Tumour necrosis factor (TNF alpha) in leishmaniasis. I. TNF alpha mediates host protection against cutaneous leishmaniasis.

Genetically resistant CBA mice developed significantly larger lesions to Leishmania major infection when they were injected with rabbit anti-tumour necrosis factor (TNF)-specific antibodies compared to control mice injected with normal rabbit immunoglobulin. BALB/c mice recovered from a previous infection following prophylactic sublethal irradiation also developed exacerbated lesions when treated with the anti-TNF antibody. Injection of TNF into the lesion of infected CBA mice significantly reduced the lesion development. Furthermore, TNF activates macrophages to kill Leishmania in vitro. These data demonstrate that TNF plays an important role in mediating host-protection against cutaneous leishmaniasis.     

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.     

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.     

Differences in Lsh gene control over systemic Leishmania major and Leishmania donovani or Leishmania mexicana mexicana infections are caused by differential targeting to infiltrating and resident liver macrophage populations.

Earlier studies had shown that the viscerotropic NIH 173 strain of cutaneous Leishmania major fails to come under Lsh gene control. Visceral Leishmania donovani LV9 and another viscerotropic cutaneous strain, Leishmania mexicana mexicana LV4, are controlled by Lsh. The results of double-infection experiments presented here show that expression of Lsh resistance against L. mexicana mexicana was enhanced in the presence of L. donovani, whereas L. major still failed to come under Lsh gene control, even in the presence of L. donovani. Prior irradiation (850 rads) of mice showed that in the absence of infiltrating monocytes, Lsh did exert some influence over L. major. The presence of a higher infiltrate of fresh monocytes after L. major infection was confirmed in liver macrophage populations isolated from mice after infection in vivo and in liver cryosections immunostained with monoclonal antibody M1/70 directed against the type 3 complement receptor CR3. The results support the hypothesis that Lsh is expressed maximally in the resident tissue macrophages and poorly in the immature macrophages preferentially infected by L. major amastigotes.     

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.     

Determination of the specificity of seric IgA produced in response to antigens of Leishmania (Leishmania) mexicana in murine leishmaniasis

In experimental leishmaniasis, the role of antibodies is not entirely clear, as some authors consider that these proteins are not involved in protection against infection. However, histopathological studies in human and experimental leishmaniasis lesions, show plasma cell infiltrates positive for IgA and secretion of IgM, IgG and IgA could mediate the formation of immune complexes with parasite antigens or self components, favoring necrosis leading to the elimination of the parasite. In this study, we determined if the serum IgA in the murine model has specific reactivity against antigens of Leishmania (Leishmania) mexicana of diagnostic utility. To do this, we used mice either susceptible or resistant to cutaneous leishmaniasis, and demonstrated by indirect ELISA that serum IgA is elevated in susceptible mice compared with that produced by resistant mice. Although other studies in murine models show that the serum IgG from mice infected with L. (L) mexicana present cross reactivity with unrelated parasite antigens derived from Trypanosoma cruzi, the analysis of the specificity of IgA by antigens of L. (L) mexicana and T. cruzi, by Western Blot, showed that the IgA serum of mice infected with T. cruzi reacts too with antigens of L. (L) mexicana. These findings suggest that IgA may be useful for the clinical management and prognosis of the disease.     

Interleukin 10- and Fcgamma receptor-deficient mice resolve Leishmania mexicana lesions

Infection of C57BL/6 (B6) mice with Leishmania mexicana is associated with a minimal immune response and chronic disease. Here we show that B6 interleukin 10-/- (IL-10-/-) mice resolve their lesions and exhibit increased gamma interferon (IFN-gamma), nitric oxide production, and delayed-type hypersensitivity. This enhanced resistance was dependent upon IL-12p40, since treatment of L. mexicana-infected IL-10-/- mice with anti-IL-12p40 monoclonal antibody abrogated healing. Antibody-opsonized L. mexicana induced IL-10 production by B6 macrophages in vitro, implicating antibody binding to Fc receptors as a mechanism involved in IL-10 production in this infection. Furthermore, B6 FcRgamma-/- mice resolve L. mexicana lesions, and lymph node cells from these mice produced less IL-10 and more IFN-gamma than cells from infected wild-type mice. These data demonstrate that removal of IL-10 or FcgammaR leads to resolution of L. mexicana disease and support a model in which ligation of FcgammaR by L. mexicana-bound immunoglobulin G promotes IL-10 production, leading to chronic disease.     

Altered virulence and vaccination properties of Leishmania parasites grown in infected vaccinated mice.

