A simple and reproducible method to obtain large numbers of axenic amastigotes of different<Emphasis Type="Italic"> Leishmania</Emphasis> species

This work describes a simple method to yield large amounts of Leishmania amastigote-like forms in axenic cultures using promastigotes as the starting population. The method described induced extracellular amastigote transformation of Leishmania amazonensis (97%), Leishmania braziliensis (98%) and Leishmania chagasi (90%). The rounded parasites obtained in axenic cultures were morphologically similar, even at the ultrastructural level, to intracellular amastigotes. Moreover, the axenic amastigotes remained viable as measured by their ability to revert back to promastigotes and to infect BALB/c mice. L. amazonensis and L. braziliensis promastigotes and axenic amastigotes differed in terms of their Western blot profiles. A 46 kDa protein was recognized by specific antibodies only in axenic and lesion-derived L. amazonensis amastigotes and not in promastigotes.     

Vaccine-induced immunity against cutaneous leishmaniasis in BALB/c mice.

Partially purified antigens, derived from Leishmania infantum or L. major promastigotes and isolated under reducing conditions, were used to immunize BALB/c mice. Three subcutaneous injections of the 64- to 97-kilodalton preparation in conjunction with muramyl dipeptide conferred long-lasting immunity against L. mexicana subsp. mexicana and L. major infection; they led to the development of antibodies neutralizing the infectiousness of promastigotes, induced specific delayed-hypersensitivity reactions, and generated populations of peritoneal macrophages capable of killing amastigotes. Vaccination resulted in no harmful effects, since these antigen neither exacerbated preexisting Leishmania infection nor impeded the formation of antibodies to other antigens administered concomitantly.     

Amastigote stage-specific monoclonal antibodies against Leishmania major.

Monoclonal antibodies were produced against gamma-irradiated amastigotes of Leishmania major. Five antibodies (T16 through T20) were selected which reacted in enzyme-linked immunoassays with the intracellular stage of the parasite. These antibodies did not react with promastigotes of L. major or Leishmania donovani. One of the monoclonal antibodies (T16) reacted with amastigotes of Leishmania mexicana amazonensis and L. donovani. Western blotting (immunoblotting) and immunoprecipitation of [35S]methionine-labeled amastigotes demonstrates that T16 reacted with multiple L. major amastigote components between 12 and 180 kilodaltons. Antibody T20 was shown to recognize a low-molecular-mass doublet (less than 26 kilodaltons) in both [14C]leucine- and [35S]methionine-labeled amastigotes. A protein of less than 180 kilodaltons was also weakly recognized by T17, T19, and T20 in metabolically labeled amastigotes. This protein reacted strongly with T16. The reactive antigens could be identified on the surface of amastigotes isolated from the lesions of infected mice and on newly transformed amastigotes within 24 h after the infection of mouse peritoneal macrophages by promastigotes. These monoclonal antibodies should prove useful for the diagnosis of L. major in human tissue biopsies.     

Effect of T-lymphocyte suppression on the parasite burden in Leishmania major-infected, genetically susceptible BALB/c mice.

Treatment of Leishmania major-infected, genetically susceptible BALB/c mice with the T-lymphocyte-immunosuppressive drug cyclosporin A (CyA) resulted in a significantly reduced parasite burden in the local site of infection and in the draining lymph nodes. These data indicate that T cells are pivotal for the propagation of L. major in vivo.     

