Specifically modified osteopontin in rheumatoid arthritis fibroblast-like synoviocytes supports interaction with B cells and enhances production of interleukin-6.
OBJECTIVE
Osteopontin (OPN) is expressed by fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA), but its pathologic role is still obscure. The present study was undertaken to analyze the role of OPN in RA by focusing on its effects on cell-cell interactions between FLS and B lymphocytes.


METHODS
FLS obtained from 10 patients with RA and 10 non-RA subjects and a B lymphocyte cell line were studied. The characteristics of OPN expression by FLS were analyzed by Western blotting, immunoprecipitation, and immunofluorescence studies. In cocultures of FLS and B lymphocytes, the effects of OPN on adhesion of B lymphocytes to FLS and the consequent production of interleukin-6 (IL-6) were analyzed in experiments involving overexpression and knockdown of OPN and inhibitory studies with an OPN-blocking antibody. In vivo, the expression of OPN in RA synovium was examined by immunohistochemistry.


RESULTS
A specifically modified 75-kd form of OPN was predominantly expressed in RA FLS, and this was associated with expression of >200-kd thrombin-cleaved OPN that was crosslinked with fibronectin and localized on the surface of the FLS. In FLS-B lymphocyte cocultures, 75-kd OPN-positive FLS produced a significantly higher amount of IL-6 than did 75-kd OPN-negative FLS. When the FLS were separated from B lymphocytes or cultured alone, the production of IL-6 was low and was not significantly different between these 2 culture conditions. Moreover, OPN overexpression enhanced production of IL-6 in 75-kd OPN-positive FLS-B lymphocyte cocultures. Addition of the OPN-blocking antibody inhibited the adhesion of B lymphocytes to FLS. Immunohistochemical analyses revealed that localization of IL-6-positive cells coincided with the sites at which OPN and B lymphocytes were colocalized.


CONCLUSIONS
Specifically modified 75-kd OPN was expressed by RA FLS. This form of OPN affected FLS-B lymphocyte interactions by supporting the adhesion of B lymphocytes to FLS and enhancing the production of IL-6.
Take
Yasuhiro
Y
Department of Orthopaedics, Osaka University Graduate School of Medicine, and Osaka Rosai Hospital, Osaka, Japan.
Nakata
Ken
K
Hashimoto
Jun
J
Tsuboi
Hideki
H
Nishimoto
Norihiro
N
Ochi
Takahiro
T
Yoshikawa
Hideki
H
D019138:Antibodies, Blocking / Q000494:pharmacology; D001172:Arthritis, Rheumatoid / Q000378:metabolism* / Q000473:pathology; D001402:B-Lymphocytes / Q000187:drug effects / Q000502:physiology; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D018920:Coculture Techniques; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D020868:Gene Silencing; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D020370:Osteoarthritis, Knee / Q000378:metabolism / Q000473:pathology; D053495:Osteopontin / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D034741:RNA, Small Interfering / Q000235:genetics; D013583:Synovial Membrane / Q000378:metabolism* / Q000473:pathology; D014162:Transfection
D019138:Antibodies, Blocking; C508600:IL6 protein, human; D015850:Interleukin-6; D010455:Peptides; D034741:RNA, Small Interfering; D053495:Osteopontin