Expression of multilectin receptors and comparative FITC–dextran uptake by human dendritic cells

Masato Kato1, Teresa K. Neil, David B. Fearnley, Alexander D. McLellan2, Slavica Vuckovic1 and Derek N. J. Hart1

Haematology/Immunology Research Group, Christchurch School of Medicine, Christchurch, New Zealand
1 Mater Medical Research Institute, Level 3, Aubigny Place, Raymond Terrace, South Brisbane, Queensland 4101, Australia

Correspondence to: M. Kato


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
Dendritic cells (DC) are potent antigen-presenting cells and understanding their mechanisms of antigen uptake is important for loading DC with antigen for immunotherapy. The multilectin receptors, DEC-205 and macrophage mannose receptor (MMR), are potential antigen-uptake receptors; therefore, we examined their expression and FITC–dextran uptake by various human DC preparations. The RT-PCR analysis detected low levels of DEC-205 mRNA in immature blood DC, Langerhans cells (LC) and immature monocyte-derived DC (Mo-DC). Its mRNA expression increased markedly upon activation, indicating that DEC-205 is an activation-associated molecule. In Mo-DC, the expression of cell-surface DEC-205 increased markedly during maturation. In blood DC, however, the cell-surface expression of DEC-205 did not change during activation, suggesting the presence of a large intracellular pool of DEC-205 or post-transcriptional regulation. Immature Mo-DC expressed abundant MMR, but its expression diminished upon maturation. Blood DC and LC did not express detectable levels of the MMR. FITC–dextran uptake by both immature and activated blood DC was 30- to 70-fold less than that of LC, immature Mo-DC and macrophages. In contrast to immature Mo-DC, the FITC–dextran uptake by LC was not inhibited effectively by mannose, an inhibitor for MMR-mediated FITC–dextran uptake. Thus, unlike Mo-DC, blood DC and LC do not use the MMR for carbohydrate-conjugated antigen uptake and alternative receptors may yet be defined on these DC. Therefore, DEC-205 may have a different specificity as an antigen uptake receptor or contribute to an alternative DC function.

Keywords: DEC-205, Langerhans cells, macrophage mannose receptor, monocyte-derived dendritic cells


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
Dendritic cells (DC) are specialist antigen-presenting cells (reviewed in 1,2). As immature cells, DC reside in non-lymphoid organs, e.g. Langerhans cells (LC) in the skin. DC are most accessible in the blood, where they circulate in low numbers, but DC can also be isolated from tissues including the skin. Alternatively, DC can be generated after 5–14 days in vitro culture of bone marrow, cord blood or blood-derived precursors including monocytes, using defined cytokine conditions (reviewed in 1,2). Cells obtained by these different methods exhibit functional and phenotypic features of the DC lineage; however, the exact relationship between these cell types has yet to be clarified. Circulating blood DC (immature blood DC) appear to represent the precursors of the tissue DC, but even a brief period of in vitro culture induces blood DC to acquire the phenotype of mature DC (3). Whilst it is well known that fresh blood DC present soluble antigens (48), studies concentrating on antigen uptake by freshly isolated human blood DC are few in number and are complicated by the fact that isolation techniques can influence DC phenotype or function (9,10). Although antigen uptake by freshly isolated blood DC has been visualized by electron microscopy (7), the cell surface molecules involved are not known. In contrast, antigen uptake by LC (1113) and monocyte-derived DC (Mo-DC) has been studied more (1418). A better understanding of the mechanisms DC use for taking up antigen may help develop better methods for loading DC with cancer or microbial antigens for clinical immunotherapy protocols.

Carbohydrate-conjugated antigen uptake by DC has been studied by eliciting an immune response against pathogenic microorganisms such as yeasts, bacteria and parasites, as well as loading artificially prepared antigens conjugated with carbohydrates (12,1419). Lectin-type receptors, especially the macrophage mannose receptor (MMR) (14,15,17,19) and DEC-205 (20), have been implicated in the uptake of carbohydrate-conjugated antigen by DC. Both molecules belongs to a family of transmembrane C-type lectins, containing a cysteine-rich (CR) domain, fibronectin type II (FNII) domain, multiple C-type carbohydrate recognition-like domains (CRD; eight for MMR and 10 for DEC-205), a transmembrane domain and a short cytoplasmic tail (2022). The MMR is found on the cell surface of macrophages and a subset of endothelial cells, and its CRD mediate the endocytosis of glycoconjugates with terminal mannose, fucose, N-acetylglucosamine or glucose residues and microorganisms with terminal mannose or N-acetylglucosamine on their cell surface (reviewed in 23). Because these terminal sugars are rarely found in mammalian cell surface or serum proteins, the MMR may be involved in discrimination of self from non-self antigens. The MMR was also found recently to bind sulfated glycoproteins (e.g. lutropin and thyrotropin) containing SO4–4GalNAcß1,4GlcNAcß1 via the CR domain and to be involved in the clearance of these hormones from the blood stream (24). Mouse DEC-205 was identified (20) as the antigen recognized by the rat mAb NLDC-145, which binds to LC and thymic epithelia (25,26) in tissue sections. Human DEC-205 has been cloned recently (10,27), but its tissue distribution and function are unclear. Despite the structural similarity between the DEC-205 and MMR, the overall amino acid similarity is only 27%, and the ligand specificity and function of DEC-205 is unknown. The MMR has been identified in Mo-DC as a major C-type lectin receptor mediating mannosylated-antigen uptake (17,18) and presentation (14,15,19). Of note, although mouse LC take up insoluble mannosylated antigens (e.g. yeast and zymosan), it was reported that mouse LC lack immunologically detectable MMR (12), suggesting that alternative receptors for mannosylated antigens are present on mouse LC.

This study provides the first comparison of the distribution of the MMR and DEC-205 on human blood DC, LC and Mo-DC, and compares this with the ability of these different DC populations to take up FITC–dextran.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
Tissue culture
L428 cells were provided by Dr V. Diehl (Klinik für Innere Medizin, Cologne, Germany), and were maintained in RPMI 1640, 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin. COS-7 cells were from ATCC (Rockville, MD) and cultured in Dulbecco's modified Eagle's MEM, 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin.

