Early in vitro priming of distinct Th cell subsets determines polarized growth of visceralizing Leishmania in macrophages

Nitza A. Gomes, Victor Barreto-de-Souza and George A. DosReis

Immunobiology Program, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Bloco G, Ilha do Fundão, Rio de Janeiro, RJ 21944-970, Brazil

Correspondence to: G. A. DosReis


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
An in vitro priming system of murine naive splenocytes was established to investigate early immune responses to Leishmania chagasi, the agent of visceral leishmaniasis in the New World. Priming of splenocytes from resistant C3H and CBA or susceptible BALB and B10 mice with L. chagasi resulted in blast transformation and in proliferating parasite-specific CD4+ T cells secreting a differential complement of cytokines (IFN-{gamma} and low IL-10 levels for resistant T cells; IFN-{gamma}, IL-4 and high IL-10 levels for susceptible T cells). After priming, intracellular parasite load was much higher in susceptible than in resistant-type splenocyte cultures. On the other hand, infection of purified splenic macrophages from either resistant or susceptible mice with live L. chagasi promastigotes, resulted in comparable parasite loads. Moreover, when early CD4+ T cell priming in splenocyte cultures was disrupted with anti-CD4 mAb, polarized parasite growth was abolished, becoming comparable in resistant and susceptible cultures. Neutralizing IL-4 activity during splenocyte priming did not affect the final parasite load in susceptible cultures. However, neutralizing IL-10 activity markedly decreased parasite load in susceptible, but not in resistant splenic macrophages. These results suggest that IL-10 plays an important role in L. chagasi infection in susceptible hosts. The results also indicate that innate control of growth of a visceralizing Leishmania in splenic macrophages results from the ability to activate different CD4+ T cell subsets.

Keywords: cytokines, differentiation, IL-10, Leishmania, Th1/Th2, visceral leishmaniasis


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The functional dichotomy between Th1 and Th2 cell subsets is central in regulating resistance to cutaneous leishmaniasis in mice (1). In the case of visceral leish- maniasis (VL), however, a counterprotective Th2 immune response is not readily apparent in mice, although it has been described in humans (2). In murine VL, regional differences in cell-mediated responses to the parasite have been described in the liver and spleen (35). Susceptible BALB strain mice resolve hepatic infection after a few weeks via an IL-12- and IFN-{gamma}-dependent Th1 response (6), but the visceralizing Leishmania later colonizes the spleen and grows indefinitely after hepatic infection has been cured (35,7). Recent studies found a mixed Th1/Th2 response by parasite-specific T cells from both acute and chronic murine VL (5,8). A shift from mixed Th1/Th2 to a pure Th1-type response was reported in mice rendered more resistant to L. donovani due to a gene-conversion mutation in the MHC class II molecule I-A (9). In addition, mice with visceral disease express both a transient increase in IL-4 and a sustained increase in IL-10 mRNA expression in spleen (10). Together, these results suggest the occurrence of Th2 responses in chronic forms of murine VL.

A genetic polymorphism conferred by the Lsh gene in mouse controls natural resistance to early growth of visceralizing Leishmania within macrophages (11). The cloned intracellular membrane transporter natural resistance- associated macrophage protein 1 (Nramp1) has been identified as the Lsh gene product, responsible for the slow parasite growth associated with the resistant allele (12). One additional genetic polymorphism controls an intrinsic propensity of primed CD4+ T cells to develop into the Th2 phenotype (13) and multiple loci affecting Th1/Th2 balance were identified in the control of resistance against leishmanial infection (14). It is unknown how different genetic factors are expressed during early responses to infection in order to determine resistant and susceptible phenotypes in VL. Here, we used genetically resistant and susceptible murine strains (15) to examine the cellular basis for the differential control of visceralizing Leishmania growth in macrophages. We established a primary in vitro system of splenic naive T cell priming (PIV) to L. chagasi. This system, originally developed for L. major (16), enables the analysis of cytokine and cellular interactions in the early response to live parasites. We demonstrate that protective CD4+ T cells are rapidly induced in resistant, and counterprotective T cells are induced in susceptible culture types, concomitant with a polarized behavior of parasite replication. Polarized parasite growth in macrophages is completely abolished by eliminating early CD4+ T cell priming. These results indicate a critical role of early acquired T cell responses in the innate host control of visceralizing Leishmania.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals and parasites
Female BALB/c and C3H/HeN mice (6–8 weeks old) were obtained from the Brazilian National Cancer Institute (INCa) animal facility, and C57Bl/10J (B10) and CBA/J mice were from Universidade Federal Fluminense, Rio de Janeiro. BALB and B10 mice are susceptible, and C3H and CBA mice are resistant respectively to in vivo infection with visceraliz- ing Leishmania (11,15). An isolate of L. chagasi (MHOM/BR/72/ strain 46) (17) was provided by Dr Carlos Corbett (University of São Paulo Medical School). Amastigotes of L. chagasi were purified from the spleens of infected Syrian hamsters, as described (2). Promastigotes were maintained in Schneider medium.

