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The Journal of Immunology, 2001, 167: 4534-4542.
Copyright © 2001 by The American Association of Immunologists

Leishmania amazonensis-Dendritic Cell Interactions In Vitro and the Priming of Parasite-Specific CD4+ T Cells In Vivo1

Hai Qi*, Vsevolod Popov* and Lynn Soong2,*,{dagger}

Departments of * Pathology and {dagger} Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The progressive disease following Leishmania amazonensis infection in mice requires functional CD4+ T cells, which are primed to a disease-promoting phenotype during the infection. To understand how these pathogenic T cells are generated and the role of dendritic cells (DCs) in this process, we use DCs of susceptible BALB/c and resistant C3H/HeJ mice to examine parasite-DC interactions in vitro as well as the effector phenotype of T cells primed by parasite-exposed DCs in vivo. Our results demonstrate that amastigotes and metacyclics efficiently enter and activate DCs of both genetic backgrounds. Infection with amastigotes fails to induce CD40-depedent IL-12 production, but rather potentiates IL-4 production in BALB/c DCs. Upon transfer into syngeneic recipients, amastigote-exposed BALB/c DCs prime parasite-specific Th cells to produce significantly higher levels of IL-4 and IL-10 than their C3H/HeJ counterparts. Transfer studies with IL-4-/- DCs indicate that this enhanced Th2 priming seen in BALB/c mice is partially due to the IL-4 production by amastigote-carrying DCs. These results suggest that L. amazonensis amastigotes may condition DCs of a susceptible host to a state that favors activation of pathogenic CD4+ T cells, and thereby provide a new perspective on the pathogenesis of cutaneous leishmaniasis and protozoan parasite-host interactions in general.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Leishmania parasites are intracellular protozoa that are transmitted to humans and other vertebrates by sandflies in the form of flagellated metacyclic promastigotes (1). After entering vertebrate hosts, the metacyclic transforms into aflagellated amastigotes, which primarily propagate inside tissue macrophages (M{phi})3 (2). Depending upon parasite species and host immune responses, Leishmania infection can cause various forms of diseases with different clinical manifestations (3). Cutaneous leishmaniasis is the most common form that can be caused by L. major in the Old World and L. mexicana or L. amazonensis (La) in the New World (3).

In experimental cutaneous leishmaniasis caused by L. major infection, the susceptibility of BALB/c mice is attributable to selective expansion of Th2 cells, whereas resistance seen in most other mouse strains, such as C57BL/6 (B6) mice, is associated with predominant Th1 responses (4). Although it is not entirely clear how these functionally distinct CD4+ T subsets are primed during L. major infection, studies with B6 mice deficient in CD4+ or CD8+ T cells indicate an essential role for MHC class II-restricted CD4+ T cells in controlling L. major infection. Disruption of the MHC class II gene renders otherwise resistant B6 mice susceptible (5, 6). In addition, nude and SCID mice cannot control L. major infection unless they are reconstituted with CD4+ T cells (7, 8).

Different from that of L. major, murine La infection leads to progressive diseases in a majority of inbred strains of mice. Furthermore, lesion development and tissue parasite load are remarkably reduced in immunodeficient MHC class II-/-, RAG2-/-, and nude mice on a B6 background (9), as well as in BALB/c SCID mice (our unpublished results). Strikingly, reconstitution of RAG2-/- mice with wild-type CD4+ T cells from syngeneic naive mice results in disease development (9). Therefore, parasite propagation and lesion pathology following La infection require CD4+ T cells that are primed to a pathogenic phenotype. Although the nature of pathogenic T cells in La-infected B6 mice is still unclear, accumulating evidence indicates that in La-infected BALB/c mice, IL-4-producing Th2 cells promote the lesion development and parasite growth. For example, administration of a neutralizing anti-IL-4 mAb ameliorates La infection in BALB/c mice (10), and adoptive transfer of La-specific Th2 cell lines exacerbates the disease.4 Yet, it is not understood how these pathogenic Th2 cells are generated. One possibility is that functional modulation of APCs by the parasite may establish a priming environment that aberrantly favors generation of pathogenic Th cells.

M{phi} are not only the primary host cell for Leishmania parasites, but also an important population of APCs. Thus, infected M{phi} could constitute the aberrant priming environment. Indeed, ample evidence has suggested that Leishmania infection impairs the ability of M{phi} to produce IL-12 (11, 12, 13). As a result, parasitized M{phi} probably cannot stimulate robust Th1 and thus inherently favor Th2 responses (14). However, Leishmania infection also significantly impairs the Ag-presenting function of M{phi} (15, 16, 17), making parasitized M{phi} an unlikely candidate to prime naive T cells.

