Natural and acquired resistance to Leishmania: cellular activation by Leishmania aethiopica of mononuclear cells from unexposed individuals is through the stimulation of natural killer (NK) cells.

Cells from normal non-Leishmania-exposed individuals could respond in vitro by proliferation and interferon-gamma (IFN-gamma) production to Leishmania aethiopica stimulation. The main cell type that appeared to be activated following such stimulation was CD3-, CD16+/56+, i.e. NK cells. Of the few CD3+ cells responding, an involvement of CD8+ cells was evident in the absence of activation of CD4+ cells in normal individuals, while a different feature was observed when patients' cells were investigated. Cells from patients with L. aethiopica infection did not show this NK response, but rather the CD4+ cells were the prominent responding cells. No evidence of the involvement of superantigens or cells utilizing the gamma delta T cell receptor (gamma delta cells) in the response of unexposed individuals was noted.     

Cytokine Responses to Parasite Antigens: In Vitro Cytokine Production to Promastigotes of L. aethiopica by Cells from Non-Leishmania Exposed Donors may Influence Disease Establishment

The various cytokine responses associated with stimulation by parasites is discussed with emphasis on Leishmania parasites.
Cells from normal individuals can respond to Leishmania antigen in vitro but the State of the antigen used for stimulation influences the outcome. We have used cells from non- Leishmania exposed donors and stimulated them in vitro with variously treated promastigotes of L. aethiopica. The levels of some cytokines released into the supernatant were measured. All the Leishmania preparations tested induced high levels of IL-6. whereas IFN-_γ production to the different stimuli was variable in the individual donors. The ability of these supernatants to inhibit intracellular forms of L. aethiopica was sometimes stronger in L. aethiopica -induced than in PHA-induced cultures. Such strong non- Leishmania specific responses, if they exist in vivo, may influence whether disease is established when the host encounters Leishmania parasites.     

Generation of species-specific DNA probes for Leishmania aethiopica

We report here the cloning of kinetoplast DNA (kDNA) sequences from Leishmania aethiopica in order to develop a specific and sensitive method for the identification of the parasite. Analysis of the cloned kDNA sequences showed different taxonomic specificities demonstrating sequence diversity within the kinetoplast DNA. Cloned whole minicircle hybridized with all Old World Leishmania species tested. Some cloned fragments of minicircle kDNA hybridized with Leishmania species causing cutaneous leishmaniasis in the Old World, but not with the viscerotropic species. Two L. aethiopica-specific clones were found. These clones hybridized with all L. aethiopica isolates tested, but did not react with other Leishmania species. The nucleotide sequence of the L. aethiopica-specific R3 clone is presented. Clones hybridizing with only some of the L. aethiopica isolates were also identified, although none of them showed specificity only for isolates causing localized (LCL) or diffuse (DCL) form of cutaneous leishmaniasis in Ethiopia.     

Induction and abrogation of LACK reactive cells in the evolution of human leishmaniasis.

Peripheral blood mononuclear cells (PBMC) from cutaneous leishmaniasis patients with ongoing Leishmania aethiopica infection and individuals cured/under treatment from L. infantum or L. donovani infection were stimulated in vitro with LACK, the Leishmania homologue of receptors for activated C kinase. The LACK protein is conserved in related leishmanial species and is expressed both in the promastigote and amastigote stages of Leishmania. Our results show that LACK induced marked NK and some CD8+ cell proliferation in PBMC from cutaneous leishmaniasis patients with active disease. These responses were coupled with high levels of IFN-gamma and IL-10 production. At the concentration tested, the proliferative responses to freeze-thawed Leishmania antigen (Ft-Leish) were higher, while the levels of IFN-gamma were consistently lower than that of LACK. Although cells from individuals cured of leishmaniasis could respond to whole Leishmania lysate by proliferation and IFN-gamma production, there was no evident response to LACK. Ethiopian controls tested at the same time also showed LACK induced proliferation with IFN-gamma and IL-10 responses. Thus LACK reactivity in terms of proliferation and cytokine induction were present in cells from some healthy donors and most of the patients with active lesions, while this response was absent in individuals cured of L. infantum or L. donovani leishmaniasis. Since cure from leishmaniasis often results in life-long protection, and active but not cured patients showed in vitro responses to LACK stimulation, questions arose as to how this highly immunodominant molecule functions during human leishmanisasis. Some possible mechanisms are discussed.     

