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The Journal of Immunology, 2008, 181, 3148 -3155
Copyright © 2008 by The American Association of Immunologists, Inc.
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The C-Type Lectin Macrophage Galactose-Type Lectin Impedes Migration of Immature APCs1

Sandra J. van Vliet2, Lutz C. Paessens2, Venice C. M. Broks-van den Berg, Teunis B. H. Geijtenbeek and Yvette van Kooyk3

Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DCs) are the most potent APCs of the immune system that seed the peripheral tissues and lymphoid organs. In an immature state, DCs sample their surroundings for incoming pathogens. Upon Ag encounter, DCs mature and migrate to the lymph node to induce adaptive immune responses. The C-type macrophage galactose-type lectin (MGL), expressed in immature DCs, mediates binding to glycoproteins carrying GalNAc moieties. In the present study, we demonstrate that MGL ligands are present on the sinusoidal and lymphatic endothelium of lymph node and thymus, respectively. MGL binding strongly correlated with the expression of the preferred MGL ligand, {alpha}-GalNAc-containing glycan structures, as visualized by staining with the {alpha}-GalNAc-specific snail lectin Helix pomatia agglutinin. MGL+ cells were localized in close proximity of the endothelial structures that express the MGL ligand. Strikingly, instead of inducing migration, MGL mediated retention of human immature DCs, as blockade of MGL interactions enhanced DC trafficking and migration. Thus, MGL+ DCs are hampered in their migratory responses and only upon maturation, when MGL expression is abolished; these DCs will be released from their MGL-mediated restraints.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The correct positioning of immune cells is crucial for the orchestration of effective immune responses. This involves not only the regulation of cell positioning at strategic sites in the body but also the control of cell mobility, allowing appropriate interactions to occur at the right time and the right place. Different protein families, including chemokines, chemokine receptors, and integrins, play crucial roles in regulating immune cell mobility. Functional defects in any of these proteins can impair the migratory capacities of dendritic cells (DCs)4 or lymphocytes and cause varied levels of immune dysregulation (1, 2, 3). In addition, members of the C-type lectin family can participate in the recruitment of immune cells into tissues or lymphoid organs. The important role for C-type lectins in leukocyte migration is exemplified by the leukocyte adhesion deficiency II (also known as congenital disorder of glycosylation IIc), in which the selectin ligand, the sialyl-Lewis X structure, is critically absent from leukocytes. The lack of sialyl-Lewis X leads to impaired tethering and rolling of immune cells on endothelial expressed selectins, thus resulting in a severe immune deficiency (4).

Professional APCs, such as DCs, are seeded throughout all peripheral tissues and in the lymph nodes (LNs) where they scan their surroundings for incoming pathogens or changes in tissue homeostasis (5). DCs play an essential role in the uptake of self and pathogenic Ags. In addition to immunity, DCs contribute to tolerance by induction of T cell unresponsiveness (6, 7), apoptosis, or induction of regulatory T cells. Recently, we reported that the C-type lectin macrophage galactose-type lectin (MGL) on immature DCs is involved in mediating down-regulation of effector T cell function and T cell death by interaction with CD45 avoiding potentially harmful T cell activation (8). Several lines of evidence suggest that MGL may also be involved in the trafficking of APC that express this {alpha}/β-GalNAc-specific lectin (9, 10, 11, 12).

In a search for novel self-ligands for MGL, we observed that sinusoidal and lymphatic endothelium of LN and thymus express MGL counterstructures containing {alpha}-GalNAc moieties. Interactions of MGL+ DCs with HMEC-1 endothelial cells did not induce DC migration; instead, the DCs were retained through {alpha}-GalNAc restraints. More strikingly, blocking MGL function increased DC emigration from skin explants. We hypothesize that in contrast to receptors that drive DC migration, MGL-{alpha}-GalNAc interactions facilitate retention and reduce the egress of immature DCs out of lymphoid organs, allowing them to take up Ags from blood or lymph.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells and reagents

Immature monocyte-derived DCs were cultured for 4–7 days from monocytes obtained from buffy coats of healthy donors (Sanquin) in the presence of IL-4 and GM-CSF (500 and 800 U/ml, respectively; Biosource). HMEC-1 cells were cultured in MCDB 131 medium (Invitrogen) supplemented with 10% FCS, 10 ng/ml epidermal growth factor, and 1 µg/ml hydrocortisone. HUVECs were isolated as previously described (13) and cultured in M199 medium (Cambrex) supplemented with 10% human serum, 10% newborn calf serum, 5 U/ml heparin, and 5 ng/ml basic fibroblast growth factor. Polyacrylamide (PAA)-coupled glycoconjugates were purchased from Lectinity. Biotinylated Helix pomatia agglutinin (HPA) was purchased form Sigma-Aldrich. Biotinylated Maackia amurensis agglutinin (MAA), Sambucus nigra agglutinin (SNA), and UEA-1 were purchased from Vector Laboratories. MGL-Fc and DC-SIGN-Fc, both containing the human IgG1 Fc domain, were generated as previously described (9). An MGL-murine Fc fusion protein was generated by cloning the extracellular part of MGL into a pcDNA3 expression vector containing exon 1–3 of murine IgG2a-Fc (14). MGL-mFc was produced by transient transfection in Chinese hamster ovary cells and MGL-mFc concentrations were determined by ELISA.

