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Infection and Immunity, February 2006, p. 1305-1312, Vol. 74, No. 2
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.2.1305-1312.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Biogenesis of Leishmania major-Harboring Vacuoles in Murine Dendritic Cells

Ulrich Körner, Veronika Fuss, Jutta Steigerwald, and Heidrun Moll^*

Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany

Received 19 August 2005/ Returned for modification 23 September 2005/ Accepted 19 November 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
In mammalian hosts, Leishmania sp. parasites are obligatory intracellular organisms that invade macrophages and dendritic cells (DC), where they reside in endocytic organelles termed parasitophorous vacuoles (PV). Most of the present knowledge of the characteristics of PV harboring Leishmania sp. is derived from studies with infected macrophages. Since DC play a key role in host resistance to leishmaniasis, there is a need to understand the properties and biogenesis of PV in Leishmania sp.-infected DC. Therefore, we determined the acquisition of endosomal and lysosomal molecules by Leishmania major-containing compartments in DC at different maturation stages, using fluorescence labeling and confocal microscopy. The results show that newly formed phagosomes in DC rapidly develop into late endosomal compartments. However, the small GTPase Rab7, which regulates late fusion processes, was found only in PV of mature bone marrow-derived DC (BMDC); it was absent in immature BMDC, suggesting an arrest of their PV biogenesis at the stage of late endosomes. Indeed, fusion assays with endocytic tracers demonstrated that the fusion activity of L. major-harboring PV toward lysosomes is higher in mature BMDC than in immature BMDC. The inhibition of PV-lysosome fusion in DC is dependent upon the viability and life cycle stage of the parasite, because live promastigotes blocked the fusion almost completely, whereas killed organisms and amastigotes induced a considerable level of fusion activity. The differences in the fusion competences of immature and mature DC may be relevant for their distinct functional activities in the uptake, transport, and presentation of parasite antigens.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Protozoan parasites of the genus Leishmania are transmitted to mammalian hosts by phlebotomine sand flies. During the blood meal of an infected insect, the flagellated promastigote form gains access to host tissues and is rapidly phagocytosed, predominantly by macrophages in which the parasites transform into amastigotes devoid of the external flagellum. This process occurs within organelles termed parasitophorous vacuoles (PV). Amastigotes are well adapted to the conditions in the PV, allowing them to survive and multiply intracellularly (7).

A critical part of the chain of events leading to degradation and death of intracellular microorganisms, including Leishmania sp. parasites, in macrophages is phagolysosome biogenesis. Newly formed phagosomes are unable to digest their contents and must therefore undergo a complex maturation process, which is driven by a series of sequential fusion events between phagosomes and early endosomes, late endosomes, and lysosomes (1, 10, 14). Each phagosome maturation stage is characterized by specific compartmental markers, such as the early endosomal markers CD71 and Rab4, the late endosomal markers Rab7 and CD68, and the late endosomal/lysosomal glycoproteins LAMP-1 and LAMP-2. These steps lead to the formation of the phagolysosome, an acidic compartment displaying the harsh and lytic environment needed to destroy microorganisms and process microbial antigens for presentation to the specific immune system.

To date, the information on PV harboring Leishmania sp. is derived almost exclusively from studies with macrophages. However, we (3, 25, 32) and others (15, 18, 21, 27, 31) have shown that dendritic cells (DC) also internalize Leishmania sp. parasites and are the most potent antigen-presenting cells for initiation and regulation of the specific T-cell response. The properties of DC vary with the different stages of their life span. DC in nonlymphoid tissues are immature cells which can phagocytose and process particles but are only weak stimulators of T-cell immune responses. The differentiation of DC is triggered by exposure to pathogens or inflammatory cytokines. It is accompanied by a loss of endocytic activity and a marked upregulation of their antigen presentation and costimulatory functions (2, 13). Although it has been demonstrated that Leishmania sp. parasites internalized by DC reside within endocytic vacuoles (11, 15, 27), the endosomal trafficking routes in DC and the remodeling of the endosome/lysosome compartment of infected DC during their maturation are largely unknown.