Immune reactivity to fractionated Leishmania aethiopica antigens during active human infection.

Fractionated antigen preparations of Leishmania aethiopica parasites were used to stimulate the peripheral blood lymphocytes of patients with active cutaneous leishmaniasis. In assays measuring lymphocyte proliferation, 9 of 10 patients with similar clinical presentations of infection responded in a similar pattern to the fractionated antigens. Marked proliferation was observed in response to antigen fractions with molecular masses of 43 to 36, 33 to 27, and less than 22 kDa. The induction of relatively high levels of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) was also observed in responses to these same three antigen fractions. In contrast, the proliferative, IFN-gamma, and TNF-alpha responses of patient lymphocytes to antigens with a molecular mass greater than 60 kDa were uniformly low. The results of this study suggest that the antigens of Leishmania parasites, which are recognized by T cells in patients with active cutaneous leishmaniasis, may be partitioned in the lower-molecular-mass antigenic determinants associated with whole-parasite preparations. The observed association between antigen-induced proliferation and IFN-gamma and TNF-alpha production may be indicative of potential disease-limiting immune effector activities which have developed during infection.     

Evaluation of amastigote reactive cells in human cutaneous leishmaniasis caused by Leishmania aethiopica

Lymphoproliferative responses to three affinity chromatography purified amastigote antigens of Leishmania pifanoi, P-2, P-4 and P-8, were evaluated in peripheral blood mononuclear cells (PBMC) from patients with Ethiopian cutaneous leishmaniasis. Antigen-stimulated cells were analysed for the percentage of CD4+, CD8+ and CD16/56+ cells and the expressed levels of gamma interferon (IFNgamma) and interleukin (IL)-10 were determined in culture supernatants. The amastigote antigens induced cellular responses in leishmaniasis patients with heterologous Leishmania parasite infection. These responses were compared to those of freeze-thawed L. aethiopica promastigote antigen stimulation. The membrane protein (P-8), and to a lesser extent the megasomal/cytoplasmic cysteine proteinase(P-2), induced proliferation with high levels of IFNgamma and IL-10 production in cells from patients with active L. aethiopica lesions. CD16/56+ NK cells were the main cell types induced to proliferate in response to P-8 and P-2 stimulation, followed by CD8+ cell populations. P-4 had no such effect. This contrasts from previous studies of New World human leishmaniasis where P-4 and P-8 were stimulatory. The success of a particular molecule in the induction of a response with a protective phenotype may be dependent on the infecting Leishmania spp. To our knowledge, there are no studies that directly compare the New versus Old World cutaneous leishmaniasis in respect of NK cell and IL-10 responses. Our studies indicate that some leishmanial molecules are recognized across the species, while others are apparently more species specific.     

Contribution of non-Leishmania-specific immunity to resistance to Leishmania infection in humans.

Lymphocytes of individuals from a country non-endemic for Leishmania (Sweden), responded with a vigorous interferon-gamma (IFN-gamma) and IL-6 response when exposed to live or dead promastigotes of Leishmania aethiopica. This response was sometimes as strong as when the same cells were exposed to the mitogen (phytohaemagglutinin (PHA)). Furthermore, supernatants of cells exposed to Leishmania promastigotes were able to inhibit the amastigote form of the same parasite. In some few instances there was no such reactivity to Leishmania parasites. It is proposed that most individuals have such a first line cytokine response which is enough to prevent further spread and growth of the parasites. In exposed individuals who display disease symptoms, this non-Leishmania-specific response is overcome (by dose) or is weak (for genetic reasons). In the latter instances curbing of parasite growth would depend on acquired immunity.     