BALB/c mice, which are normally highly susceptible to growth of Leishmania mexicana parasites in vivo, can be vaccinated with avirulent temperature-sensitive mutants of L. mexicana so that challenge with virulent organisms results in markedly diminished growth of the latter. Parasites extracted from the lesions which do appear in these mice are able to produce active infection in secondary hosts, although the rate of progression of these lesions is slower than that seen with the original virulent cloned organism. Interestingly, when irradiated parasites from the secondary hosts are themselves used to vaccinate naive BALB/c mice, less protection is seen than when irradiated virulent organisms from the initial infecting clone are used. These data suggest that when infection does take place in mice vaccinated with avirulent clones of parasite, the organisms which develop in lesions in these animals are substantially modified from those present in the initial infecting inoculum.     

Endogenous interleukin-12 is critical for controlling the late but not the early stage of Leishmania mexicana infection in C57BL/6 mice

The role of interleukin-12 (IL-12) has been clearly established in the resistance of C57BL/6 (B6) mice to Leishmania major infection, but its involvement in the control of Leishmania mexicana infection remains to be determined. Here, we show the following. (i) L. mexicana, in contrast to L. major, induces the development of nonhealing lesions in B6 mice. (ii) Cells expressing IL-12p40, gamma interferon (IFN-gamma), NOS2, and CD40L are numerous in the footpad lesion and/or the draining popliteal lymph node of animals infected for up to 14 weeks with L. mexicana. (iii) B6 mice, either IL-12p40 deficient or treated with IL-12p40-neutralizing antibodies, display a dramatic enhancement of primary and secondary lesions leading to death 10 weeks after inoculation with L. mexicana. (iv) Splenocytes harvested 4 and 8 weeks after infection of IL-12p40(-/-) B6 mice with L. mexicana are unable to produce IFN-gamma, but secrete IL-4, IL-10, and IL-18. Thus, the early control of L. mexicana infection by B6 mice is independent of IL-12, whereas IL-12 and Th1 responses play a key role in controlling the late stages of L. mexicana infection. However, they fail to resolve lesions, in contrast to L. major infection, emphasizing the different outcomes induced by these two Leishmania species in B6 mice.     

Disruption of the murine interleukin-4 gene inhibits disease progression during Leishmania mexicana infection but does not increase control of Leishmania donovani infection.

The growths of both cutaneous leishmaniasis and visceral leishmaniasis caused by Leishmania mexicana and Leishmania donovani, respectively, were measured in interleukin-4 (IL-4) knockout mice (IL-4-/-) and compared with those of similarly infected wild-type (IL-4+/+) control mice. While large, nonhealing, cutaneous lesions containing large numbers of parasites developed in IL-4+/+ mice subcutaneously infected with 5 x 10(6) L. mexicana amastigotes in the shaven rump, in IL-4-/- mice no lesions whatsoever developed and parasites were difficult to detect. Systemic spread and metastasis were also noted in IL-4+/+ but not IL-4-/- mice. In contrast, IL-4-/- mice infected intravenously with 10(7) L. donovani amastigotes were found to have consistently higher parasite burdens in their livers throughout infection than did their wild-type counterparts. However, these differences were only significant at 15 days postinfection. While the results reported here pertaining to L. donovani largely support previous studies, those related to L. mexicana provide new observations. The immunological responses of IL-4-/- and IL-4+/+ mice infected with L. mexicana were, therefore, examined both in vivo and in vitro. Although neither IL-4-/- nor IL-4+/+ mice infected with L. mexicana produced parasite-specific immunoglobulin G2a antibodies, IL-4+/+ mice, unlike IL-4-/- mice, developed significant immunoglobulin G1 antibody titers as infection progressed, indicating a Th2-influenced response in wild-type mice. In addition, IL-4-/- mice, unlike IL-4+/+ mice, developed a significant delayed-type hypersensitivity response, indicating a Th1-influenced response in IL-4-/- mice. Following in vitro stimulation, splenocytes from IL-4+/+ mice infected with L. mexicana displayed significantly higher antigen-specific proliferative responses than did IL-4-/- mice. However, gamma interferon production as measured from the supernatants of the in vitro splenocyte cultures of IL-4-/- mice was significantly higher than that from IL-4+/+ mice. This again would indicate a predominantly Th1-influenced response in the absence of a Th2 response in IL-4-/- mice infected with L. mexicana. On the other hand, at the same time point, draining lymph node cells from IL-4+/+ mice produced significantly higher quantities of IL-5 than did those from IL-4-/- mice following in vitro antigenic stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

An essential role for IL-13 in maintaining a non-healing response following Leishmania mexicana infection

