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Originally published online as doi:10.1189/jlb.1005564 on July 18, 2006

Published online before print July 18, 2006
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(Journal of Leukocyte Biology. 2006;80:838-849.)
© 2006 by Society for Leukocyte Biology

The dermal microenvironment induces the expression of the alternative activation marker CD301/mMGL in mononuclear phagocytes, independent of IL-4/IL-13 signaling

Marcel Dupasquier*, Patrizia Stoitzner{dagger}, Hui Wan*, Denise Cerqueira*, Adri van Oudenaren*, Jane S. A. Voerman*, Kaori Denda-Nagai{ddagger}, Tatsuro Irimura{ddagger}, Geert Raes§, Nikolaus Romani{dagger} and Pieter J. M. Leenen*,1

* Department of Immunology, Erasmus MC, Rotterdam, the Netherlands;
{dagger} Department of Dermatology, Innsbruck Medical University, Innsbruck, Austria;
{ddagger} Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan; and
§ Laboratory of Cellular and Molecular Immunology, Department of Molecular and Cellular Interactions, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Brussels, Belgium

1 Correspondence: Department of Immunology, Erasmus MC, Dr. Molewaterplein 50, NL-3015 GE Rotterdam, The Netherlands. E-mail: p.leenen{at}erasmusmc.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recently, we have shown that mononuclear phagocytes comprise the majority of interstitial cells in the mouse dermis, as indicated by their phenotypic and functional characteristics. In particular, these cells express the mouse macrophage galactose-/N-acetylgalactosamine-specificlectin (mMGL)/CD301, identified by the monoclonal antibody ER-MP23, as well as other macrophage markers. As expression of mMGL is induced by IL-4 or IL-13 and is therefore a marker of alternatively activated macrophages, we asked whether dermal mononuclear phagocytes are genuinely alternatively activated. We observed that these cells expressed, next to mMGL, two other alternative activation markers, namely, the mannose receptor/CD206 and Dectin-1. Yet, as this expression profile was similar in IL-4 receptor {alpha} knockout mice, neither IL-4 nor IL-13 signaling appeared to be required for this phenotype. We also found that Langerhans cells (LC), which showed only a low level of mMGL in the epidermis, up-regulated mMGL expression upon migration through the dermis, allowing these cells to internalize limited amounts of mMGL ligands. LC isolated from epidermal preparations did not show this up-regulation when cultured in standard medium, but whole skin-conditioned medium did stimulate mMGL expression by LC. The vast majority of mMGL molecules was present in the cytoplasm, however. LC, which arrived in skin-draining lymph nodes, quickly down-regulated mMGL expression, and dermally derived cells retained significant mMGL levels. Taken together, these data suggest that the dermal microenvironment induces mononuclear phagocyte subpopulations to express mMGL and possibly other markers of alternatively activated macrophages, independent of IL-4/IL-13 signaling.

Key Words: dermis • C-type lectin • Langerhans cells • macrophages


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mononuclear phagocytes are functionally versatile cells, which perform key roles in the immune system. They are traditionally separated into macrophages, the professional phagocytic cells, and dendritic cells (DC), the professional antigen-presenting cells. Nevertheless, it is becoming increasingly clear that mononuclear phagocytes perform many more functions than just taking up pathogens and presenting peptides to T cells [1 ]. They orchestrate inflammatory and immune responses and are intimately involved in maintaining tissue homeostasis by cell-cell interactions and by the production of myriad mediators and effector molecules. As a consequence, tissue damage is often connected with this function. Conversely, mononuclear phagocytes also contribute to tissue repair by breaking down granulation tissue and stimulating the production of new extracellular matrix (ECM) or by producing such components themselves [2 ]. As these functions contradict each other, they need to be controlled tightly. Environmental signals, such as cytokines, are now recognized to play pivotal roles in imposing specific phenotypes and functions in mononuclear phagocytes [3 , 4 ]. Specifically, a dichotomy of M1 and M2 macrophages, modeled after the Th1 and Th2 paradigm of Th cells, was suggested after the discovery that IL-4 stimulation drives macrophages into an alternatively activated state, which is connected with immunosuppression and tissue repair [5 ]. This state contrasts the classically activated state, which is obtained after stimulation with IFN-{gamma}, and associates with microbicidal activity and tissue degradation. However, distinguishing just two activation states is clearly an oversimplification [3 , 4 , 6 ]. Exposure of macrophages to IL-10, TGF-ß, or corticosteroids, for example, leads to a state that is connected with immunosuppression, comparable with alternatively activated macrophages, but those macrophages express phenotypes and functions that differ clearly from IL-4-treated macrophages [7 , 8 ]. As a consequence, it has been suggested that only macrophages stimulated with IL-4 or IL-13, which share a receptor chain and lead to similarly activated cells, should be called alternatively activated [6 ].

In vivo, it is difficult to demonstrate the functions of individual, differently activated macrophage populations. Consequently, identification of specific phenotypic markers, which distinguish between differentially stimulated macrophages, has been a major goal in the field. The mannose receptor (MR)/CD206 was initially found to serve this purpose for identification of alternatively activated macrophages [5 ]. More recently, other cell surface molecules, in particular, the ß-glucan receptor/Dectin-1 [9 ] and the mouse macrophage galactose-/N-acetylgalactosamine-specific C-type lectins 1 and 2 (mMGL1 and mMGL2)/CD301a and CD301b [10 ], have been described as alternative activation markers of macrophages. It is interesting that we previously found that mMGL/CD301 expression typifies mononuclear phagocytes associated with connective tissue environments, such as the dermis [11 ] or the pancreatic septa [12 ]. Therefore, we asked whether these cells might represent a population of alternatively activated macrophages in the tissue. We show in this report that although dermal mononuclear phagocytes do express mMGL/CD301, MR/CD206, and Dectin-1, they are not alternatively activated macrophages in the strict sense, as they also express these receptors in IL-4 receptor {alpha} (IL-4R{alpha}) knockout (KO) mice, in which cells are unable to respond to IL-4 and IL-13. Moreover, we demonstrate that epidermal Langerhans cells (LC) also up-regulate the expression of mMGL in response to dermal factors. Together, these results suggest that the dermal microenvironment stimulates mononuclear phagocytes to express markers of alternative activation, independent of IL-4/IL-13 signaling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Female C57BL/6J mice were obtained from Harlan (Horst, the Netherlands) and used between 10 and 16 weeks of age. IL-4R{alpha} KO mice were generated as described [13 ] and back-crossed for nine generations onto the C57BL/6 background.

