Exacerbation of murine cutaneous leishmaniasis by adoptive transfer of parasite-specific helper T cell populations capable of mediating Leishmania major-specific delayed-type hypersensitivity

The effect of adoptive transfer of in vitro-propagated Leishmania major-specific T cell populations on the course of experimentally induced cutaneous leishmaniasis was studied in mice. The L. major-specific T cells expressed the T helper/inducer phenotype and were able in vitro to a) mount a specific proliferative response, b) provide specific helper activity for antibody responses, c) activate parasitized macrophages resulting in L. major destruction, and d) secrete macrophage-activating factors as tested in a tumoricidal assay. These T cells were also found capable of transferring parasite-specific delayed-type hypersensitivity responses to normal syngeneic mice. Results indicated that the i.v. transfer of these L. major-specific T cell populations into normal syngeneic mice exacerbated cutaneous lesions induced by infection with L. major. This effect on the disease process appeared to be dependent upon recognition of parasite antigens by the injected T cells because no exacerbation of the disease process was seen after the transfer of similar T cell populations specific for an antigen unrelated to the parasite, namely ovalbumin. However, the inclusion of ovalbumin in the L. major infecting inoculum resulted in an exacerbating effect of ovalbumin-specific T cells on cutaneous leishmaniasis. These unexpected results were supported by observations showing that immunization of mice with L. major antigens in complete Freund's adjuvant 7 days before infection with L. major led to exacerbated lesions. A similar aggravation of L. major-induced cutaneous lesions was also observed in mice previously immunized with an unrelated antigen provided that this antigen was included in the L. major infecting inoculum.     

GM-CSF produced by recombinant vaccinia virus or in GM-CSF transgenic mice has no effect in vivo on murine cutaneous leishmaniasis

The hemopoietic growth and differentiation regulators, granulocyte-macrophage colony-stimulating factor (GM-CSF) and the multipotential stimulating factor (multi-CSF) have been shown to have major effects on the effector function of mature macrophages. In this study we have examined the effect of recombinant GM-CSF and multi-CSF expressed transiently from recombinant vaccinia virus, or constitutively in GM-CSF transgenic mice on the development of cutaneous leishmaniasis, caused by Leishmania major in genetically susceptible or resistant mice. We observed no effect on the development of lesions when GM-CSF or multi-CSF were administered before infection, nor on the healing of lesions when they were administered after appearance of lesions. Although only some of the GM-CSF transgenic mice or their normal littermates developed lesions after infection with L. major, there was no difference between the groups in the rate of lesion development or in the size of lesions.     

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.     

Administration of monoclonal anti-IFN-gamma antibodies in vivo abrogates natural resistance of C3H/HeN mice to infection with Leishmania major

C3H/HeN mice that are naturally resistant to cutaneous disease and systemic infections with the protozoan parasite, Leishmania major, were treated at the time of infection, and weekly thereafter, with mouse anti-rat IFN-gamma mAb or an irrelevant antibody of similar isotype. Anti-IFN-gamma-treated mice developed cutaneous lesions; parasites spread to the regional lymph nodes and then metastasized to spleens and livers. The course of disease in these animals was similar to that of genetically susceptible BALB/c mice. Two exceptions in the pathology of L. major infections were noted between BALB/c and anti-IFN-gamma-treated C3H/HeN mice: 1) BALB/c mice died of systemic complications, whereas C3H/HeN mice did not, and 2) multinucleated giant cells were observed in lymph nodes and spleens of infected BALB/c mice, whereas these cells were not observed in infected C3H/HeN mice. Control mice, those treated with either saline or irrelevant antibody of the same isotype as the anti-IFN-gamma monoclonal, showed no evidence of cutaneous disease (development of footpad lesions) or systemic infection (by histopathology). Total abrogation of the natural resistance of C3H/HeN mice could be achieved by treatment with as little as 0.5 mg/mouse/wk of anti-IFN-gamma antibody, or by a single dose of 1 mg/mouse anti-IFN-gamma antibody administered at the time of parasite inoculation. If antibody treatment was delayed for as little as 1 wk after parasite inoculation, the infections in treated animals resembled that of untreated or control antibody-treated mice: no cutaneous lesions (by footpad swelling or viable counts of leishmania in footpad tissue) or systemic disease (by microscopic analysis of touch preparations of internal organs, and histopathology of same). The production of IFN-gamma during the initial interaction of the parasite and host cells appears to be a major component of genetic control of natural resistance to infection with L. major in C3H/HeN mice.     