ELISA-based MGL-Fc binding assays

PAA-glycoconjugates were coated at 5 µg/ml on NUNC maxisorb plates overnight at room temperature. Plates were blocked with 1% BSA and MGL-mFc was added (0.5 µg/ml) for 2 h at room temperature in the presence or absence of 10 mM EGTA. Binding was detected using a peroxidase-labeled anti-mouse IgG Fc Ab (Jackson ImmunoResearch Laboratories).

Immunohistochemistry

Cryosections of healthy tissues (7 µm) were fixed with 2% paraformaldehyde. MGL-mFc (25 µg/ml), anti-MGL (18E4, 10 µg/ml), anti-HLA-DR (Santa Cruz Biotechnology), or rabbit polyclonal anti-MGL (generated by immunization with the following C-terminal peptide HWVCEAGLGQTSQESH) were added in TSM buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2) and incubated for 2 h at 37°C. Binding was detected with an Alexa 594- or Alexa 488-conjugated goat anti-mouse IgG2A-specific Ab, goat anti-rat or goat anti-rabbit Abs (Molecular Probes). To visualize the presence of {alpha}-GalNAc epitopes, sections were incubated with HPA (5 µg/ml) for 2 h at 37°C. HPA binding was detected using an Alexa 488-conjugated streptavidin (Molecular Probes). Where indicated, sections were costained using primary mouse Abs to LYVE-1 (15, 16) (lymphatic/sinusoidal endothelium) for 1 h at room temperature, followed by a secondary Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG1-specific Ab (Molecular Probes). Sections were counterstained using Hoechst.

Flow cytometry and MGL-Fc binding

MGL and MHC II protein expression was determined by incubating cells with primary Ab (18E4 and Q5/13, respectively, 5 µg/ml), followed by staining with a secondary FITC-labeled goat anti-mouse Ab (Zymed) and analyzed on FACScalibur (BD Biosciences). To assess the expression of carbohydrate epitopes on the cell surface, cells were incubated with 10 µg/ml of the biotinylated lectins in TSM supplemented with 0.5% BSA for 30 min at 37°C, followed by staining with a Alexa 488-conjugated streptavidin (Molecular Probes) and analyzed on FACSCalibur. To analyze MGL or DC-SIGN ligand expression, cells were incubated with MGL-Fc or DC-SIGN-Fc (10 µg/ml) in TSM supplemented with 0.5% BSA for 30 min at 37°C, followed by staining with a secondary FITC-labeled anti-human IgG Fc Ab (Jackson ImmunoResearch Laboratories) and analyzed on FACSCalibur. In blocking experiments, Fc-proteins were preincubated for 15 min at room temperature with 10 mM EGTA, 100 mM free carbohydrates (Sigma-Aldrich), 20 µg/ml of the lectins, or 20 µg/ml blocking Abs.

Migration assays

Transwell 24-well plates (8 µm pore; Greiner Bio-one) were coated with 1% gelatin for 1 h at 37°C. HMEC-1 cells (70,000) were seeded on the inserts and, after 24 h, 200,000 DCs was added to the monolayer of endothelial HMEC-1 cells. The lower chamber contained human RANTES (100 ng/ml; Biosource). After 2 h at 37°C, the number of transmigrated DCs (lower chamber) was determined by flow cytometry. Transendothelial migration was measured in the presence or absence of blocking or isotype-matched Abs (20 µg/ml) or free carbohydrates. DC migration on coated PAA-glycoconjugates (5 µg/ml) was studied using time-lapse video microscopy. A total of 45,000 DCs was added to the plates, allowed to settle for 30 min, and the number of migrating DC was assessed for a 30-min period. The migration assay was conducted in the absence and presence blocking Abs (20 µg/ml). Migration was scored as the percentage of cells displaying spatial movement on the coating, accompanied with changes in cell shape.

Ex vivo skin explant model

Normal healthy adult skin was obtained from plastic surgery after informed consent from all donors and used within 3 h after the operation. The 3-mm-thick slices of skin, containing both epidermis and dermis, were sliced using a dermatome. Slices of ~1 cm2 were cut and floated dermis-side down in IMDM, 10% FCS, and gentamycine. Blocking Abs to MGL or isotype control Abs were added at 100 µg/ml. After overnight incubation at 37°C, skin pieces were removed and emigrant DCs were counted by flow cytometry. Experiments were performed in triplicate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
MGL ligands can be detected in healthy human LN and thymus

To explore the distribution of MGL ligands in tissue, we generated a recombinant protein consisting of the extracellular domain of MGL fused to the mouse IgG2a Fc tail. To confirm that the recombinant MGL-mFc has similar binding properties as cellular MGL (9), MGL-mFc binding assays were performed to coated PAA-glycoconjugates. MGL-mFc recognized both {alpha}-GalNAc and the LacdiNAc (GalNAcβ1–4GlcNAc) epitope (Fig. 1). MGL-mediated binding was completely inhibited in the presence of the Ca2+-chelator EGTA, demonstrating the involvement of the MGL carbohydrate recognition domain. MGL-mFc did not interact with Lewis X or mannose, structures that were recognized by the control DC-SIGN-Fc. Thus, MGL-mFc displays an identical carbohydrate recognition profile as MGL expressed by human immature DCs (9).


