Materials and methods

Mice

Mice of inbred strains C57BL/6 and BALB/C were purchased from Charles River Germany (Sulzfeld, Germany) and used at 3–12 mo of age.

Langerin reagents

Several different antibodies against mouse Langerin were used [HD26, mouse IgG1, and a rabbit immune serum for preliminary experiments only; HD24, mouse IgG1 (^{Valladeau et al, 2002}) and 929F3, rat IgG1]. The different anti-Langerin MoAb gave identical staining results, both in terms of staining intensity and staining patterns. The anti-Langerin MoAb 929F3 was also available in FITC- and biotin-conjugated forms.

Transfection of COP5 fibroblasts for control of antibody specificity

Transfections were performed by electroporation of the mouse Langerin cDNA (^{Valladeau et al, 2002}) into the murine fibroblastic COP5 cell line (^{Tyndall et al, 1981}). Cells transfected with an empty plasmid served as mock controls.

Skin explant culture

Culture medium was RPMI-1640 supplemented with 10% fetal bovine serum, gentamycin (all from PAA, Linz, Austria), and 2-mercaptoethanol (Sigma, St Louis, MO). Mice were killed and ears were cut off at the base. Ear skin was split in dorsal and ventral halves by means of strong forceps and the dorsal halves (i.e., cartilage-free halves) were cultured in 24-well tissue culture plates (one ear per well) as described (^{Ortner et al, 1996;Romani et al, 1997;Weinlich et al, 1998}). Alternatively, epidermis and dermis were separated from each other by means of the bacterial enzyme dispase (Boehringer Mannheim, Mannheim, Germany) (^{Kitano and Okado, 1983}), and the epidermal sheets were placed in culture. In most experiments whole skin, epidermis, and dermis were cultured continuously for 48 h. Cells that had emigrated from the explants were collected, counted, and further evaluated phenotypically.

Induction of contact sensitization

Murine ear skin was painted with 15 l 1% 2,4,6-trinitro-1-chlorobenzene (TNCB), picryl chloride (Kodak Eastman, Rochester, NY) on the dorsal and ventral sides. After 24 and 48 h epidermal and dermal sheets were prepared. For this purpose, the skin was floated dermal side down on 0.5 M ammonium thiocyanate for 20 min at 37°C. The epidermis was peeled off the dermis and both parts were cut into 55 mm pieces and fixed in acetone for 20 min.

Immunohistochemistry of epidermal and dermal sheets

Epidermal and dermal sheets were incubated overnight at 4°C with anti-Langerin MoAb (HD24 and 929F3). Antibodies were visualized by using biotinylated anti-mouse and anti-rat immunoglobulin, respectively, and streptavidin-FITC or streptavidin-Texas Red (all from Amersham Life Science, Amersham, U.K.; 90 min at 37°C). Dendritic cells were counterstained with anti-I-A/E^diverse (clone 2G9, fluoresceinated, rat IgG2a; BD-Pharmingen, San Diego, CA). Specimens were viewed on a conventional Olympus epifluorescence microscope.

Isolation of Langerhans cells and dermal dendritic cells from murine skin

Epidermal cells were isolated by standard trypsinization (JHR-trypsin from Sera-lab, Crawley Down, West Sussex, U.K.) (^{Koch et al, 2001}) and dermal cells after removal of the epidermis and thorough rinsing by digestion in 0.5 mg per ml collagenase P (Roche Applied Science, Hamburg, Germany) for 30–60 min at 37°C. Both cell populations were either analyzed by fluorescence-activated cell sorter (FACS) immediately or cultured for 3–4 d in Iscove's medium (Pan, Hamburg, Germany) supplemented with 10% fetal bovine serum and 200 U per ml murine granulocyte-macrophage colony-stimulating factor using supernatants of a transfected cell line as the source of cytokine (kindly provided by A. Lanzavecchia, Bellinzona, Switzerland).

Preparation of lymph node cell suspensions

Cell suspensions from lymph nodes were obtained by digestion with 0.5 mg per ml collagenase P (Roche) for 30 min at 37°C and subsequent pressing the tissue through steel sieves, essentially as described recently for spleen cell suspensions (^{McLellan et al, 2002}). For some experiments, dendritic cells were enriched by Nycodenz gradient centrifugation (Sigma-Aldrich, Vienna, Austria) as described (^{McLellan et al, 1995}).