It is believed that naive CD4+ T cells are primed mainly in secondary lymphoid organs by dendritic cells (DCs), a unique population of professional APCs (18, 19). Immature DCs are located in most nonlymphoid tissues and can efficiently uptake soluble and particulate Ags (20, 21). Upon exposure to microbial pathogens and their components, DCs migrate to lymphoid organs where they eventually mature into potent APCs, which express high levels of MHC products and costimulatory molecules and prime naive T cells (22). Depending on maturation environment and/or lineage origin, DCs can express distinct cytokine profiles to regulate Th cell differentiation and, thereby, to ensure the induction of protective immunity (23, 24). However, certain pathogens such as HIV (25), measles virus (26), Plasmodium falciparum (27), and Trypanosoma cruzi (28) have been found to impair DC functions and the induction of effective host immune responses. Similarly, La parasites may have evolved mechanisms to modulate functions of DCs in a susceptible host to promote the generation of pathogenic T cells. To explore this possibility, we investigated parasite-DC interactions in vitro and directly examined effector phenotypes of T cells primed in vivo by parasite-exposed DCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Female wild-type, SCID, and IL-4-/- BALB/c (H-2d) mice and C3H/HeJ (HeJ) (H-2k) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were maintained under specific pathogen-free conditions and used for experiments at 6–10 wk of age with protocols approved by the Animal Care and Use Committee of the University of Texas Medical Branch (Galveston, TX).

Parasite culture and lysate preparation

La (MHOM/BR/77/LTB0016) parasites were maintained by regular passage through BALB/c mice. Promastigotes were cultured at 23°C in 20% FBS-supplemented Schneider’s Drosophila medium (Life Technologies, Rockville, MD). Stationary promastigote cultures of less than five in vitro passages were used for animal infection and purification of metacyclics by negative selection with the 3A1 mAb (a gift of Dr. D. Sacks, National Institute of Allergy and Infectious Diseases, Bethesda, MD), according to N. Courret et al. (29). Lesion-derived amastigotes were cultured at 32°C in acidified (pH 5.0) complete Schneider’s medium for 48–72 h and were used for infection of DCs. To prepare amastigote lysates, parasites (2 x 108/ml in PBS) were subjected to six freeze-thaw cycles and a 15-min sonication in an ice bath, and then were stored at -70°C in aliquot. To label metacyclics or amastigotes with the fluorescent tracking dye, CFSE (Molecular Probes, Eugene, OR), parasites were suspended at 5 x 107/ml in PBS containing 1.25 µM CFSE and incubated at room temperature for 5 min. Labeled parasites were washed four times with PBS and culture medium before infection of DCs. Amastigotes or metacyclics (8 x 107/ml in PBS) were incubated in a 60°C water bath for 15 min to prepare heat-killed parasites.

Evaluation of parasite infection of mice

Age- and sex-matched mice (5–6/group) were infected s.c. in the right hind foot with 2 x 106 stationary La promastigotes. Lesion size was monitored over time with a digital caliper (Control Company, Friendswood, TX). Tissue parasite burdens were measured via a limiting dilution assay as previously described (9). To examine parasite-specific Th cytokine production in infected mice, draining lymph node (DLN) cells (106/well in 200 µl of medium) from individual infected mice were restimulated with amastigote lysates (equivalent to 106 parasites), and supernatants were harvested at 48–72 h to determine levels of IL-4, IL-10, and IFN-{gamma} by ELISA.

Generation of DCs from the bone marrow

The protocol for generating bone marrow-derived DCs (BM-DCs) was originally described by Inaba et al. (30) and modified by Lutz et al. (31). Briefly, a single marrow cell suspension was prepared from the femurs and adjusted to 2 x 106 per 10 ml of complete IMDM (Iscove’s modified DMEM containing 10% FBS, 1 mM sodium pyruvate, 50 µM 2-ME, 50 µg/ml gentamicin, and 100 U/ml penicillin). DC culture medium was supplemented with 20 ng/ml rGM-CSF (BD PharMingen, San Diego, CA) or with 2% culture supernatants of J558L cells that were stably transfected with the murine gm-csf gene (the transfected cell line was a gift from Dr. C. Janeway, Yale University, New Haven, CT). At day 3, 10 ml of fresh GM-CSF-containing medium was added, and 10 ml of the culture medium was replaced with fresh GM-CSF-containing medium at day 6. Usually, 8-day cultures contained >70% CD11c+ cells as judged by FACS analysis and were used for all experiments. Media used for DC culture were routinely monitored for potential LPS contamination, and the LPS levels were <0.06 EU/ml as measured by the Limulus Amebocyte Lysate pyrogen test kit (BioWhittaker, Walkersville, MD).

Infection of BM-DCs with Leishmania parasites

BM-DCs were adjusted to 2.5 x 106/well in six-well plates or 106/ml in 24-well plates and incubated with parasites at a 4:1 parasite-to-DC ratio at 34°C for 12 h, and then at 37°C for an additional period of time as required in specific experiments. For the sham-exposed control, a parasite suspension was centrifuged to pellet parasites, and the resulting supernatant was added to DC cultures.