Figure 1
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  		FIGURE 1. MGL-mFc recognizes {alpha}-GalNAc and LacdiNAc structures, whereas DC-SIGN-Fc interacts with mannose and Lewis X. MGL-mFc and DC-SIGN-Fc binding was determined by an ELISA-based assay. SD < 0.05, OD 450 nm. One representative experiment of three is shown.

 
Next, cryosections of healthy human tissues were incubated with MGL-mFc to investigate the expression of potential MGL ligands in situ. In LN and thymus, MGL ligands could be identified on the lymphatic endothelium (Fig. 2). In addition to the lymph vessels in the intralobular space of the thymus, MGL-mFc also stained a few small blood vessels and a subpopulation of cells in the thymic medullar region (Fig. 2B and data not shown). No staining was observed using mouse IgG2A Abs, containing the same Fc tail as MGL-mFc (data not shown). MGL-mFc staining was completely abolished in the presence of free GalNac monosaccharides, demonstrating the dependence on the MGL carbohydrate recognition domain for binding (Fig. 2).


Figure 2
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  		FIGURE 2. MGL ligands can be detected in human LN and thymus. MGL-mFc (red) specifically interacted with the sinusoidal endothelium (LYVE-1, green) of LN (A) and with the lymphatic endothelium (LYVE-1, green) of the thymus (B). No staining was observed in the presence of free GalNAc monosaccharides, showing the specificity of the MGL binding. Nuclei are visualized in blue. One of two donors is shown. Original magnifications x200 and x400.

 
To explore a potential functional relationship between MGL protein and MGL ligand expression, the distribution of MGL was analyzed in the same tissues that harbor the MGL ligands. MGL protein expression in LN has been reported (8); however, the presence of MGL in the thymus has so far not been demonstrated. In both LN and thymus, the MGL protein and its ligands were present on different cell populations that were situated in close contact to each other (Fig. 3). In LN, MGL+ cells are located just below and in between the sinusoidal endothelial cells (Fig. 3), whereas in thymus MGL+ cells are associated with the lymph vessels in the interlobular space (Fig. 3).


Figure 3
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  		FIGURE 3. Distribution of MGL+ cells and sinusoidal or lymphatic endothelial cells in LN and thymus, respectively. Human tissue sections of LN and thymus were stained with anti-MGL (red) and LYVE-1 (green). Nuclei are visualized in blue. Sin. EC, sinusoidal endothelial cells; OZ, outer zone of the paracortex, Lymph. EC, lymphatic endothelial cells; C, cortex; IS, interlobular space. One of two donors is shown. Original magnification x400.

 
Thus, the MGL protein and its ligands are expressed in close proximity at similar anatomical locations in the LN and the thymus. The specific MGL-mFc binding to lymph vascular structures suggests a role for MGL in the adhesion and migration of DCs across the lymphatic and sinusoidal endothelium.

MGL specifically interacts with endothelial cells in an {alpha}-GalNAc-dependent manner

Previous studies have demonstrated a strong correlation between MGL binding and the expression of glycan epitopes recognized by the {alpha}-GalNAc-specific roman snail lectin HPA (9). First, we explored whether the distribution of MGL ligands in tissue also correlated with HPA reactivity. Since HPA can block MGL binding (Fig. 5D), we performed a double labeling on tissues by using HPA in combination with the specific endothelial markers. Strikingly, although HPA stained some additional vessels in LN and thymus (17), it displayed a complete overlap with the sinusoidal and lymphatic endothelium (Fig. 4). These subpopulations of endothelial cells were also recognized by MGL (Fig. 2). The GalNAc block of MGL binding to tissues (Fig. 2) combined with the specific HPA staining (Fig. 4) indicates that MGL interacts with endothelial cells in LN or thymus in an {alpha}-GalNAc-dependent manner.


Figure 5
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  		FIGURE 5. MGL specifically recognizes HMEC-1 endothelial cells. Binding of MGL-Fc and DC-SIGN-Fc to primary HUVEC (A) or HMEC-1 (B) was determined by flow cytometry. MGL-Fc binding to HMEC-1 can be blocked by EGTA, free GalNAc monosaccharides and anti-MGL Abs. C, HMEC-1 cells express {alpha}-GalNAc, {alpha}1–2 linked fucose, {alpha}2–6 linked sialic acid, and {alpha}2–3 linked sialic acid, as stained by the lectins HPA, UEA-1, SNA, and MAA, respectively. D, MGL-Fc binding can be partially blocked by the {alpha}-GalNAc-specific lectin HPA. *, Significant difference between the MGL-Fc binding and the MGL-Fc binding in the presence of HPA (p < 0.05). All results are representative for three independent experiments.

 

Figure 4
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  		FIGURE 4. Endothelial structures express GalNAc epitopes as visualized by HPA staining. HPA (green) specifically interacted with the sinusoidal endothelium (LYVE-1, red) of LN (A) and lymphatic endothelium (LYVE-1, red) in thymus (B). Original magnification x400.

