Edinburgh Napier University

  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 phosphogly-can 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 parasites through their ability to regulate the expression of iNOS by macrophages. Leishmania are digenetic parasites that alternate between the sandfly vector as a free-living flagellate form, the promasti-gote, and the macrophage phagolysosome as obligate intra-cellular amastigotes (for review, see ref. 1). Parasite survival depends on a number of factors including manipulation of the host immune system such that the host cells, macrophages, either do not receive or do not act upon appropriate signals (for reviews, see refs. 2 and 3). Resistance to 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 phos-phodisaccharide repeats (18, 19). Leishmania mexicana, Leish-mania donovani, and L. major release hydrophilic phosphoglycan (PG), and LPG PG epitopes are also found on some surface proteins such as secreted acid phosphatase from L. mexicana (20). LPG and related molecules from different Leishmania species may interact either directly with carbohydrate-binding sites of macrophage receptors (21-23) or indirectly with the complement receptors CR1 and CR3 (24, 25). Our present study demonstrates that LPG can synergize with IFN-,y for the induction of iNOS expression in murine mac-rophages in vitro. However, incubation of macrophages with LPG before activation with LPG and IFN-,y led to the inhibition of expression of iNOS. The expression of iNOS is directly correlated with leishmanicidal activity of the macro-phages. The iNOS regulatory activity of LPG is contained within the PG moiety. MATERIALS AND METHODS Materials. PGs (shown in Table 1) were synthesized as described by Nikolaev et al. (26-28). Murine recombinant IFN-,ywas provided by G. Adolf (Ernst-Boehringer-Institut fur Arzneimittel-Forschung, Vienna). LPS, from Salmonella en-teritidis, and Limulus amoebocyte lysate kit (E-Toxate) were purchased from Sigma. [methyl-3H]Thymidine (5 Ci/mmol; 1 Ci = 37 GBq) and [3H]adenine (23 Ci/mmol) were obtained 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 Lon-don). 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). J774 cells were dispensed in 24-well plates (5 x 105 cells per well; 600 ,ul per well), forming an adherent macrophage monolayer for studies of their leishmanicidal activity. NO-production by J774 cells (1 x 105 cells per well) was assessed in flat-bottomed 96-well plates (200 ,ul per well). Cell viability was determined after glycolipid/glycan treatment using Trypan blue exclusion and the [3H]adenine release assay as described by Andreoli et al. (29). Parasites. Promastigotes of L. major LV39 strain were maintained at 28°C in Schneider's Drosophila medium (GIBCO) supplemented with 10% heat-inactivated FCS. Par-Abbreviations: LPG, lipophosphoglycan; PG, phosphoglycan; sPG, synthetic phosphoglycan; iNOS, cytokine-inducible NO synthase; IFN-,y, interferon y; TNF-a, tumor necrosis factor a; LPS, lipo-polysaccharide;L-NMMA, L-NG-monomethyl-arginine; D-NMMA, D-NG-monomethyl-arginine. tPresent address: Department of Biological Sciences, Napier University , Edinburgh, EH10 5DT, Scotland. 'To whom reprint requests should be addressed. 10984 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.