Echinococcus granulosus Antigen B binds to monocytes and macrophages modulating cell response to inflammation

Background: Antigen B (EgAgB) is an abundant lipoprotein released by the larva of the cestode Echinococcus granulosus into the host tissues. Its protein moiety belongs to the cestode-specific family known as hydrophobic ligand binding protein (HLBP), and is encoded by five gene subfamilies (EgAgB8/1-EgAgB8/5). The functions of EgAgB in parasite biology remain unclear. It may play a role in the parasite's lipid metabolism since it carries host lipids that E. granulosus is unable to synthesise. On the other hand, there is evidence supporting immuno-modulating activities in EgAgB, particularly on innate immune cells. Both hypothetical functions might involve EgAgB interactions with monocytes and macrophages, which have not been formally analysed yet. Methods: EgAgB binding to monocytes and macrophages was studied by flow cytometry using inflammation-recruited peritoneal cells and the THP-1 cell line. Involvement of the protein and phospholipid moieties in EgAgB binding to cells was analysed employing lipid-free recombinant EgAgB subunits and phospholipase D treated-EgAgB (lacking the polar head of phospholipids). Competition binding assays with plasma lipoproteins and ligands for lipoprotein receptors were performed to gain information about the putative EgAgB receptor(s) in these cells. Arginase-I induction and PMA/LPS-triggered IL-1 beta, TNF-alpha and IL-10 secretion were examined to investigate the outcome of EgAgB binding on macrophage response. Results: Monocytes and macrophages bound native EgAgB specifically; this binding was also found with lipid-free rEgAgB8/1 and rEgAgB8/3, but not rEgAgB8/2 subunits. EgAgB phospholipase D-treatment, but not the competition with phospholipid vesicles, caused a strong inhibition of EgAgB binding activity, suggesting an indirect contribution of phospholipids to EgAgB-cell interaction. Furthermore, competition binding assays indicated that this interaction may involve receptors with affinity for plasma lipoproteins. At functional level, the exposure of macrophages to EgAgB induced a very modest arginase-I response and inhibited PMA/LPS-mediated IL-1 beta and TNF-alpha secretion in an IL-10-independent manner. Conclusion: EgAgB and, particularly its predominant EgAgB8/1 apolipoprotein, are potential ligands for monocyte and macrophage receptors. These receptors may also be involved in plasma lipoprotein recognition and induce an anti-inflammatory phenotype in macrophages upon recognition of EgAgB

The chimerical multi-component Q protein from Leishmania in the absence of adjuvant protects dogs against an experimental Leishmania infantum infection.

The protective potential against Leishmania infection of the Leishmania chimerical Q protein administered as a single (Q) or double dose (Q+Q) without adjuvant was analyzed in a double-blind placebo controlled experiment in dogs. During vaccination the protein induced an intense early anti-Q response but no reactivity against total Leishmania infantum proteins was detected. Several end-points were taken into consideration. In the vaccinated animals the amount and intensity of clinical symptoms was lower than in the control group. Pathological signs of disease were observed in liver, kidney and spleen of all dogs from the control group in contrast to the normal appearance of the organs of the vaccinated animals. Vaccination was able to induce parasite clearance in most dogs. Only 1/7 dog was parasite DNA positive in skin in the Q group in contrast to 6/7 dogs in control and 4/7 in Q+Q. Significant anti-SLA clearance was observed in the vaccinated animals at the end of the study. Differences between control and vaccinated animals were also observed at the biochemical level, DTH and nitrite oxide production.

The inhibition of arginase by N(omega)-hydroxy-l-arginine controls the growth of Leishmania inside macrophages.

Polyamine synthesis from l-ornithine is essential for Leishmania growth. We have investigated the dependence of Leishmania infection on arginase, which generates l-ornithine, in macrophages from BALB/c, C57BL/6, and nitric oxide synthase II (NOS II)-deficient mouse strains. We have found that N(omega)-hydroxy-l-arginine (LOHA), a physiological inhibitor of arginase, controls cellular infection and also specifically inhibits arginase activity from Leishmania major and Leishmania infantum parasites. The effect was proportional to the course of infection, concentration dependent up to 100 microM, and achieved without an increase in nitrite levels of culture supernatants. Moreover, when the l-arginine metabolism of macrophages is diverted towards ornithine generation by interleukin 4-induced arginase I, parasite growth is promoted. This effect can be reversed by LOHA. Inhibition of NOS II by N(G)-methyl-l-arginine (LNMMA) restores the killing obtained in the presence of interferon (IFN)-gamma plus lipolysaccharide (LPS), whereas the nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-3-oxide-1-oxyl (carboxy-PTIO) was without effect. However, exogenous l-ornithine almost completely inhibits parasite killing when added in the presence of LOHA to macrophages from NOS II-deficient mice or to BALB/c-infected cells activated with IFN-gamma plus LPS. These results suggest that LOHA is an effector molecule involved in the control of Leishmania infection. In addition, macrophage arginase I induction by T helper cell type 2 cytokines could be a mechanism used by parasites to spread inside the host.

A defect in interleukin-10 leads to enhanced malarial disease in Plasmodium chabaudi chabaudi infection in mice.