 
To further explore a potential role for MGL in DC migration, we examined binding of MGL to cultured endothelial cells. HUVECs, a model system for endothelial cells lining blood vessels, were not bound by MGL-Fc nor by the DC-SIGN-Fc control (Fig. 5A). TNF-{alpha}, IL-4, or IFN-{gamma} stimulation of HUVECs did not up-regulate any MGL ligand expression (data not shown). These results are in agreement with the data presented in Fig. 2, in which we did not observe any MGL-mFc binding to endothelial cells lining small blood vessels in LN. In contrast, HMEC-1 cells, a human endothelial cell line that expresses several lymphatic endothelial markers (18), strongly interacted with MGL-Fc (Fig. 5B). MGL binding could be blocked by the addition of the Ca2+-chelator EGTA, free GalNAc monosaccharides or anti-MGL Abs, demonstrating the specificity of this interaction. We observed only low binding of the DC-SIGN-Fc control to HMEC-1 cells (Fig. 5B).

To investigate the nature of the MGL ligand on these cells, we assessed by flow cytometry, using well-characterized plant/invertebrate lectins, which glycan epitopes are present on HMEC-1 cells. Indeed, HMEC-1 expressed HPA-reactive glycan structures (Fig. 5C). HPA binding could be blocked by the addition of free GalNAc, indicating that HPA recognized {alpha}-GalNAc containing glycans on the HMEC-1 cells. In addition, {alpha}1–2 linked fucose, {alpha}2–3 and {alpha}2–6 linked sialic acid structures are present on HMEC-1 cells, as visualized by the reactivity of UEA-1, SNA, and MAA, respectively (Fig. 5C). However, when these lectins were used to block MGL-Fc binding, only HPA could significantly interfere with the MGL-HMEC-1 interaction, suggesting that an {alpha}-GalNAc-containing glycoprotein or–lipid constitutes as the ligand for MGL on HMEC-1 cells (Fig. 5D). Since HPA could only inhibit MGL binding for 50%, additional MGL binding determinants on HMEC-1 cells exist that are probably composed of terminal β–GalNAc structures, the other high affinity ligand for MGL (9).

MGL mediates retention of immature DCs

To evaluate a possible involvement of MGL in DC migration, immature DCs were cultured from monocytes. Immature DCs expressed moderate levels of MGL (Fig. 6A). Next, we measured the capacity of these DCs to transmigrate across an HMEC-1 monolayer. In response to the chemokine RANTES, transmigration of immature DCs was enhanced ~3-fold and largely dependent on β2-integrins, as shown by the block using anti-β2 Abs (Fig. 6B) (19). Strikingly, anti-MGL and the addition of free GalNAc significantly increased transmigration compared with isotype control Abs and the control monosaccharide GlcNAc, respectively (Fig. 6, B and C).


Figure 6
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  		FIGURE 6. MGL mediates retention of immature DCs. A, MGL expression on immature DCs was determined by flow cytometry. Open histogram, isotype control; filled histogram, MGL staining. B and C, Transendothelial migration of immature DCs across an HMEC-1 monolayer in response to a RANTES gradient in the presence of Abs to MGL, β2-integrin, isotype control Ab or free carbohydrates. * and **, Significant difference between the isotype control and the anti-MGL (p < 0.05) and the GalNAc block and GlcNAc control (p < 0.01), respectively. D and E, DC migration on glycoconjugate-coated plates was measured using time-lapse video microscopy. Anti-MGL Abs increase DC migration on GalNAc, whereas they have no effect on the Lewis X coating. **, Significant difference between isotype control and the anti-MGL Abs (p < 0.01). All results are representative for three independent experiments.

 
To study whether {alpha}-GalNAc is sufficient to support DC migration, we followed DC mobility on glycoconjugate-coated plates with time-lapse videomicroscopy. Migration was scored as the percentage of cells displaying spatial movement on the coating, accompanied with changes in cell shape. For each condition, over 100 cells were counted. DCs did not interact with the glucitol or galactose coating (Fig. 6D). DCs firmly adhered to Lewis X and Man3, which inhibited DC mobility. In contrast, on the GalNAc-coating, DCs displayed random movement on the surface, while continuously interacting with the coating (Fig. 6D). Similar to the transmigration experiments, anti-MGL Abs significantly increased the percentage of mobile DCs on the GalNAc-coating (Fig. 6E). The increased motility was not due to a nonspecific effect of the Abs on the immature DCs, as isotype control Abs did not enhance migration. Furthermore, the anti-MGL Abs did not augment DC mobility on Lewis X (Fig. 6E). DCs incubated with anti-MGL Abs still interacted with the GalNAc-coated surface, suggesting that immature DCs express another GalNAc receptor in addition to MGL. Thus, MGL-mediated interactions impair the migratory abilities of immature DCs.

To validate our results in a more physiological setting we used the ex vivo model of spontaneous emigration of DCs from cultured skin explants. Numerous HLA-DR+MGL+ cells could be detected in the dermal layer of human skin (Fig. 7A). The MGL+ cells also expressed CD1a, CD11b, and CD11c, demonstrating that they represent dermal DCs (data not shown) (8). Also MGL ligands could be identified in the dermis (Fig. 7B, white arrows). No staining was observed in the presence of free GalNAc monosaccharides, demonstrating the specificity of the MGL binding. The reactivity of MGL-mFc with the keratinocytes in the epidermis is likely not functionally relevant, as no MGL+ cells are present in the epidermis. MGL specifically interacted with the HPA+ connective tissue surrounding dermal blood vessels (Fig. 7C).