Phenotype of Langerhans cells and dermal dendritic cells isolated from skin or lymph nodes

Cells were fixed and permeabilized with a kit (Fix & Perm^TM) from BD-Pharmingen and stained in sequence with the rat IgG anti-Langerin 929F3 MoAb or an irrelevant rat IgG as control (Jackson Immuno Research Laboratory, Avondale, PA), anti-rat immunoglobulin-phycoerythrin (BD-Pharmingen), an excess of rat IgG (100 g per ml) for blocking residual free antibody binding sites, and counterstained either with anti-MHC class II (clone 2G9), anti-CD86 (clone GL1), or anti-CD40 (clone 3/23) (all FITC conjugated and from BD-Pharmingen). Nycodenz-enriched cell populations were used for analyses of lymph node cells.

Detection of emigrated Langerhans cells in skin draining lymph nodes after contact allergen sensitization

Mice were sensitized with 200 l (abdomen) or 15 l (ears) 1% TNCB on both sides. After 24 h, single cell suspensions were prepared from skin draining lymph nodes (auricular and inguinal). Cytocentrifuge smears were prepared from these cell suspensions, as mentioned above and counterstained with 4'6-diamidino-2-phenyindole (DAPI) for visualization of nuclei. Langerin⁺ cells were counted under the microscope using 40 objective lenses and a calibrated grid (at least 20 fields). Alternatively, cells from skin draining lymph nodes (auricular and inguinal) were analyzed by FACS.

Immunohistochemistry of lymph node sections

Single enzyme labeling

Skin draining (inguinal or auricular) lymph nodes of untreated mice were frozen and cut into 10 m sections on a cryostat (Frigocut, Leitz, Vienna, Austria). The sections were briefly fixed in acetone and stained with anti-Langerin (929F3), anti-CD8 (clone TIB105, rat IgG2b), anti-CD45R (B220, TIB146, rat IgM), and irrelevant isotype-matched control rat immunoglobulin. Antibody binding was detected by alkaline phosphatase-conjugated anti-rat immunoglobulin F(ab)₂ fragments (Jackson). CD3 (clone 2C11, hamster immunoglobulin) was detected by a biotinylated anti-Syrian and Armenian hamster immunoglobulin (BD-Pharmingen) followed by alkaline phosphatase-conjugated streptavidin (Jackson). The enzyme was visualized with Vector-Blue and nuclei were counterstained with Nuclear-Fast-Red (both from Vector Laboratories, Burlingame, CA).

Double enzyme labeling

Serial frozen 5 m sections were placed on to 3-amino-propyltriethoxy-silane (Sigma) coated slides, air-dried overnight, fixed in acetone for 10 min, rehydrated in Tris-buffered saline and blocked in 5% goat and 5% rabbit serum in Tris-buffered saline for 5 min. Sections were incubated in sequence either with rat anti-CD19 and horseradish peroxidase-rabbit anti-rat immunoglobulin (DAKOCytometrics, Glostrup, Denmark) or with hamster anti-CD3, biotinylated-goat anti-hamster immunoglobulin (Jackson), and horseradish peroxidase-labeled streptavidin (Sigma). A brown color was revealed by application of the substrate diaminobenzidine. The second MoAb, rat anti-Langerin (929F3) was followed by horseradish peroxidase-rabbit anti-rat immunoglobulin (DAKO). The second enzyme reaction was developed using Histogreen, giving a blue–green color. Sections were counterstained with hematoxylin and embedded in organic mounting media (Vitro-Clud, Langenbrink, Germany).

Double immunofluorescence labeling

In some experiments sections were stained with the maturation marker 2A1 [clone 2A1, rat IgG2a, R.M. Steinman, NY (^{Inaba et al, 1992;Steinman et al, 1997})] followed by FITC-conjugated anti-rat immunoglobulin (BD-Pharmingen). After blocking residual free antibody binding sites with an excess of rat immunoglobulin (100 g per ml) counterstaining was done with a biotinylated rat anti-mouse Langerin MoAb (929F3) and streptavidin-Texas Red.

Statistics

Appropriate statistic tests (t-test, one-way ANOVA) were chosen and used to analyze data and to determine whether the differences between the results were significant.

