NF-kappaB and the MAP kinases/AP-1 pathways are both involved in interleukin-6 and interleukin-8 expression in fibroblast-like synoviocytes stimulated by protein I/II, a modulin from oral streptococci

Abstract

As in rheumatoid arthritis (RA), it was demonstrated recently that bacterial fragments of DNA or rRNA are present in the joint and therefore could play a role in inducing or perpetuating the disease, this work was initiated to define mechanisms that account for the stimulatory activities of the oral streptococcal modulin, protein I/II, on fibroblast‐like synoviocytes (FLSs) from RA patients. FLSs from RA patients were stimulated with protein I/II, and expression of interleukin (IL)‐6 and IL‐8 mRNA was evaluated by reverse transcription–polymerase chain reaction (RT–PCR). Immunoblotting by antibodies specific for activated forms of MAPKs and electrophoretic mobility shift assays (EMSAs) were performed to study downstream signalling, which allowed the synthesis of IL‐6 and IL‐8. We reported that protein I/II interactions with FLSs from RA patients trigger the synthesis and release of IL‐6 and IL‐8. We also demonstrated that protein I/II enhances the phosphorylation of ERK 1/2, p38 and JNKs and that ERK 1/2 and JNK MAPKs seem to play a more important role than p38 in protein I/II‐mediated synthesis of IL‐6 and IL‐8. Our experiments also indicated that stimulation of FLSs with protein I/II induces nuclear translocation of NF‐κB, AP‐1‐binding activity and that NF‐κB plays a major role in IL‐6 and IL‐8 secretion from activated cells.

BAFF synthesis by rheumatoid synoviocytes is positively controlled by α5β1 integrin stimulation and is negatively regulated by tumor necrosis factor α and toll‐like receptor ligands

“…The diagnosis of RA was conformed according to the revised criteria of the American College of Rheumatology (formerly, the American Rheumatism Association) (22). FLS cultures were performed as previously described (23). FLS between the third and the ninth passages were used in the experiments.…”

Cross Talk between MyD88 and Focal Adhesion Kinase Pathways

“…Human fibroblast-like synoviocytes (FLSs) were isolated from rheumatoid arthritis synovial tissues at the time of knee joint arthroscopic synovectomy and cultured as previously described (7). FLSs (5 ϫ 10 3 cells per well) were grown to confluence in 96-well plates and serum-starved for 24 h before activation experiments.…”

“…Recombinant protein I/II of S. mutans OMZ 175 was purified from pHBsr-1-transformed Escherichia coli cell extract by gel filtration and immunoaffinity chromatography as previously described (7). Potential endotoxin in protein I/II preparation was removed using polymyxin B-agarose (DetoxiGel), according to the manufacturer's recommendation (Pierce).…”

“…Protein I/II, a cell wall component from oral streptococci, binding to integrin ␣ 5 ␤ 1 induces the production of inflammatory mediators such as IL-6 and IL-8 by human monocytes, epithelial cells, endothelial cells, and fibroblasts (5,6). The signaling events leading to this cytokine release involve FAK, ERK1/2, and JNKs as well as AP-1-binding activity and nuclear translocation of NF-B (6,7). FAK is an interesting candidate to link bacterial detection, initiation of proinflammatory responses, and cell entry, because FAK is involved in invasin-mediated bacterial uptake (8).…”

Anti-Inflammatory Effects of Moxifloxacin on Activated Human Monocytic Cells: Inhibition of NF-κB and Mitogen-Activated Protein Kinase Activation and of Synthesis of Proinflammatory Cytokines

“…I-B kinases are phosphorylated by NF-B-inducing kinases, which activate various MAPKs, including the stress-activated protein kinase/c-Jun Nterminal kinase (JNK) (24). Recently, the IL-6 and IL-8 genes were identified as new targets regulated by JNK (23), while other studies have shown that ERK1/2 and JNK play an important role in protein I/II-mediated synthesis of IL-6 and IL-8 in fibroblast-like synoviocytes (29).…”