Electron microscopy (EM)

Following 24 or 48 h of coculture with parasites, BM-DCs were harvested and fixed in Ito’s fixative (1.25% formaldehyde, 2.5% glutaraldehyde, 0.03% CaCl2, and 0.03% trinitrophenol in 0.05 M cacodylate buffer, pH 7.3) at room temperature for 1 h and then overnight at 4°C. After washing, samples were postfixed in 1% osmium tetraoxide for 1 h and en bloc stained with 1% uranyl acetate in 0.1 M maleate buffer. After dehydration in a graded series of ethanol, samples were embedded in Poly/Bed 812 (Polysciences, Warrington, PA). Ultrathin sections were cut on a Sorvall MT-6000 ultramicrotome (RMC, Tucson, AZ), stained with uranyl acetate and lead citrate, and examined in a Philips 201 transmission electron microscope (Philips Electron Optics, Eindhoven, The Netherlands) at 60 kV. For each sample, ~100 cells were examined.

Cytokine assays for amastigote-exposed DCs

BM-DCs were cocultured with live or heat-killed amastigotes in 24-well plates as described above. Ammonium sulfate-purified anti-CD40 mAb was then added at a 1/10 dilution (clone FGK45, Ref. 32 ; a gift from Dr. A. Rolink, Basel Institute for Immunology, Basel, Switzerland). This dilution was chosen based on pilot experiments measuring DC cytokine production. Supernatants were harvested at 24 h and saved at -70°C until ELISA measurement. For detection of intracellular cytokines, parasite-exposed DCs were incubated with anti-CD40 mAb for 12 h before GolgiStop (BD PharMingen) was added for an additional 6 h. Cells were harvested, fixed, and stained as described below.

Flow cytometric analysis of surface and intracellular Ags

For blocking nonspecific Ab binding, normal mouse IgG (Caltag Laboratories, Burlingame, CA), hamster IgG, rat IgG (Pierce, Rockford, IL), and culture supernatants of 2.4G2 hybridoma (a gift from Dr. R. König, University of Texas Medical Branch, Galveston, TX) were used. The following specific mAbs were purchased from BD PharMingen: FITC-conjugated anti-I-Ad/Ed (2G9, also reactive to I-Ek but not I-Ak); PE-conjugated anti-CD40 (3/23), anti-CD80 (16-10A1), anti-CD86 (GL1), anti-IL-4 (BVD4-1D11), anti-IL-10 (JES5-16E3), and anti-IL-12 p40 (C15.6); and biotinylated anti-CD11c (clone HL3). Isotype control Abs included FITC-conjugated rat IgG2a; PE-conjugated rat IgG1, IgG2a, and IgG2b; PE-conjugated hamster IgG; and biotinylated hamster IgG. TriColor-labeled streptavidin, the secondary reagent for biotinylated Ab, was purchased from Caltag Laboratories.

All staining steps were performed on ice. After washing, cells were incubated for a total of 30 min with 2.4G2 hybridoma supernatants (200 µl/106 cells) to block Fc receptors and then with a mixture containing 5 µg/ml each hamster IgG, rat IgG, and mouse IgG. Thereafter, cells were stained in a final volume of 200 µl for 20 min in the presence of specific mAbs against surface Ags of interest (1 µg/106 cells). For detecting intracellular cytokines, cells were first stained for surface Ags, fixed/permeabilized with a Cytofix/Cytoperm Kit (BD PharMingen), and then incubated for 20 min with mAbs specific to cytokines in the presence of 5 µg/ml rat and mouse IgGs. Cells were washed and analyzed on a FACScan (BD Biosciences, Franklin Lakes, NJ). For characterization of DCs, at least 10,000 CD11c+ events were collected. Data were analyzed with FlowJo software (TreeStar, San Carlos, CA).

Priming T cells in vivo by DC transfer

For in vivo T cell priming by DCs, a protocol previously established by Inaba et al. (33) was used with some modifications. Briefly, 3 x 105 parasite-exposed DCs in 10 µl of PBS were injected s.c. into the right hind foot (4–5 mice/group). Popliteal DLNs were harvested at day 8. To evaluate primary T cell cytokine responses induced by DC transfer, 106 DLN cells of individual mice were cultured in 200 µl of medium containing amastigote lysates (equivalent to 106 parasites). In some cases, DLN cells from the same experimental group were pooled to purify CD4+ T cells with Dynabeads Mouse CD4 in combination with DETACHaBEAD Mouse CD4 (Dynal Biotech, Great Neck, NY) according to the manufacturer’s protocol. Purified CD4+ T cells (~95% pure) were seeded in 96-well plates at 105/well in 200 µl of medium and incubated with titrated doses of amastigote lysates together with 106 syngeneic splenocytes (pretreated with 50 µg/ml mitomycin C; Sigma, St. Louis, MO). Culture supernatants were harvested at 48 h to measure IL-4 or at 72 h to measure IL-10 and IFN-{gamma}. All media used for ex vivo assays of DLN and CD4+ T cells from DC-transferred mice were IMDM supplemented with 3% mouse serum (Gemini Bio-Products, Woodland, CA). Cytokine contents in supernatants were determined by ELISA, in which paired mAbs specific to IL-4, IL-10, and IFN-{gamma}, and their corresponding protein standards (BD PharMingen), were used following manufacturer’s suggestion. Detection limits were 16 pg/ml for IL-4, 31 pg/ml for IL-10, and 16 pg/ml for IFN-{gamma}.

