OUP user menu

Wild-type Leishmania donovani promastigotes block maturation, increase integrin expression and inhibit detachment of human monocyte-derived dendritic cells – the influence of phosphoglycans

Katarina Tejle, Margaretha Lindroth, Karl-Eric Magnusson, Birgitta Rasmusson
DOI: http://dx.doi.org/10.1111/j.1574-6968.2007.01013.x 92-102 First published online: 1 February 2008

Abstract

The protective immune response against the parasite, including the role of dendritic cells (DC) in the course of infection, plays a fundamental role. This study shows that wild-type (WT) Leishmania promastigotes and specifically the phosphoglycans family of virulence-associated antigens inhibit human monocyte-derived dendritic cells (MoDC) maturation and detachment to distinct surfaces. Immature phagocytosis of Leishmania donovani promastigotes by immature MoDC results in the increased expression of CD11b and CD51, and inhibition of cell detachment to distinct surfaces, which was dependent on the presence of phosphoglycans. These findings demonstrate that phosphoglycans of WT L. donovani might also inhibit human DC migration to lymphoid organs.

Keywords
  • dendritic cells
  • CD11b
  • CD51
  • Leishmania donovani
  • phagocytosis
  • adhesion

Introduction

Dendritic cells (DC) are professional antigen-presenting cells (APC) involved in the initiation of immune responses (Hart, 1997; Banchereau & Steinman, 1998; Banchereau, 2002). They effectively prime cytotoxic T cells for immune responses against virus, bacteria and several tumours (Hart, 1997; Lewalle et al., 2000; Sallusto & Lanzavecchia, 2002).

Upon exposure to inflammatory signals, such as lipopolysaccharide and TNF-α, immature DC up-regulate chemokine receptors that aid in the migration to lymph nodes. Costimulatory molecules (e.g. CD80, CD86) that are required for the activation of antigen-specific T cells (Banchereau & Steinman, 1998; Lee et al., 2002) are also expressed to a large extent. Mature cells are characterized by reduced phagocytic capacity and high expression of costimulatory molecules and other cellular markers like CD83 and DC–lamp (Sallusto et al., 1995; Sallusto & Lanzavecchia, 2002; Tuyaerts et al., 2002). Several observations have suggested that mature DC show a considerably reduced capacity to phagocytise exogenous antigen and a concomitant reduction in cross-presentation of exogenous antigens, when Toll-like receptors (TLRs) are engaged by for instance Malaria parasite antigens (Hickman-Miller & Yewdell, 2006; Wilson et al., 2006).

DC display significantly reduced phagocytic activity compared with macrophages (Kiama et al., 2001; Thiele et al., 2001; Moll, 2003), but recognize and process a wide range of microbial antigens via activation of cognate receptors, e.g. Fc-receptors, C-type lectin receptors (CLRs) and pattern-recognition receptor (PRRs) such as TLRs (Aderem & Ulevitch, 2000; Reis e Sousa, 2001; Takeda & Akira, 2004). Immature DC internalize antigen via macropinocytosis, receptor-mediated endocytosis and phagocytosis (Garrett et al., 2000; West et al., 2000; Nagl et al., 2002; Rescigno, 2002). From an evolutionary perspective, bacterial or particle ingestion by phagocytosis is probably the basic form of antigen handling by DC (Mellman & Steinman, 2001), while the maturation process is essential for DC to become fully capable of stimulating T cells. After antigen capture, DC are assumed to migrate into T-cells-rich areas of secondary lymphoid tissue, thereby bringing antigen from the periphery to naïve T cells (Banchereau & Steinman, 1998).

Leishmania donovani is a parasitic protozoan causing severe disease in humans (Larsen et al., 2000). Its life cycle includes an infective promastigote stage transmitted by sand fly vectors and an amastigote stage in vertebrate hosts (Pimenta et al., 1992). Current control measures for leishmaniasis in humans include testing of new diagnostics and development of affordable and effective vaccines. To achieve these goals, precise knowledge on the protective immune response against the parasite, including the role of DC in the course of infection is essential.