Antibodies
Anti-mouse CD3 {varepsilon} chain mAb 145.2C11, anti-mouse CD4 mAb GK1.5, anti-mouse IL-4 mAb BVD4-1D11 and control rat IgG2b mAb were from PharMingen (San Diego, CA). Control goat IgG and goat anti-mouse IL-10 (polyclonal antibody IgG fraction) were from Sigma (St Louis, MO). The following mAb from PharMingen were used for negative selection of splenic macrophages: anti-B220, anti-CD4 mAb GK1.5, anti-CD8 mAb 53.6.7 and anti-{gamma}{delta} TCR mAb 13D5.

Isolation and assay of purified macrophages from susceptible and resistant strains
Highly purified macrophages from normal BALB or C3H mice were obtained from spleen by magnetic cell sorting after treatment with a mixture of anti-B220, anti-CD4, anti-CD8 and anti-TCR{gamma}{delta} (all at 10 µg/ml) for 30 min at 4°C. Cells were washed and incubated with anti-rat Ig-coated magnetic beads (Bio-Mag; PerSeptive Diagnostics, Cambridge, MA) at a bead:cell ratio of 20:1 for 20 min at room temperature. Negative selection was performed on a magnetic separator and negative cells were obtained after three cycles of magnetic separation. After treatment, purified macrophages were added to 24-well culture plates at high density (107/ml), in 1 ml complete medium containing 0.5% normal mouse serum. After overnight incubation at 37°C, 7% CO2, excess non-adherent cells were discarded and macrophage monolayers were infected with 106/ml live L. chagasi promastigotes. After 7 days in culture, adherent cells were extensively washed and Schneider medium was added to the cultures. No extracellular parasite was detected at this stage.

Priming of Leishmania-specific T cells in vitro (PIV)
We used the method originally described by Shankar and Titus for priming to L. major (16). Spleens from naive mice were removed, red cells were lysed and a cell suspension was prepared. Splenocytes were resuspended at 107 cells/ml in complete culture medium containing 0.5% normal mouse serum. Culture medium consisted of DMEM supplemented with 2 mM glutamine, 5x10–5 M 2-mercaptoethanol, 10 µg/ml gentamicin, sodium pyruvate, MEM non-essential amino acids, and 10 mM HEPES buffer. Cells were cultured in 1 ml volume in 24-well culture plates (Corning, Corning, NY). Live stationary phase L. chagasi promastigotes were added at 106/ml. Anti-CD4 mAb and anti-mouse IL-10 or anti-mouse IL-4 neutralizing antibodies, and their isotype controls, were added at the dose of 10 µg/ml at the beginning of culture. After 7 days in culture at 37°C, 7% CO2, viable non-adherent cells were recovered and T cell blasts were isolated by centrifugation on a Ficoll gradient. Adherent cells were extensively washed and Schneider medium was added to the monolayer. In some experiments, proliferation was measured directly in the primary culture. In this case, non-adherent cells were recovered at day 7 in culture, transferred (200 µl/well) to 96-well plates and 0.5 µCi [3H]thymidine (5 Ci/mmol; Sigma) was added. Incorporation of [3H]thymidine onto DNA was measured by liquid scintillation spectroscopy after additional 6 h in culture. Results are mean ± SE of triplicate c.p.m. values.