Genetic variability within the species Leishmania aethiopica does not correlate with clinical variations of cutaneous leishmaniasis

Leishmania aethiopica infections in man result in a spectrum of diseases from LCL to DCL. These clinical manifestations have been attributed to genetic differences within the host or the parasites. In this study two different PCR-based methods were used to elucidate genetic variation within the species L. aethiopica. Inter- and intra-specific variations were detected in the ITS of the ribosomal operon in different strains and species of Leishmania, using a PCR-RFLP approach, and by a PCR fingerprinting technique that used single non-specific primers to amplify polymorphic regions of the genomic DNA. Both methods revealed genetic heterogeneity among ten L. aethiopica isolates examined. Unrooted distance trees separated the ten strains into two different genetic groups. This subdivision was correlated to the geographical origin of the isolates rather than to the clinical manifestation of the disease.     

Dichotomy of the human T cell response to Leishmania antigens. I. Th1-like response to Leishmania major promastigote antigens in individuals recovered from cutaneous leishmaniasis.

The T cell response to antigens from Leishmania major promastigotes was investigated in peripheral blood mononuclear cells from Sudanese individuals with a history of cutaneous leishmaniasis (CL), Sudanese individuals with positive DTH reaction in the leishmanin skin test but with no history of skin lesions, and in Danes without known exposure to Leishmania parasites. Proliferation and production of interferon-gamma (IFN-gamma) and IL-4 in antigen-stimulated cultures was measured. Lymphocytes from individuals with a history of CL proliferated vigorously and produced IFN-gamma after stimulation with either a crude preparation of L. major antigens or the major surface protease gp63. These cultures produced no or only little IL-4. Also cells from leishmanin skin test-positive donors with no history of CL produced IFN-gamma and no IL-4 in response to L. major antigens. Cells from the unexposed Danes were not activated by gp63. The cells from Danish donors produced either IFN-gamma or IL-4, but not both cytokines after incubation with the crude preparation of L. major antigens. The data show that the T cell response to Leishmania antigens in humans who have had uncomplicated CL or subclinical L. major infection is an IFN-gamma-producing Th1-like response.     

Th1 and Th2 cell-associated cytokines in experimental visceral leishmaniasis.

In experimental Leishmania donovani infection in BALB/c mice, initial susceptibility gives way to T-cell-dependent acquired resistance and eventual control over visceral infection. Since various cytokines appear to underlie the host response to Leishmania infection, we examined infected liver tissue for gene expression of cytokines associated with Th1 (gamma interferon [IFN-gamma] and interleukin-2 [IL-2]) and Th2 cells (IL-4 and IL-10). By Northern (RNA) blot analysis, only IFN-gamma mRNA expression was detected in livers of infected euthymic mice. To determine whether activation of Th1 cells develops selectively in this model, qualitative PCR analysis was used. These results indicated that mRNAs for IFN-gamma, IL-2, IL-4, and IL-10 were all induced by L. donovani infection. The potentially negative Th2 cell-associated response did not appear to play a functional role, however, since resistance was acquired, anti-IL-4 monoclonal antibody treatment did not accelerate control over visceral infection, and serum immunoglobulin E levels remained low. As judged by PCR analysis, IL-4 and IL-10 mRNAs were also expressed under three other conditions without apparent effect: in naive euthymic mice treated with IL-2, which induces leishmanicidal activity; in rechallenged immune mice, which resist reinfection; and in nude mice, which fail to control L. donovani. These results suggest that, like other Leishmania species, L. donovani infection may trigger a potentially suppressive Th2 cell-associated cytokine response. However, in T-cell-intact mice able to control L. donovani, this response either is insufficient to influence outcome or more likely is overshadowed by the Th1 cell response.     

