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Originally published online as doi:10.1189/jlb.0804473 on December 23, 2004

Published online before print December 23, 2004
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(Journal of Leukocyte Biology. 2005;77:535-543.)
© 2005 by Society for Leukocyte Biology

IL-4 supports the generation of a dendritic cell subset from murine bone marrow with altered endocytosis capacity

Mauritius Menges*, Thomas Baumeister*, Susanne Rössner*, Patrizia Stoitzner{dagger}, Nikolaus Romani{dagger}, André Gessner{ddagger} and Manfred B. Lutz*,1

* Department of Dermatology, University Hospital Erlangen, Germany;
{dagger} Department of Dermatology, Innsbruck Medical University, Austria; and
{ddagger} Institute for Clinical Microbiology, Immunology and Hygiene, University of Erlangen, Germany

1 Correspondence: Department of Dermatology, Hartmannstrasse 14, University Hospital Erlangen, 91052 Erlangen, Germany. E-mail: lutz{at}derma.imed.uni-erlangen.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DC) of myeloid origin can be generated from mouse bone marrow (BM) using granulocyte macrophage-colony stimulating factor (GM-CSF). Immature major histocompatibility complex (MHC) IIlow DC are known to bear a high endocytosis capacity, in contrast to DC precursors and mature DC. Now we found that a subset of MHC IIlow DC in BM-DC cultures is unable to exert mannose receptor-mediated endocytosis of fluorescein isothiocyanate (FITC)-dextran (DX) and resembles immature Langerhans cells (LC). The FITC-DX endocytosis activity of LC-like cells occurs at an earlier stage of development, where the surface MHC II expression is absent or very weak. This LC-like subset expresses higher levels of E-cadherin but lower amounts of the markers Gr-1, scavenger receptor 2F8, and CD11b, when compared with the highly endocytic DC subset. The latter myeloid DC resemble monocyte-derived DC (MoDC). The sorted LC-like population develops completely and exclusively into mature MHC IIhigh DC, and the MoDC-like cells remain immature MHC IIlow DC or develop into adherent MHC IIneg macrophages or mature into MHC IIhigh DC. The development of LC-like cells is promoted by interleukin-4. Thus, we show here that the simultaneous development of LC-like and MoDC-like DC subsets occurs in standard bulk cultures with GM-CSF, suggesting the existence of two different precursors for LC and MoDC in BM.

Key Words: development • myeloid • cytokines


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DC) are professional antigen-presenting cells (APC) of hematopoietic origin [1 ]. In vitro generation of DC has been reported from myeloid and lymphoid murine precursors. In addition, another subset of DC can be derived from plasmacytoid precursor cells, which show major differences to the myeloid- or lymphoid-derived DC [2 ]. Myeloid DC can be generated from murine bone marrow (BM) precursor cells with granulocyte macrophage-colony stimulating factor (GM-CSF), in parallel to macrophages and neutrophils [3 ]. In BM cultures supported by interleukin (IL)-3, DC develop together with mast cells [4 ]. Human myeloid DC can be generated from CD34+ lin– hematopoietic progenitor cells in vitro. These progenitors, cultured with GM-CSF and tumor necrosis factor (TNF), differentiate intermediately into CD1a+ CD14– or CD1a– CD14+ immature DC. The latter DC express markers related to the monocyte lineage CD11b, CD14, CD36, and M-CSF receptor and have the differentiation potential into macrophages with M-CSF or into mature DC with GM-CSF plus TNF. In contrast, the CD1a+ immature DC matures only into typical Langerhans cell (LC)-like DC [5 ]. A similar observation has been made with murine cells, where c-kit+ BM precursors gave rise to CD11c+ CD11b+ Gr-1+ monocyte-derived DC (MoDC)-like cells and CD11c+ CD11b–/dull E-cadherin+ LC-like cells [6 ].

Epidermal LC represent a DC subset that is related to myeloid DC but also show unique features [7 ]. Human and mouse LC can be generated in vitro even under serum-free conditions by culture of CD34+ cells in a cocktail consisting of GM-CSF, stem cell factor (SCF), TNF, and transforming growth factor-ß (TGF-ß) [8 , 9 ] or by fetal liver tyrosine kinase 3 ligand and TGF-ß [10 ]. Also, human CD14+ monocytes can be differentiated into a LC-like phenotype by GM-CSF, IL-4, and TGF-ß [11 , 12 ]. The addition of TGF-ß to the cultures is strictly required to achieve the LC phenotype. TGF-ß is also necessary for LC development in vivo, as LC are absent in the epidermis of TGF-ß–/– mice [13 ] and Id2–/– mice, lacking a transcription factor regulated by TGF-ß [14 ].

IL-4 is typically used in combination with GM-CSF for the generation of DC from human MoDC, where it inhibits the outgrowth of macrophages [15 ]. For the generation of murine DC from BM, IL-4 is not required when high doses of GM-CSF are used for the culture [16 ]. However, at any concentration of GM-CSF, IL-4 does not influence the simultaneous outgrowth of macrophages from mouse BM. IL-4 can also act on immature DC, in that it enhances their lipopolysaccharide (LPS)-induced IL-12 production [16 , 17 ]. So far, no data are available about the effects of IL-4 on the generation of different DC subsets.

Here, we characterize two different subsets of DC in BM-DC cultures, which can be distinguished by their endocytosis capacities of dextran (DX), which is mediated through the mannose receptor. This function also correlates with their differential expression of several endocytosis receptors. Our data indicate that these two subsets can be attributed to MoDC and LC-like DC. Furthermore, we describe that IL-4 promotes the outgrowth of LC-like DC from mouse BM.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of murine BM-DC
The preparation and culture of BM cells from C57BL/6 and BALB/c mice (Charles River, Sulzfeld, Germany) to generate DC have been described in detail before [18 ]. The dose of GM-CSF was 200 U/ml, i.e., 40 ng/ml (Peprotech/Tebu, Frankfurt, Germany). Recombinant IL-4 (100 U/ml) and TGF-ß1 (indicated doses) were purchased from Peprotech/Tebu and LPS (1 µg/ml, Escherichia coli), from Sigma (Deisenhofen, Gemany). The optimal doses of GM-CSF and IL-4 were obtained from previous studies [16 , 18 ].

Fluorescein-activated cell sorter (FACS) analysis and immunohistochemistry
FACS analysis was performed as described before [7 ]. BM-DC (1–5x105) were stained directly with phycoerythrin (PE)-conjugated monoclonal antibody (mAb) directed against major histocompatibility complex (MHC) class II (M5/114) or fluorescein isothiocyanate (FITC)-conjugated B7-1, B7-2 (GL1), CD14, Gr-1, and CD11b (all from BD PharMingen, Hamburg, Germany), F4/80 and 2F8 (CD204, from Serotec, Oxford, UK), or hybridoma supernatants from the clones 2.4G2 and TIB-219 (CD71) and NLDC145 (CD205, all from American Type Culture Collection, Manassas, VA), E-cadherin (clone ECCD-3, kind gift of Thilo Jakob, Department of Dermatology and Allergy, Biederstein Technical University, Munich, Germany), or the appropriate fluorochome-conjugated isotype control mAb at 2–5 µg/ml in phosphate-buffered saline containing 0.1% sodium azide and 5% fetal calf serum for 30 min on ice in the dark. Samples were washed once in staining buffer and were measured and analyzed with a FACScan (Becton Dickinson, Heidelberg, Germany). Cytospin specimens were labeled for immunfluorescence using a rat anti-mouse Langerin mAb [19 ] (kind gift of Dr. Saem Saeland, Schering-Plough Laboratory for Immunological Research, Dardilly, France).

Endocytosis assays
BM-DC were cultured until day 8. Then, 2 x 105 DC were incubated with 1 mg/ml FITC-conjugated DX (40 kD), FITC-conjugated ovalbumin (OVA), and Lucifer Yellow (all Molecular Probes, Leiden, The Netherlands) or FITC-conjugated Fluoresbrite latex microbeads (1 µm, Polysciences Europe, Eppelheim, Germany) for 30 min (if not otherwise indicated) on ice or at 37°C. Then, all samples were double-stained with PE-conjugated M5/114 (anti-MHC class II) mAb before FACS analysis. Quadrant markers were set according the FITC-conjugate binding observed on ice. Mean fluorescence values within the gate for endocytically active, immature DC were plotted as described previously [20 ].

Cell sorting
BM-DC from C57BL/6 mice were harvested at day 8. Then, DC were incubated for 30 min with FITC-DX at 37°C for endocytosis and were then stained on ice with M5/114-PE before sorting into the indicated subpopulations with a MoFlo high-speed cell sorter (Cytomation Bioinstruments, Freiburg, Germany).

T cell proliferation assays
Sorted BM-DC subpopulations were used from from C57BL/6 mice for incubation with FITC-DX at 37°C for 30 min and were then stained on ice with M5/114-PE before sorting into the subpopulations 3 and 4. Then, the DC were cultured in a 96-well flat-bottomed plate (Becton Dickinson) at titrated numbers, together with CD4+ T cells (3x105 cells per well) derived from allogeneic BALB/c mice or for OVA presentation from OT-II T cell receptor (TCR)-transgenic mice (kindly provided by Frances R. Carbone, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia) using the purified CD4+ T cells obtained with the CD4 magnetic cell sorter cell separation system, according to the manufacturer’s description (Miltenyi, Bergisch Gladbach, Germany). After 3 days, the triplicate cultures were pulsed with 1 µCi [3H]-thymidine (Amersham, Little Chalfont, UK) for 16 h and were harvested onto filtermats with an ICH-110 harvester (Inotech, Dottikon, Switzerland); filters were counted in a 1450 microplate counter (Wallac, Turku, Finland).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
LC-like and MoDC-like immature DC subsets in BM can be distinguished by their mannose receptor-dependent endocytosis capacity
Differentiation of murine DC from BM in vitro can be subdivided into three distinct subsets by their MHC II molecule expression at the cell surface. Several stages of myeloid precursor cells remain MHC IIneg, immature DC are MHC IIlow, and mature DC show MHC IIhigh expression [18 ]. Immature DC can endocytose different types of antigens by a large variety of mechanisms. FITC-DX is mainly taken up through the mannose receptor into clathrin-coated pits [20 , 21 ]. When the endocytosis capacity of bulk BM-DC cultures was investigated, most of the MHC IIlow expressing immature DC internalized FITC-DX rapidly, but some remained negative for FITC-DX uptake. This phenomenon could be observed with BM-DC derived from C57BL/6 or BALB/c mice (Fig. 1a ). After FACS sorting of these two MHC IIlow subsets and culture in GM-CSF for 24 h, the morphological features of the two subsets differed substantially. The MHC IIlow DXneg cells developed almost exclusively into heavily veiled cells, typical for mature DC, and no adherent macrophages could be detected (Fig. 1b) . In contrast, within the sorted MHC IIlow DXpos cultures, three cell types were visible by morphological criteria: "potato-shaped", immature DC; veiled, mature DC; and firmly adherent macrophages (Fig. 1b) . As this picture was reminiscent of the bifurcated development LC-like and MoDC-like DC described for human DC generated from CD34+ cells with GM-CSF and TNF [22 ], we further analyzed murine homologous markers that have been described to distinguish these human subsets.



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  		Figure 1. MHC IIlow immature DC consist of two subpopulations, which can be separated by their endocytosis capacity for FITC-DX. (a) BM-DC were generated from C57BL/6 or BALB/c mice and at day 8, incubated with FITC-conjugated DX for 30 min at 37°C. Then, the cells were counterstained for surface MHC II molecule expression. The numbers given represent the percentages of cells within the oval gates (n>20). (b) The populations within gates 3 and 4 from C57BL/6 mice were sorted and cultured in GM-CSF overnight. Cell morphologies differed markedly in the cultured cells. Subpopulation 4 revealed only mature DC (mDC), and in cultures from subset 3 adherent macrophages (Mph), immature DC (imDC) and mature DC were detectable (n=2).

 
FACS analysis of the sorted or gated subpopulations 3 and 4 showed a largely similar expression of low-to-absent levels of the costimulatory molecules CD80, CD86, and lymphocyte function-associated antigen-1, as well as intermediate levels of the myeloid markers F4/80, CD44, CD13, CD9, CD71, CD24, and 33D1. However, no differences were found for these markers expressed on populations 3 or 4 (not shown). The myeloid markers Gr-1 (recognizing the Ly-6C and Ly-6G antigens), the complement receptor-3 (CD11b), and the scavenger receptor (2F8, CD204) showed increased levels on the DXpos fraction, indicative of MoDC. The typical LC marker Langerin was largely absent on populations 3 and 4 only (less than 4% Langerin+). Typical Birbeck granules were occasionally present in rare cells, as detected by electron microscopy (not shown). However, the epidermal LC-typical adhesion molecule E-cadherin was expressed exclusively on the DXneg LC-like fraction of DC (Fig. 2 ). Together, the two MHC IIlow populations 3 and 4 within BM cultures show some but not all features of myeloid MoDC and LC, as described for the human counterparts generated from CD34+ cells or the ex vivo, isolated LC or other myeloid DC subsets.



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  		Figure 2. Surface markers differentially expressed on both DC subsets reveal similarities with LC (Subset 4: E-cadherin) and MoDC (Subset 3: CD11b, 2F8, Gr-1). Triple-color FACS analysis was performed with day 8 BM-DC from C57BL/6 mice. After uptake of FITC-DX at 37°C, the cells were stained on ice for MHC II and the indicated markers. Analysis gates were set for the subsets 3 and 4, similar to those in Figure 1 (n=5).

 
Divergent differentiation potential of the DXneg and DXpos cell subsets into mature DC or macrophages
To substantiate the morphological evaluation of the DXneg and DXpos fraction (Fig. 1) , we cultured the sorted DC subsets in the presence of GM-SCF for 48 h and reanalyzed them for their remaining FITC-DX content and surface MHC II expression. The DXpos cells were still FITC-fluorescent after this time, but the levels of MHC II varied. One-third of the cells remained at the MHC IIlow level, as they were at the time of sorting. Only 16% up-regulated MHC II expression (Fig. 3a ) and the costimulatory molecules CD80, CD86, and CD40 (not shown), all indicative of DC maturation. Another 22% down-regulated MHC II from the cell surface (Fig. 3a) as well as CD80, CD86, CD40, and the DC marker CD11c (not shown). Many cells adhered flatly to the culture plate (not shown).



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  		Figure 3. Differentiation and antigen presentation of the two DC subsets indicate faster maturation by LC-like cells. (a) Sorted day 8 BM-DC subsets 3 and 4 from C57BL/6 mice were cultured until day 10. Cells were removed by vigorous pipetting and stained for surface MHC II. Up- and down-regulation of MHC II could be observed differentially in both subsets. FITC-DX was only given at day 8, but fluorescence of subset 3 was still detectable at day 10. Numbers besides the analysis gates represent percentages of cells within the gate (n=2). (b) Sorted day 8 BM-DC subsets 3 and 4 were cocultured at titrated amounts with TCR-transgenic, OVA-specific CD4+ T cells, together with OVA peptide 323–339 or protein or allogeneic T cells. After 3 days, T cell proliferation was measured by [3H]-thymidine uptake (n=3). MLR, Mixed leukocyte reaction.

 
The majority of the DXneg fraction of sorted cells matured as indicated by up-regulation of MHC II (Fig. 3a) , CD80, CD86, and CD40 (not shown), and only a minority of cells remained immature, as was already indicated by the morphological evaluation. Again, these features were also observed by human DC differentiation from CD34+ cells, where LC-like cells were unable to differentiate into macrophages, in contrast to MoDC-like cells [5 ]. In conclusion and in agreement with the data shown in Figure 1b , the subset 3 bears a broader potentiel to differentiate/mature into macrophages and DC or remain unaltered, as the subset 4 almost completely undergoes maturation without macrophage differentiation potential.

Antigen processing and presentation capacities of MoDC-like and LC-like DC
Although DX is taken up through a mannose receptor-mediated mechanism, immature DC also use macropinocytosis as a major mechanism of fluid-phase endocytosis [20 , 21 , 23 ]. As DX cannot be used as an antigen recognized by T cells, we used OVA as a read-out system. To test which DC subtype is superior in antigen presentation after OVA peptide loading and OVA protein processing, OVA-specific CD4+ T cells from OT-II TCR-transgenic mice were cultured with sorted MHC IIlow DXpos cells (subset 3) or MHC IIlow DXneg cells (subset 4) together with OVA peptide or protein. As the LC-like DC were only slighly more efficient in OVA peptide presentation, they showed much higher efficiency to take up OVA protein, process it, and present it for presentation (Fig. 3b) . This indicates that subset 4 cells, despite their inability to take up DX through mannose receptor-medicted endocytosis, are more efficient in macropinocytosis than the MoDC-like subset 3. When no antigen loading on MHC II molecules is required for T cell activation, as for the stimulation of T cells by allogeneic DC, both DC subsets were equally potent (Fig. 3b) .

Immature LC-like DC endocytose DX at a MHC IIneg/low differentiation stage
As immature LC-like cells did not endocytose FITC-DX at the MHC IIlow stage, we investigated whether these cells were completely lacking this function, or this ability was active at a different stage of development, i.e., lower MHC II expression. We observed that a proportion of cells within the MHC IIneg fraction was able to ingest FITC-DX. Therfore, we sorted the MHC IIneg DXpos cells (subset 2) and followed their differentiation in parallel to the MHC IIlow DXpos cells (subset 3) at day 8 of culture. At day 10, the subset 2 gained MHC II at the cell surface and at day 13, additionally lost FITC fluorescence (Fig. 4a ). The DX molecules cannot be processed enzymatically by DC, but the FITC fluorescence is quenched by low pH in endosomal compartments [20 ]. This indicates again that LC-like cells are directing endocytosed materials to intracellular antigen processing compartments more rapidly. In contrast, at day 13, the sorted subset 3 cells still remained FITCpos (Fig. 4a) , indicating that the tracer is not localized in acidic compartments. The sorted subset 3 cells lost its FITC fluorescence only at its MHC IIhigh stage at day 14. It is interesting that the percentage of mature antigen-presenting MHC IIhigh expressing DC was identical in subsets 3 and 2 at days 10, 13, and 14 (Fig. 4a) .



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  		Figure 4. The non-DX-endocytosing LC-like subset 4 derives from the endocytic subset 2. (a) Subsets 2 and 3 from day 8 BM-DC from C57BL/6 mice were sorted and cultured with GM-CSF. Still-retained FITC-DX content within the cells was analyzed with additional staining for surface MHC II molecules at the indicated days. Here, mild pipetting was used to remove only weakly adherent cells but not stronger adherent cells down-regulating CD11c and MHC II, which then develop into macrophages that are obtained after vigorous pipetting, as shown in Figure 3a . (b) Schematic model proposing the differential pathways of DC development for subsets 3 (upper panel) and 2 (lower panel). Numbers besides the analysis gates represent percentages of cells within the gate (n=2).

 
Together, the cells of the MHC IIneg DXpos subset 2 differentiated into MHC IIlow DXneg subset 4. This indicated that the MHC IIneg DXpos population 2 is the endocytically active precursor population of the MHC IIlow DXneg LC-like cells. Sorted population 3 MoDC-like cells retained their receptor-mediated endocytosis capacity until a relatively high MHC II expression level, where the LC-like cells already lost this function but were more effective in receptor-independent macropinocytosis for the uptake of FITC-OVA. Thus, the two sorted subsets follow distinct routes of antigen uptake and expression of MHC II surface molecules (Fig. 4b) .

Maturation preferences of MoDC on culture passaging and LC on LPS
DC maturation occurs spontaneously in BM-DC cultures [18 ]. Immature LC-like cells seem to mature much faster than MoDC (Figs. 3a and 4a) . Immediate maturation of DC is required to defend pathogens and can therefore be forced by microbial products (e.g., LPS) or mimicked in vitro by passaging DC to fresh culture dishes, so-called "endogenous danger signals" [24 ].

To test whether the partially matured day 14 cultures of subsets 2 and 3 (Fig. 4a) could be stimulated further, we transferred the cells to fresh culture plates with or without LPS treatment for 16 h. In the MoDC-like cell cultures of subset 3, the proportion of mature DC increased only moderately upon cell transfer (12%>22%), and this could not be enhanced further by LPS (22%; Fig. 5 , upper row). In contrast, the day 14 cell cultures of subset 2 did not respond to cell transfer (12%>14%) but matured readily on additional LPS treatment (41%; Fig. 5 , lower row). This indicates that the MoDC- and LC-like DC subsets also differ in their capacity to respond to exogenous or endogenous danger signals [24 ].



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  		Figure 5. BM-DC subsets 3 and 4 from C57BL/6 mice show different maturation potential on transfer and LPS. Although sorted gate 3 MoDc-like cells respond maximally to transfer alone, the LC-like subset 2 cells mature on LPS but not on transfer. The day 14 populations from sorted cell gates 2 and 3 (same cells as Fig. 4 ) were transferred to fresh culture dishes only or together with LPS. Numbers besides the analysis gates represent percentages of cells within the gate (n=2).

 
IL-4 supports immature and mature LC-like cell differentiation
TGF-ß has been reported to support the development of LC [8 ] but also to inhibit DC maturation and convert them into tolerogenic DC [25 ]. We found that the continuous presence of low doses of TGF-ß only moderately increased the subset of LC-like MHC IIlow DXneg cells in BM cultures without inhibiting their spontaneous DC maturation. High doses of TGF-ß, as described, blocked DC maturation without polarizing for either of the two immature DC subsets (Fig. 6 ).



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  		Figure 6. High doses of TGF-ß inhibit mature DC generation, and low doses show a mild polarization toward subset 4 cells. BM-DC were generated from C57BL/6 mice for 8 days with GM-CSF, with or without TGF-ß at the indicated doses throughout the culture. Then, FITC-DX was added for 30 min at 37°C before staining for surface MHC II molecules was performed, and the cells were analyzed by FACS (n=6).

 
In the following experiments, we tested whether IL-4 would influence the LC-like or the MoDC-like subset generation. When BM was generated with GM-CSF in the continuous presence of IL-4, the subset 3, expressing the scavenger receptor 2F8 and CD14 but not other receptors involved in antigen uptake such as the Fc receptor for immunoglobulin G II/III (2.4G2), complement receptor-3 (CD11b), was clearly reduced. For other endocytosis receptors, the situation inverted as the transferrin receptor (CD71) and the C-type lectin CD205 (DEC205, NLDC145) were increased with IL-4 (Fig. 7a ). Functional analyses showed a clear reduction of the MoDC-like subset 3 and increasing percentages of LC-like cells of subset 4 (Fig. 7b) . Invesigation of different endocytosis mechanisms revealed that macropinocytosis (Lucifer Yellow, FITC-OVA), mannose-receptor-mediated endocytosis (FITC-DX), and phagocytosis of FITC-conjugated latex beads were reduced to ~50% by DC cultured in GM-CSF plus IL-4 rather than by DC grown in GM-CSF alone (Fig. 7c) . This indicates that several types of endocytosis are reduced at the MHC IIlow stage under conditions that promote LC-like cells when compared with conditions that favor MoDC.



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  		Figure 7. IL-4 shifts BM-DC to less endocytic LC-like cell populations. BM-DC from C57BL/6 mice were cultured for 8 days in GM-CSF ± IL-4. (a) Then, cells were stained for MHC II and other indicated markers. Numbers within the quadrants represent percentages of cells (n=3). (b) Similar to the surface staining, uptake of FITC-DX was performed for 30 min with BM-DC grown in GM-CSF alone or GM-CSF plus IL-4 (n=4). (c) To day 8 BM-DC, FITC-DX was added for the indicated time periods. Then, cells were counterstained for MHC II and analyzed by FACS. Mean fluorescence values for FITC are shown for the MHC IIlow-gated cells (n=4). LY, Lucifer Yellow.

 
These differences in antigen uptake could not be attributed to a direct effect of IL-4 treatment on DC, as observed for the IL-12 secretion by DC [16 ], as short-term, overnight treatment with IL-4 could not influence the potential to endocytose FITC-DX of either subset (not shown). Together with the data shown in Figure 2 , this indicates that the rapid up- or down-regulation of the markers is not influenced by IL-4 but rather argues for a selective, long-term differentiation program into separate lineages of DC with different expression levels of endocytosis receptors and antigen-uptake functions.

Thus, IL-4 and to a lesser extent, low doses of TGF-ß support the outgrowth of the LC-like DC from murine BM cultures. These findings are in agreement with the literature about the functions of TGF-ß. The polarizing effect of IL-4 in supporting LC growth and suppressing MoDC generation from murine BM has not been desccribed before.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The generation of DC from BM precursors represents a major source of this cell type [18 , 26 ]. Most methods use GM-CSF as a growth factor, which drives the development of a common myeloid precusor cell into granulocytes, macrophages, and DC [3 ]. Although these bulk culture systems reveal high cell yields, the generation of DC is not synchronized, and after about 1 week of culture, a mixture of precursor cells, granulocytes, macrophages, immature and mature DC, and few remaining B cells is detectable. Within the BM, committed precursors for granulocytes and a common macrophage and DC precursor can be distinguished [27 ]. In culture, the peak of granulocyte development (day 5) precedes the peak for macrophages and DC (days 8–10). At the latter time-points, a population of MHC IIlow cells is designated as immature DC, although they are also a source for macrophages. Upon activation by proinflammatory or pathogen-derived substances, macrophages adhere firmly to the culture plastic, and DC remain nonadherent with their typical veiled morpholgy [18 ].

It is common that the nonadherent population of mature BM-DC is believed to originate from a unique type of BM precursor. Here, we show that by using standard methods, two types of DC develop: one with more LC-like characteristics and the other with a more myeloid, MoDC-like phenotype. In cultures of purified human CD34+ and murine Lin– c-kit+ early hematopoietic stem cells, it has been observed that two types of DC resembling LC and MoDC grow out with GM-CSF, SCF, and TNF as growth factors [5 , 6 ]. Our data are in agreement with these previous reports and extend these data with respect to the expression of endocytosis receptors, antigen processing, and regulation by IL-4 of the two DC subsets.

Similar to the previous reports, we found that within BM, two different precursors exist that give rise to LC-like or MoDC-like DC subsets. The differences to the previous reports are that we could functionally define these subsets by their mannose receptor-mediated uptake of FITC-DX and their antigen-processing kinetics. The differentiation into the two DC subsets could be demonstrated by separate cultures of the sorted subsets 3 and 4 in GM-CSF. Cells that expressed equally low levels of MHC II at their surface could be separated into FITC-DXpos and FITC-DXneg cells. The surface marker analysis revealed that both subsets expressed most markers in common and at comparable levels. Only few differences were found that are nevertheless indicative of LC-like or rather MoDC-like cells.

The FITC-DXneg subset expressed E-cadherin and exclusively developed into mature DC. In contrast, the FITC-DXneg cells expressed higher levels of CD11b, 2F8, and Gr-1 and remained immature or developed into macrophages or into mature DC. Analysis of the two most typical markers for LC revealed no differences. Langerin expression and Birbeck granules were rarely detectable in populations 3 and 4. Although these markers represent the best markers for LC in situ or ex vivo, they might not be genetically regulated by the individual cell type but rather induced by peripheral factors such as TGF-ß or maturation. In fact, when we performed FACS analysis on TGF-ß-cultured, BM-derived LC, Langerin was expressed exclusively on the MHC IIhigh, expressing mature LC fraction, but not on immature LC (our unpublished observations). Thus, IL-4 might contribute to a more LC-like phenotype by driving E-cadherin expression but not the expression of other typical features of LC. The low level of Birbeck granula we detected on the Mo-DC-like cells might support the latter hypothesis.

Sorted subsets 3 and 4 were equally potent in stimulating an allogeneic MLR as reported before [6 ]. Marked differences were observed when the two subsets were pulsed with OVA peptide or protein for presentation to naive TCR-transgenic, OVA-specific T cells. Here, the LC-like cells were superior to MoDC-like cells in both cases. Peptide loading depends on the amount of MHC II expression, and the OVA protein requires macropinocytosis and antigen processing in addition. A higher MHC II expression for peptide loading by LC-like cells can be explained by its faster up-regulation than on the MoDC subset as shown in Figure 3a . Also, isolated LC were always much more efficient in antigen presentation on a per-cell basis than isolated DC from other organs such as the spleen [28 ]

The more efficient presentation of OVA protein by the LC-like subset occurs at a stage (gate 4), where they switched off FITC-DX uptake already. Although FITC-DX can be taken up at an earlier stage (gate 2), the LC-like cells seem to retain their capacity for macropinocytosis of OVA. Isolated LC can retain their macropinocytosis capacity but do not seem to process these proteins, as MHC II synthesis is shut down already [28 ]. In contrast, LC, matured in vivo by contact sensitization, are able to take up, process, and present OVA even at mature stages after migration into the lymph nodes [29 ]. It is remarkable that LC-like cells seem to exert different types of endocytosis mechanisms at different stages of their development/maturation, i.e., receptor-mediated uptake in an early, restricted window, and macropinocytosis is maintained throughout their development. This feature is clearly distinct from the MoDC subset, where endocytosis occurs during a defined MHC IIlow stage (gate 3). In addition, the artifical antigen processing of FITC-DX occurs much faster by the LC subset when compared with MoDC (Fig. 4a) .

As for antigen processing and presentation, both DC subsets also show differences in their maturation potential. Although day 14 DC sorted from subset 3 did respond with maturation to transfer into fresh culture dishes, the subset 2 remained unchanged. The addition of LPS to the transferred cultures inverted the situation in that the LC-like subset responded with vigorous maturation, and the MoDC subset did not mature further than by the transfer alone. This might indicate that LC, residing in the first line to oppose pathogens, might be more susceptible to microbial products than endogenous danger signals. The reverse situation might apply to monocytes, which circulate in the blood and upon endogenous inflammatory signals, extravasate into the tissue. Finally, in the tissue, these cells might contact the pathogen and differentiate into macrophages or DC [30 ].

GM-CSF promotes the myeloid DC development, but other cytokines influence the outgrowth of certain subsets. TGF-ß has been described to inhibit DC maturation [25 ] but also to be an absolute requirement for LC development [13 ]. In our bulk cultures, we could block the spontaneous DC maturation by high doses of TGF-ß, but the LC-polarizing effects of low doses of TGF-ß were less pronounced. The lack of strong effects by low doses of TGF-ß on LC generation might be caused by other factors produced in the bulk culture system.

IL-4 is a prototype T helper cell type 2 cytokine, but major sources are also mast cells and natural killer T cells. Acting on cells of the myeloid lineage, IL-4 has diverse functions. The effects on human monocytes and macrophages have been studied extensively. IL-4 increases fluid-phase pinocytosis and receptor-mediated uptake in human monocytes [31 ] and macrophages [32 , 33 ], which functionally leads to enhanced parasite-killing activity [34 ]. In DC, it enhances the IL-12 production induced by maturation factors such as LPS [16 , 17 ].

Despite the common use of IL-4 together with GM-CSF for protocols to generate MoDC from human peripheral blood monocytes, little information is available about the role of IL-4 in promoting the generation of DC subsets. Human LC development from CD34+ cells is promoted by TGF-ß, but IL-4 promoted the differentiation of DC, which resemble MoDC and inhibt LC growth [35 ]. Here, we provide evidence that the effect of IL-4 on mouse DC subset development is opposite that observed on human LC. In our bulk cultures with IL-4, less cells expressed the scavenger receptor (2F8) and CD14 but more transferrin receptor CD71 and the lectin-like endocytosis receptor CD205. The capacities of bulk MHC IIlow cells to take up tracer molecules via macropinocytosis, mannose receptor, or phagocytosis were reduced by DC grown with GM-CSF plus IL-4. As mentioned above, this effect of IL-4 could not be observed by short-term treatment and therefore, most likely is the result of an increased generation of the LC-like subset 4 in the cultures with IL-4.

This shifting capacity of IL-4 toward the more potent antigen processing and presenting LC-like subset when compared with the MoDC subset might account for the phenomenon that BM-DC generated with GM-CSF plus IL-4 are more efficient APC in vitro and in vivo than BM-DC generated with GM-CSF alone [36 37 38 39 40 ].

Thus, the features in common between BM-derived, LC-like and MoDC-like subsets in culture as well as differences between BM-derived, LC-like or MoDC-like subsets and their in vivo counterparts might indicate the regulation of these markers by environmental factors. BM-DC always express the macrophage marker F4/80, which is not found in vivo. BM macrophages express low levels of CD11c, which is also not observed in vivo. Another example for this might be the general absence of CD4 and CD8{alpha} expression on BM-DC. In contrast, other characteristics such as the differentiation potential (Fig. 1) , the developmental stage at which they can endocytose OVA (stage 2 for LC-like and stage 3 for MoDC-like cells), the capacity to express certain markers (2F8, CD11b, Gr-1), mannose-receptor-mediated endocytosis (DX), a differential responsiveness to maturation stimuli (Fig. 5) , or the responsiveness to IL-4 (Fig. 7) , which can be found under the same culture conditions, might rather indicate their genetically determined regulation in the individual cell types.

Together, we found that bulk BM-DC cultures with GM-CSF contain LC-like and MoDC-like subsets. The subsets can be distinguished by the expression of surface receptors and their endocytosis capacities (Fig. 4b) . As both types of cells are grown under the same culture conditions, this method allows optimal, functional comparison of the two DC types in vitro. Both subsets show profound differences in antigen processing and presentation. The LC-like subset seems to represent the more efficient antigen-presenting cell type, and MoDC maintain the capacity to develop into macrophages or DC. Although the LC-promoting effect of TGF-ß is observed for human and murine DC, we report here that the capability of IL-4 to favor murine LC development remains unique for mouse BM-derived cells.


   ACKNOWLEDGEMENTS
 
This work was supported by the Deutsche Forschungsgemeinschaft (DFG Lu 851/1-1, Lu 851/2-1, SFB643) and the ELAN Fonds of the University Hospital Erlangen. We are grateful to Gerold Schuler for generous support of our projects. We thank Thilo Jakob for the E-cadherin antibody, Francis Carbone for the OT-II mice, and Peter Rohwer for cell sorting.

Received August 26, 2004; revised November 12, 2004; accepted December 8, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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