Statistical analysis

For comparison of mean values of different experimental groups, the two-tailed t test was used and p values were calculated in SigmaPlot software (SPSS, Chicago, IL). A difference in mean values was deemed significant when p < 0.05 or very significant when p < 0.01.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Differential susceptibilities of BALB/c and HeJ mice to La infection

BALB/c mice are known to be susceptible to La infection. As shown in Fig. 1GoA, these mice developed progressive lesions when challenged with 2 x 106 promastigotes. However, similarly infected HeJ mice showed no measurable lesions during the observation period. Analysis of the tissue parasite burden at 10 wk postinfection revealed that the number of parasites per foot was ~1700-fold lower in HeJ mice than in BALB/c mice (Fig. 1GoB), indicating that HeJ mice are resistant to the infection. To determine parasite-specific cytokine production in these mice, DLN cells were restimulated in vitro with amastigote lysates. At 10 wk postinfection, significantly higher levels of IL-4 and IL-10 were detected in BALB/c mice than in HeJ mice (p < 0.01), whereas IFN-{gamma} production was similar in both strains of mice (Fig. 1GoC). Therefore, the susceptibility of BALB/c mice to La infection is linked to an enhanced production of IL-4 and IL-10, whereas the resistance of HeJ mice is associated with markedly reduced Th2 responses. These data are consistent with previous findings that Th2 cells promote disease progression in La-infected BALB/c mice (10).4



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  		FIGURE 1. Differential susceptibilities of BALB/c and HeJ mice to La infection. A, Lesion development in BALB/c and HeJ mice following infection with 2 x 106 promastigotes was monitored by measuring the thickness of the infected foot. Each data point represents the mean ± SD for the respective experimental group. Results represent three independent experiments. B, Parasite burdens of infected BALB/c and HeJ mice were examined in a limiting dilution assay at 10 wk postinfection. The mean ± SD of parasite number per foot is shown for the two strains of mice (**, p < 0.01, n = 5). C, At 10 wk postinfection, 106 DLN cells from individual BALB/c or HeJ mice were collected and cultured in the presence of amastigote lysates (equivalent to 106 parasites). Culture supernatants were harvested for 48–72 h to measure concentrations of IL-4, IFN-{gamma}, and IL-10. For each cytokine, the mean concentration ± SD is shown for the two strains of mice (**, p < 0.01, n = 5). For B and C, results are representative of two independent experiments.

 
Efficient uptake of La parasites by DCs of susceptible and resistant mice

To explore the possibility that La parasites may modulate DC functions to favor the priming of pathogenic Th2 cells in susceptible BALB/c mice, we compared parasite-induced responses in DCs of BALB/c and HeJ backgrounds. Immature BM-DCs were derived from BALB/c and HeJ bone marrow. As shown in Fig. 2GoA, ~80% of the total BALB/c BM population are CD11c+ DCs, of which ~83% express various levels of MHC class II molecules. Same results were also consistently obtained with HeJ BM cells (data not shown). Because immature DCs are phagocytic (21) and capable of engulfing L. major parasites (34, 35), we first examined whether DCs of susceptible and resistant backgrounds differ in parasite uptake. BM-DCs from BALB/c or HeJ mice were cocultured with CFSE-labeled La amastigotes or metacyclics that were purified from promastigote bulk culture. Flow cytometric analyses revealed that the parasite-associated CFSE fluorescence colocalized with a majority of CD11c+ DCs (62–80%) of both BALB/c and HeJ backgrounds. The increased side scattering intensity in CFSE+ DCs indicated the presence of intracellular parasites (Fig. 2GoB). The percentages of fluorescent DCs were reduced by merely 5% when heat-killed amastigotes or metacyclics were used (data not shown), consistent with the passive nature of parasite entry to phagocytic host cells (36). To further examine the parasite inside DCs, we conducted EM studies. After exposure to amastigotes for 24 h, DCs of BALB/c and HeJ mice typically contained several ultrastructurally intact parasites within one or two parasitophorous vacuole(s) (Fig. 3Go, A and B). The structural integrity of intracellular parasites was maintained after 48 h of coculture (Fig. 3GoC; HeJ DCs, data not shown). Similar studies were conducted with purified metacyclics or promastigote bulk culture, which contains ~10% highly infectious metacyclics. Although intact amastigote-like parasites were found inside vacuoles of metacyclic-exposed DCs, most parasites were destroyed by 24 h in bulk culture-exposed DCs of both strains of mice. Taken together, these data demonstrate that both BALB/c and HeJ DCs can efficiently uptake La parasites and that amastigotes and metacyclics are capable of surviving inside DCs.



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  		FIGURE 2. Phenotyping BM-DCs and their infection by La parasites. A, BM-DC population (8 day, BALB/c) was stained for surface expression of CD11c and MHC class II molecules. The quadrant gate was set up according to isotype control staining, and numbers represent the percentage of cells within each gate. B, DCs of BALB/c and HeJ mice were cocultured for 24 h with CFSE-labeled amastigotes or purified metacyclic promastigotes at a 4:1 parasite-to-DC ratio (see Materials and Methods for parasite-labeling procedure). Cells were stained for CD11c before being subjected to FACS analysis. Contour plots for CD11c+ cells are shown. The y-axis represents the side scattering intensity; the x-axis represents green fluorescence intensity due to CFSE-labeled parasites. The autofluorescence level of DCs exposed to unlabeled parasites is shown at the bottom (Control). Similar results were obtained from more than three independent experiments.

