Proteasome inhibition induces apoptosis in primary human natural killer cells and suppresses NKp46-mediated cytotoxicity

Abstract

Bortezomib is a selective and potent inhibitor of the proteasome and has prominent effects in vitro and in vivo against tumors. Very recently, cytotoxic effects of bortezomib on immune-competent cells such as T cells and dendritic cells were also revealed. The aim of the study was to investigate the effects of this agent on natural killer cell survival and function. We investigated cytotoxic properties of bortezomib on natural killer cell apoptosis and function. Primary resting natural killer cells were purified from peripheral blood mononuclear cells of healthy donors by negative selection. The apoptotic cells were quantified by dual labeling of recombinant annexin V and propidium iodide. Mitochondrial membrane potential and expression of natural killer cell activating receptors were also quantified by flow cytometry. Natural killer cell cytotoxicity against murine and human tumor cells was tested by chromium 51 release assay. Our results demonstrate that bortezomib induces apoptosis in resting natural killer cells in a dose- and time-dependent manner. Glutathione, a reactive oxygen species scavenger, prevented the loss of mitochondrial membrane potential and conferred protection against bortezomib-induced apoptosis in resting natural killer cells, indicating a role for oxidative stress. Additionally, bortezomib significantly decreased expression of the natural killer activating receptor NKp46 in non-apoptotic resting natural killer cells in a dose-dependent manner, and as a result the redirected cytotoxicity mediated via NKp46 activation was diminished. Bay 11-7082, a pharmacological inhibitor of NF-kappaB activation, also reduced NKp46 expression and suppressed redirected cytotoxicity. Bortezomib induces apoptosis in primary resting natural killer cells in a dose- and time-dependent manner, and reduces NKp46 receptor expression as well as natural killer cell cytotoxicity mediated by the NKp46 activation pathway, suggesting that bortezomib may disrupt natural killer cell-mediated immunity through at least two different mechanisms: induction of natural killer cell apoptosis, and suppression of NKp46 receptor-mediated cytotoxicity.