GENERATION OF LYMPHOKINE-ACTIVATED KILLER ACTIVITY IN RODENTS BUT NOTIN HUMANS IS NITRIC-OXIDE DEPENDENT

The role of nitric oxide (NO) in the generation of lymphokine-activate d killer (LAK) cells was investigated. Here we report that L-arginine analog N-G-monomethyl-L-arginine (NMMA), a specific inhibitor of nitri c oxide synthase, prevents LAK cell generation from cultured rat splen ic cells. Accumulated NO ndproduct 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 CD 25 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 per formed in the presence or absence of NMMA. LAK cell generation could n o longer be blocked if NMMA was added to the rat cell cultures 24 hr a fter rIL-2 stimulation. To further confirm the role of NO in LAK cell generation, rat splenic cells were cultured in medium without L-argini ne. Under such conditions rIL-2 could not induce LAK cell generation. Hemoglobin, which is a scavenger of NO, also inhibited LAK cell genera tion. Finally, addition of sodium nitroprusside (SNP) which releases N O in cultures was able to overcome blocking effects of NMMA. To attemp t the identification of NO-producing cells, lysosomotropic agent, L-le ucine methyl ester (LME), was used. Generation of LAK cell activity wa s virtually abolished in cell cultures treated with LME. Addition of S NP to cultures, however, sufficed to restore LAK cell generation. Thes e results suggest that LAK cell precursors depend on a exogenous NO su pply from other cell types in order to display their full cytotoxic po tential. Similar results were also obtained by using mouse splenocytes as responder cells. In contrast, NMMA did net affect generation of LA K cells from human peripheral blood or spleen mononuclear cells. (C) 1 994 Academic Press, Inc.