Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0904529 on February 22, 2005

Published online before print February 22, 2005
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(Journal of Leukocyte Biology. 2005;77:699-709.)
© 2005 by Society for Leukocyte Biology

Regulated recruitment of DC-SIGN to cell–cell contact regions during zymosan-induced human dendritic cell aggregation

Gonzalo de la Rosa*, María Yáñez-Mó{dagger}, Raphael Samaneigo*, Diego Serrano-Gómez{ddagger}, Laura Martínez-Muñoz, Elena Fernández-Ruiz{dagger}, Natividad Longo*, Francisco Sánchez-Madrid{dagger}, Ángel L. Corbí{ddagger} and Paloma Sánchez-Mateos*,1

* Servicio de Inmunología, Hospital General Universitario Gregorio Marañón, Madrid, Spain;
{dagger} Servicio de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid, Spain; and
{ddagger} Departamento de Inmunología, Centro de Investigaciones Biológicas, Madrid, Spain

1 Correspondence: Hospital Gregorio Marañón, Servicio de Inmunología, C/ Dr. Esquerdo 46, 28007 Madrid, Spain. E-mail: rsanchezma.hgugm{at}salud.madrid.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Zymosan is a ß-glucan, mannan-rich yeast particle widely used to activate the inflammatory response of immune cells. We studied the zymosan-binding potential of human dendritic cells (hDCs) by using specific carbohydrate inhibitors and blocking monoclonal antibodies. We show that DC-specific intercellular adhesion molecule-grabbing nonintegrin (DC-SIGN) is a major nonopsonic recognition receptor for zymosan on hDCs. Indeed, blocking of DC-SIGN inhibited the inflammatory response of DCs to zymosan. We compared the zymosan-binding capacity of hDC-SIGN to that of Dectin-1 and complement receptor 3 (CR3), which are receptors involved in the nonopsonic recognition of these yeast-derived particles. Dectin-1- and DC-SIGN-K562 cells bound to zymosan particles, whereas CR3-K562 cells did not. DC-SIGN and Dectin-1 were also expressed in COS cells to compare their ability to trigger particle internalization in a nonphagocytic cell line. DC-SIGN transfectants were unable to internalize bound particles, indicating that DC-SIGN is primarily involved in recognition but not in particle internalization. Zymosan induced a rapid DC aggregation that was accompanied by a dramatic change of DC-SIGN distribution in the plasma membrane. Under resting conditions, DC-SIGN was diffusely distributed through the cell surface, displaying clusters at the free leading edge. Upon zymosan treatment, DC-SIGN was markedly redistributed to cell–cell contacts, supporting an adhesion role in DC–DC interactions. The mechanism(s) supporting DC-SIGN-mediated intercellular adhesion were further investigated by using DC-SIGN-K562 aggregation. DC-SIGN was highly concentrated at points of cell–cell contact, suggesting a role for enhanced avidity during DC-SIGN-mediated intercellular adhesion.

Key Words: phagocytosis • inflammatory response • pathogen recognition • C-type lectins


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Direct recognition of microbial components by innate-immune cells is essential to capture pathogens invading sites that are poor in serum factors (opsonins). Binding to structural components of the pathogen surface itself (nonopsonic phagocytosis) is mediated mainly by surface lectins that bind complex polysaccharides exposed on the surface of microbes [1 ]. Mannosylated proteins are particularly abundant in a variety of microorganisms, and therefore, mannose-binding receptors may play an important role in the initiation of immune responses against these organisms. The insoluble yeast cell particle, termed zymosan, which contains mainly {alpha}-mannan/mannoproteins and ß-glucans, is currently used to explore phagocytosis mediated by surface lectins [2 , 3 ]. Zymosan can activate a broad range of cell types, such as macrophages, polymorphonuclear leukocytes, and natural killer cells [4 5 6 ]. In macrophages, zymosan induces inflammatory signals through Toll-like receptors (TLR)2 and TLR6 [7 , 8 ]. In cooperation with TLR, the surface lectin Dectin-1 (that binds ß-glucan) has been demonstrated to mediate phagocytosis of unopsonized zymosan by murine macrophages, whereas neither the macrophage mannose receptor (MMR) nor complement receptor 3 (CR3; {alpha}m/ß2 integrin, CD11b/CD18, or membrane-activated complex-1) appears to play a significant role [9 10 11 ].

Dendritic cells (DCs) are professional antigen-presenting cells that play a critical role in the induction of primary adaptive-immune responses [12 ]. Immature DCs distributed through peripheral tissues are specialized sentinels for antigen capture. Antigen uptake is mediated by several mechanisms depending on antigen size, such as macropinocytosis for soluble material, receptor-mediated endocytosis for small antigen complexes or viruses, and phagocytosis for larger particles, usually above 0.5 µm in diameter [13 14 15 ]. Phagocytosis can be mediated by immunoglobulin G (IgG) or CRs that bind to opsonized microbes or by direct recognition of pathogen surface ligands. Upon pathogen recognition, DCs are activated and migrate to secondary lymphoid organs, where they present pathogen-derived antigens to naive T cells. Knowledge about the DC receptors involved in recognition of pathogens is starting to emerge and includes TLRs and C-type lectins [16 , 17 ]. TLRs form a complex pattern recognition system for microbial ligands that discriminates infectious agents from self to trigger inflammatory responses [18 , 19 ]. C-type lectins are characterized by a carbohydrate recognition domain, which interacts with pathogen-derived carbohydrate structures in a calcium-dependent manner [20 ]. DCs express several members of the C-type lectin superfamily, namely Langerin (CD207), MMR (CD206), DEC-205 (CD205), Dectin-1, and DC-specific intercellular adhesion molecule (ICAM)-grabbing nonintegrin (SIGN; CD209) [17 ]. Upon soluble ligand binding, C-type lectins are rapidly internalized from the cell surface to undergo antigen presentation to T cells [21 22 23 24 ]. The C-type lectin DC-SIGN is mainly expressed on DCs and binds with high affinity to synthetic mannose- and fucose-containing glycoconjugates [25 26 27 ]. DC-SIGN acts as a pathogen receptor of a wide variety of microbes including fungi, protozoa, bacteria, and viruses and also interacts with carbohydrate-bearing self-glycoproteins (ICAM-2 and ICAM-3) to mediate cellular adhesion processes [25 , 28 , 29 ].