Figure 7
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  		FIGURE 7. DC emigration from skin explants. A and B, Distribution of HLA-DR+ (A, green) and MGL+ cells (A, red) and MGL ligands (B, red) in skin was determined by immunohistochemistry. No staining was observed in the presence of free GalNAc monosaccharides, showing the specificity of the MGL binding. Nuclei are visualized in blue. D, Dermis; ED, epidermis. C, MGL ligands surround endothelial structures in human skin. MGL-mFc (red) and GalNAc epitopes (HPA staining, green) specifically enclose endothelial cells (green/red). All original magnifications x400. D, DCs emigrated from skin explants express MHC II and MGL. MGL and MHC II expression were determined by flow cytometry and immunohistochemistry on cytospins of emigrated skin DCs. Open histogram, isotype control; filled histogram, MGL or MHC II staining. E, Emigration of skin DCs was significantly enhanced in the presence of anti-MGL Abs. *, Significant difference between isotype control and the anti-MGL Abs (p < 0.05). Results are representative for three independent experiments.

 
After overnight culture, DCs that emigrated 1 cm2 skin pieces were counted and characterized. All emigrated DCs expressed high levels of MHC class II and a subset of the DCs was also MGL positive (Fig. 7D). Although we could detect MGL+ DC in the emigrated population, their numbers were slightly lower than expected. This can be explained by the DC maturation that accompanies the emigration from skin explants (20). DC maturation abrogates MGL expression and thus reduces the final numbers of DCs that are still MGL positive after completing the emigration from skin (8). Similar to our in vitro migration assays, blocking MGL function by the addition of blocking Abs significantly enhanced the number of emigrating DCs (Fig. 7E). No significant differences could be observed if the skin pieces were incubated with isotype control Abs, anti-DC-SIGN or anti-LFA-1 Abs (Fig. 7E and data not shown). The emigration of skin-resident T cells was not altered in the presence of anti-MGL Abs (data not shown).

Based on our in vitro migration experiments, it is likely that expression of MGL on immature DCs and the presence of GalNAc moieties hamper DC migration and favor DC retention in lymphoid tissues. Blocking MGL function or down-regulation of expression, due to DC maturation (8), can relieve DCs from the GalNAc constraints and improve the migratory capacities of DCs.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In recent years, our understanding on DC trafficking has greatly increased, yet little is known about which mechanisms actively retain immature DCs in tissue (21, 22). One molecule that has been implicated in selective DC retention is the junctional adhesion molecule-A. Junctional adhesion molecule-A–/– DCs showed an increase in random migration and transmigration across lymphatic endothelial cells (23). In this study, we demonstrate that the C-type lectin MGL facilitates {alpha}-GalNAc-dependent retention of immature DCs. Blocking MGL function enhanced the migratory capacities of DCs. It is tempting to speculate that MGL-GalNAc interactions are involved in maintaining the localization of immature DCs at strategic sites and thus prevent DC egress.

Several C-type lectins have been shown to be involved in the migration of leukocytes. DC-SIGN binding to endothelial ICAM-2 mediates recruitment of DC precursors to the peripheral tissues (24). Similarly, the C-type lectin family of selectins facilitates lymphocyte homing and entry into lymphoid tissues and sites of inflammation (25). MGL interactions do not facilitate migration; instead, MGL seems to mediate retention of DCs in tissue, a function previously unknown to C-type lectin family members. Strikingly, bone marrow-derived macrophages from mannose receptor-deficient mice display an enhanced random motility (26), similar to immature DCs on which MGL binding has been blocked. Thus, both MGL and mannose receptor have an inhibitory role during the migration of APCs.

Our data seems to contradict previous reports showing that in mice, mMGL can support migration of mouse mMGL+ DCs. Although we observed increased DC emigration from human skin explants in the presence of blocking anti-MGL Abs, anti-mouse MGL Abs blocked the migration of mMGL+ dermal cells from skin (12). Since mMGL possesses a broader carbohydrate recognition profile compared to human MGL (9, 27), mMGL might possess additional ligands that support the migratory function of mMGL. Migrating mouse Langerhans cells up-regulate mMGL expression during lymphatic transit, suggesting a role for mMGL in lymph vessel migration (10). However, the interaction between mMGL and the lymphatics could also slow down the Langerhans cells, as we have observed for human immature DCs, allowing full maturation to occur before these cells reach the skin-draining LN. In accordance with such process, it seems logical that mature Langerhans cells in the LN have lost all surface expression of mMGL (10).

