Generation of lymphokine-activated killer activity in rodents but not in humans is nitric oxide dependent

The role of nitric oxide (NO) in the generation of lymphokine-activated killer (LAK) cells was investigated. Here we report that L-arginine analog NG-monomethyl-L-arginine (NMMA), a specific inhibitor of nitric oxide synthase, prevents LAK cell generation from cultured rat splenic cells. Accumulated NO endproduct nitrite (NO2-), as measured in the supernatants of rat splenic cells, correlated well with the generation of LAK cells. In contrast, cell proliferation induced by rIL-2 or by Con A was not affected by NMMA. Similarly, phenotypic expression of CD25 in rIL-2-stimulated cultures was unaffected. Furthermore, we could not observe differences in percentages of CD5-CD8+ cells (NK and LAK cell phenotype markers in rats) between rIL-2-stimulated cultures performed in the presence or absence of NMMA. LAK cell generation could no longer be blocked if NMMA was added to the rat cell cultures 24 hr after rIL-2 stimulation. To further confirm the role of NO in LAK cell generation, rat splenic cells were cultured in medium without L-arginine. Under such conditions rIL-2 could not induce LAK cell generation. Hemoglobin, which is a scavenger of NO, also inhibited LAK cell generation. Finally, addition of sodium nitroprusside (SNP) which releases NO in cultures was able to overcome blocking effects of NMMA. To attempt the identification of NO-producing cells, lysosomotropic agent, L-leucine methyl ester (LME), was used. Generation of LAK cell activity was virtually abolished in cell cultures treated with LME. Addition of SNP to cultures, however, sufficed to restore LAK cell generation. These results suggest that LAK cell precursors depend on a exogenous NO supply from other cell types in order to display their full cytotoxic potential. Similar results were also obtained by using mouse splenocytes as responder cells. In contrast, NMMA did not affect generation of LAK cells from human peripheral blood or spleen mononuclear cells.