In this work, phagocytosis was studied by immature human monocyte-derived DC (MoDC) of wild-type (WT) promastigotes or a mutant lacking phosphoglycan-specific carbohydrate repeats, Gal–Man–PO4 (lpg2−KO) (Desjardins & Descoteaux, 1997).

The results show that interaction with WT L. donovani promastigotes cause an increase in total F-actin in immature MoDC. In this study it was also found that such cells increase their surface level of CD11b and CD51 following uptake of WT promastigotes, and that these WT promastigotes inhibit the detachment of maturing MoDC to distinct surfaces, effects that were dependent on the presence of phosphoglycans on L. donovani.

Materials and methods

Culture medium and cytokines

Iscove's modified Dulbecco's medium (IMDM) was supplemented with 4 mM l-glutamine, 10% heat-inactivated foetal calf serum (FCS), 100 U mL−1 penicillin and 100 μg mL−1 streptomycin (all from Gibco BRL/Invitrogen). Recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF), IL-4 and TNF-α were purchased from R&D Systems.

The Leishmania culture medium consisted of M199 supplemented with 10% heat-inactivated (56 °C, 45 min), sterile-filtered FCS, 40 mM HEPES, 100 U mL−1 penicillin, 100 μg mL−1 streptomycin, 0.1 mM adenine, 5% hemin, 0.0001% biotin, 0.001 mg mL−1 biopterine and 500 μg mL−1 G418 (all from Gibco BRL/Invitrogen).

Cell isolation

Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coats from healthy donors and separated according to Böyum (1968). After sedimentation on a gradient with dextran (Lymphoprep; Axis-Shield PoC AS), centrifugation in a swing-out rotor for 30 min at 400 g at 4 °C and brief hypotonic lyses, the cells were harvested and washed repeatedly in ice-cold calcium-free KRG (120 mM NaCl, 4.9 mM KCl, 1.2 mM MgSO4, 8.3 mM KH2PO4 and 10 mM glucose) to remove density gradient residue and platelets. After the final wash, PBMCs were isolated by negative selection using a cocktail of biotin-conjugated antibodies to CD3, CD7, CD19, CD45RA, CD56 and IgE, respectively, and MACS CD14 micro beads coupled to antibiotin monoclonal antibodies (Miltenyi Biotec). The resulting monocyte-enriched fractions were used to generate DC.

Human MoDC

Cells from the monocyte-enriched fractions were seeded onto sterile glass cover slips in cell culture plates (Nunc) at 4 × 106 cells per well and left to adhere for 2 h at 37 °C in 5% CO2. Nonadherent cells were removed by washing with KRG with 1 mM CaCl2, at 37 °C. DCs were generated by culturing the cells in 1 mL of IMDM containing 1000 U mL−1 GM-CSF and 500 U mL−1 IL-4. The cytokines were added to the cultures at Day 0 and Day 3 to generate immature DC at Day 5–6. 1000 U mL−1 TNF-α was added at Day 6 for procession of the cells into mature DC at Day 9–10 (Romani et al., 1996; Bender et al., 1998; Banchereau et al., 2000; Fonteneau et al., 2001; Tuyaerts et al., 2002).

Flow cytometry and antibodies

Fluorescence-activated cell sorting (FACS) analysis (FACS-Calibur, BD, Biosciences) was used to analyze cell-surface antigen expression of a variety of leukocyte markers on both adherent and nonadherent cells. Adherent cells were harvested using a cell scraper (Becton-Dickinson). 1 × 106 cells were washed in phosphate buffered-saline (PBS) pH 7.3 and incubated for 30 min on ice with either fluorescein-isothiocyanate (FITC)-conjugated monoclonal anti-human antibodies to CD68, CD86, HLA-DP, DQ, DR, CD11c (all from Dakopatts) or CCR5 (BD, Biosciences), or phycoerythrin-conjugated monoclonal anti-human antibody to CD80 (Becton-Dickinson) or CCR7 (R&D Systems). Control cells were processed similarly using FITC- or phycoerythrin-matched mouse isotype control IgG1 or IgG2. The cells were pelleted and resuspended in 2% paraformaldehyde (Sigma-Aldrich). A gate based on forward and side scatters was set to exclude cell debris. The mean fluorescence of at least 1000-gated cells was determined. Results were analyzed using the winmdi-program (Version 2.8, Scripps Research Institute, La Jolla, CA).