Secondary proliferation and cytokine production by PIV T cells
Before re-stimulation, isolated T cell blasts were rested in DMEM, 0.5% normal mouse serum, for 3 days. After rest, triplicate wells were prepared on flat-bottom 96-well microtiter plates (Corning) containing 106 irradiated (3000 rad) normal syngeneic splenocytes, 5x104 isolated T cell blasts and graded numbers of live L. chagasi promastigotes were added in 200 µl culture medium with 10% FCS. Proliferation was assessed by uptake of [3H]thymidine onto DNA after 3 days in culture, following a pulse with 0.5 µCi [3H]thymidine during the last 18 h. In order to measure lymphokine production, the same rested PIV T cells (106/well) were re-stimulated in 1 ml complete culture medium, with 5x106/well irradiated (900 rad) normal syngeneic splenocytes, with or without 106/well live L. chagasi promastigotes, or with or without soluble anti-CD3 mAb (5 µg/ml). Supernatants from triplicate cultures were individually collected after 48 h. The contents of IL-10, IL-4 and IFN-{gamma} in supernatants were evaluated by sandwich ELISA, according to a protocol provided by the manufacturer, using pairs of cytokine-specific mAb, one of which was biotinylated (capture and detection mAb pairs for IL-10, IL-4 and IFN-{gamma}; PharMingen). The reaction was revealed with alkaline phosphatase-conjugated streptavidin (Southern Biotechnology Associates, Birmingham, AL) using p-nitrophenol phosphate (Sigma) as substrate. Recombinant murine IL-10, IFN-{gamma} and IL-4 (PharMingen) were used as positive controls.

Determination of intracellular parasite burden at the end of PIV
After a primary 7-day culture, non-adherent cells were removed, adherent cells were extensively washed, and 1 ml of Schneider medium containing 20% FCS and 2% human urine as described (18) was added to the wells. After washing, no extracellular parasites were detected. After additional 5–7 days at 26°C, the growing extracellular motile L. chagasi promastigotes derived from infected macrophages were counted. Data show individual values or mean ± SE of triplicate cultures.

Statistical analysis
Proliferation data and parasite counts were first normalized by a log transformation. Transformed data and ELISA OD readings were then compared by paired Student's t-test. Significance is indicated in legends. All experiments shown are representative of at least two independent experiments, with similar results.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
PIV with susceptible and resistant splenocytes generates L. chagasi-specific proliferating CD4+ T cells, and leads to polarized outcomes of parasite load
Following in vitro priming with L. chagasi, activated T cells from both susceptible (BALB) and resistant (C3H) phenotypes were isolated and rested, and proliferation was induced with live L. chagasi promastigotes and fresh antigen-presenting cells (APC). Both resistant and susceptible T cells proliferated to parasites in a dose-related manner (Fig. 1AGo). Secondary proliferation was abolished with anti-CD4 mAb and did not occur with epimastigotes of the unrelated parasite Trypanosoma cruzi (not shown). To investigate whether splenocyte priming of naive CD4+ T cells reproduces resistance/susceptibility against infection with visceralizing Leishmania (15), the intracellular load of L. chagasi was measured in adherent cells at the end of primary culture, after removal of lymphocytes and any residual extracellular parasite (Fig. 1BGo). In this and other experiments, parasite growth was always polarized at the end of PIV, ranging from 60- to 3000-fold higher in susceptible, compared to resistant macrophages (Fig. 1BGo). The absolute number of intracellular parasites produced in PIV cultures varied between different experiments, specially in susceptible cells, presumably due to variations in promastigote infectivity. However, intracellular loads taken from replicates within the same experiment were very similar.



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Fig. 1. (A) Parasite-induced proliferation in PIV T cells. Splenocyte cultures from either susceptible BALB or resistant C3H strains were primed with live L. chagasi. Activated T cells were isolated, rested and re-stimulated with the indicated numbers of live L. chagasi promastigotes/culture, plus fresh APC. Proliferation was assessed by [3H]thymidine uptake after 3 days. Data are mean ± SE of triplicate cultures. (B) PIV mimics the in vivo behavior of parasite growth. Splenocytes from naive C3H and BALB mice were primed for 7 days with live L. chagasi promastigotes. After culture, non-adherent cells and parasites were removed, and Schneider medium was added to the cultures. Intracellular amastigote burden was determined in splenic adherent cells by counting the number of resulting viable promastigotes after 7 days at 26°C. Data shown are the triplicate values of a representative experiment. (C) Purified splenic macrophages from BALB and C3H mice are equally susceptible to infection. Highly purified macrophages from normal C3H and BALB spleens were cultured for 7 days in the presence of live L. chagasi promastigotes. After culture, monolayers were washed and Schneider medium added to the cultures. Intracellular amastigote burden was determined as described in (B).