A comparison of the growth of Leishmania mexicana in IL-4(-/-), IL-4Ralpha(-/-) and wild-type BALB/c mice demonstrated a disease exacerbative role for IL-13 as well as IL-4. Thus, while both IL-4(-/-) and IL-4Ralpha(-/-) mice were more resistant than wild-type controls to infection with L. mexicana, IL-4Ralpha(-/-) mice, which are unresponsive to IL-13 as well as IL-4, were significantly more resistant to parasite growth than their IL-4(-/-) counterparts. Cytokine and antibody analysis revealed a Th1-biased specific response in both infected IL-4(-/-) and IL-4Ralpha(-/-) mice compared with wild-type animals. Reconstituting SCID mice with IL-4(-/-), IL-4Ralpha(-/-) or wild-type splenocytes prior to infection demonstrated that the early onset of lesion growth was dependent on the presence of lymphocytes responding to IL-4 and/or IL-13, as lesions failed to develop in only the SCID IL-4Ralpha(-/-) reconstituted mice. An independent role for IL-13 in L. mexicana infection was demonstrated by comparing disease progression in IL-13(-/-), IL-4(-/-)/IL-13(-/-) and wild-type B6/129 mice. In contrast to IL-4(-/-)/IL-13(-/-) mice, which were resistant, IL-13(-/-) mice developed lesions similar in size to wild-type animals up to week 8 post-infection. However, in contrast to wild-type mice in which disease continued to progress, lesions eventually healed in IL-13(-/-) mice, in association with the development of a Th1 response. Collectively our results suggest that IL-4 plays a critical role in early lesion development, and that IL-13 plays a crucial part in maintaining a chronic non-healing infection.     

Leishmania mexicana amazonensis infections in ''resistant'' inbred mice following removal of the draining lymph node.

Highly resistant (C57BL/10) and intermediately resistant (DBA/2) mice were infected subcutaneously with Leishmania mexicana amazonensis in a hind footpad subsequent to removal of the draining popliteal node. These mice developed greatly exacerbated Leishmania infections as compared to sham-operated controls or to mice infected in the contralateral footpad. The majority of mice in which the draining lymph nodes were removed prior to infection developed metastases, lost their delayed hypersensitivity responses to Leishmania, and some died. Significantly fewer metastases and no deaths were observed in the control groups. The results emphasize the importance of lymphatic control of Leishmania m. amazonensis infection in relatively resistant mouse strains.     

IL-13 gene-deficient mice are susceptible to cutaneous L. mexicana infection.

Recent studies have demonstrated that IL-13 mediates susceptibility to cutaneous L. major infection via IL-4-independent pathway. To determine whether IL-13 also plays a similar role in pathogenesis of cutaneous L. mexicana infection, we analyzed the course of L. mexicana infection in IL-13(-/-) and IL-4/IL-13(-/-) C57BL/6x129sv/Ev mice and compared with that in similarly infected wild-type mice. IL-13(-/-) mice were as susceptible as the wild-type mice to L. mexicana and developed rapidly progressing, large non-healing lesions following cutaneous L. mexicana infection. In contrast, similarly infected IL-4/IL-13(-/-) mice were highly resistant and developed either no lesions or small lesions containing few parasites that totally resolved by 12 weeks following infection. Throughout the course of infection IL-13(-/-) and the wild-type mice produced significantly more Th2-associated L. mexicana antigen (LmAg)-specific IgG1 than IL-4/IL-13(-/-) mice. All three groups produced comparable levels of Th1-associated IgG2a. At week 12 post infection, LmAg-stimulated spleen cells from L. mexicana-infected IL-4/IL-13(-/-) produced significantly higher levels of IL-12 and IFN-gamma as compared to those from similarly infected wild-type and IL-13(-/-) mice. Although both IL-13(-/-) and the wild-type spleen cells produced IL-4 following in vitro antigenic stimulation, the wild-type mice produced significantly more. These findings demonstrate that IL-13 is not involved in mediating susceptibility to L. mexicana. Moreover, they also indicate that IL-4 not IL-13 is a dominant cytokine involved in pathogenesis of cutaneous L. mexicana infection.     

Generalized infection and lack of delayed hypersensitivity in BALB/c mice infected with Leishmania tropica major.

The susceptibility of a few strains of mice to a subcutaneous injection of Leishmania tropica major, the causative agent of cutaneous leishmaniasis in humans, was studied. The infection in six strains (CBA, AKR/J, AKR/cu, C57BL/6, A/J, and C3H) remained cutaneous, and the animals recovered within 3 to 4 months. In contast, the infection in BALB/c became generalized and killed 1005 of infected animals. Intraperitoneal injection of infected liver of BALB/c to A/J and syngeneic mice produced a lethal disease in BALB/c but no infection in A/J mice. Lower doses of the parasite produced a lethal infection in BALB/c but no apparent disease in A/J. Hence, the host rather than the parasite is responsible for the outcome of the disease. The peak antibody titer of BALB/c mice was not significantly higher than that of A/J mice. However, BALB/c failed to show any delayed hypersensitivity to leishmania tested by footpad reaction, whereas A/J mice showed a strong response.     

