Multiple mechanisms of target cell disintegration are employed in cytotoxicity reactions mediated by human natural killer cells.

The rate of disintegration of target cells subsequent to lytic programming by human peripheral blood natural killer (NK) cells was investigated using a quantitative calcium pulse technique. The rate of this initial calcium-independent target cell disintegration was indicative of a first-order decay process for programmed target cells with a calculated half-life of less than 3 min. This initial, rapid disintegration phase was independent of the overall cytotoxic activity of the lymphocyte preparation tested. Moreover, initial rates of target cell disintegration were comparable for target cell lines that exhibit up to 6-fold differences in overall susceptibility to natural cytotoxicity. In these studies we also consistently observed very slow, calcium-independent disintegration of additional target cells following apparent completion of the rapid disintegration process. Using a 51Cr release assay and K-562 target cells, the kinetics of this slow disintegration process were examined and found to be similar for donors exhibiting up to a 2-fold difference in overall cytotoxic activity and independent of the concentration of programed target cells. Whereas the initial rapid disintegration mechanism was independent of temperature over the range of 10-37 degrees C, the slow disintegration mechanism exhibited a direct dependence on the incubation temperature. Furthermore, we observed that supernatants obtained after the termination of lytic programing by ethylene diaminetetraacetic acid could effect the slow lysis of fresh NK-susceptible target cell lines. These results support the utilization of at least two distinct mechanisms for target cell lysis by human NK cells.

Experimental application of a multistep kinetic model for natural cytotoxicity: determination of rate constants for lytic programming and killer-cell-independent lysis.

A multistep kinetic model for natural cytotoxicity reactions in vitro is described that includes consideration of the effect of nonlytic target-binding lymphocytes on the experimentally determined kinetic parameters. The expression for the maximal velocity, Vmax, obtained using this model is essentially identical to that obtained using simpler models, whereas the expression for the apparent Michaelis constant, KappM, is considerably more complex. As a first step in the application of this model, experiments were designed to determine the relative contribution of some of the component terms to the value of KappM. Methods were developed that allow for experimental determination of the rate constants for both lytic programming and killer-cell-independent lysis (KCIL) steps in the cytolytic process. The results obtained support lytic programming as the rate-determining step in natural cytotoxicity reactions and demonstrate that terms related to nonlytic target-binding lymphocytes contribute significantly to experimentally determined values for KappM. In addition, the methods developed for the determination of the rates of lytic programming and KCIL should prove useful for various studies of the mechanism of cytotoxicity and the effects of drugs and disease on such phenomena.