Antibodies, conjugates, and other reagents
The following antibodies have been used in this study: rat anti-mMGL1 and -mMGL2, clone ER-MP23, IgG2a, as hybridoma culture supernatant or biotinylated, and rat control IgG2a, clone PH2-99, were generated in our lab. Rat anti-mouse Langerin (Clone 929F3, IgG1, purified or Alexa Fluor 488-labeled) was kindly provided by Dr. Sem Saeland (INSERM U 503/IFR, Lyon, France); purified rat anti-MR (Clone MR5D3, IgG2a) and rat anti-Dectin-1 (Clone 2A11, IgG2b) were from Hycult Biotechnology (Uden, the Netherlands); and phycoerythrin (PE)-labeled rat anti-I-A/I-E (Clone M5/114.15.2, IgG2b), PE-labeled rat anti-CD86 (Clone GL1, IgG2a), allophycocyanin (APC)-labeled rat anti-CD11b (Clone M1/70, IgG2b), and APC-labeled streptavidin (SA) were from BD PharMingen (San Diego, CA). FITC- and HRP-labeled rabbit anti-rat Igs were from Dako (Glostrup, Denmark), and Texas Red-labeled SA and PE-labeled goat anti-rat Igs were from Caltag (San Francisco, CA). Culture medium was RPMI 1640 (Cambrex, Verviers, Belgium), supplemented with 10% FCS (HyClone, Kansas City, MO), 100 U/ml penicillin, and 100 µg/ml streptomycin (both from Cambrex). FITC was from Sigma Chemical Co. (St. Louis, MO), whereas biotinylated {alpha}-N-acetylgalactosaminide ({alpha}-GalNAc), ß-GalNAc, ß-N-acetylglucosaminide (ß-GlcNAc), and LewisX (LeX)-conjugated polyacrylamide carriers were obtained from GlycoTech (Rockville, MD).

Solid-phase binding assay
Adsorption of the purified, soluble recombinant mMGL1 [14 ] or mMGL2 [15 ] onto ELISA plates (655061, Greiner, Kremsmünster Austria) was carried out by adding 100 µl protein solution [2.5 µg/ml in Dulbecco’s PBS (DPBS)] to each well and incubating the plates for 18 h at 4°C. After blocking nonspecific binding using 3% BSA in DPBS for 2 h at room temperature, a dilution series of ER-MP23 hybridoma culture supernatant or purified control antibodies (stock concentration: 10 µg/ml) in DPBS plus 1% BSA was added. After incubation for 2 h at room temperature, wells were washed three times with DPBS to remove unbound materials, and then 100 µl HRP-conjugated goat anti-rat IgG solution (0.75 µg/ml in DPBS) was added to detect bound materials. After incubation for 1 h at room temperature, the wells were washed three times with DPBS. Subsequently, 100 µl 1 mM 2,2'-amino-bis(3-ethylbenzthiazoline-6-sulfonic acid; ABTS) solution containing 0.34% H2O2 in 0.1 M sodium citrate buffer (pH 4.3) was added, and the absorbance was measured at 405 nm on a microplate reader (Model 550, Bio-Rad, Hercules, CA).

To detect blocking ability of the ER-MP23 antibody, 300 ng recombinant mMGL1 or mMGL2 was adsorbed per well onto plates. Inhibition of nonspecific binding and subsequent washing was done as described before. Thereafter, increasing concentrations of ER-MP23 or a rat control antibody were added to the wells and incubated for 2 h at room temperature. Subsequently, biotinylated LeX polymers (for mMGL1) or ß-GalNAc polymers (for mMGL2) were added to the wells and incubated for 1.5 h at 4°C. Bound polymers were detected by incubation with 100 µl HRP-conjugated SA solution (1.25 µg/ml in DPBS) for 0.5 h at 4°C and visualized with ABTS as before.

Binding of desialylated erythrocytes to macrophage cell lines
Macrophage cell lines RAW309Cr.1 (expressing significant levels of mMGL) and RAW264.7 (mMGL–/low), grown in a 96-well culture plate (Nunc, Roskilde, Danmark), were preincubated with 50 µg/ml-purified ER-MP23, 200 mM galactose, or 200 mM mannose for 15 min at room temperature. Thereafter, human erythrocytes, desialylated by neuraminidase treatment as described [16 ], were added and incubated for 30 min at 37°C. Subsequently, plates were washed five times with PBS plus Ca2+ and Mg2+, remaining erythrocytes were lysed with 10 mg/ml Na2CO3, and hemoglobin absorbance was measured at 414 nm on a Titertek Multiscan (Flow Labs, Redwood City, CA).

Staining of tissue sections
Back-skin or skin-draining lymph nodes were frozen in Tissue-Tek O.C.T. compound (Sakura Finetek, Zoeterwoude, the Netherlands) and cut into 6 µm-thick sections on a cryostat (Leitz, Wetzlar, Germany). Subsequently, sections were fixed for 4 min (skin) or 10 s (lymph nodes) in acetone, rehydrated in PBS, pH 7.8, plus 0.05% Tween-20 (Fluka, Buchs, Switzerland), incubated with avidin/biotin blocking kit (Vector Labs, Burlingame, CA), blocked with 10% normal rabbit serum, and incubated with purified antibodies. Thereafter, antibody binding was detected with FITC (for Langerin)- or HRP-labeled anti-rat Igs (for mMGL, MR, and Dectin-1). HRP-labeled antibodies were visualized by incubating slides with nickel-3,3'-diaminobenzidine tetrahydrochloride (Ni-DAB; Sigma Chemical Co.) plus 1% H2O2 for 3 min. Slides were subsequently embedded in Entellan mounting media (Merck, Darmstadt, Germany). For fluorescence stainings, sections were subsequently blocked with 5% normal rat serum and incubated with biotinylated ER-MP23 antibodies. After visualization with Texas Red-labeled SA, sections were embedded with Vectashield mounting media, which contained 4',6-diamidino-2-phenylindole (DAPI) to counterstain nuclei (Vector Labs). Incubation steps were all performed in the dark at room temperature for 30 min; sections were washed twice between incubations with PBS supplemented with 0.05% Tween-20. Thereafter, sections were examined using a Zeiss Axioscop or a Zeiss Axioplan 2 fluorescence microscope (Zeiss, Göttingen, Germany).