Immunofluorescence staining

Immature and mature MoDC were fixed for 15 min at room temperature in 2.0% (w/v) paraformaldehyde (Sigma-Aldrich) in KRG, washed three times in PBS (pH 7.6) and incubated for 45 min on ice with an FITC-conjugated monoclonal anti-human antibody to CD86 (Dakopatts) diluted 1 : 100 in PBS (pH 7.6) with 1% bovine serum albumin (BSA) (Boehringer-Mannheim) and 10% normal goat serum (Dakopatts). Control cells were processed similarly using mouse isotype control IgG1. After washing three times in PBS, incubation was continued for 45 min on ice with Alexa594 Fluor-conjugated goat anti-mouse antibody (Molecular Probes) diluted 1 : 200 in PBS with 1% BSA (200 μL per  cover slip). The labelled cells were washed three times in PBS and mounted in an anti-fading medium supplemented with 20% Airvol 203 (Air Products) and 4% Citifluor/Glycerol (Citifluor Ltd) in 20 mM Tris buffer (pH 8.5).

Parasites

WT L. donovani promastigotes (Sudanese strain 1S) were derived from amastigotes isolated from the spleen of an infected hamster and grown at 26 °C in modified M199 medium at pH 7.0–7.4 (McNeely & Turco, 1990). The WT promastigotes and the mutant strain lpg2−KO, lacking the disaccharide-phosphate repeats of phosphoglycan (Descoteaux et al., 1995), were both transfected with green fluorescent protein (GFP) (Scianimanico et al., 1999). The promastigotes were cultured in tightly sealed, sterile tissue culture flasks (Nunc) and were allowed to reach the stationary phase before use. The day before an experiment, the promastigotes were spun down at 350 g for 5 min, resuspended in the same volume of fresh growth medium and cultured over night at 26 °C.

IgG-opsonization of yeast

Fresh baker's yeast (Saccharomyces cervisiae; 108 mL−1) was labelled for 60 min at 37 °C with 0.25 mg mL−1 FITC in 0.2 M carbonate buffer (pH 9.5), washed several times in PBS (pH 7.3), resuspended in PBS, and stored at −20 °C. The yeast was opsonized for 30 min at 37 °C with a rabbit anti-yeast antibody (20 μg mL−1) in 25% heat-inactivated (30 min, 56 °C) normal human serum (IgG opsonization), washed twice and resuspended to 107 mL−1 in KRG (pH 7.3) (Hed & Stendahl, 1982).

Type I collagen-coated surfaces

Collagen R (type I rat, SERVA) was diluted 1 : 10 (2 mg mL−1) in distilled water, added to sterile glass cover slips in cell culture plates (Nunc) and left to adsorb over night at 4 °C, followed by washing in PBS.

Phagocytosis and labelling of F-actin

Yeast particles were suspended in MoDC culture medium at 37 °C and added to the DC cells at a ratio of 1 : 1. Before incubation with cells, the promastigotes were spun down and resuspended in fresh DC culture medium at 37 °C. A parasite: cell ratio of 10 : 1 for WT and 5 : 1 for lpg2−KO promastigotes was used to compensate for the reduced uptake of WT promastigotes compared with the mutant (Holm et al., 2001). The cells were first incubated for 20 min with yeast and for 30 min with promastigotes, respectively, at 37 °C and 5% CO2 (pulse) and washed three times to remove unbound prey. Incubation was continued at 37 °C for the indicated times (chase), followed by fixation for 15 min at room temperature in 2.0% (w/v) paraformaldehyde (Sigma-Aldrich) in KRG and washing in PBS (pH 7.3). The fixed cells were washed three times in PBS supplemented with 1% BSA (Boehringer-Mannheim), designated PB, and then incubated in PB for 30 min at room temperature to block the unspecific binding of phalloidin. The cells were subsequently incubated for 30 min at room temperature with Alexa594 Fluor-phalloidin (Molecular Probes) to stain F-actin. Phalloidin from a stock solution (200 U mL−1 in methanol, kept at −20 °C) was diluted 1 : 40 in PBS (pH 7.3) supplemented with 100 μg mL−1 lysophosphatidylcholine (Sigma-Aldrich) as a membrane permeabilizing agent. After labelling, the cells were washed three times in PB and mounted in an anti-fading medium supplemented with 20% Airvol 203 (Air Products) and 4% Citifluor/Glycerol (Citifluor Ltd) in 20 mM Tris buffer (pH 8.5).