In the present study, we used L. major, a cause of human cutaneous leishmaniasis, to analyze the properties of parasite-containing PV in DC of different maturation stages. Phagolysosome biogenesis was assessed by fluorescence labeling of endocytic marker proteins and confocal laser microscopy. Furthermore, we characterized the fusion competence of PV harboring L. major in DC, and we examined whether different life cycle stages of L. major induce distinct pathways of phagosome maturation in DC. The results show that L. major-harboring PV in DC did not acquire the whole array of molecules typical for lysosomal organelles and had a reduced capacity to fuse with lysosomes. The impaired fusion activity was most pronounced in immature DC. Moreover, our data demonstrate that the highest level of inhibition of endosome-lysosome fusion in immature DC was induced by live promastigotes, whereas killed organisms were not effective and amastigotes established only a partial block of PV fusion with lysosomes.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation and culture of DC. Freshly prepared DC were generated from the bone marrow of 6- to 8-week-old BALB/c mice (Charles River Breeding Laboratories, Sulzfeld, Germany) as described previously (20, 28). Briefly, 2 x 10⁶ bone marrow cells derived from the tibia and femur of the hind legs were incubated in 10 ml RPMI 1640 medium (Invitrogen, Karlsruhe, Germany) supplemented with 10% (vol/vol) heat-inactivated fetal calf serum, 2 mM L-glutamine, 10 mM HEPES buffer, 0.05 mM ß-mercaptoethanol, 100 U/ml penicillin, and 20 µg/ml gentamicin (culture medium) in the presence of 200 U/ml granulocyte-macrophage colony-stimulating factor (GM-CSF; PeproTech, London, United Kingdom). Cultures were fed with GM-CSF on day 3 if maintained for a period of 6 days and were fed again on day 6 or on days 6 and 8 if maintained for a period of 8 or 10 days, respectively. The bone marrow-derived DC (BMDC) were harvested on day 6, 8, or 10 and were allowed to adhere to glass chamber slides (Nunc, Wiesbaden, Germany) for 3 to 4 h. The cells were shown to have a typical DC morphology with a myeloid DC phenotype (MHC class II⁺, CD80⁺, CD86⁺, CD11c⁺, CD205⁺, B220^–, CD8^–). For activation, BMDC harvested on day 8 were incubated overnight with lipopolysaccharide (LPS; Sigma-Aldrich, Taufkirchen, Germany) at 1 µg/ml, CpG oligodeoxynucleotide (ODN) 1668 (5'-TCC ATG ACG TTC CTG ATG CT-3') or the control AT-rich ODN (non-CpG ODN, 5'-ATT ATT ATT ATT ATT ATT AT-3') (QIAGEN Operon, Cologne, Germany) at 100 µg/ml, CD40 ligand (PeproTech) at 1 µg/ml, or interleukin 10 (IL-10; PeproTech) at 20 µg/ml. A mouse DC line derived from fetal skin (FSDC) (12) was passaged in culture medium. All experiments were done with subconfluent cultures.