Results

Specificity of novel anti-Langerin/CD207 antibodies

Specificity of MoAb HD24 against murine Langerin was shown on mouse Langerin-transfected COP fibroblasts (^{Valladeau et al, 2002}). Other anti-Langerin antibodies were generated by immunization of rats and the specificity of the particular rat MoAb used in this study (929F3) is shown in Figure 1. Langerin-transfected fibroblasts stained clearly with the 929F3 MoAb in contrast to mock-transfected cells as analyzed by FACS (Figure 1).

Figure 1.

The new rat MoAb (929F3) is specific for Langerin. Fibroblasts were transfected either with a mock gene or the mouse Langerin gene. In FACS analyses the new MoAb 929F3 stained specifically the Langerin-transfected cells (right panel) and not the control cells (left panel). Thin line, isotype control; bold line, Langerin staining.

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Langerhans cells express Langerin in the steady state in the skin

MoAb against mouse Langerin specifically stain Langerhans cells. This is best visualized in epidermal sheets as first shown by^{Valladeau et al (2002)}. Here, we confirm that virtually all Langerhans cells, as identified by their MHC class II expression, display strong Langerin reactivity in intracellular granular structures. Inversely, all Langerin⁺ cells expressed MHC II (Figure 2a). In native dermis we observed only some rare Langerin⁺ cells, possibly Langerhans cells in transit (data not shown).

Figure 2.

Langerin is expressed in resident and migrating Langerhans cells. In epidermal (rows A, B) and dermal sheets (rows C, D, E + F), Langerhans cells were stained with anti-Langerin HD24 MoAb (middle column) and with anti-MHC class II (left column) or the maturation marker 2A1 as indicated. Double exposures of both colors are depicted in the right column. Langerin is expressed in all MHC class II⁺ cells, i.e., Langerhans cells, in both native (A) and cultured (B) epidermis. About 60% of migrating MHC class II⁺ cells in the lymphatic vessels in the dermis express Langerin (C). In an in vivo inflammatory situation after application of contact allergen lymphatic vessels also contained Langerin (929F3 MoAb)/MHC class II-double positive cells (D). Almost all Langerin⁺ cells (green fluorescence in E, F; using a FITC-conjugated 929F3 anti-Langerin) in the lymphatic vessels expressed the maturation marker 2A1 (red fluorescence in E, F). Mature, Langerin^– cells, i.e., migrating dermal dendritic cells are marked with arrowheads. Isotype controls were always clearly negative. Scale bar = 20 m for all pictures.

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Migratory Langerhans cells express Langerin in situ

Murine skin explants were cultured for 48 h and epidermal and dermal sheets were prepared for immunohistochemistry at the end of the culture period. Inspection of epidermal sheets confirmed the previously described phenomenon (^{Larsen et al, 1990;Lukas et al, 1996;Weinlich et al, 1998}): the density of Langerhans cells remaining in the epidermis was greatly reduced. The few remaining Langerhans cells appeared activated by their strongly increased MHC class II expression. They uniformly expressed Langerin at a staining intensity indistinguishable to that of Langerhans cells in untreated epidermis (Figure 2b). The corresponding dermal sheets showed migratory dendritic cells in "cords" (^{Larsen et al, 1990}), i.e., string-like accumulations of strongly MHC class II⁺ cells. Approximately 60% of the MHC class II⁺ cells in the "cords" expressed Langerin (Figure 2c), indicating that they were Langerhans cells from the epidermis. Langerin staining was predominantly intracellular. This percentage varied to a large extent from "cord" to "cord". The vast majority of migrating dendritic cells were mature as determined by two criteria. First, even conventional fluorescence microscopy revealed the prominent cell surface localization and the absence of intracellular MHC II molecules, which may be regarded as a sign of maturation (^{Pierre et al, 1997}). Secondly, and more specifically, most emigrating Langerin⁺ Langerhans cells in the "cords" coexpressed the maturation marker 2A1 (Figure 2e,f).

Migratory Langerhans cells express Langerin in vivo

An inflammatory situation in the skin in vivo was induced by the epicutaneous application of the contact allergen TNCB. After 24 h only few lymph vessels filled with emigrating cutaneous dendritic cells were visible. "Cords" were more pronounced after 48 h, albeit to a lesser degree as compared with the skin explant culture model. Again, a large part of the migratory cells were Langerhans cells as defined by their marked expression of Langerin (Figure 2d).