Labelling of CD86, CD11b or CD51

After phagocytosis, the MoDC were fixed and washed three times in PBS (pH 7.6). The preparation was labelled for 45 min on ice with monoclonal mouse antibodies against CD11b and CD51 (both from Boehringer-Mannheim) diluted 1 : 50 in PBS (pH 7.6) with 1% BSA (Boehringer-Mannheim) and 10% normal goat serum. The cells were washed three times in PBS and incubated for 45 min on ice with Alexa594 Fluor-conjugated goat anti-mouse antibodies (Molecular Probes) diluted 1 : 200 in PBS with 1% BSA, washed three times in PBS and mounted as described above.

Confocal microscopy

Imaging was performed using a Sarastro 2000 confocal microscope (Molecular Dynamics) equipped with a Nikon microscope and a × 60 (NA 1.4) oil immersion objective. The 514 nm line of the Argon laser was used for simultaneous excitation of FITC/GFP and Alexa594 Fluor. Dichroic mirrors with cut-off wavelengths of 535 and 595 nm were used for the excited and emitted light, respectively. A 545DF30 nm band-pass filter was employed for the green signal (FITC/GFP) and a 600 nm long-pass emission filter for the red signal (Alexa594Fluor). This filter set-up ensured no detection of the red signal in the green channel, or vice versa.

Scanning electron microscopy (SEM)

Immature MoDC were grown on cover slips (Ø=13 mm) and used on Day 6. The cells were first incubated for 20 min with promastigotes at 37 °C and 5% CO2 (pulse) and washed three times to remove unbound prey. Incubation was continued at 37 °C for 10 min or 60 min followed by rinsing twice in 0.15 M sodium cacodylate buffer (Link Nordiska). The cells were fixed in 2% glutharaldehyde (Link Nordiska) diluted in 0.15 M sodium cacodylate buffer, rinsed several times in the same buffer, and postfixed for 1 h in 1% osmium tetroxide (Link Nordiska) diluted in 0.15 M sodium cacodylate buffer. The specimens were dehydrated in a graded series of 50–100% ethanol, critical point dried from CO2, and sputter-coated with platinum. Digital micrographs were obtained from a LEO 1550 GEMINİ FEG high-resolution scanning electron microscope (LEO Electron Microscope) operated at 5 kV and a tilt of 30°.

Quantification of phagocytosis

Phagocytic capacity was assessed after a 20-min chase to allow maximal internalization of the prey. Scanned confocal sections of MoDC labelled with Alexa594 Fluor phalloidin were randomly examined and the number of FITC-labelled yeast cells ingested per MoDC was counted. In all, 122–124 MoDC were investigated in each group. The results of two experiments performed on different days were combined.

Quantification of total F-actin and expression of CD 86, CD11b and CD51

Analysis of total F-actin and expression of integrins was made in a standard fluorescence microscope (Zeiss Axioskop) using an × 63 oil immersion objective with a numerical aperture of 1.4. Light microscopy images were captured with a CCD camera with a ZVS-47E amplifier (Zeiss) and visualized by easy image analysis 2000 (Version 2.7.2.2, Tekno Optik AB), and saved in the tiff format. A 496DF10 nm (Texas Red Chroma) band-pass filter was used for detection of the red signal (Alexa594 Fluor). All images were recorded with exactly the same instrument settings and acquisition parameters. Images were digitally analysed using scion image software for Windows (Version Beta 4.0.2, Scion Corporation). The rim around each MoDC was traced manually with an Intuos3 Pen Tablet (WACOM) to acquire the cell area and mean staining intensity. The area of the MoDC was multiplied with the mean F-actin or integrin staining intensity. These calculations were carried out on 181–247 cells from duplicate preparations. The results were compiled from data on samples from three separate experiments performed on different days. To compensate for possible day-to-day variation in instrument performance, all results were normalized against parallel data from controls.