 
To investigate whether intracellular parasite load in macrophages was dependent or not on the presence of co-cultured T cells, highly purified macrophages from both susceptible BALB and resistant C3H mice were infected in vitro with L. chagasi. After 7 days in culture, the resulting parasite load did not differ between BALB and C3H splenic macrophages (Fig. 1CGo), suggesting that CD4+ T cells were required for polarized parasite growth.

PIV with susceptible and resistant splenocytes leads to priming of distinct T cell subsets
Although PIV T cells from either BALB or C3H mice proliferated to L. chagasi, the resulting primed T cells could be functionally distinct. To investigate this possibility, supernatants from secondary cultures were collected and assayed for activity of the cytokines IFN-{gamma}, IL-4 and IL-10 (Fig. 2Go). PIV T cells from susceptible BALB and B10, and from resistant C3H and CBA strains (11,15), secreted a comparable amount of type 1 cytokine IFN-{gamma} in response to L. chagasi (Fig. 2Go, top), but secretion of type 2 cytokines was different between strains. Secretion of IL-10 was much higher (4.3- to 8.7-fold) in PIV T cells from susceptible BALB and B10 strains, than in cells from resistant C3H and CBA strains (Fig. 2Go, middle). In addition, parasite-induced secretion of IL-4 was detected only in susceptible BALB and B10 T cells, but not in resistant C3H and CBA T cells (Fig. 2Go, bottom), although CBA T cells gave a relatively higher background secretion of this cytokine. These results indicate rapid induction of different CD4+ T cell subsets during priming of resistant or susceptible splenocytes. To investigate whether differential secretion of type 2 cytokines was selective for L. chagasi stimulation, parasite-activated T cells from susceptible and resistant types were polyclonally re-stimulated with anti-CD3 mAb and APC (Table 1Go). Secretion of IFN-{gamma}, IL-10 and IL-4 was similar in both BALB and C3H T cells. These results indicate that distinct T cell subsets are selectively activated by antigenic stimulation with L. chagasi.



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Fig. 2. Different CD4+ T cell subsets are induced in resistant and susceptible splenocytes. Naive splenocytes from susceptible BALB and B10, and from resistant C3H and CBA strains were primed with L. chagasi. After priming, rested T cell blasts were re-stimulated with irradiated syngeneic APC in the presence or absence of L. chagasi promastigotes for 2 days. Supernatants were assayed for content of IFN-{gamma} (top), IL-10 (middle) or IL-4 (bottom), by sandwich ELISA. Results are mean ± SE of triplicate cultures. P < 0.01 for all cytokine levels in the presence of parasites, except IL-10 and IL-4 in C3H, and IL-4 in CBA cultures, which were NS.

 

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Table 1. Anti-CD3-induced cytokine secretion by parasite-activated T cells from susceptible and resistant strains
 
Blockade of CD4+ T cell priming abolishes polarized parasite growth
To investigate the involvement of T cells in induction of polarized parasite growth, PIV cultures were established in the presence of anti-CD4 mAb or an isotype control. At the end of the PIV, lymphocyte proliferation and resulting intracellular load of L. chagasi in adherent cells were measured (Fig. 3Go). Addition of anti-CD4 did not affect viable T cell recovery, but blast transformation in primary cultures was completely inhibited in both susceptible and resistant cultures (Fig. 3A and CGo). More important, following disruption of parasite-specific CD4+ T cell activation, parasite load decreased 10-fold in susceptible BALB macrophages, from 150.0 ± 21.6x106 to 14.3 ± 2.9x106/culture (Fig. 3BGo). Conversely, after CD4 blockade in the primary culture, parasite load increased nearly 5-fold in resistant C3H macrophages, from 2.5 ± 0.3x106 to 11.3 ± 1.8x106/culture (Fig. 3BGo). Therefore, in the absence of CD4+ T cell activation, the intracellular load of L. chagasi became essentially similar in both BALB and C3H macrophages. These results were confirmed in two additional experiments and indicate that the T cell subsets generated by PIV were functionally opposed in the two strains. The results also indicate that polarized parasite growth in macrophages is due to the activity of distinct CD4+ T cell subsets.