 


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  		FIGURE 3. Transmission EM of parasite-carrying DCs. La amastigotes were cocultured with DCs of BALB/c (A and C) or HeJ (B) background at a 4:1 parasite-to-DC ratio. At 24 h, structurally intact amastigotes (marked with arrows) were found inside a parasitophorous vacuole of a BALB/c DC (A) or HeJ DC (B). The structural integrity of the parasites was maintained for at least 48 h (C). Results represent >10 samples prepared from DCs of different bone marrow donors. Approximately 100 cells were examined for each sample. The original magnification for these pictures was x5600. PV, Parasitophorous vacuole; N, DC nuclei. Bar = 1 µm.

 
La parasites activate both BALB/c and HeJ DCs

Upon exposure to microbes or their products, DCs usually undergo an activation/maturation process characterized by up-regulation of MHC products and costimulatory molecules (19). Thus, we examined whether differential activation of DCs following exposure to La parasites could contribute to the development of distinct Th responses observed in BALB/c and HeJ mice (Fig. 1Go). We found that exposure to amastigotes or metacyclic promastigotes resulted in up-regulation of MHC class II, CD40, CD80, and CD86 in DCs of both genetic backgrounds (Fig. 4Go). Up-regulation of these molecules was not seen in sham-exposed DCs and could not be blocked by LPS-neutralizing polymyxin B (data not shown). Given that DCs might be exposed to dead parasites in vivo and that they can engulf heat-killed parasites in vitro (our unpublished data), we also examined whether heat-killed parasites could activate DCs. Heat-killed amastigotes activated DCs of both mouse strains in patterns similar to those of their live counterparts (Fig. 4GoA). However, heat inactivation significantly attenuated the ability of metacyclics to activate DCs (Fig. 4GoB). Regardless of developmental stages of the parasite, DCs from both susceptible and resistant backgrounds are comparable in surface expression of MHC class II and costimulatory molecules after parasite exposure. Thus, it is unlikely that the susceptibility to La infection in BALB/c mice is due to gross defects in parasite-induced DC activation.



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  		FIGURE 4. La parasites up-regulate expression of MHC class II and costimulatory molecules on DCs. BALB/c and HeJ DCs were cocultured with live or heat-killed amastigotes (A) or metacyclic promastigotes (B) for 24 h before FACS analysis for surface expression of MHC class II and costimulatory molecules (CD40, CD80, and CD86). Histograms depict expression profiles of the indicated molecules on gated CD11c+ cells (see Fig. 2GoA for CD11c staining). Blocking steps described in Materials and Methods essentially brought nonspecific staining of DCs by isotype control Abs to the level of autofluorescence, which was always adjusted to the first decade on the 4-decade log scale. For clarity, staining profiles of isotype controls were not included in the histogram overlay. The difference in MHC class II staining intensity between BALB/c and HeJ DCs was due to the fact that FITC-conjugated 2G9 mAb reacts to the I-Ad, I-Ed, and I-Ek, but not to the I-Ak, molecules. Results represent three independent experiments on promastigote-exposed DCs and more than five on amastigote-exposed DCs.

 
Differential effects of La amastigotes on cytokine production by BALB/c or HeJ DCs

Activated DCs can produce a variety of cytokines (e.g., IL-12, IL-10, and IL-4) that polarize Th subset development (37, 38). Therefore, it is possible that differential production of DC cytokines following parasite exposure could contribute to distinct Th2 responses in BALB/c and HeJ mice. To test this possibility, DCs were exposed to parasites for 12 h and then treated with an agonistic anti-CD40 mAb (32) for another 18 h. This was to mimic the engagement of CD40 molecules on DCs by T cell-derived CD40 ligands, which can greatly augment DC cytokine production (39, 40).

As revealed in flow cytometric analyses, only a small fraction (~1–4%) of BALB/c or HeJ DCs produce IL-12 following amastigote exposure without anti-CD40 treatment (data not shown). When further treated with the anti-CD40 mAb, amastigote-exposed HeJ DCs consistently exhibited a 2-fold increase in the frequency of IL-12 producers, as compared with the control (Fig. 5Go). However, pre-exposure of BALB/c DCs to live or heat-killed amastigotes did not lead to increased CD40-dependent IL-12 production (Fig. 5Go). Because IL-4 or IL-10 production by DCs was not detectable by intracellular staining (data not shown), we sought to measure these two cytokines by ELISA. As shown in Fig. 6Go, CD40 engagement was sufficient to induce IL-10 secretion in both BALB/c and HeJ DCs, and this was not affected by pre-exposure to the parasite. IL-4 production was detected in BALB/c DCs that were exposed to amastigotes and then treated with the anti-CD40 mAb. However, IL-4 was not detectable in HeJ DCs under the same conditions (Fig. 6Go). Additionally, exposure to amastigotes for 24 h led to a 2-fold increase of IL-4 mRNA levels in BALB/c but not HeJ DCs, as measured by RT-PCR (data not shown). Collectively, these data show that La amastigotes fail to enhance CD40-dependent IL-12 production, but rather potentiate IL-4 production in BALB/c DCs.



