Tumour necrosis factor alpha promotes secretion of 14-3-3? by inducing necroptosis in macrophages

Background 14-3-3? is an intracellular protein also detected in the serum and synovial fluid of patients with rheumatoid arthritis (RA). It is closely related to disease activity and anti-cyclic citrullinated peptide antibody levels. However, the main source of 14-3-3? and the mechanism of its release into the extracellular space remain unclear. Addressing these two points was the main goal of the current study. Methods The source of 14-3-3? was investigated by immunostaining RA synovial tissue. Fibroblast-like synoviocytes, CD4.sup.+ cells, and macrophages were selected as candidates among the various cell types in the synovial tissue. Phosphorylation of mixed-lineage kinase domain-like pseudokinase (MLKL) and cell death of macrophages were studied by phalloidin staining and electron microscopy after stimulation with an oxidative stress inducer (diamide) or tumour necrosis factor (TNF)-[alpha]. Extracellular 14-3-3? protein levels were examined by western blotting. Results Macrophages from the synovial tissue from RA, but not osteoarthritis, showed dense and widespread cytoplasmic staining for the 14-3-3? protein, co-localized with peptidylarginine deiminase 4. Swelling and membrane rupture of macrophages were induced by treatment with TNF-[alpha], but not interleukin (IL) 6/soluble IL-6 receptor (sIL-6R). Increased MLKL phosphorylation followed by necroptosis was also induced in TNF-[alpha]-stimulated macrophages. Necrostatin-1, a necroptosis inhibitor, antagonized MLKL phosphorylation. High levels of 14-3-3? were detected in the culture supernatants of macrophages stimulated with diamide and TNF-[alpha], but not IL-6/sIL-6R. Conclusions Macrophages that highly express 14-3-3? undergo TNF-[alpha]-induced necroptosis with damage to the cellular structure, resulting in the secretion of 14-3-3? into the extracellular space. The current study provides a novel mechanism for 14-3-3? level increase in the RA synovial fluid. Keywords: Rheumatoid arthritis, 14-3-3?, TNF-[alpha], Necroptosis, Macrophage