Epidermal cell and lymph node cell suspensions
Ears of killed mice were cleaned with 70% ethanol and cut off at the base. Thereafter, they were split in dorsal and ventral halves by means of strong forceps; both halves were incubated at 37°C in 0.8% trypsin (Merck) in PBS for 25 min (cartilage-free half) or 45 min. Epidermis was peeled off and shaken in a water bath for 30 min, and cell suspension was filtered through a 70-µm nylon cell strainer (BD Falcon, Bedford, MA). The resulting epidermal cell suspensions contained 1–3% LC. Epidermal cells were stained directly for flow cytometry or cultured for 1 day in culture medium, supplemented with 4 ng/ml murine GM-CSF (BioSource, Camarillo, CA). In some experiments, epidermal cells were cultured for 1 day in conditioned medium obtained from a Day-3 skin explant culture (see below), which had also been supplemented with GM-CSF. Cells were stained for ER-MP23-bio, followed by SA-APC and I-A/I-E-PE to detect extracellular mMGL or only for I-A/I-E-PE. Thereafter, cells were fixed with 2% paraformaldehyde (PFA; Merck), permeabilized with 0.25% saponin (Sigma Chemical Co.), and subsequently, stained with anti-Langerin-Alexa Fluor 488 antibodies or to detect intracellular mMGL, with anti-Langerin and ER-MP23-bio antibodies, followed by SA-APC.

Axillary, brachial, and inguinal skin-draining lymph nodes were collected from untreated mice or from mice, which were treated with 250 µl 1% FITC in 1:1 acetone:dibutylphtalate (Sigma Chemical Co.) on their shaved back skin 24 h before they were killed. Skin-draining lymph nodes were cut into small pieces and subsequently filtered through a 70-µm nylon cell strainer. Cells were fixed and permeabilized as described above. Thereafter, they were stained with purified anti-Langerin antibodies, which were visualized with PE-labeled anti-rat Igs. Cells were subsequently stained with ER-MP23-bio and SA-APC. Fluorescence of all cells was measured on a FACSCalibur (Becton Dickinson, San Jose, CA) and analyzed using WinMDI 2.8 software.

Skin explant cultures
Dorsal and ventral ear halves were cultured on 1 ml culture medium in 24-well plates for 1, 2, 3, or 4 days. Cells, which had emigrated out from the explants, were collected afterward and stained for flow cytometry as described for epidermal cell suspensions. For analysis of emigrant cells by confocal microscopy, cells were allowed to adhere for 6 h on Lab-Tek II chamber slides (Nunc). Thereafter, they were fixed and permeabilizied with PFA and saponin and stained for Langerin-AF488 and ER-MP23-bio, followed by SA-TxR, as described for tissue slides. Slides were analyzed with a Zeiss LSM 510 Meta confocal microscope. For the endocytosis experiments, 10 µg/ml biotinylated {alpha}-GalNAc, ß-GalNAc, or ß-GlcNAc polymers were added to the cultured medium as indicated during the 2-day culture period. In preliminary experiments, we determined that matured LC and mMGL+ dermal-derived cells expressed similar high levels of CD86 (data not shown). Therefore, collected emigrated cells were fixed, permeabilized, and stained with anti-Langerin-Alexa Fluor 488, CD86-PE, and SA-APC to detect the endocytosed sugar polymers. In some wells, 50–100 µg/ml-purified ER-MP23 anti-mMGL antibodies were added during the culture period to block binding of the glycosylated polymers (no difference in blocking efficiency was observed between the two concentrations). For the emigration experiments, 100 µg/ml purified ER-MP23 antibodies or control rat IgG2a antibodies were added to the medium during the 2-day culture period. Thereafter, cells were fixed, permeabilized, and stained with anti-Langerin-Alexa Fluor 488 and CD11b-APC. Cells were analyzed by flow cytometry as indicated above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ER-MP23 binds mMGL1 and mMGL2
The antimononuclear phagocyte mAb ER-MP23 previously described [12 , 17 ] appears to recognize the mMGL/CD301. This notion is based on the following observations. In preliminary experiments, we found that ER-MP23 precipitated a 43-kDa antigen from a J774-1.6 macrophage cell line homogenate. Protein sequencing revealed the nonblocked N terminus MEYENLQNIRIE, which corresponds to the described N terminus of the mouse macrophage mMGL1 [18 ], with the exception of two amino acids (underscored). Recently, mMGL2 has been discovered, which shows 79.0% nucleotide identity and 91.5% amino acid identity to mMGL1 [15 ]. As shown in Figure 1A and 1B , we found now by ELISA assays with recombinant mMGL1 and mMGL2 that ER-MP23 bound to extracellular domains of mMGL1 and mMGL2. We confirmed this finding by positive flow cytometry stainings of Chinese hamster ovary cell clones, which had been transfected with full-length mMGL1 or mMGL2 (data not shown). To investigate whether ER-MP23 interferes with the carbohydrate ligand-binding function of the lectins, we incubated immobilized, recombinant mMGL1 and mMGL2 with increasing concentrations of ER-MP23. Thereafter, we assessed the remaining sugar-binding capacity of the two recombinant lectins. As shown in Figure 1C and 1D , ER-MP23 could indeed inhibit the binding of ligands to mMGL1 and mMGL2, although the inhibition of mMGL1 ligand binding is possibly more efficient. To confirm that this blocking also occurs at the cellular level, we incubated two macrophage cell lines, RAW309Cr.1 and RAW264.7, being high and low mMGL-expressing cell lines, respectively (Fig. 1E) , with desialylated erythrocytes. Desialylation of erythrocytes exposes nonreduced terminal galactose residues at N-linked sugar chains of erythrocytes, thereby forming high-affinity ligands for the mMGL lectins [15 ]. As shown in Figure 1F , erythrocyte binding could indeed be observed for the RAW309Cr.1 but not for the RAW264.7 cells. This binding could be inhibited by preincubating the macrophages with galactose but not with mannose. Moreover, preincubation with ER-MP23 also inhibited erythrocyte binding to RAW309Cr.1 cells, thus confirming that ER-MP23 blocks the binding of carbohydrate ligands to the mMGL lectins. Probably, ER-MP23 recognizes an epitope inside the carbohydrate recognition domains of mMGL1 and mMGL2.