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  		FIGURE 5. IL-12 production by amastigote-exposed DCs. BALB/c and HeJ DCs were cocultured with live or heat-killed La amastigotes or left untreated for 12 h, and were further incubated with an anti-CD40 mAb for 18 h. Monensin was present during the last 6 h. Cells were stained for surface CD11c and then for intracellular IL-12 p40. The IL-12 staining pattern of CD11c+ cells is shown. Because autofluorescent levels varied among DCs of different forward scattering intensities, we set up the IL-12+ gate in bivariate contour plots instead of histograms. Results were representative of three independent experiments. Live, DCs exposed to live amastigotes. Heat-killed, DCs exposed to heat-killed amastigotes. Control, DCs not exposed to parasites. Isotype, DCs stained with isotype-matched control Ab.

 


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  		FIGURE 6. IL-4 and IL-10 production by amastigote-exposed DCs. BALB/c and HeJ DCs were cocultured with live or heat-killed La amastigotes or left untreated for 12 h, then were further incubated with an anti-CD40 mAb for 24 h. Culture supernatants were harvested to determine cytokine concentrations by ELISA. Results for one of three independent experiments were shown. Control, DCs not exposed to parasites. Heat-killed, DCs exposed to heat-killed amastigotes. Live, DCs exposed to live amastigotes. ND, Not detectable.

 
Amastigote-carrying BALB/c or HeJ DCs prime distinct effector Th cells in vivo

IL-12 and IL-4 can skew Th responses toward a type-1 or type-2 phenotype, respectively. To test whether amastigote-exposed BALB/c or HeJ DCs prime distinct Th responses in vivo, a DC transfer protocol was used (21, 33). Pilot studies revealed that at 7–9 days after s.c. transfer with 3 x 105 amastigote-carrying DCs (62–80% carrying the parasite, Fig. 2Go), DLNs reached a peak size of 1.5–3 x 107 cells/node and displayed a strong T cell-proliferative response in vitro (data not shown). Subsequently, DLN cells from individual mice were collected at 8 days posttransfer to examine cytokine production by Th cells in vitro. Similar studies were conducted with DCs derived from IL-4-/- BALB/c mice to directly evaluate the significance of DC-derived IL-4 in priming parasite-specific Th cells.

As shown in Fig. 7GoA, when restimulated in vitro with amastigote lysates, DLN cells from DC-transferred BALB/c mice produced significantly higher levels of IL-4 and IL-10 than did the HeJ counterpart (p < 0.01 for both cytokines, n = 9). When the IL-4-/- DCs were used in the transfer, the level of parasite-specific IL-4 production by BALB/c DLN cells was significantly reduced (p < 0.05, n = 9), but that of IL-10 was not (p = 0.08, n = 9). Levels of IFN-{gamma} production by DLN cells did not significantly differ among the three groups (p > 0.5, n = 9) (BALB/c recipients given wild-type DCs: 2.7 ± 1.1 ng/ml; BALB/c recipients given IL-4-/- DCs: 3.1 ± 1.3 ng/ml; HeJ recipients given wild-type DCs: 3.0 ± 1.4 ng/ml). Furthermore, CD4+ T cells were purified from recipient DLNs and restimulated with amastigote lysates in the presence of syngeneic mitomycin-treated splenocytes. Culture supernatants were harvested to examine the cytokine profile of Th cells. As shown in Fig. 7GoB, CD4+ T cells from BALB/c mice given wild-type DCs produced higher levels of IL-4 and IL-10 than did those transferred with IL-4-/- DCs or those of the HeJ counterpart. In addition, CD4+ T cells from BALB/c mice transferred with IL-4-/- DCs produced a higher level of IFN-{gamma} than did cells from the other two groups. These data demonstrate that amastigote-carrying BALB/c DCs are significantly more potent in priming Th2 responses than their HeJ counterparts, and that DC-derived IL-4 is partially responsible for enhanced IL-4 and IL-10 production in Th cells.