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Fig. 3. Natural resistance to intracellular L. chagasi growth requires CD4+ T cell priming. Naive splenocytes from BALB (A and B) and C3H (C and D) mice were cultured with L. chagasi promastigotes in the presence of anti-CD4 or isotype control rat mAb. After 7 days , primary T cell proliferation (A and C) and resulting intracellular parasite load (B and D) were assessed. Proliferation was measured by [3H]thymidine uptake into DNA and results are the mean ± SE of triplicate cultures. P < 0.01 for treatments with anti-CD4. Amastigote burden in splenic adherent cells was determined after 7 days of transfer to Schneider medium, as in Fig. 1Go. Results are the mean ± SE of triplicate cultures. P < 0.01 for treatments with anti-CD4.

 
We also assessed the resulting intracellular parasite load following neutralization of IL-4, IL-10 or IFN-{gamma} in susceptible and resistant splenocytes (Fig. 4Go). Neutralizing IL-4 activity during priming with L. chagasi had no effect on the final parasite load in susceptible splenocyte cultures (Fig. 4Go, top). On the other hand, neutralizing IL-10 activity in the primary culture markedly reduced parasite load in susceptible (Fig. 4Go, middle), but not in resistant splenocytes (Fig. 4Go, bottom). Both polyclonal and monoclonal (not shown) anti-IL-10 antibodies reduced the parasite load by 10- to 100-fold, compared with control antibodies. As expected, neutralizing IFN-{gamma} activity markedly increased parasite growth in resistant splenocytes (not shown). These results indicate that differential cytokine secretion by T cell subsets accounts for the polarized behavior of parasite growth in splenic macrophages, with an important role for IL-10 in establishment of the susceptible phenotype. Finally, we investigated whether the protective effect of anti-IL-10 was mediated through enhanced nitric oxide production by macrophages. After 7 days in culture, the intracellular parasite load of susceptible BALB splenocytes dropped from 26.0 ± 4.7x105/culture (isotype control) to 2.1 ± 0.26x105/culture in the presence of anti-IL-10 antibody, but remained at 2.1 ± 0.70x105/culture in the presence of anti-IL-10 plus 1000 µM of the iNOS inhibitor L-NMMA. This dosage of L-NMMA blocked >80% of NO production in macrophages stimulated by LPS plus IFN-{gamma} (not shown). These results suggest that NO production is not involved in the protective effect of anti-IL-10 antibody on the growth of L. chagasi.



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Fig. 4. Neutralization of IL-10, but not IL-4 activity eliminates parasite load in macrophages from susceptible-type PIV cultures. Naive splenocytes from susceptible BALB mice, were cultured with L. chagasi promastigotes in the presence, either of neutralizing anti-IL-4 or isotype control mAb (top), or neutralizing anti-IL-10 or control goat IgG (middle). In addition, naive splenocytes from resistant C3H mice were also cultured with neutralizing anti-IL-10 or control IgG (bottom). After 7 days, non-adherent cells and parasites were removed, and Schneider medium was added to adherent cells. Endogenous L. chagasi amastigote burden was determined after an additional 5 days. Results are the mean ± SE of triplicate cultures. P < 0.01 for anti-IL-10 in BALB, and NS for anti-IL-4 in BALB and for anti-IL-10 in C3H cultures.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Murine VL is a complex disease characterized by regional differences in the ability to control infection. After hepatic infection is cured in the susceptible host, a second wave of splenic infection ensues, disrupting cytoarchitecture (35,7). Therefore, mechanisms down-regulating effector T cell function in spleen must be present. We have previously demonstrated that splenic CD4+ T cells from mice chronically infected with L. chagasi express an impairment in proliferation and cytokine production to either anti-CD3 or L. chagasi antigen Lcr1 and that this functional suppression is mediated by CTLA-4 signaling (5), a known negative co stimulatory receptor. Unresponsiveness is promptly reversed by B7-1 or CTLA-4 blockade, allowing CD4+ T cells to become activated, to secrete type 1 and type 2 cytokines, and to mediate parasite killing (5). Recently, we have demonstrated that CTLA-4 suppression of cellular immunity against L. chagasi is mediated by secretion of transforming growth factor-ß (19). A second factor limiting the immune response in spleen could be an imbalance in Th1/Th2 cell subsets in susceptible hosts. Recent studies found that a mixed Th1/Th2 response correlates with susceptibility to L. donovani infection (9). In addition, IL-4 is produced transiently and IL-10 accumulates in the spleen in the course of visceral disease (10). These results indicate that visceralizing Leishmania can induce Th2 T cells under certain genetic and environmental conditions.