Parasites and treatment of DC with L. major. The cloned virulent L. major isolate (MHOM/IL/81/FE/BNI) was maintained by passage in BALB/c mice. Amastigotes were isolated from skin lesions as described previously (4). Promastigotes were grown in vitro in blood agar cultures. For analysis by confocal microscopy, parasites were stained with green fluorescent cell tracker 5-chloromethylfluorescein diacetate (CMFDA) or red fluorescent cell tracker 5- and 6-4-chloromethylbenzoylaminotetramethylrhodamine (CMTMR) (Molecular Probes, Leiden, Netherlands) prior to their use for infection according to the manufacturer's instructions. Briefly, stationary-phase promastigotes were incubated at 27°C and amastigotes at 37°C for 45 min with 30 µM CMFDA or 10 µM CMTMR. After being washed, they were incubated for an additional 30 min at the appropriate temperature in fresh culture medium. The promastigotes were not complement opsonized. For some experiments, stationary-phase promastigotes were heat killed at 80°C for 30 min after the staining procedure. FSDC and BMDC were treated with stationary-phase promastigotes at a ratio of 2:1 or with amastigotes at a ratio of 4:1. After 30 min of exposure to parasites at 37°C in a humidified atmosphere with 5% CO₂, unbound extracellular parasites were removed by washing the cells three times with phosphate-buffered saline (PBS). To analyze PV biogenesis, DC were examined for the expression of endosomal marker proteins either immediately (0.5-h value) or after the addition of fresh culture medium and incubation for a further 0.5 to 23.5 h (1-, 2-, 4-, and 24-h values), using intracellular immunostaining and confocal microscopy. The average number of parasites per infected DC was determined by counting 100 to 200 immature DC (6 days) or mature DC (10 days) at 24 and 72 h after pulse infection with stationary-phase promastigotes for 30 min. The parasite load was quantified by staining with acridine orange (Sigma-Aldrich) and ethidium bromide (Sigma-Aldrich) and analysis by fluorescence microscopy at 495 nm (26).

Loading of DC with endocytic tracers. For analysis of the fusion ability of PV with lysosomes, DC seeded into chamber slides (6 x 10⁵ cells in 0.4 ml of culture medium per chamber) were exposed to CMFDA-stained L. major parasites. After 30 min, free extracellular parasites were removed by repeated washing with PBS, and 1 µM LysoTracker Red DND-99 (Molecular Probes), preferentially labeling lysosomes as the main acidic compartments, was added for 30 min. Thereafter, the supernatants were removed from each well, and fresh culture medium was added for the remaining culture period of 23 h. Alternatively, DC were incubated for 30 min at 37°C with 1 mg/ml lysine-fixable tetramethylrhodamine-coupled dextran (M_r, 10,000 or 70,000) (Molecular Probes), washed, and then cultured overnight in tracer-free culture medium to allow the accumulation of fluorescent tracer molecules in lysosomes. Subsequently, the cells were exposed to CMFDA-stained parasites at 37°C for 30 min. Thereafter, they were washed with PBS to remove free parasites and were cultured for a further 23.5 h.

Intracellular immunostaining of proteins of the PV and analysis by confocal microscopy. At different time points (0.5, 1, 2, 4, or 24 h) after DC exposure to stationary-phase promastigotes for 30 min (see above), the cells were washed with PBS containing 0.25 mM MgCl₂ and 0.35 mM CaCl₂, fixed with 4% paraformaldehyde, and then permeabilized with 0.2% saponin in blocking solution (10% goat serum in PBS containing Mg²⁺ and Ca²⁺). Thereafter, DC were incubated for 45 min at room temperature in a humid chamber with one of the following primary antibodies in saponin-containing blocking solution: monoclonal antibody (MAb) C2, a rat immunoglobulin G1 (IgG1) recognizing mouse CD71 (transferrin receptor) (BD Pharmingen, Heidelberg, Germany), fluorescein isothiocyanate-conjugated rat anti-mouse LAMP-2 MAb (BD Pharmingen), rabbit anti-mouse Rab4 antibodies (Santa Cruz, Santa Cruz, Calif.), rabbit anti-mouse Rab7 antibodies (Santa Cruz), phycoerythrin-conjugated rat anti-mouse LAMP-1 MAb (Santa Cruz), and biotin-conjugated rat anti-mouse CD68 (macrosialin) MAb (Serotec, Oxford, United Kingdom). Isotype-matched control antibodies were purchased from BD Pharmingen and DakoCytomation (Glostrup, Denmark). After treatment with the primary antibodies, the cells were washed three times with PBS containing Ca²⁺ and Mg²⁺ and were stained with the secondary reagents streptavidin-Alexa Fluor 546 (Molecular Probes) or rhodamine-conjugated goat anti-rat or goat anti-rabbit antibody (Dianova, Hamburg, Germany) in saponin-containing blocking solution for 45 min at room temperature. Cells were mounted in fluorescent mounting medium (DakoCytomation) before observation with a LSM 510 Zeiss confocal laser scanning microscope. Confocal microscopy images were acquired by use of LSM 510 software and analyzed by use of LSM 3.2 image browser before being exported to Adobe PhotoShop. For each experiment and at each time point, the percentages of parasite-containing compartments displaying endocytic marker molecules or having fused with lysosomes were determined after 50 to 300 PV were counted.

