Release mechanism of high mobility group nucleosome binding domain 1 from lipopolysaccharide-stimulated macrophages

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.