Cytokines, antibodies and chemicals
rhIL-4 was obtained from Sigma (St Louis, MO), rhGM-CSF was from Sandoz-Pharma (Auckland, New Zealand) and rh TNF-{alpha} was from Hoffman-La Roche (Basel, Switzerland). mAb used were the following: the 15-2 (IgG1, anti-MMR) (28) was a gift from Dr C. Rijken (TNO Prevention and Health, Leiden, Netherlands). The Na 1/34 (IgG2a, CD1a) was a gift from Dr. A. McMichael (Institute of Molecular Medicine, Oxford, UK). Phycoerythrin (PE)-conjugated CD3 (Leu4, IgG1), CD14 (LeuM3, IgG2b), CD16 (Leu11c, IgG1), CD19 (LeuM12, IgG1), and HLA-DR (L243, IgG2a) with appropriate subclass controls were from Becton Dickinson (San Jose, CA). PE–Cy5-conjugated anti-HLA-DR (Imm-357, IgG1) and a subclass control was from Coulter-Immunotech (Sydney, NSW, Australia). PE–CD83 (HB15a, IgG2b) was from Immunotech (Marseille, France). CMRF-44 (IgM) was made in-house (29). FITC-conjugated sheep anti-mouse Ig (FITC–SAM) and FITC-conjugated sheep anti-rabbit (FITC–SAR) Fab were from Amrad Biotech (Amrad Biotech, Boronia, VIC, Australia). Goat anti-human MMR was a gift from Dr. P. Stahl (Washington University, St Louis, MO) (30). Unless specified all chemicals were purchased from Sigma (Castle Hill, NSW, Australia) or BDH Chemicals (Palmerston North, New Zealand).

Preparation of human DEC-205–Ig fusion protein
A ~800 bp PCR fragment containing the human DEC-205 signal peptide, CR domain and FNII domain was amplified from pBK14-1 (27) with a T3 sequencing primer (5'-AATTAACCCTCACTAAAGGG-3') and a DEC-205 gene-specific primer (5'-ACGGATCCACTTACCTGTAGGCTTTAAGCAGATGCCCCA-3' at nucleotide 668–698) containing a BamHI site (underlined) and a splicing donor sequence (in italics) using the Expand PCR system (Roche Molecular Biochemicals, Auckland, New Zealand). The fragment was digested with BamHI and EcoRI, cloned into the pIg1 vector (31), and the DNA sequence confirmed. The resultant plasmid (pCR/FN–Ig) was transfected to COS-7 cells using Fugene 6 (Roche Molecular Biochemicals) according to the manufacturer's recommendation. The conditioned medium was collected 5–7 days after transfection, and subjected to a HiTrap Protein A column (Amersham Pharmacia Biotech, Auckland, New Zealand) and a DEC-205 Ig fusion protein (CR/FN–Ig) purified according to the manufacturer's recommendation. To confirm the identity, the CR/FN–Ig was subjected to N-terminus amino acid sequencing by the Protein Microchemistry Facility (University of Otago, Dunedin, New Zealand). This was SGRAA, corresponding to the predicted amino acid sequence of human DEC-205 at positions 28–32 (27), confirming the identity of this fusion protein as well as the predicted signal peptide cleavage site, based on the mouse DEC-205 data (32 and data not shown).

Preparation of rabbit anti-human DEC-205
Two New Zealand rabbits were immunized 4 times intradermally with ~50 µg of CR/FN–Ig according to the conventional immunization protocol using both incomplete (for primary immunization) and complete Freund's adjuvant (Sigma). After checking the titer against CR/FN–Ig by ELISA, the rabbits were exanguinated to collect the sera. The sera were pooled and the anti-human IgG was absorbed by passing the sera through a normal human Ig-conjugated NHS-activated HiTrap column (Amersham Pharmacia Biotech). The flow-through fraction was subjected to a HiTrap Protein A column and anti-DEC-205 purified. This antibody recognized human DEC-205 monospecifically as it reacted to a single ~200 kDa band in the extract of Hodgkin's disease-derived cell line L428 (a DEC-205 mRNA-positive cell line) by Western blot analysis (data not shown).

Cell labeling and flow cytometry
Labeling was performed at 4°C using standard techniques. For staining with mouse mAb, cells were incubated with saturating concentrations of primary antibody for 30 min, washed twice, incubated with FITC–SAM for 30 min, washed twice, blocked with 10% mouse serum for 5 min, and then incubated with PE-conjugated second antibody for 20 min, washed and analyzed. For staining with rabbit anti-DEC-205, cells were incubated with 1:300 diluted anti-DEC-205 IgG for 30 min, washed, incubated with FITC–SAR for 20 min, washed and analyzed. The non-immune rabbit serum was used as a negative control. Analysis of labeled cells was performed using a FACS Vantage flow cytometer (Becton Dickinson) equipped with a 488 nm argon ion laser (Ion Laser Technologies, Salt Lake City, UT). The instrument was calibrated daily using standardized fluorescent beads (CaliBRITE; Becton Dickinson). When possible, at least 10,000 events were acquired from each sample and analyzed using CellQuest software (Becton Dickinson). Live cells were gated on the basis of forward and side scatter characteristics. In some experiments propidium iodide exclusion was used to gate live cells.

Cell preparation
Both normal human blood and normal breast skin were obtained locally with an appropriate informed consent and local Ethical Committee approval.

Immature and activated blood DC was prepared from human peripheral blood as described previously (3,33). Briefly, peripheral blood mononuclear cells (PBMC) were prepared by isolation over Ficoll-Paque gradients (Amersham Pharmacia Biotech). T lymphocytes were removed by resetting with neuraminidase-treated sheep erythrocytes, followed by Ficoll-Paque separation. Contaminating erythrocytes were removed by ammonium chloride lysis. T lymphocyte-depleted PBMC were labeled with a mixture of anti-CD3, -CD14, -CD16 and -CD19. After incubation with goat anti-mouse Ig-coated magnetic beads (Miltenyi Biotech, Gladbach, Germany), labeled cells were removed by magnetic immunodepletion. The resulting lineage-depleted cells were subsequently labeled with FITC–SAM and PE–HLA-DR, and further purified by FACS. Immature blood DC were identified among the lineage-negative cells on the basis of HLA-DR+ staining. For activated blood DC, T lymphocyte-depleted PBMC were cultured at 2x107 cells/ml in RPMI 1640 and 10% FCS for 12–15 h. Low density cells were isolated by separation over Nycodenz gradient (Nycomed Pharma, Oslo, Norway), and labeled sequentially with CMRF44, FITC–SAM, PE–CD14 and PE–CD19. Activated blood DC were sorted by FACS as CD14CD19CMRF44+ cells or gated as lineage-negative cells for antigen uptake experiments. For DEC-205 staining, fresh or overnight cultured T lymphocyte-depleted PBMC were subjected to depletion of CD3+, CD14+, CD16+ and CD19+ cells by magnetic separation as above, and stained with PE-HLA-DR and rabbit IgG anti-human DEC-205.