Figure 1
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  		Figure 1. ER-MP23 binds to recombinant mMGL1 and mMGL2 and blocks the function of the lectin on the surface of macrophages. The binding of ER-MP23 antibodies to immobilized recombinant mMGL1 (A) and mMGL2 (B) was measured by ELISA in a dilution series of hybridoma supernatant. As controls, binding of purified LOM-14 (recognizing mMGL1 and mMGL2) and LOM-8.7 (mMGL1-specific) to recombinant mMGL1 (A) and mMGL2 (B) was included as a dilution series of a 10-µg/ml stock solution. The binding of mAb was detected using HRP-conjugated goat mAb specific for rat IgG (H+L). Absorbance at 405 nm was measured on a microplate reader. (C, D) Blocking ability of ER-MP23 was assessed by incubating recombinant mMGL1 and mMGL2 with increasing concentrations of ER-MP23. Thereafter, remaining binding was assessed by incubating plates with biotinylated ligands for mMGL1 (LeX, C) or mMGL2 (ß-GalNAc, D), followed by detection with HRP-labeled SA. (E) A mixture of the macrophage cell lines RAW309Cr.1 (H-2b/d) and RAW264.7 (H-2d) was stained for H-2b and for mMGL. Note that the H-2b-positive RAW309Cr.1 cells express a considerable amount of mMGL, whereas the RAW264.7 cells do not. (F) The binding of desialylated erythrocytes to both macrophage cell lines was quantified by lysing bound erythrocytes and measuring hemoglobin absorbance at 414 nm. Macrophages were not pretreated or preincubated with 50 µg/ml ER-MP23, 200 mM galactose, or 200 mM mannose.

 
Dermal mononuclear phagocytes express markers of alternatively activated macrophages
Previously, we characterized antigen-presenting cell populations in the mouse dermis and observed that the majority of all dermal cells represents mononuclear phagocytes expressing mMGL [11 , 15 , 19 ]. As the mMGL lectins have been identified as markers for alternatively activated macrophages [10 ], we approached the question of whether resident dermal mononuclear phagocytes might represent alternatively activated macrophages. To that end, we stained skin sections for two other well-established markers of alternatively activated macrophages, the MR/CD206 [5 , 20 ] and the ß-glucan receptor/Dectin-1 [9 ]. As shown in Figure 2 , we found that a similarly large population of dermal cells also expressed MR (Fig. 2B) . Linehan [21 ] identified these cells recently as mononuclear phagocytes. Dectin-1 staining showed a less uniform profile, indicating that this marker was expressed at a high level only by clusters of cells in the dermis (Fig. 2C) , reminiscent of the expression of MHC Class II in the ear dermis [11 ]. Nevertheless, all cells were observed to express at least a low level of Dectin-1. In the ear dermis, we found that MHC Class II and high Dectin-1 expression were indeed colocalized on the same cells (data not shown). Therefore, we conclude that dermal mononuclear phagocytes indeed express multiple markers of alternatively activated macrophages, a phenotype that can be induced by IL-4 or IL-13 signaling [6 ]. Macrophages that lack the IL-4/IL-13 common receptor {alpha} chain (IL-4R{alpha}) cannot develop into alternatively activated macrophages [22 ]. To assess whether IL-4 or IL-13 is required in the induction of the phenotype of the resident dermal macrophages, we stained skin sections of IL-4R{alpha}–/– mice [13 ] for the same markers. As shown in Figure 2 , we found that dermal macrophages in IL-4R{alpha}–/– mice still expressed all these markers in similar patterns. Therefore, the expression of alternative activation markers on dermal macrophages does not depend on IL-4 or IL-13 signaling.


Figure 2
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  		Figure 2. Dermal mononuclear phagocytes express mMGL, MR, and Dectin-1, independent of IL-4-/IL-13 signaling. C57BL/6J mice (left column) or IL-4R{alpha} KO mice (right column), steady-state back-skin sections, were stained for mMGL (A), MR (B), or Dectin-1 (C). Antibody staining was visualized with Ni-DAB. Whereas all dermal mononuclear phagocytes were observed to express mMGL and MR homogenously, different subpopulations were observed to express different levels of Dectin-1. Cells expressing a high Dectin-1 level were observed to be organized in clusters (half-filled arrow), and the remaining dermal mononuclear phagocytes were found to express only lower levels of Dectin-1 (solid arrow).

 
LC migrating through the dermis express mMGL
In our analyses of dermal mononuclear phagocyte populations, we also identified LC in the dermis on the basis of their expression of Langerin/CD207 as a unique marker for LC. They most probably represent migrating cells on their way to skin-draining lymph nodes [23 ]. Quantitative determinations revealed that approximately 6% of all interstitial cells in the C57BL/6J mouse dermis expressed Langerin [11 ], which were previously found to be negative for mMGL [19 , 24 ], and in agreement with these reports, we found no mMGL expression on epidermal LC in our in situ stainings (Fig. 3A ). In contrast, approximately 2% of all dermal cells, corresponding to approximately one-third of all dermal LC, coexpressed mMGL and Langerin (Fig. 3B) , and the other dermal LC were mMGL-negative by immunofluorescence. When we studied dermal LC in the IL-4R{alpha}–/– mouse dermis, we found a comparable subpopulation of these cells expressing mMGL (Fig. 3C) . Therefore, mMGL expression appears to be induced on at least a subset of migrating LC located in the dermis, which is similar to other resident dermal mononuclear phagocytes, independent of IL-4 or IL-13 signaling.


