TNF but not Fas ligand provides protective anti-L. major immunity in C57BL/6 mice

The pro-inflammatory cytokine TNF is essential for a protective immune response to some but not all strains of Leishmania major. TNF-deficient mice of a resistant genetic background succumbed rapidly to an infection with L. major BNI. Another member of the TNF superfamily, Fas ligand (FasL), has also been reported to be critical for the immune response to L. major. To test the relative importance of TNF versus FasL for the control of L. major BNI, we infected wildtype C57BL/6 (B6.WT), B6.TNF(-/-), B6.gld and C57BL/6.gld x TNF(-/-) (B6.gld.TNF(-/-)) double-negative mice. Visceral, fatal disease was only observed in B6.TNF(-/-) mice, but not in B6 gld mice. The course of infection and the immune response of B6.gld.TNF(-/-) mice were similar to those of B6.TNF(-/-) mice. B6.gld.TNF(-/-) mice had a high tissue parasite burden and expressed prominent amounts of inducible nitric oxide synthase (iNOS) in the skin, the lymph nodes (LN) and the spleen as previously reported for B6.TNF(-/-) mice, whereas the tissue parasite load and the iNOS expression of B6.gld mice resembled that of B6.WT controls. Neither the TNF- nor the FasL-deficiency exerted a detectable intrinsic effect on the proliferation of T cells. Thus, TNF, but not FasL is essential for the control of L. major BNI. The discrepancy between these and other published data are most likely due to the use of different strains of the pathogen.     

Loss of TNF signaling facilitates the development of a novel Ly-6C(low) macrophage population permissive for Leishmania major infection

In the absence of TNF, the normally resistant C57BL/6 (B6.WT) strain develops a fatal, progressive form of leishmaniasis after infection with Leishmania major. It is not yet understood which TNF activity or the lack thereof is responsible for the dramatic progression of leishmaniasis in TNF-negative (B6.TNF(-/-)) mice. To elucidate the underlying mechanisms resulting in the fatal outcome of L. major infection in this gene-deficient mouse strain, we analyzed the monocytic component of the inflammatory infiltrate in the draining popliteal lymph node and the site of the infection using multicolor flow cytometry. The leukocytic infiltrate within the draining lymph node and footpad of B6.TNF(-/-) mice resembled that of B6.WT mice over the first 2 wk of cutaneous L. major infection. Thereafter, the B6.TNF(-/-) mice showed an increase of CD11c(+)Ly-6C(+)CCR2(+) monocytic dendritic cells within the popliteal lymph node in comparison with B6.WT mice. This increase of inflammatory dendritic cells was paired with the accumulation of a novel CD11b(+)Ly-6C(low)CCR2(low) population that was not present in B6.WT mice. This B6.TNF(-/-)- and B6.TNFR1(-/-)-specific cell population was CD115(+)Ly-6G(-)iNOS(-), not apoptotic, and harbored large numbers of parasites.     

Rapidly fatal leishmaniasis in resistant C57BL/6 mice lacking TNF

The resolution of infections with the protozoan parasite Leishmania major in mice requires a Th1 response that is closely associated with the expression of IL-12, IFN-gamma, and inducible NO synthase. Previous Ab neutralization studies or the use of mice deficient for both TNF receptors suggested that TNF plays only a limited role in the control of parasite replication in vivo. In this study we demonstrate that resistant C57BL/6 (B6.WT) mice locally infected with L. major rapidly succumb to progressive visceral leishmaniasis after deletion of the TNF gene by homologous recombination. A reduction of the parasite inoculum to 3000 promastigotes did not prevent the fatal outcome of the disease. An influence of the altered morphology of secondary lymphoid organs in C57BL/6-TNF(-/-) (B6.TNF(-/-)) mice on the course of disease could be excluded by the generation of reciprocal bone marrow chimeras. Although infected B6.TNF(-/-) mice mounted an L. major-specific IFN-gamma response and expressed IL-12, the onset of the immune reaction was delayed. After in vitro stimulation, B6.TNF(-/-) inflammatory macrophages released 10-fold less NO in response to IFN-gamma than B6.WT cells. However, in the presence of a costimulus, e.g., L. major infection or LPS, the production of NO by B6.WT and B6.TNF(-/-) macrophages was comparable. In vivo, inducible NO synthase protein was readily detectable in skin lesions and draining lymph nodes of B6.TNF(-/-) mice, but its expression was more disperse and less focal in the absence of TNF. These are the first data to demonstrate that TNF is essential for the in vivo control of L. major.     