Origin and phenotype of dendritic cells that emigrate out of skin explants

As opposed to the human system (^{Lenz et al, 1993;Larregina et al, 2001}) it has not been possible so far to draw an unequivocal distinction between Langerhans cells and dermal dendritic cells that migrate from murine whole skin explants. We therefore used Langerin antibodies to analyze dendritic cells that had "crawled out" from murine whole skin explants over a period of 48 h. Double-labeling of cytospins revealed that virtually all Langerin-expressing cells were also strongly MHC class II⁺ and expressed the maturation marker 2A1 (^{Inaba et al, 1992}) (data not shown). Conversely, however, not all MHC class II⁺ cells were anti-Langerin reactive. Control specimens of emigrant dendritic cells from epidermal sheets that had been obtained by dispase treatment before the onset of culture, were virtually all Langerin⁺ (Figure 3a). Dendritic cells from the corresponding dermal sheets were largely Langerin^– (Figure 3a), yet, a substantial subpopulation of Langerin⁺ dendritic cells was consistently found. As expected, dendritic cells from whole skin explants represented a mixture of Langerin⁺ (i.e., Langerhans cells) and Langerin^– (i.e., dermal dendritic cells) MHC class II⁺ cells (Figure 3a). Both populations occurred in approximately equal proportions, Langerhans cells being slightly more frequent. A mean of 62% of all MHC class II⁺ cells coexpressed Langerin on cytospins. By FACS, similar percentages of intracellularly Langerin⁺ cells were determined for populations of dendritic cells (i.e., MHC class II⁺ cells) emigrated from dispase-procured epidermal sheets, corresponding dermal sheets and from whole skin explants (Figure 3b). In preparations of dermal cells isolated by collagenase digestion of native dermis we found that an average of 10% of the MHC class II-expressing cells were positive for Langerin (Figure 3c). Interestingly, after a culture period of 2 d the proportion of Langerin-expressing cells in the MHC class II⁺ fraction rose significantly over 50% and decreased afterwards.

Figure 3.

Origin of migratory skin dendritic cells. From skin explant cultures of epidermis, dermis, and whole skin, cutaneous dendritic cells emigrated into the medium during a 48 h incubation. (A) Emigrated cells were double labeled for Langerin and MHC class II on cytospins and double-positive cells were scored (mean SD, n = 4, ^*p<0.01, ^**p<0.001). (B) By FACS, these migratory cells were double labeled for Langerin and MHC class II and analyzed (one representative experiment of at least three). Nearly all epidermal cells were double positive for both markers (left panel) in contrast to the dermal cells (middle panel). Mixed populations of Langerhans cells and dermal dendritic cells emigrated from complete skin (right panel). (C) Dermal cells were prepared by collagenase digestion and cultured up to 3 d stained with anti-Langerin and MHC class II MoAb. By FACS Langerin⁺ cells of all MHC class II expressing cells were determined and are presented in percent (meanSD, n=5, ^*p<0.001). An increase in Langerin⁺ cells in the dendritic cell population in the dermis could be best observed after 2 d of culture. For all experiments we used 929F3 MoAb.

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Retention of Langerin in Langerhans cells during maturation

Based on its specificity it is tempting to use Langerin as a tracer for Langerhans cells from the epidermis. Yet, Langerin is endocytosed quickly after antigen uptake (^{Valladeau et al, 1999}) and is downregulated upon Langerhans cell culture (^{Valladeau et al, 2002}). Birbeck granules disappear at the ultrastructural level (^{Schuler and Steinman, 1985}). We therefore wondered for how long Langerhans cells, that were generated by different isolation procedures, would express Langerin. Emigrated Langerhans cells obtained by epidermal explant culture and Langerhans cells isolated by trypsinization of native epidermis and cultured together with keratinocytes were stained side by side for Langerin and MHC II and analyzed on cytospins or by FACS. On cytospins, migratory Langerhans cells identified by their MHC II expression, retained Langerin expression during 4 d of skin explant culture and only few Langerhans cells completely lost their Langerin expression (data not shown). Cytospins of day 4 Langerhans cells obtained by trypsinization of native epidermis showed a more pronounced decrease in Langerin expression with more cells actually losing anti-Langerin reactivity, as described by^{Valladeau et al (2002)}. Time-course experiments were evaluated by more sensitive FACS analyses. We observed a decrease in intracellular Langerin staining intensity in the Langerhans cells generated both ways (Figure 4) with a more pronounced reduction in the Langerhans cells obtained by trypsinization.