LC were isolated from human breast skin as described previously (34). Briefly, epidermal sheets were prepared by digesting split thickness breast skin with dispase II (Roche Molecular Biochemicals). Epidermal cell suspension was obtained by mechanical and enzymatic (trypsin and DNase I for 20 min at 37°C) dissociation of the epithelial sheets, and LC were enriched over Lymphoprep gradient (Amersham Pharmacia Biotech). Cells were labeled with PE–HLA-DR, and HLA-DR+ LC were sorted by FACS or gated for antigen uptake experiments.

Immature and mature Mo-DC were prepared as described previously (35). Briefly, T lymphocyte-depleted PBMC were plated on Falcon tissue culture plastic (Becton Dickinson Labware, Franklin Lakes, NJ) and allowed to adhere for 2 h. The non-adherent cells were removed, and the adherent cells cultured for 7 days in RPMI 1640, 10% FCS, 50 U/ml rhIL-4 and 200 U/ml rhGM-CSF. The cells were labeled sequentially with CD1a, FITC–SAM and PE–CD14, and immature Mo-DC were sorted by FACS as CD1a+CD14 cells. For mature Mo-DC, lipopolysaccharide (LPS) (Sigma; 1 µg/ml) was added at day 5 to the culture described above, and the cells cultured for further 2 days. The cells were labeled subsequently with CD83, FITC–SAM and PE–CD14, and mature Mo-DC were sorted by FACS as CD14CD83+ cells.

Macrophages were prepared from human PBMC as described previously (36). Briefly, tissue culture plasticadherent cells in T lymphocyte-depleted PBMC were cultured for 4 days in RPMI 1640 and 10% pooled AB serum. Adherent cells were harvested in ice-cold PBS, 0.1% BSA and 5 mM EDTA by pipetting. Macrophages were gated on the basis of forward and 90° light scatter characteristics by FACS.

RT-PCR analysis
Total RNA was isolated from FACS-purified cells (104–105 cells/preparation, purity >95%) using the RNeasy kit (Qiagen, Hilden, Germany). The RNA was treated with RNase-free DNase I (Life Technologies, Gaithersburg, MD) and reverse transcribed with SuperScript II (Life Technologies) with an oligo d(T)18 primer. The resultant cDNA was subjected to PCR using primers specific for human DEC-205 (27) and MMR (21,22) (Table 1Go). Human GAPDH (37) was used for normalization of the cDNA input. To confirm no contamination of genomic DNA in the DNase I-digested RNA, the RNA preparation was subjected to PCR without reverse transcription for GAPDH which primers could utilize genomic DNA as a template if contaminated. The 40 µl PCR reactions consisted of 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 200 µM dNTPs, 0.5 µM primers, 20% (v/v) Q solution (Qiagen) plus 1.5 mM (for DEC-205 and GAPDH) or 2.5 mM (for MMR) MgCl2 and 1 U Taq polymerase (Qiagen or Roche Molecular Biologicals). The PCR conditions were: an initial denaturation at 94°C for 5 min, 32 (for GAPDH) or 37 cycles (for DEC-205 and MMR) of thermal cycling at 94°C for 15 s, 57°C for 15 s and 72°C for 30 s, and final extension at 72°C for 5 min. The PCR reactions were fractionated with 2% agarose gel (BioRad, Hercules, CA) and transferred to a charged nylon membrane (Micron Separations INC, Westbrough, MA) using a vacuum blotting apparatus (BioRad) and UV cross-linked (Stratagene, La Jolla, CA). The membrane was probed with the digoxigenin-labeled internal primers specific for DEC-205, MMR and GAPDH (see Table 1Go), and the specific signals were detected by chemiluminescence according to the manufacturer's recommendation (Roche Molecular Biologicals).


View this table:
[in this window]
[in a new window]
 
Table 1. Gene-specific primers used in RT-PCR for DEC-205, MMR and GAPDH
 
FITC–dextran and lucifer yellow uptake
Cells were suspended in RPMI 1640, 10% FCS and 25 mM HEPES, pH 7.4, and incubated with 1 mg/ml of FITC–dextran (Mr = 40,500; Sigma) or lucifer yellow CH (Sigma) for 20 min at 4 or 37°C. In some experiments, cells were preincubated with inhibitors (1 mg/ml of galactose, mannose or mannan; all from Sigma) for 10 min at 37°C and then used in uptake experiments. Cells were washed 3 times with ice-cold PBS, 0.1% BSA and 0.01% NaN3, and labeled on ice with PE-conjugated appropriate mAb. The uptake was calculated as the change in MFI between cell samples incubated at 37 and 4°C.

The uptake by immature blood DC was assessed by a three-color FACS protocol developed to minimize the possible changes in their phenotype and function during the purification procedure. Briefly, blood was collected and PBMC were isolated immediately by Ficoll-Paque gradient separation. The cells were directly subjected to FITC–dextran and lucifer yellow uptake as described above, and labeled with PE–CD3, PE–CD14, PE–CD16, PE–CD19 and PE–Cy5-HLA-DR or with appropriate control antibodies for 15 min on ice. Cells were washed and analyzed immediately by FACS. Immature blood DC were identified as linHLA-DR+ cells. The obtained results were similar to those with our conventional immature blood DC preparation as described above (data not shown).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
DEC-205 is expressed on blood and Mo-DC
We prepared the monospecific IgG anti-human DEC-205 by immunizing rabbits with the DEC-205–Ig fusion protein (CR/FN–Ig) as described in the Methods. Using this antibody we examined the cell surface expression of DEC-205 on immature and activated blood DC, immature and mature Mo-DC, and macrophages by FACS analysis (Fig. 1Go). Low-level expression of DEC-205 was present on linHLA-DRlow immature blood DC (Fig. 1AGo). After overnight culture in the presence of FCS, the differentiated/activated lineage- negative blood DC formed two subsets based on HLA-DR positivity. However, the DEC-205 expression on both linHLA-DRhigh (a mixture of CD11c+ and CD11c) and linHLA-DRlow (CD11c) blood DC subsets was similar to that of immature blood DC, indicating that this differentiation/activation of blood DC had minimal effect on the cell surface DEC-205 expression. Immature Mo-DC (CD1a+CD14) expressed a low levels of DEC-205; however, surface DEC-205 increased markedly on mature Mo-DC (C14CD83+) after maturation with LPS (Fig. 1BGo). No cell-surface DEC-205 was detected on macrophages.



