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Originally published online as doi:10.1189/jlb.1202608 on May 22, 2003

Published online before print May 22, 2003
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(Journal of Leukocyte Biology. 2003;74:206-215.)
© 2003 by Society for Leukocyte Biology

Mannose receptor contribution to Candida albicans phagocytosis by murine E-clone J774 macrophages

Isabelle Porcaro*, Michel Vidal{dagger}, Sylvie Jouvert*, Philip D. Stahl{ddagger} and Jean Giaimis*

* Laboratoire d’Immunologie et de Parasitologie EA 2413, Université Montpellier I, France;
{dagger} UMR CNRS 5539, Université Montpellier II, France; and
{ddagger} Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri

Correspondence: Jean Giaimis, Faculté de Pharmacie, 15 av. Charles Flahault, BP 14 491, 34 093 Montpellier Cedex 5, France. E-mail: jgiaimis{at}iup.pharma.univ-montp1.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mannoproteins, as the main constituents of the outer layer of yeast cell walls, are able to interact with phagocytic cells in an opsonin-independent manner through the mannose receptor (MR) and to induce yeast ingestion by the professional phagocytes. Moreover, the MR also mediates endocytosis of soluble ligands through clathrin-coated pits. Here, we studied some aspects of the interaction between the MR and Candida albicans using murine E-clone macrophages and the consequences on MR trafficking. Using a pull-down assay involving mixture E-clone macrophage detergent lysate with mannosylated Sepharose beads and glutaraldehyde-fixed, heat-killed (HK) C. albicans, we found that binding of solubilized MR to mannosylated particles occurred with characteristics similar to the receptor’s cell-surface mannose-binding activity. We then demonstrated that MR expressed on E-clone macrophages contributed to phagocytosis of unopsonized, HK C. albicans and that yeast phagocytosis induced a decrease in MR endocytic activity without concomitant degradation of the receptor in the time lapse studied.

Key Words: receptor-mediated endocytosis • ß-glucan receptors • mannan


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mannose receptor (MR) was originally discovered as a macrophage membrane lectin-like receptor involved in receptor-mediated endocytosis [1 2 3 ]. Recent studies indicate that MR is expressed on most tissue macrophages [1 ], in vitro-derived dendritic cells (DC) [4 , 5 ], retinal pigment epithelium [6 ], selected lymphatic or hepatic endothelial cells, and mesangial cells [7 , 8 ]. Mature MR is a 180-kDa transmembrane receptor, displaying N- and O-linked oligosaccharides in its extracellular domain [9 , 10 ]. MR cloning revealed a type I transmembrane protein, presenting five different domains: an amino-terminal extracellular cysteine-rich domain, a fibronectin type II domain, a succession of eight lectin-like carbohydrate recognition domains (CRDs), a single transmembrane domain, and a short, carboxy-terminal cytoplasmic tail [11 12 13 ].

Recent studies have revealed at least two distinct binding domains for this receptor. One of these domains allows recognition of sulfated carbohydrates such as the Gal-NAC-4-SO4 motif, binds ligands in a calcium-independent manner, and is localized within the cysteine-rich domain [14 15 16 ]. Another calcium-dependent binding domain displays specificity for glycoconjugates expressing mannose/fucose residues and has been localized within CRDs, especially CRDs 4, 5, and 7, which are necessary for the binding of multivalent ligands [17 , 18 ]. For this later binding site, many ligands have been described and include horseradish peroxidase (HRP) [19 20 21 ], neutrophil-derived myeloperoxidase [22 ], and most lysosomal hydrolases [2 ].

The presence of MR in macrophages indicates that in steady-state conditions, 10–20% of the receptors are found on the cell surface and 80–90%, intracellularly [23 , 24 ]. A characteristic MR feature is the constitutive ability to be rapidly internalized from the plasma membrane via clathrin-coated vesicles for delivery into the endosomal system. An examination of the cytoplasmic domain of the MR revealed that it contains two potential endocytosis motifs: a motif based on a conserved tyrosine residue and another one based on a dihydrophobic motif [25 ]. The MR is also found on an intracellular compartment with characteristics of early endosomes [26 , 27 ]. From this endosomal compartment, the MR rapidly recycles back to the cell surface. Kinetic studies have shown that the MR cycles approximately three times an hour [23 ].

In addition to endocytosis, the MR mediates nonopsonic phagocytosis by macrophages from a wide variety of microbes, including yeast, protozoa, and bacteria, and is therefore considered to be an important molecule of innate immunity [3 , 28 ]. MR mediation of microorganism uptake has been indicated in numerous studies that demonstrate the ability of soluble ligands such as yeast mannan, well-known MR ligands [19 ], to reduce the macrophage phagocytic activity of unopsonized microorganisms [29 30 31 32 33 34 35 36 ]. Additional evidence of MR involvement in a phagocytic process was obtained after transfection of normally nonphagocytic COS cells with the MR cDNA. After transfection, COS cells express MR and acquire the ability to bind and internalize microorganisms such as Pneumocystis carinii [34 ]. Conversely, expression of a tailless mutant MR allows yeast adhesion but not ingestion [12 ].

