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Lipophosphoglycan (LPG) glycoconjugates from promastigotes of Leishmania were not able to induce the expression of the cytokine-inducible nitric oxide synthase (iNOS) by the murine macrophage cell line, J774. However, they synergize with interferon y to stimulate the macrophages to express high levels of iNOS. This synergistic effect was critically time-dependent. Preincubation of J774 cells with the LPG glycans 4-18 h before stimulation with interferon y resulted in a significant reduction in the expression of iNOS mRNA and of NO synthesis, compared with cells preincubated with culture medium alone. The regulatory effect on the induction of iNOS by LPG is located in the LPG phosphoglycan disaccharide backbone. Synthetic fragments of this backbone had a similar regulatory effect on NO synthesis. Further, the production of NO by activated macrophages in the present system was correlated directly with the leishmanicidal capacity of the cells. These data therefore demonstrate that LPG glycoconjugates have a profound effect on the survival of Leishmania Leishmania major infection in the murine model is directly associated with the expression of cytokine-inducible NO synthase (iNOS) (4-7).Macrophages express iNOS following activation by a variety of immunological stimuli such as interferon y (IFN-'y), tumor necrosis factor a (TNF-a), and bacterial lipopolysaccharide (LPS) (for reviews, see refs. 8-10). iNOS catalyzes the synthesis of high concentrations of NO from L-arginine and molecular oxygen (for review, see ref. 11), and NO is involved in the killing of a range of microorganisms (for reviews, see refs. 12-14), of which L. major is an example (15-17). We report here that lipophosphoglycan (LPG), a predominant surface molecule of promastigotes, can regulate the expression of iNOS and influence the survival of the parasites.The basic LPG structure of all Leishmania species consists of four domains: (i) a 1-O-alkyl-2-lysophosphatidyl(myo)inositol anchor; (ii) a hexasaccharide core; (iii) a polymer of repeating phosphodisaccharides of galactose and mannose; and (iv) a neutral mannose cap (see Fig. 1), with some species specific differences in the carbohydrate side-chains of the helical phosphodisaccharide repeats (18,19 from Amersham. Phosphatidylinositol-specific phospholipase C (PI-PLC) was purchased from Oxford Glycosystems (Abingdon, U.K.). L-NG-monomethyl-arginine (L-NMMA), an inhibitor of NO synthase, and D-NG-monomethyl-arginine (D-NMMA), its inert enantiomer, were kindly provided by S. Moncada (The Cruciform Project, University College London). All other reagents were of analytical grade.Cell Culture. The murine macrophage cell line J774 was obtained from the American Type Culture Collection and was passaged in DMEM containing 2 mM L-glutamine, 100 units/ml penicillin, 100 ,tg/ml streptomycin, and 10% heat-inactivated fetal calf serum (FCS) 'To whom reprint requests should be addressed. 10984The publication costs of this article were defrayed in part by page charge payment. This article must the...

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SU M M A RYNitric oxide (NO) derived from L-arginine by the catalytic action of inducible NO synthase (iNOS) plays an important role in killing parasites. Many cell types express high levels of iNOS when activated by a number of immunological stimuli which include interferon-gamma (IFN-), tumour necrosis factor alpha, and lipopolysaccharide. IFN-is typically produced by the Th1 subset of CD4+ T cells, whose di¡erentiation depends on interleukin-12 (IL-12) produced by macrophages. Mice with a disrupted iNOS gene were highly susceptible to Leishmania major infection compared with similarly infected control wild-type mice.The mutant mice developed signi¢cantly higher levels of TH1-cell response compared with the control mice, suggesting that NO is likely to be the e¡ector molecule in the immunological control of this and other intracellular parasitic infections. To ensure their survival, the Leishmania parasites have evolved e¡ective means to inhibit NO synthesis. The highly conserved major surface glycolipids, glycoinositol-phospholipids and lipophosphoglycan (LPG), of Leishmania are potent inhibitors of NO synthesis. Furthermore, LPG can also inhibit IL-12 synthesis, thereby indirectly blocking the induction of iNOS. The evolutionary and therapeutic implications of these ¢ndings are discussed. I N T RODUC T IO NThere is considerable current interest in nitric oxide (NO) which plays an important role in a variety of biological functions (Moncada & Higgs 1993; Liew & Cox 1991;Nathan & Xie 1994;Bredt & Snyder 1994;Marletta 1994;). These include platelet aggregation, neurotransmission and cytotoxicity. NO is derived from L-arginine together with molecular oxygen in a reaction catalysed by the enzyme NO synthase (NOS), with NADPH and tetrahydrobiopterin as cofactors. NO is very unstable with a half-life between 3 and 15 s, and it is usually measured as its oxidative products, nitrite and nitrate, in culture supernatants or serum. NOS can be competitively inhibited by L-arginine analogues such as L-N G -monomethyl arginine (L-NMMA).NO is derived from the guanidino nitrogen of Larginine. L-NMMA has a methyl group at the guanidino nitrogen, therefore preventing the utilization of this nitrogen for NO synthesis. The inhibition is highly speci¢c in that the D-enantiomer, D-NMMA, is completely inert. The availability of NOS speci¢c inhibitors such as L-NMMA has contributed greatly to study in the NO ¢eld, helping it to become one of the most exciting and proli¢c areas of biomedical research.There are three isoforms of NOS, the neuronal form (nNOS), the endothelial form (eNOS), and the inducible form (iNOS). They share a number of common structural features such as NADPH-, FAD-, FMNand calmodulin-binding domains, but otherwise have little amino acid sequence homology. These are relatively large molecules, with molecular weights of 150 kDa for nNOS, and 130 for eNOS and iNOS. Furthermore, homodimerization is required for full biological activity. So far, the 3D structure of NOS is not available, nor is the precise L-arginine...