Leishmania major parasite stage-dependent host cell invasion and immune evasion

Leishmania pathogenesis is primarily studied using the disease-inducing promastigote stage of Leishmania major. Despite many efforts, all attempts so far have failed to culture the disease-relevant multiplying amastigote stage of L. major. Here, we established a stably growing axenic L. major amastigote culture system that was characterized genetically, morphologically, and by stage-specific DsRed protein expression. We found parasite stage-specific disease development in resistant C57BL/6 mice. Human neutrophils, as first host cells for promastigotes, do not take up amastigotes. In human macrophages, we observed an amastigote-specific complement receptor 3-mediated, endocytotic entry mechanism, whereas promastigotes are taken up by complement receptor 1-mediated phagocytosis. Promastigote infection of macrophages induced the inflammatory mediators TNF, CCL3, and CCL4, whereas amastigote infection was silent and resulted in significantly increased parasite numbers: from 7.1 ± 1.4 (after 3 h) to 20.1 ± 7.9 parasites/cell (after 96 h). Our study identifies Leishmania stage-specific disease development, host cell preference, entry mechanism, and immune evasion. Since the amastigote stage is the disease-propagating form found in the infected mammalian host, the newly developed L. major axenic cultures will serve as an important tool in better understanding the amastigote-driven immune response in leishmaniasis.     

Macrophage interactions with neutrophils regulate Leishmania major infection

Macrophages are host cells for the pathogenic parasite Leishmania major. Neutrophils die and are ingested by macrophages in the tissues. We investigated the role of macrophage interactions with inflammatory neutrophils in control of L. major infection. Coculture of dead exudate neutrophils exacerbated parasite growth in infected macrophages from susceptible BALB, but killed intracellular L. major in resistant B6 mice. Coinjection of dead neutrophils amplified L. major replication in vivo in BALB, but prevented parasite growth in B6 mice. Neutrophil depletion reduced parasite load in infected BALB, but exacerbated infection in B6 mice. Exacerbated growth of L. major required PGE(2) and TGF-beta production by macrophages, while parasite killing depended on neutrophil elastase and TNF-alpha production. These results indicate that macrophage interactions with dead neutrophils play a previously unrecognized role in host responses to L. major infection.     

Leishmania amazonensis: humoral response to amastigote excreted-antigens in murine leishmaniasis

With the purpose of studying the antigenic role that factors excreted by Leishmania amastigotes might have during murine infection, immunoblots were carried out with sera from C57BL/6 and BALB/c mice infected with two strains of Leishmania (L.) amazonensis, NR and IFLA/BR. Both strains differ widely in virulence in BALB/c mice. BALB/c but not C57BL/6 sera recognized several excretion products. The excreted antigens showed a strong response towards IgG1 and IgG2a isotypes whilst they reacted only weakly against IgG2b and IgG3. A low-molecular weight antigen (about 20 kDa) excreted by both Leishmania strains was strongly recognized by IgG1 from BALB/c mice sera infected with IFLA/BR, the most virulent strain. Sera from NR infected mice were incapable of recognizing this antigen in spite of its presence in NR excreted products. The results indicate that the humoral immune response to excreted antigens of amastigotes depends on both the host genetic background and the parasite strain.     

Cysteine protease B of Leishmania mexicana inhibits host Th1 responses and protective immunity

C3H mice infected with Leishmania mexicana fail to develop a protective Th1 response, and are unable to cure. In this study, we show that L. mexicana cysteine proteases suppress the antileishmanial immune response. Previous studies demonstrated that deletion of the entire multicopy cysteine protease B (CPB) gene array in L. mexicana is associated with decreased parasite virulence, potentially attributable to factors related to parasite fitness rather than to direct effects on the host immune response. We now show that C3H mice infected with the L. mexicana deletion mutant (Deltacpb) initially develop lesions that grow at rates comparable to those of wild-type L. mexicana-infected mice. However, in contrast to controls, Deltacpb-induced lesions heal with an accompanying Th1 immune response. Lesion resolution was Th1 dependent, as Deltacpb-infected IL-12p40(-/-) and STAT4(-/-) mice developed high parasite burdens and progressive disease. Moreover, when L. major was transfected with a cosmid expressing multiple L. mexicana CPB genes, this parasite induced a significantly lower IFN-gamma response compared with wild-type L. major. These data indicate that cysteine proteases of L. mexicana are critical in suppressing protective immune responses and that inhibition of CPB may prove to be a valuable immunomodulatory strategy for chronic forms of leishmaniasis.     