Figure 4.

Loss in intensity of Langerin expression in Langerhans cells during prolonged culture periods. Langerhans cells were generated either by trypsinization of the epidermis and subsequent culture for 4 d (upper panels) or by migration out of epidermal sheets during 4 d (lower panels). Cells were double labeled for Langerin (929F3 MoAb) and MHC class II and analyzed by FACS. This experiment is representative of four independent experiments.

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Langerhans cells can be detected in the T cell area of skin draining lymph nodes in the steady state

As intracellular Langerin expression is retained in migratory Langerhans for some time, we used it as a Langerhans cell tracer molecule and searched for Langerin⁺ cells in the lymph nodes. Langerin-expressing cells can be found in skin draining lymph nodes from untreated mice as recently shown by^{Henri et al (2001)}. Therefore, we first compared Langerin expression in skin-draining vs mesenteric lymph nodes of untreated mice. Lymph node cells were prepared, enriched for dendritic cells by a Nycodenz gradient, stained for Langerin and analyzed by FACS. We found a significant population of Langerin⁺ cells in skin draining lymph nodes (Figure 5a). By contrast, in mesenteric lymph nodes only very few Langerin⁺ cells occurred (Figure 5a). By immunohistochemistry of lymph node sections this difference was confirmed in that much higher numbers of Langerin-expressing cells were visible in the skin-draining nodes than in the mesenteric lymph nodes (Figure 5b).

Figure 5.

Langerin⁺ cells are found in skin draining lymph nodes. (A) Cells from skin-draining and mesenteric lymph nodes were isolated, enriched on a Nycodenz-gradient and stained for Langerin. In skin draining lymph nodes (left panel) we found substantial numbers of Langerin⁺ cells in contrast to mesenteric lymph nodes (right panel). Langerin staining bold line, isotype staining, dashed line. One representative experiment of three is shown. (B) By immunohistochemistry high numbers of Langerin⁺ cells (blue color) can be appreciated in an auricular node (left panel) but only few scattered Langerin⁺ cells in a mesenteric node (right panel). One representative experiment of three is shown. Panel B, original magnification 60. For all experiments we used 929F3 MoAb.

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To locate more precisely these migratory skin dendritic cells in situ, we stained sections of skin draining lymph nodes of untreated mice with anti-Langerin and markers that define the T and B cell areas (CD3 for T cells and CD45R/B220 for B cells). Langerin⁺ cells were found in the T cell areas. By contrast, B cell follicles did not harbor Langerin-reactive cells in general but rarely a few cells could be observed (Figure 6 a–c). On consecutive sections CD11c and CD205/DEC-205 localized to the same areas (data not shown). To underscore these findings we conducted double-labeling of lymph node sections. Langerin-expressing cells colocalized with CD3⁺ T cells (Figure 6d) but were excluded from B cell areas (defined by anti-CD19 labeling) (Figure 6e).

Figure 6.

Langerin⁺ cells localize to the T cell areas of skin draining lymph nodes. Lymph node sections were immunolabeled to determine the localization of Langerin⁺ cells (929F3 MoAb). On consecutive sections (top row) it becomes apparent that Langerin⁺ cells (b) are in the T cell area as defined by anti-CD3 reactivity (c) but not in the B cell follicles as defined by B220 labeling (a). Double labeling of Langerin (blue cells) and CD3 (d) and CD19 (e) (brown cells) confirms the mutual exclusion of B cells and Langerin-reactive cells (e). Original magnification: (a–c) 40; (d,e) 240.