As a model to study microbial phagocytosis by human DCs (hDCs), we have examined the contribution of various receptors to the nonopsonic recognition of zymosan. In this article, we show that binding of zymosan to hDCs is inhibited by mannan and ß-glucan. Dectin-1 appears to be the ß-glucan receptor involved in zymosan binding by murine DCs [11 ]. We have identified DC-SIGN as the mannan-inhibitable receptor for zymosan on primary DCs. We analyzed by confocal microscopy the subcellular localization of DC-SIGN during the phagocytic process and the role of DC-SIGN in modulating zymosan-induced proinflammatory cytokine release. It is interesting that we show that zymosan binding induces a strong relocalization of DC-SIGN to DC–DC intercellular contacts, supporting its role in intercellular adhesion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Culture medium, cytokines, and reagents
RPMI and fetal calf serum (FCS) were from Gibco-BRL, Life Technologies (Paisley, Scotland); interferon-{gamma} (IFN-{gamma}; PeproTech, London, UK) was used at 0.2 ng/ml; and cytochalasin D (CytD; used at 5 µM), laminarin (from Laminaria digitata, used at 500 µg/ml), mannan (from Saccharomyces cerevisiae, used at 100 µg/ml), methylglucoside (used at 100 µg/ml), and sucrose (0.6 M) were from Sigma Chemical Co. (St. Louis, MO). Human immunodeficiency virus type 1 (HIV-1) gp120 recombinant antigen, clone env 166C1AH, was from Boehringer Ingelheim (Barcelona, Spain). Mycobacterium bovis [Bacillus Calmette Guerin (BCG) vaccine, Pharmacia Spain S.A., Barcelona] was used at five particles/DC. ICAM-3-Fc was from R&D Systems (Minneapolis, MN). Lipopolysaccharide (LPS; from Escherichiacoli, 026.B6, Difco, Detroit, MI) was used at 100 ng/ml. N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-seryl-(lysyl)(3)-lysine (PAM3CSK4) synthetic lipopeptide (EMC Microcollections, Germany) was used at 100 ng/ml.

Antibodies
Anti-DC-SIGN monoclonal antibody (mAb) clone MR-1 was described previously [30 ] and was used as purified or as ascytic fluid; anti-DC-SIGN polyclonal antibody (pAb) C-20 was from Santa Cruz Biotechnology (CA). Anti-FLAG.M2 mAb was from Sigma Chemical Co.; anti-human MMR, clone 19.2, IgG1, and anti-hDC-SIGN mAb, clone DCN46, were from BD PharMingen (San Diego, CA); anti-ICAM-2 mAb, BT-1, was from Serotec (Oxford, UK). The DC-SIGN mAb clone 120526 was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health (Bethesda, MD).

Cell cultures
Monocyte-derived, immature DCs were generated as described previously [31 ]. Briefly, peripheral blood mononuclear cells were obtained from healthy donors by centrifugation over Ficoll-hypaque. After three washing steps, monocytes were isolated using CD14-labeled magnetic microbeads (Miltenyi Biotec, Bergish Gladblach, Germany) and cultured in RPMI 10% FCS in the presence of 5 ng/ml interleukin-4 (R&D Systems) and 50 ng/ml granulocyte macrophage-colony stimulating factor (PeproTech) for 5–7 days. K562-DC-SIGN, CD11b, and Dectin-1 transfectants were generated as described previously [30 , 32 , 33 ]. Cells were grown on RPMI 10% FCS supplemented with 400 µg/ml G418 sulfate (Gibco-BRL, Life Technologies) to maintain stable transfectant expression.

Binding assays
K562 transfectants or DCs were collected, washed with cold phosphate-buffered saline (PBS), and resuspended in prechilled RPMI 0.1% bovine serum albumin (Sigma Chemical Co.). For blocking experiments, mAb (10 µg/ml), mannan (100 µg/ml), laminarin (500 µg/ml), and methylglucoside (100 µg/ml) were incubated for 30 min at 4°C. Cells were then incubated with Zimosan A BioParticles, fluorescein isothiocyanate (FITC)-conjugated (Molecular Probes Europe BV, Leiden, The Netherlands) at 50 particles/cell ratio, unless indicated otherwise. Cells were labeled with CD45-allophycocyanin (APC) to discriminate cells binding to FITC-labeled zymosan from yeast aggregates. After washing to remove unbound particles, cells were analyzed on a FACScan cytometer (Becton Dickinson, San Jose, CA). To test the binding ability of K562-DC-SIGN transfectants, cells were incubated with ICAM-3-Fc (2 µg/ml) for 30 min at 4°C, washed in cold PBS, and incubated with anti-human Fc-FITC-conjugated pAb for 30 min at 4°C. Cells were then washed and analyzed on a FACScan cytometer.