In the thymus and LN, MGL specifically recognized the lymphatic and sinusoidal endothelium, respectively, indicating that in situ those endothelial cells express GalNAc structures that serve as MGL binding determinants. Normally, MGL expression can only be detected on immature DCs (8). Immature DCs are highly abundant in lymphoid organs, as all thymic and half of the LN-resident DC populations display an immature phenotype (28). These immature DCs have been implicated in maintaining peripheral tolerance in steady-state and the induction of appropriate immune responses during infections. Recently, Bonasio et al. (29) showed that circulating DCs home to the thymus via the blood and there they contribute to the induction of central tolerance. Moreover, a small percentage of peripheral DCs can re-enter circulation through efferent lymphatics (30). Thus, MGL binding to lymphatic vessels in the thymus could impair DCs from exiting the thymus, allowing these immature DCs to interact with developing thymocytes undergoing antigenic selection.

LN sinuses have been proposed to function as molecular sieves for filtering lymph-borne soluble or shed Ags (16, 31). Lymph-borne Ags appear to be excluded from the paracortex, thus requiring a cell type that captures and transports these Ags to the T cell compartment. The close proximity of MGL+ cells to the sinusoidal endothelial cells suggests that these immature MGL+ APCs are ideally positioned for capturing Ags from lymph. Upon DC maturation, MGL expression is lost (8), thus releasing the DC from its GalNac restraints. The maturing DCs are then able to migrate to the T cell area for efficient Ag presentation to T cells. Alternatively, Ag could be carried by migratory DCs and subsequently be transferred to LN-resident DCs (32). For some Ags the migratory DCs seem to act as simple ferries, while efficient CD8+ T cell priming requires the transfer of Ag to LN-resident potentially MGL+ DCs (33, 34). Furthermore, we demonstrated that MGL can also function as an genuine Ag uptake receptor that facilitates Ag presentation in MHC class II (35). Thus, the MGL-sinus interaction would certainly retain the immature DCs at their strategic localization.

In skin, MGL interacted with the connective tissue surrounding the small blood vessels. Precursor DCs enter the skin via the bloodstream. Binding of these extracellular matrix components might avert DCs from transmigrating back into blood. The exact nature of the adhesive processes modulated by MGL is currently unclear. Clearly, MGL-mediated retention can counteract β2-integrin function, as the transmigration of DCs across HMEC-1 was completely β2-integrin dependent. One might speculate that MGL-GalNAc interactions invoke outside-in signaling, thus modulating integrin affinity or avidity. However, the MGL Abs used did not alter LFA-1-mediated adhesion to ICAM-1 (data not shown). Further studies are needed to identify the endothelial glycoproteins or lipids that carry the GalNAc determinants for MGL binding and how these retention molecules are regulated. These insights will also provide more knowledge on how the MGL-GalNAc interaction influences the adhesive properties of DCs.

In contrast, MGL-mediated retention could also hamper the induction of appropriate immune responses. Several adenocarcinomas show aberrant glycosylation patterns, whereby short O-glycans, such as the Tn Ag ({alpha}-GalNAc-Ser/Thr), are highly up-regulated (36). This Tn Ag is one of the high affinity carbohydrate ligands for human MGL. Thus, the tumor microenvironment might prevent egress of MGL+ APCs and indeed we observed large infiltrates of MGL+ DCs within human colon carcinomas (37). The defective migration of DCs could hamper effective cross-priming of tumor Ags. Moreover, the remaining MGL+ DCs would then be capable of blocking the cytolytic function of incoming tumor-specific cytotoxic T cells (8).

In summary, our results provide evidence for an interaction between MGL+ immature DCs and GalNac epitopes present on lymphatic endothelial structures in LN and thymus that point to an impairment of DC exit from these tissues mediated by MGL. Further studies will be needed to identify the endothelial glycoproteins or lipids that carry these GalNAc determinants for MGL binding and how the expression of these retention molecules and carbohydrate determinants is regulated.


   Acknowledgments
 
We thank S. van der Pol for her assistance with the time-lapse video microscopy and Dr. L. de Witte and Drs. M. de Jong for their collaboration on the skin explant model.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by an Netherlands Organization for Scientific Research Pionier Grant 900-02-002. Back

2 S.J.v.V. and L.C.P. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Yvette van Kooyk, Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Center, PO Box 7057, 1007MB Amsterdam, The Netherlands. E-mail address: y.vankooyk{at}vumc.nl Back

4 Abbreviations used in this paper: DC, dendritic cell; HPA, Helix pomatia agglutinin; LN, lymph node; MAA, Maackia amurensis agglutinin; MGL, macrophage galactose-type lectin; SNA, Sambucus nigra agglutinin; PAA, Polyacrylamide. Back

Received for publication December 18, 2007. Accepted for publication July 3, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Gunn, M. D., S. Kyuwa, C. Tam, T. Kakiuchi, A. Matsuzawa, L. T. Williams, H. Nakano. 1999. Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization. J. Exp. Med. 189: 451-460. [Abstract/Free Full Text]
  2. Martin-Fontecha, A., S. Sebastiani, U. E. Hopken, M. Uguccioni, M. Lipp, A. Lanzavecchia, F. Sallusto. 2003. Regulation of dendritic cell migration to the draining lymph node: impact on T lymphocyte traffic and priming. J. Exp. Med. 198: 615-621. [Abstract/Free Full Text]
  3. Corbi, A. L., A. Vara, A. Ursa, M. C. Garcia Rodriguez, G. Fontan, F. Sanchez-Madrid. 1992. Molecular basis for a severe case of leukocyte adhesion deficiency. Eur. J. Immunol. 22: 1877-1881. [Medline]
  4. Yakubenia, S., M. K. Wild. 2006. Leukocyte adhesion deficiency II. FEBS J. 273: 4390-4398. [Medline]
  5. Banchereau, J., R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392: 245-252. [Medline]
  6. Steinman, R. M., D. Hawiger, M. C. Nussenzweig. 2003. Tolerogenic dendritic cells. Annu. Rev. Immunol. 21: 685-711. [Medline]
  7. Steinman, R. M.. 2003. The control of immunity and tolerance by dendritic cell. Pathol. Biol. 51: 59-60.
  8. van Vliet, S. J., S. I. Gringhuis, T. B. Geijtenbeek, Y. van Kooyk. 2006. Regulation of effector T cells by antigen-presenting cells via interaction of the C-type lectin MGL with CD45. Nat. Immunol. 7: 1200-1208. [Medline]
  9. van Vliet, S. J., E. van Liempt, E. Saeland, C. A. Aarnoudse, B. Appelmelk, T. Irimura, T. B. Geijtenbeek, O. Blixt, R. Alvarez, I. van Die, Y. van Kooyk. 2005. Carbohydrate profiling reveals a distinctive role for the C-type lectin MGL in the recognition of helminth parasites and tumor antigens by dendritic cells. Int. Immunol. 17: 661-669. [Abstract/Free Full Text]
  10. Dupasquier, M., P. Stoitzner, H. Wan, D. Cerqueira, A. van Oudenaren, J. S. Voerman, K. Denda-Nagai, T. Irimura, G. Raes, N. Romani, P. J. Leenen. 2006. The dermal microenvironment induces the expression of the alternative activation marker CD301/mMGL in mononuclear phagocytes, independent of IL-4/IL-13 signaling. J. Leukocyte Biol. 80: 838-849. [Abstract/Free Full Text]
  11. Sato, K., Y. Imai, T. Irimura. 1998. Contribution of dermal macrophage trafficking in the sensitization phase of contact hypersensitivity. J. Immunol. 161: 6835-6844. [Abstract/Free Full Text]
  12. Chun, K. H., Y. Imai, N. Higashi, T. Irimura. 2000. Migration of dermal cells expressing a macrophage C-type lectin during the sensitization phase of delayed-type hypersensitivity. J. Leukocyte Biol. 68: 471-478. [Abstract/Free Full Text]
  13. Beekhuizen, H., R. van Furth. 1994. Growth characteristics of cultured human macrovascular venous and arterial and microvascular endothelial cells. J. Vasc. Res. 31: 230-239. [Medline]
  14. Backstrom, M., T. Link, F. J. Olson, H. Karlsson, R. Graham, G. Picco, J. Burchell, J. Taylor-Papadimitriou, T. Noll, G. C. Hansson. 2003. Recombinant MUC1 mucin with a breast cancer-like O-glycosylation produced in large amounts in Chinese-hamster ovary cells. Biochem. J. 376: 677-686. [Medline]
  15. Banerji, S., J. Ni, S. X. Wang, S. Clasper, J. Su, R. Tammi, M. Jones, D. G. Jackson. 1999. LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J. Cell Biol. 144: 789-801. [Abstract/Free Full Text]
  16. Engering, A., S. J. van Vliet, K. Hebeda, D. G. Jackson, R. Prevo, S. K. Singh, T. B. Geijtenbeek, H. van Krieken, Y. van Kooyk. 2004. Dynamic populations of dendritic cell-specific ICAM-3 grabbing nonintegrin-positive immature dendritic cells and liver/lymph node-specific ICAM-3 grabbing nonintegrin-positive endothelial cells in the outer zones of the paracortex of human lymph nodes. Am. J. Pathol. 164: 1587-1595. [Abstract/Free Full Text]
  17. Paessens, L. C., J. J. Garcia-Vallejo, R. J. Fernandes, Y. van Kooyk. 2007. The glycosylation of thymic microenvironments: a microscopic study using plant lectins. Immunol. Lett. 110: 65-73. [Medline]
  18. Nisato, R. E., J. A. Harrison, R. Buser, L. Orci, C. Rinsch, R. Montesano, P. Dupraz, M. S. Pepper. 2004. Generation and characterization of telomerase-transfected human lymphatic endothelial cells with an extended life span. Am. J. Pathol. 165: 11-24. [Abstract/Free Full Text]
  19. D'Amico, G., G. Bianchi, S. Bernasconi, L. Bersani, L. Piemonti, S. Sozzani, A. Mantovani, P. Allavena. 1998. Adhesion, transendothelial migration, and reverse transmigration of in vitro cultured dendritic cells. Blood 92: 207-214. [Abstract/Free Full Text]