In comparison to the well-described recycling pathway of the MR after endocytosis, less is known about the intracellular trafficking of this receptor after a phagocytic process. Previous studies have demonstrated that MR activity was decreased when macrophages were infected for 24–48 h with microorganisms such as Leishmania donovani [37 ] or Candida albicans [38 ], microorganisms for which MR involvement during the ingestion process has been documented [32 , 39 ]. These studies have shown that MR down-regulation may be imputable to an increase of the receptor degradation [36 , 37 ]. Conversely, it was found that polystyrene microspheres covered with lipoarabinomannan (LAM), a lipoglycan-containing terminal mannose oligosaccharides, were phagocytosed by macrophages through MR without interfering with its recycling [40 ]. Similarly, using antibody-coated Staphylococcus aureus as a phagocytic probe, the results of another study suggested that MR could be rapidly recycled from the phagosome membrane [41 ].

To investigate the behavior of the MR implicated in a phagocytic process, we analyzed its ability to mediate an endocytosis process after having or not been previously involved in phagocytosis. For these studies, we used J774 E-clone macrophages that express the MR [42 ] and C. albicans, a yeast particle that when unopsonized, has been reported to be phagocytosed by macrophages through the MR [12 , 32 , 43 44 45 ]. Our results demonstrate that the MR of E-clone macrophages specifically binds unopsonized heat-killed (HK) C. albicans and that phagocytosis of this yeast particle is partially mediated by the MR. We show that after uptake of unopsonized HK C. albicans, the ability of the E-clone to perform MR-mediated endocytosis is partially reduced, apparently without degradation of this receptor. Different possible explanations for these results are discussed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
The J774 E-clone macrophage cell line used throughout this study was selected on the basis of its MR expression [42 ]. These macrophages were grown in 75 cm2 plastic flasks [Techno Plastic Products AG (TPP), Switzerland] in RPMI-1640 medium (Eurobio, Les Ulis, France) containing 7.5% heat-inactivated fetal calf serum, glutamine (2 mM), penicillin (100 UI/ml), and streptomycin (20 µg/ml; all from Gibco, Cergy Pontoise, France). The cells were incubated at 37°C in an air (95%)-CO2 (5%) humidified incubator. Cells were removed from the flasks by gentle pipetting. We usually recovered 5–10 x 105 cells/ml with viability exceeding 98%, as determined by trypan blue exclusion. The recovered cells adhered to a support and spread very rapidly. E-clone macrophages for phagocytic experiments were adjusted to 1 x 105 cells/ml with culture medium and were allowed to adhere (1x105 cells/well) to 14-mm diameter sterile glass coverslips (Poly Labo, Strasbourg, France) placed in 24-well plates (TPP). E-clone macrophages for fluid phase or MR-mediated endocytosis experiments were adjusted to 2 x 105 cells/ml with culture medium and were placed in 24-well plates (4x105 cells/well). E-clone macrophages for transferrin receptor (TfR)-mediated endocytosis were adjusted to 2 x 105 cells/ml with culture medium and were placed in 12-well plates at 4 x 105 cells/well. For phagosome experiments, E-clone macrophages were adjusted to 2 x 106 cells/ml with culture medium and distributed in cell-culture dishes at 5 x 107 cells/dish. All cells were used in the experiments no more than 24 h after plating.

Yeast
Stock cultures of C. albicans (IVP 3153) were maintained on Sabouraud’s dextrose agar (Biomérieux, Craponne, France) at 4°C. Stationary growth-phase yeast was prepared by placing stock cultures in Sabouraud’s dextrose broth and culturing for 48 h at 28°C under agitation. C. albicans was recovered and washed three times in calcium- and magnesium-free phosphate-buffered saline (PBS-, Eurobio). HK yeast was prepared by autoclaving (30 min, 120°C) in PBS-. The resulting HK yeast was washed three times with PBS-, and tested for viability by counting colonies after incubation for 24–48 h at 37°C on Sabouraud’s dextrose agar. Over 99% of the HK yeast had a blastospore morphology. HK yeast suspensions were stored at 4°C in RPMI medium without serum (4x109 yeast/ml). HK yeast was washed twice with PBS- and diluted in RPMI immediately before use.

Phagocytosis
E-clone macrophages were washed once with serum-free RPMI medium and then incubated with 350 µl of this medium, with or without laminarin [a soluble ß-glucan prepared from Laminarin digitata (Sigma Chemical Co., St. Louis, MO)] and/or {alpha}-mannans prepared from Saccharomyces cerevisiae (Sigma Chemical Co.) or C. albicans {provided by BioRad Laboratory (Hercules, CA) and prepared as described by Fradin et al. and Kocourek and Ballou [45 46 47 ]}. The HK C. albicans suspension was then added to the macrophages (50 µl/well of 2x108 yeast/ml) to give a HK C. albicans:macrophage ratio of ~100:1. Phagocytosis was allowed to proceed for 1 h at 37°C without stirring in an air-CO2 humidified incubator. Unassociated macrophage HK yeast was removed by washing twice with warm RPMI-1640 medium. Ingested and adherent yeast was identified using an isotonic tannic acid solution [48 ]. Briefly, the cells were incubated with 1% (w/v) tannic acid solution in RPMI-1640 medium for 1 min, washed twice with RPMI, and dried. The preparations were stained with May-Grünwald-Giemsa and mounted cell-side down on glass slides. Preincubation with mannans and laminarin or their presence during phagocytosis did not disturb cell adherence to coverslips (data not shown). Each experiment was performed in triplicate, and 100 macrophages were scored for each coverslip under a light microscope (magnification, x1000). Data are expressed as the phagocytosis index (mean number of ingested HK C. albicans per cell multiplied by the percentage of cells having ingested at least one HK C. albicans). Standard deviations were 5–15%. Each type of experiment was repeated at least three times.