Leishmania major: low infection dose causes short-lived hyperalgesia and cytokines upregulation in mice

Neural involvement was traditionally associated with leprosy. However, more recent studies have shown the presence of a persistent hyperalgesia in cutaneous leishmaniasis caused by the infection of BALB/c mice with a high dose of Leishmania major. In this study, we report the presence of hyperalgesia within the first two weeks of infection caused by a low dose of the parasite. Using BALB/c mice, we demonstrate the presence of hyperalgesia during the first 10 days of infection as assessed by thermal pain tests. After 10 days these decreased pain thresholds start to recover resulting in similar levels to those in uninfected controls during the third week of infection. This hyperalgesia is accompanied by a sustained upregulation of interleukin-1beta (IL-1beta) and an early upregulation of interleukin-6 (IL-6) which is restored to normal levels after five days of infection. In conclusion, this study shows that, during early infection, the low dose of L. major causes hyperalgesia accompanied by an upregulation of IL-1beta and IL-6 and that these effects are reversed within the first two weeks of infection.     

IL-10 and TGF-beta control the establishment of persistent and transmissible infections produced by Leishmania tropica in C57BL/6 mice

Leishmania tropica is the causative agent of Old World anthroponotic cutaneous leishmaniasis, which is characterized by lesions that take an extended period of time to heal, often resulting in disfiguring scars, and are more refractory to treatment than leishmaniasis caused by Leishmania major. Immunologic studies involving experimental animal models of L. tropica infection are virtually nonexistent. In the current study, infectious-stage L. tropica were used to establish dermal infections in C57BL/6 and BALB/c mice. In both strains, the lesions were slow to develop and showed minimal pathology. They nonetheless contained a stable number of between 10(4) and 10(5) parasites for over 1 year, which were efficiently picked up by a natural sand fly vector, Phlebotomus sergenti. Control of parasite growth depended on the development of a Th1 response, as C57BL/6 mice genetically deficient in Th1 cytokines and BALB/c mice treated with Abs to IFN-gamma harbored significantly more parasites. By contrast, IL-10-deficient mice harbored significantly fewer parasites throughout the infection. To further study the immunologic mechanisms that may prevent efficient clearance of the parasites, IL-10 and TGF-beta signaling were abrogated during the chronic phase of infection in wild-type C57BL/6 mice. Distinct from chronic L. major infection, IL-10 blockade alone had no effect on L. tropica, but required simultaneous treatment with anti-TGF-beta Abs to promote efficient parasite clearance from the infection site. Thus, chronic infection with L. tropica appears to be established via multiple suppressive factors, which together maintain the host as a long-term reservoir of infection for vector sand flies.     

The control of Leishmania (Leishmania) major by TNF in vivo is dependent on the parasite strain

Previous studies provided evidence that the role of TNF in the control of Leishmania (Leishmania) major might vary with the parasite strain. Here, we analyzed the development and outcome of cutaneous leishmaniasis in C57BL/6 wild-type (B6.WT) and TNF-deficient (B6. TNF(-/-)) mice infected with two different isolates of L. (L.) major (FRIEDLIN vs. BNI). L. (L.) major BNI caused progressive, fatal disease in B6.TNF(-/-) mice. In contrast, B6.TNF(-/-) mice infected with the L. (L.) major FRIEDLIN strain exhibited partial resistance characterized by chronic, non-healing skin lesions without lethality. Analysis of the tissue parasite numbers showed that the numbers of L. (L.) major FRIEDLIN and BNI parasites were comparable in footpads and lymph nodes of B6.TNF(-/-) mice, whereas in the spleen the parasite numbers were strikingly lower in the case of L. (L.) major FRIEDLIN. In vitro, cytokine-activated inflammatory macrophages killed L. (L.) major FRIEDLIN more efficiently than L. (L.) major BNI. These results suggest that in the absence of TNF, the course of leishmaniasis depends on the biology of the inoculated L. (L.) major strain, which most likely explains the previously published discrepant results on the role of TNF in leishmaniasis.     

