Regulation of the expression of nitric oxide synthase and leishmanicidal activity by glycoconjugates of Leishmania lipophosphoglycan in murine macrophages.

Abstract

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...

The role(s) of lipophosphoglycan (LPG) in the establishment ofLeishmania majorinfections in mammalian hosts

“…Possibly, the role of LPG in macrophage deactivation could be redundant with GIPLs or other parasite molecules such as PPG, which has also been implicated in parasite virulence (13,14,43). To resolve these questions, it will be necessary to generate other L. major mutants defective in individual or multiple classes of candidate molecules.…”

“…Purified LPG inhibits macrophage signaling and activation in vitro (13,26,27), as revealed by monitoring key effectors such as NO and IL-12. However, infection of BALB͞c PEMs by lpg1 Ϫ did not lead to significant induction of either effector, despite destruction of Ͼ70% of the invading parasites (Fig.…”

“…However, concerns have been raised: LPG may be applied in routes and amounts that may not be physiologically relevant, and the sharing or similarity of LPG domains to those of other parasite molecules discussed above raises the issue of specificity and the possibility of cross-activity. For example, many of the functions attributed to LPG above have been ascribed also to PPG, GIPLs, and͞or GPI-anchored proteins (3,6,9,(12)(13)(14). Thus, teasing out the specific contributions of LPG within the complex milieu of the parasite glycocalyx remains a significant challenge.…”

An Effect of Parasite-Encoded Arginase on the Outcome of Murine Cutaneous Leishmaniasis

“…To test this conjecture, the amount of cellular NO ⅐ released into culture supernatants of BMMs infected with wild-type, ⌬arg, or ⌬arg[pArg] L. mexicana was assessed by measuring the amount of nitrite ion produced. Because IFN-␥ is necessary for leishmanicidal activity and transcription of iNOS (29,30), some of the cells were preincubated for 24 h with 100 U recombinant murine IFN-␥/ml. Forty-eight hours after infection was initiated, nitrite was significantly increased in IFN-␥-primed BMMs infected with the ⌬arg knockout compared with IFN-␥-primed BMMs infected with either wild-type or ⌬arg[pArg] parasites (Fig.…”

Immunization with a Recombinant Stage-Regulated Surface Protein fromLeishmania donovaniInduces Protection Against Visceral Leishmaniasis

“…rHASPB1 was then dialyzed against sterile PBS and subsequently purified on a polymyxin B agarose column (Sigma, Poole, U.K.), to eliminate possible LPS contamination. Before vaccination, batches were tested for functionally relevant LPS contamination, by assaying their ability to synergize with IFN-␥ for the induction of inducible NO synthase (31). No activity was detectable in such assays (sensitivity Ͻ1 ng/ml LPS; data not shown).…”