Synovial fluid induced nuclear factor-kappaB DNA binding in a monocytic cell line

To determine the effects of synovial fluids (SF) on DNA binding activity of transcription factor nuclear factor-kappaB (NF-kappaB) in the Mono Mac 6 monocytic/macrophage cell line as a model for the interaction between SF and synovial tissue macrophages in arthritis.

Mono Mac 6 cells were incubated with SF from the knee joints of human subjects with rheumatoid arthritis (RA), undifferentiated seronegative oligoarthritis, and osteoarthritis (OA). Nuclear extracts prepared from the Mono Mac 6 cells and RA synovial tissue were analyzed by electrophoretic mobility shift analysis (EMSA) for NF-kappaB DNA binding proteins.

Induction of NF-kappaB DNA binding by the p65(RelA)/p50 heterodimer was observed in response to incubation of Mono Mac 6 cells with SF (20% in culture medium) from 5 of 8 subjects with RA, 4 of 5 with OA, and none of 3 with undifferentiated seronegative oligoarthritis. Incubation of SF with neutralizing antibodies against tumor necrosis factor-alpha (TNF-alpha), but not antibodies against interleukin 6 (IL-6), significantly reduced the induction of p65/p50 binding activity in SF from subjects with RA and OA. Unexpectedly, a slowly migrating SF inducible NF-kappaB-binding complex was observed in EMSA of Mono Mac 6 cells after incubation with SF from 5 of 8 RA and 2 of 5 OA subjects. The induction of this complex by SF was not affected by neutralization of TNF-alpha or IL-6 in SF, and the complex was not inducible by TNF-alpha, IL-1beta, TNF-alpha/IL-1beta, IL-6, platelet derived growth factor, lipopolysaccharide, or tetradecanoyl phorbol acetate. The slowly migrating complex could not be supershifted with antibodies against NF-kappaB, Jun, or the transcriptional coactivators p300 or CBP. A NF-kappaB-binding complex with similar slow mobility was observed in nuclear extracts prepared from fresh human RA synovial tissue.

Biological activity of TNF-alpha in SF from RA and OA subjects is capable of inducing p65/p50 NF-kappaB DNA binding activity in macrophages. A property of SF that is independent of TNF-alpha and other cytokines is responsible for the induction of a novel slowly migrating NF-kappaB-binding complex. Soluble mediators in SF of subjects with RA and OA can therefore modulate binding of nuclear proteins to the NF-kappaB binding site in macrophages and may play a role in inflammatory gene expression in arthritis.