In order to investigate early Th1/Th2 differentiation in spleen, we used a PIV system similar to that described for L. major (16). Here, we demonstrate for the first time, that early in vitro priming of naive CD4+ T cells with visceralizing Leishmania, results in rapid functional dichotomy regarding genetically resistant and susceptible (11,15) host strains. Similar to L. major (20), the PIV response mimicked the in vivo response to infection by L. chagasi, since spleen cells from resistant C3H and CBA mice mounted a type 1 response (IFN-{gamma} and low IL-10 levels), while susceptible BALB and B10 splenocytes produced a mixed type 1/type 2 response (IFN-{gamma}, IL-4 and high IL-10 levels). Rapid induction of this functional dichotomy was concomitant with establishment of a polarized behavior of parasite growth in splenic macrophages. Polarized parasite growth was absent when highly purified macrophages from resistant and susceptible strains were infected in vitro with L. chagasi. More important, blockade of early CD4+ T cell activation with anti-CD4 mAb completely abolished polarized growth of L. chagasi. Thus, splenic macrophages from either resistant or susceptible strains became equally permissive to parasite growth in the absence of T cell activation/differentiation. Neutralization of IL-4 during priming had no effect on the final parasite load of susceptible splenocytes. Lack of an effect of IL-4 could be due to intrinsic characteristics of L. chagasi growth or to the kinetics of IL-4 production, i.e. either transient or delayed. On the other hand, early induction of IL-10 secretion by CD4+ T cells in susceptible splenocytes is required for the exacerbated parasite growth in macrophages. Neutralizing IL-10 activity markedly reduced parasite growth in susceptible-type splenocytes, without any effect on parasite replication in cells of the resistant type. These results agree with a previously proposed deleterious role for IL-10 in immunopathogenesis of human kalazar (21,22). On the other hand, IL-10 production does not appear to play a significant role in the PIV system against L. major (20), or in L. major infection in vivo (23), indicating important differences regarding the Leishmania species involved. We also investigated whether IL-10 affected parasite growth through downregulation of NO production. However, we found that an excess (1 mM) dosage of the iNOS inhibitor L-NMMA had no effect on the reduction of parasite load attained with anti-IL-10 in susceptible cultures. These results suggest that, different from L. major (20,24), NO production does not appear to be important for the control of intracellular growth of L. chagasi, at least in susceptible-type splenocytes. As expected, neutralizing IFN-{gamma} activity exacerbated parasite growth in resistant splenocytes. However, we did not investigate the role of NO secretion or other mediators in macrophages from resistant strains. Further studies are necessary to identify intracellular mediators controlling L. chagasi growth, and their possible cross-regulation by IFN-{gamma} and IL-10.

Our studies demonstrate that, in the absence of CD4+ T cell activation, macrophages from polarized susceptible and resistant strains are equally permissive for growth of L. chagasi, indicating that expression of the Lsh phenotype by splenic macrophages requires CD4+ T cell functioning. Our results agree with an earlier study with L. donovani, where only liver, but not splenic or other tissue macrophages expressed the Lsh phenotype (25). Even liver macrophages express the Lsh phenotype only if they are obtained after at least 2 days of infection with L. donovani (26), which suggests a need for previous macrophage contact with T cells. Six different loci were identified in the multigenic control of Th1/Th2 differentiation in response to infection with L. major (14). There is also a polymorphic locus at chromosome 16 that controls the intrinsic propensity of BALB CD4+ T cells to commit to the Th2 phenotype (13). Some of these loci could play a role in infection by visceralizing Leishmania, as well. In addition, we cannot discard that Nramp1 controls differential Th cell development. Control of intravacuolar environment by Nramp1 could lead to differential patterns of antigen processing and presentation by infected macrophages, and to differential Th1/Th2 T cell priming. In agreement with this possibility, macrophage cell lines transfected with the products of Nramp1-resistant and -susceptible alleles show differences in MHC class II expression and in antigen- presenting function for CD4+ T cells (27). In summary, our results demonstrate that macrophage innate resistance or susceptibility to infection with visceralizing Leishmania depends on early adaptive T cell responses in order to be functionally expressed. This finding indicates that genetic resistance to infection depends on intricate and contingent relations between innate and acquired immune mechanisms early after infection. The PIV system we employed could be helpful for dissecting the molecular and cellular requirements of differential induction of Th1/Th2 T cells in VL.