Quantification of MoDC adhesion to different surfaces

The adhesion of MoDC, which had engulfed WT promastigotes and lpg2−KO promastigotes, was investigated after 24 h incubation using a standard fluorescence microscope. The preparations were labelled with fluorescent phalloidin to visualize the cells. The mean number of adherent MoDC in 20 random fields of view was assessed. The experiment was repeated three times on separate days.

Statistical methods

Statistical analyses were carried out on the results from measurement of total F-actin, expression of CD86, CD11b and CD51, and the mean number of adherent MoDC in 20 random fields of view. Error bars indicate SEM. The significance levels of differences between control and experimental groups were evaluated with the Student's t-test. A P-value of <0.01 was considered significant.

Results

Main features of the human buffy coat MoDC according to the developmental stage

To investigate the effect of maturation on the phagocytic capacity of MoDC, purified monocytes were cultured with GM-CSF plus IL-4 for 6 days to yield immature MoDC, followed by TNF-α for 3 additional days to yield mature cells. Specific marker expression was studied with flow cytometry and/or indirect immunofluorescence. During maturation, an increased expression of the costimulatory molecule CD86 was observed. Maturation induced by TNF-α also resulted in an up-regulation of CD80, HLA-DP, DQ, DR, CD11c, CCR7 and a concomitant down-regulation of CD68 as assessed by flow cytometry. For CCR5 there was also an increase from a very low value. It was found that 42% of immature MoDC were able to internalize unopsonized yeast, while 54% of the cells internalized opsonized yeast. Only 10% of mature MoDC were able to internalize prey (Data not shown).

Scanning electron and confocal imaging of MoDC 24 h post coincubation with stationary phase L. donovani promastigotes

The interaction of immature MoDC and L. donovani promastigotes was studied using SEM. A series of SEMs demonstrated lpg2−KO and WT promastigotes attaching to the cells at various stages of internalization. During engulfment of lpg2−KO promastigotes (Fig. 1b and c), a parasite sometimes appeared to be ‘sinking’ into the MoDC (Fig. 1d). WT promastigotes were internalized by immature MoDC in at least three different ways, by formation of a narrow tube-like pseudopodium around the parasite (Fig. 2a and b), by covering the parasite with a smooth membrane (Fig. 2c and d) or by wrapping the parasite in a broad pseudopodium (Fig. 2e and f).

1

SEMs of immature human MoDC, interacting with lpg2−KO mutant Leishmania donovani promastigotes for 24 h. Control cell (immature MoDC) (a). MoDC incubated with lpg2−KO mutant promastigotes, lacking functional LPG (b–d). Scale bars are 1 μm.

2

SEMs of immature human MoDC, interacting with WT Leishmania donovani promastigotes for 24 h. MoDC incubated with promastigotes (a–f). Scale bars are 1 μm.

F-actin imaging and quantification in MoDC 24 h post co-incubation with stationary phase L. donovani promastigotes or TNF-α

The surface expression of the phosphoglycan family of phosphoglycans has previously been shown to increase periphagosomal F-actin in macrophages (Holm et al., 2001). When similar experiments were performed on immature MoDC, no such effect on peri-phagosomal F-actin was observed (Fig. 3). To further investigate the effect of phosphoglycans on the total F-actin content of immature MoDC, the cells were incubated with WT and lpg2−KO promastigotes, respectively. After initial 30-min incubation followed by washing (pulse), most remaining promastigotes were found attached to cells. After further 24-h incubation (chase) the cells were fixed, labelled with fluorescent phalloidin and total F-actin was measured. The measurement revealed an increase in total F-actin in MoDC incubated with WT promastigotes compared with DC incubated with lpg2−KO (P<0.001) (Fig. 4). The level of F-actin in DC containing lpg2−KO mutants did not differ from controls without promastigotes. Similar to WT promastigotes, F-actin was increased by adding TNF-α for 24 h as a positive control (P<0.001) (Fig. 4).