Experimental cutaneous leishmaniasis. II. A possible role for prostaglandins in exacerbation of disease in Leishmania major-infected BALB/c mice

Leishmania major infection in genetically susceptible BALB/c mice is associated with the development of chronic primary lesions as well as multiple metastatic lesions. Spleen cells from these mice were shown to have depressed in vitro responses to concanavalin A (Con A) that coincided with the development of indomethacin-sensitive suppressor cells. Depressed responses to Con A were noted as early as 1 wk after parasite inoculation and correlated with the increased production of prostaglandin E2 (PGE2) by spleen cells from infected mice. Mice induced by prior irradiation (550 rad) to heal infection did not develop indomethacin-reversible depression in responsiveness to Con A. Although macrophages appear to be the major source of PGE2 production, in vitro studies indicate that infection per se is not a sufficient stimulus to initiate prostaglandin (PG) synthesis, suggesting the involvement of other cell types. Mice treated in vivo with indomethacin exhibited significantly fewer metastatic lesions than control mice, suggesting that PG may play a role in the exacerbation of cutaneous disease in these animals.     

The potency and durability of DNA- and protein-based vaccines against Leishmania major evaluated using low-dose, intradermal challenge

DNA- and protein- based vaccines against cutaneous leishmaniasis due to Leishmania major were evaluated using a challenge model that more closely reproduces the pathology and immunity associated with sand fly-transmitted infection. C57BL/6 mice were vaccinated s.c. with a mixture of plasmid DNAs encoding the Leishmania Ags LACK, LmSTI1, and TSA (AgDNA), or with autoclaved L. major promastigotes (ALM) plus rIL-12, and the mice were challenged by inoculation of 100 metacyclic promastigotes in the ear dermis. When challenged at 2 wk postvaccination, mice receiving AgDNA or ALM/rIL-12 were completely protected against the development of dermal lesions, and both groups had a 100-fold reduction in peak dermal parasite loads compared with controls. When challenged at 12 wk, mice vaccinated with ALM/rIL-12 maintained partial protection against dermal lesions and their parasite loads were no longer significantly reduced, whereas the mice vaccinated with AgDNA remained completely protected and had a 1000-fold reduction in dermal parasite loads. Mice vaccinated with AgDNA also harbored few, if any, parasites in the skin during the chronic phase, and their ability to transmit L. major to vector sand flies was completely abrogated. The durable protection in mice vaccinated with AgDNA was associated with the recruitment of both CD8(+) and CD4(+) T cells to the site of intradermal challenge and with IFN-gamma production by CD8(+) T cells in lymph nodes draining the challenge site. These data suggest that under conditions of natural challenge, DNA vaccination has the capacity to confer complete protection against cutaneous leishmaniasis and to prevent the establishment of infection reservoirs.     

Prophylactic treatment of BALB/c mice with cyclosporine A and its analog B-5-49 enhances resistance to Leishmania major

The effect of cyclosporine A (Cs A) and its analog B-5-49 on Leishmania major in vitro and in vivo in the highly susceptible BALB/c mouse strain has been investigated. In vitro, both of these drugs showed significant toxicity toward L. major, but only at relatively high levels (greater than 25 micrograms/ml). However, at 5 and 10 micrograms/ml, levels which correspond more closely to physiologically achievable concentrations, no growth-inhibitory effect in vitro was observed. On administration of the drugs to animals with established lesions, no beneficial effect was observed and, in fact, some exacerbation of lesion development and disease progression was noted. Surprisingly, a majority of the mice treated prophylactically with Cs A for a period of 7 consecutive days beginning 1 day before infection with L. major did not develop ulcerated cutaneous lesions, although some footpad swelling was observed 10 days to 2 wk after infection. These resistant animals displayed a sustained DTH after infection, and were resistant to further challenge with virulent L. major. Prophylactic treatment with the B-5-49 analog of Cs A was also effective in enhancing resistance to L. major infection in BALB/c mice, although to a somewhat lesser degree. Because the cyclosporines tested do not appear to be directly toxic nor inhibitory in vivo for established L. major infections, it appears that these drugs may be effective in modulating the induction stage of the immune response toward the parasites in the BALB/c mouse in such a way as to allow a protective immunity to develop.     

Granulocyte-macrophage colony-stimulating factor is a growth-factor for promastigotes of Leishmania mexicana amazonensis

In this paper we show that murine lung conditioned medium (LCM) displays, in addition to its already described colony-stimulating activity on bone marrow cells, a potent growth-stimulating activity on promastigotes of Leishmania mexicana amazonesis. Immunoprecipitation of LCM with an antibody specific for murine granulocyte-macrophage colony stimulating factor (GM-CSF) abrogates both activities, indicating that the leishmanial growth-promoting activity is due to the presence of GM-CSF on LCM. Furthermore, recombinant GM-CSF (rGM-CSF) added to the culture medium or to the immunoprecipitated LCM is able to respectively induce or to partially recover the growth-promoting activity of the LCM. Sequential in vitro passages of the parasite induces a progressive loss of sensitivity to the growth-factor. Parasite forms recently collected from lesions are significantly more responsive to the growth-factor than forms already adapted to grow in culture. Since it has been shown that several different microorganisms display receptors for vertebrate-like hormones and that GM-CSF is able to enhance a cutaneous leishmanial lesion, our results permit us to raise the hypothesis that a direct interaction between a host-derived hormone and a pathogenic microorganism can be of importance in defining the fate of an infection. The fact that GM-CSF is produced by cells that actively participate in a leishmanial infection (T-lymphocytes and macrophages) reinforces our hypothesis.     