    Acknowledgments
 
This work was supported by the Brazilian National Research Council (CNPq), Programa de Apoio ao Desenvolvimento Científico e Tecnológico (PADCT/CNPq/World Bank), Financing Agency of Studies and Projects (FINEP), Rio de Janeiro State Financing Agency (FAPERJ), and PRONEX Initiative of Brazilian Ministry of Science and Technology. N. A. G. is supported by CAPES doctoral fellowship.


    Abbreviations
 
APC antigen-presenting cell
Nramp 1 natural resistance-associated macrophage protein 1
PIV priming in vitro
VL visceral leishmaniasis

    Notes
 
Transmitting editor: R. L. Coffmann

Received 18 June 1999, accepted 9 May 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Heinzel, F. P., Sadick, M. D., Mutha, S. S. and Locksley, R. M. 1991. Production of interferon {gamma}, interleukin 2, interleukin 4, and interleukin 10 by CD4+ lymphocytes in vivo during healing and progressive murine leishmaniasis. Proc. Natl Acad. Sci. USA 88:7011.[Abstract]
  2. Kaye, P. M., Curry, A. J. and Blackwell, J. M. 1991. Differential production of Th1- and Th2-derived cytokines does not determine the genetically controlled or vaccine-induced rate of cure in murine visceral leishmaniasis. J. Immunol. 146:2763.[Abstract/Free Full Text]
  3. Wilson, M. E., Sandor, M., Blum, A. M., Young, B. M., Metwali, A., Elliott, D., Lynch, R. G. and Weinstock, J. V. 1996. Local suppression of IFN-{gamma} in hepatic granulomas correlates with tissue-specific replication of Leishmania chagasi. J. Immunol. 156:2231.[Abstract]
  4. Smelt, S. C., Engwerda, C. R., McCrossen, M. and Kaye, P. M. 1997. Destruction of follicular dendritic cells during chronic visceral leishmaniasis. J. Immunol. 158:3813.[Abstract]
  5. Gomes, N. A., Barreto-de-Souza, V., Wilson, M. E. and DosReis, G. A. 1998. Unresponsive CD4+ T lymphocytes from Leishmania chagasi-infected mice increase cytokine production and mediate parasite killing after blockade of B7-1/CTLA-4 molecular pathway. J. Infect. Dis. 178:1847.[ISI][Medline]
  6. Murray, H. W. and Hariprashad, J. 1995. Interleukin 12 is effective treatment for an established systemic intracellular infection: experimental visceral leishmaniasis. J. Exp. Med. 181:387.[Abstract]
  7. Saha, B., Nanda-Roy, H., Pakrashi, A., Chakrabarti, R. N. and Roy, S. 1991. Immunobiological studies on experimental visceral leishmaniasis. I. Changes in lymphoid organs and their possible role in pathogenesis. Eur. J. Immunol. 21:577.[ISI][Medline]
  8. Murphy, M. L., Cotterell, S. E. J., Gorak, P. M. A, Engwerda, C. R. and Kaye, P. M. 1998. Blockade of CTLA-4 enhances host resistance to the intracellular pathogen, Leishmania donovani. J. Immunol. 161:4153.[Abstract/Free Full Text]
  9. Sen, E. and Roy, S. 1998. Immunobiological studies on experimental visceral leishmaniasis. V. The I-A (Bm12) mutation specifies resistance to infection. Scand. J. Immunol. 47:431.[ISI][Medline]
  10. Melby, P. C., Yang, Y. Z., Cheng, J. and Zhao, W. 1998. Regional differences in the cellular immune response to experimental cutaneous or visceral infection with Leishmania donovani. Infect. Immun. 66:18.[Abstract/Free Full Text]
  11. Bradley, D. J. 1977. Genetic control of Leishmania populations within the host. II. Genetic control of acute susceptibility of mice to L. donovani infection. Clin. Exp. Immunol. 30:130.[ISI][Medline]