View larger version (48K):
[in this window]
[in a new window]
 
Fig. 1. FACS analysis of DEC-205 expression in various preparation of DC. (A) Immature (top panels) and activated blood DC (bottom panels) were stained with PE-conjugated HLA-DR and the rabbit anti-DEC-205, and analyzed by FACS. The rabbit non-immune serum was used as a negative control. The results are representative of four similar experiments. (B) immature Mo-DC, mature Mo-DC and macrophages were stained with the rabbit anti-DEC-205 (solid line), and analyzed by FACS. The non-immune rabbit serum was used as a negative control (dashed line). The results are representative of three similar experiments.

 
Human blood DC and LC lack immunologically detectable MMR
Using the 15-2 mAb against human MMR, we examined the expression of the MMR on immature and differentiated/activated blood DC, LC, immature and matured Mo-DC, and macrophages (Fig. 2Go). FACS analysis with the 15-2 did not detect any surface MMR on either immature, differentiated/activated blood DC or LC, whereas both immature Mo-DC and macrophages showed high levels of MMR expression as expected (17,18). The MMR expression diminished upon maturation of Mo-DC induced by LPS. It was noted that the MMR expression on immature Mo-DC was ~10-fold higher than that of macrophages, probably due to the presence of IL-4 during culture (38). Similar results were obtained with the polyclonal goat anti-MMR (data not shown), indicating that the lack of MMR staining on blood DC and LC was not due to selectable loss or blockade of the 15-2 epitope.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 2. FACS analysis of MMR expression in various preparations of DC. Blood DC, LC, Mo-DC and macrophages were labeled with anti-MMR mAb 15-2 (solid line) or negative control (shaded) and analyzed by FACS. The results are representative of three similar experiments.

 
Expression of DEC-205 and MMR mRNA in DC
RT-PCR analysis was performed to assess the expression of human DEC-205 and MMR at the transcriptional level within highly purified blood DC, LC, Mo-DC and macrophages (purity >95%) (Fig. 3Go). A low level of DEC-205 mRNA was detected in freshly isolated, immature blood DC, LC, immature Mo-DC and macrophages. The signal level increased markedly in differentiated/activated blood DC and in mature Mo-DC, indicating again that DEC-205 is a differentiation/activation-associated antigen. No MMR mRNA was detected in immature and differentiated/activated blood DC and LC. An apparently high level of MMR mRNA was detected in immature Mo-DC, but this decreased to undetectable levels upon their maturation. A low level of the transcript was detected in macrophages. These results confirmed that blood DC and LC do not express the MMR, and the expression of both DEC-205 and MMR is, at least in a part, transcriptionally regulated.



View larger version (77K):
[in this window]
[in a new window]
 
Fig. 3. RT-PCR analysis for DEC-205 and MMR in DC and macrophages. Genomic DNA-free total RNA was purified from FACS-purified DC and macrophages (purity >95%), reverse-transcribed with a oligo d(T)18 primer, and subjected to RT-PCR and Southern blot analysis for DEC-205 and MMR. GAPDH was used for normalization of cDNA input.

 
Comparison of FITC–dextran and lucifer yellow uptake by immature blood DC with other cell types
Cells representing various preparation of DC (immature and differentiated/activated blood DC, LC, and immature Mo-DC) and macrophages were compared for their ability to take up FITC–dextran (Fig. 4AGo, left panel). Lucifer yellow was used to assess the level of pinocytosis that would also contribute FITC–dextran uptake (Fig. 4AGo, right panel). LC, immature Mo-DC and macrophages all showed high levels of endocytoxic activity. The FITC–dextran uptake by LC, immature Mo-DC and macrophages showed {Delta}MFI = 486 ± 190, 470 ± 52 and 640 ± 284 (n = 3) (mean ± SD) respectively, and the lucifer yellow uptake {Delta}MFI = 195 ± 98, 164 ± 82 and 116 ± 49 (n = 3) respectively. In contrast, immature blood DC showed minimal activity for both FITC–dextran uptake ({Delta}MFI = 7.3 ± 7.5, n = 3) and lucifer yellow ({Delta}MFI = 0.3 ± 0.6, n = 3). The differentiated/activated blood DC were slightly more active in FITC–dextran uptake ({Delta}MFI = 30.3 ± 10.0, n = 3) than immature blood DC; however, the lucifer yellow uptake ({Delta}MFI = 1.0 ± 0.4, n = 3) remained minimal, suggesting that the FITC–dextran uptake by differentiated/activated blood DC was mediated by receptor-dependent endocytosis, rather than pinocytosis. Both FITC–dextran and Lucifer yellow uptake by immature and differentiated/activated blood DC was markedly less than that of either LC immature Mo-DC or macrophages.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 4. Comparison of FITC–dextran and lucifer yellow uptake by immature blood DC with other cell types (A). Immature and activated blood DC, monocytes, LC, Mo-DC and macrophages were prepared and compared in their ability to uptake FITC–dextran (left panel) and lucifer yellow (right panel). The results depict mean {Delta}MFI(37 – 4°C) ± SD (n = 3). (B) The effect of sugar inhibitors (galactose, mannose or mannan at 1 mg/ml) on FITC–dextran uptake. Cells with high FITC–dextran uptake were preincubated with inhibitors and further incubated with FITC–dextran. The results were normalized using the uptake without inhibitor as 100% (mean ± SD, n = 3).