Cytokine interactions in experimental cutaneous leishmaniasis. Interleukin 4 synergizes with interferon-gamma to activate murine macrophages for killing of Leishmania major amastigotes

We investigated the effect of recombinant murine interleukin 4 (IL 4) in the absence or presence of recombinant murine interferon-gamma (IFN-gamma) on adherent bone-marrow macrophages (M phi), peritoneal exudate and resident peritoneal M phi from susceptible BALB/c M phi, which were pulse-infected with Leishmania major amastigotes (AM), IL 4 (5-100 U/ml) failed to activate any of these M phi populations for killing of intracellular AM. However, in the presence of low concentrations of IFN-gamma (10-20 U/ml), which alone caused only a slight or intermediate reduction of the number of intracellular parasites. IL 4 led to a dramatic increase of the parasite elimination by all M phi populations. In the case of resident peritoneal M phi, the synergism of IFN-gamma and IL 4 required the incubation of the M phi with both cytokines or with IFN-gamma alone for at least 10 h prior to infection; adding both cytokines after infection of the M phi did not cause a significant reduction of the intracellular parasite burden. The synergistic effect of IL 4 and IFN-gamma was completely abrogated in the presence of anti-IL 4 antibodies. Furthermore, there was no significant difference between M phi derived from either susceptible BALB/c or from resistant C57BL/6 mice. Evidence is presented that the synergistic action of IL 4 and IFN-gamma occurs via an L-arginine-dependent killing pathway. From these data we conclude that IL 4 provides a strong stimulus for the killing of intracellular L. major AM provided low concentrations of IFN-gamma are present. Also, IFN-gamma is apparently an important priming signal for the activation of resident M phi to eliminate intracellular AM.     

Tumor necrosis factor-alpha in combination with interferon-gamma, but not with interleukin 4 activates murine macrophages for elimination of Leishmania major amastigotes

We have previously shown that during an infection with Leishmania major, susceptible BALB/c mice, as opposed to mice of a resistant strain (C57BL/6), are primed by lipopolysaccharide for the production of high levels of tumor necrosis factor-alpha (TNF-alpha) which is known to be a potent macrophage (M phi) stimulator in other parasitic diseases. In the present study we investigated whether TNF-alpha activates M phi for killing of L. major parasites. In the absence of interferon-gamma (IFN-gamma) or lipopolysaccharide, TNF-alpha (0.025-25,000 U/ml) failed to activate peritoneal exudate M phi from BALB/c mice for killing of L. major amastigotes. In the presence of suboptimal doses of IFN-gamma (5 or 10 U/ml), however, TNF-alpha mediated a rapid elimination of intracellular parasites, which was highly significant compared to IFN-gamma alone. The combination of TNF with interleukin 4, in contrast, was inactive in this respect and allowed survival of intracellular parasites. From these data we conclude that the presence of IFN-gamma is crucial for TNF-alpha-mediated killing of L. major parasites by M phi. Disease progression in susceptible mice therefore seems to be a consequence of a deficiency of IFN-gamma and a predominance of interleukin 4 rather than the result of an excess amount of TNF-alpha.     

Mycobacterium bovis BCG-induced protection against cutaneous and systemic Leishmania major infections of mice.

We examined the protective effects of Mycobacterium bovis bacillus Calmette-Guérin (BCG) administration on Leishmania major infections of BALB/c and P/J mice. There were two treatment protocols. In the first, the footpads of naive animals were inoculated with mixtures of L. major and BCG (viable or heat killed) or the soluble mycobacterial antigen, purified protein derivative. Viable BCG, but not heat-killed BCG or purified protein derivative, inoculated with L. major amastigotes into the footpads of naive BALB/c or P/J mice protected these animals from the metastatic spread of parasites to the viscera and from ensuing lethal systemic infection. This treatment also induced cures of the cutaneous lesions of P/J mice but not of BALB/c mice. In the second protocol, we induced an immune response to BCG before inoculation of L. major. BCG given intraperitoneally 10 days before infection of footpads with leishmania offered protection against the metastatic spread of amastigotes in both P/J and BALB/c mice, regardless of intralesional treatment, and modulated the severity of cutaneous infection by 30 to 50%. Inoculation of a mixture of viable BCG and L. major amastigotes into BCG-immune mice completely protected both BALB/c and P/J strains from cutaneous disease; we recovered no parasites from the inoculated footpads of these animals. Furthermore, each of the nonspecifically protected mice of both the BALB/c and P/J strains developed immunity to rechallenge with viable L. major. Injection of amastigotes at a site remote from the original lesion, the contralateral footpad, resulted in the complete clearance of parasites in the inoculum with no evidence of either cutaneous or systemic disease over an extended observation period.     

