Eda‐containing fibronectin is synthesized from rheumatoid synovial fibroblast‐like cells

Abstract

Objective. To identify the cells that synthesize EDA-containing fibronectin (FN) and examine the role of EDA+ FN in the pathogenesis of rheumatoid joint lesions.Methods. Localization of EDA+ FN and c-Fos protein in rheumatoid joints was studied immunohistochemically by utilizing antibodies for EDA+ FN and c-Fos. Expression of EDA+ FN was studied by immunoelectron microscopy and in situ hybridization. The amount of EDA+ FN was measured by enzymelinked immunosorbent assay.Results. EDA+ FN was specifically localized in the synovial lining layer of synovium with active rheumatoid arthritis (RA) (n = 17), but not in that with osteoarthritis (n = 4) or with inactive fibrous RA (n = 2).EDA+ FN messenger RNA was localized in the synovial lining layer. EDA+ FN was immunoelectron microscopically localized in the synovial lining fibroblast-like (type B) cells. EDA+ FN was also detected at the cartilagepannus junction and on the surface of RA cartilage. Double staining showed that EDA+ FN colocalized with c-Fos protein in the rheumatoid synovial lining layer. Quantification of EDA+ FN showed that it was highly concentrated in rheumatoid synovial fluids.Conclusion. EDA+ FN is synthesized by the synovial lining fibroblast-like (type B) cells in situ in rheumatoid synovium, and appears to be expressed in association with activated or transformed states of synovium.Fibronectin (FN), synthesized in rheumatoid synovium (1) and specifically located in rheumatoid pannus and on cartilage surface (2,3), may be important in rheumatoid joint destruction (4). FN is composed of more than 2 subunits of protein with molecular weights of 220-250 kd (5). Multiple isoforms of FN are produced from a single gene by alternative splicing at 3 distinct sites: EDA, EDB, and IIICS (5). Recent studies indicate that isoforms containing the EDA or EDB regions are expressed in early stages of fetal development, malignant transformation, and wound healing ( 5 ) , which suggests that EDAcontaining or EDB-containing FN may be expressed in association with cellular proliferation and transformation. The EDA-or EDB-containing FN is found in the synovial lining cells and small blood vessels of rheumatoid joints (6,7), and plasma levels of EDA+ FN in rheumatoid arthritis (RA) patients are higher than those in healthy individuals (8).We used immunohistochemistry techniques to show that EDA+ FN, which is specifically localized in the rheumatoid synovial lining layer, is synthesized by the synovial lining fibroblast-like (type B) cells. EDA+ FN is also detected in invasive fronts of active cellular pannus and on rheumatoid cartilage surfaces that are free of pannus. EDA+ FN is also colocalized with c-Fos protein in the rheumatoid synovial lining layer.Kazuo Hino, BS, Shunichi Shiozawa, MD, Yasuo Kuroki, MD, Hitoshi Ishikawa, MD, Kazuo Chihara, MD: Kobe University, Kobe, Japan; Kazuko Shiozawa, MD: Kakogawa National Hospital, Kakogawa, Japan; Kiyotoshi Sekiguchi, PhD: Osaka Medical Center for Maternal and Child Health, Osaka, Japan; Hisanobu Hirano, MS, Eiji Sakash...

Increased macrophage activation mediated through toll‐like receptors in rheumatoid arthritis

“…We suggest that once inflammation within the joint causes joint destruction, a variety of molecules are released, such as ED-A of fibronectin, HSPs, and HMGB-1. Each of these molecules is highly expressed in the rheumatoid joint, and each is capable of activating TLR-2 and/or TLR-4 (22)(23)(24)(25)(26)(27)(28)(29). Therefore, once joint damage occurs, a selfperpetuating process, mediated by TLR ligation by endogenous ligands, may be established that promotes the chronic, progressive destruction mediated by the continued activation of macrophages.…”

“…Bacterial PG was identified in RA synovial tissue by a monoclonal antibody, particularly in macrophages and antigen-presenting cells (20,21). Additionally, endogenous mammalian TLR agonists, including fibrinogen, extra domain A (ED-A) of fibronectin, Hsp60 and Hsp70, low molecular weight fragments of hyaluronic acid, and high mobility group box chromosomal protein 1 (HMGB-1), a highly conserved nuclear protein that stabilizes nucleosome formation, are expressed in the RA joint, and each has been shown to activate NF-B through TLR-4 and/or TLR-2 (22)(23)(24)(25)(26)(27)(28)(29).…”

Human wee1 kinase is directly transactivated by and increased in association with c-Fos/AP-1: rheumatoid synovial cells overexpressing these genes go into aberrant mitosis

“…Synovial cells were dispersed as described (Hino et al, 1995), placed in a 10 cm dish (Asahi Techno Glass Corp, Tokyo, Japan), and cultured for 3 h in DMEM (Nissui Pharmaceutical Co., Ltd, Tokyo, Japan) containing 10% FCS (Biosciences Pty Ltd, Lome, Australia) supplemented with 50 U/ml penicillin and 50 mg/ml streptomycin (complete DMEM). Nonadherent cells were discarded, and the remaining adherent synovial cells were cultured for 3 days.…”

Identification of the Peptide Sequences within the EIIIA (EDA) Segment of Fibronectin That Mediate Integrin α9β1-dependent Cellular Activities

“…EIIIA is prominently expressed during disease states, such as rheumatoid arthritis (18), wound healing (19 -21), vascular intimal proliferation (22,23), cardiac transplantation (24), and in invasive tumors (25). The temporal expression of the EIIIA and EIIIB segments differ, suggesting an active role for these extra domains (21,24).…”