) supporting B cell autoreactivity toward locally generated citrullinated (cit) antigens, including those contained in neutrophil extracellular traps (NETs). However, only a minority of RA-rmAbs from B cells isolated from ELS(+) RA tissues react against NETs. Thus, alternative cellular sources of other potential autoantigens targeted by locally differentiated B cells remain undefined. RA fibroblast–like synoviocytes (FLS) have been implicated in the release of RA-associated autoantigens. In this study, we aimed to define stromal-derived autoantigens from RA-FLS targeted by RA-rmAbs. Seventy-one RA-rmAbs were screened toward RA-FLS by living-cell immunofluorescence (IF). Western blotting was used to identify potential autoantigens from RA-FLS protein extracts. Putative candidates were validated using colocalization immunofluorescence confocal microscopy, ELISA, immunoprecipitation assay, and surface plasmon resonance on unmodified/cit proteins. Serum immunoreactivity was tested in anti-citrullinated peptide/protein Abs (ACPA)(+) versus ACPA(−) RA patients. Ten out of 71 RA-rmAbs showed clear reactivity toward RA-FLS in immunofluorescence with no binding to NETs. One stromal-reactive RA-rmAb (RA057/11.89.1) decorated a ∼58-kDa band that mass spectrometry and Western blotting with a commercial Ab identified as calreticulin (CRT). Confocal microscopy demonstrated significant cellular colocalization between anti-CRT RA057/11.89.1 in RA-FLS. RA057/11.89.1 was able to immunoprecipitate rCRT. Deimination of CRT to cit-CRT moderately increased RA057/11.89.1 immunoreactivity. cit-CRT displayed increased blocking capacity compared with unmodified CRT in competitive binding assays. Finally, anti–cit-CRT Abs were preferentially detected in ACPA(+) versus ACPA(−) RA sera. We identified a synovial B cell–derived RA-rmAb locally differentiated within the ELS(+) RA synovium reacting toward CRT, a putative novel autoantigen recently described in RA patients, suggesting that FLS-derived CRT may contribute to fuel the local autoimmune response.