3

Confocal imaging of immature human MoDC, interacting with Leishmania donovani promastigotes, for measuring the amount of F-actin (red) around individual phagosomes containing GFP-transfected preys (green): MoDC infected with lpg2−KO mutant promastigotes (a, b) or WT promastigotes (c, d). All images are 35 μm × 35 μm.

4

F-actin in MoDC after interaction with of Leishmania donovani promastigotes for 24 h, i.e. of WT promastigotes, lpg2−KO mutant promastigotes or TNF-α. The graph shows the normalized mean values of the fluorescence of single cells labelled with fluorescent phalloidin and all results were normalized against control cells. Each group contains data from a total of 181–247 MoDC from three separate experiments; error bars indicate SEM; **P<0.01; ***P<0.001.

Effect of phosphoglycans on CD86 expression on immature MoDC

CD86 is a recognized marker of MoDC maturation (Banchereau & Steinman, 1998; Lee et al., 2002). To assess the effect of L. donovani promastigotes on MoDC maturation, analysis of CD86 expression was made on fixed immature MoDC. The cells had been incubated for 24 h with WT promastigotes, lpg2−KO mutants or TNF-α. The results show an up-regulation of CD86 by TNF-α and lpg2−KO mutants, indicating the initiation of maturation. By contrast, WT L. donovani promastigotes able to express phosphoglycans, failed to induce maturation, as assessed by the expression of CD86 (Fig. 5).

5

Expression of CD86 in MoDC after interaction with Leishmania donovani promastigotes for 24 h, i.e. with WT promastigotes, lpg2−KO mutant promastigotes or TNF-α. The graph shows the normalized mean values of the fluorescence of single cells labelled with anti-CD86 and a fluorescent secondary antibody and all results were normalized against control cells. Each group contains data from a total of 217–245 MoDC from three separate experiments; error bars indicate SEM; ***P<0.001.

Effect of phosphoglycans on adhesion and expression of CD11b and CD51 integrins in MoDC

Maturation of MoDC is accompanied by a loss of adhesion to surrounding tissue (Holm et al., 2003). To assess the effect of phosphoglycan expression on adhesion, maturing MoDC were allowed to adhere to glass or collagen. After incubation of immature MoDC with L. donovani promastigotes for 24 h, it was found that the cell adherence remained high when incubated with WT promastigotes, but it was reduced with the lpg2−KO mutant (158±0.1 vs. 70±0.2; SEM) or TNF-α as a positive control (Fig. 6). There was furthermore significantly higher expression of the integrins CD11b and CD51 on MoDC containing WT L. donovani promastigotes compared with lpg2−KO mutants (P<0.001). Incubation with TNF-α increased expression of CD11b, but not of CD51 (Fig. 7).

6

Adherence of MoDC to glass (light gray bars) or Type 1 collagen (dark gray bars) following interaction with Leishmania donovani promastigotes for 24 h, i.e. with WT promastigotes, lpg2−KO mutant promastigotes or TNF-α. The graph shows the mean number of fluorescent phalloidin-stained cells per field of view and all results were normalized against control cells. Each group contains data from three separate experiments; error bars indicate SEM; ***P<0.001.

7

Expression of CD11b (light gray bars) and CD51 (dark gray bars) in MoDC following uptake of Leishmania donovani promastigotes for 24 h, i.e. with WT promastigotes, lpg2−KO mutant promastigotes or TNF-α. The graph shows the normalized mean values of the fluorescence of single cells labelled with anti-CD11b or anti-CD51 and a fluorescent secondary antibody and all results were normalized against control cells. Each group contains data from a total of 195–240 DC from three separate experiments; error bars indicate SEM; **P<0.01;***P<0.001.

