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  3. Quantitative analysis of hemin-induced neutrophil extracellular trap formation and effects of hydrogen peroxide on this phenomenon

Quantitative analysis of hemin-induced neutrophil extracellular trap formation and effects of hydrogen peroxide on this phenomenon

  • Biochem Biophys Rep. 2017 Jul 24;11:147-153. doi: 10.1016/j.bbrep.2017.07.009.
Ayako Ohbuchi 1,  Mari Kono 2,  Kaihei Kitagawa 1,  Mariko Takenokuchi 1,  Shion Imoto 3,  Katsuyasu Saigo 1
Affiliations

Affiliations

  • 1 Faculty of Pharmacological Sciences, Himeji Dokkyo University, 7-2-1 Kamiono, Himeji, Hyogo 670-8524, Japan.
  • 2 Scientific Research Division, Scientific Affairs, Sysmex Corporation, 1-3-2 Murotani, Nishi-ku, Kobe, Hyogo 651-2241, Japan.
  • 3 Department of Health Science, Kobe Tokiwa University, 2-6-2 Otani-cho, Nagata-ku, Kobe, Hyogo 653-0838, Japan.
PMID: 28955779 DOI: 10.1016/j.bbrep.2017.07.009
Abstract

Formation of neutrophil extracellular traps (NETs) can perpetuate sterile inflammation; thus, it is important to clarify their pathophysiological characteristics. Free heme, derived via hemolysis, is a major contributor to organ damage, and reportedly induces neutrophil activation as well as Reactive Oxygen Species (ROS) production and NET formation. For this study, we examined hemin (Fe3+ -protoporphyrin IX)-induced NET formation quantitatively in vitro as well as the effects of oxidative stress. NETs formed in vitro from cultured neutrophils were quantitatively detected by using nuclease treatment and Sytox Green, a nucleic acid stain. Hemin-induced NET production was found to be in a dose-dependent manner, NADPH oxidase-dependent and Toll-like Receptor (TLR)-4 independent. Additionally, the iron molecule in the porphyrin ring was considered essential for the formation of NETs. In the presence of low concentrations of hydrogen peroxide, low concentrations of hemin-induced NETs were enhanced, unlike those of phorbol myristate acetate (PMA)-induced NETs. Quantitative analysis of NET formation may prove to be a useful tool for investigating NET physiology, and hemin could function as a possible therapeutic target for hemolysis-related events.

Keywords

DPI, diphenyleneiodonium; ELISA, Enzyme-Linked Immuno-Sorbent Assay; Extracellular trap; HO-1, heme oxygenase-1; Hemin; Hydrogen peroxide; LPS, lipopolysaccharide; MPO, myeloperoxidase; NADPH oxidase, nicotinamide adenine dinucleotide phosphate oxidase; NET, neutrophil extracellular traps; Neutrophil; PAD4, peptidylarginine deiminases 4; PMA, phorbol myristate acetate; Quantitative detection; ROS, reactive oxygen species; TAK-242 (PubChem CID: 11703255); TLR, toll-like receptor; diphenylene iodonium (PubChem CID: 3101); hemin (PubChem CID: 121225420); hydrogen peroxide (PubChem CID: 784); phorbol myristate acetate (PubChem CID: 22833501); polymyxin B (PubChem CID: 4868); protoporphyrin IX (PubChem CID: 4971); sytox green (PubChem CID: 46863923).

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