Department of Public Health and Pediatric Sciences, University of Turin, Medical School, Turin, Italy.
PLoS One. 2013 May 14;8(5):e63045. doi: 10.1371/journal.pone.0063045. Print 2013.
IFI16, a nuclear pathogenic DNA sensor induced by several pro-inflammatory cytokines, is a multifaceted protein with various functions. It is also a target for autoantibodies as specific antibodies have been demonstrated in the sera of patients affected by systemic autoimmune diseases. Following transfection of virus-derived DNA, or treatment with UVB, IFI16 delocalizes from the nucleus to the cytoplasm and is then eventually released into the extracellular milieu. In this study, using an in-house capture enzyme-linked immunsorbent assay we demonstrate that significant levels of IFI16 protein can also exist as circulating form in the sera of autoimmune patients. We also show that the rIFI16 protein, when added in-vitro to endothelial cells, does not affect cell viability, but severely limits their tubulogenesis and transwell migration activities. These inhibitory effects are fully reversed in the presence of anti-IFI16 N-terminal antibodies, indicating that its extracellular activity resides within the N-terminus. It was further demonstrated that endogenous IFI16 released by apoptotic cells bind neighboring cells in a co-culture. Immunofluorescence assays revealed existence of high-affinity binding sites on the plasma membrane of endothelial cells. Free recombinant IFI16 binds these sites on HUVEC with dissociation constant of 2.7 nM, radioiodinated and unlabeled IFI16 compete for binding sites, with inhibition constant (Ki) of 14.43 nM and half maximal inhibitory concentration (IC50) of 67.88 nM; these data allow us to estimate the presence of 250,000 to 450,000 specific binding sites per cell. Corroborating the results from functional assays, this binding could be completely inhibited using anti-IFI16 N-terminal antibody, but not with an antibody raised against the IFI16 C-terminal. Altogether, these data demonstrate that IFI16 may exist as circulating protein in the sera of autoimmune patients which binds endothelial cells causing damage, suggesting a new pathogenic and alarmin function through which this protein triggers the development of autoimmunity.
IFI16 是一种核内致病性 DNA 传感器，可被多种促炎细胞因子诱导，是一种具有多种功能的多功能蛋白。它也是自身抗体的靶标，因为已经在受系统性自身免疫性疾病影响的患者的血清中证实了特异性抗体。在病毒衍生的 DNA 转染后或用 UVB 处理后，IFI16 从核内易位到细胞质中，然后最终释放到细胞外环境中。在这项研究中，我们使用内部捕获酶联免疫吸附测定法证明，自身免疫患者的血清中也存在作为循环形式的显著水平的 IFI16 蛋白。我们还表明，当 rIFI16 蛋白在体外添加到内皮细胞中时，不会影响细胞活力，但严重限制它们的管状形成和 Transwell 迁移活性。这些抑制作用在存在抗 IFI16 N 端抗体时完全逆转，表明其细胞外活性位于 N 端。进一步表明，凋亡细胞释放的内源性 IFI16 在共培养物中与相邻细胞结合。免疫荧光测定显示内皮细胞的质膜上存在高亲和力结合位点。游离重组 IFI16 与 HUVEC 上的这些结合位点以解离常数 2.7 nM 结合，放射性碘标记和未标记的 IFI16 竞争结合位点，抑制常数 (Ki) 为 14.43 nM，半最大抑制浓度 (IC50) 为 67.88 nM；这些数据使我们能够估计每个细胞存在 250,000 到 450,000 个特异性结合位点。与功能测定结果相吻合，这种结合可以完全被抗 IFI16 N 端抗体抑制，但不能被针对 IFI16 C 端的抗体抑制。总之，这些数据表明，IFI16 可能作为自身免疫患者血清中的循环蛋白存在，与内皮细胞结合导致损伤，提示该蛋白通过触发自身免疫的新的致病和警报功能。