TGF-beta-associated enhanced susceptibility to leishmaniasis following intramuscular vaccination of mice with Leishmania amazonensis antigens

Leishmania amazonensis and Leishmania braziliensis are the main causal agents of anergic diffuse cutaneous leishmaniasis and hyperergic mucosal leishmaniasis in man, respectively. In this work we demonstrate that intramuscular vaccination of BALB/c mice with whole antigens of L. amazonensis (LaAg) but not L. braziliensis (LbAg) results in increased susceptibility to cutaneous leishmaniasis. LaAg vaccination resulted in an increased capacity of the draining lymph nodes to produce IL-10 and TGF-beta during antigen recall responses. In vitro cultivation with LaAg but not LbAg induced increased apoptosis of CD8+ T cells. Following infection with L. amazonensis, LaAg-vaccinated mice produced significantly more TGF-beta and a higher serum IgG1/IgG2a antibody ratio compared with LbAg-vaccinated and non-vaccinated animals. The association of TGF-beta with enhanced susceptibility to infection was confirmed in mice co-vaccinated with LaAg and neutralizing anti-TGF-beta antibodies. Upon parasite challenge, these animals developed much smaller lesion sizes and parasite burdens, comparable with non-vaccinated controls. The disease-promoting effect of LaAg vaccination is not a general event, as in contrast to BALB/c, the disease outcome in C57Bl/6 mice was unaltered. Together, these findings indicate that species-specific components of L. amazonensis activate overt TGF-beta production that predisposes more susceptible individuals to aggravated disease following vaccination.     

Leishmania major: nature of immunity induced by immunization with a mutagenized avirulent clone of the parasite in mice

A chemically mutagenized avirulent form of Leishmania major was used to immunize BALB/c and C57B1/6 mice against challenge with virulent L. major. Immunity was elicited when the avirulent parasite was injected intravenously or intraperitoneally, but not subcutaneously. In fact, the latter route of immunization sometimes resulted in exacerbation of a subsequent infection with virulent L. major. Mice immunized with avirulent L. major developed upon challenge with virulent L. major cutaneous lesions which were significantly smaller and contained substantially fewer parasites than lesions on control nonimmune animals. Finally, the protection conferred by immunization with avirulent L. major could be adoptively transferred with T cells of the CD4+ lineage but not the CD8+ lineage.     

Resolution of an infection with Leishmania braziliensis confers complete protection to a subsequent challenge with Leishmania major in BALB/c mice

Both Leishmania major and L. braziliensis induce cutaneous leishmaniasis in BALB/c mice. Whereas BALB/c mice die of infection with L. major, they cure an infection with L. braziliensis. We report here that after curing an infection with L. braziliensis, BALB/c mice are resistant to challenge with L. major. When challenged with L. major, L. braziliensis pre-treated BALB/c mice mounted a delayed-type hypersensitivity response to L. major and produced high amounts of interferon-gamma (IFN-gamma) but low amounts of interleukin-4. The IFN-gamma produced by the L. braziliensis pre-infected mice was involved in the protection seen against L. major challenge since treating the mice with a neutralizing anti-IFN-gamma abrogated the protection. This suggests that cross-reactive antigen epitopes exist between L. braziliensis and L. major and that pre-infection with L. braziliensis primes BALB/c mice to epitopes on L. major that can elicit a protective Th1 response to the parasite.     