Measurement of fluid-phase and MR-mediated endocytosis
Fluid-phase endocytosis and MR activity were measured by the uptake of HRP (Sigma Chemical Co.). E-clone macrophages in 24-well plates were washed once with RPMI-0.5% bovine serum albumin (BSA) and were incubated for 1 h in the same medium containing HRP at 37°C. Yeast mannan (from S. cerevisiae, 1.25 mg/ml) were added to companion wells to measure nonspecific MR-mediated endocytosis. The cells were then washed four times with PBS-EGTA (2 mM) and lysed in PBS- Triton X-100 (0.1%). The amount of HRP endocytosed by E-clone macrophages was measured as described [49 ] using O-phenylenediamine-1.2-benzenediamine-dihydrochloride (Sigma Chemical Co.) as substrate. A standard curve of known HRP concentrations was used to determine the amounts of peroxidase internalized.

Measurement of TfR-mediated endocytosis
Confluent monolayers of E-clone macrophages were washed twice with serum-free RPMI medium containing 0.2% BSA and were incubated for 1 h at 37°C to deplete endogenous transferrin. Iron-loaded human transferrin (Sigma Chemical Co.) was labeled with 125I by the Iodo-Gen method [50 ]. E-clone macrophages were incubated with 125I-transferrin (20 µg/ml) in serum-free RPMI medium containing 0.2% BSA for 2 or 8 min at 37°C. Nonspecific uptake was determined using excess, unlabeled transferrin (1 mg/ml). Endocytosis was stopped by placing the plates on ice. Excess 125I-transferrin was removed by washing the wells three times with buffered saline (150 mM NaCl, 1 mM CaCl2, 5 mM KCl, 1 mM MgCl2, 20 mM HEPES, pH 7.4). The cells were solubilized in 0.1 N NaOH containing 0.1% (v/v) Triton X-100, and the amount of radioactivity in the lysate was determined in a {gamma}-counter (Packard).

Pull-down assays and Western blotting
Using mannosylated-Sepharose (Man-Sepharose)
E-clone macrophages were washed twice with PBS supplemented with Ca++ and Mg++ (PBS+) and were lysed in PBS+ containing protease inhibitors (Sigma Chemical Co.) and 1% Triton X-100 (107 macrophages per 1 ml lysis solution, ~1 mg protein/ml). Lysates (250 µl) were incubated overnight at 4°C with 50 µl Sepharose or Man-Sepharose, prepared according to ref. [51 ]. In some experiments, the incubation mixture contained {alpha}-mannans (1.25 mg/ml), laminarin (1.25 mg/ml), or EGTA (5 mM). Supernatant and Sepharose were separated by centrifugation, and the recovered Sepharose was washed twice with 1 ml PBS+ and 0.1% Triton X-100. Bound proteins were eluted from Sepharose beads by mixing with 30 µl sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer [52 ].

Using HK C. albicans
HK yeast particles were dispersed in PBS- (2x109/ml) and fixed overnight with glutaraldehyde (1%). The yeast was washed thoroughly and incubated twice in a 100 mM glycine solution, washed with PBS+, and diluted to 2 x 109/ml in PBS+. Some fixed HK yeast was treated with Concanavalin A (Con A; Sigma Chemical Co.) [53 ]. E-clone macrophage lysate (250 µl) was incubated overnight with 108 yeast particles and was washed, and the protein bound to the HK yeast was subsequently eluted with 30 µl sample buffer.

Eluted proteins were run on 8% SDS-PAGE, and separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Schleicher & Schuell, Dassel, Germany). The membrane was blocked by incubation in 5% (w/v) nonfat milk in Tris-buffered saline and 0.1% Tween 20 for 1 h and was probed with polyclonal rabbit anti-murine MR antibody [54 ]. Binding was detected with peroxidase-coupled anti-rabbit immunoglobulin G (Dako S. A., Trappes, France) and chemiluminescence (Amersham Pharmacia Biotech, Orsay, France).

Phagosome isolation using latex beads
Preparation of latex beads conjugated with mannosylated BSA
Mannosylated BSA (EY Laboratories, San Mateo, CA) was covalently coupled to carboxylated latex beads (Polysciences, Warrington, PA) using water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC). Briefly, carboxylated latex beads (1 ml, 2.5% solids) were washed with 2-morpholinoethane sulfonic acid (50 mM, pH 6) buffer and incubated with 600 µg mannosylated BSA in the presence of 10 mg EDAC for 15 h at room temperature. The reaction was stopped by centrifugation, and the particles were thoroughly washed and resuspended in 1% PBS-BSA.

Phagosome preparation
Beads conjugated with mannosylated BSA (~109 beads) were incubated with cells (bead:cell ratio, approximately 20:1) for 1 h at 4°C. Plates were then quickly warmed and incubated at 37°C for a 15-min pulse. The cells were then washed three times at 4°C and chased for 15 or 60 min at 37°C in culture medium in the presence of 25 mM NH4Cl when indicated. After chase, plates were washed once with cold PBS-EDTA (5 mM), and cells were scraped off with a rubber policeman and resuspended in homogenization buffer (250 mM sucrose, 3 mM imidazole, 5 mM EGTA, pH 7.4) containing protease inhibitors (2 µg/ml aprotinin, 2 µg/ml antipain, and 1 mM phenylmethylsulfonyl fluoride). The cells were then homogenized on ice using a Dounce homogenizer. Unbroken cells were pelleted, and the supernatants were used to isolate phagosomes by flotation on sucrose gradient, as described by Desjardins et al. [55 ]. Latex beads collected at the 10–25% sucrose interface were resuspended in ice-cold homogenization buffer and pelleted (15 min, 40,000 g). Identical protein amounts of phagosomes were loaded on SDS-PAGE and analyzed for the presence of MR and Lamp-1 [1D4B monoclonal antibody (mAb), Southern Biotechnology Associates, Birmingham, AL] by Western blot.

