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First published online 2 December 2003
doi: 10.1242/jcs.00860

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Dendritic cells as host cells for the promastigote and amastigote stages of Leishmania amazonensis: the role of opsonins in parasite uptake and dendritic cell maturation

¹ Unité d'Immunophysiologie et Parasitisme Intracellulaire, Institut Pasteur, Paris, France ² Centre d'Imagerie Dynamique, Institut Pasteur, Paris, France and ³ Unité de Génétique Mycobactérienne, Institut Pasteur, Paris, France

View larger version (87K): [in a new window]   Fig. 1. Redistribution of MHC class II and H-2M molecules during dendritic cell (DC) maturation. Double-colour confocal laser scanning microscopy analysis of MHC class II and H-2M molecule distributions in (A) immature (i) DCs and (B) BCG-treated DCs. In iDCs (A), intracellular class II (green) and H-2M (red) molecules are mostly co-located within vesicles (yellow). Contact with BCG for 21 hours induces DC maturation, leading to the redistribution of class II molecules to the cell surface and of H-2M-containing organelles to the cell centre (B). For each panel, a three-dimensional representation is shown. Scale bar: 5 µm.

View larger version (23K): [in a new window]   Fig. 2. Infection of dendritic cells (DCs) with different developmental stages of L. amazonensis. Fourteen day-old DCs were co-incubated for 21 hours with amastigotes (Am) from different origins (nude Swiss or BALB/c mice) or with either stationary phase promastigotes (SPm) or metacyclic Pm (MPm) at a 4:1 parasite to DC ratio. MPm were purified by centrifugation on Ficoll gradients (MPm Ficoll) or by negative selection using the mAb 3A1 (MPm 3A1). Before contact with DCs, some Am isolated from nude mice and MPm Ficoll were opsonized with Abs by incubation with an Am- or a Pm-specific immune serum (IS), respectively. After staining of the fixed and permeabilised cells with the Am-specific mAb 2A3-26 and an appropriate fluorochrome conjugate, the percentages of infected DCs were determined either by (A) fluorescence microscopy (500 cells counted) or by (B) flow cytometry (25,000 cells counted). (A) Results are expressed as the means + 1 s.d. of two or three different experiments. (B) Representative dot plot of an overall Leishmania-infected DC population after overnight exposure to MPm Ficoll. Almost all cells express the CD11c molecule (y-axis) and the infected cells express the epitope recognised by the 2A3-26 mAb (x-axis).

View larger version (15K): [in a new window]   Fig. 3. Determination of the number of parasites present in dendritic cells (DCs) infected with various Leishmania developmental stages opsonized or not with specific Abs. DCs were incubated with either amastigotes from nude mice (Am nude) covered or not with Abs, or stationary phase promastigotes (SPm) or with metacyclic promastigotes (MPm Ficoll) previously coated or not with Abs. Twenty-one hours (white bars) or 69 hours (grey bars) after adding parasites, DCs were recovered, fixed, permeabilised and stained as in Fig. 2. Intracellular parasites were detected by fluorescence microscopy. For each determination, the number of parasites present in at least 100 infected DCs were counted.

View larger version (81K): [in a new window]   Fig. 4. Characterisation of parasitophorous vacuoles (PVs) by immunofluorescence confocal microscopy. After 21 to 26 hours of infection with either stationary phase promastigotes (SPm; A-C) or metacyclic promastigotes (MPm Ficoll; D-I or MPm 3A1; J-L), dendritic cells (DCs) were fixed, permeabilised and double-stained with primary Abs as indicated below and appropriate red and green fluorochrome conjugates. Each horizontal row successively represents the red and green images of an infected DC 0.2 µm section, followed by the superimposition of these two images. A yellow colour indicates a co-localisation or a very tight association of the molecules concerned. (A-C) To detect acidic compartments, cells were incubated with DAMP before fixation. Cells were then double-stained with the amastigote-specific 2A3-26 mAb (A) and rabbit anti-dinitrophenol Abs (B). (D-F) Double staining of parasites using the mAb 2A3-26 (D) and lamp-2 molecules (E). (G-I and J-L) Double staining of H-2M (G, J) and MHC class II (H, K) molecules. In H and J, the PV membrane surrounding intracellular parasites is indicated by arrows. In G, H and I, H-2M (G) and MHC class II (H) molecules within parasites are shown by arrowheads. n, DC nucleus. Scale bars: 5 µm.

View larger version (28K): [in a new window]   Fig. 5. Percentages of PVs able to accumulate the weak base DAMP or containing various host cell molecules from the endocytic route in Leishmania-infected DCs. Twenty-one to 26 hours after infection with amastigotes from nude mice (Am nude; white bars) or metacyclic promastigotes (MPm Ficoll; grey bars), DCs were fixed, permeabilised and incubated with immunological reagents before analysis by fluorescence microscopy. Association of the following molecules with parasite-containing parasitophorous vacuoles (PVs) was examined: lamp-1, lamp-2, rab7p, MHC Class II and H-2M molecules. To determine the association of 3-(2,4-dinitroanilino)-3' amino-N-methyldipropylamine (DAMP) with PVs, infected DCs were processed as described in the legend of Fig. 4. About 100-200 organelles were counted for each molecule examined. Results are representative of two or three experiments.

View larger version (33K): [in a new window]   Fig. 6. Expression of co-stimulatory and adhesion molecules at the surface of dendritic cells (DCs) incubated or not with BCG or Leishmania parasites. Fourteen days after the initiation of the culture, DCs were incubated for 21 hours or not with various developmental stages of Leishmania or with BCG and then stained with Abs directed against DC molecules before fixation and flow cytometry analysis. Data obtained with iDCs not incubated with either micro-organism are indicated by black lines and those obtained with BCG-stimulated DCs (positive controls) are shown as blue lines. DCs incubated with amastigotes from nude mice (Am nude; upper panels) or metacyclic promastigotes (MPm Ficoll; lower panels) are represented by red lines for parasites devoid of bound Abs and green lines for Ab-opsonized parasites. All histograms were based on 25,000 ungated events. Relevant isotype controls were included in all experiments (not shown). These results are representative of three or four experiments.

View larger version (43K): [in a new window]   Fig. 7. Kinetics of CD86 cell surface expression in the uninfected and Leishmania-infected dendritic cells (DCs) present in the same cell populations. On day 14, DCs were cultured for an additional 21 hours (upper panels) or 69 hours (lower panels) (i) in the absence of parasites (no parasites) or with (ii) BCG or (iii) parasites devoid of Abs (Am nude, MPm Ficoll), or (iv) Ab-coated parasites (Am nude + IS, MPm Ficoll + IS). DCs were double labelled with a CD86-specific mAb (FL2) and the 2A3-26 mAb (FL1). In each dot plot, the cells expressing high levels of CD86 molecules were subdivided into FL1 low (left region corresponding to uninfected DCs) and FL1 high (right region corresponding to infected DCs) DCs. The values indicated in the left and right regions are the percentages of CD86high DCs related to uninfected and Leishmania-containing DC sub-populations, respectively. The results shown are representative of three similar experiments.

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