Reduced pathology following infection with transgenic Leishmania major expressing murine CD40 ligand

Leishmanization is the inoculation of live Leishmania into the host to vaccinate against subsequent infections. This approach has been largely discontinued due to safety concerns. We have previously shown that combining CD40 ligand (CD40L) with Leishmania antigen preferentially induces a type 1 immune response and provides some protection to vaccinated mice (G. Chen, P. A. Darrah, and D. M. Mosser, Infect. Immun. 69:3255-3263, 2001). In the present study, we developed transgenic L. major organisms which express and secrete the extracellular portion of CD40L (L. major CD40LE). We hypothesized that these organisms would be less virulent but more immunogenic than wild-type organisms and therefore be more effective at leishmanization. Transgenic parasites expressing CD40L mRNA and protein were developed. BALB/c mice infected with these parasites developed significantly smaller lesions containing fewer parasites than animals infected with wild-type organisms. Infection of resistant C57BL/6 mice with low doses of transgenic parasites induced a significant amount of protection against subsequent high-dose infection with wild-type organisms. These results demonstrate that transgenic organisms expressing CD40L are less virulent than wild-type organisms while retaining full immunogenicity.     

Antileishmanial activity of a naphthoquinone derivate against promastigote and amastigote stages of Leishmania infantum and Leishmania amazonensis and its mechanism of action against L. amazonensis species

Parasitology Research - Leishmaniasis has become a significant public health issue in several countries in the world. New products have been identified to treat against the disease; however,...     

CD40-CD40 ligand costimulation is not required for initiation and maintenance of a Th1-type response to Leishmania major infection

Although previous studies demonstrated a requirement for CD40-CD40 ligand (CD40L) interaction in the development of resistance to Leishmania infection, we recently showed that mice lacking the gene for CD40L (CD40L(-/-) mice) can control Leishmania major infection when they are infected with reduced numbers of parasites. In this study, we examine the cytokine pattern in healing versus nonhealing CD40L(-/-) mice and investigated whether CD40 activation is required for resistance to reinfection. We observed that CD4(+) cells in healed CD40L(-/-) mice produce high levels of gamma interferon compared to cells from nonhealing, high-dose-inoculated mice. In addition, we observed a higher frequency of interleukin-12 (IL-12)- producing cells and a reduced number of IL-4-producing cells in mice infected with reduced numbers of parasites. Importantly, we found that healed CD40L(-/-) mice are highly resistant to reinfection with a large parasite inoculum. In addition, by comparing the cytokine patterns at an early and late stage of infection in nonhealing CD40L(-/-) mice, we demonstrated that nonhealing CD40L(-/-) mice produce a weak Th1-type response during the early stage of infection, but this response wanes as a Th2-type response emerges during late stages of infection. Anti-IL-4 antibody treatment, starting either at the beginning of infection or at week 4 postinfection enabled CD40L(-/-) mice to control a high-dose infection. Together, these results show that CD40-CD40L interaction, although important for IL-12 production in high-dose infections, is not required for either the development or maintenance of resistance in mice infected with reduced numbers of parasites.     

Cyclosporin A enhances elimination of intracellular L. major parasites by murine macrophages.

In this study we analyzed the influence of cyclosporin A (CyA) on the process of phagocytosis of L. major promastigotes and amastigotes by inflammatory peritoneal macrophages (MP) from BALB/c mice. Our data clearly demonstrate that CyA profoundly enhanced the degradation by peritoneal MP of both intracellular L. major promastigotes and amastigotes. This effect was T cell-independent and specifically associated with CyA, since the similarly structured cyclosporin F (CyF) was ineffective. CyA did not alter the replication and infectivity of extracellular parasites. From these results we conclude that the inhibition of intracellular parasite replication in the presence of CyA substantially contributes to the previously described suppressive effect of CyA on the development of L. major-induced lesions in BALB/c mice.     

Catalase inhibits nitric oxide synthesis and the killing of intracellular Leishmania major in murine macrophages.