Phagosome isolation using HK yeast particles
Preparation of yeast particles conjugated with magnetic beads
HK S. cerevisiae were biotinylated by incubating approximately 2 x 107 particles in PBS with 20 mg sulfo-NHS-biotin (Pierce, Rockford, IL) for 1 h at room temperature with continuous stirring. After thoroughly washing by centrifugation, biotinylated yeast particles were incubated with streptavidin superparamagnetic microbeads (50 nm diameter; magnetic cell sorter, Miltenyi Biotec, Paris, France) for 30 min at room temperature. About 80% of yeast particles were retained on a mass spectroscopy (MS) separation column placed in the magnetic field of a MiniMACS separator (Miltenyi Biotec) when an aliquot of the preparation was analyzed to control its magnetic-binding capacity.

Phagosome isolation
Magnetic yeast particles were incubated with 2 x 106 E-clone macrophages (bead:cell ratio, approximately 3:1) for 2 h at 37°C. Cells were then washed twice with ice-cold PBS and once with homogenization buffer, scraped off with a rubber policeman, and homogenized in 2 ml using a cell cracker. The homogenate was then loaded on a MS separation column placed on its MiniMACS magnetic support (Miltenyi Biotec), and the flow-through was collected. The column was washed three times with 1 ml PBS before removing the magnetic support, and the microbead-labeled subcellular fraction was eluted from the column using SDS-PAGE loading buffer. The proteins of the collected fractions were separated by SDS-PAGE and transferred on a PVDF membrane to analyze the presence of MR (see above) and flotillin-1 (mAb clone 18, BD Biosciences, Pont de Claix, France) by Western blot.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Binding of MR solubilized from E-clone macrophages to Man-Sepharose and HK C. albicans
MR expression appears to be confined to primary macrophages, but a few cell lines, such as E-clone J774 macrophages, express this protein [42 ]. After E-clone macrophage solubilization using 1% Triton X-100, a band was detected on Western blots (Fig. 1A , lane 1) at the molecular weight (MW) expected for MR [10 , 11 ]. A pull-down assay was developed in which E-clone macrophage lysate was mixed with Sepharose beads (mannosylated or not) and HK C. albicans particles to characterize MR binding to ligands. The amounts of MR in the resulting pellet (bound fraction) and in the first supernatant (unbound fraction) were analyzed by Western blot. Man-Sepharose bound the MR, but the crude beads did not (Fig. 1A) .



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  		Figure 1. Solubilized MR binds to Man-Sepharose. (A) E-clone macrophages (106) were lysed using 1% Triton X-100 and incubated overnight at 4°C with Sepharose beads derived with mannose (lanes 4 and 5) or unconjugated resin (lanes 2 and 3), as described in Materials and Methods. After pelleting and washing the beads, Sepharose-bound (lanes 3 and 5) and unbound (lanes 2 and 4) proteins were run on 8% SDS-PAGE and transferred on to PVDF membrane, and MR was detected by Western blotting. Crude cell lysate (20 µl) was loaded on the gel (lane 1). (B) MR was detected in the protein fraction bound to Man-Sepharose, as described in A. Cell lysate incubation with beads (lane 1) was performed in the presence of S. cerevisiae {alpha}-mannan (lane 2), laminarin (lane 3), or EGTA (lane 4), as described in Materials and Methods.

 
We assessed the specificity of this binding using {alpha}-mannan, a soluble MR ligand that binds to CRDs of MR (especially CRDs 4, 5, and 7; ref. [18 ]). S. cerevisiae {alpha}-mannan (1.25 mg/ml) significantly reduced MR binding to Man-Sepharose (Fig. 1B , lane 2), indicating that CRDs are involved in this binding. The interaction between {alpha}-mannan and CRDs is Ca2+-dependent and blocked by Ca2+ chelators [23 ]. As shown in Figure 1B , lane 4, EGTA completely abolished the binding of solubilized MR to Man-Sepharose. These results demonstrate that solubilized MR binds specifically to Man-Sepharose via the receptor CRDs. The soluble ß-glucan (laminarin, 1.25 mg/ml) did not affect the binding of MR to Man-Sepharose (Fig. 1B , lane 3), indicating that this polyoside interacts poorly with the MR (see below).

Glutaraldehyde-treated HK C. albicans particles also bound MR (Fig. 2 ). The binding of solubilized MR to HK C. albicans was completely blocked by EGTA and partially affected by S. cerevisiae {alpha}-mannan (data not shown). Con A, a soluble lectin that recognizes terminal mannose residues [53 ], was preincubated with glutaraldehyde-fixed C. albicans to specifically mask the MR ligands. As shown in Figure 2 , lane 5, solubilized MR did not bind to Con A-treated HK C. albicans. The overall results of these experiments with HK C. albicans indicate that binding with MR involves the recognition of mannosylated patterns by the receptor CRDs.