Phagocytic assays
DC-SIGN-expressing COS cells were prepared as described previously. [30 ] Dectin-1-FLAG was inserted into a pcDNA3.1/v5-his plasmid. COS cells were transfected using the Superfect transfection reagent (Qiagen GmbH, Hilden, Germany), as described in the protocol for transient transfection of adherent cells. Cells were used within 36 h after transfection. Cells were incubated with zymosan for 2 h, washed, fixed, and stained for confocal microscopy. Transfected Dectin-1 COS cells were detected using anti-FLAG mAb followed by a Cy3-conjugated goat anti-mouse pAb. Phagocytic assays in K562-transfected cells were performed as described in binding assays but with minor changes: Briefly, cells were incubated with zymosan for 20 min on ice and subsequently, on a 37°C prewarmed bath for 30 min. After 10 min back on ice, cells were pretreated for 1 min with ice-cold quenching solution (Phagotest kit, Orpegen Pharma, Heidelberg, Germany) and washed with pH 7 RPMI or washed with pH 5 RPMI for suppressing fluorescence of the yeast attached to the cell membrane.

Cytokine assays
Immature DCs were preincubated in the presence of the corresponding blocking reagents at room temperature in polypropylene tubes for 30 min prior to incubation with 13 µg/ml unlabeled Zimosan (Sigma Chemical Co.) and then plated onto 96-well plates (Costar, Costar Europe, Badhoevedorp, The Netherlands) at a final concentration of 5 x 105 cells/ml at 37°C for 3 h. The level of tumor necrosis factor {alpha} (TNF-{alpha}) and in-culture supernatants was determined by the human TNF-{alpha} enzyme-linked immunosorbent assay (Pierce Endogen, Rockford, IL). All experiments were performed six times using duplicates of each sample.

Immunofluorescence assays and confocal microscopy
Cells were layered over poly-L-lysine-coated glass coverslips and incubated for 1 h at 37°C and subsequently incubated with the desired stimuli for a time ranging from 30 min to 3 h. Cells were fixed with 2% paraformaldehyde in PBS for 15 min at room temperature and washed three times with 25 mM Tris-buffered saline. When permeabilization was needed, samples were incubated with 3% Triton X-100 for 5 min at room temperature. After a blocking step in blocking reagent (Boehringer Mannheim, Germany)/0.5% azide/100 µg/ml poly-human Ig for 10 min at room temperature, cells were labeled with the desired antibody for 30 min at 37°C and subsequently incubated with either anti-goat or anti-mouse fluorochrome-conjugated, cross-absorved secondary antibodies. Laser-scanning confocal microscopy was performed with argon and helio/neonlaser beams and attached to an Ultra-spectral Leica TCS-SP2-AOBS inverted epifluorescence microscope using x60 and x100 oil immersion objectives.

Flow cytometry
Cells were preincubated with poly-human Ig for 10 min at room temperature and then incubated with the appropiate fluorochrome-conjugated mAb for 30 min at 4°C. Cells were then washed and analyzed on a FACScan cytometer (Becton Dickinson) using CellQuest software.

Aggregation assays
K562-DC-SIGN transfectants were washed with PBS 1 mM EDTA, resuspended in RPMI 10% FCS, and incubated onto a 24-well plate under the described conditions at a final concentration of 105 cell/ml for 20 min at 37°C. Before and after aggregation, cells were collected gently and counted using a FACScan cytometer.

Aggregation between untransfected K562 and K562-DC-SIGN was performed labeling the cells with 1 µM 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF; Molecular Probes) or 10 µM seminaphthorhodafluor (SNARF; Molecular Probes), respectively. After a final washing step, protocol was followed as explained previously.

Western blot
Western blotting was carried out as follows: Cells were stimulated or not with 13 µg/ml zymosan particles for 1 h at 37°C, collected, washed with PBS, and lysed with ice-cold 0.5% Triton buffer containing 1 mM EDTA, 250 mM NaCl, 50 mM Hepes, pH 7, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (Sigma Chemical Co.) for 15 min. Cellular debris was removed by centrifugation at 13,000 rpm for 15 min at 4°C. Protein concentration was measured using the Bio-Rad (Hercules, CA) DC protein assay. Samples were boiled in sodium dodecyl sulfate (SDS)-bromophenol blue and resolved in a SDS-polyacrylamide gel electrophoresis gel, and the proteins were transferred into polyvinylidene difluoride membranes and revealed with anti-DC-SIGN pAb C-20.

Statistical analysis
Data are expressed as means ± SD. The statistical significance of differences between means was assessed by using Student’s t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DC-SIGN is a major, nonopsonic zymosan recognition receptor on hDCs
To investigate the functional role of various receptors in recognition of unopsonized zymosan by hDCs, we examined the contribution of receptor-specific carbohydrates in inhibiting zymosan binding in the absence of serum (Fig. 1A ). Soluble, fungal-derived mannan exerted the strongest inhibitory effect on zymosan binding, whereas ß-glucan inhibition with laminarin was lower. Furthermore, a combination of mannan and ß-glucan largely abrogated the nonopsonic recognition of zymosan by DCs. This suggests that binding of zymosan by DCs is predominantly mannan-dependent, although ß-glucan receptors also contribute. In accordance with previous results [9 ], methylglucoside, an inhibitor of the CD11b lectin-binding domain, had no effect, suggesting that CR3 is not involved in the recognition of unopsonized zymosan by DCs. To assess the role of mannan-inhibitable receptors in zymosan binding, DCs were pretreated with specific mAb against DC-SIGN, MMR, or an isotypic control IgG. Anti-DC-SIGN mAb prevented binding of zymosan to DCs, nearly up to that obtained with mannan, whereas mAb against MMR showed a minimal effect (Fig. 1A) . Furthermore, the combination of anti-DC-SIGN with ß-glucan blocked the recognition of zymosan in a comparable way with mannan and ß-glucan. This demonstrates that DC-SIGN is a major nonopsonic pattern recognition receptor on DCs that cooperates in an additive manner with the recognition of zymosan.