Comparison of receptors required for entry of Leishmania major amastigotes into macrophages.

We investigated the mechanisms of entry of amastigotes of Leishmania major from two different sources into macrophages by comparing their use of the Fc receptor (FcR), complement receptor type 3 (CR3), and mannose-fucose receptor (MFR). Amastigotes were obtained from BALB/c mice and SCID mice. FcR involvement was examined by opsonizing L. major with parasite-specific immunoglobulin G (IgG). Antiparasite IgG did not alter the uptake of amastigotes from BALB/c mice since these amastigotes had antibody bound to their surface: IgG1 was the most predominant antibody, followed by IgG2b, IgM, and IgG2a. However, opsonization with antiparasite IgG enhanced the entry of amastigotes that lacked antibody on their surface, namely, amastigotes obtained from SCID mice or from macrophages infected in vitro. These results indicate that the FcR is important for amastigote entry into macrophages. Down-modulation of FcRs onto immune complexes, however, did not reduce the entry of amastigotes containing surface-bound IgG into macrophages. Monoclonal antibodies against the CR3 inhibited the entry of amastigotes from either BALB/c or SCID mice into J774A.1 macrophage-like cells. Simultaneous blocking of FcR and CR3 further increased the inhibition of phagocytosis. Treatment of macrophages with soluble mannan or down-modulating the MFR onto mannan-coated coverslips had no effect on the entry of amastigotes from BALB/c or SCID mice. Thus, the MFR does not appear to be used by amastigotes of L. major. We show that ingestion of amastigotes appears to occur primarily through the FcR and CR3; however, additional receptors may also participate in the uptake of amastigotes.     

Depletion of peritoneal CD5+ B cells has no effect on the course of Leishmania major infection in susceptible and resistant mice.

The mouse peritoneal cavity contains a unique self-renewing population of B cells (B-1) derived from fetal liver precursors and mainly producing polyreactive antibodies. Since B-1 cells are a potential source of IL-10, it has been suggested that these cells may contribute to the susceptibility of BALB/c mice to Leishmania major infection by skewing the T helper cell network towards a Th2 phenotype. Accordingly, L. major infection of B cell-defective BALB/c Xid mice (lacking B-1 cells) induces less severe disease compared with controls. However, in addition to the lack of B-1 cells, the Xid immune deficiency is characterized by high endogenous interferon-gamma (IFN-gamma) production. In the present study, the role of B-1 cells during L. major infection was investigated in mice experimentally depleted of peritoneal B-1 cells. Six weeks old C57Bl/6 and BALB/c mice were lethally irradiated and reconstituted with autologous bone marrow which allows systemic depletion of B-1 cells. Untreated BALB/c, C57Bl/6 as well as BALB/c Xid mice were used as controls. After reconstitution, mice were injected with L. major amastigotes and progression was followed using clinical, parasitological and immunological criteria. As previously reported, BALB/c Xid mice showed a significant reduction in disease progression. In contrast, despite the dramatic reduction of B-1 cells, B-1-depleted BALB/c mice showed similar or even worse disease progression compared with control BALB/c mice. No differences were found between B-1-depleted or control C57Bl/6 mice. Our data suggest that the B-1 cells do not contribute to the susceptibility of BALB/c mice to L. major infection.     