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  		Figure 2. Solubilized MR binds to HK C. albicans. MR was detected in the protein fraction bound with the particle pellets, i.e., Man-Sepharose beads (lane 2), unconjugated resin (lane 3), or HK gluteraldehyde C. albicans (lane 4), which was opsonized with Con A (lane 5). Crude cell lysate (20 µl) was loaded on the gel (lane 1).

 
These results demonstrate that solubilized MR from E-clone macrophages interacts with cell-wall components of C. albicans with mannose-binding activity typical of these receptors. However, only a fraction of the solubilized MRs obtained from these macrophages bound to particulate ligands. Unbound MR remained in the supernatant after incubation of E-clone macrophage lysate with Man-Sepharose (Fig. 1A , lane 4) or with HK C. albicans (data not shown). Moreover, incubation of constant volumes of macrophage lysate with increasing amounts of Man-Sepharose led to comparable amounts of unbound, solubilized MR (data not shown). This suggests the presence of a MR pool with low-binding capacity to mannose residues.

Endocytosis mediated by MR on E-clone macrophages
MR endocytosis on the E-clone macrophage surface was measured by the uptake of HRP, a highly mannosylated enzyme (Fig. 3 ). As HRP can be taken up by a MR-mediated process and fluid-phase endocytosis, we analyzed the residual ability of E-clone macrophages to endocytose HRP in the presence of excess {alpha}-mannan. Under these latter conditions, HRP endocytosis was almost totally abrogated when the HRP concentration was below 50 µg/ml. Using 300 µg/ml HRP, the amount endocytosed by E-clone macrophages in the absence of {alpha}-mannan (reflecting MR and fluid-phase endocytosis) was almost twice that endocytosed in the presence of {alpha}-mannan, when only fluid-phase endocytosis occurred (Fig. 3) . As described in other studies (reviewed in ref. [2 ]), endocytosis was mainly MR-mediated when the HRP concentration did not exceed 100 µg/ml. MR-mediated endocytosis by E-clone macrophages led to the accumulation of ~1.2 x 106 HRP molecules/cell-1 h-1. As E-clone macrophages express ~4 x 105 MR/cell [54 ], this amount of accumulated HRP indicates that the MR of E-clone macrophages is rapidly recycled after endocytosis. Each receptor cycles every 15–20 min, as in alveolar macrophages [23 ].



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  		Figure 3. HRP endocytosis by E-clone macrophages. Macrophages were incubated with the indicated HRP concentrations in the absence (•) or presence ({circ}) of S. cerevisiae {alpha}-mannans, as described in Materials and Methods. The difference between the two curves ({triangleup}) corresponds to HRP internalized through MR. The values are means of determinations of triplicate cultures. Vertical bars indicate standard deviations for each value. The results shown are representative of three independent experiments.

 
Role of E-clone macrophage MR in the phagocytosis of unopsonized HK C. albicans
E-clone macrophages can phagocytose unopsonized HK C. albicans. Adherent and intracellular-ingested HK C. albicans were differentiated: Those adhering to the cell surface were stained purple, and the ingested yeast cells were pink (Fig. 4 ). Phagocytosis for 1 h with a yeast:cell ratio of 100:1 prompted almost all E-clone macrophages to ingest at least one HK C. albicans. Moreover, under these experimental conditions, more than 75% of cell-associated HK C. albicans were ingested (data not shown).



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  		Figure 4. Phagocytosis of C. albicans by E-clone macrophages. After phagocytosis was allowed to proceed as described in Materials and Methods, cells were treated with tannic acid (A) or not (B) before May-Grünwald-Giemsa staining. Note that tannic acid treatments allowed detection of surface-bound yeast (stained purple), contrary to internalized yeast (stained pink). Original bars, 4 µm.

 
Mannan are the main constituents of C. albicans cell walls [56 ]. {alpha}-Mannan from S. cerevisiae or C. albicans inhibited HK C. albicans phagocytosis by E-clone macrophages in a concentration-dependent manner (Fig. 5 ). The MRs are thus involved in HK C. albicans phagocytosis by E-clone macrophages, as shown with DC [44 ] and bone marrow macrophages [38 ]. C. albicans and S. cerevisiae mannans (1 mg/ml) strongly inhibited phagocytosis. C. albicans mannans were more effective than those of S. cerevisiae. Indeed, C. albicans mannan (5 µg/ml) inhibited HK C. albicans ingestion by 50%. The same concentration of S. cerevisiae mannan was about tenfold less effective than C. albicans mannan.



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  		Figure 5. Concentration curve for mannan inhibition of C. albicans phagocytosis by E-clone macrophages, which were incubated with HK C. albicans in the presence of increasing concentrations of mannans from S. cerevisiae (•), from C. albicans ({circ}), or from S. cerevisiae plus laminarin (50 µg/ml; {blacksquare}). The data are expressed as a phagocytosis index (as defined in Materials and Methods) and are mean determinations of triplicate cultures. Vertical bars indicate standard deviations for each value. The results shown are representative of five independent experiments.

