Modeling nuclear molecule release during in vitro cell death.
The extracellular release of nuclear molecules occurs prominently during cell death and can determine the immune properties of dead and dying cells. Depending on inciting events and environmental conditions, cells can die by apoptosis or necrosis, although these processes differ in their immunological consequences. Whereas apoptosis is immunologically silent, necrosis leads to inflammation, possibly reflecting the array of "danger" molecules released. To investigate these processes, the extracellular release of HMGB1 during necrosis was characterized in vitro using Jurkat T cell leukemia cells treated to induce necrosis by freeze-thaw, heat, hydrogen peroxide or ethanol. HMGB1 is a non-histone nuclear protein that represents a prototype alarmin; the pro-inflammatory activity of HMGB1, however, depends on past-translational modifications and association with other molecules including cytokines. As results of these studies showed, the extent and kinetics of HMGB1 release from necrotic cells varies markedly depending on its induction. Among treatments tested, freeze-thaw produced the highest levels of extracellular HMGB1; levels with ethanol treatment were very low and in the range of untreated controls. Similar results were obtained with DNA, with freeze-thaw leading to significant amounts of extracellular DNA although this DNA was subject to rapid nuclease digestion. Together, these findings suggest that the rapid release of nuclear molecules is not an invariable feature of necrosis and that the characterization of the immune properties of dead and dying cells should use systems in which the content and stability of extracellular components are well defined.
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