Definition of a secondary target cell trigger during natural killer cell cytotoxicity: possible role of phospholipase A2.

Phospholipase A2 (PA-2) is known to be involved in many calcium-dependent cellular processes and inhibitors of PA-2 have been shown to inhibit natural killer cell-mediated cytotoxicity (NK CMC). Since the trigger stage is calcium dependent, it was postulated that this effector cell-associated enzyme may play a role in early calcium-dependent processes. To define how PA-2 might be involved in NK lysis, the effect of both PA-2 inhibitors and exogenous PA-2 on the stages of NK lysis was examined.

Studies on the mechanism of the human natural killer cell lethal hit: a new method for rapid physical isolation of lethally hit K562 targets and inhibition of cytolysis by reduced temperature or trypsin.

A new method was developed which allows for rapid (2 min) physical isolation of viable K562 target cells after being programmed to lyse (lethally hit) by purified human natural killer (NK) cells (LGL). To achieve this K562 cells which were obtained from the 34-36% interface of discontinuous Percoll gradients and purified human NK cells (LGL) which were obtained from the (43-45% Percoll) interface were employed. Using a Ca2+ pulse method and the separation of NK-K562 conjugates with EDTA and rapid centrifugation on Percoll gradients at 4 degrees C we could physically isolate the lethally hit K562 cells from the LGL allowing the study of the events leading to their subsequent lysis.

Studies on the mechanism of the human natural killer cell lethal hit: evidence for transfer of protease-sensitive structures requisite for target cell lysis.

These studies analyze the effects of various enzymes on the terminal, killer cell-independent (KCIL) stages of the human natural killer (NK) cytolytic mechanism. The addition of trypsin (T), chymotrypsin (CT), or papain (P) to standard NK reaction mixtures (PBL or LGL and K562 cells) completely ablated cytolytic activity, whereas collagenase was ineffective. Inhibition by T was reversed by preincubation with soybean trypsin inhibitor (SBTI) or fetal calf serum, indicating that the inhibition was indeed due to T.