 
FITC–dextran uptake by immature Mo-DC is predominantly mediated by MMR and the binding of MMR to FITC–dextran can be inhibited by mannose or a mannan, but not by galactose (17,18,39). Therefore, the inhibition study with these sugars was performed to determine the contribution of mannose-specific receptors for the FITC–dextran uptake (Fig. 4BGo). Both mannose and mannan substantially inhibited the uptake of FITC–dextran (70 and 68% respectively) by Mo-DC compared to the minimal effect seen with galactose (15%), indicating that Mo-DC employed a mannose-specific receptor (most likely to be MMR) to mediate FITC–dextran uptake. In contrast, these sugars had little or no effect on FITC–dextran uptake by macrophages, indicating either pinocytosis or a receptor system with different specificity such as the lipoprotein-related protein (40) is the predominant mechanism for the uptake. The uptake by LC was slightly inhibited by galactose or mannose (20 and 25% respectively) and to a greater extent by the mannan (48%). This may indicate that LC utilize a receptor system with different ligand specificity from that of mannose-specific receptors in addition to their high pinocytotic activity. After 48 h of in vitro culture in media, LC uptake of FITC–dextran was reduced by >50% (data not shown). The small level of FITC–dextran uptake by both immature and activated blood DC was not affected by the presence of mannose or mannan (data not shown), suggesting that mannose-specific receptors are not involved.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
Two cell-surface multilectins, DEC-205 and MMR have been identified in various DC preparations (17,25,27,41). Both molecules belong to the MMR family and are structurally similar. However, the overall amino acid identity between the DEC-205 and the MMR is only 27% (20,27), suggesting that they have distinct functions, which may be related to their expression and ligand specificity. This study provides the first comparison of DEC-205 and MMR expression with FITC–dextran uptake by various populations of human DC.

Immature Mo-DC utilize the MMR as a high capacity and broad specificity antigen-uptake receptor for FITC–dextran (17) or mannosylated antigens (18), and this results in 100- to 1000-fold more efficient presentation of mannosylated antigens than non-mannosylated antigens to T lymphocytes (14,21). However, our data suggest that neither LC nor blood DC express the MMR. Mouse LC take up zymosan, and the uptake can be inhibited with mannan and ß-glucan (12). This indicates that a mannan/ß-glucan receptor exist in LC. It was reported that human LC expressed the MMR detected by polyclonal anti-MMR (42) but mAb against MMR failed to detect MMR on LC (43,44). Furthermore, recent in situ studies using immunological and in situ hybridization methods showed that the expression of MMR is restricted to macrophages and a subset of vascular and lymphatic endothelial cells (45,46). Our results support the latter based on three experiments: (i) mAb 15-2 and polyclonal anti-MMR fail to stain LC (Fig. 2Go), (ii) RT-PCR failed to detect MMR mRNA in LC (Fig. 3Go) and (iii) FITC uptake by LC was not inhibited with mannose (Fig. 4Go), a potent inhibitor of MMR-dextran binding (39). We noted, however, that mannan could partly inhibit the FITC–dextran uptake by LC more efficiently than mannose, suggesting that a mannan receptor exists on LC, even though the majority of FITC–dextran uptake appeared to be mediated by pinocytosis as shown by lucifer yellow uptake (Fig. 4Go).

Recently, Milone and Fitzgerald-Bocarsly reported that the MMR present on blood DC mediated the uptake of enveloped RNA and DNA viruses and induction of IFN-{alpha} (47). Interestingly, the IFN-{alpha} production of DC by virus infection could be markedly inhibited with galactose and N-acetylgalactosamine, sugars which do not influence MMR–mannose binding (39). Further, it appeared that a subset of blood DC (ILT3+ILT1+) expressed MMR at a low level (48). However, our results established that blood DC do not express the MMR at both the mRNA and the protein levels (Figs 2 and 3GoGo). There may be differences in the blood DC preparations, however, and further investigation is required to clarify this possibility. This data also raises the possibility of an alternative receptor with specificity for carbohydrate ligands, perhaps DEC-205, being involved in antigen uptake by blood DC.

DEC-205 has been implicated as an antigen-uptake receptor because it is structurally similar to the MMR and DC incubated with a rabbit anti-mouse DEC-205 could elicit the proliferation of rabbit IgG specific T lymphocytes (20). Recently, the antigen detected by mAb MR6 (gp200-MR6) was identified as DEC-205 (49) and the adjuvant effect of mAb MR6 (50) also suggests that DEC-205 is involved in antigen uptake. MR6 was raised against human thymic stromal cells and stains thymic cortical epithelium intensely with weak staining of T and B lymphocytes, macrophages, dendritic cells and some non-lymphoid tissues such as urothelium and mammary epithelium (51,52). Interestingly, expression of gp200-MR6 in breast carcinoma appeared to decrease with malignant progression (53). These reports indicate that human DEC-205 is expressed broadly in lymphoid and non-lymphoid tissues as shown in mice using rabbit anti-mouse DEC-205 and NLDC-145 (the mAb against mouse DEC-205) (25,26,41). Although MR6 behaved as either an agonist or antagonist for IL-4 and this function was linked to IL-4 receptor, the ligand and function of DEC-205 is unknown. We asked whether DEC-205 was involved in carbohydrate-conjugated antigen uptake by assessing the expression of DEC-205 on potential antigen-presenting cells and by correlating the FITC–dextran uptake in various preparations of DC.

The rabbit IgG anti-DEC-205 detected only a low level of DEC-205 in immature linHLA-DRlow blood DC (Fig. 1AGo). This blood DC population can be divided into a CD11c+ subset (myeloid DC) and a CD11c subset (plasmacytoid DC) (48), thus it appears that both DC subsets expressed low levels of DEC-205. After overnight culture in the presence of FCS, the lineage-negative blood DC split into a HLA-DRhigh subset and a HLA-DRlow subset. Both subsets expressed similar levels of DEC-205 on their surface or showed minimal increase compared to immature blood DC. The majority of lin HLA-DRlow subset was CD11c and linHLA-DRhigh subset was the mixture of CD11c+ and CD11c population (S. Vuckovic, unpublished results). The marked increase of DEC-205 mRNA during differentiation/maturation of blood DC (Fig. 3Go) did not correlate to the cell-surface staining of DEC-205, indicating that there may be a large DEC-205 intracellular pool or post-transcriptional regulation of surface DEC-205 controlled by other signals. Interestingly, DEC-205 expression increased upon activation of Mo-DC at the protein and mRNA levels (Figs 1 and 3GoGo), a process usually regarded as being associated with a decreased antigen uptake. Importantly, this increase of DEC-205 was associated with reduced MMR levels on the Mo-DC. As MMR-mediated FITC–dextran uptake diminishes upon activation of Mo-DC (17), it is unlikely that DEC-205 is involved in FITC–dextran uptake by Mo-DC. The data also suggests that ligand specificity of the DEC-205 differs from that of MMR. The increase of DEC-205 upon DC differentiation/activation has been noted in murine DC (54,55), encouraging speculation that DEC-205 may have additional functions, unrelated to antigen uptake.