Axenic promastigote forms of Leishmania (Viannia) lainsoni as an alternative source for Leishmania antigen production

The present study demonstrates that axenic cultures of Leishmania (Viannia) lainsoni produce larger cell masses in NNN-LIT medium, as well as higher amounts of total proteins in cell extracts, than Leishmania (Leishmania) amazonensis. Antigenicity of L. (V.) lainsoni whole promastigotes is similar to that of L. (L.) amazonensis, as demonstrated by an indirect immunofluorescence diagnostic test using sera from human patients and dogs infected with visceral leishmaniasis. Infectivity of the L. (V.) lainsoni strain used in the present work was demonstrated by the detection by transmission-electron microscopy of tissue amastigotes in skin lesion samples from an experimentally infected hamster. Incubation of lesion fragments in NNN-LIT medium allowed us to obtain promastigote forms, which could be cultivated successfully in vitro. lsoenzyme analysis of such promastigotes confirmed the parasite strain as L. (V.) lainsoni, as compared to other Leishmania reference strains. Our data indicate that L. (V.) lainsoni is a useful alternative source for antigen production as well for use in assays that depend on large cell volumes of Leishmania spp. parasites.     

Viscerotropic growth pattern of Leishmania tropica in BALB/c mice is suggestive of a murine model for human viscerotropic leishmaniasis

Leishmania (L.) tropica is a causative agent of cutaneous leishmaniasis, and occasionally of visceral or viscerotropic leishmaniasis in humans. Murine models of Leishmania infection have been proven to be useful for elucidation of mechanisms for pathogenesis and immunity in leishmaniasis. The aim of this study was to establish a murine model for human viscerotropic leishmaniasis, and the growth pattern of L. tropica was studied in different tissues of BALB/c mice in order to find out whether the parasite visceralizes in this murine model. L. major was used as a control as this species is known to cause a progressive infection in BALB/c mice. L. tropica or L. major was injected into the footpad of mice, and thickness of footpad, parasite loads in different tissues, and the weight of the spleen and lymph node were determined at different intervals. Results showed that L. tropica visceralizes to the spleen and grows there while its growth is controlled in footpad tissues. Dissemination of L. tropica to visceral organs in BALB/c mice was similar to the growth patterns of this parasite in human viscerotropic leishmaniasis. The BALB/c model of L. tropica infection may be considered as a good experimental model for human diseases.     

Low and high-dose intradermal infection with Leishmania major and Leishmania amazonensis in C57BL/6 mice

A model of skin infection with Leishmania amazonensis with low doses of parasites is compared to infection with high doses of L. amazonensis and low and high doses of Leishmania major. C57BL/6 mice were infected with 103 or 10(6) parasites in the ear and the outcome of infection was assessed. The appearance of lesions in mice infected with 103 parasites was delayed compared to mice infected with 10(6) Leishmania and parasites were detectable at the infection site before lesions became apparent. Mice infected with L. amazonensis displayed persistent lesions, whereas infection with L. major spontaneously healed in all groups, although lymphocytes persisted at the site of infection after healing. Macrophages persisted only in L. amazonensis-infected mice. High-dose L. amazonensis-infected mice produced lower levels of IFN-γ and TNF than mice infected with L. major. No correlation between the persistence of parasites and IL-10 levels and the production of nitric oxide or urea by macrophages was found. We conclude that infection with low doses of L. amazonensis in the dermis changes the course of infection by delaying the appearance of lesions. However, low-dose infection does not change the outcomes of susceptibility and cytokine production described for subcutaneous infection with high numbers of parasites.     

