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The Journal of Immunology, 2007, 179, 5701 -5710
Copyright © 2007 by The American Association of Immunologists, Inc.
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Sialyl-Lewisx on P-Selectin Glycoprotein Ligand-1 Is Regulated during Differentiation and Maturation of Dendritic Cells: A Mechanism Involving the Glycosyltransferases C2GnT1 and ST3Gal I1

Sylvain Julien*, Matthew J. Grimshaw*, Mark Sutton-Smith{dagger}, Julia Coleman*, Howard R. Morris{dagger},{ddagger}, Anne Dell{dagger}, Joyce Taylor-Papadimitriou* and Joy M. Burchell2,*

* Breast Cancer Biology Group, King’s College London, Guy’s Hospital, London, United Kingdom; {dagger} Division of Molecular Biosciences, Imperial College, London, United Kingdom; and {ddagger} M-Scan, Wokingham, Berks, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
To fulfil their function as APCs, dendritic cells (DC) and their precursors need to travel from blood to the peripheral tissues and, upon activation, migrate from tissues to draining lymph nodes. Because O-glycans play a role in T cell trafficking, we investigated the O-glycosylation profile of human monocyte-derived DC. Sialyl-Lewisx (sLex), a glycan involved in extravasation via selectin binding, was found to be expressed exclusively on P-selectin glycoprotein ligand-1 in monocytes and immature DC. However, sLex was lost from mature DC even though these cells retained expression of P-selectin glycoprotein ligand-1. Maturation of DC led to a rapid change in the expression of glycosyltransferases involved in O-linked glycosylation. A down-regulation of C2GnT1 mRNA and enzymatic activity was observed with a concurrent up-regulation of ST3Gal I and ST6GalNAc II mRNA resulting in a loss of the core 2 structures required for sLex expression as a P-selectin ligand. Interestingly, the early regulation of these glycosyltransferases was mediated by PGE2, which is known to be required for human DC migration. The pattern of O-glycosylation seen in mature cells was very similar to that expressed by naive T cells, which home to lymph nodes. Our data show that the regulation of O-glycosylation controls sLex expression, and also suggest that O-glycans may have a function in DC migration.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DC)3 are pivotal to the immune response as they play a central role in innate immunity and in initiating the adaptive response (1). To drive the immune system to target cancer-associated Ags, many attempts have been made to use DC, often those derived from monocytes, for the immunotherapy of cancers (2). However, the limited results indicate that the biological properties of monocyte-derived DC need to be defined in more detail, particularly concerning their migration abilities.

To physiologically fulfil their function, DC or their precursors need to accomplish two crucial migration steps: first, they have to leave the blood to enter peripheral tissues; second, mature DC need to leave these tissues to home to the draining lymph nodes. DC and DC precursors, including monocytes circulating in the blood extravasate to tissues, such as epidermis, dermis, tonsils, or gastrointestinal mucosa, by crossing the endothelial layer. In peripheral tissues, DC can be activated by the recognition and uptake of foreign Ags and/or by proinflammatory cytokines. During the maturation of DC the expression of molecules involved in Ag uptake is decreased, while expression of those involved in Ag presentation to T cells is increased. Maturation stimuli also trigger the regulation of several genes involved in migration to the peripheral lymph nodes (3), where DC will initiate the adaptive response by presenting processed Ag to naive or central memory T cells (4).

Of the immune effector cells, lymphocyte trafficking has been most extensively studied (5). Selectin ligands expressed by lymphocytes bind E- and P-selectin expressed by endothelial cells, thereby mediating the rolling step that precedes the tight attachment of lymphocyte to the endothelial lining (6). This process is necessary for lymphocytes to be able to leave the blood stream and migrate to peripheral tissues such as skin (6). Selectins also play an important role in the trafficking of DC to tissues. Immature, but not mature, murine DC, bind to E- and P-selectins and this is a requirement for the migration of immature DC into inflamed skin in the murine system (7).

The E- and P-selectin ligand is the carbohydrate determinant known as sialyl-Lewis x (sLex; Fig. 1). sLex is a peripheral carbohydrate structure generated by the sequential action of various glycosyltransferases and can be found linked to different types of glycoconjugates, such as O-glycans (6), complex N-glycans (8, 9), or glycolipids (10).