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Increased numbers of Langerhans cells can be detected in the skin draining lymph nodes after contact sensitization

Next, we investigated Langerin expression in skin draining lymph nodes 24 h after application of a contact sensitizer (1% TNCB) to abdominal and ear skin. Cytospins of unenriched suspensions of lymph node cells were prepared and stained for Langerin. In lymph nodes from control mice that had been treated with the vehicle only (acetone/olive oil) few Langerin⁺ cells were detected by immunofluorescence microscopy. The numbers of Langerin⁺ cells per inguinal lymph node in vehicle-treated mice was on average 48602700. In inguinal lymph nodes from sensitized animals the number of Langerin-expressing cells increased up to an average 5435022960 (Figure 7a) thus inducing an 11-fold increase in immigrated Langerhans cells. In FACS analyses of skin draining lymph node cells (auricular and inguinal) from vehicle- or TNCB-treated mice we observed a pronounced increase in the percentages of Langerin⁺ cells (Figure 7b). When these percentages were multiplied with the total cell numbers obtained in the hemocytometer we observed again a rise in absolute Langerhans cell numbers in skin draining lymph nodes after TNCB application (Figure 7c); however, this was not as pronounced, which may be explained by the different experimental settings.

Figure 7.

Increased numbers of Langerin⁺ cells in skin draining lymph nodes after contact sensitization with TNCB. Mice were treated with the contact sensitizer TNCB or vehicle (acetone/olive oil) on the shaved abdominal or ear skin. After 24 h skin draining lymph node cells were prepared and stained for Langerin (929F3 MoAb). (A) On cytospins, nuclei were counterstained with DAPI and the Langerin⁺ cells were counted (meanSD, n=5, ^*p<0.001). (B) By FACS, the percentage of Langerin⁺ cells in total lymph node suspensions was determined. One representative experiment of 11. (C) Absolute numbers of Langerhans cells determined by relating FACS-derived percentages with hemocytometer counts (meanSD, n=11, ^*p<0.0001).

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Phenotype of Langerin⁺ cells in skin draining lymph nodes

Langerin⁺ cells in the nodes of untreated mice were further analyzed, particularly with regard to their state of maturation. Cells were isolated from skin draining lymph nodes, enriched for dendritic cells with a Nycodenz-gradient. CD11c⁺ and CD11c^– cell populations were analyzed for Langerin and MHC class II expression by FACS. No Langerin staining was found in the CD11c^– fraction emphasizing the specificity of Langerin for dendritic cells. All Langerin-expressing cells were detectable in the CD11c⁺ fraction representing lymph node dendritic cells. Furthermore, Langerin⁺ cells expressed high levels of MHC class II, which has been described to be a marker for migratory Langerhans cells in skin draining lymph nodes (^{Ruedl et al, 2000;Henri et al, 2001}) (Figure 8a). Almost all migrating Langerin⁺ cells in the lymphatic vessels of the skin expressed the maturation marker 2A1 (Figure 2e,f). We were therefore interested whether the Langerin⁺ cells that arrive in the lymph node in the "steady state" were already mature. Indeed, by immunofluorescence on lymph node sections we noted that most, but not all, Langerin⁺ cells coexpressed the maturation marker 2A1^- (Figure 8b). Thus, a distinct minor (< 3% of Langerin⁺ cells) subset of immature (i.e., 2A1) Langerhans cells was consistently detected in the lymph nodes. FACS analyses confirmed that almost all Langerin⁺ cells in the skin draining lymph node coexpressed the costimulatory molecules CD86 and to a lesser extent CD40 (Figure 8c). For comparison, we induced a strong inflammation in the skin by epicutaneous application of the contact allergen TNCB and examined the maturation state of immigrated Langerhans cells in the skin draining lymph nodes 24 and 48 h thereafter. We observed a slight but consistent increase in cells coexpressing Langerin and CD86 or Langerin and CD40; however, this was not statistically significant (Figure 8c). Additionally, the intensity of these two costimulatory molecules on Langerin⁺ cells in the lymph nodes was consistently enhanced after application of contact allergen (Figure 8d).

Figure 8.

Phenotype of Langerin⁺ cells in skin draining lymph nodes. (a) Skin draining lymph node cells were isolated, enriched with a Nycodenz-gradient, and analyzed by FACS for MHC class II and Langerin expression. Langerin⁺ cells were only found in the CD11c⁺, i.e., dendritic cell fraction and expressed high levels of MHC class II. (b) Double labeling of skin draining lymph node sections with the maturation marker 2A1 (green fluorescence) and Langerin (red fluorescence) shows that nearly all Langerin⁺ cells are mature (yellow fluorescence in A). (A) Overview, double exposure. Some mature non-Langerhans cells are marked with arrowheads(B,C) Same field, single labeling. (D,E) higher magnification of 2A1 (E), Langerin (F), and overlap (D). Scale bars: (A) 50 m; (B,C) 100 m; (D–F) 20 m. (c) Skin draining lymph node cells were enriched with a Nycodenz-gradient and examined for expression of costimulatory molecules CD86 and CD40. A high percentage of Langerin⁺ cells expressed these markers and numbers increased after induction of a contact hypersensitivity reaction (meanSD, n=5). (d) By FACS analysis an increase in intensity of CD86 (left panel) and CD40 (right panel) expression was observed in TNCB-treated mice (bold line) in contrast to untreated mice (thin line). Dotted lines are isotype controls. One representative experiment of five is shown. For all experiments we used 929F3 MoAb.