Granulocyte-Macrophage Colony-Stimulating Factor is a Growth-Factor for Promastigotes of Leishmania mexicana amazonensis

ABSTRACT. In this paper we show that murine lung conditioned medium (LCM) displays, in addition to its already described colony-stimulating activity on bone marrow cells, a potent growth-stimulating activity on promastigotes of Leishmania mexicana amazonensis . Immunoprecipitation of LCM with an antibody specific for murine granulocyte-macrophage colony stimulating factor (GM-CSF) abrogates both activities, indicating that the leishmanial growth-promoting activity is due to the presence of GM-CSF on LCM. Furthermore, recombinant GM-CSF (rGM-CSF) added to the culture medium or to the immunoprecipitated LCM is able to respectively induce or to partially recover the growth-promoting activity of the LCM. Sequential in vitro passages of the parasite induces a progressive loss of sensitivity to the growth-factor. Parasite forms recently collected from lesions are significantly more responsive to the growth-factor than forms already adapted to grow in culture. Since it has been shown that several different microorganisms display receptors for vertebrate-like hormones and that GM-CSF is able to enhance a cutaneous leishmanial lesion, our results permit us to raise the hypothesis that a direct interaction between a host-derived hormone and a pathogenic microorganism can be of importance in defining the fate of an infection. The fact that GM-CSF is produced by cells that actively participate in a leishmanial infection (T-lymphocytes and macrophages) reinforces our hypothesis.     

Nonhealing infection despite Th1 polarization produced by a strain of Leishmania major in C57BL/6 mice

Experimental Leishmania major infection in mice has been of immense interest because it was among the first models to demonstrate the importance of the Th1/Th2 balance to infection outcome in vivo. However, the Th2 polarization that promotes the development of nonhealing cutaneous lesions in BALB/c mice has failed to adequately explain the mechanisms underlying nonhealing forms of leishmaniasis in humans. We have studied a L. major strain from a patient with nonhealing lesions that also produces nonhealing lesions with ulcerations and high parasite burden in conventionally resistant C57BL/6 mice. Surprisingly, these mice develop a strong, polarized, and sustained Th1 response, as evidenced by high levels of IFN-gamma produced by Leishmania-specific cells in the draining lymph node and in the ear lesion, and an absence of IL-4 or IL-13. The parasites fail to be effectively cleared despite high level induction of inducible NO synthase in the lesion, and despite their sensitivity to killing by IFN-gamma-activated macrophages in vitro. Infection of IL-10(-/-) mice, blockade of the IL-10R, or depletion of CD25(+) cells during the chronic phase promotes parasite killing, indicating that IL-10 and regulatory T cells play a role in rendering the Th1 responses ineffective at controlling infection in the skin. Mice with nonhealing primary lesions are nonetheless resistant to reinfection in the other ear. We suggest that nonhealing infections in animal models that are explained not by aberrant Th2 development, but by overactivation of homeostatic pathways designed to control inflammation, provide better models to understand nonhealing or reactivation forms of leishmaniasis in humans.     

Intradermal inoculations of low doses of Leishmania major and Leishmania amazonensis metacyclic promastigotes induce different immunoparasitic processes and status of protection in BALB/c mice

In order to simulate the natural long term parasitisms which may occur in mammals infected with Leishmania, cutaneous leishmaniases due to Leishmania major or Leishmania amazonensis were induced using a model based on the inoculation of 10-1000 metacyclic promastigotes into the ear dermis of BALB/c mice. The final outcome of these parasitisms was dependent upon the number of inoculated parasites. Only some of the mice inoculated with ten parasites displayed cutaneous lesions, whereas most mice infected with 100 metacyclics and all mice infected with 1000 metacyclics developed progressive lesions. We found, using the latter experimental conditions, that the onset of the pathology was associated with: (a) parasite multiplication in the inoculation site and the draining lymph node correlating with an increase of the lymph node cell number, especially in L. major-infected mice; and (b) the detection of lymph node cells, at least in part CD4(+) T lymphocytes, able to produce high levels of interferon-gamma, interleukin (IL)-4, IL-10 and IL-13. Thereafter, mice infected by L. major harboured few parasites in the ear and had a 100-fold reduction in lymph node parasite load between 23 and 40 weeks post-inoculation. In contrast, the parasite loads of L. amazonensis-infected mice remained stable in the ear and increased in nodes during the same period of time. Only L. major-infected mice that exhibited cutaneous lesions in the primary site were resistant to the re-inoculation of 1000 metacyclic promastigotes, whereas all L. amazonensis-primary infected mice remained susceptible to a second homologous challenge. These results are the first to document that a status of resistance to re-infection, referred to concomitant immunity, is coupled to the development of primary progressive lesions in L. major-infected BALB/c mice. Such a protective status is absent in L. amazonensis-infected BALB/c mice.     