CD8+ T cells are required for primary immunity in C57BL/6 mice following low-dose, intradermal challenge with Leishmania major

Standard murine models of cutaneous leishmaniasis, involving s.c. inoculation of large numbers of Leishmania major promastigotes, have not supported an essential role for CD8(+) T cells in the control of primary infection. Recently, a L. major model combining two main features of natural transmission, low parasite dose and inoculation into a dermal site, has been established in resistant C57BL/6 mice. In the present studies, C57BL/6 mice with CD8(+) T cell deficiencies, including CD8(-/-) and CD8-depleted mice, failed to control the growth of L. major following inoculation of 100 metacyclic promastigotes into the ear dermis. The resulting dermal pathology was minor and delayed. Lesion formation in wild-type mice was coincident with the killing of parasites in the inoculation site. Both events were associated with the accumulation of CD8(+) T lymphocytes in the skin and with the capacity of CD8(+) T cells recovered from draining lymph nodes or infected dermis to release IFN-gamma following coculture with infected dendritic cells. Reconstitution of resistance to L. major in RAG(-/-) mice using T cells from naive donors was optimal when both CD4(+) and CD8(+) T cells were transferred. Primed CD8(+) T lymphocytes obtained from C57BL/6 mice during the acute stage of infection were able to mediate both pathology and immunity when transferred alone. The low dose, intradermal challenge model reveals that CD8(+) T cells play an essential role in both pathogenesis of and immunity to primary infection with L. major in the skin.     

Leishmania mexicana: effect of concomitant malaria on cutaneous leishmaniasis. Development of lesions in a Leishmania-susceptible (BALB/c) strain of mouse

Effect of concomitant malaria on cutaneous leishmaniasis. Development of lesions in a Leishmania-susceptible (BALB/c) strain of mouse. Experimental Parasitology 65, 269-276. Symptoms of human leishmaniasis vary greatly, ranging from cryptic infections to cases with fatal sequelae. Factors regulating the severity of the disease are largely undetermined. Malaria coincides geographically with leishmaniasis in many areas and the immunosuppressive effects of malaria are well documented. It is therefore plausible that malaria could enhance the course of concomitant leishmaniasis. Interactions between Leishmania mexicana and Plasmodium yoelii were examined in BALB/c mice. Percentage of blood cells infected with P. yoelii and diameter of footpad lesions caused by L. mexicana were the criteria used to assay for disease severity. L. mexicana and P. yoelii infections were each significantly enhanced in dually infected mice when compared to mice infected with either parasite alone. Mortality rates due to the normally nonlethal P. yoelii were high during concurrent infections.     

Macrophages induce neutrophil apoptosis through membrane TNF, a process amplified by Leishmania major

Neutrophils are recruited to the site of parasite inoculation within a few hours of infection with the protozoan parasite Leishmania major. In C57BL/6 mice, which are resistant to infection, neutrophils are cleared from the site of s.c. infection within 3 days, whereas they persist for at least 10 days in susceptible BALB/c mice. In the present study, we investigated the role of macrophages (MPhi) in regulating neutrophil number. Inflammatory cells were recruited by i.p. injection of either 2% starch or L. major promastigotes. Neutrophils were isolated and cultured in the presence of increasing numbers of MPhi. Extent of neutrophil apoptosis positively correlated with the number of MPhi added. This process was strictly dependent on TNF because MPhi from TNF-deficient mice failed to induce neutrophil apoptosis. Assays using MPhi derived from membrane TNF knock-in mice or cultures in Transwell chambers revealed that contact with MPhi was necessary to induce neutrophil apoptosis, a process requiring expression of membrane TNF. L. major was shown to exacerbate MPhi-induced apoptosis of neutrophils, but BALB/c MPhi were not as potent as C57BL/6 MPhi in this induction. Our results emphasize the importance of MPhi-induced neutrophil apoptosis, and membrane TNF in the early control of inflammation.     

Non-sand fly transmission of a North American isolate of Leishmania infantum in experimentally infected BALB/c mice

Leishmania infantum, an etiologic agent of zoonotic visceral leishmaniasis, is endemic in the foxhound population in the United States and Canada. Leishmaniasis is usually transmitted by blood-feeding sand flies; however, epidemiological data do not support a significant role for sand flies in the maintenance of foxhound infections in North America, and an alternate mode of transmission may exist. The present study was conducted to determine if transplacental or direct transmission occurs in pregnant BALB/c mice experimentally infected with L. infantum isolated from a naturally infected foxhound from Virginia as well as to determine if the parasite was directly transmitted to the males used to breed the mice. Female BALB/c mice were intravenously inoculated with 1 x 10(6) promastigotes of the LIVT-1 strain of L. infantum. Mice were bred to uninfected male BALB/c mice 2 mo postinoculation. Pregnant mice were killed between days 13 and 18 of gestation. Pups and placentas were collected at necropsy, divided, and used for parasite culture and polymerase chain reaction (PCR) analyses. Culture and PCR analyses were performed on spleens from the male mice to determine the possibility of sexual transmission. Leishmania sp. DNA was detected in 4 of 88 pups and 3 of 16 placentas from LIVT-1-inoculated mice. One male mouse used to breed infected females was PCR positive. This work provides evidence for a low level of nonvector transmission of North American L. infantum in a mouse model.     

