Tunicamycin‐induced neutrophil extracellular trap (NET)‐like structures in cultured human myeloid cell lines

Abstract

The mechanism of neutrophil extracellular trap cell death (NETosis), a regulated cell death pathway relevant to infection, autoimmunity and sepsis, is not completely known. The reason for this, at least in part, is the lack of an in vitro system that recapitulates the NETosis pathway using established human cell lines. We show that exposure of a human promyelocytic leukemia cell line HL-60 to the glycosyltransferase inhibitor tunicamycin (TM) resulted in extrusion of decompacted genomic DNAs to extracellular space, morphologically similar to NETs. Immunostaining using antibodies against NET marker proteins and bacterial trapping assay showed biochemical similarities between the TM-induced extracellular DNA structures and NETs. The NET-like structures were also generated on exposure of TM to other myeloid cell lines, such as U937 and THP-1. Thus, our findings provide an experimental setting to induce NET-like structures using cultured human myeloid cell lines, which may help our understanding of the regulation and function of NETosis.

Nuclear extrusion precedes discharge of genomic DNA fibers during tunicamycin‐induced neutrophil extracellular trap‐osis (NETosis)‐like cell death in cultured human leukemia cells

“…As previously reported (Nakayama and Saitoh, ), images of HL‐60 cells exposed to TN for 15 min showed unaltered cellular morphology, whereas cells at 6 h of incubation showed largely extended extracellular DNA fibers seemingly similar to NETs (Supplementary Figure S1A). Using time‐lapse microscopic techniques, we investigated alterations of cellular morphology between 15 min and 6 h of TN exposure.…”

“…Control cells treated with DMSO underwent no such changes during the 6‐h observation period (Supplementary Figure S2). Thus, these findings suggested that TN‐induced extracellular DNA formation occurred following marked morphological alterations, including appearance of ovoid‐shaped dark objects and relocation of the objects to extracellular space, which were somehow overlooked in our previous study (Nakayama and Saitoh, ).…”

The influence of agents differentiating HL‐60 cells toward granulocyte‐like cells on their ability to release neutrophil extracellular traps

“…An increasing number of studies have pointed to neutrophils releasing NETs as contributors to many diseases, therefore current studies on NETs formation constitute a substantial part of investigations directed toward neutrophil biology . Unfortunately, much progress in this field has been constrained due to the lack of available models for genetic studies, which are usually necessary to fully understand mechanisms at the molecular level . We propose that HL‐60 cells may be a model for genetic modifications and further differentiation into granulocyte‐like cells.…”

DNase γ, DNase I and caspase‐activated DNase cooperate to degrade dead cells

“…). The extracellular extrusion of decompacted DNA fibers, which are susceptible to DNase I treatment, is a hallmark of NET observed in several myeloid lineages, including neutrophils, macrophages, mast cells and eosinophils (Nakayama & Saitoh ). Although it is known that the inhibition of DNase I activity is essential for the generation of NET, there are no reports regarding other DNases.…”