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  		FIGURE 7. Distinct T cell cytokine profiles induced in vivo by amastigote-carrying DCs of resistant or susceptible background. DCs that had been cocultured with live amastigotes for 24 h were s.c. transferred (3 x 105 DC/mouse) to the right hind foot of syngeneic wild-type mice. The cytokine profiles of DLN cells were examined in three groups of mice: BALB/c mice receiving wild-type BALB/c DCs ({circ}), BALB/c mice receiving IL-4-/- BALB/c DCs (•), and HeJ mice receiving HeJ DCs ({square}). A, DLN cells (106) from individual mice were restimulated with amastigote lysates (equivalent to 106 parasites). The levels of IFN-{gamma} (not plotted), IL-4, and IL-10 in the supernatants were determined by ELISA. Data from two independent experiments were pooled, and each data point represents an individual recipient. Two-tailed t tests were conducted for comparison between groups, and p values are given in the inset. B, DLN cells of DC recipients (four to five per group) were pooled for isolation of CD4+ T cells using magnetic beads. CD4+ T cells (105, ~95% pure) were restimulated with 106 mitomycin-treated syngeneic splenocytes in the presence of indicated doses of amastigote lysates (in parasite equivalent). IL-4, IL-10, and IFN-{gamma} in the supernatants were measured by ELISA. In the absence of amastigote lysates and/or feeder splenocytes, cytokine concentrations in the supernatants were minimal (<16 pg/ml for IL-4, <50 pg/ml for IL-10, and <100 pg/ml for IFN-{gamma}). Similar results were obtained from two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies have indicated that IL-4-producing Th2 cells mediate the pathogenesis following La infection in BALB/c mice (10).4 In correlation, we have found that the magnitude of parasite-specific Th2 responses is a major distinction between susceptible BALB/c and resistant HeJ mice following the infection. Specifically, DLN cells from La-infected BALB/c mice produce moderate levels of both Th1 (IFN-{gamma}) and Th2 (IL-4 and IL-10) cytokines, whereas resistant HeJ mice exhibit significantly reduced Th2 responses without developing stronger Th1 responses (Fig. 1GoC). It is known that an otherwise susceptible host can be rendered resistant to La infection by virtue of strong Th1 induction through vaccination (41) or direct transfer of Th1 cells.4 In light of these findings, data presented in Fig. 1GoC suggest that the magnitude of Th2 responses could determine the outcome of La infection when the level of Th1 responses is only moderate.

It is noticeable that amastigote-carrying BALB/c DCs prime CD4+ T cells to produce much higher levels of IL-4 and IL-10 than their HeJ counterparts (Fig. 7Go). This observation correlates with our data describing cytokine profiles of DLN cells from infected mice (Fig. 1GoC), implying that the effector phenotype of CD4+ T cells activated during La infection may be determined by amastigote-exposed DCs. Accumulating evidence has indicated the existence of direct interactions between Leishmania amastigotes and DCs in vivo. In genetically resistant B6 mice, viable L. major amastigotes have been found to persist in DCs, and these DCs could stimulate a vigorous T cell recall response (42). Dr. Sacks and colleagues (43, 44) have shown that in a natural L. major infection model, the DC compartment is mobilized to initiate T cell responses only after tissue parasitization by amastigotes reaches a threshold level. Furthermore, we have detected parasite-carrying DCs in DLNs within 3 days of s.c. inoculation with CFSE-labeled La amastigotes (our unpublished data). Therefore, future studies are warranted to determine how amastigote-DC interactions in vivo can influence T cell effector phenotypes and shape the outcome of Leishmania infection.

La parasites could induce different responses in BALB/c and HeJ DCs, and thus potentially account for the different abilities of these DCs to prime Th2 cells (Fig. 7Go). First, the ability to engulf La parasites could be different between the two DCs. However, the observation that BALB/c and HeJ DCs similarly take up not only La amastigotes but also metacyclics (Fig. 2Go) clearly argues against this possibility. Furthermore, EM studies reveal no major differences between BALB/c and HeJ DCs in terms of quantity, location, ultrastructure, and apparent fate of intracellular parasites (Fig. 3Go and our unpublished data). Thus, no gross difference in parasite engulfment by DCs of these two backgrounds could explain their distinct abilities to prime Th2 cells.

Second, enhanced Th2 priming by amastigote-carrying BALB/c DCs could result from defects in parasite-induced DC activation, as the strength and nature of costimulation provided by DCs to T cells could skew the balance of Th1/Th2 subset development (45). In particular, DCs that are not optimally activated and consequently provide insufficient costimulation can preferentially prime IL-10-producing regulatory T cells that suppress bystander Th1 development, and thereby favor Th2 responses (46, 47). Although some protozoan parasites suppress DC activation (27, 28), data reported herein (Fig. 4Go) and results from others (34, 35) demonstrate that Leishmania parasites can activate DCs. Regardless of whether parasites are metacyclics or amastigotes, live or dead, they up-regulate surface expression of MHC class II and costimulatory molecules in BALB/c and HeJ DCs in a similar fashion. These in vitro studies indicate no major difference at the level of parasite-induced DC activation in these two strains of mice. Bennett et al. (48) have recently shown that L. mexicana parasites that carry the gene for a green fluorescent protein do not activate DCs of the CBA background. The difference between this report and our study (Fig. 4Go) could be due to the Leishmania species used, potential distinctions between freshly prepared wild-type parasites vs a genetically engineered parasite clone, and/or differences in responsiveness to the parasite among DCs of different genetic backgrounds.

