Preparation of histones and their effects on macrophages

AIM: To prepare histones with different methods and to observe their effects on cell viability and cytokine release by macrophages in vitro.
METHODS: Prokaryotic expression plasmids of histone H3 and histone H4 were constructed by cloning methods. The histones containing GST-tag or His-tag (GST-H3, GST-H3, His-H3, His-H4) were expressed and purified. The histones from eukaryotic cells (RAW264.7 and 293F cells) were extracted with the high salt method. RAW264.7 cells were treated with histones (7.5~50 mg/L) for 4 h and the cell vitality was examined by MTT assay and flow cytometry. The concentrations of IL-6 and TNF-α in the culture supernatants of RAW264.7 cells treated with histones were also detected.
RESULTS: His-H3/H4 and the histones from eukaryotic cells significantly reduced the viability of RAW264.7 cells and induced apoptosis. The effects of histones from different sources on the releases of IL-6 and TNF-α were different.
CONCLUSION: His-H3/His-H4 expressed by prokaryotic plasmids and the histones extracted from eukaryotic cells affect the vitality of macrophages as well as induce late apoptosis and necrosis. Histone may involve in the inflammatory process by promoting the release of inflammatory cytokines.