Cells from Healthy Non-Exposed Individuals Produce Cytokines to Selected Fractions of Leishmania Promastigotes

We are interested in cellular responses to antigens of parasites to which the cell donor has not been previously exposed and how such responses may influence parasite establishment. In order to characterize such responses we have used cells from unexposed healthy donors and analysed the lymphoproliferative response to various Leishmania aethiopica antigen preparations and the cytokines produced in the process. Peripheral blood lymphocytes were stimulated with SDSPAGE separated L. aethiopica antigen coupled to nitrocellulose particles. Fifteen of the 16 unexposed individuals tested had proliferative responses to either the whole or and the antigen-bearing nitrocellulose fractions (NC fractions). Although the degree of response to the fractionated antigen varied in individuals, major stimulatory fractions were found in the high molecular weight region of 110-80 kDa (fractions 3-6) and low molecular weight region of 46-18 kDa (fractions 12-16). Substantial amounts of interferon gamma (IFN-γ) and interleukin 2 (IL-2) were present in the supernatants of cells stimulated with the whole unfractionated antigen. The potential relevance of such responses in resistance to Leishmania infection is discussed.     

Responsiveness in Diffuse versus Local Cutaneous Leishmaniasis is Due to Parasite Differences

Leishmania aethiopica infection results in two main clinical entities, diffuse disease (DCL) and localized ulcers (LCL). The lack of reactivity to leishmanial antigens has been attributed, among other things, to some inherent immunological defect of the host or considered as a consequence of the initial site of infection. Properties unique to the infecting parasite have been said to contribute little if anything to the differences between DCL and LCL found in the same areas of Ethiopia. Data are given to show that infected individuals respond by higher production of IL-2 to antigens from LCL isolates (Icl antigen), than to antigens derived from DCL isolates (dcl antigen). Furthermore, dcl antigen induced less gamma interferon from lymphocytes of all individuals tested than did lcl antigen. Lymphocytic proliferation of cells from control individuals working in the endemic area was higher in response to lcl isolates than to del isolates. These findings suggest that some differences in the parasites may contribute to the clinical outcome of infection with L. aethiopica .     

In vivo and in vitro control of Leishmania mexicana due to garlic-induced NO production

Leishmania mexicana is the main causal agent of cutaneous leishmaniasis in the Yucatán peninsula in Mexico. Control of this disease is associated with a Th1-type immune response and garlic extract has been reported as a Th1 immunomodulator in BALB/c mice infected with Leishmania major. In this study, we investigated the effect of garlic extracts on L. mexicana infection in vivo and in vitro. Garlic extract reduced footpad lesions in L. mexicana-infected BALB/c mice by inducing IFN-gamma production from T cells. In vitro, garlic extract reduced macrophage infection through induction of nitric oxide (NO) production. Garlic extract may thus act on both T cells and macrophages to stimulate IFN-gamma production and NO synthesis for parasite killing. A 10- to 14-kDa fraction was identified as responsible for the in vitro effect of the whole extract and may lead to the identification of novel immunomodulating drugs and therapeutic alternatives for the treatment of leishmaniasis.     

Leishmania promastigotes release a granulocyte chemotactic factor and induce interleukin-8 release but inhibit gamma interferon-inducible protein 10 production by neutrophil granulocytes

Recent data from our laboratory suggest that neutrophil granulocytes (polymorphonuclear leukocytes [PMN]) can serve as host cells for Leishmania major in the early phase of infection. In line with these findings, an early influx of PMN to the infected tissues was shown by others to be associated with susceptibility to infection with L. major. The mechanisms underlying the initial PMN recruitment to the site of infection is poorly understood. In the present study we investigated whether Leishmania can influence PMN migration. Supernatants of Leishmania promastigotes were tested for their chemotactic activity using an in vitro chemotaxis assay. All Leishmania species tested (L. major, L. aethiopica, and L. donovani) displayed a marked chemotactic effect on human PMN. However, no effect on the migration of macrophages and NK cells was observed. Checkerboard analysis revealed that the observed PMN migration was due to chemotaxis rather than chemokinesis. Most of the chemotactic activity was found in fractions containing molecules with sizes between 10 and 50 kDa. Pretreatment of PMN with N-formyl-methionyl-leucyl-phenylalanine blocked the chemotactic activity of Leishmania supernatants up to 75%. In addition, we found that leishmanial contact induced the release of interleukin-8 (IL-8) and inhibited the production of gamma interferon-inducible protein 10 (IP-10) by PMN. These data suggest that infection with Leishmania promastigotes leads to PMN accumulation via the production of a chemotactic factor by the parasites, and this effect is amplified by the induction of IL-8 production in PMN. On the other hand, the inhibition of IP-10 production can lead to prevention of NK cell activation.     

