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

  • John R. Ortaldo
  • Isaac Blanca
  • Ronald B. Herberman
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 187)


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.


Target Cell Natural Killer Activity Large Granular Lymphocyte Target Cell Lysis Strontium Chloride 
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  1. 1.
    R. B. Herberman (Ed.), “NK Cells and Other Natural Effector Cells,” Academic Press, New York (1982).Google Scholar
  2. 2.
    T. Timonen, J. R. Ortaldo, and R. B. Herberman, Characteristics of human large granular lymphocytes and relationship to natural- killer and K cells, J. Exp. Med. 153:569 (1981).PubMedCrossRefGoogle Scholar
  3. 3.
    C. W. Reynolds, T. Timonen, and R. B. Herberman, Natural killer (NK) cell activity in the rat. I. Isolation and characterization of the effector cells, J. Immunol. 127:282 (1981).PubMedGoogle Scholar
  4. 4.
    J. C. Roder and T. Haliotis, A comparative analysis of the NK cytolytic mechanism and regulatory genes, in: “Natural Cell- Mediated Immunity Against Tumors,” R. B. Herberman, ed., Academic Press, New York (1980).Google Scholar
  5. 5.
    P. C. Quan, T. Ishizaka, and B. R. Bloom, Studies on the mechanism of NK cell lysis, J. Immunol. 128:1786 (1982).PubMedGoogle Scholar
  6. 6.
    C. S. Henney, On the mechanism of T-cell mediated cytolysis, Transplant. Rev. 17:37 (1973).PubMedGoogle Scholar
  7. 7.
    S. C. Wright and B. Bonavida, Selective lysis of NK-sensitive target cells by a soluble mediator released from murine spleen cells and human peripheral blood lymphocytes, J. Immunol. 126:1516 (1981).PubMedGoogle Scholar
  8. 8.
    S. C. Wright and B. Bonavida, Studies on the mechanism of natural killer (NK) cell-mediated cytotoxicity (CMC). I. Release of cytotoxic factors specific for NK-sensitive target cells (NKCF) during coculture of NK effector cells with NK target cells, J. Immunol. 129:433 (1982).PubMedGoogle Scholar
  9. 9.
    S. C. Wright, M. L. Weitzen, R. Kahle, G. A. Granger, and B. Bonavida, Studies on the mechanism of natural killer cytotoxicity. II. Coculture of human PBL with NK sensitive or resistant cell lines stimulates release of natural killer cytotoxic factor (NKCF) selectively cytotoxic to NK-sensitive target cells, J. Immunol. 130:2479 (1983).PubMedGoogle Scholar
  10. 10.
    E. Farram and S. R. Targan, Identification of human natural killer soluble cytotoxic factor (s) NKCF derived from NK- enriched lymphocyte populations: specificity of generation and killing, J. Immunol. 130:1252 (1983).PubMedGoogle Scholar
  11. 11.
    S. Wright and B. Bonavida, YAC1 variant clones selected for resistance to NKCF are also resistant to natural killer cell- mediated cytotoxicity. Proc. Natl. Acad. Sci. USA 80(6):1688 (1983).PubMedCrossRefGoogle Scholar
  12. 12.
    J. T. Forbes and T. N. Oeltmann, Carbohydrate receptors in natural cell-mediated cytotoxicity, in: “NK Cells and Other Natural Effector Cells,” R. B. Herberman, ed., Academic Press, New York (1982).Google Scholar
  13. 13.
    J. R. Ortaldo, T. T. Timonen, and R. B. Herberman, Inhibition of activity of human NK and K cells by simple sugars: Discrimination between binding and post-binding events, Clin. Immunol. Immunopathol. 31(3):439 (1984).CrossRefGoogle Scholar
  14. 14.
    O. Stutman, P. Dien, R. E. Wisum, and E. C. Lattime, Natural cytotoxic cells against solid tumors in mice: blocking of cytotoxicity by D-mannose, Proc. Natl. Acad. Sci. USA 77:2895 (1980).PubMedCrossRefGoogle Scholar
  15. 15.
    J. S. Tkacz and J. O. Lampen, Tunicamycin inhibition of polyisoprenyl-N-acetylglucosamyl pyrophosphate formation in calf liver microsomes, Biochem. Biophys. Res. Commun. 65:248 (1975).CrossRefGoogle Scholar
  16. 16.
    P. K. Keller, D. Y. Boon, and F. C. Crum, N-acetyl glucosamine- 2-phosphate transferase from hen oviduct: Solubilization, characterization, and inhibition by tunicamycin, Biochemistry 18:3946 (1979).PubMedCrossRefGoogle Scholar
  17. 17.
    G. W. Hart, The role of asparagine-linked oligosaccharides in cellular recognition by thymic lymphocytes. Effects of tunicamycin on the mixed lymphocyte reaction, J. Biol. Chem. 257:151 (1982).PubMedGoogle Scholar
  18. 18.
    J. A. Werkmeister, J. C. Roder, C. Curry, and H. F. Pross, The effect of unphosphorylated and phosphorylated sugar moieties on human and mouse natural killer cell activity: is there selective inhibition at the level of target recognition and lytic acceptor site?, Cell. Immunol. 80:172 (1983).PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1985

Authors and Affiliations

  • John R. Ortaldo
    • 1
  • Isaac Blanca
    • 1
  • Ronald B. Herberman
    • 1
  1. 1.Biological Therapeutics Branch, Biological Response Modifiers Program, Frederick Cancer Research FacilityNational Cancer InstituteFrederickUSA

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