Figure 3
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  		Figure 3. LC migrating through the dermis show an up-regulated expression of mMGL. Steady-state back-skin sections from C57BL/6J mice (A, B) or IL-4R{alpha} KO mice (C) were stained for Langerin (green) and mMGL (red) and counterstained for DAPI (blue). (A) LC in the epidermis do not express mMGL at a detectable level. Note the high background fluorescence of the keratin layers. (B) In contrast, approximately one-third of all dermal LC, thus LC migrating to skin-draining lymph nodes, was found to be mMGL-positive. (C) Comparable with dermal macrophages in IL-4R{alpha} KO mice, dermal LC also express mMGL.

 
mMGL induction on LC is not just a consequence of maturation
Upon migration through the dermis, LC mature, which is reflected in the up-regulation of markers such as CD40, CD80, CD86, CCR7/CD197, CD205, MHC Class I, and MHC Class II [25 ]. Hence, expression of mMGL by LC might be merely a consequence of LC maturation. To investigate this possibility, we determined the immunophenotype of freshly prepared and 24 h-cultured epidermal single-cell suspensions by flow cytometry. As shown in Figure 4C and 4E , we found that immature LC in a fresh epidermal cell suspension already expressed a low level of mMGL, detectable on their surface and after permeabilization of the cells. This is in contrast to our in situ findings, where we found no mMGL expression on LC (Fig. 3A) and most probably, reflects the higher sensitivity level of flow cytometry. After 1 day of culture, LC expressed a much higher level of MHC Class II molecules, indicating that they indeed possessed a more mature phenotype (Fig. 4B) . Whereas intracellular mMGL detectability did not change during the 1-day culture period (Fig. 4F) , we found that the cell surface mMGL expression by LC was down-regulated completely (Fig. 4D) . Therefore, mMGL expression by in vitro maturing LC appears to be down-regulated, as is the case for bone marrow-derived DC [26 ] and as has been shown for other endocytic receptors. The observed, increased mMGL expression on LC in the dermis is thus not simply induced by LC maturation but possibly relates to the dermal microenvironment and might represent a model for regulation of alternative activation marker expression by dermal mononuclear phagocytes in general. Therefore, we decided to investigate this in more detail.


Figure 4
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  		Figure 4. In vitro-matured LC do not up-regulate mMGL expression. LC, which had been obtained by trypsinization of the epidermis, were processed freshly (A, C, E) or after 1 day of culture in culture medium (B, D, F). Cells were stained intracellularly for Langerin, extracellularly for MHC Class II, and extracellularly (C, D) or intracellularly (E, F) for mMGL. Whereas fresh LC show a low level of extracellular and intracellular mMGL expression, 1 day-matured LC remain only intracellularly positive at a low level for mMGL staining. Shaded histogram, Isotype control; bold-lined histogram, ER-MP23.

 
LC, which have migrated through the dermis, express a high intracellular level of mMGL
To assess whether the dermal microenvironment induces mMGL expression on LC, we performed ear skin explant cultures for 1, 2, 3, or 4 days. During this procedure, LC will emigrate from the epidermis, interact with the dermal microenvironment, and end up in the culture medium, where they can be collected easily for further analyses [23 , 27 ]. Whereas we found reasonable amounts of dermal emigrants already after 24 h, only few LC were found at this early time-point. This is most probably explained by the notion that they have to leave the epidermal environment and cross the basal membrane before accessing the dermis. After 48 h, however, LC could be recovered efficiently from the medium, and their number did not increase significantly anymore thereafter. Analysis of emigrated LC from all time-points by flow cytometry indicated that these cells hardly showed mMGL on their surface (Fig. 5A 5C , 5E and 5G ). Yet, they expressed a significantly increased level of intracellular mMGL, compared with uncultured cells or cells matured in vitro (Figs. 4E and 4F and 5B 5D 5E and 5H) . In line with the in situ profile in the steady-state dermis, we found that approximately half of the LC would express a high level of intracellular mMGL, and the other half showed only a low level (Fig. 5B 5D 5E 5H) . This expression pattern did not change essentially during the culture (Fig. 5) . We also analyzed emigrant cells with a confocal microscope to confirm the intracellular expression of mMGL by LC (Fig. 6 ). These analyses revealed that mMGL is mainly but not fully colocalized with Langerin in the cytoplasm of mature LC. Langerin has been shown previously to be internalized upon LC maturation [28 ]. Therefore, these findings suggest that the dermal microenvironment indeed induces (intracellular) mMGL expression in LC.


Figure 5
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  		Figure 5. mMGL expression by LC is associated with exposure to dermal factors. Ear halves were cultured for 1 (A, B), 2 (C, D), 3 (E, F), or 4 days (G, H) in culture medium. Thereafter, cells that had migrated out were collected and stained intracellularly for Langerin, extracellularly for MHC Class II, and extracellularly (A, C, E, G) or intracellularly (B, D, F, H) for mMGL. Emigrated LC showed a limited increase of extracellular mMGL, while intracellular mMGL expression levels were clearly up-regulated. The vertical line represents the maximum staining of isotype control antibodies. (I, J) To assess the influence of soluble dermis-derived factors, epidermal cell suspensions were cultured 1 day in regular culture medium (bold line) or in medium obtained after a 3-day skin-explant culture (thin line, open histogram). Filled histograms show isotype control profiles. LC, which had been cultured in skin explant medium, showed an up-regulated, intracellular mMGL expression level comparable with whole skin emigrants and retained (or up-regulated) extracellular mMGL expression. Cells were gated as shown in Figure 4 .

 

Figure 6
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  		Figure 6. mMGL colocalizes significantly with Langerin inside mature LC. Whole-skin emigrants were allowed to adhere on slides. Thereafter, they were fixed and permeabilized, stained for mMGL and Langerin, and analyzed by confocal microscope. Note the colocalization of Langerin and mMGL in the cytoplasm of LC.

 
To determine whether a soluble factor derived from the dermis induces mMGL expression in LC, we cultured epidermal cell suspensions in whole skin-conditioned medium. In these cultures, we found that isolated LC cultured in skin-conditioned medium showed an intracellular mMGL expression pattern comparable with that of whole-skin emigrants, with a subpopulation of cells expressing higher mMGL levels than uncultured cells or cells cultured in standard culture medium (Fig. 5J) . Moreover, LC, which had been cultured in skin-conditioned medium, expressed mMGL on their surface (Fig. 5I) . Therefore, we conclude that a soluble factor from the dermis is indeed important in the induction of mMGL expression in and on LC. As we routinely added GM-CSF to the culture media for epidermal cell suspensions to ensure LC survival, this factor apparently is not instrumental for mMGL induction, although it does induce Dectin-1, another marker of alternative activation [9 ].