 
Other lectin-like receptors, such as ß-glucan receptors (BGRs), have been described as being involved in the phagocytosis of unopsonized yeast [45 , 57 ]. ß-Glucans are also major constituents of the C. albicans cell wall and are exposed at the yeast surface [58 ]. We therefore assessed the contribution of BGRs in HK C. albicans phagocytosis of E-clone macrophages by comparing the phagocytic activities in the presence of {alpha}-mannan and a soluble ß-glucan (laminarin). {alpha}-Mannan (50 µg/ml) partially reduced HK C. albicans phagocytosis, and {alpha}-mannan plus laminarin (50 µg/ml each) led to almost complete inhibition (Fig. 5) .

Decreased E-clone macrophage MR endocytic activity after phagocytosis of HK C. albicans
We compared the MR-mediated endocytic activities of E-clone macrophages before and after they had ingested HK C. albicans to analyze the behavior of MR in phagocytosis. HRP (50 µg/ml) was used to measure MR-mediated endocytosis. After 1 h of C. albicans phagocytosis, the cells were washed to remove nonadhering HK C. albicans and were chased for 1 h to ensure the ingestion of adhering yeast cells. As shown in Figure 6 , E-clone macrophages that had ingested HK C. albicans were less able to sustain MR-mediated endocytosis. This reduced activity was correlated with the number of yeast cells offered to the macrophages. The greatest reduction in MR endocytosis was obtained when the HK yeast:macrophage ratio was close to 100:1. This incubation reduced the capacity of E-clone macrophages to carry out MR-mediated endocytosis of HRP by 50% (Fig. 6) . Conversely, similar amounts of HRP were recovered when the MR was initially involved in endocytosis followed by phagocytosis (data not shown), thus ruling out a nonspecific effect of yeast phagocytosis on HRP detection. Moreover, we obtained similar results using the same procedure with human monocyte-derived macrophages (data not shown), for which MR involvement during unopsonized C. albicans phagocytosis has been described [32 ].



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  		Figure 6. HRP endocytosis by E-clone macrophages before and after C. albicans phagocytosis. Macrophages (5x105cells) were incubated with C. albicans at the indicated yeast:cell ratio for 1 h at 37°C, washed, and chased for 1 h at 37°C to allow the ingestion of adherent yeasts. HRP (50 µg/ml) uptake was then assessed as described in Materials and Methods in the presence (•) or absence ({square}) of S. cerevisiae {alpha}-mannan (1.25 mg/ml). The difference between the two curves ({circ}) corresponds to HRP internalized through MR. The values are mean determinations of triplicate cultures. Vertical bars indicate standard deviations for each value. The results shown are representative of three independent experiments.

 
The presence of two different pathways for the same receptor, depending of whether it was involved in the phagocytosis or endocytosis process, was previously characterized for Fc{gamma} receptors (Fc{gamma}R). Mellman et al. [59 ] demonstrated that the ligand-Fc{gamma}R complex is internalized and recycled intact back to the cell surface after monovalent ligand endocytosis. Conversely, after receptor-mediated phagocytosis, Fc{gamma}R are not recycled back to the cell surface but stay in the phagolysosomal compartment where they are degraded. We attempted to determine if the decrease in MR-mediated endocytosis after C. albicans phagocytosis was linked to MR degradation. E-clone macrophages that had and had not phagocytosed HK C. albicans were thus lysed, and the amounts of MR they contained were compared by Western blot. As shown in Figure 7 , we did not note any significant difference in the amounts of MR in our experimental conditions.



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  		Figure 7. C. albicans phagocytosis by E-clone macrophages does not induce MR degradation. MRs were submitted (lanes 2 and 4) or not (lanes 1 and 3) to yeast phagocytosis at a yeast:cell ratio of 30:1, with (lanes 3 and 4) or without (lanes 1 and 2) 1 h pretreatment with cycloheximide (10 µg/ml). Macrophages were then lysed using 1% Triton X-100, and equal cell lysate volumes were analyzed by SDS-PAGE and Western blot for the presence of MR, as described in Materials and Methods.

 
The MR, like other receptors, e.g., the TfR, is endocytosed through clathrin-coated pits and recycled back to the plasma membrane [60 ]. We thus assessed TfR endocytosis in E-clone macrophages after yeast phagocytosis. No difference was found in transferrin uptake by macrophages that had and had not phagocytosed yeast at the highest yeast:macrophage ratio (100:1; Fig. 8 ), indicating that this phenomenon is specific to the MR and is not a result of impairment of the entire endocytic process.



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  		Figure 8. Transferrin uptake by the E-clone cells before and after phagocytosis of C. albicans. Macrophages were incubated with 125I-transferrin for 2 or 8 min, before (open bars) or after (solid bars) phagocytosis of C. albicans. The results represent means ± SD of triplicate cultures. The results are representative of three independent experiments.

 
To further characterize MR traffic after phagocytosis, we prepared phagosomes using the procedure described by Desjardins et al. [55 ] using mannosylated BSA coupled to latex beads, as described in Materials and Methods. As presented in Figure 9A , MR was detected in phagosomes from cells having internalized beads for 15 min without chase (lane 1). Significant amounts of MR remained associated with the phagosomal compartment after a 15 min pulse with 15 or 60 min chase (Fig. 9A , lanes 2 and 3). Concomitantly, Lamp-1 acquisition (Fig. 9B) was taken as an indication of phagosome maturation, as already reported [41 ]. It is interesting that when the chase was performed in the presence of NH4Cl for impairing phagosome maturation [61 ], as indicated by the absence of Lamp-1 acquisition (Fig. 9B , lane 4), the amount of MR associated with phagosomes was comparable with that detected without chase (Fig. 9A , lanes 1 and 4).