Extracellular staining of DC. In freshly prepared BMDC populations, DC were identified by labeling with MAb HL3, a hamster IgG1 directed against mouse CD11c (BD Pharmingen) and cytochrome5-conjugated goat anti-hamster IgG (Dianova) before permeabilization for intracellular staining.

Statistical analysis. Unpaired Student's t test was used for statistical analyses. Differences were considered significant when P was <0.05.

Top Abstract Introduction Materials and Methods Results Discussion References

 
L. major promastigotes reside in late endosomal compartments of DC precursors and immature DC. The phagocytic activity of DC varies with their maturation stage. While mature DC have a strongly reduced ability to internalize particles, DC precursors and immature DC efficiently take up microorganisms, such as L. major. For the characterization of L. major-containing compartments in DC, we therefore used the cell line FSDC (representing myeloid DC progenitors arrested at an early stage of development) and freshly prepared immature DC (BMDC that had been cultured in the presence of GM-CSF for 8 days). The features of PV harboring L. major in DC were evaluated according to the presence of endosomal marker proteins using intracellular immunostaining and confocal immunofluorescence microscopy. Freshly prepared DC were identified by cell surface labeling of CD11c, and parasites were visualized after prestaining with CMFDA or CMTMR.

After infection of DC with L. major promastigotes, all parasites were located in membrane-bound compartments. The outline of the phagosome membrane was largely congruent with the shape of the parasites. Late endosomal/lysosomal molecules could be detected in the majority of PV membranes of both DC precursors and immature DC (Fig. 1). Distinct red rims indicated the presence of CD68 (Fig. 1A, 1 to 5, and Fig. 1B, 1 to 4) and LAMP-1 (Fig. 1A, 6 to 9, and Fig. 1B, 5 to 8) in the membranes of L. major-containing organelles in FSDC and BMDC. For examination of the PV biogenesis, BMDC and FSDC were evaluated at 0.5, 1, 4, or 24 h after infection with L. major. The results of the immunofluorescence analysis showed that the early endosomal marker CD71 was detectable in few PV (about 10%) only within 30 min (BMDC) or 4 h (FSDC) after infection (Fig. 2). At 24 h, virtually none of the PV in BMDC (<1%) and less than 3% of the PV in FSDC contained this early marker. In contrast, the majority of PV in both FSDC and BMDC expressed LAMP-1 and LAMP-2 proteins already in the early phase of infection. Thus, PV maturation in L. major-infected DC seems to occur very fast. The rapid formation of late endosomal compartments was most pronounced in BMDC, as indicated by the early expression of high levels of LAMP-1, LAMP-2, and CD68 (Fig. 2A), while lower amounts of these markers were detected in FSDC (Fig. 2B). On the other hand, a higher proportion of PV in FSDC was positive for Rab7, another late endosomal molecule, which is involved in the fusion of late endosomes with lysosomes (5, 22). Of note, the immortalized line FSDC has been shown to display the morphology and surface marker profile suggestive of a myeloid progenitor cell with features of both DC and macrophages (12).