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  		Figure 1. DC-SIGN mediates binding and TNF-{alpha} production in zymosan-treated DCs. (A) DCs were preincubated in the absence or presence of the following carbohydrates: mannan (100 µg/ml), laminarin (500 µg/ml), and methylglucoside (Metyl Glu; 100 µg/ml); or mAb (10 µg/ml): anti-DC-SIGN, anti-MMR, or isotype control. Cells were then incubated with FITC-Zimosan at 50 particles/cell ratio. Cells were labeled with CD45-APC to discriminate cells binding FITC-labeled zymosan from yeast aggregates and analyzed by flow cytometry. The binding to zymosan in the absence of inhibitors (with isotype-matched control) ranged between 40% and 52% and was arbitrarily set as 100% to compare inhibition in distinct experiments. (B) DCs were preincubated with isotype control, anti-DC-SIGN mAb, laminarin, or anti-MMR as indicated. After 30 min, cells were plated at 106 cell/ml in RPMI 10% FCS, with or without 13 µg/ml zymosan for 3 h. Culture supernatants were recovered and analyzed for TNF-{alpha} production. The media ± SDof three to six independent experiments is shown (*, P<0.001, vs. basal; **, P<0.05, vs. basal).

 
We then examined, on DCs, the ability of anti-DC-SIGN mAb to block the zymosan-induced production of inflammatory mediators, such as TNF-{alpha} (Fig. 1B) . The addition of zymosan to DCs induced the release of TNF-{alpha} into the culture supernatants within 3 h. Blocking of ß-glucan receptors and MMR partially inhibited TNF-{alpha} production, whereas additional blocking of DC-SIGN significantly inhibited the TNF-{alpha} response to zymosan. These data indicate that DC-SIGN contributes to the magnitude of the DC inflammatory response to zymosan.

Zymosan is a ligand of DC-SIGN
We further analyzed the potential of DC-SIGN to bind zymosan, using K562 transfectants stably expressing DC-SIGN (Fig. 2A ). K562 cells do not express any other known zymosan receptors, as demonstrated by the absence of zymosan binding to untransfected cells. Binding of K562-DC-SIGN to ICAM-3-Fc was used as a positive control for DC-SIGN function. K562 cells transfected with Dectin-1 and CR3 were used to compare the ability of DC-SIGN to bind zymosan with other receptors. Binding studies demonstrated that DC-SIGN as well as Dectin-1 clearly mediate adhesion to zymosan. By contrast, K562-CR3 did not bind unopsonized zymosan. This lack of binding was not a result of experimental conditions, as the ability of the lectin domain of CR3 to bind carbohydrate ligands at 4°C, in the absence of integrin activation, has been shown previously [34 ]. Blocking antibodies against DC-SIGN completely inhibited binding of zymosan by K562-DC-SIGN (Fig. 2B) . In addition, soluble fungal-derived mannan significantly abrogated binding, whereas laminarin, a soluble, fungal-derived ß-glucan, failed to inhibit the binding of zymosan to K562-DC-SIGN. This mannan dependence is in agreement with the high affinity of DC-SIGN for mannose-containing glycoconjugates [27 ]. By contrast, ß-glucan but not mannan inhibited the binding of zymosan to K562-Dectin-1, in agreement with previous studies [9 ]. Binding of zymosan by K562-DC-SIGN was dose- and time-dependent (Fig. 2C and 2D) .



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  		Figure 2. Zymosan is a ligand of DC-SIGN. (A) K562 DC-SIGN-transfected cells bind to soluble ICAM-3-Fc (left panel). Transfectants (K562) stably expressing CR3, DC-SIGN, or Dectin-1 were incubated at 4°C with zymosan BioParticles, FITC-conjugated at 50 particles/cell ratio and analyzed by flow cytometry. (B) Specific adhesion was determined by preincubating the cells with mAb anti-DC-SIGN, mannan, or laminarin (a soluble ß-glucan from L. digitata that blocks ß-glucan receptor) for 30 min at 4°C prior to addition of zymosan-FITC-conjugated particles per cell. A representative experiment out of five is shown. (C and D) Zymosan binding to DC-SIGN increases with increasing zymosan concentration and is time-dependent.

 
DC-SIGN is not a phagocytic receptor for zymosan
In addition to particle binding, some receptors may trigger particle internalization directly. To determine the phagocytic capacity of DC-SIGN, it was expressed in the nonphagocytic COS cell line, and the ability to internalize zymosan particles was analyzed. COS cells transfected with Dectin-1, which was reported to confer phagocytic capacity by itself [35 ], were used as positive control. Dectin-1 and DC-SIGN-transfected COS cells bound zymosan (Fig. 3A ). Zymosan interaction with Dectin-1-transfected cells induced a rapid relocalization of Dectin-1 to particle-binding sites (Fig. 3B , arrows). In addition, Dectin-1 clusters colocalized with actin-rich phagocytic cups around particles. By contrast, neither DC-SIGN nor F-actin was redistributed to sites of zymosan binding in DC-SIGN-transfected cells (Fig. 3B) . Particle internalization could not be detected by Z-scan confocal microscopy, even after prolonged time-periods (data not shown). These data indicate that DC-SIGN expression in a nonphagocytic cell does not confer phagocytic ability to the cell. Therefore, DC-SIGN appears primarily to participate in particle binding but does not seem to be capable of transmitting intracellular signals that trigger phagocytosis of large particles.



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  		Figure 3. DC-SIGN is not a phagocytic receptor for zymosan. COS cells were transfected as described in Materials and Methods with Dectin-1 (left column) or DC-SIGN (right column), incubated or not with zymosan FITC-conjugated bioparticles (A) or with unconjugated zymosan particles (B) at a ratio of 50 particles/cell for 2 h at 37°C to allow phagocytosis, then washed and fixed, stained with anti-FLAG mAb or anti-DC-SIGN mAb, and analyzed by confocal microscopy. Projections of confocal sections are depicted. Zymosan particles bind to both transfectants but only induce actin polymerization on Dectin-1-COS cells (arrows). Original bar = 25 µm. (C) K562 transfectants were incubated at 37°C in the presence of FITC-zymosan for 1 h and subsequently washed with pH 7 or pH 5 buffer or incubated for 1 min with quenching solution. Data represent mean with SD of at least three independent experiments (**, P<0.05, vs. pH 7 washing).