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Discussion

Until now, the hallmark for murine Langerhans cells was the tennis-racket-shaped Birbeck granule that could only be visualized by transmission electron microscopy (^{Birbeck et al, 1961;Wolff, 1967;Hashimoto, 1971}). Langerin/CD207, a type II transmembrane protein belonging to the family of Ca²⁺-dependent (C-type) lectin receptors, was identified as a main molecular component of Birbeck granules, first in humans (^{Valladeau et al, 1999, 2000}) and then in the mouse (^{Valladeau et al, 2002}). The development of monoclonal anti-mouse Langerin antibodies (^{Valladeau et al, 2002}) made it possible for the first time to trace murine Langerhans cells along their migration pathway. We show here that Langerhans cells express Langerin during the entire migration process from the epidermis through lymphatic vessels into the lymph nodes.

Origin of dendritic cells emigrating out of whole skin explants

Novel anti-mouse Langerin MoAb (^{Valladeau et al, 2002}) make it possible for the first time to determine the relative proportions of Langerhans cells and dermal dendritic cells in murine whole skin explant cultures. As expected, dendritic cells "crawling out" from epidermal sheets are virtually all Langerin⁺, and dendritic cells originating from dermal sheets are largely Langerin^–. Still, surprisingly many Langerin⁺ cells occurred in the dermal emigrant populations as compared with human dermal populations where this percentage rarely exceeded 5% of all dendritic cells (^{Lenz et al, 1993}). This is in contrast to the finding that immunofluorescence of native dermis revealed only few scattered Langerin⁺ cells. One explanation for this discrepancy might be the minute pieces of epidermis that inevitably adhere to the dermal sheet after dispase separation or hair follicles as observed by histologic hematoxylin and eosin staining. Another source for Langerin⁺ Langerhans cells in the dermis could be emigrating Langerhans cells already on their way out of the skin in the steady state, but one might not expect such high numbers. A further possibility is that Langerin⁺ cells in the dermal preparations are immigrating Langerhans cell precursors, expressing already MHC class II but not yet Langerin. They would develop into Langerin-expressing cells during the emigration out of the dermal explants. This possibility is underscored by our observation that dermal cell suspensions isolated by enzymatic digestion and cultured for 2 d contained over five times more Langerin⁺ cells as compared with the onset of culture. Such a precursor population migrating out of human skin explants expressing CD14⁺ and Langerin has been described by^{Larregina et al (2001)}; however, preliminary experiments did not reveal the presence of CD14⁺ cells in freshly isolated dermal cell suspensions.

Langerhans cells can be traced to the draining lymph nodes by their Langerin expression

As Langerin is expressed intracellularly in Langerhans cells for quite a long time, we were able to track migrating Langerhans cells to their destination in the lymphoid organs.^{Henri et al (2001)} observed that migratory skin dendritic cells still express Langerin in the skin draining lymph nodes. Here we extend these initial findings in that Langerin⁺ cells were found in the T cell areas and were almost always absent from B cell follicles. No Langerin expression could be detected in the CD11c^– cells in the lymph nodes underlining that Langerin is an exclusive marker for dendritic cells. Further characterization of Langerin-expressing cells in the lymph nodes revealed that these cells express high levels of MHC class II as has been described for immigrated Langerhans cells in the lymph nodes (^{Ruedl et al, 2000;Henri et al, 2001}). Furthermore, we demonstrate that substantial numbers of Langerin⁺ cells were only found in skin draining lymph nodes, whereas just few scattered cells were visible in the mesenteric lymph nodes. This emphasizes the validity of Langerin as a marker for epidermis-derived Langerhans cells. Weak Langerin staining in the mesenteric lymph nodes was shown by^{Valladeau et al (2002)}. They also described Langerin⁺ cells in epithelia of the lung and the tonsils. Similar Langerin-expressing cells in the epithelium of the gut (A. Kaser, personal communication) would be a possible source for the Langerin⁺ cells in the mesenteric lymphoid tissue.

