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Updated: Jun 6, 2025
Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood
Published on: September 12, 2016
Oxysterols Suppress Release of DNA from Granulocytes into Extracellular Space After Stimulation with Phorbol
Yuichi Watanabe1, Takashi Obama1, Tomohiko Makiyama1
1Division of Biological Chemistry, Department of Pharmaceutical Sciences, Showa University Graduate School of Pharmacy, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan.
Background:
Neutrophils eject their DNA strings and cellular proteins into the extracellular space upon treatment with various stimulants. In the present study, we examined the effects of four major oxidized cholesterol metabolites on DNA release from granulocytes.
Methods And Results:
When oxysterols were added to HL-60-derived granulocytes stimulated with phorbol 12-myristate 13-acetate (PMA), they suppressed the release of DNA and myeloperoxidase from the cells. Among the four oxysterols tested, 7-ketocholesterol was the most effective. Addition of the same concentration of 7-ketocholesterol did not induce any cytotoxic effects, as evaluated based on the release of lactate dehydrogenase and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) assays. DNA release from human peripheral blood neutrophils after PMA stimulation was also suppressed by 7-ketocholesterol. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis was used to quantify sterol content in the cells. The addition of oxysterols increased the cellular content of the corresponding compounds by more than 10-fold compared to those at baseline. Treatment of HL-60-derived granulocytes with methyl-β-cyclodextrin that removes sterol compounds from the membranes increased DNA release from the cells in a dose-dependent manner.
Conclusions:
These results suggest that oxysterols have suppressive effects on DNA release from granulocytes stimulated with PMA.
