Amplification of the synovial inflammatory response through activation of mitogen‐activated protein kinases and nuclear factor κB using ligation of CD40 on CD14+ synovial cells from patients with rheumatoid arthritis

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Abstract
Objective To determine the signal transduction pathways in CD14+ synovial cells from patients with rheumatoid arthritis (RA) after CD40 ligation, and to examine their role in amplifying synovial inflammation in affected joints. Methods Expression of messenger RNA was analyzed using quantitative reverse transcription–polymerase chain reaction. Cytokines and chemokines were measured using enzyme‐linked immunosorbent assay. Activation of kinases was detected using Western blotting. Nuclear translocation of NF‐κB was examined using immunohistochemistry. CD14+ synovial cells were enriched using magnetic cell sorting. Fibroblast‐like synoviocytes (FLS) were obtained by passaging primary synovial cell culture. Results Stimulation of CD14+ synovial cells from RA patients by recombinant soluble CD154 (rsCD154) significantly induced expression of tumor necrosis factor α (TNFα), interleukin‐1α (IL‐1α), and IL‐1β. CD14+ RA synovial cells stimulated with rsCD154 plus interferon‐γ (IFNγ) induced significantly higher production of IL‐6, IL‐8, and monocyte chemoattractant protein 1 by FLS compared with unstimulated CD14+ synovial cells, through TNFα‐, IL‐1α−, and IL‐1β–mediated pathways. Stimulation with rsCD154 plus IFNγ induced the activation of ERK‐1/2, p38 MAPK, and NF‐κB. Specific inhibitors of MAPK/ERK‐1/2 kinases and p38 MAPK significantly reduced the production of TNFα and IL‐1β by rsCD154 plus IFNγ–stimulated CD14+ synovial cells, and also inhibited production of these cytokines by freshly isolated synovial cells from RA patients. Conclusion These data indicate that the CD40–CD154 interaction activates the ERK, p38, and NF‐κB pathways in CD14+ synovial cells from RA patients to produce TNFα, IL‐1α, and IL‐1β, which in turn amplifies inflammatory responses by stimulating FLS. Inhibition of the CD40–CD154 interaction or its signal transduction pathways would be a strong and efficient strategy for the management of synovial inflammation in RA.