 
Phagocytosis assays were also carried out in the K562 transfectants to exclude the possibility that COS cells lack the signaling components required for internalization by DC-SIGN. Untransfected, DC-SIGN- and Dectin-1-K562 were incubated for 1 h at 37°C with FITC-zymosan, and then, the zymosan particles that remained bound to the cell surface were removed by washing the cells in acid medium, or the particle fluorescence was quenched by incubating the cells with trypan blue (Fig. 3C) . Most of FITC-zymosan particles bound to DC-SIGN-K562 cells were removed by acidic washing or fluorescence quenching, indicating that particles were bound to the cell surface. By contrast, the FITC-zymosan captured by Dectin-1-K562 was resistant to both treatments, indicating that these cells were able to internalize most of the bound particles.

Subcellular localization of DC-SIGN during phagocytosis of zymosan by DCs
DC-SIGN was distributed homogenously through the surface of immature DCs stained in suspension (data not shown). Upon DC adhesion to various substrata (fibronectin, poly-L-lysine), there was a clear relocalization of DC-SIGN to membrane ruffles located at both cellular poles (Fig. 4A ). DC-SIGN clusters were observed at the cell edges and not at cell-to-cell contacts (Fig. 4A , detailed inset). Within 1 h of zymosan stimulation, DCs became aggregated around zymosan particles, displaying small, cellular aggregates firmly adherent to substratum (data not shown). It is interesting that staining of zymosan-stimulated DCs revealed a strong redistribution of DC-SIGN toward DC–DC intercellular contact regions (Fig. 4B) , which were established between adjacent cell bodies or through cellular processes such as filopodia or lamellipodia. The accumulation of DC-SIGN at intercellular contacts was observed as soon as 20 min after zymosan stimulation and disappeared after 24 h (data not shown). DC-SIGN clustering at intercellular regions was not only the result of membrane overlapping, as demonstrated by comparative analysis with other membrane markers such as CD45 (Fig. 4C) . To quantify this effect, the ratio of the labeling intensities at sites of cell–cell contact and at noncontact areas of the plasma membrane was measured in confocal sections. Therefore, line scans were performed through several zymosan-stimulated DCs double-stained for DC-SIGN and CD45 as shown in Figure 4C , and the mean intensity values for each fluorescence channel were measured at cell–cell contact and at noncontact areas. Evaluation of 17 line scans resulted in cell–cell contacts versus cell body ratios of 6.1 ± 2.3 for DC-SIGN and 1.9 ± 0.8 for CD45. The contact/noncontact ratio of CD45 corresponded to the effect of overlapping cell membranes, and therefore, the threefold enhancement of DC-SIGN signal versus CD45 indicates that DC-SIGN is actively concentrated at contact sites.



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  		Figure 4. DC-SIGN relocalizes at DC–DC contacts in zymosan-treated DCs. Immature DCs, at a concentration of 5 x 105 cells/ml, were layered over poly-L-lysine-coated glass coverslips, unlabeled zymosan (13 µg/ml) particles were added (B–D) or not (A), and cells were incubated for 1 h at 37°C. (A and B) DCs were stained with anti-DC-SIGN mAb (red) and analyzed by confocal microscopy. Projections of confocal sections are depicted. Insets display the amplified single confocal section of the selected regions. Note that upon zymosan stimulation, DC-SIGN was redistributed at intercellular DC–DC contacts (B, detailed inset) but was not in resting cells (A, inset). (C) Quantification of the membrane distribution of DC-SIGN compared with CD45. A representative confocal section and scan profile are shown. The ratio of labeling intensities at cell–cell contact versus noncontact sites was determined for each channel (green CD45 and red DC-SIGN) in the same line scans. Analysis of 17 line scans resulted in cell–cell contacts versus cell body ratios of 6.1 ± 2.3 for DC-SIGN and 1.96 ± 0.8 for CD45. (D) DC-SIGN is localized at cell–cell contacts, whereas FITC-zymosan is within intracellular phagosomes. (E and F) Zymosan-stimulated DCs were double-stained with anti-DC-SIGN pAb (green) and anti-ICAM-3 mAb (E, red) or anti-CD11b (F, red); a colocalization histogram is inserted in the merged panel. There was no colocalization of DC-SIGN with ICAM-3 or with CD11b. Original bar = 10 µm.

 
To address the possibility that zymosan particles could be mediating DC–DC interactions, we used FITC-zymosan to better visualize the particle in relation to DC-SIGN clusters at intercellular junctions (Fig. 4D) . After 1 h of phagocytosis, most FITC-zymosan particles were completely engulfed and did not colocalize with DC-SIGN clusters. We used dual-label immunofluorescence confocal microscopy to examine the distribution of ICAM-3 and CD11b integrin in zymosan-treated DCs, as ICAM-2, another DC-SIGN ligand, is not expressed by DCs (Fig. 4E and 4F , respectively). There was no colocalization of DC-SIGN with ICAM-3 or with CD11b at intercellular contacts (Fig. 4E and 4F , colocalization histograms). These results globally suggest that zymosan particles are not mediating DC–DC cross-linking, and an alternative ligand could be interacting with DC-SIGN during zymosan-induced intercellular adhesion.