Resistance to macrophage-mediated killing as a factor influencing the pathogenesis of chronic cutaneous leishmaniasis

Cutaneous leishmaniasis can be either a spontaneously healing or chronic disease, depending upon the strain of parasite and the immunological status of the host. We have investigated parasite factors responsible for the variable pathogenesis observed in leishmanial infections by testing the sensitivity of several leishmanial strains to intracellular killing in lymphokine (LK) activated mouse macrophages. Significant microbicidal activity against Leishmania tropica, a strain which heals in C57BL/6 (B6) mice, was found. In contrast, a strain (Maria) which has previously been shown to induce chronic nonhealing cutaneous lesions in B6 mice was resistant to killing in activated macrophages. This resistance to killing was observed in macrophages activated by LK obtained from either Bacille Calmette-Guérin-, L. tropica, or the Maria strain infected mice. The inability of LK activated macrophages to kill the Maria strain was shown not to be due to parasite induced inhibition of killing mechanisms, since Maria strain infected, LK treated macrophages exhibited tumoricidial activity similar to uninfected macrophages. Furthermore, LK activated macrophages simultaneously infected with the Maria strain and another intracellular pathogen, Toxoplasma gondii, killed Toxoplasma, but not the Maria strain. Temperature was also found to significantly influence the multiplication and killing of Leishmania parasites. As would be expected from their cutaneous nature, L. tropica and Maria strain parasites multiplied better at 35 degrees C than at 37 degrees C. Also consistent with the failure of cutaneous strains to visceralize in immunocompetent mice was the observation that the killing of leishmanial parasites was enhanced at the higher temperature. Thus, the temperature dependent growth capacity and sensitivity to killing of a given leishmanial strain in macrophages may be important factors influencing the pathogenesis of cutaneous leishmaniasis.     

The role of interleukin-10 in susceptibility of BALB/c mice to infection with Leishmania mexicana and Leishmania amazonensis

Recent studies have demonstrated the critical role of IL-10 in susceptibility to cutaneous and visceral leishmaniasis caused by Leishmania major and Leishmania donovani, respectively. To determine whether IL-10 also plays a similar role in the susceptibility and pathogenesis of cutaneous leishmaniasis caused by the New World species, L. mexicana and L. amazonensis, we analyzed their course of infection in IL-10-deficient BALB/c mice and their wild-type counterparts. Although IL-10-deficient mice infected with either L. mexicana or L. amazonensis failed to control the lesion progression, we did observe consistently lower levels of infection in IL-10(-/-) mice compared with wild-type BALB/c mice. We also observed increased IFN-gamma and NO production and higher levels for IL-12p40 and IL-12Rbeta(2) mRNA in cells from IL-10(-/-) mice compared with cells from BALB/c mice. The mRNA levels for IL-4, which increased significantly in both IL-10(-/-) and BALB/c mice, were comparable. When treated with anti-IL-4 mAb, IL-10(-/-) mice resolved the infection more effectively and had significantly fewer parasites in their lesions compared with similarly treated BALB/c mice. These findings suggest that IL-10, although not the dominant mediator of susceptibility of BALB/c mice to infection with L. mexicana and L. amazonensis, does play a significant role in regulating the development of a protective Th1-type response. However, effective resolution of infection with these New World parasites requires neutralization of both IL-4 and IL-10.     

Characterisation of Bangladeshi Leishmania isolated from kala-azar patients by isoenzyme electrophoresis

