NITRIC OXIDE AND HOST DEFENCE 573 Cytokines that regulate macrophage production of nitrogen oxides and expression of antileishmanial activities C.A. Nacy, B.J. Nelson, M.S. Meltzer and S.J. Green Department of Cellular Immunology, Walter Reed Army Institute of Research, 9620 Medical Center Drive, Rockville, MD 20850 (USA) Resident tissue and inflammatory macrophages produce low but detectable levels of nitrogen oxides de novo, and serve successfully as host cells for Leish- mania major, a protozoan parasite that replicates in phagolysosomes (Fortier et al., 1983). In contrast, activated macrophages release markedly increased levels of NO 2 and NO~ (Stuehr and Marietta, 1987; Drapier et al., 1988), and the intracellular en- vironment of these cells is extremely inhospitable for L. major survival (Nacy et al., 1981). We recently correlated the intracellular microbicidal capacity of activated macrophages with production of nitrogen oxides (Green et al., 1990a): in all cases, macro- phages that kill intracellular amastigotes release high levels of NO 2 into the culture medium. The effec- tor molecule for destruction of the parasite is NO, the highly reactive and unstable intermediate of the nitrogen oxidation of L-arginine (Hibbs eta!., !987; Green et al., 1990a; Liew et al., 1990a). A competi- tive inhibitor of L-arginine, N~'-monomethyl-L - arginine (NGMMLA), completely blocks synthesis of NO and inhibits killing of L. major (Green et al., 1990a). Nitrogen oxidation as a requisite for destruction of intracellular Leishmania in activated macrophages is confirmed by different methodologies in several laboratories: (a) specificity of NGMMLA inhibition of killing is verified through competition with excess L-arginine (Green et al., 1990a); (b) macrophage an- tileishmarial activity is directly dependent upon the concentration of extracellular L-argin~ne: culture medium depleted of L-arginine by arginase does not sustain this macrophage effector function (Green et al., 1990a; Mauel et al., 1991); (c) levels of macro- phage cytotoxicity against Leishmania strongly corre- late with release c f NO 2 (Green et al., 1990a; Liew et al., 1990a; Mal.el et al., 1991 ; Roach et al., 1991); (d) NO gas reduces viability of L. major promastigotes more than 100-fold (Liew et al., 1990a) and NO chemically generated from NaNO 2 at pH 5 suppresses replication of L. enriettii promastigotes (Mauel et al., 1991); and (e) exogenously added iron (in the form of FeSO4) inhibits macrophage an- tileishmanial activity (Mauel et ai., 1991). Excess iron may protect iron-dependent enzymes susceptible to NO inactivation. Target enzymes in Leishmania, enzymes with labile iron prosthetic groups necessary for DNA replication, the citric acid cycle and mitochondrial respiration, are likely to be similar to those of tumour target cells. Leishmania also have at least one other enzyme with an iron group susceptible to NO inactivation" superoxide dis- mutase (SOD; Meshnick and Eaton, 1981). The SOD of most eukaryotic cells requires manganese, not iron. NO inactivation of SOD in Leishmania could markedly increase parasite vulnerability to attack with reactive oxygen intermediates. In this instance, NO would be an accessory factor for the more prox- imate effector molecules, H202 and O 2 . Both synthesis of NO and induction of macro- phage cytotoxic activity require multiple signals deli- vered to macrophages in a defined sequence (Ruco and Meltzer, 1978; Drapier et al., 1988). Resident peritoneal macrophages from untreated mice exposed only to IFNT release little NO 2 and are not tumori- cidal. IFNT-treated cells respond, however, to small quantities of a second signal (such as muramyl dipep- tide, MDP) for tumoricidal activity and production of NO 2 equivalent to that released by BCG- activated macrophages (Drapier et al., 1986, 1988). No activity is observed with macrophages treated with MDP alone. Resident peritoneal macrophages treated with IFNT or bacterial endotoxic lipopolysac- charities (LPS) alone also produce minimal levels of NO 2 (<5 ~.M/106cells/72h) and fail to develop tumoricidal activity. The same cells exposed to both IFN~ and LPS produce high levels of NO 2 (> > 30 ~M/106cells/72h) and develop potent cyto- toxic activity (Green et al., 1990b). Induction of mac- rophage microbicidal activity by IFNT exposed an apparent paradox" resident peritoneal maccrophages treated with IFNT alone and then exposed to L. major amastigotes produce high levels of NO 2