Only recently immigrating LC in skin-draining lymph nodes are mMGL+
After migration through the dermis and transport via afferent lymph, LC reach their destination in vivo, i.e., the skin-draining lymph nodes. To investigate the mMGL expression profile during this development, we obtained lymph node single-cell suspensions and analyzed Langerin-positive cells for the detectability of intracellular mMGL. As represented in Figure 7C , we found that only a minor fraction of Langerin-positive cells in the steady-state skin-draining lymph nodes showed intracellular mMGL, suggesting that mMGL is down-regulated again in LC once these cells have arrived in lymph nodes. Labeling steady-state skin-draining lymph node tissue sections for Langerin and mMGL by immunofluorescence confirmed this notion (Fig. 7E) . Whereas we identified mMGLhigh-expressing cells scattered throughout the paracortex, which are presumably dermis-derived mononuclear phagocytes [29 ], we did not find coexpression of mMGL on Langerin-positive cells. To study the mMGL phenotype of recently immigrating LC, we painted FITC onto shaved back-skin and analyzed prior (FITC–) and recent immigrant (FITC+) LC in skin-draining LN 24 h later. As depicted in Figure 7D , we found that FITC– LC were again virtually mMGL-negative, whereas FITC+ LC showed the same mMGL profile as LC in skin explant cultures: one subpopulation of high mMGL-expressing cells and one of low mMGL-expressing cells. These results confirm the notion that LC, which arrive freshly in LN, still have detectable mMGL levels, which are down-regulated once they have arrived in the skin-draining lymph nodes.


Figure 7
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  		Figure 7. Only recent immigrant LC in skin-draining lymph nodes are mMGL-positive. Skin-draining lymph nodes from untreated mice (A, C) or from mice that had been treated with FITC solution 24 h prior to their euthanization (B, D) were collected and homogenized to obtain a single-cell suspension. Thereafter, cells were stained intracellularly for Langerin and mMGL. LC in skin-draining lymph nodes from untreated mice or FITC– LC (thus, LC, which had left the skin more than 24 h earlier) from FITC-treated mice showed negligible intracellular mMGL levels. In contrast, recently immigrated FITC+ LC from FITC-treated mice still showed a significant mMGL expression. The increased fluorescence level (FL-1) of FITC– LC compared with untreated mice is probably explained by the uptake of soluble FITC transported via afferent lymph. The vertical line represents the maximum staining of isotype control antibodies. (E) Skin-draining lymph node sections from an untreated mouse were stained for Langerin (green) and mMGL (red) and counterstained for DAPI (blue). Note that no double-positive cells are present. B, B cell area; T, T cell area.

 
LC use mMGL only as a minor receptor to take up ligands
As the dermal microenvironment induced mMGL expression on the surface of LC, we wondered whether LC would use the lectins as endocytic receptors to take up antigens. To approach this question, we performed skin-explant cultures with soluble mMGL ligands present in the medium (polyacrylamide conjugated to {alpha}-GalNAc or to the ß-enantiomer, ß-GalNAc) or a control-glycosylated polymer, which is not a ligand for the mMGL lectins (ß-GlcNAc-conjugated polyacrylamide). It has been shown previously that the two GalNAc glycoconjugates are bound preferentially by mMGL2 and can be taken up by immature bone marrow-derived DC and that this ingestion can be blocked by adding mMGL-blocking antibodies [26 ]. After 2 days of culture, we harvested the skin emigrant cells in the medium and analyzed whether LC had taken up the ligands and whether ER-MP23 antibodies in the medium blocked it. As shown in Figure 8 , we found that LC endocytosed {alpha}-GalNAc and ß-GlcNAc but barely ß-GalNAc. Only the {alpha}-GalNAc uptake by LC could be inhibited to some degree by ER-MP23 antibodies, suggesting that a small amount of this ligand was taken up via mMGL2. In comparison, dermal cells endocytosed {alpha}-GalNAc and ß-GalNAc to a much larger extent than LC, and this ingestion could be decreased efficiently by ER-MP23 antibodies. Therefore, we conclude that LC use mMGL only as minor receptors to take up antigens.


Figure 8
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  		Figure 8. LC use mMGL as a minor endocytic receptor for ligand uptake. Skin-explant cultures were performed for 2 days with two biotinylated mMGL2 ligands ({alpha}-GalNAc, ß-GalNAc) or a control polymer (ß-GlcNAc), with or without ER-MP23 present in the medium. Thereafter, cells were harvested and stained for Langerin, CD86, and SA-APC to detect endocytosed ligands. Epidermal (Langerin+, CD86+) and dermal cells (Langerin–, CD86+) were gated, and their mean fluorescence intensity (MFI) was determined. Dermal mMGL+ cells took up mMGL2 ligands proficiently, and this incorporation could be blocked efficiently by adding ER-MP23 antibodies. However, LC endocytosed the {alpha}-GalNAc enantiomer only to a lower level and did not take up the ß-GalNAc form at all via a mMGL-dependent manner. Depictured is the result of one experiment of two performed experiments with similar outcomes.

 
Inhibition of mMGL does not detectably affect LC migration
Previously, it has been shown that blocking mMGL through addition of antibodies in skin-explant cultures inhibits the migration of dermal macrophages, suggesting that mMGL function is required for cellular migration [19 ]. To assess whether the mMGL lectins would also play a role in LC migration, we performed skin-explant cultures with ER-MP23 or control rat IgG2a antibodies in the culture medium. Thereafter, we assessed the number of Langerin+ and Langerin– CD11b+ cells that had emigrated into the medium. These analyses indicated that the number of Langerin-positive emigrants did not differ between the two culture conditions (Fig. 9 ). In agreement with the earlier findings [19 ], we observed that the number of dermal mononuclear phagocytes, identified as CD11b-positive, Langerin-negative cells, was decreased ~25% in the presence of ER-MP23 antibody during the culture period (Fig. 9) . From this result, we conclude that inhibition of mMGL by ER-MP23 can indeed decrease the migration of dermal mMGL-positive cells to some extent, but the migration of epidermal LC is not detectably affected.