To identify the prevalent Leishmania species in Bangladesh, a total of nine patients aged 4-35 years, were studied; six (66.7%) of them were below 20 years of age. All the patients were clinically diagnosed to have visceral leishmaniasis; their haematological profile was in accordance with leishmaniasis and all were improved after treatment with sodium stibogluconate. All the aspirated materials (eight bone marrows and one splenic aspirate) yielded growth of Leishmania parasite in NNN media; Leishman-Donovan bodies were found in seven (77.8%) of them in a Giemsa stained smear. Aldehyde test (AT) was positive in all the nine cases examined, whereas, complement fixation test (CFT) was positive in seven (77.8%) and indirect fluorescent antibody test (IFAT) in eight (88.9%) cases. In this study, five of the nine isolates from kala-azar patients were characterised by isoenzyme analysis comparing with five WHO reference strains viz., Leishmania (Leishmania) donovani (DD8), L.(L.) donovani (HU3), L.(L.) infantum (IPT-1), L.(L.) tropica (K-27) and L.(L.) major (5-ASKH) using cellulose acetate electrophoresis. By analysing 11 soluble isoenzymes it was found that all five WHO reference strains had distinct electrophoretic mobility of the isoenzymes studied. No interspecies difference was observed amongst the five isolates from kala-azar patients examined and their isoenzyme profiles were consistent with WHO reference strain of L.(L.) donovani (DD8) but different from L.(L.) donovani (HU3).     

In vitro and in vivo activity of a palladacycle complex on Leishmania (Leishmania) amazonensis

Background

Antitumor cyclopalladated complexes with low toxicity to laboratory animals have shown leishmanicidal effect. These findings stimulated us to test the leishmanicidal property of one palladacycle compound called DPPE 1.2 on Leishmania (Leishmania) amazonensis, an agent of simple and diffuse forms of cutaneous leishmaniasis in the Amazon region, Brazil.

Methodology/Principal Findings

Promastigotes of L. (L.) amazonensis and infected bone marrow-derived macrophages were treated with different concentrations of DPPE 1.2. In in vivo assays foot lesions of L. (L.) amazonensis-infected BALB/c mice were injected subcutaneously with DPPE 1.2 and control animals received either Glucantime or PBS. The effect of DPPE 1.2 on cathepsin B activity of L. (L.) amazonensis amastigotes was assayed spectrofluorometrically by use of fluorogenic substrates. The main findings were: 1) axenic L. (L.) amazonensis promastigotes were destroyed by nanomolar concentrations of DPPE 1.2 (IC50 = 2.13 nM); 2) intracellular parasites were killed by DPPE 1.2 (IC50 = 128.35 nM), and the drug displayed 10-fold less toxicity to macrophages (CC50 = 1,267 nM); 3) one month after intralesional injection of DPPE 1.2 infected BALB/c mice showed a significant decrease of foot lesion size and a reduction of 97% of parasite burdens when compared to controls that received PBS; 4) DPPE 1.2 inhibited the cysteine protease activity of L. (L.) amazonensis amastigotes and more significantly the cathepsin B activity.

Conclusions/Significance

The present results demonstrated that DPPE 1.2 can destroy L. (L.) amazonensis in vitro and in vivo at concentrations that are non toxic to the host. We believe these findings support the potential use of DPPE 1.2 as an alternative choice for the chemotherapy of leishmaniasis.     

Lymphotoxin alpha beta 2 (membrane lymphotoxin) is critically important for resistance to Leishmania major infection in mice

Although the essential role of TNF-alpha in the control of intracellular pathogens including Leishmania major is well established, it is uncertain whether the related cytokine lymphotoxin alphabeta2 (LTalpha1beta2, membrane lymphotoxin) plays any role in this process. In this study, we investigated the contribution of membrane lymphotoxin in host response to L. major infection by using LTbeta-deficient (LTbeta(-/-)) mice on the resistant C57BL/6 background. Despite mounting early immune responses comparable to those of wild-type (WT) mice, LTbeta(-/-) mice developed chronic nonhealing cutaneous lesions due to progressive and unresolving inflammation that is accompanied by uncontrolled parasite proliferation. This chronic disease was associated with striking reduction in IL-12 and Ag-specific IFN-gamma production by splenocytes from infected mice. Consistent with defective cellular immune response, infected LTbeta(-/-) mice had significantly low Ag-specific serum IgG1 and IgG2a levels compared with WT mice. Although administration of rIL-12 to L. major-infected LTbeta(-/-) mice caused complete resolution of chronic lesions, it only partially (but significantly) reduced parasite proliferation. In contrast, blockade of LIGHT signaling in infected LTbeta(-/-) mice resulted in acute and progressive lesion development, massive parasite proliferation, and dissemination to the visceral organs. Although infected LTbeta(-/-) WT bone marrow chimeric mice were more resistant than LTbeta(-/-) mice, they still had reduced ability to control parasites and showed defective IL-12 and IFN-gamma production compared with infected WT mice. These results suggest that membrane lymphotoxin plays critical role in resistance to L. major by promoting effective T cell-mediated anti-Leishmania immunity.