Transmembrane signaling during natural killer cell-mediated cytotoxicity. Regulation by protein kinase C activation.
NK cells can mediate either FcR-dependent cytotoxicity against antibody-coated target cells or direct cytotoxicity against a variety of tumor cells. We used homogeneous, cloned populations of CD16+/CD3- human NK cells to characterize and compare the transmembrane signaling mechanisms used during these alternative forms of cytotoxicity. Cross-linkage of NK cell FcR with anti-FcR (anti-CD16) mAb or direct binding to NK-sensitive tumor targets resulted in a rapid release of inositol phosphates and increases in [Ca2+]i. The receptor-dependent [Ca2+]i increase (as monitored in indo-1 loaded NK cells by flow cytometry) consisted of an initial release of calcium from intracellular stores, followed by a sustained influx of calcium across the plasma membrane. To assess the potential regulatory feedback role of protein kinase C (PKC) activation in these proximal signaling events, NK cells were pretreated with either PKC-activating phorbol esters, nonactivating phorbol ester homologs, or synthetic diacylglycerols. Brief pretreatment with activating phorbol esters rapidly inhibited, in a concentration-dependent manner, both phosphoinositide hydrolysis and increases in [Ca2+]i induced by FcR ligation, whereas pretreatment with an inactive phorbol ester had no effect. This acute inhibitory effect was not explained by FcR down-regulation, which occurred with more prolonged exposure to phorbol esters. In contrast, the phosphoinositide turnover and [Ca2+]i increase in NK cells stimulated with NK-sensitive tumor targets were not affected by prior exposure to PKC-activating phorbol esters. This differential regulatory effect of phorbol ester on proximal signaling was paralleled by a corresponding effect on cytotoxicity, i.e., phorbol ester-induced activation of PKC inhibited FcR-dependent cytotoxicity, but did not alter direct cytotoxicity against NK-sensitive tumor cells. These results indicate that PKC activation can differentially regulate alternative forms of NK cell-mediated cytotoxicity by rapidly and specifically desensitizing the FcR.
Leibson
P J
PJ
Department of Immunology, Mayo Clinic, Rochester, MN 55905.
Midthun
D E
DE
Windebank
K P
KP
Abraham
R T
RT
D000943:Antigens, Differentiation / Q000502:physiology*; D002118:Calcium / Q000502:physiology; D002460:Cell Line; D003602:Cytotoxicity, Immunologic; D004075:Diglycerides / Q000494:pharmacology; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D007694:Killer Cells, Natural / Q000502:physiology*; D008213:Lymphocyte Activation; D010716:Phosphatidylinositols / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology*; D011961:Receptors, Fc / Q000502:physiology*; D017452:Receptors, IgG; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000943:Antigens, Differentiation; D004075:Diglycerides; D010716:Phosphatidylinositols; D011961:Receptors, Fc; D017452:Receptors, IgG; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium