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(Journal of Leukocyte Biology. 2002;71:845-853.)
© 2002 by Society for Leukocyte Biology

Antagonistic effects of IL-4 and TGF-ß1 on Langerhans cell-related antigen expression by human monocytes

G. Guironnet, C. Dezutter-Dambuyant, C. Vincent N. Bechetoille, D. Schmitt and J. Péguet-Navarro

Department of Dermatology, INSERM U346, Hôpital E. Herriot, Lyon, France

Correspondence: J. Péguet-Navarro, Department of Dermatology, INSERM U346, Hôpital E. Herriot, Lyon, France. E-mail: peguet{at}lyon151.inserm.fr


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ABSTRACT
 
In this study, we analyzed the specific effects of transforming growth factor ß (TGF-ß1) and/or IL-4 on monocyte-derived cells. Monocytes were cultured with GM-CSF, GM-CSF/TGF-ß1, GM-CSF/IL-4, or GM-CSF/IL-4/TGF-ß1 before cell morphology, phenotype, and function were assessed. As expected, interleukin-4 is mandatory for monocyte differentiation into potent allostimulatory DC. In its absence, monocyte-derived cells share many phenotypic and functional features with macrophages. However, it is interesting that the cells express E-cadherin, independent of exogenous TGF-ß1, and addition of the cytokine induced CCR6 expression. Most importantly, a subset of monocytes cultured with GM-CSF/TGF-ß1 expresses Langerin, as confirmed by electron microscopy analysis. Langerin engagement with specific monoclonal antibodies induces its internalization and the formation of typical Birbeck granules. Monocytes cultured in GM-CSF/IL-4 did not express the LC markers E-cadherin, CCR6, or Langerin. The simultaneous addition of TGF-ß1 allows most of the cells to express E-cadherin but rarely CCR6 and Langerin. Taken together, the results add further evidence that LC can derive from monocytes and demonstrate an antagonistic effect of IL-4 and TGF-ß1 on monocyte differentiation toward the LC pathway.

Key Words: IL-4 • dendritic cells • E-cadherin • langerin


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INTRODUCTION
 
Dendritic cells (DC) are antigen-presenting cells (APC) characterized by their unique ability to initiate primary T-cell responses in vitro and in vivo [1 , 2 ]. Many types of DC with subtle differences in phenotype have been described in peripheral blood and in most lymphoid or nonlymphoid organs. The heterogeneity of DC relates to different developmental pathways and activation/maturation stages. Although each of these myeloid DC subsets derives from hematopoietic cells and displays the ability to activate naive T cells, it is not yet clear whether they represent different stages of maturation of a unique DC lineage or whether they stem from different progenitors.

In vitro, DC can be generated from CD34+ cord blood or bone marrow progenitors in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) and tumor necrosis factor {alpha} (TNF-{alpha}). At an early stage of differentiation, two distinct DC populations emerged: the CD1a+ DC precursors, which differentiate into typical Langerhans cells (LC), Birbeck granules+, Langerin+, and E-cadherin+, and the CD14+ DC precursors, which differentiate into interstitial DC, expressing CD68 and factor XIIIa as dermal DC. Several studies have identified transforming growth factor ß (TGF-ß1) as a mandatory factor for LC development in vivo and in vitro. In particular, TGF-ß1 null mice lack LC, whereas other DC populations appear normal [3 ]. In humans, TGF-ß1 is required for the development of CD1a+ and CD14+ populations from CD34+ progenitors [4 5 6 7 8 ]. Furthermore, addition of exogenous TGF-ß1 polarized the CD14+ population toward the LC pathway. It is interesting that addition of interleukin (IL)-4, even in the presence of TGF-ß1, results in the development of DC lacking LC features.

Immature DC displaying dermal DC features can be generated in vitro from peripheral blood monocytes. IL-4 combined with GM-CSF appears as a mandatory cytokine for this differentiation pathway. In a previous study, Geissmann et al. [9 ] demonstrated that addition of TGF-ß1 to the culture medium results in cells with typical LC features.

In the present study, we analyzed the effects of IL-4 and TGF-ß1, alone or in combination, on the morphology, phenotype, and function of peripheral blood CD14+ cells cultured with GM-CSF. We found that in the absence of IL-4, the cells express E-cadherin independent of exogeneous TGF-ß1, and addition of the cytokine induced CCR6 expression. It is most interesting that a part of cells cultured with GM-CSF and TGF-ß1 expresses Langerin and typical Birbeck granules upon cross-linking. IL-4 down-regulates expression of all these LC-related markers even in the presence of TGF-ß. The results add further evidence that LC can derive from monocytes and demonstrate an antagonistic effect between IL-4 and TGF-ß1 on monocyte differentiation toward the LC pathway.


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MATERIALS AND METHODS
 
Culture medium
Culture medium was RPMI 1640-supplemented with L-glutamine (Gibco-BRL, Grand Island, NY), 1% antibiotic solution (Sigma Chemical Co., St. Louis, MO), and 10% heat-inactivated fetal calf serum (Myoclone, Gibco-BRL), thereafter called complete medium.

Monocyte isolation and culture
Mononuclear cells were obtained from peripheral blood of healthy donors by centrifugation on Ficoll-Hypaque (Pharmacia, St. Quentin en Yvelines, France). Cells (40x106) were layered sequentially on a discontinuous Percoll gradient [50% and 40% in phosphate-buffered saline, 5% fetal calf serum (FCS)] and centrifuged at 450 g for 25 min at 4°C. The monocyte-enriched fraction (nearly 70%) was collected from the interface over the 50% Percoll solution, and lymphocytes were recovered from the cell pellet. Then, monocytes were purified by negative magnetic depletion using hapten-conjugated anti-CD3, -CD7, -CD19, -CD45RA, and -CD56 (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany) and a magnetic cell separator (Midi MACS), according to the manufacturer’s instructions. The technique resulted routinely in >85% purity, as assessed by flow cytometry.

Purified monocytes were cultured for 6 days in six-well tissue-culture plates (Costar Corp., Cambridge, MA) in complete medium supplemented with different combinations of the following cytokines: recombinant human (rh)GM-CSF (200 ng/ml; specific activity: 2x106 U/mg, Schering-Plough Research, Kenilworth, NJ), IL-4 (33 ng/ml; specific activity: 5x106–20x106 U/mg, R&D Systems, Minneapolis, MN), and rhTGF-ß1 (10 ng/ml; specific activity: 16x106–50x106 U/mg, R&D Systems). The different cytokine combinations were GM-CSF alone, GM-CSF + TGF-ß1, GM-CSF + IL-4, or GM-CSF + IL-4 + TGF-ß1. At days 2 and 4, cells were fed with fresh medium and cytokines. To develop macrophages, some monocytes were cultured in RPMI 1640 supplemented with 40% human AB serum.

CD40 ligand (CD40L)-mediated activation
For study of CD40L-mediated activation, fibroblastic L cells transfected with CD40L or CD32 as controls (kindly provided by Schering-Plough Laboratories) were irradiated at 80 Gy and added to the monocyte cultures in a proportion of 1/10. Cells were collected 40 h later.

Flow cytometry
Briefly, 1 x 105 cells were incubated for 20 min at 4°C with affinity-purified mouse monoclonal antibodies (mAb) at the appropriate concentration or with irrelevant isotype-matched mouse immunoglobulins (Igs) at the same concentration. Cells were washed and for indirect staining, further incubated for 20 min at 4°C with fluorescein isothiocyanate (FITC)-conjugated F(ab)'2 fragments of goat anti-mouse Ab. The following mAb were used: anti-human leukocyte antigen (HLA)-DR-FITC, B8.12.2 (IgG1); anti-CD54-FITC, 84H10 (IgG1); anti-CD80-FITC, mAb 104 (IgG1); anti-CD83, HB15A (IgG1); and anti-CD40, mAb 89 (IgG1; all from Immunotech, Marseille, France); anti-CD1a-FITC, BB-1; anti-CD64-FITC, 10.1; and anti-CD86, IT2.2 (IgG1; from Pharmingen, San Diego, CA); anti-CD14-FITC, TUK 4 (IgG1; from Dako, Glostrup, Denmark); anti-E-cadherin, HECD-1 (IgG1; from Takara, Shiga, Japan); anti-CD16-phycoerythrin (PE), B73.1 (IgG1); and anti-Langerin (DCGM4, IgG1; a generous gift from Schering-Plough, Dardilly, France); anti-CD71-FITC, Lo1.1 (IgG2a; from Becton Dickinson, San Jose, CA); and anti-CCR6-PE (IgG2b; from R&D Systems).

For double-color fluorescence, cells were incubated with FITC-labeled, anti-HLA-DR, CD1a, CD54, CD80, or CD86, washed, and stained further with Langerin-PE (DCGM4, IgG1; Immunotech).

Intracellular staining for Factor XIIIa (polyclonal rabbit antiserum, Behringwerke, Marburg, Germany), CD68, KP1, IgG1 (Dako), or Langerin was carried out using the fix and perm cell permeabilization kit (Caltag Laboratories, S. San Francisco, CA), according to the manufacturer’s instructions. Cells were washed twice, and primary antibodies were visualized with FITC-conjugated goat anti-rabbit or goat anti-mouse Ig.

Fluorescence analysis was performed on a FACScan using the LYSYS II software (Becton Dickinson, Le Pont de Claix, France).

Mixed lymphocyte reaction
Allogeneic T cells were isolated from the lymphocyte pellet obtained after Percoll gradient by rosetting with sheep red blood cells as described previously [10 ]. The T-cell population contained >95% CD3+, as assessed by flow cytometry. Mixed lymphocyte reactions were carried out in round-bottomed microtiter plates (105 allogeneic T cells and graded numbers of DC per well). Triplicate cultures were maintained for 5 days at 37°C in a humidified atmosphere of 5% CO2. T-cell proliferation was measured by pulsing the cells with 0.4 µCi 3H-methylthymidine (25 Ci/mmol, Amersham, Les Ulis, France) for the final 18 h of culture. Cells were then harvested, and incorporated thymidine was quantitated in a direct beta-counter (Matrix 96, Packard Instruments Co., Meriden, CT).

Electron microscopy
Fixed cells
Cells were fixed in 2% glutaraldehyde in cacodylate buffer at 4°C for at least 3 days. The samples were post-fixed for 60 min with 1% osmium tetroxide in cacodylate buffer with sucrose, dehydrated, and embedded in Epoxy medium. Ultrathin sections were examined after post-staining with uranyl acetate and lead citrate on a JEOL 1200 EX electron microscope (CMEABG, Université Lyon, France).

Vital cells
Cells were incubated with DCGM4 mAb followed by goat anti-mouse IgG conjugated with 5 nm gold particles (Amersham, Arlington Heights, IL). Cells were fixed immediately or warmed up to 37°C for 10 min before fixation.


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RESULTS
 
Monocytes cultured with GM-CSF and TGF-ß1 develop into nonadherent cells that express the LC-related molecules E-cadherin and Langerin
In earlier studies, monocytes cultured with GM-CSF alone have been shown to develop into adherent macrophagic cells [11 , 12 ]. In our isolation and culture conditions, most monocytes became nonadherent cells when cultured for 6 days with GM-CSF or GM-CSF/TGF-ß1 (Fig. 1B and 1C ). Cells supplied with exogenous TGF-ß1 exhibited a characteristic crescent shape (Fig. 1C and 1E) , and cells cultured in GM-CSF alone were more round (Fig. 1B) . Both populations appeared quite different from typical macrophages differentiated from monocytes in the presence of 40% human AB serum (Fig. 1A) . As demonstrated earlier, addition of IL-4 generated nonadherent, dendritic-shaped cells (Fig. 1D and 1E) .



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  		Figure 1. Morphology of day 6 monocyte-derived cells cultured under the different cytokine combinations. Isolated monocytes were cultured for 6 days in RPMI medium supplemented with 40% human AB serum (A) and 10% FCS and GM-CSF (B), GM-CSF/TGF-ß1 (C), GM-CSF/IL-4 (D), or GM-CSF/IL-4/TGF-ß1 (E). Cells were then examined under an inverted microscope (original magnification, x200).

Phenotypic analysis was carried out, and Figure 2 gives the results from a representative experiment. In any cytokine condition, monocyte-derived cells expressed HLA-DR, CD54, and CD40, and CD80, CD86, and CD83 were faintly or not expressed at the cell surface (Fig. 2A) . For most of these markers, a similar pattern of expression was observed, independent of the presence of IL-4 in the culture medium. However, a decrease in HLA-DR and CD54 staining was observed consistently on monocytes cultured with GM-CSF/IL-4/TGF-ß1.



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  		Figure 2. Phenotypic analysis of monocyte-derived cells cultured under the different cytokine combinations. Monocytes were cultured for 6 days in RPMI supplemented with 40% human AB serum (SAB) or 10% FCS and the indicated combinations of cytokines. Cells were then labeled with a panel of antibodies specific to DC-related molecules (A), monocyte/macrophage-related molecules (B), or LC-related molecules (C) and examined by flow cytometry. Results with irrelevant isotype-matched control mAb were overlaid (empty histograms). In some cases, the MFI followed by the percentage of stained cells was indicated. Data are representative of 10 experiments.

In the absence of IL-4, cells expressed high levels of CD14 (Fig. 2B) and almost no CD1a (Fig. 2C) , whereas the opposite pattern was observed on cells supplied with the cytokine (Fig. 2B) . The monocyte/macrophage markers CD16 and CD71 were found exclusively on cells cultured with GM-CSF or GM-CSF/TGF-ß1 (Fig. 2B) . CD68 was highly expressed on cells cultured without IL-4, and the staining intensity was weaker in the presence of the cytokine. All the cells expressed FXIIIa, but the expression level was far higher when IL-4 was supplied.

Unexpectedly, we found that monocytes cultured in GM-CSF or GM-CSF/TGF-ß1 expressed the LC-related molecule E-cadherin (Fig. 2C ; Table 1 ). E-cadherin was absent on freshly isolated monocytes but already expressed at a lower level after a 3-day culture. Importantly, a similar pattern of E-cadherin expression was found on typical macrophages generated from monocytes cultured in the presence of human AB serum (Fig. 2C) or M-CSF (unpublished results), as well as on monocytes cultured in serum-free medium in the absence of any exogenous cytokines (unpublished results).


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  		Table 1. Antagonist Effects of IL-4 and TGF-ß1 on LC-Related Antigen Expression by Cultured Monocytes

It is most interesting that a minor population of GM-CSF/TGF-ß1-treated monocytes expressed Langerin at the membrane as well as the intracellular level (Fig. 2C ; Table 1 ). Langerin expression could be detected already after a 4-day culture (unpublished results). As revealed by double-staining experiments, all the Langerin-positive cells coexpressed CD1a, HLA-DR, CD54, and CD80 but not CD86 antigens (Fig. 3 ), suggesting an immature phenotype.



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  		Figure 3. Phenotype of the Langerin-positive cells. Monocytes were cultured for 6 days with GM-CSF and TGF-ß1 and were double-stained with FITC-conjugated mAb followed with anti-Langerin-PE. Cells were analyzed on a FACScan. Numbers indicate the percentage of cells in the quadrant.

By contrast, the GM-CSF/IL-4-derived DC never expressed the LC-related markers E-cadherin and Langerin (Fig. 2C ; Table 1 ). Similar results were obtained when using far lower IL-4 concentrations (5 or 15 ng/ml; unpublished results). As described previously [9 ], addition of TGF-ß1 into the culture medium allowed a significant proportion of cells to express E-cadherin after a 6-day culture (Fig. 2C ; Table 1 ). The pattern of E-cadherin expression was similar whether the cells had been cultured with low (5 ng/ml) or high IL-4 concentrations (30 ng/ml; unpublished results). Langerin expression was observed rarely (Table 1) , and it coincided with a high expression of E-cadherin. Globally, the percentage of positive cells and the mean fluorescence intensity (MFI) for both markers were always far lower than those observed on GM-CSF/TGF-ß1-derived cells (Fig. 2C) .

The expression of CCR6, the chemokine receptor specific to the LC lineage, was shown in Table 1 . Although CCR6 expression could be noticed on a significant proportion of the cells (25.5±14.4%) when cultured with GM-CSF/TGF-ß1, a dramatic down-regulation of the percentage of stained cells was observed in the presence of IL-4 (0.7±1.5%).

Electron microscopy analysis
In any culture conditions, monocyte-derived cells displayed immature cell features such as large cytoplasm, numerous organelles, and short villosities (not shown). The cells exhibited numerous small clear vacuoles in their cytoplasm, rare class II compartments, and phagosomes were absent (not shown). All these features are characteristic of immature myeloid DC at an early stage of differentiation.

As illustrated in Figure 4 , some of the cells contained intracytoplasmic granules that varied in size and appearance according to the culture conditions. Ring-shaped granules were observed in about 15% of the cells cultured in TGF-ß1-free medium, independent of the presence of IL-4 (9 out of 65 cells cultured with GM-CSF and 14 out of 76 cells with GM-CSF/IL-4; Fig. 4A ). They were identical to those described previously by Grassi et al. [13 ]. Conversely, TGF-ß1 induced the formation of rod-shaped Birbeck granule-like structures (Fig. 4B) , although in a lower proportion of cells when culture was carried out in the simultaneous presence of IL-4 (4 out of 73 cells, i.e., 5%, vs. 15 out of 101 cells). Elongated and electron-lucent structures were observed while forming from the plasma membrane or while scattered in the cytoplasm (Fig. 4B , panels 2 and 4). Some short structures similar to Birbeck granules were also noted, although their central lamella were interrupted or absent and were rarely striated (Fig. 4B , panels 1 and 3).



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  		Figure 4. Depending on TGF-ß1 supply, cells harbor rod-shaped or ring-shaped intracytoplasmic granules. Monocytes were cultured for 6 days with the different cytokine combinations, fixed at day 6, and processed for electron microscopy analysis (more than 70 cells were examined for each experimental conditions). GM-CSF (A, panels 1 and 2) or GM-CSF/IL-4-derived cells (A, panels 3 and 4) displayed ring-shaped granules. By contrast, TGF-ß1 supply to GM-CSF (B, panels 1–3) or GM-CSF/IL-4 (B, panel 4) induced rod-shaped granules. Note that the central lamella was absent (solid arrows) or not periodically striated (open arrows). Original scale bars, 200 nm.

Cross-linking Langerin induces its internalization and the formation of Birbeck granules
GM-CSF/TGF-ß1-treated monocytes were stained with DCGM4 mAb followed by goat anti-mouse Igs conjugated with colloidal gold particles. Cells were fixed immediately after staining (time=0) or warmed up to 37°C for 10 min before fixation. At time = 0, a varying density of gold particles was observed at the cell surface, ranging from 27 to 133 gold particles/100 µm membrane. In cells displaying weak membrane staining, clusters of gold particles bound to the cell membrane or concentrated into coated pits and coated vesicles (Fig. 5A , panels 1–3). Some peculiar, curved, or rod-shaped, endocytic structures fused with gold particles containing coated vesicles (Fig. 5A , panel 4). By contrast, in cells displaying a high density of gold particles at the cell surface, Langerin cross-linking induced the formation of typical Birbeck granules displaying periodical striation (Fig. 5B , panel 1).



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  		Figure 5. Langerin cross-linking on GM-CSF/TGF-ß1-derived cells. Cells were cultured for 6 days with GM-CSF/TGF-ß1 and stained with DCGM4 mAb followed by goat anti-mouse Igs conjugated with gold particles. Cells were fixed immediately or warmed up to 37°C for 10 min before fixation. At time = 0, a various density of gold particles was detected at the cell surface (small, solid arrows). In cells displaying weak membrane staining (A), anti-Langerin mAb access coated pits (cp), coated vesicles (cv), and rod-shaped structures fused with endocytic vesicles (open arrow). By contrast, in cells displaying high membrane staining (B, panel 1), typical Birbeck granules developed from the plasma membrane (open arrow). At time = 10 mn, more numerous, typical Birbeck granules were observed (B, panels 2 and 3, open arrows), some of them containing rare gold particles (small, solid arrows). Original scale bars, 100 nm.

When cells were warmed up to 37°C for 10 min, more numerous, short Birbeck granules could be detected in the cytoplasm, some of them containing rare gold particles (Fig. 5B , panels 2 and 3).

Allostimulatory function
The allostimulatory function of monocyte-derived cells was compared after the 6-day culture in the different cytokine conditions (Fig. 6A ). Results showed that monocytes differentiated in the absence of IL-4 were unable to stimulate allogeneic T cells independent of exogenous TGF-ß1.