Figure 9
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  		Figure 9. mMGL is not involved in LC migration. Skin-explant cultures were performed for 2 days with ER-MP23 or an isotype control antibody in the medium. Thereafter, emigrated cells were harvested, stained for Langerin and CD11b, and quantified by flow cytometry. Total acquired events in the control medium were set to 100%, and the amount of total events in the ER-MP23-containing medium as well as the amount of the gated cells were thereafter related to this number. Shown are the average cell numbers of four experiments. Addition of ER-MP23 could inhibit the emigration of ~25% of dermal cells but did not affect emigration of LC.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown here that resident mononuclear phagocytes in the steady-state dermis express a phenotype, which is strongly reminiscent of alternatively activated macrophages: They express mMGL/CD301, MR/CD206, and Dectin-1—three surface lectins known to be induced by IL-4/IL-13 stimulation of macrophages and DC and considered to be markers of alternative activation [5 , 9 , 10 , 20 ]. Nevertheless, dermal mononuclear phagocytes express a similar phenotype in IL-4R{alpha} KO mice, in which cells cannot respond to IL-4 or IL-13. Therefore, dermal mononuclear phagocytes do not represent alternatively activated macrophages in the strict sense, implying that the phenotype is a result of IL-4 or IL-13 signaling [6 ]. In this respect, our data agree with the results found by Linehan et al. [30 ], who showed that MR expression in IL-4R{alpha} KO mice was not affected in resident tissue macrophages. Granuloma macrophages induced by schistosome eggs, however, were MR-negative in IL-4R{alpha} KO mice, demonstrating that these cells represent true alternatively activated macrophages in wild-type mice [30 ]. Nevertheless, we consider it likely that the gene expression profile of dermal and possibly also other connective tissue macrophages will show similarity with truly alternatively activated macrophages, as we will discuss below. Epidermal LC, which express a low level of extra- and intracellular mMGL in their immature state, strongly up-regulate intracellular mMGL when they migrate through the dermis. Maturation of LC in vitro, in the absence of dermal factors, did not cause such an up-regulation; therefore, LC maturation in itself is not sufficient to increase mMGL levels. Once LC have left the dermis and reach skin-draining lymph nodes, mMGL is down-regulated quickly again in LC. Therefore, dermal factors appear to be responsible for the induction of mMGL expression in LC.

Previously, mMGL expression has been used to discriminate between dermal macrophages and LC [11 , 29 ], as LC seemed to be mMGL-negative [24 ]. Our current findings show, however, that immature LC in the epidermis do express mMGL, although at levels that are too low to be detected by immunofluorescence on tissue sections. Migrating through the dermis, one-third to one-half of the LC expresses higher mMGL levels, which allow detection by this method. Once these cells reach skin-draining lymph nodes, mMGL levels are down-regulated again so that LC appear negative for mMGL on stained sections. Therefore, mMGL remains useful to discriminate LC from dermally derived mononuclear phagocytes using immunofluorescence or immunohistochemistry on steady-state lymph nodes, as cells originating from the dermis retain mMGL positivity [29 ]. This quantitative difference in mMGL levels also enables the flow cytometric distinction between the two lymph node antigen-presenting cell populations (unpublished results). However, this difference in mMGL expression may not be sufficient to distinguish between LC and dermally derived cells in conditions where dermal factors influence LC profoundly. Therefore, mMGL expression is not a universal marker to distinguish between epidermal LC and dermally derived mononuclear phagocytes but is useful in conditions where mMGL expression profiles have been defined, such as described here.

The transient induction of mMGL in LC raises the question of whether this lectin serves particular functions in LC. For dermal macrophages, several mMGL functions have been established. For instance, this lectin functions as an endocytic receptor (this study; ref. [26 ]) and as a signaling receptor involved in cellular migration induced by cytokine production (this study; ref. [18 ]). However, mMGL seems to play only a minor role, if any, in endocytosis and migration by LC. At least two reasons can be envisaged that explain this difference. First, it might be that we used an ineffective ligand to test lectin-mediated endocytosis. The applied single residue-conjugated polymers have a higher affinity for mMGL2 compared with mMGL1; the latter lectin preferentially recognizes galactose and N-acetylgalactosamine residues in more complex structures such as the terminal LeX antigen [15 ]. It has been shown that dermal macrophages express mMGL1 and mMGL2 [15 ], but we cannot exclude that LC only express mMGL1. Therefore, conclusions can be drawn only to a limited extent, and testing LC with mMGL1- and mMGL2-specific antibodies will be necessary to resolve the issue of whether LC express mMGL1, mMGL2, or both. Second, the two mMGL proteins might execute other functions, specifically inside LC, as we found LC-associated mMGL to be expressed predominately intracellularly. It has been shown for two lectin families, galectins and annexins, that they perform intracellular functions [31 ]. Other C-type lectins, which have been shown to disappear from the surface of mature DC but have been found to persist intracellularly, include Langerin (this study; ref. [28 ]) and the MR (unpublished observation), thus two other well-known endocytic receptors. Hypothetically, these lectins might assist in accumulating and storing antigens in retention compartments [32 ] in order to break them down later and present them in MHC Class II context, to transfer them into the cytoplasm to cross-present them in MHC Class I context, or to transfer them to other DC subpopulations. In this respect, it is conceivable that the mMGL lectins are only expressed briefly on the LC surface, are quickly internalized again, and are delivered then to retention compartments. Our findings of extracellular mMGL expression on in vitro-matured LC in skin-conditioned medium would fit into this picture. Future determinations of other mMGL functions might shed more light on the functions of mMGL expression in LC, in particular, and in mononuclear phagocytes in general.