To enhance the migration of Langerhans cells into the lymph nodes we induced contact hypersensitivity reactions with TNCB and observed an increase in Langerin⁺ cells in skin draining lymph nodes. Other studies have shown that higher numbers of dendritic cells can be found in the lymph nodes after application of different contact sensitizers (^{Knight et al, 1985;Macatonia et al, 1986;Botham et al, 1987}). Dendritic cells were identified by their forward/side scatter profiles in the FACS, their expression of MHC class II and other dendritic cell markers (CD11c), or by the presence of sensitizing trackers such as FITC. These assays did not allow an unequivocal discrimination between migratory Langerhans cells and migratory dermal dendritic cells and their relative contributions to sensitization. Additionally, the assays may not always permit a clear-cut distinction between migratory cells or lymph node-resident MHC class II⁺ cells because free FITC may diffuse through blood and lymph to spleen and lymph nodes, respectively, where it would stain resident dendritic cells (^{Pior et al, 1999}; and own observations). The MoAb against Langerin used here are tools to visualize selectively immigrated Langerhans cells in the skin draining lymph nodes and to study the relative contributions of Langerhans cells to the induction of immune responses.

Immigrated Langerhans cells in the skin draining lymph nodes are largely mature in the steady-state situation

In the steady-state, immature or partially mature Langerhans cells are thought to carry self antigens to the lymph nodes thereby maintaining peripheral tolerance (^{Steinman and Nussenzweig, 2002}). Because of the lack of a Langerhans cell-specific marker it was not possible to define the maturation stage of immigrated Langerhans cells in the steady-state vs the inflammatory situation. Here we show for the first time that Langerhans cells trafficking into the lymph node in the steady state already express maturation marker 2A1, costimulatory molecules CD86 and CD40, and high levels of MHC class II. This is in some contrast to recent observations by^{Geissmann et al (2002)} who reported that Langerin⁺ cells were largely immature in human lymph nodes, albeit in a different situation, i.e., a chronic inflammation. We observed a small but consistent increase in the expression of the CD86 and CD40 after application of contact allergen, i.e., in response to inflammation. In keeping with our data,^{Ruedl et al (2001)} showed that CD40⁺/CD11c⁺ sorted lymph node cells, possibly Langerhans cells, expressed CD80 and CD86 in the steady state and upregulated them after application of a fluorescent tracer, i.e., in response to inflammation, too. In addition, the lymph node dendritic cells still took up and processed antigens to some degree also indicating an incomplete maturation state (^{Koch et al, 1995;Ruedl et al, 2001}).

In a mouse model of tolerance induction by injection of the anti-DEC-205 MoAb coupled to a protein, tolerance was broken after simultaneous injection of anti-CD40 MoAb, which increased surface expression of CD86 and CD40 on CD11c⁺ lymph node dendritic cells (^{Hawiger et al, 2001}).^{Menges et al (2002)} demonstrated that partially mature dendritic cells expressed lower levels of CD40 molecules and prevented the development of experimental autoimmune encephalomyelitis in mice in contrast to fully matured dendritic cells when adoptively transferred.

Migration is closely linked to the maturation process. Upregulation of CCR7, for instance, is a prerequisite for migration to occur (^{Caux et al, 2002}). Therefore, it is unlikely that Langerhans cells arriving in the lymph node in the steady state would still express an immature phenotype, even though^{Geissmann et al (2002)} recently suggested a way of uncoupling migration (CCR7 expression) from maturation (CD83, CD86, and CD208 expression). We observed here that, in response to inflammation, higher numbers of more mature Langerhans cells arrive in the lymph node. This would increase the frequency of encounters with T cells and thus the generation of immunity. Thus, a combination of migration levels and the stage of dendritic cell maturation might determine the outcome of the response in the lymph node. This new antibody will allow to gain further insights into these functional states of Langerhans cells.

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Acknowledgments

The continued support of Dr Peter Fritsch, Chairman of the Department of Dermatology is greatly appreciated. This work was supported by a grant from the Austrian Science Fund to N. Romani (FWF grant no. P-14949-MED).