Analysis of variables associated with promotion of resistance and its abrogation in T cell-reconstituted nude mice infected with Leishmania major

Upon intradermal challenge with the protozoan parasite Leishmania major, some mouse strains develop chronic cutaneous lesions, whereas other mouse strains show a resolving pattern of disease. The importance of T cell-dependent immunity in resistance to cutaneous leishmaniasis is substantiated by the susceptibility to infection of athymic nude mice of both resistant and susceptible strains. Small numbers of T lymphocytes from uninfected euthymic mice promote resistance in nude mice but T cells from chronically infected mice can impair this protective effect. In the present study we used an adoptive transfer system in which nude mice were reconstituted with T cells from normal or chronically infected mice in order to further investigate protection against disease or disease promotion. The results supported the following conclusions: (a) the host-protective activity of T cells from uninfected mice is highly effective even in long-term chronically infected nude mice, (b) T cell-mediated exacerbation of cutaneous disease does not involve enhancement of lesion development and is thus unlikely to be based on an accelerated proliferation of parasites in the lesion, (c) disease-promoting cells are not only found in genetically susceptible mice but can also be induced in genetically resistant mice, and (d) lymphoid organs of genetically susceptible mice chronically infected with L. major contain resistance-promoting cells in addition to disease-promoting cells. The data, together with those of others, continue to support the notion that recruitment with expansion and/or activation of different T cell subsets underlies genetically based resistance and susceptibility of mice to L. major.     

The relative contribution of IL-4 receptor signaling and IL-10 to susceptibility to Leishmania major

The roles of IL-10 and IL-4 receptor signaling were evaluated in a murine model of Leishmania major infection. In previous studies the L. major substrain LV39 caused progressive, nonhealing lesions in BALB/c mice deficient for IL-4R alpha-chain (IL-4R alpha), while substrain IR173 was highly controlled. To explore whether IL-10 is responsible for inducing susceptibility to LV39, wild-type and IL-4R alpha(-/-) mice were treated with anti-IL-10R mAb, and in a genetic approach, the IL-4R alpha(-/-) mice were crossed with BALB/c IL-10(-/-) mice. In contrast to the lack of resistance conferred by IL-4R alpha gene deletion, partial resistance to LV39 was conferred by IL-10 gene deletion or treatment of BALB/c mice with anti-IL-10R mAb. Lesion sizes and LV39 parasite numbers were further and dramatically reduced in both anti-IL-10R-treated IL-4R alpha(-/-) mice and IL-4R alpha x IL-10 double knockouts. Anti-IL-10R mAb treatment further suppressed parasite growth in IL-4R alpha(-/-) mice infected with L. major IR173. Production of IFN-gamma was only increased relative to wild-type or littermate controls in IL-4R alpha(-/-) mice with complementary defects in IL-10. Comparisons of IFN-gamma-treated infected macrophages in vitro indicated that LV39 required 25- to 500-fold greater concentrations of IFN-gamma than IR173-infected macrophages to achieve a similar efficiency of parasite killing. These studies suggest that regardless of parasite substrain, IL-10 is as important as IL-4/IL-13 in promoting susceptibility to L. major and even more so for those substrains that are relatively resistant to IFN-gamma mediated killing.     

Infection-induced respiratory burst in BALB/c macrophages kills Leishmania guyanensis amastigotes through apoptosis: possible involvement in resistance to cutaneous leishmaniasis

The immune mechanisms that underlie resistance and susceptibility to leishmaniasis are not completely understood for all species of Leishmania. It is becoming clear that the immune response, the parasite elimination by the host and, as a result, the outcome of the disease depend both on the host and on the species of the infecting Leishmania. Here, we analyzed the outcome of the infection of BALB/c mice with L. guyanensis in vivo and in vitro. We showed that BALB/c mice, which are a prototype of susceptible host for most species of Leishmania, dying from these infections, develop insignificant or no cutaneous lesions and eliminate the parasite when infected with promastigotes of L. guyanensis. In vitro, we found that thioglycollate-elicited BALB/c peritoneal macrophages, which are unable to eliminate L. amazonensis without previous activation with cytokines or lipopolysaccharide, can kill L. guyanensis amastigotes. This is the first report showing that infection of peritoneal macrophages with stationary phase promastigotes efficiently triggers innate microbicidal mechanisms that are effective in eliminating the amastigotes, without exogenous activation. We demonstrated that L. guyanensis amastigotes die inside the macrophages through an apoptotic process that is independent of nitric oxide and is mediated by reactive oxygen intermediates generated in the host cell during infection. This innate killing mechanism of macrophages may account for the resistance of BALB/c mice to infection by L. guyanensis.