Studies of Human Natural Killer Cytotoxic Factor (NKCF): Characterization and Analysis of its Mode of Action

Summary

Soluble natural killer cytotoxic factors (NKCF) have been detected in the supernatant of normal mouse, rat, and human lymphocytes stimulated in vitro for 1 to 3 days in serum-free medium. Stimulation of large granular lymphocytes (LGL) with NK-sensitive targets or mitogens has resulted in high levels of NKCF production. Previous studies in the mouse and human systems have analyzed the cells responsible for production, specificity, and general characteristics of NKCF. In the present study, using human NKCF as a model for cytolysis by LGL, we have analyzed a variety of agents previously demonstrated to inhibit NK activity. These have included: (i) phosphorylated sugars; (ii) protease inhibitors; (iii) antibodies to rat LGL granules; (iv) Ca⁺⁺, and Mg⁺⁺; (v) lipomodulin; (vi) nucleotides; (vii) prostaglandins; and (viii) inhibitors of lysosomal enzymes. All inhibitors were tested for their effects on production of NKCF after target cell interaction, binding of NKCF to target cells, and target cell lysis (after 6-hour NKCF absorption and washing of targets). Phosphorylated sugars and antibodies to rat LGL granules were found to inhibit the lysis of targets by NKCF, whereas the other agents tested had no detectable effect (ATP, cyclic AMP, protease inhibitors, prostaglandin E2). In regard to the production of NKCF, the data indicated that (i) the absence of calcium and magnesium, (ii) prostaglandin E2, and (iii) ATP inhibited production, whereas phosphorylated sugars did not. Studies with these types of agents will now enable us to dissect the sites at which these agents function within the lytic process. In addition to the above studies, purification studies were performed using tritiated arginine to label NKCF to begin biochemical characterization of human NKCF. The results indicated that radiolabeled NKCF has an apparent molecular weight between 20,000 and 40,000. This material demonstrated a pattern of binding to target cells which was similar to the pattern of lysis by NKCF. In addition, the binding of this material was competitively inhibited by unlabeled NKCF preparations. Such approaches with radiolabeled NKCF should be useful for the further study of the biochemical characteristics of human NKCF and of its mechanism of action.