In comparison to immature Mo-DC or LC, immature blood DC are markedly less active in both FITC–dextran and lucifer yellow uptake. Upon differentiation/activation by overnight culture, blood DC slightly increased their endocytic activity; however, the activity never reached the level of Mo-DC or LC (Fig. 4Go). Our observation that blood DC do not take up large amount of soluble antigens has to be viewed in the light of their known ability to efficiently process and present antigens to T lymphocytes. Electron microscopic studies showed that freshly isolated blood DC are able to take up colloidal gold-conjugated BSA (7). The fact that these blood DC are more potent APC than blood monocytes, which are capable of taking up 10–20 times more antigens in our system (Fig. 4Go), suggests that blood DC require only small amount of soluble antigens to induce immune responses as previously described (56,57). Thus, their antigen uptake receptors might be predicted to direct very efficient trafficking of antigen to the relevant processing compartments.


    Note added in proof
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
DEC-205 and MMR have been classified as CD205 and CD206, respectively, at the 7th International Workshop and Conference on Human Leucocyte Differentiation Antigens.


    Acknowledgments
 
This work was supported by grants from Health Research Council of New Zealand, and the Foundation for Research Science and Technology. We thank Amanda Boyce for assistance with cell culture, Lisa Whyte for operating our FACS facility and Kirsten Taylor for general laboratory assistance. We also acknowledge Diana Carne for performing protein microsequencing.


    Abbreviations
 
CRD carbohydrate recognition domain
CR cysteine-rich
DC dendritic cells
FNII fibronectin type II
LC Langerhans cells
LPS lipopolysaccharide
MMR macrophage mannose receptor
Mo-DC monocyte-derived dendritic cells
PBMC peripheral blood mononuclear cells
PE phycoerythrin
SAM sheep anti-mouse
SAR sheep anti-rat

    Notes
 
2 Current address: Department of Dermatology, University of Würzburg, 97080 Würzburg, Germany Back

Transmission editor: A. McMichael

Received 30 January 2000, accepted 13 July 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 