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FIG. 1. Analysis of late endosomal/lysosomal proteins in phagosomes containing L. major promastigotes. BMDC (A) or FSDC (B) were treated with CMFDA-labeled parasites (green staining) for 30 min and, 24 h later, the cells were fixed, permeabilized, and stained for CD68 or LAMP-1 (red staining). Freshly prepared DC were identified by the expression of CD11c (blue staining). The optical sections (0.5- to 0.8-µm thickness) obtained by confocal fluorescence microscopy illustrate the association of PV in immature BMDC with CD68 (A, 1 to 5) and LAMP-1 (A, 6 to 9) and of PV in FSDC with CD68 (B, 1 to 4) and LAMP-1 (B, 5 to 8). Colocalizations of CD68 or LAMP-1 with the PV are indicated by arrows. Panels A4, A8, B3, and B7 are differential interference contrast images. Panels A5, A9, B4, and B8 represent the superimposition of the fluorescence images.

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FIG. 2. Biogenesis of PV in immature BMDC (A) and FSDC (B) infected with CMFDA- or CMTMR-labeled L. major promastigotes. At 0.5, 1, 4, or 24 h after pulse infection, the cells were fixed, permeabilized, and stained with antibodies and appropriate secondary reagents to examine the association of CD71, CD68, Rab7, LAMP-1, or LAMP-2 with L. major-containing PV by confocal fluorescence microscopy. Freshly prepared BMDC were identified by CD11c labeling. For each time point and each molecule, the percentages of parasite-containing compartments that colocalized with endosomal/lysosomal proteins were determined after 50 to 300 PV were counted. The data are mean values ± standard errors of the means for two to six independent experiments.

Influence of the maturation and activation status of DC on PV biogenesis. The results described above demonstrated that L. major-containing PV in a DC precursor line resemble those in immature DC with regard to their ability to fuse rapidly with late endocytic compartments. Next, we compared the characteristics of PV in freshly prepared immature and mature BMDC at different times after infection with L. major.

High levels of the late endosomal markers CD68, LAMP-1, and LAMP-2 were present in the parasite-containing organelles of both immature and mature DC (Fig. 3), even at the earliest time points after infection, indicating that the rapid formation of late endosomal compartments harboring L. major in DC is independent of the stage of DC maturation. The most notable difference between mature and immature BMDC was the rapid acquisition of Rab7 by L. major-containing PV in mature DC (Fig. 3B). While Rab7, a major effector of late endosome/lysosome transformation, could not be detected in PV of immature DC at any of the studied time points after infection (30 min to 4 h), 22% of the parasite-harboring compartments in mature DC displayed this molecule already at 30 min postinfection. At 1 h, the percentage of Rab7-positive PV had decreased considerably, and only background levels were detectable thereafter (Fig. 3B). The absence of Rab7 in immature BMDC suggests an arrest of their PV biogenesis at the stage of late endosomes.

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FIG. 3. Biogenesis of PV in immature BMDC (A) and mature BMDC (B) infected with CMFDA- or CMTMR-labeled L. major promastigotes. At 0.5, 1, 2, or 4 h after pulse infection, the cells were fixed, permeabilized, and stained with antibodies and appropriate secondary reagents to examine the association of CD68, LAMP-1, LAMP-2, CD71, Rab7, or Rab4 with L. major-containing PV by confocal fluorescence microscopy. DC were identified by CD11c labeling. For each time point and each molecule, the percentages of parasite-containing compartments that colocalized with endosomal/lysosomal proteins were determined after 100 to 200 PV were counted. The data are mean values ± standard deviations for two independent experiments.

The properties of DC not only change during their maturation but also vary depending on their activation status. To determine the effect of DC activation on the biogenesis of L. major promastigote-containing PV, BMDC were exposed to the stimulus LPS, CpG ODN, or CD40 ligand and were compared with nonstimulated BMDC with regard to the presence of endosomal/lysosomal marker proteins in the PV at 24 h after infection. Furthermore, the effect of IL-10, a cytokine promoting the development of tolerance-inducing DC, was examined. The results demonstrated that none of these DC treatments modified the biogenesis of L. major-harboring PV in BMDC (data not shown). Together, our findings document that PV biogenesis is influenced by the stage of maturation but not the activation status of DC.