Mouse peritoneal macrophages activated with interferon-gamma (IFN-gamma) and lipopolysaccharide produce substantial amounts of nitric oxide (NO), which correlates with the elimination of the intracellular protozoan parasite Leishmania major. Both the production of NO and the leishmanicidal function of the activated macrophages can be significantly inhibited by catalase in a dose- and time-dependent manner. These results could not be interpreted by the reduction of H2O2 by catalase since the removal of H2O2 by the addition of glutathione peroxidase had no effect on the NO synthesis or the leishmanicidal function of activated macrophages. Furthermore, catalase did not affect the induction of NO synthase in IFN-gamma-activated macrophages. In contrast, the inhibition of NO synthesis and leishmanicidal activity by catalase was reversed in a dose-dependent manner by the addition of tetrahydrobiopterin, a cofactor of NO synthase. Taken together, these results not only further support the central role of NO as the cytotoxic moiety, but also suggest that hydrogen peroxide may interfere with NO production by affecting the levels of cofactor needed for its synthesis.     

Interleukin-7 enhances antimicrobial activity against Leishmania major in murine macrophages.

Recently, it has been shown that interleukin-7 (IL-7) is able to induce secretion of cytokines and tumoricidal activity by human monocytes. This study shows that treatment of murine macrophages infected with Leishmania major with IL-7 without any other stimulus reduced the percentage of infected cells, as well as the parasite burden per cell, in a dose-dependent manner to a limited degree (45% reduction of the number of amastigotes per 100 macrophages). Simultaneous treatment of macrophages with gamma interferon and IL-7 led to nearly complete (> 99%) elimination of amastigotes. Addition of anti-tumor necrosis factor alpha or N omega-monomethyl-L-arginine acetate reversed the leishmanicidal effects of IL-7, and production of nitric oxide was induced in the presence of IL-7.     

Leishmania amazonensis impairs DC function by inhibiting CD40 expression via A2B adenosine receptor activation

Dendritic cells (DCs) play an essential role in the modulation of immune responses and several studies have evaluated the interactions between Leishmania parasites and DCs. While extracellular ATP exhibits proinflammatory properties, adenosine is an important anti-inflammatory mediator. Here we investigated the effects of Leishmania infection on DC responses and the participation of purinergic signalling in this process. Bone marrow-derived dendritic cells (BMDCs) from C57BL/6J mice infected with Leishmania amazonensis, Leishmania braziliensis or Leishmania major metacyclic promastigotes showed decreased major histocompatibility complex (MHC) class II and CD86 expression and increased ectonucleotidase expression as compared with uninfected cells. In addition, L. amazonensis-infected DCs, which had lower CD40 expression, exhibited a decreased ability to induce T-cell proliferation. The presence of MRS1754, a highly selective A(2B) adenosine receptor antagonist at the time of infection increased MHC class II, CD86 and CD40 expression in L. amazonensis-infected DCs and restored the ability of the infected DCs to induce T-cell proliferation. Similar results were obtained through the inhibition of extracellular ATP hydrolysis using suramin. In conclusion, we propose that A(2B) receptor activation may be used by L. amazonensis to inhibit DC function and evade the immune response.     

Heme requirement and acquisition by extracellular and intracellular stages of Leishmania mexicana amazonensis

The inability to synthesize heme, a well known metabolic defect of trypanosomatid protozoa, accounts for their growth requirement for heme compounds in vitro. We now extend this finding to a pathogen Leishmania mexicana amazonensis, especially the intracellular replicative stage of amastigotes in the macrophage. We measured the level of heme and its biosynthetic enzymes, aminolevulinate dehydratase and porphobilinogen deaminase in the parasites and in infected and non-infected macrophages of J774G8 line. Succinylacetone was used to inhibit heme biosynthesis. Leishmanias transform and grow only in medium containing either heme (usually supplied as hemin) or protoporphyrin IX (the latter is leishmanicidal at high concentrations). We detected 1.2, 8.5 and 25 pmol mg-1 protein of heme in amastigotes, promastigotes and macrophages, respectively. The activities of porphobilinogen deaminase and aminolevulinate dehydratase in macrophages were 70 and 2400 pmol h-1 mg-1 protein, respectively. Leishmania-infected macrophages gave the same results and leishmanias had negligible activities of these enzymes. Succinylacetone at 10(-9)-10(-3) M had no effect on leishmanias, but dose-dependently inhibited the activity of aminolevulinate dehydratase to a negligible level and lowered that of porphobilinogen deaminase in macrophages, resulting in a maximum of 66% reduction in intracellular heme. Amastigotes grew equally well in succinylacetone-treated and control untreated macrophages. The results suggest that L. m. amazonensis, incapable of heme biosynthesis, acquires heme exogenously from the culture medium, i.e., fetal bovine serum, independent of the heme synthesized by the macrophages.     