  1. Hart, D. N. J. 1997. Dendritic cells: unique leucocyte populations which control the primary immune response. Blood 90:3245.[Free Full Text]
  2. Banchereau, J. and Steinman, R. M. 1998. Dendritic cells and the control of immunity. Nature 392:245.[ISI][Medline]
  3. Fearnley, D. B., McLellan, A. D., Mannering, S. I., Hock, B. D. and Hart, D. N. J. 1997. Isolation of human blood dendritic cells using the CMRF-44 monoclonal antibody: implications for studies on antigen presenting cell function and immunotherapy. Blood 89:3708.[Abstract/Free Full Text]
  4. Van Voorhis, W. C., Valinsky, J., Hoffman, E., Luban, J., Hair, L. S. and Steinman, R. M. 1983. Relative efficacy of human monocytes and dendritic cells as accessory cells for T cell replication. J. Exp. Med. 158:174.[Abstract]
  5. Mehta-Damani, A., Markowicz, S. and Engleman, E. G. 1995. Generation of antigen-specific CD4+ T cell lines from naive precursors. Eur. J. Immunol. 25:1206.[ISI][Medline]
  6. Macatonia, S. E., Patterson, S. and Knight, S. C. 1991. Primary proliferative and cytotoxic T-cell responses to HIV induced in vitro by human dendritic cells. Immunology 74:399.[ISI][Medline]
  7. Nijman, H. W., Kleijmeer, M. J., Ossevoort, M. A., Oorschot, V. M. J., Vierboom, M. P. M., van de Keur, M., Kenemans, P., Kast, W. M., Geuze, H. J. and Melief, C. J. M. 1995. Antigen capture and major histocompatibility class II compartments of freshly isolated and cultured human blood dendritic cells. J. Exp. Med. 182:163.[Abstract]
  8. Thomas, R. and Lipsky, P. E. 1994. Human peripheral blood dendritic cell subsets—isolation and characterization of precursor and mature antigen-presenting cells. J. Immunol. 153:4016.[Abstract/Free Full Text]
  9. Fanger, N. A., Voigtlaender, d., Liu, C., Swink, S., Wardwell, K., Fisher, J., Graziano, R. F., Pfefferkorn, L. C. and Guyre, P. M. 1997. Characterization of expression, cytokine regulation, and effector function of the high affinity IgG receptor FcgRI (CD64) expressed on human blood dendritic cells. J. Immunol. 158:3090.[Abstract]
  10. McCarthy, D. A., Macey, M. G., Bedford, P. A., Knight, S. C., Dumonde, D. C. and Brown, K. A. 1997. Adhesion molecules are upregulated on dendritic cells isolated from human blood. Immunology 92:422.[ISI][Medline]
  11. Caux, C., Massacrier, C., Dezutter-Dambuyant, C., Vanbervliet, B., Jacquet, C., Schmitt, D. and Banchereau, J. 1995. Human dendritic Langerhans cells generated in vitro from CD34+ progenitors can prime naive CD4+ T cells and process soluble antigen. J. Immunol. 155:5427.[Abstract]
  12. Reis e Sousa, C., Stahl, P. D. and Austyn, J. M. 1993. Phagocytosis of antigens by Langerhans cells in vivo. J. Exp. Med. 178:509.[Abstract]
  13. Nestle, F. O., Filgueira, L., Nickoloff, B. J. and Burg, G. 1998. Human dermal dendritic cells process and present soluble protein antigens. J. Invest. Dermatol. 110:762.[Abstract]
  14. Tan, M. C. A., Mommaas, A. M., Drijfhout, J. W., Jordens, R., Onderwaer, J. J. M., Verwoerd, D., Mulder, A. A., ven der Heiden, A. N., Scheidegger, D., Oomen, L. C. J. M., Ottenhoff, T. H. M., Tulp, A., Neeljes, J. J. and Koning, F. 1997. Mannose receptor-mediated uptake of antigens strongly enhances HLA class II restricted antigen presentation by cultured dendritic cells. Eur. J. Immunol. 27:2426.[ISI][Medline]
  15. Engering, A. J., Cella, M., Fluitsma, D., Brockhaus, M., Hoefsmit, E. C. M., Lanzavecchia, A. and Pieters, J. 1997. The mannose receptor functions as a high capacity and broad specificity antigen receptor in human dendritic cells. Eur. J. Immunol. 27:2417.[ISI][Medline]
  16. Sallusto, F. and Lanzavecchia, A. 1994. Efficient presentation of soluble antigen by cultured human dendritic cells in maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J. Exp. Med. 179:1109.[Abstract]
  17. Sallusto, F., Cella, M., Danieli, C. and Lanzavecchia, A. 1995. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial product. J. Exp. Med. 182:389.[Abstract]
  18. Avraméas, A., McIlroy, D., Hosmalin, A., Autran, B., Debré, P., Monsigny, M., Claude Roche, A. and Midoux, P. 1996. Expression of a mannose/fucose membrane lectin on human dendritic cells. Eur. J. Immunol. 26:394.[ISI][Medline]
  19. Prigozy, T. I., Sieling, P. A., Clemens, D., Stewart, P. L., Behar, S. M., Porcelli, S. A., Brenner, M. B., Modlin, R. L. and Kronenberg, M. 1997. The mannose receptor delivers lipoglycan antigens to endosomes for presentation to T cells by CD1b molecules. Immunity 6:187.[ISI][Medline]
  20. Jiang, W., Swiggard, W. J., Heufler, C., Peng, M., Mirza, A., Steinman, R. M. and Nussenzweig, M. C. 1995. The receptor DEC-205 expressed by dendritic cells and thymic epithelial cells is involved in antigen processing. Nature 375:151.[ISI][Medline]
  21. Ezekowitz, R. A. B., Sastry, K., Bailly, P. and Warner, A. 1990. Molecular characterization of the human macrophage mannose receptor: demonstration of multiple carbohydrate recognition-like domains and phagocytosis of yeasts in Cos-1 cells. J. Exp. Med. 172:1785.[Abstract]
  22. Taylor, M. E., Conary, J. T., Lennartz, M. R., Stahl, P. D. and Drickamer, K. 1990. Primary structure of the mannose receptor contains multiple motifs resembling carbohydrate-recognition domains. J. Biol. Chem. 265:12156.[Abstract/Free Full Text]
  23. Stahl, P. D. and Ezekowitz, R. A. B. 1998. The mannose receptor is a pattern recognition receptor involved in host defense. Curr. Opin. Immunol. 10:50.[ISI][Medline]
  24. Fiete, D. J., Beranek, M. C. and Baenziger, J. U. 1998. A cysteine-rich domain of the `mannose' receptor mediates GalNAc-4-SO4 binding. Proc. Natl Acad. Sci. USA 95:2089.[Abstract/Free Full Text]
  25. Witmer-Pack, M. D., Swiggard, W. J., Mirza, A., Inaba, K. and Steinman, R. M. 1995. Tissue distribution of the DEC-205 protein that is detected by the monoclonal antibody NLDC-145, II. Expression in situ in lymphoid and nonlymphoid tissues. Cell. Immunol. 163:157.[ISI][Medline]
  26. Kraal, G., Breel, M., Janse, M. and Bruin, G. 1986. Langerhans cells, veiled cells, and interdigitating cells in the mouse recognized by a monoclonal antibody. J. Exp. Med. 163:981.[Abstract]
  27. Kato, M., Neil, T. K., Clark, G. J., Morris, C. M., Sorg, R. V. and Hart, D. N. J. 1998. cDNA cloning of human DEC-205, a putative antigen-uptake receptor on dendritic cells. Immunogenetics 47:442.[ISI][Medline]
  28. Barrett-Bergshoeff, M., Noorman, F., Bos, R. and Rijken, D. C. 1997. Monoclonal antibodies against the human mannose receptor that inhibit the binding of tissue-type plasminogen activator. Thromb. Haemost. 77:718.[ISI][Medline]
  29. Hock, B. D., Starling, G. C., Daniel, P. B. and Hart, D. N. J. 1994. Characterization of CMRF-44, a novel monoclonal antibody to an activation antigen expressed by the allostimulatory cells within peripheral blood, including dendritic cells. Immunology 83:573.[ISI][Medline]