  20. Angel, C. E., E. George, A. E. Brooks, L. L. Ostrovsky, T. L. Brown, P. R. Dunbar. 2006. Cutting edge: CD1a+ antigen-presenting cells in human dermis respond rapidly to CCR7 ligands. J. Immunol. 176: 5730-5734. [Abstract/Free Full Text]
  21. Randolph, G. J., V. Angeli, M. A. Swartz. 2005. Dendritic-cell trafficking to lymph nodes through lymphatic vessels. Nat. Rev. Immunol. 5: 617-628. [Medline]
  22. Cavanagh, L. L., U. H. von Andrian. 2002. Travellers in many guises: the origins and destinations of dendritic cells. Immunol. Cell Biol. 80: 448-462. [Medline]
  23. Cera, M. R., A. Del Prete, A. Vecchi, M. Corada, I. Martin-Padura, T. Motoike, P. Tonetti, G. Bazzoni, W. Vermi, F. Gentili, et al 2004. Increased DC trafficking to lymph nodes and contact hypersensitivity in junctional adhesion molecule-A-deficient mice. J. Clin. Invest. 114: 729-738. [Medline]
  24. Geijtenbeek, T. B., D. J. Krooshoop, D. A. Bleijs, S. J. van Vliet, G. C. van Duijnhoven, V. Grabovsky, R. Alon, C. G. Figdor, Y. van Kooyk. 2000. DC-SIGN-ICAM-2 interaction mediates dendritic cell trafficking. Nat. Immunol. 1: 353-357. [Medline]
  25. Ley, K., G. S. Kansas. 2004. Selectins in T-cell recruitment to non-lymphoid tissues and sites of inflammation. Nat. Rev. Immunol. 4: 325-335. [Medline]
  26. Sturge, J., S. K. Todd, G. Kogianni, A. McCarthy, C. M. Isacke. 2007. Mannose receptor regulation of macrophage cell migration. J. Leukocyte Biol. 82: 585-593. [Abstract/Free Full Text]
  27. Tsuiji, M., M. Fujimori, Y. Ohashi, N. Higashi, T. M. Onami, S. M. Hedrick, T. Irimura. 2002. Molecular cloning and characterization of a novel mouse macrophage C-type lectin, mMGL2, which has a distinct carbohydrate specificity from mMGL1. J. Biol. Chem. 277: 28892-28901. [Abstract/Free Full Text]
  28. Wilson, N. S., D. El Sukkari, G. T. Belz, C. M. Smith, R. J. Steptoe, W. R. Heath, K. Shortman, J. A. Villadangos. 2003. Most lymphoid organ dendritic cell types are phenotypically and functionally immature. Blood 102: 2187-2194. [Abstract/Free Full Text]
  29. Bonasio, R., M. L. Scimone, P. Schaerli, N. Grabie, A. H. Lichtman, U. H. von Andrian. 2006. Clonal deletion of thymocytes by circulating dendritic cells homing to the thymus. Nat. Immunol. 7: 1092-1100. [Medline]
  30. Bonasio, R., U. H. von Andrian. 2006. Generation, migration, and function of circulating dendritic cells. Curr. Opin. Immunol. 18: 503-511. [Medline]
  31. Gretz, J. E., C. C. Norbury, A. O. Anderson, A. E. Proudfoot, S. Shaw. 2000. Lymph-borne chemokines and other low molecular weight molecules reach high endothelial venules via specialized conduits while a functional barrier limits access to the lymphocyte microenvironments in lymph node cortex. J. Exp. Med. 192: 1425-1440. [Abstract/Free Full Text]
  32. Inaba, K., S. Turley, F. Yamaide, T. Iyoda, K. Mahnke, M. Inaba, M. Pack, M. Subklewe, B. Sauter, D. Sheff, et al 1998. Efficient presentation of phagocytosed cellular fragments on the major histocompatibility complex class II products of dendritic cells. J. Exp. Med. 188: 2163-2173. [Abstract/Free Full Text]
  33. Allan, R. S., J. Waithman, S. Bedoui, C. M. Jones, J. A. Villadangos, Y. Zhan, A. M. Lew, K. Shortman, W. R. Heath, F. R. Carbone. 2006. Migratory dendritic cells transfer antigen to a lymph node-resident dendritic cell population for efficient CTL priming. Immunity 25: 153-162. [Medline]
  34. Belz, G. T., C. M. Smith, L. Kleinert, P. Reading, A. Brooks, K. Shortman, F. R. Carbone, W. R. Heath. 2004. Distinct migrating and nonmigrating dendritic cell populations are involved in MHC class I-restricted antigen presentation after lung infection with virus. Proc. Natl. Acad. Sci. USA 101: 8670-8675. [Abstract/Free Full Text]
  35. van Vliet, S. J., C. A. Aarnoudse, V.C. Broks-van den Berg, M. Boks, T. B. Geijtenbeek, Y. van Kooyk. 2007. MGL-mediated internalization and antigen presentation by dendritic cells: a role for tyrosine-5. Eur. J. Immunol. 37: 2075-2081. [Medline]
  36. Brockhausen, I.. 1999. Pathways of O-glycan biosynthesis in cancer cells. Biochim. Biophys. Acta 1473: 67-95. [Medline]
  37. Saeland, E., S. J. van Vliet, M. Backstrom, V. C. van den Berg, T. B. Geijtenbeek, G. A. Meijer, Y. van Kooyk. 2007. The C-type lectin MGL expressed by dendritic cells detects glycan changes on MUC1 in colon carcinoma. Cancer Immunol. Immunother. 56: 1225-1236. [Medline]




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