In vitro and in vivo activity of major constituents from Pluchea carolinensis against Leishmania amazonensis

The search for new therapeutic agents from natural sources has been a constant for the treatment of diseases such as leishmaniasis. Herein, in vitro and in vivo pharmacological activities of pure major phenolic constituents (caffeic acid, chlorogenic acid, ferulic acid, quercetin, and rosmarinic acid) from Pluchea carolinensis against Leishmania amazonensis are presented. Pure compounds showed inhibitory activity against promastigotes (IC₅₀?=?0.2–0.9 μg/mL) and intracellular amastigotes (IC₅₀?=?1.3–2.9 μg/mL). Four of them were selected after testing against macrophages of BALB/c mice: caffeic acid, ferulic acid, quercetin, and rosmarinic acid, with selective indices of 11, 17, 10, and 20, respectively. Ferulic acid, rosmarinic acid, and caffeic acid controlled lesion size development and parasite burden in footpads from BALB/c experimentally infected mice, after five injections of compounds by intralesional route at 30 mg/kg every 4 days. Pure compounds from P. carolinensis demonstrated antileishmanial properties.     

Differential production of granulocyte-macrophage colony-stimulating factor by macrophages from mice susceptible and resistant to Leishmania mexicana amazonensis.

The present results demonstrate that macrophages from mice susceptible to infection with Leishmania mexicana amazonensis sustain a higher production of granulocyte-macrophage colony-stimulating factor (GM-CSF) throughout the in vitro infection than macrophages from a resistant strain. Resident macrophages from BALB/c and C57B1/10 mice were infected with promastigotes of L. mexicana amazonensis and the amount of biologically active GM-CSF was measured in the supernatants collected at different times of infection. Measurements were made by bone marrow and GM-CSF/interleukin-3 addicted cell proliferation. Because GM-CSF is a disease-exacerbating cytokine, its differential production by infected macrophages may be one of the mechanisms defining resistance or susceptibility to a leishmanial infection.     

Interleukin-10 and interleukin-4 inhibit intracellular killing of Leishmania infantum and Leishmania major by human macrophages by decreasing nitric oxide generation

The host response to Leishmania infection is regulated by a specific pattern of local cytokine production. We investigated the effect of interleukin (IL)-10 and IL-4 on the leishmanicidal activity of human macrophages (M?). As with L. major, intracellular killing of L. infantum by human M? was obtained following ligation of surface CD23 or cell treatment with Interferon-γ (IFN-γ). This leishmanicidal activity required nitric oxide (NO) generation by activated M?, and it was partially mimicked by cell treatment with chemical NO donors. Addition of recombinant human IL-10 or IL-4 to CD23 mAb or IFN-γ decreased L. infantum and L. major killing by infected M?. IL-10 was more potent than IL-4 in inhibiting the leishmanicidal activity of human M?. Inhibition of Leishmania killing by IL-4 and IL-10 correlated with decreased NO generation from M?, and was reversed when exogenous NO was added to cell cultures. Therefore, IL-10 and IL-4 down-regulate leishmanicidal activity of human M?, in part by inhibiting NO generation by these cells.     

Lipophosphoglycan blocks attachment of Leishmania major amastigotes to macrophages.