  30. Lennartz, M. R., Cole, F. S., Shepherd, V. L., Wileman, T. E. and Stahl, P. D. 1987. Isolation and characterization of a mannose-specific endocytosis receptor from human placenta. J. Biol. Chem. 262:9942.[Abstract/Free Full Text]
  31. Fawcett, J., Holness, C. L. L., Needham, L. A., Turley, H., Gatter, K. H., Mason, D. Y. and Simmons, D. L. 1992. Molecular cloning of ICAM-3, a third ligand for LFA-1, constitutively expressed on resting leukocytes. Nature 360:481.[ISI][Medline]
  32. Swiggard, W. J., Mirza, A., Nussenzweig, M. C. and Steinman, R. M. 1995. DEC-205, a 205-kDa protein abundant on mouse dendritic cells and thymic epithelium that is detected by the monoclonal antibody NLDC-145: purification, characterization, and N-terminal aminoacid sequence. Cell. Immunol. 165:302.[ISI][Medline]
  33. McLellan, A. D., Starling, G. C. and Hart, D. N. J. 1995. Isolation of human blood dendritic cells by discontinuous Nycodenz gradient centrifugation. J. Immunol. Methods 184:81.[ISI][Medline]
  34. McLellan, A. D., Heiser, A., Sorg, R. V., Fearnly, D. B. and Hart, D. N. J. 1998. Dermal dendritic cells associated with T lymphocytes in normal human skin display an activated phenotype. J. Invest. Dermatol. 111:841.[Abstract]
  35. Vuckovic, S., Fearnley, D. B., Mannering, S. I., Dekker, J., Whyte, L. F. and Hart, D. N. J. 1998. Generation of CMRF-44+ monocyte-derived dendritic cells: insights into phenotype and function. Exp. Hematol. 26:1255.[ISI][Medline]
  36. Andreesen, R., Brugger, W., Scheibenbogen, C., Kreutz, M., Leser, H.-G., Rehm, A. and Lohr, G. W. 1990. Surface phenotype analysis of human monocyte to macrophage maturation. J. Leuk. Biol. 47:490.[Abstract]
  37. Tokunaga, K., Nakamura, Y., Sakata, K., Fujimori, K., Ohkubo, M., Sawada, K. and Sakiyama, S. 1987. Enhanced expression of a glyceraldehyde-3-phosphate dehydrogenase gene in human lung cancers. Cancer Res. 47:5616.[Abstract]
  38. Stein, M., Keshav, S., Harris, N. and Gordon, S. 1992. Interleukin 4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunologic macrophage activation. J. Exp. Med. 176:287.[Abstract]
  39. Taylor, M. E. and Drickamer, K. 1993. Structural requirements for high affinity binding of complex ligands by the macrophage mannose receptor. J. Biol. Chem. 268:399.[Abstract/Free Full Text]
  40. Noorman, F., Braat, E. A. and Rijken, D. C. 1995. Degradation of tissue-type plasminogen activator by human monocyte- derived macrophages is mediated by the mannose receptor and by the low- density lipoprotein receptor-related protein. Blood 86:3421.[Abstract/Free Full Text]
  41. Inaba, K., Swiggard, W. J., Inaba, M., Meltzer, J., Mirza, A., Sasagawa, T., Nussenzweig, M. C. and Steinman, R. M. 1995. Tissue distribution of the DEC-205 protein that is detected by the monoclonal antibody NLDC-145, I. Expression on dendritic cells and other subsets of mouse leukocytes. Cell. Immunol. 163:148.[ISI][Medline]
  42. Condaminet, B., Peguet-Navarro, J., Stahl, P. D., Dalbiez-Gauthier, C., Schmitt, D. and Berthier-Vergnes, O. 1998. Human epidermal Langerhans cells express the mannose-fucose binding receptor. Eur. J. Immunol. 28:3541.[ISI][Medline]
  43. Noorman, F., Braat, E. A. M., Barrett-Bergshoeff, M., Barbé, E., van Leeuwen, A., Lindeman, J. and Rijken, D. C. 1997. Monoclonal antibodies against the human mannose receptor as a specific marker in flow cytometry and immunohistochemistry for macrophages. J. Leuk. Biol. 61:63.[Abstract]
  44. Mommaas, A. M., Mulder, A. A., Jordens, R., Out, C., Tan, M. C., Cresswell, P., Kluin, P. M. and Koning, F. 1999. Human epidermal Langerhans cells lack functional mannose receptors and a fully developed endosomal/lysosomal compartment for loading of HLA class II molecules. Eur. J. Immunol. 29:571.[ISI][Medline]
  45. Linehan, S. A., Martinez-Pomares, L., Stahl, P. D. and Gordon, S. 1999. Mannose receptor and its putative ligands in normal murine lymphoid and nonlymphoid organs: in situ expression of mannose receptor by selected macrophages, endothelial cells, perivascular microglia, and mesangial cells, but not dendritic cells. J. Exp. Med. 189:1961.[Abstract/Free Full Text]
  46. Takahashi, K., Donovan, M. J., Rogers, R. A. and Ezekowitz, R. A. 1998. Distribution of murine mannose receptor expression from early embryogenesis through adulthood. Cell Tissue Res. 292:311.[ISI][Medline]
  47. Milone, M. C. and Fitzgerald-Bocarsly, P. 1998. The mannose receptor mediates induction of INF-alpha in peripheral blood dendritic cells by enveloped RNA and DNA viruses. J. Immunol. 161:2391.[Abstract/Free Full Text]
  48. Cella, M., Jarrossay, D., Facchetti, F., Alebardi, O., Nakajima, H., Lanzavecchia, A. and Colonna, M. 1999. Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type I interferon. Nat. Med. 5:919.[ISI][Medline]
  49. Mckay, P. F., Imami, N., Johns, M., Taylor-Fishwick, D. A., Sedibane, L. M., Totty, N. F., Hsuan, J. J., Palmer, D. B., George, A. J. T., Foxwell, B. M. J. and Ritter, M. A. 1998. The gp200-MR6 molecule which is functionally associated with the IL-4 receptor modulates B cell phenotype and is a novel member of the human macrophage mannose receptor family. Eur. J. Immunol. 28:4071.[ISI][Medline]
  50. Sivolapenko, G. B., Imami, N., Larché, M., Epenetos, A. A. and Ritter, M. A. 1996. Enhanced in vivo immunogenicity induced by an antibody to the IL-4 receptor-associated gp200-MR6 molecule. Scand. J. Immunol. 44:135.[ISI][Medline]
  51. Al-Tubuly, A. A., Luqmani, Y. A., Shousha, S., Melcher, D. and Ritter, M. A. 1996. Differential expression of gp200-MR6 molecule in benign hyperplasia and down-regulation in invasive carcinoma of the breast. Br. J. Cancer 74:1005.[ISI][Medline]
  52. Tungekar, M. F., Gatter, K. C. and Ritter, M. A. 1996. Bladder carcinomas and normal urothelium universally express gp200-MR6, a molecule functionally associated with the interleukin 4 receptor (CD 124). Br. J. Cancer 73:429.[ISI][Medline]
  53. Kaklamanis, L., Koukourakis, M. I., Leek, R., Giatromanolaki, A., Ritter, M., Whitehouse, R., Gatter, K. C. and Harris, A. L. 1996. Loss of interleukin 4 receptor-associated molecule gp200-MR6 in human breast cancer: prognostic significance. Br. J. Cancer 74:1627.[ISI][Medline]
  54. De Smedt, T., Pajak, B., Muraille, E., Lespagnard, L., Heinen, E., De Baetselier, P., Urbain, J., Leo, O. and Moser, M. 1996. Regulation of dendritic cell numbers and maturation by lipopolysaccharide in vivo. J. Exp. Med. 184:1413.[Abstract]
  55. Vremec, D. and Shortman, K. 1997. Dendritic cell subtypes in mouse lymphoid organs, Cross-correlation of surface markers, changes with incubation, and differences among thymus, spleen, and lymph nodes. J. Immunol. 159:565.[Abstract]
  56. Steinman, R. M. and Swanson, J. 1995. The endocytic activity of dendritic cells. J. Exp. Med. 182:283.[ISI][Medline]
  57. Inaba, K., Metlay, J. P., Crowley, M. T. and Steinman, R. M. 1990. Dendritic cells pulsed with protein antigens in vitro can prime antigen-specific, MHC-restricted T cells in situ. J. Exp. Med. 172:631.[Abstract]