Presentation of the Leishmania antigen LACK by infected macrophages is dependent upon the virulence of the phagocytosed parasites

We have previously demonstrated that murine macrophages (Mphi) infected with Leishmania promastigotes, in contrast to Mphi infected with the amastigote stage of these parasites, are able to present the Leishmania antigen LACK (Leishmania homologue of receptors for activated C kinase) to specific, I-Ad-restricted T cell hybrids and to the T cell clone 9.1-2. These T cells react with the LACK (158-173) peptide, which is immunodominant in BALB/c mice. Here, we show that the level of stimulation of the LACK-specific T cell hybridoma OD12 by promastigote-infected Mphi is clearly dependent upon the differentiation state of the internalized parasites. Thus, shortly after infection with log-phase or stationary-phase promastigotes of L. major or of L. amazonensis, Mphi strongly activated OD12. The activity was transient and rapidly lost. However, under the same conditions, activation of OD12 by Mphi infected with metacyclic promastigotes of L. major or of L. amazonensis was barely detectable. At the extreme, Mphi infected with amastigotes were incapable to stimulate OD12. Thus, the presentation of LACK by infected Mphi correlates with the degree of virulence of the phagocytosed parasites, the less virulent being the best for the generation/expression of LACK (158-173)-I-Ad complexes. While the intracellular killing of the parasites appears to be an important condition for the presentation of LACK, it is not the only requisite. The partial or total destruction of intracellular L. amazonensis amastigotes does not allow the presentation of LACK to OD12. A preferential interaction of LACK (158-173) with recycling rather than newly synthesized MHC class II molecules does not explain the transient presentation of LACK by Mphi infected with log-phase or stationary-phase promastigotes because brefeldin A strongly inhibited the presentation of LACK to OD12. Taken together, these results suggest that virulent stages of Leishmania, namely metacyclics and amastigotes, have evolved strategies to avoid or minimize their recognition by CD4+ T lymphocytes.     

Homologues of LMPK, a mitogen-activated protein kinase from Leishmania mexicana, in different Leishmania species

LMPK, a mitogen-activated protein (MAP) kinase homologue of Leishmania mexicana, is essential for the proliferation of the amastigote, the mammalian stage of the protozoan parasite. This has been demonstrated using deletion mutant promastigotes, the insect stage of the parasite: first, in vitro after differentiation to amastigotes, which subsequently lost their potential to proliferate; second, by infection of peritoneal macrophages, which were able to cope with the infection and cleared the parasites; third, by infection of BALB/c mice, which showed no lesion development. The lmpk deletion mutant promastigotes are a potential live vaccine because they infect macrophages, transform to amastigotes and deliver amastigote antigens to raise an immune response without causing the disease. In addition, inhibition of LMPK in a wild-type infection is likely to resolve the disease and as such, is an ideal target for drug development against leishmaniasis. Here we investigated the presence and copy number of lmpk homologues in Leishmania amazonensis, L. major, L. tropica, L. aethiopica, L. donovani, L. infantum, and L. braziliensis and discuss the results with regard to drug development and vaccination using kinase deletion mutants.     

Enhanced replication of Leishmania amazonensis amastigotes in gamma interferon-stimulated murine macrophages: implications for the pathogenesis of cutaneous leishmaniasis

During Leishmania major infection in mice, gamma interferon (IFN-gamma) plays an essential role in controlling parasite growth and disease progression. In studies designed to ascertain the role of IFN-gamma in Leishmania amazonensis infection, we were surprised to find that IFN-gamma could promote L. amazonensis amastigote replication in macrophages (Mphis), although it activated Mphis to kill promastigotes. The replication-promoting effect of IFN-gamma on amastigotes was independent of the source and genetic background of Mphis, was apparently not affected by surface opsonization of amastigotes, was not mediated by interleukin-10 or transforming growth factor beta, and was observed at different temperatures. Consistent with the different fates of promastigotes and amastigotes in IFN-gamma-stimulated Mphis, L. amazonensis-specific Th1 transfer helped recipient mice control L. amazonensis infection established by promastigotes but not L. amazonensis infection established by amastigotes. On the other hand, IFN-gamma could stimulate Mphis to limit amastigote replication when it was coupled with lipopolysaccharides but not when it was coupled with tumor necrosis factor alpha. Thus, IFN-gamma may play a bidirectional role at the level of parasite-Mphi interactions; when it is optimally coupled with other factors, it has a protective effect against infection, and in the absence of such synergy it promotes amastigote growth. These results reveal a quite unexpected aspect of the L. amazonensis parasite and have important implications for understanding the pathogenesis of the disease and for developing vaccines and immunotherapies.     