Infection of interleukin-10 (IL-10)-nonexpressing (IL-10(-/-)) mice with Plasmodium chabaudi chabaudi (AS) leads to exacerbated pathology in female mice and death in a proportion of them. Hypoglycemia, hypothermia, and loss in body weight were significantly greater in female IL-10(-/-) mice than in male knockout mice and all wild-type (WT) mice during the acute phase of infection. At this time, both female and male IL-10(-/-) mice produced more gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and IL-12p40 mRNA than their respective WT counterparts. Inactivation of IFN-gamma in IL-10(-/-) mice by the injection of anti-IFN-gamma antibodies or by the generation of IL-10(-/-) IFN-gamma receptor(-/-) double-knockout mice resulted in reduced mortality but did not affect body weight, temperature, or blood glucose levels. The data suggest that IFN-gamma-independent pathways may be responsible for these pathological features of P. chabaudi malaria and may be due to direct stimulation of TNF-alpha by the parasite. Since male and female knockout mice both produce more inflammatory cytokines than their WT counterparts, it is likely that the mortality seen in females is due to the nature or magnitude of the response to these cytokines rather than the amount of IFN-gamma or TNF-alpha produced.

Involvement of protein kinase A in the induction of arginase in murine bone marrow-derived macrophages.

Arginase is induced in bone marrow-derived macrophages by agents that increase the intracellular concentrations of cAMP (Br-cAMP, prostaglandin E2) and, in their presence, the LPS induced NO synthesis is down regulated. Moreover, interleukin 10 which induces arginase in macrophages is able to increase the cAMP-dependent protein kinase A activity. In contrast, suppressors of NOS synthesis like protein kinase C inhibitors and calmodulin antagonists (W7), or NO activators (A23187) have no effect on the induction of arginase by LPS. These results strongly suggest that PKA is involved in the induction of arginase and supports the hypothesis that there is a reciprocal regulation of these two enzymes that drives the macrophages towards opposite functional states.

Reciprocal regulation of the nitric oxide synthase/arginase balance in mouse bone marrow-derived macrophages by TH1 and TH2 cytokines.

Activation with lipopolysaccharide induces macrophages to produce the enzymes arginase and nitric oxide (NO) synthase. Both enzymes use as a substrate the amino acid L-arginine, which can be either hydrolyzed by arginase to urea and ornithine or oxidized by NO synthase to NO and citrulline. NO is important in the bactericidal and cytotoxic activities of macrophages. An equivalent functional role of arginase and its products is not known. We tested the induction of arginase in bone marrow-derived macrophages by endogenous mediators that are known to induce NO synthase, such as interferon-gamma (IFN-gamma), or suppress the induction of this enzyme, such as interleukin (IL)-4, IL-10, and prostaglandin E2 (PGE2). We find that PGE2 and the TH2 cytokines IL-4 and IL-10 are potent inducers of arginase. In contrast, the TH1 cytokine IFN-gamma does not induce arginase. Simultaneous application of both types of mediators leads to reduced induction of both arginase and NO synthase. Exposure of macrophage cultures to inducers of NO synthase exhausts their ability to respond subsequently to inducers of arginase. Conversely, exposure of the cells to inducers of arginase exhausts their ability to respond subsequently to inducers of NO synthase. The results are consistent with a competition of both enzymes for their substrate, L-arginine, with a reciprocal inhibition in the induction of both enzymes, or a combination of both phenomena. The enzymes NO synthase and arginase appear to define two alternate functional states of macrophages, induced by TH1 and TH2 cytokines, respectively.

Arginase induction by suppressors of nitric oxide synthesis (IL-4, IL-10 and PGE2) in murine bone-marrow-derived macrophages.

The present study addresses the regulatory mechanisms involved in the arginine metabolism of macrophages by arginase and nitric oxide synthase. Induction of both enzymes with LPS or by mixed lymphocyte reaction has been reported. Here, we demonstrate that these enzymes can be independently induced in murine bone-marrow-derived macrophages with the appropriate agonists. Arginase expression is specifically triggered by IL-4, IL-10, PGE2 as well as non-toxic or detoxified LPS. Conversely, IFN gamma induces only NO synthesis in these cells. The results demonstrate that the metabolism of arginine in macrophages is controlled by TH1/TH2-dependent cytokines and suggest a regulatory role of arginase on the NO synthesis by intracellular substrate depletion.

Determination of arginase activity in macrophages: a micromethod.

We propose a modification of Schimke's method for urea determination as a valuable micromethod for measuring arginase in activated macrophages. The method exhibits the following advantages: (a) it uses small amounts of samples (approximately 25,000 macrophages per assay); (b) it does not interfere with other related metabolites that are also present in the activated macrophage such as citrulline or arginine; (c) saturating concentrations of the substrate arginine can be used; and (d) it is much more sensitive than Schimke's method and can detect small amounts of urea, in the order of 0.02 mumol.

Parallel induction of nitric oxide and glucose-6-phosphate dehydrogenase in activated bone marrow derived macrophages.

The production of nitric oxide (NO.) and the induction of glucose-6-phosphate dehydrogenase by lipopolysaccharides (LPS) from different sources was studied in bone marrow derived macrophages (BMM phi). NO. production was found to be linked to the induction of glucose-6-phosphate dehydrogenase, suggesting the possible involvement of this enzyme in the cytotoxic mechanism resulting from the release of NO. by activated macrophages.