Molecular karyotype and chromosomal localization of genes encoding two major surface glycoproteins, gp63 and gp46/M2, hsp70, and beta-tubulin in cloned strains of several Leishmania species.

The molecular karyotypes of several Leishmania isolates (Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Leishmania panamensis, Leishmania donovani, Leishmania major, Leishmania aethiopica, Leishmania tropica, Leishmania enriettii) have been analyzed by clamped homogeneous electric field (CHEF) gel electrophoresis. The chromosomal localization of genes encoding 2 major surface glycoproteins, gp63 and gp46/M2, heat shock protein 70 (hsp70), and beta-tubulin was determined for cloned isolates of 8 of these Leishmania species. The chromosome size class assignment of hsp70 genes was most conserved in that all species contained a single hybridizing DNA band of approximately 1200 kb. The beta-tubulin gene probe hybridized predominantly to large (1600-1750 kb) chromosome-size DNA and to 1-5 additional bands, the number of which depended on the species. The number and size of DNA bands hybridizing to gp63 or gp46/M2 gene probes were not uniformly conserved among species. In contrast to previous reports of gp63 genes being located on a single chromosome, using various CHEF gel conditions we observed a Leishmania major gp63 gene probe hybridizing to at least 2 chromosomal DNA bands in the New World species and in L. tropica. Gp46/M2 genes were located on 1 band in L. donovani, L. major, and L. aethiopica or 2 bands in L. tropica and L. amazonensis, but surprisingly, do not hybridize to any chromosomal DNA of species in the L. braziliensis complex or in L. enriettii. Whenever both genes were present in a species, gp63 and gp46/M2 genes were located on different chromosomal DNA bands.     

Leishmania donovani-reactive Th1- and Th2-like T-cell clones from individuals who have recovered from visceral leishmaniasis.

Infections in humans by Leishmania donovani parasites can result in a fatal disease, visceral leishmaniasis (VL), or in a self-limiting asymptomatic infection. In murine models of the infection employing Leishmania major, the course of the disease can be directed into a VL-like syndrome by interleukin-4 (IL-4)-producing Th2 cells, or cure may result by Th1 cells secreting gamma interferon (IFN-gamma). The present study examined the potential of human T cells to generate Th1 or Th2 responses to L. donovani. The profiles of IFN-gamma, IL-4, and lymphotoxin secretion after antigen stimulation were analyzed in a panel of L. donovani-reactive CD4+ human T-cell clones generated from individuals who had recovered from VL after antimonial treatment. Two of the T-cell clones produced large amounts of IL-4 without production of IFN-gamma, seven clones produced both IFN-gamma and IL-4, and eight produced only IFN-gamma. This is the first report of a Th1- and Th2-type response in human leishmaniasis. These results suggest that in analogy with murine models, there is a dichotomy in the human T-cell response to L. donovani infections. Preferential activation of IL-4-producing Th2-like cells may be involved in the exacerbation of human VL, whereas activation of IFN-gamma-producing Th1 cells may protect the host from severe disease. Identification of leishmanial antigens activating one or the other type of T cells will be important in the development of vaccines against leishmaniasis.     

The Pathogenesis of Leishmania aethiopica Infection in BALB/c Mice

A mouse model for L. aethiopica infection is described. BALB/c mice were unable to clear an infection with 1 x 10(7) promastigotes injected into the hind footpad. However, there was no ulceration of the lesion and no development of overt clinical symptoms after 203 days of infection. Spread of viable organisms was evident in the draining lymph node but not in the spleen or liver. The control of the infection was associated with the development of classical delayed hypersensitivity responses to phenolized promastigotes and appeared as a localized granulomtaous infiltration. The infiltration had features of classical tuberculoid granulomas, but superimposed on it was a strong eosinophilic infiltration. The relevance of such cells though unclear is discussed.     

