We have previously reported that mice lacking inducible nitric oxide synthase (NOS2) developed enhanced Th1 cell responses. We now investigated the mechanism by which NO modulates Th1 cells differentiation. Peritoneal macrophages from NOS2-deficient mice infected with Leishmania major in vivo or stimulated with IFN-gamma or lipopolysaccharide (LPS) in vitro produced significantly higher levels of IL-12 than those from heterozygous or wild-type mice. A macrophage cell line, J774, produced significant amounts of IL-12 following activation with LPS, or LPS plus IFN-gamma. This could be markedly enhanced by the NOS inhibitor L-NG monomethyl arginine (L-NMMA), but profoundly inhibited by the NO-generating compound S-nitroso-N-acetyl-penicillamine (SNAP). The effect of NO in this system is selective, since SNAP enhanced and L-NMMA decreased TNF-alpha synthesis by LPS-activated J774 cells. The differential effect of NO on IL-12 and TNF-alpha is at the transcriptional level and is activation dependent. Since IL-12 is a major inducer of Th1 cells which produce IFN-gamma that can activate macrophages to produce IL-12, our data demonstrate that NO can be an inhibitor of this feedback loop, preventing the excessive amplification of Th1 cells which are implicated in a range of immunopathologies.
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...
Summary MR_L/MP-lpr/lpr (MRL/lpr)NO is a critical mediator of a variety of biological functions, including vascular relaxation, platelet aggregation, neurotransmission, tumoricidal and microbicidal activity, and immunosuppression (6-10). It is also implicated in a range of immunopathologies (11)(12)(13). NO is derived from the guanidino nitrogen atom(s) (14) and molecular oxygen (15, 16) in a reaction catalyzed by the enzyme NOS. There are three major isoforms of NOS (17): the neuronal form
It is now generally accepted that IFN-gamma, secreted by Th1 cells, is the most potent cytokine leading to macrophage activation and host resistance against infection with the intracellular protozoan parasite Leishmania. It is also established that IL-12 is a critical cytokine involved in the differentiation and expansion of Th1 cells. Therefore, the ability of Leishmania parasites to actively suppress IL-12 production by host macrophages may be an important strategy for parasite survival. Here we report that a major parasite cell surface molecule, phosphoglycan (PG), of Leishmania could selectively inhibit the synthesis of IL-12(p40, p70) by activated murine macrophages. Furthermore, synthetic PG (sPG) was able to inhibit IL-12 release in a dose-dependent manner. Inhibition was dependent on the galactose(beta1-4)mannose(alpha1)-PO4 repeating units and not the glycophosphoinositol lipid anchor of lipophosphoglycan. At the concentration used, sPG had no effect on the release of TNF-alpha or IL-6 in activated macrophages. The inhibition of IL-12(p40) production was at the transcriptional level, but was not mediated through NF kappaB inhibition. These data demonstrate that PG may be an important molecule for the establishment and survival of the parasite in permissive hosts.
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