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  		Figure 9. Association of MR with phagosomes containing mannosylated BSA-latex beads, which were pulsed (15 min) for phagocytosis by E-clone macrophages (lanes 1) and chased for 15 min (lanes 2) or 60 min (lanes 3 and 4). The chase was performed in the presence of NH4Cl (lanes 4). Phagosomes were then prepared by sucrose flotation and analyzed by Western blot for the presence of MR (A) and Lamp-1 (B), as described in Materials and Methods. E-clone macrophage lysate was loaded in lanes 5 as a control. MW standards are indicated on the left.

 
To examine the fate of MR after yeast phagocytosis, HK yeast particles coupled to superparamagnetic microbeads were used to prepare phagosomes as described in Materials and Methods. As shown in Figure 10A , we detected the presence of MR in the phagosomal compartment after a 2-h pulse phagocytosis when using this procedure. Note that the isolated compartment displayed mature phagosome characteristics, as indicated by the presence of flotillin-1 (Fig. 10B) , which is known to be acquired by phagosomes during maturation, probably through fusion with late endosomes [62 ].



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  		Figure 10. Isolation of E-clone phagosomes using yeast particles coupled to superparamagnetic microbeads. HK S. cerevisiae particles coupled to paramagnetic microbeads were phagocytosed for 2 h as described in Materials and Methods. The cell lysate was then loaded on a separation column placed in a magnetic field, and the flow-through (lanes 1) and three successive washes (lanes 2–4) were collected. The magnet support was then taken out, and phagosomes containing paramagnetic yeast particles were eluted using SDS-PAGE loading buffer (lanes 5). Collected fractions were loaded on SDS-PAGE, electrotransferred on a PVDF membrane, and analyzed by Coomassie staining (C) and by Western blot for the presence of MR (A) and flotillin-1 (B).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The MR binds and mediates the ingestion of a wide range of microorganisms (bacteria, fungi, and protozoa) expressing the appropriate oligosaccharide ligands. This receptor may be considered as a prototypical cell-surface pattern-recognition receptor. This transmembrane receptor is present on resident macrophages, DC, and on subsets of endothelial cells [7 ]. The MR appears to be confined to primary macrophages, but a few cell lines such as the E-clone macrophage derived from the mouse J774 cell line also bear this protein [44 ]. Like Fc{gamma}R, MR mediates the internalization of particulate ligands by phagocytosis and soluble ones by endocytosis. Many studies have shown that C. albicans phagocytosis by macrophages involves the MR [12 , 29 , 32 , 38 , 43 ]. C. albicans cell walls contain {alpha}-mannans, a complex assembly of {alpha}-1,6- and {alpha}-1,3-linked mannose residues [56 ]. The MR interacts with {alpha}-mannans, resulting in C. albicans binding and ingestion by macrophages [12 , 29 , 32 ]. The MR extracellular region has two domains that recognize different carbohydrates. The amino-terminal CRD interacts with sulfated glycoproteins, and the CRDs bind unsulfated polysaccharide chains such as mannan [15 , 16 ]. The carbohydrate recognition properties of the MRs are somewhat exclusive, as the MR on hepatic endothelial cells can apparently bind sulfated ligands but not mannosylated ligands. Conversely, the MRs on murine alveolar macrophages bind mannosylated ligands but not sulfated ligands [14 ].

We first examined the MR binding capacity of these cells using a pull-down assay and Western blotting. These studies were conducted with E-clone macrophages solubilized with Triton X-100, similar to the procedure described by Baenziger and co-workers [14 , 15 ]. E-clone macrophage lysates were incubated with HK C. albicans. We found that MR binds this particulate ligand but only when the yeast has been fixed in glutaraldehyde, which probably stabilizes mannoglycoproteins on the yeast cell wall; otherwise, they would be labile and readily extracted by Triton X-100 [53 ]. Moreover, we have shown that the binding of solubilized MR to glutaraldehyde-fixed C. albicans can be inhibited or blocked by various conditions such as the presence of EGTA, competition by {alpha}-mannan, and masking of mannosylated motifs by Con A. Overall, these arguments highly suggest an interaction between CRDs of the receptor and mannosylated ligands on the yeast surface, as confirmed by the similar binding characteristics of solubilized, MR with Man-Sepharose particles.

However, we were unable to pellet the entire MR pool by mannosylated particulate ligands using this pull-down assay. There was always free MR, even when an excess amount of Man-Sepharose was used. There are several possible explanations for this. One could be related to our experimental conditions. Macrophage lysis could affect the MR binding capacity because of a conformational problem of the solubilized receptor or by releasing competitive, neosynthesized glycoproteins. Conversely, there may be a pool of MR that is unable to bind mannosylated ligands. It was recently suggested that the binding of MR to sulfated glycoprotein-bearing ligands may involve dimerization of this receptor. Dimerization could prevent mannan from reaching their binding sites within CRDs. In contrast, monomeric forms of the MR allow mannose-bearing ligands to reach CRDs [63 ]. Additional experiments using sulfated glycoprotein-bearing ligands could reveal whether the MR on E-clone macrophages also binds these ligands.

