Derived from honeybees, melittin is a 26–amino acid, α-helical, membrane-attack protein that efficiently kills mammalian cells. To investigate the contribution of colloid-osmotic effects to the mechanism of cell death, we studied the effect of melittin on lymphocyte membrane permeability and cell volumes. Melittin concentrations of 0.5 to 2.0 μM induced release of membrane permeability markers without total disruption of the cell membrane. At these melittin concentrations, electrical-impedance cytometry demonstrated melittin-induced changes in red blood cell volumes (P < 0.01), but no change in lymphocyte cell volumes (P > 0.05). Streaming video microscopy, obtaining images of melittin-treated lymphocytes at 80-ms intervals, demonstrated a loss of optical density (P < 0.001) suggesting a flattening of the cell but no significant increase in cell perimeter (P > 0.05). Real-time multiparameter flow cytometry of melittin-treated lymphocytes confirmed simultaneous loss of the cytoplasmic marker, calcein, and uptake of the DNA dye, ethidium homodimer, but demonstrated no increase in forward light scatter. Transmission-electron microscopy of melittin-treated lymphocytes showed normal cell volumes but discontinuities in the cell membrane suggesting direct membrane toxicity. We conclude that melittin causes lymphocyte death by a “leaky patch” mechanism that is independent of colloid-osmotic effects.
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Vol. 41 • No. 10