Studies on the mechanism for natural killer cell cytotoxicity and its regulation

Sammanfattning: Studies on the Mechanism for Natural Killer Cell Cytotoxicity and Its Regulation Lin-Sheng Xiao Dissertation from the Unit for Applied Biochemistry, Clinical Research Center, Karolinska Institute, Stockholm, Sweden Since the proposed perforin-granzymes exocytosis can not sufficiently account for NK cell mediated cytolysis, the existence of a new and yet unknown mechanism for induction of the damage to targets by NK cells has been suggested. Our data illustrates that NK cell oncolytic activity has a requirement for L-arginine. The concurrent determination of NO and citrulline production in the presence of L-arginine during the NK cell-mediated killing indicates that NK cell activity is directly associated with a higher L-arginine metabolism resulting in NO formation. NO and citrulline generation declined in proportion to decreased NK cell cytotoxicity in the absence of L-arginine in the medium or in the presence of L-NMMA, an inhibitor of NO synthase. These results could thus define a L-arginine/NO-linked pathway in the human NK cell effector mechanism and could be further verified by the demonstration of NO synthase activity in homogenised NK cells by using a highly sensitive and specific [14C]L-arginine conversion assay. NOS activity was found to be mainly linked to the membrane of NK cells and principally in an inducible as well as Ca++ - independent isoform. The seemingly low output of NOS activity in NK cell fractions may suggest that NO seems to work as a signal rather than being a killer molecule and that NO formation could be a compartmentalized process by which relatively a higher NO content may be obtained in target cells. In contrast, exogenous NO produced by NO donors may inhibit NK cell activity via an increase in intracellular cGMP. Such an effect of exogenous NO seems to open the possibility that NK cells might as well be under the regulation in vivo by NO-releasing cell types. The marked alternation of NK cell activity during emotional stress has received intensive attention. Our data showed that psychotropic molecules may interfere with NK cells. The antidepressant-caused inhibition of NK cell activity was found to be related to an early post binding event and associated with a slight increase in intracellular cGMP in NK cells without apparent relation to target cell killing. These results indirectly provide evidence that human neurons and NK cells may share some regulatory mechanism(s). Activation-induced apoptosis in immune effector cells has been widely studied and appeares to be a regulatory mechnism in T cell-mediated immune response. Our study regarding the bilateral reaction between NK cells and tumor cells revealed a feedback-like inhibition in both recycling capability and cytolytic activity of NK effector cells detached from targets. However there was no evidence that target cells may induce NK cell death by apoptosis. The paralysis of NK cells after target cell stimulation may be mediated by an increase in intracellular cAMP. In conclusion, NK cells seem to posess primarily a membrane bound NOS. NO of NK cell origine participates, at least partially, in target cell killing. However, exogenous NO may down-regulate NK activity possibly through the high level of cGMP. The regulation of human NK cells by target cells and psychotropic molecules was also illustrated in the present study. key words: natural killer cell, nitric oxide, cytotoxicity, nitric oxide synthase, cyclic GMP, cyclic AMP, immunol regulation