Leishmania pifanoi amastigote antigens protect mice against cutaneous leishmaniasis.

In the search for a leishmaniasis vaccine, extensive studies have been carried out with promastigote (insect stage) molecules. Information in this regard on amastigote (mammalian host stage) molecules is limited. To investigate host immune responses to Leishmania amastigote antigens, we purified three stage-specific antigens (A2, P4, and P8) from in vitro-cultivated amastigotes of Leishmania pifanoi by using immunoaffinity chromatography. We found that with Corynebacterium parvum as an adjuvant, three intraperitoneal injections of 5 micrograms of P4 or P8 antigen provided partial to complete protection of BALB/c mice challenged with 10(5) to 10(7) L. pifanoi promastigotes. These immunized mice developed significantly smaller or no lesions and exhibited a 39- to 1.6 x 10(5)-fold reduction of lesion parasite burden after 15 to 20 weeks of infection. In addition, P8 immunization resulted in complete protection against L. amazonensis infection of CBA/J mice and partial protection of BALB/c mice, suggesting that this antigen provided cross-species protection of mice with different H-2 haplotypes. At different stages during infection, vaccinated mice exhibited profound proliferative responses to parasite antigens and increased levels of gamma interferon production, suggesting that a Th1 cell-mediated immune response is associated with the resistance in these mice. Taken together, the data in this report indicate the vaccine potential of amastigote-derived antigens.     

Specific suppressor T cells for delayed-type hypersensitivity in susceptible mice immunized against cutaneous leishmaniasis.

BALB/c mice injected intravenously with 10(6) or higher doses of formaldehyde-fixed promastigotes (ffp) of Leishmania major developed significantly lower levels of delayed-type hypersensitivity (DTH) compared with uninjected control mice when they were subsequently immunized intradermally with ffp. The suppression of DTH was antigen specific and was also inducible with lethally irradiated promastigotes or soluble parasite antigens. The suppressive effect was adoptively transferable with splenic T cells which express the Lyt-1+2+ and L3T4+ phenotypes. These specific suppressor T cells were active against both the inductive and expressive phases of DTH. They were sensitive to 200 rads of gamma-irradiation in vitro and appeared to manifest the suppressive activity via soluble factors. In spite of this profound suppression of DTH, BALB/c mice injected intravenously with 4 X 10(7) ffp were substantially protected against a challenge infection with L. major promastigotes. The possible relationship between the suppressor T cells for DTH and prophylactic immunization against fatal cutaneous leishmanial infection in susceptible BALB/c mice is discussed.     

Cyclosporin A treatment converts Leishmania donovani-infected C57BL/10 (curing) mice to a noncuring phenotype.

Cyclosporin A prevents visceralization of Leishmania major infection of BALB/c mice (N. C. Behforouz, C. D. Wenger, and B. A. Mathison, J. Immunol. 136:3067-3075, 1986; W. Solbach, K. Forberg, E. Kammerer, C. Bogdan, and M. Rollinghoff, J. Immunol. 134:702-707, 1986). We report that cyclosporin A exacerbates disseminated leishmaniasis caused by L. donovani in C57BL/10 mice. Normal mice challenged with 5 x 10(6) amastigotes intravenously cleared the infection within several months by spontaneous acquisition of cell-mediated immunity. In contrast, cyclosporin A administered daily intraperitoneally at a dose of 1.25 mg per mouse prevented development of curative immunity and converted C57BL/10 (curing) mice to a noncuring phenotype. A rationale for the contrasting effects of cyclosporin A in the two murine models of leishmaniasis is provided.     