The fusion activity of L. major-containing PV with lysosomes is strongly reduced in immature DC. The finding that Rab7, a key molecule controlling the formation of phagolysosomes, is not present in PV of immature BMDC and can be detected only during the initial phase of infection in PV of mature BMDC (see above) (Fig. 3) suggested that L. major modulates the fusogenic activity of phagosomes in DC. To test this assumption, BMDC at different stages of maturation were infected with CMFDA-labeled L. major promastigotes, and the lysosomes were stained with LysoTracker dye. Subsequently, the fusion activity of the PV with lysosomes was analyzed by confocal microscopy at 24 h after infection. L. major-harboring PV from immature BMDC rarely fused with lysosomes (Fig. 4, panels A and B1); only about 5% of the lysosomal compartments in BMDC that had been cultured for 6 days colocalized with promastigotes. During further DC maturation, the percentage of fusion events increased and reached a level of about 18% (Fig. 4, panels A and B2 to B5). The poor fusion activity of promastigote-containing organelles with lysosomes in immature DC cannot be explained by a delay in PV maturation, because fusion events were almost absent even after 2 and 3 days of infection (data not shown).

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FIG. 4. Fusion properties of L. major-containing PV in BMDC at different maturation stages. BMDC that had been cultured for 6, 8, or 10 days were exposed to CMFDA-labeled L. major promastigotes for 30 min. Subsequently, LysoTracker dye was added to stain the lysosomes. After 24 h, the cells were fixed and analyzed for lysosomes colocalizing with parasites by confocal fluorescence microscopy. DC were identified by CD11c labeling (blue). (A) For each BMDC population, the percentage of parasite-containing lysosomal compartments (fusion events) was determined after 50 to 200 organelles were counted. Data are the mean values ± standard deviations for two independent experiments. Panel B1, L. major-containing PV (green staining) in immature BMDC (cultured for 6 days) not colocalizing with lysosomes (red staining). Panels B2 to B5, L. major-containing PV (green staining) in mature BMDC (cultured for 10 days) that colocalizes with a lysosomal compartment (arrows). Panel B3 is a differential interference contrast image; panel B5 represents the superimposition of the fluorescence images shown in panels B2 and B4. Yellow color indicates colocalization. Optical sections (1-µm thickness) are shown.

Interestingly, the number of parasites per infected DC at 72 h after pulse infection with promastigotes was slightly higher in immature BMDC than in mature BMDC (immature BMDC, 2.56 parasites per DC; mature BMDC, 2.23 parasites per DC), while it was almost identical at 24 h (immature BMDC, 1.62 parasites per DC; mature BMDC, 1.66 parasites per DC). This result indicates that the maturation stage of DC affects the intracellular survival of parasites.

Only live promastigotes, not heat-killed promastigotes or amastigotes, cause a block of fusion between PV and lysosomes in DC. To evaluate whether the incompetence of L. major-harboring PV to fuse with lysosomes depends on parasite viability or life cycle stage, immature BMDC or FSDC were exposed to live promastigotes, heat-killed promastigotes, or live amastigotes. In contrast to PV containing live promastigotes, a large proportion of PV containing heat-killed promastigotes (about 70%) colocalized with lysosomes in both BMDC and FSDC (Fig. 5). Moreover, the fusogenic activity of PV containing amastigotes was much higher than that of PV containing live promastigotes (34% compared to 10% in freshly prepared DC). The percentage of fusion events was higher (20%) in the DC progenitor line, FSDC, that had been exposed to live promastigotes, a finding that correlated with the higher level of Rab7 in the PV of these cells (see above) (Fig. 2). These observations suggest that after internalization of live promastigotes, the maturation of PV in DC is strongly reduced and the vast majority of PV is arrested at the stage of late endosomes. In contrast, a considerable proportion of PV containing amastigotes fuse with lysosomes.