Leishmania-specific T cells expressing interferon-gamma (IFN-gamma) and IL-10 upon activation are expanded in individuals cured of visceral leishmaniasis.

Peripheral blood mononuclear cells (PBMC) from patients who have recovered from visceral leishmaniasis often respond to Leishmania antigens in vitro by production of both IL-4, IFN-gamma and IL-10. In order to establish the cellular sources of these cytokines, we activated cells from individuals with a history of visceral leishmaniasis with Leishmania antigen for 6 days in culture, and identified cytokine production at the single-cell level by flow cytometry. The cytokines were only found in CD3+ cells and among these mainly within the CD4+ subset. The percentage of cytokine-producing cells was compared in Leishmania-activated PBMC cultures from the previous patients and from individuals living in a village where leishmaniasis does not occur. The percentage of IL-10- and IFN-gamma-containing cells was significantly higher in the previous patients than in the controls, indicating that Leishmania-specific T cells producing IL-10 and/or IFN-gamma had been expanded as a result of the infection. The cytokine-producing cells in the previous patients could be divided into three types: (i) cells producing IFN-gamma only; (ii) cells producing IL-4 only; and (iii) cells producing IFN-gamma and IL-10 simultaneously. The first and second group of cells can be described as Th1- and Th2-type cells, respectively. The third group could be a regulatory subset of T cells important for maintaining a balance between Th1- and Th2-type cells in these individuals.     

Differences in Gamma Interferon Production In Vitro Predict the Pace of the In Vivo Response to Leishmania amazonensis in Healthy Volunteers

The initial encounter of Leishmania cells and cells from the immune system is fundamentally important in the outcome of infection and determines disease development or resistance. We evaluated the anti-Leishmania amazonensis response of naive volunteers by using an in vitro priming (IVP) system and comparing the responses following in vivo vaccination against the same parasite. In vitro stimulation allowed us to distinguish two groups of individuals, those who produced small amounts of gamma interferon (IFN-gamma) (n = 16) (low producers) and those who produced large amounts of this cytokine (n = 16) (high producers). IFN-gamma production was proportional to tumor necrosis factor alpha and interleukin 10 (IL-10) levels but did not correlate with IL-5 production. Volunteers who produced small amounts of IFN-gamma in vitro remained low producers 40 days after vaccination, whereas high producers exhibited increased IFN-gamma production. However, 6 months after vaccination, all individuals tested produced similarly high levels of IFN-gamma upon stimulation of their peripheral blood mononuclear cells with Leishmania promastigotes, indicating that low in vitro producers respond slowly in vivo to vaccination. In high IFN-gamma producers there was an increased frequency of activated CD8(+) T cells both in vitro and in vivo compared to the frequency in low producers, and such cells were positive for IFN-gamma as determined by intracellular staining. Such findings suggest that IVP responses can be used to predict the pace of postvaccination responses of test volunteers. Although all vaccinated individuals eventually have a potent anti-Leishmania cell-mediated immunity (CMI) response, a delay in mounting the CMI response may influence resistance against leishmaniasis.     

Molecular epidemiology and population genetics in Leishmania

Polymorphic DNA sequences have been amplified using different PCR-based techniques and used for species identification, strain discrimination and population genetic studies in Leishmania. A PCR fingerprinting method that uses single non-specific primers generates species-specific banding patterns with some intraspecies variation. This approach can be used to identify Leishmania species and also to discriminate strains of different Leishmania species. Cultivation of the parasites is, however, mandatory. PCR-restriction fragment length polymorphism of the internal transcribed spacer (ITS) in the ribosomal operon differentiates all Leishmania species, except members of the L. donovani and L. brasiliensis complexes. ITS-single-strand conformation polymorphism or ITS sequencing can detect strain specific-variation (except in L. infantum); culturing is not required. Species of Leishmania exhibit different degrees of genetic variation (L. tropica > L. aethiopica > L. major > L. donovani). Population analysis using co-dominant DNA markers developed by sequence-confirmed amplified region analysis revealed a primarily clonal structure in a L. donovani population from Sudan and suggested that occasional recombination events may occur in this population.