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Generation of a Lipid-like Cytotoxin from Human CD16+ Natural Killer Cells¹

Department of Immunology and Cancer, Research Institute, Cleveland Clinic Foundation, and Department of Regulatory Biology, Cleveland State University, Cleveland, Ohio 44106

Supernatants from unstimulated CD16+ natural killer (NK) cells or from CD16+ NK cells cocultured with K562 tumor cells (to generate NK cytotoxic factor) were both cytotoxic to target cells. Interleukin 2 stimulation of the CD16+ NK cells in the absence of tumor cell stimulation resulted in supernatants which mediated an increased cytotoxicity as compared to the unstimulated supernatants. The cytotoxic activity was recovered in the chloroform fraction of a Bligh-Dyer lipid extraction suggesting that the toxic moiety in the CD16+ NK cell-derived supernatants might be a lipid. Separation of the cytotoxic supernatants into M_r < 10,000 and M_r > 10,000 fractions revealed that the M_r < 10,000 fraction of both supernatants had no detectable protein but retained cytotoxicity equal to that of the matched unfractionated supernatant. For convenience, we refer to this lipid-like cytotoxin in the M_r < 10,000 fraction of the supernatants from unstimulated CD16+ NK cells as lipotoxin (LTX) and the cytotoxin in the M_r < 10,000 fraction of supernatant from interleukin 2 stimulated CD16+ NK cells as LTX*. Increasing concentrations of LTX and LTX* caused a dose related increase in cytotoxicity. Both LTX and LTX* mediated killing as early as 18 h and their cytotoxicity was not significantly affected by heating at 56°C for 2 h or by freezing and thawing. Heating at 63°C resulted in a decrease in cytotoxic activity of 10 to 20%. The <10,000 dalton fraction of supernatants from both unstimulated and interleukin 2 stimulated CD3- cells (a crude NK cell population) mediated greater cytotoxicity than the CD3+ cell supernatants, and the majority of cytotoxicity from the CD3- cell supernatants was recovered in this fraction. Thus, NK cells were more efficient producers of the lipid-like cytotoxin than T-cells but whether LTX made by NK cells can also be made by T-cells remains to be determined. We propose that lipotoxin: (a) coexists with protein cytotoxins in NK cell supernatant preparations; (b) mediates significant cytotoxicity when separated from proteinaceous cytotoxins; (c) is responsible for the spontaneously secreted cytotoxic activity observed by others; (d) is distinct from previously reported proteinaceous cytotoxins, e.g., NK cytotoxic factor, tumor necrosis factor , and cytolysin/perforin; (e) accounts for the lipophilic nature of cytotoxic factor activity in NK cell supernatants; and (f) causes the cytotoxic activity observed in a small molecular weight fraction of stimulated NK cell supernatants.

¹ This work was supported by a grant from the Mudge Foundation and a fellowship to L. M. C. from the American Podiatric Medical Association.

² Present address: Georgetown University School of Medicine, Department of Microbiology, Division of Molecular Virology and Immunology, 3900 Reservoir Road, Washington, DC 20007.

³ To whom requests for reprints should be addressed, at Immunology and Cancer, Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44106.

Received 1/27/89. Revised 8/ 7/89. Accepted 9/ 2/89.