Mechanism of lysis by large granular lymphocyte granule cytolysin: generation of a stable cytolysin-RBC intermediate

The effect of ionic strength and pH on the hemolytic activity of large granular lymphocyte granule cytolysin was examined in detail. Cytolysin-mediated lysis of RBC was inhibited by either low ionic strength or low pH. Under these conditions a nonlytic cytolysin-RBC intermediate was formed as revealed by hemolysis when cytolysin pretreated cells were washed and resuspended at physiologic ionic strength and pH. Formation of the cytolysin-RBC intermediate at low ionic strength (250 mM sucrose), pH 7.3, required greater than 0.1 mM calcium. In contrast, formation of the intermediate at physiologic ionic strength (150 mM NaCl), pH 6.0, was calcium independent. Both types of intermediates were stable at 37 degrees C and required calcium to induce subsequent lysis. The degree of lysis of the intermediate generated at low ionic strength was similar to that measured under standard conditions with the use of either whole granule preparations or purified cytolysin. However, lysis of intermediates formed at pH 6.0 was much less efficient. Our data indicate that a stable cytolysin-RBC intermediate can be formed in which cytolysin is present in an unreactive state on the RBC surface; under conditions of physiologic ionic strength and calcium concentrations this intermediate rapidly lyses.