Promastigotes of the intracellular protozoan parasite Leishmania major invade mononuclear phagocytes by a direct interaction between the cell surface lipophosphoglycan found on all Leishmania species and macrophage receptors. This interaction is mediated by phosphoglycan repeats containing oligomers of beta (1-3)Gal residues specific to L. major. We show here that although amastigotes also use lipophosphoglycan to bind to both primary macrophages and a cell line, this interaction is independent of the beta (1-3)Gal residues employed by promastigotes. Binding of amastigotes to macrophages could be blocked by intact lipophosphoglycan from L. major amastigotes as well as by lipophosphoglycan from promastigotes of several other Leishmania species, suggesting involvement of a conserved domain. Binding of amastigotes to macrophages could be blocked significantly by the monoclonal antibody WIC 108.3, directed to the lipophosphoglycan backbone. The glycan core of lipophosphoglycan could also inhibit attachment of amastigotes, but to a considerably lesser extent. The glycan core structure is also present in the type 2 glycoinositolphospholipids which are expressed on the surface of amastigotes at 100-fold-higher levels than lipophosphoglycan. However, their inhibitory effect could not be increased even when they were used at a 300-fold-higher concentration than lipophosphoglycan, indicating that lipophosphoglycan is the major macrophage-binding molecule on amastigotes of L. major. In the presence of complement, the attachment of amastigotes to macrophages was not altered, suggesting that lipophosphoglycan interacts directly with macrophage receptors.     

CD40/CD40 ligand interactions are required for T cell-dependent production of interleukin-12 by mouse macrophages

We have previously shown that T cell receptor-activated mouse T helper (Th)1 clones induce the production of interleukin (IL)-12 by splenic antigen-presenting cells (APC). Here, we show that the expression of CD40L by activated T cells is critical for T cell-dependent IL-12 production by mouse macrophages. IL-12 was produced in cultures containing alloreactive Th1 clones stimulated with allogeneic peritoneal macrophages, or in cultures of splenocytes stimulated with anti-CD3. Anti-CD40L monoclonal antibodies (mAb) inhibited the production of IL-12, but not IL-2, in these cultures by ?90% and had dramatic inhibitory effects on antigen-dependent proliferation of Th1 clones. In addition, both activated T cells and a Th1 clone derived from CD40L knockout mice failed to induce IL-12 production from splenic APC or peritoneal macrophages. Finally, macrophages cultured in the absence of T cells produced IL-12 upon stimulation with soluble recombinant CD40L in combination with either supernatants from activated Th1 clones or with interferon-γ and granulocyte/macrophage colony-stimulating factor. Thus, both CD40L-dependent and cytokine-mediated signals from activated T cells are required to induce the production of IL-12 by macrophages. A blockade at the level of IL-12 production may explain, at least in part, the dramatic ability of anti-CD40L mAb to inhibit disease in animal models that are dependent upon the generation of a cell-mediated immune response. Moreover, a defect in T cell-dependent induction of IL-12 may contribute to the immune status of humans that lack functional CD40L.     

Localization and activity of different lysosomal proteases in rat macrophages infected by Leishmania amazonensis

Leishmania are protozoans of the trypanosomatidae family that cause human infections. The amastigote form of Leishmania is an obligate intracellular parasite of mononuclear phagocytes that multiplies within parasitophorous vacuoles (pv) of phagolysosomal origin. To investigate the strategies which allow Leishmania to withstand these potentially cytotoxic conditions, the distribution and activities of various lysosomal peptidases in rat macrophages infected or uninfected with Leishmania amazonensis amastigotes were studied. Specific immunoglobulins against cathepsins (cat.) B, H, L and D were used to localize these endopeptidases by immunocytochemistry. Results showed that most or even all of the secondary lysosomes in the host cell fuse with parasite-filled phagosomes, leading to translocation of the proteases in the parasitophorous vacuoles. A further study consisted in assays of five protease activities: dipeptidylpeptidases (DPP) I and II (exopeptidases), cat. B, cat. H and cat. D. Infection of macrophages was followed by a gradual increase in all these protease activities except for DPP II. These increases were apparently not related to parasite protease activities. It seems that infection by Leishmania amazonensis is followed by increased synthesis and/or reduced catabolism of host cell lysosomal proteases or alternatively by inactivation of endogenous inhibitors. Amastigote infectivity may be related, at least in part, to the development of mechanisms that allow the parasite to withstand unfavorable environmental conditions.     

