The mechanism of human NK cell-mediated cytotoxicity. Mode of action of surface-associated proteases in the early stages of the lytic reaction

The possible involvement of cell surface-associated proteolytic enzymes in human NK cell-mediated cytotoxicity and the mechanism by which such enzymes exert their activity have been studied. The treatment of intact cells with 3H-DFP under restricted conditions that predominantly bind surface-associated enzymes resulted in the labeling of five to six enzyme bands. Among these were a 35,000-dalton enzyme, which may be a previously identified trypsin-like proteinase engaged in cytotoxicity, and a 58,000-dalton elastase. The latter seems not to be involved in the reaction, as potent inhibitors of this enzyme have negligible effect on cytotoxicity. Of the membrane-associated enzymes, those engaged in cytotoxicity seem to be concealed from the external environment, as pretreatment of the effector cells with protease inhibitors such as trasylol and PMSF have no effect on the reaction. Immediately upon addition of the target cells and the initiation of cytotoxicity, the reaction becomes highly sensitive to inhibitors for a limited time interval of 2 to 5 min when trasylol is employed and 5 to 10 min when TPCK is the inhibitor, suggesting that target cell binding triggers the exposure of the enzymes to the external environment, rendering them accessible to the inhibitors. This short sensitivity period parallels the interval in which the reaction is sensitive to the microfilament inhibitor cytochalasin B. As the reaction proceeds, it becomes increasingly resistant to inhibitors of both proteolysis and cytoskeleton, at the same time suggesting that microfilament action and the unraveling of proteases may be processes that bear a close linkage with one another. The surface-associated elastase on the other hand maintains a constitutive mode of activity distinctive and unrelated to that of enzymes engaged in cytotoxicity. These findings suggest the existence on the surface of the NK lymphocyte of a mechanism that associates the receptor for target cells with an array of enclaved proteolytic enzymes via microfilaments. The resting cytotoxic structures become activated as the receptor attaches to the target cell, triggers the exposure of the proteolytic moiety, and initiates the lytic phase of the reaction.