Discussion

Skin DCs are among the first phagocytes to encounter Leishmania parasites, and inhibition of DC activation by the parasite is of obvious benefit for the establishment of infection. In line with this, DC from mice with chronic L. donovani infection display defective migration to lymphatic tissue related to the inhibition of CCR7 expression (Ato et al., 2002), which has also been seen for Leishmania major promastigotes (Ponte-Sucre et al., 2001; Jebbari et al., 2002). This effect was conceivably dependent on a product excreted/secreted by L. major, putatively LPG (Jebbari et al., 2002). In this study, the effect of WT L. donovani promastigotes and phosphoglycan – deficient mutants (lpg2−KO) on human MoDC maturation and detachment to distinct surfaces was investigated.

The presence of the surface phosphoglycans has proved crucial for preventing phagolysosome fusion and killing of the parasite (Rasmusson & Descoteaux, 2004). This is achieved by blocking peri-phagosomal F-actin remodelling (Holm et al., 2001) through small G-proteins (Lodge & Descoteaux, 2005a, b, 2006; Lerm et al., 2006) and PKCα (Holm et al., 2003). Lodge & Descoteaux (2006) have also demonstrated that the amastigotes, and in particular LPG, destroy the integrity and signalling of lipid rafts, and thereby also the formation of a functional NADPH oxidase; the latter is essential for the killing of the parasite(Lodge et al., 2006).

The details of how immature DC detect and internalize intact pathogens, such as Leishmania parasites, remain to be investigated. Previous studies show conflicting results on whether DC can phagocytise Leishmania (Marovich et al., 2000; Brandonisio et al., 2004).

The results show that immature human MoDC, differentiated in vitro, can be infected with live, infectious stage L. donovani promastigotes (De Trez et al., 2004). Using SEM and confocal microscopy, it was determined that the extracellulary located WT promastigotes and lpg2−KO mutants were taken up and internalized by the cells (Figs 13). The MoDC engulfed Leishmania in various ways, via coiling phagocytosis, where the parasites are wrapped by a broad pseudopodium, and via tube phagocytosis, where the parasites are surrounded by a narrow tube-like pseudopodium. Other investigators have shown that DC engulf Borreliae in similar ways (Suhonen et al., 2003). The previously established reduced phagocytic capacity of mature MoDC, compared with immature cells (Banchereau & Steinman, 1998), was also evident from these data and observed for both unopsonized and IgG-opsonized prey.

Several methods to isolate blood monocytes and induce monocyte differentiation into DC have been reported (Bender et al., 1998; Banchereau et al., 2000; Fonteneau et al., 2001; Tuyaerts et al., 2002). To obtain comparable DC populations, human monocytes were cultured for 6 days with GM-CSF and IL-4. At this time, cells were CD68 positive and CD86 negative, which is indicative of immature cells (Bender et al., 1996; Banchereau & Steinman, 1998). After 3–4 days in culture with TNF-α the cells were CD80, CD86, HLA-DP, DQ, DR, CD11c, CCR7 positive and CD68 negative, suggesting maturation (Banchereau & Steinman, 1998). Moreover, the maturation process induced concomitant changes in DC morphology and F-actin structure.

For macrophages it has been demonstrated convincingly that the effect on the F-actin structure can be attributed to the presence of phosphoglycans, and more specifically LPG, either by adding LPG to an lpg2− KO mutant or by coating the prey with isolated LPG (Lodge & Descoteaux, 2005a, b, 2006; Lodge et al., 2006). Thus it was found previously that both virulent L. donovani promastigotes expressing full-length LPG and yeast particles coated with LPG are phagocytosed to a lesser extent compared with control prey (Holm et al., 2001). Moreover, the LPG-positive prey induced significantly more polymerization of F-actin close to the plasma membrane during the uptake process (Holm et al., 2001). It is likely that hyper-polymerization of cortical F-actin renders the cells less flexible, and therefore also less motile. Such a cell would, hypothetically, also have decreased phagocytic capacity.

