Inhibition of the calpain-mediated proteolysis of protein kinase C enhances lytic activity in human NK cells.
Recent evidence from our laboratory has demonstrated that NK/LAK cell activation of human lymphocytes is protein kinase C (PKC)-dependent. Here, we have investigated the translocation of PKC in human NK cells exposed to sensitive targets or to PMA, a phorbol ester. In NK cells exposed to K562 for 6 hr, we observed a weak translocation of PKC whereas in NK cells exposed to PMA more than 90% of cytosolic PKC was translocated to the membrane in less than 5 min. Stimulation of NK cells with an NK-resistant target, however, did not translocate PKC even after 6 hr. Translocation of PKC to the membrane was followed by the appearance of PKM, the cytosolic calcium/phospholipid (Ca2+/PL)-independent form of PKC. The conversion of PKC to PKM was mediated by calpain, an intracellular calcium-dependent thiol proteinase. When we used two inhibitors of calpain, calpain inhibitor I (CI-I) and calpain inhibitor II (CI-II), both caused a dose-related enhancement of NK-CMC when the inhibitors were present throughout the 3-hr chromium release assay. This enhancement could be circumvented by PMA or by the PKC inhibitor H-7. CI-I and CI-II added together caused a greater increase in NK-CMC than when each was added alone. CI-I and CI-II also enhanced antibody-dependent cell-mediated cytotoxicity (ADCC), substantiating further our previous contention that the activation of both NK-CMC and ADCC may involve a common lytic pathway. Activation of NK cells with IL-2 for 18 hr at 37 degrees C was inhibited in the presence of CI-I. To investigate a possible feedback inhibition mechanism due to the buildup of PKC, we examined phosphatidylinositol (PI) metabolism in NK cells activated by IL-2 in either the presence or the absence of CI-I. We observed a significant decrease in PI turnover when NK cells, activated in the presence of IL-2 and CI-I, were stimulated with K562 as compared to NK cells activated by IL-2 alone, then stimulated with K562.
Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis 46202-5128.
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Shenoy
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Brahmi
D000328:Adult; D000920:Antibody-Dependent Cell Cytotoxicity; D001692:Biological Transport; D002154:Calpain / Q000037:antagonists & inhibitors*; D006023:Glycoproteins / Q000494:pharmacology*; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology; D007694:Killer Cells, Natural / Q000276:immunology*; D008213:Lymphocyte Activation / Q000187:drug effects; D010716:Phosphatidylinositols / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D006023:Glycoproteins; D007376:Interleukin-2; D010716:Phosphatidylinositols; C039304:calpain inhibitors; D011493:Protein Kinase C; D002154:Calpain; D013755:Tetradecanoylphorbol Acetate