We next tested whether other phagocytic or maturative stimuli were able to induce DC-SIGN redistribution to intercellular contacts (Table 1 ). Opsonized zymosan, engulfed mainly by CR3, did not induce DC-SIGN intercellular relocalization. Maturative stimuli such as LPS, TNF-{alpha}, IFN-{gamma}, or the pure TLR-2 stimulus, PAM3CSK4 lipopeptide [8 ] also failed to induce DC-SIGN relocalization. Only DC-SIGN ligands such as M. bovis and soluble fungal-derived mannans from S. cerevisiae induced DC-SIGN intercellular accumulation. However, other DC-SIGN ligands such as HIV gp120 or ICAM-3-Fc did not induce DC-SIGN intercellular relocalization. These results suggest that direct binding to DC-SIGN is important but not sufficient to mediate its intercellular redistribution. In addition, it seems to be independent of DC maturation, as mannan did not induce DC maturation while inducing cell–cell relocalization of DC-SIGN.


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  		Table 1. DC-SIGN Redistribution by Distinct Stimuli

 
Clustering of DC-SIGN at intercellular contacts may be mediated through cytoskeletal interactions. Therefore, we used dual-label confocal microscopy to study the distribution of the actin cytoskeleton during zymosan stimulation. Under baseline conditions (Fig. 5A ), there was a clear colocalization of DC-SIGN with F-actin into membrane ruffles located at both poles of DCs. DC-SIGN also colocalized with actin-rich phagocytic cups at the beginning of the process (early phagosomes, Fig. 5C , white). By contrast, upon longer zymosan activation, there was no redistribution of F-actin to DC-SIGN domains (cell–cell contacts), and DC-SIGN was not observed around late phagosomes (Fig. 5B) . During phagosome maturation, internalized plasma membrane receptors may be recycled back to the cell surface, using a protein recycling mechanism similar to that used by the endocytic pathway.



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  		Figure 5. DC-SIGN colocalizes with F-actin at phagocytic cups. Untreated DCs (A) or zymosan-stimulated DCs for 1 h at 37°C (B) were costained with anti-DC-SIGN mAb (red) and FITC-falloidin (green) and analyzed by confocal microscopy. Projections of confocal sections are depicted (A and B). (C) DCs were treated with zymosan for 20 min to investigate the presence of DC-SIGN on early phagosomes. Two confocal planes corresponding to DC-SIGN relocalization at cell–cell contact and at phagocytic cups where it colocalized with F-actin (first two columns) are shown. (Inset) A colocalization histogram of green and red signals; the corresponding colocalization dots are represented in white in the merging panel. Original bar = 10 µm.

 
DC-SIGN-mediated intercellular adhesion
To study the role of DC-SIGN in intercellular adhesion, we used the ability of K562-DC-SIGN to aggregate spontaneously in culture (Fig. 6A ) compared with untransfected cells, which do not form cellular aggregates (data not shown). K562-DC-SIGN cell aggregation is DC-SIGN-specific, as it was inhibited by anti-DC-SIGN mAb and mannan (Fig. 6B and 6C) . It was also completely inhibited by EDTA, indicating that it is calcium-dependent. Similarly to other adhesive lectins, DC-SIGN-mediated cell aggregation was independent of temperature and active metabolism (not shown). We observed clustering of DC-SIGN molecules at cell–cell contacts of K562-DC-SIGN cellular aggregates (Fig. 6D) suggesting a role for enhanced avidity as one of the mechanisms that controls DC-SIGN-mediated intercellular adhesion. K562 cells do not express ICAM-3 but do express ICAM-2. K562-DC-SIGN aggregation was not inhibited by anti-ICAM-2 mAb; however, it is not known that this antibody blocks DC-SIGN-ICAM-2 interactions. To determine whether a homophilic interaction between DC-SIGN molecules could be mediating K562 aggregation, we performed aggregation assays using a mixture of K562-DC-SIGN with untransfected K562, labeled with SNARF or BCECF, respectively (Fig. 6E) . Cell aggregates contained K562-DC-SIGN cells and untransfected cells and were blocked by anti-DC-SIGN mAb (data not shown), indicating that it is a DC-SIGN-specific, heterophilic-binding reaction.



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  		Figure 6. DC-SIGN-mediated intercellular adhesion. (A) K562-DC-SIGN-transfected cells form aggregates, (B) which were disrupted by mAb anti-DC-SIGN. (C) K562-DC-SIGN were pretreated with anti-DC-SIGN or anti-ICAM-2 mAb, mannan (100 µg/ml), CytD (5 µM), and EDTA (1 mM), as indicated in Materials and Methods and were allowed to aggregate for 20 min. Cell aggregation was quantified by flow cytometry. A representative experiment out of four is shown. (D) DC-SIGN was stained in K562-DC-SIGN aggregates, finding out that it was concentrated at intercellular contacts. (E) Untransfected K562 and K562-DC-SIGN cells were marked with BCECF (white) and SNARF (gray), respectively, mixed, and allowed to aggregate; gray and white cells were present in aggregates. (F) Untransfected K562, K562-DC-SIGN, unstimulated DCs, or zymosan-stimulated DCs were lysated and analyzed by Western blotting with anti-DC-SIGN pAb; DC-SIGN multimeric forms were not enhanced upon zymosan stimulation. Same protein amounts were loaded for every lane.