Although the quality and strength of costimulatory signals delivered by DCs to CD4+ T cells can affect development of distinct Th subsets, the cytokine environment during T cell priming can be a dominant factor shaping the cytokine profile of activated T cells (49). Thus, another possibility to explain the differential Th2 priming by amastigote-carrying BALB/c or HeJ DCs is that La amastigotes may condition these DCs to produce different cytokines. IL-12 is a potent Th1-driving factor indispensable in the initiation of protective immunity against L. major infection (50, 51). Given that IL-12 production in M{phi} is selectively inhibited by various Leishmania species (11, 12, 13), it is generally believed that DCs are the major source of IL-12 during L. major infection (34, 35). In this study, we have shown that CD40 engagement enhances IL-12 production in amastigote-exposed HeJ but not BALB/c DCs (Fig. 5Go). This is consistent with our finding that amastigote-carrying BALB/c DCs prime stronger Th2 responses as compared with the HeJ counterparts (Fig. 7Go). Because La amastigotes do up-regulate CD40 expression in BALB/c DCs (Fig. 4Go), current efforts are directed to investigate whether amastigotes could render a portion of those CD40+ DCs incapable of producing IL-12 in response to CD40 engagement. Of note, despite producing higher levels of IL-12, amastigote-exposed HeJ DCs do not induce stronger Th1 responses than their BALB/c counterparts. This might relate to the finding that IL-12R expression in CD4+ T cells is down-regulated during La infection (52).

DCs may induce Th2 development by producing IL-10 and IL-4 (53, 54). Of particular interest, d’Ostiani et al. (54) have shown that hyphae of Candida albicans directly stimulate murine DCs to produce a high level of IL-4. Our data indicate that amastigote exposure potentiates IL-4 production from BALB/c but not HeJ DCs (Fig. 6Go). However, exposure to La amastigotes alone is not sufficient; subsequent CD40 engagement is also needed for IL-4 secretion from BALB/c DCs. These observations suggest that direct DC-T cell interactions may enhance IL-4 production from amastigote-carrying BALB/c DCs. We are currently investigating whether factors other than CD40 are also involved in signaling these DCs to produce IL-4. Although the level of IL-4 produced by amastigote-exposed BALB/c DCs is low under our in vitro experimental conditions, this DC-derived IL-4 contributes to the enhanced Th2 response in vivo. The transfer study reveals that CD4+ LN T cells produce significantly lower levels of IL-4 but higher levels of IFN-{gamma} in IL-4-/- DC-transferred mice than in mice given wild-type amastigote-carrying DCs (Fig. 7Go). Importantly, IL-4-/- and wild-type DCs show no major differences in amastigote uptake, surface marker expression (CD11c, MHC class II, CD40, CD80, and CD86), and IL-12 production after exposure to amastigotes (data not shown). Therefore, production of IL-4 by amastigote-carrying DCs could be a significant Th2-biasing mechanism during La infection of BALB/c mice.

A lack of the IL-4 gene in BALB/c DCs does not totally abrogate priming of IL-4-producing CD4+ T cells in vivo (Fig. 7Go). This could be because some amastigotes escape from carrier DCs to induce host responses independent of transferred DCs, or because amastigotes potentiate DCs to produce other Th2-driving factors in addition to IL-4. The observation that IL-4-/- and wild-type DCs indeed induce distinct Th cytokines in vivo (Fig. 7Go) suggests that donor DCs rather than cells of the recipient initiate T cell responses. Therefore, potential production of other Th2-skewing cytokines/chemokines by amastigote-exposed DCs warrants further investigation.

Inappropriate T cell responses could exacerbate diseases caused by Leishmania parasites and other pathogens. Given the crucial role of DCs in T cell-mediated immune responses, it is tempting to speculate that a pathogen may modulate the DC compartment, and thereby the host response to the pathogen. Our studies of murine leishmaniasis have suggested that Leishmania parasites can modulate DCs to skew CD4+ T cell responses toward a phenotype that could lead to pathogenesis and uncontrolled parasite growth. This study would also improve our general understanding of the complex interactions among intracellular pathogens, DCs, and T cells.


   Acknowledgments
 
We are grateful to Drs. Antonius Rolink, Rolf König, David Sacks, and Charles Janeway for kindly sharing reagents, and Drs. Jiaren Sun, Vivian Braciale, and David Mosser for critical comments on the manuscript. We thank Dr. Jiaxiang Ji for helpful discussion, and Violet Han for expert assistance in EM studies.


   Footnotes
 
1 This study was supported in part by National Institute of Allergy and Infectious Diseases Grant AI43003 (to L.S.). H.Q. is supported by the James W. McLaughlin Fellowship Fund. Back

2 Address correspondence and reprint requests to Dr. Lynn Soong, 301 University Boulevard, Medical Research Building 3.132, University of Texas Medical Branch, Galveston, TX 77555-1070. E-mail address: lysoong{at}utmb.edu Back

3 Abbreviations used in this paper: M{phi}, macrophage; DC, dendritic cell; DLN, draining lymph node; La, Leishmania amazonensis; EM, electron microscopy; BM-DC, bone marrow-derived DC. Back

4 J. Ji, J. Sun, H. Qi, and L. Soong. Selective expansion of T helper subsets in vitro and in vivo: its impact on cutaneous lesions following infection with Leishmania amazonensis. Submitted for publication. Back

Received for publication June 5, 2001. Accepted for publication August 14, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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