Figure 1
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  		FIGURE 1. Biosynthesis of core 1 and core 2 O-linked glycans. The name of the Ag corresponding to a carbohydrate structure is indicated on the top left of the structure. As shown at the bottom of the figure, an extended O-glycan contains a core domain (here core 2), a stem region of variable length due to repetition (n ≥ 0) of a N-acetyl-lactosamine disaccharide motif (brackets), and a peripheral terminal structure, illustrated here is sialyl Lewisx, which is the tetrasaccharide motif surrounded by the dashed line. R, Serine or threonine; GalNAc, N-acetylgalactosamine; Gal, galactose; Neu5Ac, N-acetylneuraminic acid (sialic acid); Fuc, fucose. Glycosyltransferases involved in the biosynthesis are indicated by letters next to the arrows such as a, core 1 beta3GalT; b, C2GnT1 (core 2-synthase); c, ST3Gal I; d, ST6GalNAc I and II; e, ST6GalNAc I, II, and IV.

 
High-affinity binding to P-selectin is observed when sLex is carried on core 2-based O-linked glycan chains and these ligands have been demonstrated to have functional relevance in the rolling step of lymphocytes (6). The high-affinity counterreceptor for P-selectin has been identified as P-selectin glycoprotein ligand-1 (PSGL-1), expressed by most leukocytes, carrying sLex on a core 2 O-glycan attached to a specific amino acid (Thr57) of the protein (11). Recently, sLex present on CD43, another O-glycoprotein expressed by most hemopoietic cells, has been identified as a functional ligand for E-selectin, and again the requirement of core 2 O-glycans has been shown (12).

The glycosylation profiles of resting and activated T cells are different (13), leading to expression of different glycoforms of CD43 and PSGL-1. An important change upon T cell activation is the up-regulation of the C2GnT1 glycosyltransferase responsible for the synthesis of core 2 O-glycans. This change allows the expression of high-affinity selectin ligands which bind to selectins and are necessary for activated T cells to extravasate from the blood to the tissues (14, 15).

The ability of mature DC to traffic to the draining lymph nodes is vital for the execution of their function. Chemokines and their receptors, such as CCL21 and CCR7 (16), play a dominant role in this migration (3, 4). It has been recently reported that PSGL-1 may act as a secondary receptor for CCL21 in mouse T cells, and play a role in the T cell homing to lymph node (17). This function has been shown to be dependent on the O-glycosylation status of PSGL-1. Upon maturation, DC also express adhesion molecules such as CD44 variants (18) and extracellular proteases such as matrix metalloprotease (MMP)-9 and MMP-2 (19). Of note, CD44 and MMP-9 are known to be O-glycosylated and the pattern of O-linked glycosylation could alter their function (20, 21). However, nothing is known about the O-glycosylation profile of mature DC.

In the present study, we show that upon differentiation and maturation, monocyte-derived DC dramatically alter their expression of sLex carried by PSGL-1. These changes are consistent with the regulation of expression of relevant glycosyltransferases involved in O-linked glycosylation, resulting in changes in the expression of the whole O-glycoprofile, as assessed by mass spectrometry. The data strongly indicate that the profile of O-glycans expressed by mature DC may influence their migratory ability.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of human monocyte-derived DC

DC were generated from CD14+ cells as published previously (22). Briefly, PBMC were obtained from buffy coat preparations by density-gradient centrifugation on Ficoll-Paque. The CD14+-expressing cells were positively selected using CD14 microbeads (MACS; Miltenyi Biotec) following the manufacturer’s instructions. These cells were cultured for 6 days in AIM-V (Invitrogen Life Technologies), 400 U/ml recombinant human (rhu) GM-CSF (R&D Systems), and 2000 U/ml rhuIL-4 (R&D Systems) at a cell concentration of 106 cells/ml at 37°C and 5% CO2. For the maturation of DC, 1000 U/ml TNF-{alpha} (R&D Systems) plus 18 µg/ml–1 PGE2 (Sigma-Aldrich), or 100 ng/ml LPS (Sigma-Aldrich) were added on day 6. When necessary, cells were treated with 2 mM 1-benzyl-2-acetamido-2-deoxy-{alpha}-D-galactopyranoside (BGN; Sigma-Aldrich) during the 48 h of maturation. On day 8, the cells were harvested and characterized by flow cytometry before further analyses.