Only recently, mMGL expression was found to be induced upon alternative activation of macrophages in vitro and in vivo by the Th2-associated inflammatory cytokines IL-4 and/or IL-13 [10 ]. In the steady-state mouse, however, mMGL is expressed almost exclusively by connective tissue macrophages but not by other populations including splenic macrophages, Kupffer cells, or peripheral blood monocytes (ref. [24 ]; unpublished observations). Hence, also, the connective tissue microenvironment appears to provide factors that induce the expression of mMGL and most probably, other markers on resident mononuclear phagocytes and cells in transit. We have shown that this dermis-associated mMGL expression by macrophages and LC was independent of IL-4 and IL-13, excluding these as factors responsible for imposing an alternative-like activation phenotype.

Can inflammatory mediators other than IL-4 and IL-13 be held responsible for induced mMGL expression in migrating LC? Some of our experimental conditions, in which we found mMGL in and on LC, are clearly connected with inflammation, such as in vivo skin painting with FITC and adjuvant or in vitro skin explant cultures. However, different arguments plea against a predominant role of inflammatory mediators in dermal mMGL induction in LC. First, we have found that LC in the undisturbed steady-state dermis are also mMGL-positive in situ. Second, obtaining epidermal cell suspensions activates keratinocytes to produce an array of different mediators, including proinflammatory factors such as GM-CSF [33 ]. In in vitro studies, it has been shown already that GM-CSF is capable of inducing the expression of Dectin-1 on macrophages [9 ]. However, as we generally included GM-CSF in our epidermal cell suspension cultures to ensure LC viability and found no mMGL induction, we can also exclude this cytokine. Only when epidermal cell suspensions were cultured in whole skin-conditioned medium, up-regulation of mMGL in and on LC was observed. In line with this, we regard the detectable mMGL level in recent immigrant FITC+ LC in skin-draining lymph nodes a reflection of the relative immaturity and recent dermal sojourn of inflammatory LC [34 ] rather than an enhancing effect of inflammatory mediators on mMGL expression. Taken together, connective tissue factors present in the steady-state and not inflammation-related mediators seem to be most important for the induction of mMGL expression in LC.

In skin-draining lymph nodes, mMGL expression can be found on two subpopulations of mononuclear phagocytes. Cells that have immigrated from the dermis are present in the paracortical T cell area and express a high level of mMGL (ref. [29 ] and unpublished observations), and macrophages in subcapsular and trabecular sinuses have only low levels of mMGL [24 ]. As these latter cells filter the lymph fluid, it seems likely that these cells are exposed to the dermal factors and/or their breakdown products, which are brought into the lymph nodes via the afferent lymph flow. Therefore, subcapsular sinus macrophages might be induced to express mMGL in a similar way as LC, which have been cultured in skin-conditioned medium. Resident mononuclear phagocytes in the dermis will be exposed constantly to a high concentration of the dermal factors. Although intriguing, the notion that this exposure induces the expression of mMGL and possibly also other alternative activation markers on dermal resident cells remains to be proven. As the dermis predominately consists of ECM, we tested various ECM components for their ability to induce mMGL in preliminary in vitro experiments. Nevertheless, culturing bone marrow-derived DC on collagen type I (ECM component of the dermis), fibronectin (ECM component of the epidermal basement membrane and the dermis), or collagen type VII (ECM of the epidermal basement membrane) did not induce an increased mMGL expression in these cells, whereas IL-4 as positive control treatment did (unpublished observations). Therefore, the identity of the dermal factors, which induce the expression of mMGL, and possibly other alternative activation markers on mononuclear phagocytes in the steady-state dermis and lymph nodes remains unknown.

Alternatively activated macrophages are often associated with tissue remodeling or tissue repair, thus with the breakdown and de novo synthesis of ECM components [6 , 7 ]. Despite the fact that the dermal mononuclear phagocytes are not IL-4/IL-13-activated, a major role of these cells in connective tissue maintenance is likely. We have shown recently that the majority of dermal interstitial cells are macrophages and not fibroblasts as widely believed. Synthesis of ECM components by macrophages is well-documented [2 ], and as such, the notion that the majority of interstitial cells synthesizes the ECM of the dermis might nevertheless turn out to be true. Alternatively activated macrophages are also associated with tolerance and suppression of immune responses. The dermis and other connective tissues might thus be places where an immunosuppressive microenvironment is supported. Tumor-associated macrophages, which mostly reside in and regulate supporting connective tissue [35 ], have been shown consistently to express mMGL [36 37 38 ]. It is feasible that the ECM production stimulated by the tumor generates a microenvironment, which activates tumor-infiltrating macrophages along an alternative route with related connective tissue, maintaining an immune-suppressive function. Identifying the factors that induce macrophages to adopt phenotypes similar to alternatively activated macrophages and counteracting these might thus also benefit cancer treatments.

To summarize, we have shown that resident dermal mononuclear phagocyte subpopulations express several markers of alternatively activated macrophages, for which IL-4 or IL-13 signaling is not required. In addition, LC migrating through the dermis in vivo or cultured in vitro in medium from skin-explant cultures contain higher mMGL levels than immature, epidermal LC or LC cultured in standard medium. LC, which arrive freshly in skin-draining lymph nodes, still show intracellular mMGL, whereas LC, which have remained for a prolonged time in the lymph nodes, did not. Therefore, the expression of mMGL in LC was linked closely to the exposure to the dermal microenvironment and factors derived thereof.


   ACKNOWLEDGEMENTS
 
This study was supported by the J. A. Cohen Institute/Interuniversitary Research Institute for Radiopathology and Radioprotection (IRS; Leiden, The Netherlands). P. S. and N. R. were supported by Grant P-14949 of the Austrian Science Fund, and G. R. was supported by a grant from the "Institute for Promotion of Innovation by Science and Technology in Flanders" (IWT-Vlaanderen) for "Generisch Basisonderzoek aan de Universiteiten" (IWT-GBOU). We express our appreciation for the contribution of the following colleagues to the realization of this paper: Dr. Timo van den Berg for his help in the erythrocyte binding studies, Dr. Sem Saeland for making the anti-Langerin antibodies available, Dr. Frank Brombacher for the permission to use material from IL-4R{alpha} KO mice, Dr. Gert van Cappellen for help with the confocal microscope, and Tar van Os for preparation of the (photo)graphs in this work.

Received October 4, 2005; revised May 20, 2006; accepted June 12, 2006.


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 DISCUSSION
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