BACKGROUND 14-3-3η 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 14-3-3η 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 14-3-3η was investigated by immunostaining RA synovial tissue. Fibroblast-like synoviocytes, CD4+ 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)-α. Extracellular 14-3-3η 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 14-3-3η protein, co-localized with peptidylarginine deiminase 4. Swelling and membrane rupture of macrophages were induced by treatment with TNF-α, but not interleukin (IL) 6/soluble IL-6 receptor (sIL-6R). Increased MLKL phosphorylation followed by necroptosis was also induced in TNF-α-stimulated macrophages. Necrostatin-1, a necroptosis inhibitor, antagonized MLKL phosphorylation. High levels of 14-3-3η were detected in the culture supernatants of macrophages stimulated with diamide and TNF-α, but not IL-6/sIL-6R. CONCLUSIONS Macrophages that highly express 14-3-3η undergo TNF-α-induced necroptosis with damage to the cellular structure, resulting in the secretion of 14-3-3η into the extracellular space. The current study provides a novel mechanism for 14-3-3η level increase in the RA synovial fluid.

中文翻译：

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肿瘤坏死因子α通过诱导巨噬细胞坏死性坏死而促进14-3-3η的分泌。

背景技术14-3-3η是在类风湿性关节炎（RA）患者的血清和滑液中也检测到的细胞内蛋白。它与疾病活性和抗环瓜氨酸肽抗体水平密切相关。然而，尚不清楚14-3-3η的主要来源及其释放到细胞外空间的机制。解决这两点是当前研究的主要目标。方法通过对RA滑膜组织进行免疫染色研究了14-3-3η的来源。在滑膜组织的各种细胞类型中，选择成纤维细胞样滑膜细胞，CD4 +细胞和巨噬细胞作为候选。在用氧化应激诱导剂（二酰胺）或肿瘤坏死因子（TNF）-α刺激后，通过鬼笔环肽染色和电子显微镜研究了混合谱系激酶域样假激酶（MLKL）的磷酸化和巨噬细胞的细胞死亡。通过蛋白质印迹检查细胞外14-3-3η蛋白水平。结果来自RA的滑膜组织的巨噬细胞，而非骨关节炎，显示了与肽酰精氨酸脱亚氨酶4共定位的浓密且广泛的14-3-3η蛋白胞浆染色。TNF-α治疗可引起巨噬细胞肿胀和膜破裂。 ，但不是白介素（IL）6 /可溶性IL-6受体（sIL-6R）。在TNF-α刺激的巨噬细胞中，还诱导了MLKL磷酸化增加，然后坏死性坏死。Necrostatin-1，一种坏死病抑制剂，拮抗MLKL磷酸化。在用二酰胺和TNF-α刺激的巨噬细胞的培养上清液中检测到高水平的14-3-3η，但未检测到IL-6 / sIL-6R。结论高表达14-3-3η的巨噬细胞经历TNF-α诱导的坏死性坏死，破坏细胞结构，导致14-3-3η分泌到细胞外空间。当前的研究提供了RA滑液中14-3-3η水平增加的新机制。