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Signal transduction in activated natural killer cells and natural killer cells inactivated with sensitive targets
J J Gibboney1, A M Shenoy, X Jin
1Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis 46202-5200.
Abstract:
We have recently demonstrated that the activation of NK cells involves a protein-kinase-C (PKC)-dependent step as well as a phosphatidylinositol (PI)-linked signal transduction system. We have also shown that when NK cells are incubated with sensitive targets for up to 6 h they lose their lytic potential and require IL-2 to regain their lytic activity. PKC is involved in many cell processes such as the transduction of hormonal signals and the machinery of cellular secretion and is activated by diacylglycerol and by a number of phorbol esters, including phorbol myristic acid (PMA). To reinforce the role of PKC in NK-cell-mediated cytotoxicity, we first showed that NK-CMC is inhibited by two agents that inhibit PKC activation, staurosporine and sphingosine. Next, we showed that antibody-dependent cellular cytotoxicity of NK-resistant targets was PKC dependent and Ca2+ dependent. Further, we showed that PMA plus ionophore alone could induce NK killing of resistant targets and that this killing was PKC and Ca2+ dependent. Finally, we contrasted the role of PKC in these activated cells to the role of PKC in cells that have been inactivated with a sensitive target, K562. We showed that PKC is not required for the IL-2-dependent reactivation of NK cells but that Ca2+ is required. To further determine what event in signal transduction is inactivated by K562, we showed that inactivated NK cells do not turnover PI in response to K562 stimulation.
Insights
Natural killer (NK) cell cytotoxicity relies on protein-kinase-C (PKC) and phosphatidylinositol (PI) signaling. While PKC is crucial for NK cell activation, it
Area of Science:
- Immunology
- Cellular Signaling
- NK Cell Biology
Background:
- Natural killer (NK) cell activation involves protein-kinase-C (PKC) and phosphatidylinositol (PI) signal transduction.
- NK cells lose lytic potential after prolonged incubation with targets and require IL-2 for reactivation.
- PKC plays a role in various cellular processes, including hormonal signal transduction and secretion.
Purpose of the Study:
- To reinforce the role of PKC in NK cell-mediated cytotoxicity.
- To investigate the involvement of PKC and calcium (Ca2+) in antibody-dependent cellular cytotoxicity (ADCC).
- To differentiate the role of PKC in activated versus inactivated NK cells.
Main Methods:
- Inhibition of NK cell-mediated cytotoxicity (NK-CMC) using PKC inhibitors (staurosporine, sphingosine).
- Assessment of PKC and Ca2+ dependence in antibody-dependent cellular cytotoxicity (ADCC) against NK-resistant targets.
- Induction of NK cell killing using phorbol myristic acid (PMA) plus ionophore and subsequent analysis of PKC and Ca2+ roles.
- Comparison of PKC involvement in IL-2-dependent reactivation of inactivated NK cells and phosphatidylinositol (PI) turnover.
Main Results:
- NK-CMC was inhibited by staurosporine and sphingosine, confirming PKC involvement.
- ADCC against NK-resistant targets was dependent on both PKC and Ca2+.
- PMA plus ionophore induced NK killing of resistant targets in a PKC and Ca2+-dependent manner.
- PKC was not required for IL-2-dependent NK cell reactivation, but Ca2+ was necessary.
- Inactivated NK cells failed to exhibit PI turnover upon K562 stimulation, indicating a defect in this signaling pathway.
Conclusions:
- PKC is a critical mediator of NK cell activation and cytotoxicity.
- Ca2+ plays a significant role in both NK cell activation and reactivation.
- K562 cell interaction inactivates NK cells by disrupting PI turnover, a key signaling event.