Interleukin-3 induces antimicrobial activity against Leishmania amazonensis and Trypanosoma cruzi and tumoricidal activity in human peripheral blood-derived macrophages.

The ability of interleukin-3 (IL-3) to induce antimicrobial and tumoricidal activity was evaluated. Macrophages infected with two intracellular protozoa, Leishmania amazonensis or Trypanosoma cruzi, were treated with cytokines. IL-3 induced a dose-dependent enhancement of microbistasis against leishmanias, and the activity of IL-3 (100 ng/ml) was comparable to that of gamma interferon (IFN-gamma) (1,000 U/ml). In addition, IL-3 in combination with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage CSF (M-CSF) or with IFN-gamma reduced infection and lowered the required dose. IL-3 similarly activated macrophages to inhibit intracellular replication of T. cruzi. Furthermore, IL-3 induced antibody-independent tumoricidal activity against melanoma cells that was dose dependent and comparable to that of lipopolysaccharide and GM-CSF. The mechanisms by which IL-3 induced antimicrobial activity may involve at least the augmentation of oxidative capacity. IL-3, at concentrations of 0.5 ng/ml or greater, led to a significantly increased oxidative burst which paralleled the inhibition of protozoan replication. The enhancement of oxidative capacity by IL-3 (5 ng/ml or higher) was comparable to that of IFN-gamma. The induction of tumoricidal activity was associated with the production of tumor necrosis factor alpha (TNF-alpha), which in this system may feed back to enhance the macrophage inhibition of leishmanias, as demonstrated by neutralization of IL-3 activation by anti-TNF-alpha antibody. Thus, peripheral blood macrophages remain responsive to IL-3, as demonstrated by enhanced antimicrobial and tumoricidal activity. IL-3 may have potential clinical applications because of these properties and its effect on myelopoiesis.     

Vaccine-induced protection against Leishmania amazonensis is obtained in the absence of IL-12/23p40

Protozoa of the genus Leishmania are intracellular parasites of macrophages and may cause diverse clinical forms of leishmaniasis, including cutaneous, diffuse cutaneous, mucocutaneous and visceral leishmaniasis. Infection with L. major in mice indicates that a protective immune response is achieved when Th1 cells are developed. Thus, adoptive or vaccine-induced protection against leishmaniasis is largely dependent on cell-mediated immunity and IFN-gamma production. Induction of a Th1 response is dependent on the presence of IL-12 whilst lymphocytes are activated. This study was aimed at evaluating the role of IL-12 during infection with L. amazonensis and after vaccination with Leishvacin (killed Leishmania amazonensis promastigotes), since the role of this cytokine in vaccine-induced immunity with this preparation in experimental models or in humans is not yet elucidated. Hence, C57BL/6 interleukin-12-deficient mice (IL-12p40(-/-)) and wild-type controls (wt) were infected with L. amazonensis and the course of infection, parasite burden and cytokine production were compared. IL-12p40(-/-) mice were more susceptible to L. amazonensis than wt: lesions and parasite burden were larger in IL-12p40(-/-) when compared to wt. Interestingly, IL-4 was not produced in the absence of IL-12 in response to infection with L. amazonensis. To evaluate the role of IL-12 in the vaccine-induced immunity against L. amazonensis infection, IL-12p40(-/-) wt mice were vaccinated in the base of the tail and subsequently challenged with L. amazonensis in the footpads. Surprisingly, vaccinated IL-12p40(-/-) mice developed smaller lesions and had fewer parasites in footpads than non-vaccinated controls. Lymph node and spleen cells from vaccinated IL-12p40(-/-) mice did not produce high levels of IFN-gamma in response do in vitro stimulus with antigen. Hence, partial protection against infection with L. amazonensis could be obtained in the absence of functional IL-12 and a typical Th1 response.     