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  		Figure 6. Allostimulatory capacity of monocyte-derived cells. (A) Monocytes were cultured for 6 days in RPMI/10% FCS supplemented with GM-CSF, GM-CSF/TGF-ß1, GM-CSF/IL-4, or GM-CSF/IL-4/TGF-ß1. (B) Cells were cultured as described in (A) and then plated for 40 h on CD40L-transfected fibroblasts. For both conditions, cells were recovered, and graded numbers were added to 105 allogeneic T cells. T-cell proliferation was assessed by 3H-thymidine incorporation during the final 18 h of culture. Results are expressed as the mean cpm ± SD from triplicate wells. T cells alone gave less than 30 cpm. Data are representative of five experiments.

CD40 ligation promotes the differentiation of GM-CSF/TGF-ß1-derived cells into allostimulatory DC
Monocyte-derived cells were collected after a 6-day culture with the different cytokines and then were exposed to CD40L for 2 days. The cell phenotype and allostimulatory function were then analyzed.

As illustrated in Figure 7 , CD40 ligation induced the down-regulation of CD14 on the cells precultured with GM-CSF or GM-CSF/TGF-ß1. Most of these cells acquired CD80 and CD86 although at a lower level than IL-4-treated cells. Furthermore, only a small subpopulation acquired CD83 at the cell surface.



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  		Figure 7. Phenotypic analysis of CD40L-stimulated cells. Day 6 monocyte-derived cells obtained from GM-CSF, GM-CSF/TGF-ß1, GM-CSF/IL-4, or GM-CSF/IL-4/TGF-ß1-supplemented cultures were incubated for 40 h with CD40L-transfected fibroblasts. Harvested cells were stained and analyzed by flow cytometry. Results with irrelevant isotype-matched control mAb were overlaid (empty histograms). Data are representative of five experiments.

However, as illustrated in Figure 6B , monocytes precultured in the absence of IL-4 acquired nearly as strong allostimulatory property as their counterparts treated with the cytokine, providing the primary culture had been carried out in the presence of TGF-ß1.


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DISCUSSION
 
The present study was aimed at analyzing the effects of TGF-ß1 and/or IL-4 on monocytes cultured with GM-CSF.

Here, we demonstrated that monocytes cultured in the absence of IL-4 shared many phenotypic and functional features with monocyte/macrophages, such as strong expression of CD14, CD16, CD64, and CD71, and lack of allostimulatory function. However, it is interesting that the cells expressed E-cadherin, a molecule considered as a typical LC marker [14 , 15 ]. Within epidermis, E-cadherin is involved in LC adhesion to keratinocytes [15 16 17 ]. In vitro, E-cadherin is expressed on the CD34+-derived CD1a population that displays all the characteristics of LC [18 ]. Furthermore, the CD34+-derived CD14+ population as well as GM-CSF/IL-4-derived immature DC were shown to acquire E-cadherin expression, provided exogenous TGF-ß1 was added to the culture medium [7 8 9 ]. It is interesting that we found here that E-cadherin is strongly expressed on monocytes cultured in GM-CSF, independent of exogenous TGF-ß1. Furthermore, a similar staining pattern was noticed on monocytes cultured in serum-free medium in the absence of any cytokines (not shown), as well as on typical macrophages developed in human serum-supplemented cultures. Taken together, the results suggest that E-cadherin expression on monocyte-derived cells is independent of exogenous TGF-ß1, and E-cadherin is not only expressed on the LC-related DC but at least in vitro, on many cells committed to tissue homing.

In addition to E-cadherin expression, monocytes cultured in GM-CSF/TGF-ß1 express CCR6, the macrophage-inflammatory protein-3{alpha} (MIP-3-{alpha}) receptor specific for the immature state of the LC lineage [19 , 20 ]. It is most interesting that a subpopulation of cells displays Langerin expression and rod-shaped, intracytoplasmic granules. Langerin is a novel, C-type lectin, specific to cells of the LC pathway first described on CD34+-derived LC, especially upon addition of TGF-ß1 to the culture medium. In situ, Langerin expression is highly restricted on LC in skin and mucosa [21 ]. Langerin is an endocytic receptor that functions as a potent inductor of Birbeck granules [22 ]. Indeed, binding Langerin to ligand promotes membrane superimposition via interaction of its extracellular domains. This interaction seems to be the basis for the membrane zippering leading to the characteristic Birbeck granule structure [22 ]. In the present study, Langerin expression on GM-CSF/TG-ß1-derived cells was assessed by flow cytometry and confirmed by electron microscopy. Langerin expression was low, as compared with that found on LC from cord blood CD34+ progenitors or epidermis [21 , 22 ]. However, as described for LC, we found that Langerin engagement, as mimicked by DCGM4 mAb, induced a very rapid internalization of the molecule into coated pits and coated vesicles. Furthermore, on cells displaying high Langerin density, cross-linking induced the formation of typical Birbeck granules. That cells displayed Birbeck granule-like organelles in the absence of cross-linking might be relevant to the low density of Langerin molecules at the cell surface. Indeed, high local concentration of Langerin was shown to be an important parameter for membrane superimposition and zippering [22 ]. Thus, the low density of Langerin would be sufficient here to allow membrane superimposition but would be insufficient to allow crystal-like central structure to be formed.