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FIG. 5. Fusion activities of L. major-containing PV in DC that had been exposed to live or heat-killed promastigotes or live amastigotes of L. major. Lysosomal compartments in immature BMDC (cultured for 8 days) or FSDC were labeled by loading with rhodamine-coupled dextran before incubation of the cells with CMFDA-stained parasites for 30 min. After 24 h, the cells were fixed and analyzed for lysosomes colocalizing with PV by confocal fluorescence microscopy. Freshly prepared BMDC were identified by CD11c labeling. For each experimental group, the percentage of lysosomal compartments colocalizing with PV (fusion events) was determined after 100 organelles were counted. The results are representative of three or four independent experiments.

Top Abstract Introduction Materials and Methods Results Discussion References

 
DC are extraordinarily efficient in processing microbial antigen and presenting it to T cells, thus linking the innate and adaptive arms of the immune system. In leishmaniasis, the initiation and instruction of the specific immune response by DC is critical for the outcome of the disease. An intriguing feature of DC is the tight control of their antigen processing and presentation functions according to their maturation status. Although it has been shown previously that Leishmania sp. parasites reside in endosomal compartments of DC (11, 15, 27), the characteristics of Leishmania sp.-containing PV in DC remain poorly understood, and it is unknown whether they are subject to changes during DC maturation. In the present study, the major new findings are that the fusogenic activity of L. major-harboring compartments toward lysosomes is strongly impaired in immature DC and that this inhibition of phagosome-lysosome fusion is dependent on the viability and life cycle stage of the parasite.

Both macrophages and DC serve as host cells for Leishmania sp. parasites, but the two cell types differ in important ways. DC have unique surveillance and migratory properties, enabling them to carry parasite antigens captured in the infected skin to the draining lymph nodes for presentation to naïve T cells, thus initiating the specific immune response (23, 24). A critical specialization of DC is the process of maturation (2). Immature DC efficiently take up antigen but express relatively low levels of major histocompatibility complex (MHC) and costimulatory molecules on their surface and, thus, have a reduced capacity for T-cell stimulation. In contrast, mature DC downregulate their endocytic activity, while the transport of immunogenic peptide-MHC class II complexes from the lysosomal compartment to the plasma membrane, together with increased surface expression of costimulatory molecules, enables them to activate even resting T cells (6, 16, 30). Using L. major-infected Langerhans cells, DC derived from the skin, we previously demonstrated that the MHC class II levels associated with L. major-containing PV decrease with DC maturation (11). We also showed that immunogenic peptide-MHC class II complexes in L. major-infected DC are remarkably long-lived (11), supporting the concept that DC are highly specialized for the prolonged retention and efficient presentation of parasite antigen in leishmaniasis.

In the present study, we analyzed the biogenesis of PV housing L. major, as characterized by the acquisition of endosomal and lysosomal marker proteins, and the fusion activity of the L. major-containing PV with lysosomes in DC at different maturation stages. The determination of the kinetics of endosome/lysosome marker recruitment into the PV revealed that L. major-containing phagosomes in all types of DC tested, a DC precursor line as well as freshly prepared immature and mature DC, can fuse with late endocytic compartments very quickly after their formation, as hallmarked by the rapid acquisition of high levels of LAMP-1, LAMP-2, and CD68 (Fig. 2 and 3). On the other hand, none or only very small amounts of the PV were found to display the early endosomal molecules CD71 and Rab4, indicating low or very transient interactions with early endocytic organelles just upon parasite uptake. Interestingly, very similar patterns of endosomal/lysosomal marker expression were observed with DC that, in addition to culture in the presence of GM-CSF, were treated with the stimuli LPS, CpG ODN, or CD40 ligand. This finding supports the view that activation of DC, although greatly enhancing their T-cell-stimulatory capacity via induction of IL-12 expression (17, 21), does not influence the biogenesis of L. major-containing phagosomes.