 
Finally, as it has been described that DC-SIGN forms oligomers [27 ], we explored whether assembly of monomeric receptors into multimers could contribute to regulate DC-SIGN-mediated intercellular interactions. We used Western blotting to determine whether oligomerization of DC-SIGN was constitutive or could be enhanced following receptor binding (Fig. 6F) . In untreated and zymosan-stimulated DC lysates, multiple forms of DC-SIGN were detected, including a low molecular weight 44-kDa monomer and others of higher molecular weight, 90 kDa or larger, presumptive multimers. After ß-mercaptoethanol treatment, the majority of DC-SIGN protein migrated as a monomer (data not shown). These data indicate that DC-SIGN multimerization appears to be constitutive, and it is not enhanced upon zymosan stimulation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
DC-SIGN mediates DC attachment to a diversity of pathogens including viruses, bacteria, fungi, and protozoa, although little is known about the ability of DC-SIGN to mediate phagocytosis. Here, we show that in hDCs, DC-SIGN collaborates with Dectin-1 in the recognition as well as in the inflammatory response to zymosan. By contrast, when DC-SIGN is expressed in nonphagocytic cells, it is only involved in particle binding to the cell surface and does not trigger a phagocytic response. Upon zymosan phagocytosis by DCs, DC-SIGN was redistributed to cell–cell contact regions, suggesting a major role in DC–DC intercellular adhesion. Therefore, we used K562 cells transfected with DC-SIGN to further analyze the molecular mechanisms involved in DC-SIGN-mediated intercellular adhesion. Our data indicate that DC-SIGN clustering may enhance receptor avidity and facilitate intercellular adhesion.

The mode of internalization of pathogens depends on their size, as viruses are internalized by receptor-mediated endocytosis, whereas internalization of large microorganisms occurs via phagocytosis. The molecular mechanisms underlying both internalization processes differ markedly. Endocytosis requires assembly of clathrin at the site of receptor clustering, whereas phagocytosis involves acute assembly of F-actin [36 ]. DCs express several receptors implicated in the phagocytosis of nonopsonized yeast particles, such as MMR, Dectin-1, and CR3. However, we did not observe CR3 and MMR-specific binding of unopsonized zymosan by DCs or by K562-CR3. This is in accordance with a previous study, which indicated that MMR and CR3 are not involved in the nonopsonic recognition of zymosan by macrophages [9 ]. In addition to Dectin-1, we show that DC-SIGN is a major recognition receptor involved in zymosan binding by DCs. It has been suggested that DC-SIGN is a phagocytic receptor capable of mediating the internalization of large particles such as Candida albicans [37 ]; however, this study was performed on DCs that express a wide variety of phagocytic receptors for this yeast. Therefore, the most reliable method for establishing the phagocytic capacity of a specific receptor is to express it in a nonphagocytic cell line [1 ]. Although MMR and Dectin-1 confer on COS cells the ability to internalize yeast particles [35 , 38 ], DC-SIGN failed to do it. Thus, DC-SIGN may primarily participate in binding but not in internalization of large particles. Consistent with our results, a recent report showed that a related mouse homologue of DC-SIGN, DC-SIGN related-1 (SIGNR-1), was involved in zymosan binding by macrophages but did not appear to mediate particle internalization in nonphagocytic cells [39 ]. The DC-SIGN family of C-type lectins in mouse is composed of five members (mouse-DC-SIGN and the SIGNR-1–4 molecules) [40 ], whereas in humans, it has only two members (DC-SIGN and DC-SIGNR/L-SIGN) [41 ]. Mouse SIGN-R1 is more closely related to human L-SIGN than to DC-SIGN, as it is specifically expressed by liver sinusoidal endothelial cells and not by DCs [42 ].

It has been reported that heat-killed yeasts provide a powerful activation stimulus to DCs, and whole recombinant S. cerevisiae yeast expressing tumor antigens was used as a novel vaccine strategy to target DCs and successfully induced tumor protection [43 ]. Zymosan is a complex microbial surface that can simultaneously activate many parallel signaling pathways on DCs. Therefore, to study the functional role of a single molecule, it may be important to block other receptors signaling the inflammatory response to zymosan. We have shown that the addition of anti-DC-SIGN significantly inhibited the binding of zymosan to DCs, although it did not block TNF-{alpha} production. Similarly, the ß-glucan receptor blocker laminarin or anti-MMR mAb alone did not inhibit the zymosan-induced TNF-{alpha} response (not shown). Addition of laminarin plus anti-MMR mAb had no effect but in combination with anti-DC-SIGN, significantly reduced TNF-{alpha} production. These data indicate that DC-SIGN consistently contributes to the magnitude of the TNF-{alpha} response to zymosan by DCs. Only few studies have highlighted that members of the C-type lectin family may participate in signal transduction events, leading to cell activation or inhibition through interference with TLR-mediated signaling [10 , 11 , 44 , 45 ]. However, there is no evidence that DC-SIGN signals TNF-{alpha} directly other than by increased zymosan binding.

DC-SIGN was first described for its binding affinity to ICAM-3-coated beads, suggesting a role in DC-lymphocyte interactions, and later for its binding affinity to ICAM-2-coated beads, suggesting a role during DC transendothelial migration [25 , 29 ]. The marked clustering of DC-SIGN to DC–DC contacts upon zymosan stimulation points out a role of this molecule during DC–DC intercellular adhesion. By contrast, we could not detect any DC-SIGN clustering during DC-lymphocyte interactions, even when a clear redistribution of lymphocyte ICAM-3 indicated an active polarization of lymphocyte adhesion receptors toward the DC (data not shown). The physiological role of DC–DC interactions is less known; however, the process of DC maturation involves some degree of cell aggregation, and it was described that stimulation through adhesion receptors, such as CD43 and CD44, induces intercellular adhesion and maturation of DCs [46 , 47 ]. Zymosan phagocytosis by DC enhanced adhesion to extracellular matrix ligands and intercellular adhesion ligands, resulting in the rapid formation of small cellular aggregates firmly adhered to the substratum (data not shown). Multiple adhesion receptors may be involved in zymosan-induced DC aggregation, which was not inhibited by anti-DC-SIGN mAb and anti-ß2 mAb (data not shown). As ICAM-3 was not clustered at DC–DC intercellular contacts, it does not appear to be the DC-SIGN ligand, suggesting that an alternative, unknown ligand might be involved. We carefully investigated the presence of zymosan particles at intercellular contact sites, where DC-SIGN is clustered to exclude the possibility of particle-mediated, DC–DC cross-linking. DCs are highly efficient phagocytic cells, and after 1 h of phagocytosis, most of the zymosan particles were engulfed completely. Zymosan particles were never observed colocalizing with DC-SIGN clusters at intercellular junctions, suggesting that the particles are not mediating DC–DC interactions directly. However, as soluble mannans also induced DC-SIGN intercellular redistribution, it cannot be formally excluded that soluble materials produced during zymosan phagocytosis could mediate cross-linking of DCs. This possibility can be excluded in K562-DC-SIGN cell aggregation that is constitutive, suggesting a direct interaction of DC-SIGN with a cell ligand. However, K562-DC-SIGN intercellular aggregation may be distinct to zymosan-induced DC–DC interactions. The ability of untransfected K562 to be aggregated together with K562-DC-SIGN strongly suggests that DC-SIGN is involved in a heterophilic interaction, although we cannot completely exclude the possibility of a DC-SIGN–DC-SIGN interaction.

