Mechanism of norepinephrine-mediated inhibition of human NK cytotoxic functions: inhibition of cytokine secretion, target binding, and programming for cytotoxicity

Abstract

Norepinephrine (NE) has been shown to inhibit human peripheral blood-derived natural-killer (NK) cell cytotoxicity (NKCC) in vitro. We demonstrate in this study that NE not only inhibits IL-2-activated NKCC but antibody-dependent cellular cytotoxicity (ADCC) as well. NK cytotoxicity by purified NK cells against K562 (NKCC) and against Raji cells (ADCC) were inhibited by NE (1-100 microM) by more than 50% in a 4-h (51)Cr release assay. The mechanism underlying the inhibition has been examined. NK cytotoxicity is dependent on target recognition and formation of NK-target conjugates, and activation by IL-2 is dependent on the secretion of cytokines (such as TNF-alpha) by NK cells. We hypothesized that the inhibition of NK functions by NE may be due to disruption of NK-target conjugation, blocking programming for lysis, and/or inhibition of cytokine secretion. Pretreatment of human peripheral blood mononuclear cells (PBMC) with NE for 15 min significantly reduced the binding to K562 cells by CD16(+) NK lymphocytes. In the presence of K562 cells, NE down-regulated the expression of CD16 (FcgammaRIII) by human PBMC, an NK cell receptor responsible and necessary for ADCC and cytokine secretion. We also demonstrate that NE inhibited the IL-2-mediated up-regulation of the activation marker CD69. At concentrations of 10(-6) to 10(-5) M, NE inhibited TNF-alpha, IFN-gamma, and GM-CSF secretion by NK cells, which are essential for IL-2-driven NK maturation and functions. In addition, using single-cell analysis, NE pretreatment of lymphocytes reduced the frequency of killer cells in the NK-K562 conjugate population in a concentration-dependent manner, indicating an inhibition of the programming for lysis by NK cells. In summary, these data demonstrate that NE-induced inhibition of NK cytotoxicity is manifested at multiple levels, including a modification of NK cell receptor ligation to target cells, blockade of NK cytokine secretion necessary for NK maturation and differentiation, and inhibition of the target-induced activation of the cytotoxic mechanism(s) in NK cells. Thus, sympathetic activation, as often induced experimentally, may profoundly impair natural cellular immunity through varied measurable pathways.