  12. Vidal, S., Gros, P. and Skamene, E. 1995. Natural resistance to infection with intracellular parasites: molecular genetics identifies Nramp1 as the Bcg/Ity/Lsh locus. J. Leuk. Biol. 58:382.[Abstract]
  13. Bix, M., Wang, Z. E., Thiel, B., Schork, N. J. and Locksley, R. M. 1998. Genetic regulation of commitment to interleukin 4 production by a CD4+ T cell-intrinsic mechanism. J. Exp. Med. 188:2289.[Abstract/Free Full Text]
  14. Beebe, A.M., Mauze, S., Schork, N. J. and Coffman, R. L. 1997. Serial backcross mapping of multiple loci associated with resistance to Leishmania major in mice. Immunity 6:551.[ISI][Medline]
  15. Bradley, D. J 1974. Genetic control of natural resistance to Leishmania donovani. Nature 250:353.[ISI][Medline]
  16. Shankar, A. H. and Titus, R. G. 1993. Leishmania major-specific, CD4+, major histocompatibility complex class II-restricted T cells derived in vitro from lymphoid tissues of naive mice. J. Exp. Med. 178:101.[Abstract]
  17. Laurenti, M. D., Corbett, C. E. P., Sotto, M. N, Sinhorini, I. L. and Goto, H. 1996. The role of complement in the acute inflammatory process in the skin and in host-parasite interaction in hamsters inoculated with Leishmania (Leishmania) chagasi. Int. J. Exp. Pathol. 77:15.[ISI][Medline]
  18. Lima, H. C., Bleyenberg, J. A. and Titus, R. G. 1997. A simple method for quantifying Leishmania in tissues of infected animals. Parasitol. Today 13:80.[ISI]
  19. Gomes, N. A., Gattass, C. R., Barreto-de-Souza, V., Wilson, M. E. and DosReis, G. A. 2000. Transforming growth factor-ß mediates CTLA-4 suppression of cellular immunity in murine kalaazar. J. Immunol. 164:2001.[Abstract/Free Full Text]
  20. Soares, M. B. P., David, J. R. and Titus, R. G. 1997. An in vitro model for infection with Leishmania major that mimics the immune response in mice. Infect. Immun. 65:2837.[Abstract]
  21. Karp, C. L., El-Safi, S. H., Wynn, T. A., Satti, M. H., Kordofani, A. M, Hashim, F. A., Hag-Ali, M., Neva, F. A., Nutman, T. B. and Sacks, D. L. 1993. In vivo cytokine profiles in patients with kala-azar. J. Clin. Invest. 91:1644.[ISI][Medline]
  22. Ghalib, H. W., Piuvezam, M. R., Skeiky, Y. A., Siddig, M, Hashim, F. A., el-Hassam, A. M., Russo, D. M. and Reed, S. G. 1993. Interleukin 10 production correlates with pathology in human Leishmania donovani infections. J. Clin. Invest. 92:324.[ISI][Medline]
  23. Chatelain, R., Mauze, S. and Coffman, R. L. 1999. Experimental Leishmania major infection in mice: role of IL-10. Parasite Immunol. 21:211.[ISI][Medline]
  24. Li, J., Hunter, C. A. and Farrell, J. P. 1999. Anti-TGF-ß treatment promotes rapid healing of Leishmania major infection in mice by enhancing in vivo nitric oxide production. J. Immunol. 162:974.[Abstract/Free Full Text]
  25. Olivier, M. and Tanner, C. E. 1987. Susceptibilities of macrophage populations to infection in vitro by Leishmania donovani. Infect. Immun. 55:467.[ISI][Medline]
  26. Crocker, P. R., Blackwell, J. M. and Bradley, D. J. 1984. Expression of the natural resistance gene Lsh in resident liver macrophages . Infect. Immun. 43:1033.[ISI][Medline]
  27. Lang, T., Prina, E., Sibthorpe, D. and Blackwell, J. M. 1997. Nramp1 transfection transfers Ity/Lsh/Bcg-related pleiotropic effects on macrophage activation: influence on antigen processing and presentation. Infect. Immun. 65:380.[Abstract]




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