Requirement of thiol compounds as reducing agents for IL-2-mediated induction of LAK activity and proliferation of human NK cells.
Thiol-related compounds, such as L-cystine, 2-ME or reduced glutathione (GSH), are important in many lymphoid cell activation pathways. We investigated their role in IL-2-generated lymphokine-activated killer (LAK) activity in human NK (CD16+ and/or CD56+) cells. Depletion of GSH and L-cystine, but not L-leucine or glycine, from medium used during culture of NK cells with IL-2 inhibited LAK and proliferative activities, whereas IL-2-independent lytic function of NK cells remained intact. Addition of L-cysteine, 2-ME or GSH, but not methylated analogs of these compounds (which cannot function as proton donors) to L-cystine/GSH-depleted medium restored proliferative response of NK cells and LAK generation. In the presence of L-buthionine-(S,R)-sulfoximine, an inhibitor of GSH synthesis, IL-2-induced LAK activity and proliferation of NK cells in medium without L-cystine and GSH, could be restored, at least in part, by addition of GSH, but not 2-ME or L-cystine. Furthermore, intracellular GSH levels were depressed in cells cultured in L-cystine/GSH-depleted medium but could be restored by the addition of 2-ME. The results suggest that (1) L-cystine or thiol containing compounds such as L-cysteine, 2-ME, or GSH are necessary for effective IL-2-activation of human NK cells, (2) these compounds must be functional proton-donors, i.e., reducing agents, implying regulation of the IL-2 activation pathway by oxidation-reduction, and (3) GSH synthesis is necessary for the activation. Experiments were performed to begin to dissect the redox-sensitive step(s) in LAK development. Depletion of reducing agents had no effect on internalization of rIL-2. In contrast, intracellular granzyme A activity was significantly depressed in NK cells cultured with rIL-2 in L-cystine/GSH-depleted medium compared with those cultured in medium in which L-cystine levels had been replenished. The findings suggest that step(s) in the transduction of the IL-2 signal in NK cells, between the internalization of IL-2 and the maturation of the lytic mechanism, are subject to regulation by oxidation-reduction.
Yamauchi
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Division of Cellular and Gene Therapies, Food and Drug Administration, Bethesda, Maryland 20892.
Bloom
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D000818:Animals; D002478:Cells, Cultured; D003553:Cystine / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D053804:Granzymes; D007376:Interleukin-2 / Q000494:pharmacology*; D015979:Killer Cells, Lymphokine-Activated / Q000187:drug effects / Q000502:physiology*; D007694:Killer Cells, Natural / Q000276:immunology*; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000494:pharmacology*
D007376:Interleukin-2; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D003553:Cystine; D053804:Granzymes; D012697:Serine Endopeptidases; C509212:GZMA protein, human; D005978:Glutathione