Effect of protein kinase inhibitors on the growth, morphology, and infectivity of Leishmania promastigotes

The effect of two protein kinase inhibitors, staurosporine and H-7, on the growth, morphology and infectivity of Leishmania major and Leishmania amazonensis promastigotes was examined. Incubation with H-7 (600 μM) for up to one hour had no effect on parasite growth, morphology or infectivity. Staurosporine, however, was cytotoxic for promastigotes and incubation for 1, 5 or 15 minutes with 10 μM inhibitor killed 19, 34 and 59 %, respectively, of the parasites. Longer incubations, up to one hour, at this concentration did not increase parasite killing. However, treatment with 25 μM staurosporine for one hour was highly toxic, only 4 % of the promastigotes surviving after 72 h. Lower concentrations of staurosporine, 0.25 and 2.5 μM, had only minor effects on parasite growth. Incubation of either L. major or L. amazonensis with staurosporine (10 μM for 10 minutes) caused marked morphological changes in the size and appearance of the flagellar pocket, and/or cytoplasm of the viable parasites. Treated parasites were still capable of infecting mouse peritoneal macrophages and causing disease in BALB/c mice, though the treated parasites were less virulent than control promastigotes. These results indicate that staurosporine, while inhibiting promastigote growth, does not prevent differentiation to amastigotes and amastigote replication. Received: 26 June 1996 / Accepted: 20 August 1996     

T-cell responses to immunodominant LACK antigen do not play a critical role in determining susceptibility of BALB/c mice to Leishmania mexicana

Although BALB/c mice develop lesions when infected with Leishmania mexicana, the mechanisms which are responsible for susceptibility to this parasite have not been elucidated. In contrast, susceptibility of BALB/c mice to Leishmania major has been shown to depend on the early production of interleukin-4 (IL-4) by T cells which react to the parasitic LACK antigen. Here, we demonstrate that the lesions induced by L. mexicana are delayed compared to those induced by L. major but rapidly develop at later time points. Interestingly, while LACK-tolerant BALB/c-derived IE-LACK transgenic mice were resistant to L. major, they were susceptible to L. mexicana and developed lesions similar to those observed in wild-type BALB/c mice. The latter result was observed despite the fact that (i) LACK was expressed by L. mexicana, (ii) splenocytes from BALB/c mice were able to stimulate LACK-specific T-cell hybridoma cells when incubated with live L. mexicana promastigotes, and (iii) LACK-specific T cells contributed to IL-4 production in L. mexicana-infected BALB/c mice. Thus, in contrast to what was observed for L. major-infected mice, LACK-specific T cells do not play a critical role in determining susceptibility to L. mexicana. Although BALB/c mice are susceptible to both L. major and L. mexicana, the mechanisms which are responsible for susceptibility to these parasites are likely to be different.     

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.     

Effect of cyclosporin A on the T-effector and T-suppressor cell response in contact sensitivity.

The effect of cyclosporin A (CyA) on the induction, expression and regulation of the immune response to the contact sensitivity agent 2,4-dinitroflouro-benzene, (DNFB) has been studied in BALB/c mice. CyA was administered orally during the sensitization phase or during the elicitation phase. The following results were obtained. CyA administered orally during the sensitization phase at a high dose resulted in a reduction of the contact sensitivity response. Feeding lower doses had no effect on the ear swelling response. CyA inhibited the ear swelling response if administered during the elicitation phase. In transfer experiments it was shown that the induction of the T-effector cell of delayed hypersensitivity (TDH) was insensitive to the suppressive action of CyA, while the effector function of this T-cell subpopulation was CyA-sensitive. T-suppressor (TS) cell response was studied in animals by inducing tolerance through epicutaneous application of an allergen overload and by transfer of TS cells induced by intravenous injection of the contact sensitizer. Tolerance induction in vivo and TS-cell induction and function were inhibited in CyA-treated animals.