High mobility group box 1 protein (HMGB1) is a non-histone nuclear protein with dual function. Inside the cell, HMGB1 binds DNA and regulates transcription, whereas outside the cell, it serves as a cytokine and mediates the late effects of LPS. The movement of HMGB1 into the extracellular space has been demonstrated for macrophages stimulated with LPS as well as cells undergoing necrosis but not apoptosis. The differential release of HMGB1 during death processes could reflect the structure of chromatin in these settings as well as the mechanisms for HMGB1 translocation. Since apoptotic cells can release some nuclear molecules such as DNA to which HMGB1 can bind, we therefore investigated whether HMGB1 release can occur during apoptosis as well as necrosis. For this purpose, Jurkat cells were treated with chemical inducers of apoptosis (staurosporine, etoposide, or camptothecin), and HMGB1 release into the medium was assessed by Western blotting. Results of these experiments indicate that HMGB1 appears in the media of apoptotic Jurkat cells in a time-dependent manner and that this release can be reduced by Z-VAD-fmk. Panc-1 and U937 cells treated with these agents showed similar release. In addition, HeLa cells induced to undergo apoptosis showed HMGB1 release. Furthermore, we showed using confocal microscopy that HMGB1 and DNA change their nuclear location in Jurkat cells undergoing apoptosis. Together, these studies indicate that HMGB1 release can occur during the course of apoptosis as well as necrosis and suggest that the release process may vary with cell type.

Rheumatoid arthritis is a chronic inflammatory disease characterized by destruction of cartilage and bone that is mediated by synovial fibroblasts. To determine the mechanisms by which these cells are activated to produce matrix metalloproteinases (MMPs), the effects of microparticles were investigated. Microparticles are small membrane-bound vesicles whose release from immune cells is increased during activation and apoptosis. Because microparticles occur abundantly in the synovial fluid in rheumatoid arthritis, they could represent novel stimulatory agents.

To investigate the potential of DNA to elicit immune responses in man, we examined the capacity of a variety of oligodeoxynucleotides (ODNs) to stimulate highly purified T cell-depleted human peripheral blood B cells.

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Microparticles are small membrane vesicles released from the cell membrane by exogenous budding. To elucidate the interactions of microparticles with macrophages, the effect of microparticles released from Jurkat T cells on RAW 264.7 cells was determined. Microparticles were isolated by differential centrifugation, using FACS analysis with annexin V and cell surface markers for identification. Various inducers of apoptosis increased the release of microparticles from Jurkat cells up to 5-fold. The released microparticles were then cultured with RAW 264.7 cells. As shown by confocal microscopy and FACS analysis, RAW 264.7 macrophages cleared microparticles by phagocytosis. In addition, microparticles induced apoptosis in RAW 264.7 cells in a dose-dependent manner with up to a 5-fold increase of annexin V positive cells and 9-fold increase in caspase 3 activity. Cell proliferation as determined by the MTT test was also reduced. Furthermore, microparticles stimulated the release of microparticles from macrophages. These effects were specific for macrophages, since no apoptosis was observed in NIH 3T3 and L929 cells. These findings indicate that microparticles can induce macrophages to undergo apoptosis, in turn resulting in a further increase of microparticles. The release of microparticles from apoptotic cells may therefore represent a novel amplification loop of cell death.