Murakami Taisuke, Hu Zhongshuang, Tamura Hiroshi, Nagaoka Isao
Department of Host Defense and Biochemical Research, Juntendo University, Graduate School of Medicine, Tokyo 113‑8421, Japan.
Laboratory Program Support Consulting Office, Tokyo 160‑0023, Japan.
Mol Med Rep. 2016 Apr;13(4):3115-20. doi: 10.3892/mmr.2016.4893. Epub 2016 Feb 16.
Alarmins are identified as endogenous mediators that have potent immune-activating abilities. High mobility group nucleosome binding domain 1 (HMGN1), a highly conserved, non-histone chromosomal protein, which binds to the inner side of the nucleosomal DNA, regulates chromatin dynamics and transcription in cells. Furthermore, HMGN1 acts as a cytokine in the extracellular milieu by inducing the recruitment and maturation of antigen-presenting cells (dendritic cells) to enhance Th1-type antigen-specific immune responses. Thus, HMGN1 is expected to act as an alarmin, when released into the extracellular milieu. The present study investigated the release mechanism of HMGN1 from macrophages using mouse macrophage‑like RAW264.7 cells. The results indicated that HMGN1 was released from lipopolysaccharide (LPS)‑stimulated RAW264.7 cells, accompanied by cell death as assessed by the release of lactate dehydrogenase (LDH). Subsequently, the patterns of cell death involved in HMGN1 release from LPS‑stimulated RAW264.7 cells were determined using a caspase‑1 inhibitor, YVAD, and a necroptosis inhibitor, Nec‑1. YVAD and Nec‑1 did not alter LPS‑induced HMGN1 and LDH release, suggesting that pyroptosis (caspase‑1‑activated cell death) and necroptosis are not involved in the release of HMGN1 from LPS‑stimulated RAW264.7 cells. In addition, flow cytometric analysis indicated that LPS stimulation did not induce apoptosis but substantially augmented necrosis, as evidenced by staining with annexin V/propidium iodide. Together these findings suggest that HMGN1 is extracellularly released from LPS‑stimulated RAW264.7 macrophage‑like cells, accompanied by unprogrammed necrotic cell death but not pyroptosis, necroptosis or apoptosis.
警报素被鉴定为具有强大免疫激活能力的内源性介质。高迁移率族核小体结合域1（HMGN1）是一种高度保守的非组蛋白染色体蛋白，它与核小体DNA的内侧结合，调节细胞中的染色质动力学和转录。此外，HMGN1在细胞外环境中作为一种细胞因子，通过诱导抗原呈递细胞（树突状细胞）的募集和成熟来增强Th1型抗原特异性免疫反应。因此，当HMGN1释放到细胞外环境中时，有望作为一种警报素发挥作用。本研究使用小鼠巨噬细胞样RAW264.7细胞研究了HMGN1从巨噬细胞中的释放机制。结果表明，HMGN1从脂多糖（LPS）刺激的RAW264.7细胞中释放，伴随着乳酸脱氢酶（LDH）的释放所评估的细胞死亡。随后，使用半胱天冬酶-1抑制剂YVAD和坏死性凋亡抑制剂Nec-1确定了LPS刺激的RAW264.7细胞中HMGN1释放所涉及的细胞死亡模式。YVAD和Nec-1并没有改变LPS诱导的HMGN1和LDH释放，这表明细胞焦亡（半胱天冬酶-1激活的细胞死亡）和坏死性凋亡不参与LPS刺激的RAW264.7细胞中HMGN1的释放。此外，流式细胞术分析表明，LPS刺激并未诱导细胞凋亡，但显著增加了坏死，这通过膜联蛋白V/碘化丙啶染色得以证明。这些发现共同表明，HMGN1从LPS刺激的RAW264.7巨噬细胞样细胞中细胞外释放，伴随着非程序性坏死性细胞死亡，而不是细胞焦亡、坏死性凋亡或凋亡。