A most important feature was the developmental regulation of Rab7 expression in L. major-containing phagosomes of DC. This protein, which controls the fusion of late endosomes with lysosomes, was virtually absent in PV of immature DC, but significant amounts were detected in those of DC precursors and mature DC. It is noteworthy that the maximal levels of Rab7 expression in DC were consistently lower than those reported for macrophages (7). An analysis of the fusion activity of L. major-containing PV with lysosomes demonstrated that the fusogenic property was strongly impaired in freshly prepared immature DC infected with promastigotes, while it increased steadily during DC maturation (Fig. 4). Interestingly, the decreased fusion activity of PV with lysosomes in immature DC correlated with enhanced parasite survival in these cells. Thus, L. major promastigotes reduce lysosomal degradation in immature DC. In vivo, DC in the periphery, such as skin, generally exhibit an immature phenotype. The observation that PV of immature DC, relative to those of mature DC, fuse poorly with lysosomes provides an explanation for how immature DC, while migrating from the infected skin to the draining lymph node and undergoing the maturation process, can preserve parasite antigen for the generation of peptide-MHC complexes and efficient presentation to T cells.

The results of the present study also show that the inhibition of PV-lysosome fusion in L. major-infected DC is dependent upon the viability and life cycle stage of the parasite. Live promastigotes blocked the phagolysosome generation in immature BMDC almost completely (Fig. 4 and 5), whereas phagosomes containing killed organisms showed extensive fusion with lysosomes. Furthermore, the low level of Rab7 expression in the L. major-containing PV of DC was a specific effect of live parasites. While Rab7 could not be detected in PV of immature DC and was present in 22% of the PV in mature DC treated with live promastigotes, a large proportion of PV in immature DC (53%) and mature DC (63%) that had been exposed to heat-killed parasites expressed Rab7 (Fig. 3 and data not shown). L. major amastigotes induced a considerably higher level of fusion activity than promastigotes. These findings are reminiscent of the situation with macrophages, for which it has been demonstrated that promastigotes, unlike amastigotes, are located in phagosomes that poorly fuse with late endocytic/lysosomal compartments. This phenomenon could be linked to the stage-specific expression of lipophosphoglycan, the predominant surface glycoconjugate of promastigotes (8, 9, 19). The promastigote-containing phagosomes with low fusogenic activity were shown to display an impaired recruitment of Rab7 (29). Again, the latter finding with infected macrophages is analogous to our observation with DC and suggests that, in both types of host cells, the fusion restriction allows the differentiation of promastigotes into amastigotes which are more adapted to the lysosomal compartment. However, an important characteristic of the inhibition of phagosome-lysosome fusion in DC is its regulation according to the stage of DC maturation.

In conclusion, the findings of the present study extend our understanding of the interaction of DC with Leishmania sp. and the mechanisms underlying the unique ability of DC to initiate the parasite-specific T-cell response so efficiently. Our data are consistent with the concept that immature DC in the skin, upon ingestion of the promastigote form deposited by the sand fly vector, can sequester antigen by reducing lysosomal degradation until the maturing cells have migrated to the draining lymph node. When reaching the lymph node, lysosomal proteolysis in mature DC mobilizes the formation of immunogenic peptides to be loaded onto MHC class II molecules and presented to resting T cells. Immature DC that remain in the skin and mature in response to local inflammatory signals may also present antigenic peptides derived from killed promastigotes or amastigotes that are released by macrophages, thus contributing to the instruction of the local immune response.

 
This work was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG Priority Programme 1131).

We thank Christine Hambrecht for technical assistance and Hilde Merkert for help with confocal microscopy.

 
* Corresponding author. Mailing address: Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany. Phone: 49 931 312627. Fax: 49 931 312578. E-mail: heidrun.moll{at}mail.uni-wuerzburg.de.

Editor: W. A. Petri, Jr.

U.K. and V.F. contributed equally as first authors of this work.

Present address: Micromet AG, Staffelseestrasse 2, D-81477 München, Germany.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Infection and Immunity, February 2006, p. 1305-1312, Vol. 74, No. 2
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.2.1305-1312.2006
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