Regulation of Resting and IL-2-Activated Human Cytotoxic Lymphocytes by Exogenous Nucleotides: Role of IL-2 and Ecto-ATPases

In this investigation we studied the modulation of human NK- and CTL-mediated cytotoxicity in response to extracellular nucleotides. NK cell-mediated cytotoxicity (CMC) was inhibited in a dose-dependent manner by ATP/ADP. GTP/GDP, and by pentasodium triphosphate (PST), whereas MHC-restricted CTL, were inhibited by GTP/GDP and PST, but not by ATP/ADP. Triphosphates were the most potent inhibitors, followed by diphosphates and monophosphates which were the least effective, suggesting that inhibition was not due to the sugars nor adenosine and guanosine nucleotides, but rather to the increasing negative charges. Cultured CTL, fresh NK cells that had been incubated with IL-2 for 18 hr and IL-2-dependent NK 3.3 cells were all inhibited by GTP, but not by ATP. This differential regulation of fresh NK cells and CTL by exogenous nucleotides is dependent upon the presence of IL-2, but IL-4, IL-6, and IL-8 did not have any effect. Mouse CTL are resistant to ATP presumably because they contain high levels of ecto-ATPases. Different levels of ecto-ATPase activity in human CTL and NK cells may therefore explain the difference in the responses of these effector cells to extracellular nucleotides. To test this possibility we determined the levels of ecto-ATPases in human CTL and NK cells and showed that CTL contained five times more ecto-ATPases than NK cells. Incubation of NK cells with IL-2 or IL-4 did not significantly change the level of ecto-ATPase activity on NK cells. Treatment of NK cells with IL-2 also did not significantly change the substrate specificity or NK-ecto-ATPases toward the extracellular ATP and GTP. Furthermore, treatment of CTL and NK cells with a potent ecto-ATPase inhibitor. 5′-fluorosulfonylbenzoyladenosine (FSBA), did not significantly alter the effect of exogenous nucleotides on the lytic potential of CTL and NK cells. Our results demonstrate that (1) exogenous nucleotides are inhibitors rather than mediators of NK-CMC; (2) the inhibition of NK-CMC increases with the increase in localized negative charges and is independent of the nucleoside structure; (3) NK- and CTL-CMC are differentially inhibited by exogenous nucleotides; (4) the differential inhibition of NK- and CTL-CMC is dependent upon IL-2; (5) ecto-ATPases are responsible neither for the protection of EC from the lytic effect of ATP nor for the differential regulation of NK- and CTL-CMC by nucleotides; and (6) inhibition is a postbinding event and is not caused by inactivation of serine esterases or blockage of exocytosis in activated EC.