As shown previously [13 , 23 , 24 ], we found that IL-4 was mandatory for monocyte differentiation into typical CD1a+, CD14- DC displaying strong allostimulatory property. However, conversely to TGF-ß1, IL-4 acts as a divergent factor in the commitment of monocytes toward the LC pathway. Indeed, monocytes cultured in GM-CSF/IL-4 never expressed the LC markers E-cadherin, Langerin, or CCR6. In the simultaneous presence of TGF-ß, monocytes have been shown to acquire E-cadherin [9 ] and CCR6 [25 ]. The present results demonstrated that IL-4 induced a down-regulation of all these TGF-induced LC markers. This agrees with recent findings showing that IL-4 blocks the expression of CCR6 and MIP-3{alpha} responsiveness at different steps of LC development [26 ]. Thus, TGF-ß1 counteracts, only to a certain extent, the IL-4-provided signal. It is noteworthy, however, that Birbeck granule-like structures were observed in the presence of TGF-ß1, whereas in its absence, the cells developed ring-shaped, intracytoplasmic structures.

Finally, the results add further evidence that LC can derive from monocytes, as suggested by many clinical observations. After allogeneic bone-marrow transplantation, Murphy et al. [27 ] observed that within the superficial dermis, cells with surface antigens of monocyte/macrophages were replaced gradually by dermal and epidermal cells exhibiting coexpression of monocyte/macrophage and LC antigens. As assessed by electron microscopy analysis, many of these cells expressed prominent phagolysosomes and Birbeck granules. In mycosis fungoides, electron microscopy analysis of skin section revealed the presence of monocytes in the basal layer, LC in the stratum spinosum, and intermediate cells, i.e., monocytes with Birbeck granule-like structures, in the interposed layers [28 ]. In the literature, some cases of associated histiocytosis X and monocyte leukemia provide additional evidence of a close relationship between LC and monocytes [29 ]. Recently, it was shown that reticular dysgenesis, which is characterized by the lack of blood monocytes, is devoid of LC [30 ].

In conclusion, a subset of monocytes cultured with GM-CSF/TGF-ß1 differentiates into typical LC. The cells are very immature cells, as shown by their morphology, phenotype, and lack of allostimulatory function. They have the potential to develop into potent, allostimulatory DC upon CD40-ligation, and it remains to be determined if the presence of TGF-ß1 prevents their full maturation upon addition of inflammatory stimuli, as described previously [31 ]. That only a subset of monocytes cultured with GM-CSF and TGF-ß1 expresses Langerin may suggest that these selective culture conditions suboptimized the percentage of cells committed to the LC pathway. Other cytokines may be needed to enhance this process. Indeed, in addition to TGF-ß, several cytokines have been shown recently to skew monocyte differentiation into LC lineage. Independent of TGF-ß1, more than 50% of monocytes cultured with GM-CSF and IL-15 developed into E-cadherin, CCR6, and Langerin-positive DC, which failed unexpectedly to express the bona fide Birbeck granules [32 ]. Furthermore, IL-10 induced the expression of CCR6 and responsiveness to MIP-3{alpha} in differentiated monocytes [26 ]. Alternatively, the Langerin-positive cells may represent a subset of monocytes aimed to colonize the dermis as resident LC precursors before entering epidermis. In this context, among the cells migrating spontaneously out of human skin explants, a CD14+ Lang+ CCR6+ subset has been identified recently [33 ]. It is interesting that the cells differentiate into typical LC in the presence of TGF-ß, supplied or not with GM-CSF, and addition of IL-4 down-regulates expression of the LC markers.

Received February 21, 2001; revised December 17, 2001; accepted December 19, 2001.


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