Our results indicate that the solubilized MR of E-clone macrophages (or at least some of them) interacts with mannosylated patterns on the surface of HK C. albicans. We then analyzed this interaction using macrophages and yeast and found that E-clone macrophages bound and phagocytosed HK C. albicans and that this capacity was significantly reduced by {alpha}-mannan. Similarly, {alpha}-mannan has been shown to decrease the phagocytosis of a wide spectrum of unopsonized microorganisms such as Mycobacterium tuberculosis, P. carinii, L. donovani, and recently Borrelia burgdorferi, suggesting that the MR is sufficient for binding and subsequent ingestion of these microorganisms [34 35 36 , 39 ].

{alpha}-Mannan partly blocks the phagocytosis of HK C. albicans, whereas {alpha}-mannan plus the soluble ß-glucan, laminarin, almost completely prevents it. As laminarin is bound by BGRs but poorly by the MR (Fig. 1B) , the BGR may also be involved in HK C. albicans phagocytosis by E-clone macrophages. Recent studies have identified dectin-1 as the main BGR in mouse macrophage but also in macrophage-like cell lines such as J774 [64 ]. Additional studies have reported another ß-glucan binding site on CR3. It is interesting that C. albicans mannoproteins, unlike those of S. cerevisiae, contain ß-1.2-oligomannosides, which bind to the MR and also to CR3 [45 ]. Interaction with these lectin-like receptors may explain why C. albicans mannan interferes to a greater extent with HK C. albicans phagocytosis than does S. cerevisiae mannan. The phagocytosis of unopsonized C. albicans by E-clone macrophages could thus involve the MR and the BGR, as noted with other yeast particles (e.g., S. cerevisiae or P. carinii) [33 , 65 ] and other microorganisms (e.g., Mycobacterium species) [66 ]. As the heat treatment of C. albicans altered its cell walls, especially the labile mannoprotein layer, thus exposing the more stable ß-glucan layer [56 ], we were unable to determine more precisely the actual contributions of the MR and the BGR to the phagocytosis of C. albicans by these macrophages.

We measured the ability of E-clone macrophages to carry out MR-mediated endocytosis using the highly mannosylated glycoprotein, HRP [38 ]. The results of Figure 6 indicate that prior phagocytosis of HK C. albicans inhibits the capacity of the macrophages to mediate MR endocytosis. This decrease varies with the number of HK C. albicans ingested by E-clone macrophages, suggesting a correlation with the amount of MR engaged in HK C. albicans phagocytosis. Moreover, the fact that we obtained similar results using human macrophages (data not shown) indicates that this process is not unique to E-clone macrophages.

The amount of HRP endocytosed via MR varies with the number of MR on the macrophage cell surface and the internalization efficiency. A general defect in receptor-mediated endocytosis could be ruled out, as transferrin uptake is not affected by the phagocytosis of yeast particles. Conversely, MR immobilization on the macrophage cell surface is not caused by the persistence of adherent yeast particles, as HRP endocytosis was tested after almost all the cell-associated HK C. albicans had been ingested.

We cannot exclude the possibility that the endocytic capacity of MR was specifically impaired after its involvement in phagocytosis. For example, some metabolites such as nitric oxide and tumor necrosis factor produced during phagocytosis have been reported to affect its recycling properties [67 ]. However, our results indicate a decrease in the MR present on the cell surface after phagocytosis. Two mechanisms could account for this: degradation of receptors in a phagolysosomal compartment and slow recycling to the plasma membrane. Our data, obtained using mannosylated BSA coupled to latex beads, showed that impairing phagosome maturation increased the amount of MR associated with phagosomes, probably by inhibiting its degradation (Fig. 9A , lane 4 vs. 3). The presence of MR in mature phagosomes was confirmed using HK yeast particles (Fig. 10) . However, it is likely that a pool of MR was recycled back to the cell surface from maturing phagosomes, as the total amount of MR did not markedly decrease (Fig. 7) . Mannose receptor recycling from this "deeper" compartment would, however, occur at a slower rate compared with recycling from the endocytic pathway. At this point, it is interesting to note that even if TfR is rapidly and constitutively recycled, the number of TfRs on the surface of Chinese hamster ovary cells is reduced after endocytosis of multivalent transferrin prepared by chemical cross-linking [68 ]. Multivalent transferrin selectively retains TfRs in long-standing endocytic recycling compartments. Multivalent mannosylated particles such as HK C. albicans may therefore also slow down MR recycling to the plasma membrane after phagocytosis. The apparent discrepancy with data obtained using LAM microspheres, showing that phagocytosis does not interfere with MR recycling [40 ], could be a result of the fact that LAM is rapidly shed from microspheres after phagocytosis. Another possibility is that as recent studies have reported in DC [69 ], DC-specific intercellular adhesion molecule-3-grabbing nonintegrin and MR (marginally) could mediate LAM binding.

Therefore, our data suggest that the ability of MR to mediate endocytosis is impaired after its involvement in phagocytosis because of different routing as compared with its endocytic pathway. Intracellular trafficking of the MR may be important in regulating its biological activity, as it has been found that signal transduction mainly occurs when the MR is involved in phagocytosis [70 ].


   ACKNOWLEDGEMENTS
 
This work was supported by grants from Montpellier Universities I and II and by a grant from the Philippe Foundation (to J. G.). We are indebted to K. Funato for advice on phagosome preparation experiments. We thank Frederic Boudard for carefully reading the manuscript and providing helpful discussion. We also thank Marc Tabouret (Research and Development, Bio-Rad, France) for providing the C. albicans mannans.

Received December 16, 2002; revised February 28, 2003; accepted March 19, 2003.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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