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Current standard methods for the measurement of cell-mediated cytotoxicity rely on radioactive tracers, which either detect the release of cytoplasmic contents after plasma membrane disintegration by dying cells (51Cr release), or retained DNA by living cells (the JAM test). In this study, the annexin V binding assay of early apoptosis was applied to measure cell-mediated cytotoxicity. Primed human lymphocytes were examined for their ability to lyse either xenogeneic pig endothelial or allogeneic human PBMC target cells by assaying annexin V binding and the results compared with those obtained by the JAM test. Assaying annexin V binding by indirect immunofluorescence was demonstrated to be more sensitive and faster than the JAM test, which is a well-described, sensitive and simple assay for DNA fragmentation and cell death. However, the annexin V binding method was considered a more accurate measurement of absolute cytotoxicity as individual cell lysis was detected directly. In other methods, cytotoxic activity was calculated indirectly as a percentage of retained or released radioactive label. In addition, the apoptosis induced by the cell-mediated cytotoxicity can be visualized by this method thereby allowing a more accurate and sensitive quantitation of the number of apoptotic cells present when low effector to target ratios are used. These advantages make the annexin V binding method superior to other conventional cytotoxicity assays, particularly in situations where effector cells can be easily distinguished or separated from target cells.

The influence of histamine at various concentrations on the cell cycle state of hematopoietic stem cells (CFU-s) was investigated. CFU-s were triggered from the G0 state into the S phase of the cell cycle by in vitro treatment of mouse bone marrow cells with high concentrations of histamine. This effect could be antagonized by a histamine H2 receptor blocking agent. When bone marrow cells were treated with a histamine H1 receptor antagonist prior to histamine treatment, low concentrations of histamine also triggered the entrance of CFU-s into the DNA synthetic phase. Our findings further suggest the existence of histamine H1 and H2 receptors on the surface of CFU-s cells and the antagonistic effect of these two histamine receptor subtypes on the cell cycle state of CFU-s. Our results also suggest that histamine may participate in regulating the proliferation of hematopoietic stem cells in vivo during immune or inflammatory responses.