Freeze-fracture and cytochemistry study of the interaction between Leishmania mexicana amazonensis and macrophages

The process of interaction between macrophages and promastigote and amastigote forms of Leishmania mexicana amazonensis was analyzed using freeze fracture and cytochemistry. The promastigotes inside endocytic vacuoles of macrophages presented an altered distribution of intramembranous particles and a wavy aspect of the plasma membrane. However, amastigotes did not show such alterations. The membrane alterations are probably caused by intracellular cell lysis of the promastigotes by the macrophages. An accumulation of intramembranous particles was seen in the plasma membrane of amastigote forms in the area of adhesion to the macrophages. The parasitophorous vacuole membrane had intramembranous particles randomly distributed. The enzyme activity of Mg++-ATPase, 5'-nucleotidase and NAD(P)H-oxidase was cytochemically detected, at the ultrastructural level, in normal mouse peritoneal macrophages and in macrophages infected with Leishmania mexicana amazonensis. Mg++-ATPase and 5'-nucleotidase are uniformly distributed throughout the macrophage's plasma membrane but were not detected in the membrane lining endocytic vacuoles containing ingested parasites (parasitophorous vacuole). NAD(P)H-oxidase activity was seen in those portions of the macrophage's plasma membrane which enter in direct contact with parasites and also in association with the membrane of the parasitophorous vacuole. The amount of reaction product, indicative of NAD(P)H-oxidase activity, was larger in macrophages which interacted with the promastigote than in those which interacted with the amastigote form of L. mexicana amazonensis. Concanavalin A binding sites and anionic sites of the macrophage's surface, labeled before the interaction, are not interiorized together with the parasites, however, are observed in endocytic vacuoles which do not contain parasites.     

Altered transport properties of pentamidine-resistant Leishmania donovani and L. amazonensis promastigotes

Pentamidine-resistant clones of Leishmania donovani and L. amazonensis promastigotes were developed by increase of the drug pressure in the culture medium and characterized. The resistant clones could grow in 40 and 20?μM pentamidine as determined for L.?donovani and L. amazonensis, respectively, with 50% inhibitory concentrations (IC₅₀ values) being 140 and 60?μM, which were 18 and 75 times higher than those recorded for the parental clones, respectively. Biochemical analysis of the clones showed that the acquired pentamidine resistance was specific (no cross-resistance to unrelated drugs and no reversibility with verapamil) and stable in vitro and in vivo. Pentamidine resistance is related to decreased drug uptake and highly increased efflux in both clones of Leishmania spp., accompanied by an alteration in polyamine carriers. Furthermore, a modification of the uptake of pyrimidine nucleosides and several amino acids by these resistant clones indicates alterations in the surface membrane.     

Impairment of the oxidative metabolism of mouse peritoneal macrophages by intracellular Leishmania spp.

When stimulated in vitro with macrophage-activating factor or lipopolysaccharide, mouse peritoneal macrophages acquire the capacity to develop a strong respiratory burst when they are triggered by membrane-active agents. The presence of intracellular parasites of the genus Leishmania (L. enriettii, L. major) significantly inhibited such activity, as measured by chemiluminescence, reduction of cytochrome c and Nitro Blue Tetrazolium, and hexose monophosphate shunt levels. On the contrary, inert intracellular particles such as latex beads strongly increased the macrophage respiratory burst, suggesting that the Leishmania-linked inhibition resulted from a specific parasite effect. Impairment of macrophage oxidative metabolism by intracellular Leishmania spp. was a function of the number of infecting microorganisms and was more pronounced in macrophages infected with living than with dead parasites. Moreover, the metabolic inhibition was less apparent in L. enriettii-infected macrophages that were exposed to both macrophage-activating factor and lipopolysaccharide, i.e., conditions leading to complete parasite destruction. The mechanisms of respiratory burst inhibition by intracellular Leishmania spp. are unclear, but these observations suggest that such effects may contribute significantly to intracellular survival of the microorganisms.