Re-examination of the immunosuppressive mechanisms mediating non-cure of Leishmania infection in mice

Summary: The interleukin (IL)‐4 driven, polarized T‐helper 2 cell (Th2) response that controls non‐healing infection with Leishmania major in BALB/c mice has long been embraced as the underlying principle with which to consider the pathogenesis of non‐healing and systemic forms of leishmaniasis in humans. The inability, however, to reveal a Th2 polarity associated with non‐curing clinical disease has suggested that alternative cells and cytokines are involved in susceptibility. In this review, various mouse models of non‐curing infection with L. major and other Leishmania species are re‐examined in the context of the suppression mediated by IL‐10 and regulatory T (Treg) cells. These activities are revealed in L. major‐infected BALB/c IL‐4 knockout (KO) and IL‐4Rα KO mice and especially in non‐cure resistant mice that do not default to a Th2 pathway as a result of inherent defects in Th1 differentiation. In contrast to the extreme BALB/c susceptibility arising from an aberrant Th2 response, non‐cure in resistant mice arises from an imbalance in Treg cells that are activated in the context of an ongoing Th1 response and whose primary function may be to suppress the immunopathology associated with persistent antiparasite responses in infected tissues.     

Effect of CD4 monoclonal antibody in vivo on lesion development, delayed-type hypersensitivity and interleukin 3 production in experimental murine cutaneous leishmaniasis

Highly susceptible BALB/c mice subjected to adult thymectomy followed by prolonged (15 weeks), twice-weekly injections of a low dose (100 micrograms) of CD4 monoclonal antibody (MoAb) develop resistance to otherwise uniformly fatal and disseminating Leishmania major infection. In contrast, similar treatment with CD8 MoAb has no effect on the course of L. major infection. CD4 MoAb administered after the lesion establishment also has no effect. Mice which become resistant following CD4 MoAb treatment developed the classical delayed-type hypersensitivity (DTH) which persisted at 72 h after footpad injection with killed L. major promastigotes. A substantial degree of resistance can also be induced in the BALB/c mice with two i.v. high doses of 500 micrograms of CD4 MoAb given immediately prior to L. major infection. The treated mice developed classical DTH and significantly smaller lesions. The spleen cells from these mice also produced significantly lower levels of IL-3 compared to that of untreated control mice when cultured with L. major antigens in vitro. In contrast, genetically resistant CBA mice treated with CD4 MoAb developed significantly larger lesions but lower levels of classical DTH compared to untreated mice. These data confirmed and extended earlier reports on the prophylactic effect of CD4 MoAb in susceptible BALB/c mice and the disease exacerbative effect in the resistant CBA mice. The data also further illustrate the direct correlation between classical DTH and resistance, and the inverse correlation between IL-3 production and resistance in experimental cutaneous leishmaniasis.     

Coexistence of antigen-specific TH1 and TH2 cells in genetically susceptible BALB/c mice infected with Leishmania major

CD4-positive T cell clones with specificity for the protozoan parasite Leishmania major (L. major) of both the protective TH1 and the disease-exacerbating TH2 subtype were isolated from a diseased L. major-infected mouse of the susceptible BALB/c strain. In addition, TH2 cells were isolated from the lesion-draining lymph nodes of an animal clinically healed nine months after sublethal irradiation and subsequent infection. Our data support the notion that the differential outcome of the disease in non-irradiated versus irradiated BALB/c mice reflects the regulation of the two CD4+ T cell subsets. These data also argue against the possibilities that: 1) TH2 cells and their precursors are totally eliminated by irradiation and that 2) TH2 cells are capable of completely hindering the expansion of TH1 cells in diseased animals.     

Histopathologic changes induced by vaccination in experimental cutaneous leishmaniasis of BALB/c mice.

Highly susceptible BALB/c mice became partially resistant to Leishmania mexicana amazonensis infection after intravenous immunization with solubilized homologous promastigote antigen. Immunized BALB/c mice exhibited mixed mononuclear cell reactions, with granulomatous inflammation, collagen deposition, and fibrinoid necrosis at the site of infection. In contrast, naive animals displayed a monomorphic picture composed of largely vacuolated and parasitized macrophages with areas of coagulative necrosis. Electron microscopy revealed an increased number of eosinophils, sometimes in close contact with parasitized macrophages, in immunized animals. These findings illustrate that histologic changes reflect host immune status in cutaneous leishmaniasis, and that susceptibility of BALB/c mice to L m amazonensis, although dependent on genetic background, can be artificially modified.