Our data indicate that DC-SIGN-mediated intercellular adhesion might be regulated by a mechanism of enhanced avidity by clustering DC-SIGN molecules at the cell surface. Clustering adhesion receptors through cytoskeletal interactions appears to be a general mechanism used to dynamically regulate cell adhesion. For example, integrin clustering by the actin cytoskeleton or P-selectin in clathrin-coated pits may promote multivalent interactions that stabilize adhesion [48 , 49 ]. However, our data on DC-SIGN-expressing cell lines do not support that DC-SIGN may induce actin rearrangements, in accordance with the lack of actin relocalization to DC-SIGN domains (cell–cell contacts) observed on DCs. Lateral mobility of plasma membrane proteins may also depend on local organization of some types of domains, usually termed lipid rafts [50 ]. In this regard, the distribution of DC-SIGN in microdomains that reside within lipid rafts [51 ] has been reported recently, with an average diameter of 200 nm, which may promote multivalent interactions that further stabilize adhesion. Indeed, we observed colocalization of DC-SIGN with raft markers at DC–DC contacts (data not shown). Finally, another mechanism that may control formation and stabilization of cellular interactions is the assembly of monomeric receptors into oligomers, which has been reported for selectins [52 , 53 ]. Our data do not support this mechanism, as homo-oligomerization of DC-SIGN was constitutive and did not change following zymosan stimulation.

In summary, this study clearly shows that in hDCs, DC-SIGN is a major, nonopsonic recognition receptor for zymosan that collaborates with Dectin-1 to elicit an inflammatory response. DC-SIGN is involved in the binding of zymosan but not in particle phagocytosis. Upon zymosan stimulation, DC-SIGN is redistributed to cell-to-cell contact regions, suggesting an adhesive, functional role in DC–DC interactions. Clustering of DC-SIGN appears to play an important role in regulating DC-SIGN-mediated intercellular adhesion.


   ACKNOWLEDGEMENTS
 
Grant SAF2002-04615-C02-02 was from the Ministerio de Ciencia y Tecnología to P. S-M. G. d. l. R. was supported by Fellowship 01/9241 from Instituto de Salud Carlos III. We thank J. Villarejo and I. Treviño for their excellent technical assistance, the members of the Department of Hematology of the Hospital General Universitario Gregorio Marañón for the material provided, and Dr. S. Sanchez-Ramón for helpful discussions.

Received September 17, 2004; revised January 12, 2005; accepted January 24, 2005.


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 RESULTS
 DISCUSSION
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D. Serrano-Gomez, E. Sierra-Filardi, R. T. Martinez-Nunez, E. Caparros, R. Delgado, M. A. Munoz-Fernandez, M. A. Abad, J. Jimenez-Barbero, M. Leal, and A. L. Corbi
Structural Requirements for Multimerization of the Pathogen Receptor Dendritic Cell-specific ICAM3-grabbing Non-integrin (CD209) on the Cell Surface
J. Biol. Chem., February 15, 2008; 283(7): 3889 - 3903.
[Abstract] [Full Text] [PDF]

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 J. Virol.Home page
J. Sabatte, A. Ceballos, S. Raiden, M. Vermeulen, K. Nahmod, J. Maggini, G. Salamone, H. Salomon, S. Amigorena, and J. Geffner
Human Seminal Plasma Abrogates the Capture and Transmission of Human Immunodeficiency Virus Type 1 to CD4+ T Cells Mediated by DC-SIGN
J. Virol., December 15, 2007; 81(24): 13723 - 13734.
[Abstract] [Full Text] [PDF]

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 Antimicrob. Agents Chemother.Home page
D. Serrano-Gomez, R. T. Martinez-Nunez, E. Sierra-Filardi, N. Izquierdo, M. Colmenares, J. Pla, L. Rivas, J. Martinez-Picado, J. Jimenez-Barbero, J. L. Alonso-Lebrero, et al.
AM3 Modulates Dendritic Cell Pathogen Recognition Capabilities by Targeting DC-SIGN
Antimicrob. Agents Chemother., July 1, 2007; 51(7): 2313 - 2323.
[Abstract] [Full Text] [PDF]

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 BloodHome page
A. Dominguez-Soto, L. Aragoneses-Fenoll, E. Martin-Gayo, L. Martinez-Prats, M. Colmenares, M. Naranjo-Gomez, F. E. Borras, P. Munoz, M. Zubiaur, M. L. Toribio, et al.
The DC-SIGN-related lectin LSECtin mediates antigen capture and pathogen binding by human myeloid cells
Blood, June 15, 2007; 109(12): 5337 - 5345.
[Abstract] [Full Text] [PDF]

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 J. Immunol.Home page
J. V. Nayak, D. A. Hokey, A. Larregina, Y. He, R. D. Salter, S. C. Watkins, and L. D. Falo Jr
Phagocytosis Induces Lysosome Remodeling and Regulated Presentation of Particulate Antige