This study showed that WT L. donovani induce increased levels of F-actin in immature MoDC at 24 h following uptake (P<0.001) compared with controls (Fig. 4). The authors cannot solely attribute the effect to LPG, because the lpg2−– mutation affects the expression of other phosphoglycans as well. To follow up on this, the effect of promastigotes and the role of phosphoglycans for cell maturation and detachment were investigated by assessing the expression of functional adhesion molecules (integrins) and attachment to different surfaces. A low level of CD86 expression was found in cells containing WT promastigotes compared with lpg2−KO mutants (P<0.1) (Fig. 5). The inhibition of CD86 up-regulation was thus dependent on the presence of phosphoglycans. In addition, immature MoDC containing WT promastigotes showed 81% higher degree of adhesion to glass compared with positive controls, i.e. cells induced to mature by TNF-α after 24 h (Fig. 6). The results also show that MoDC adhesion to glass and type I collagen was higher to either substrate after 24-h incubation with WT promastigotes compared with lpg2−KO mutants (P<0.001) (Fig. 6).

In a very recent investigation Donovan (2007) demonstrated that infections of monocytes during differentiation to become MoDC resulted in a distinct phenotype, similar to that of undifferentiated monocytes. Moreover, infection of MoDC with either L. major or L. donovani caused a clear down-regulation of CD1 molecules, normally involved in the presentation of lipid antigens to T lymphocytes (Porcelli & Modlin, 1999). These data strengthen the author's findings that Leishmania infections interfere with the differentiation from monocytes to MoDC. Some differences were also seen in the expression of the CD1 family of molecules and of other markers (CD40, HLA-DR, CD80 and CD86) between cells infected with wild-type parasites and LPG- or phosphoglycan-deficient parasites, but these were not statistically justified (Donovan et al., 2007). For CD86, however, a significant difference between wild type and lpg2−KO parasite infections was observed.

Integrin-mediated adhesion and detachment are important features in cell migration. It has thus been observed that lipopolysaccharide can stimulate immature DC to adhere rapidly, develop polarity and assemble actin-rich filopodia or podosomes at the leading edge of the cells (Burns et al., 2004). Podosome assembly was associated with β2-integrins expression and the recruitment of CD11b to podosomes and required collagen I. This suggests a specific role for CD11b/CD18 and CD11c/CD18 in DC adhesion and motility (Burns et al., 2004). In the present study, immature MoDC were seeded onto glass cover slips and the expression of CD11b and CD51 on individual cells incubated for 24 h with TNF-α, WT or lpg2−KO promastigotes was investigated. The results revealed an increased exposure of CD11b and CD51 on the surface of MoDC post interaction with WT promastigotes (P<0.001) (Fig. 7).

In conclusion, this study shows that WT L. donovani interferes with MoDC maturation and increases the expression of the integrins CD11b and CD51, thereby affecting cell detachment to distinct surfaces. In the future direction of this research the authors would like to discuss several very topical questions concerning for instance the cytokine and chemokine signalling between DC and other cells promoting an inflammatory response, and how that is modified by distinct Leishmania parasites. The specific role of LPG could be tested using a lpg2−KO add-back L. donovani mutant, which in the interaction with J774 macrophages restored a wild-type phenotype recording F-actin accumulation around the phagosome (Holm et al., 2001). Finally, the dynamics of F-actin remodelling in live cells during phagocytosis is interesting to elucidate further, which could be addressed by live confocal imaging and fluorescence photobleaching recovery of fluorescent G-actin incorporated into growing or disassembling F-actin filaments. This could elucidate why L. donovani promastigotes can survive and multiply within phagocytic cells.

Acknowledgements

The authors are very grateful to Albert Descoteaux (Université du Québec) for the gift of lpg2−KO plpg2 and WT promastigotes. The authors thank Dr Marie Larsson for stimulating discussions. The project was supported financially by the Lions Foundation, the Faculty of Health Sciences at Linköping University, the Swedish Research Council – Medicine, the Swedish Society for Medical Research, the Swedish Medical Association, Magn. Bergvalls Stiftelse and Stiftelsen Lars Hiertas Minne.

Footnotes

  • Editor: Derek Wakelin

References

View Abstract