Flow cytometry analysis

Cells were stained with Abs to CD1a, CD14, CD40, CD80, CD83, CD86, and HLA-DR (Beckman Coulter) and CCR7 (BD Biosciences). sLex Ag was detected using the Ab CSLEX1 (BD Biosciences), culture supernatant from hybridomas secreting the IgM FH6 (a gift from Prof. H. Clausen, University of Copenhagen, Copenhagen, Denmark), or CH0-131 mAbs specific for core 2-associated sLex (23) (a gift from Dr. B. Walcheck, University of Minnesota, St. Paul, MN), or the Ab HECA-452 (BD Biosciences). Samples (105 cells) were incubated with Abs for 30 min on ice in PBS containing 0.5% BSA. Cells were then fixed in 1% formaldehyde. Appropriate isotype controls were used. Samples were analyzed using an EPICS XL Flow Cytometer (Beckman Coulter) and WinMDA 2.8 software (The Scripps Research Institute).

Electrophoresis and Western blotting

Cell pellets (5–30 x 106 cells) were lysed in 50 mM Tris-HCl, 150 mM NaCl buffer containing 1% Triton X-100. Proteins (100 µg) were loaded onto 4–12% gradient acrylamide gel, submitted to SDS-PAGE electrophoresis under reducing conditions, and electrotransferred on nitrocellulose membranes (Biotrace NT; Gelman Science) in accordance with standard procedures (24). Membranes were blocked 1% BSA in TBS and incubated with anti-CD44 (HCAM (F-4); Santa Cruz Biotechnology), anti-CD43 (84-3C1; Santa Cruz Biotechnology), anti-MUC1 (HMFG1), anti-PSGL-1 (KPL-1; BD Biosciences), or anti-sLex (HECA-452) Abs in 0.05% TBST, for 1 h. After washing, labeled proteins were revealed using appropriate secondary Abs conjugated to alkaline-phosphatase and NBT/X-phosphate revelation reagent (Roche).

Immunoprecipitation

A total of 250 µg of total cell lysate proteins was incubated (4 h, 4°C) with 5 µg of the anti-PSGL-1 mAb (KPL-1; BD Biosciences) or anti CD43 mAb (84-3C1; Santa Cruz Biotechnology). Cell lysates were then incubated with 100 µl of G-protein coupled to Sepharose beads (overnight, 4°C). Immunoprecipitated proteins were collected by centrifugation (5 min, 12,000 rpm), washed, and subjected to SDS-PAGE as described above.

RNA extraction, reverse transcription, and quantitative real-time PCR (qRT-PCR)

Cell samples were snap frozen using RNA Later reagent (Qiagen). Total RNA was extracted from cells using the Nucleospin RNA II kit (Macherey Nagel), according to the manufacturer’s instructions. DNased RNA (2 µg) was reverse transcribed using random hexamer primers and Superscript III reverse transcriptase (Invitrogen Life Technologies). qRT-PCR was performed using the Opticon qRT-PCR Analysis System (MJ Research), a hot-start PCR that contained the double strand-specific DNA-binding dye SYBR Green I (Sigma-Aldrich), and 10 pM of the forward and reverse primers (Table I). After 5 min at 95°C, 40 cycles were performed: 15 s denaturation at 94°C, 30 s annealing at 60°C, 30 s extension at 72°C, and fluorescence detection at 78°C. A melting curve fluorescence analysis was performed on each sample once the amplification cycles were completed to verify that a single product had been amplified. Each sample was normalized to the housekeeping gene beta-actin by removing the cycle threshold (Ct) value of beta-actin from the Ct value of the gene under investigation ({Delta}Ct). The fold difference was calculated by subtracting the {Delta}Ct of the test sample from the control sample to give {Delta}{Delta}Ct, and then fold difference = 2–{Delta}{Delta}Ct (25).


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  		Table I. Pairs of primers used in qRT-PCR

 
Mass spectrometry (MS)

O-glycans were isolated from trypsinized detergent extracts of cell pellets by reductive elimination, permethylated using the sodium hydroxide procedure, and purified on a Sep-Pak C-18 cartridge, as previously described (26, 27). MALDI-TOF data were acquired using a Perseptive Biosystems Voyager DE-STR with delayed extraction. MS/MS data were acquired using a 4800 MALDI TOF/TOF (Applied Biosystems) mass spectrometer. The collision energy was set to 1 kV and argon was used as collision gas. Samples were dissolved in 10 µl of methanol and mixed with a 1:1 ratio (v:v) with 2,5-dihydroxybenzoic acid as matrix.

C2GnT activity assay

C2GnT activities were determined as described previously (28). Briefly, cells were lysed in 120 mM NaCl, 40 mM Tris, 1% Triton X-100 and 20% glycerol, with Mini Complete Protease Inhibitor Mixture (Roche), 30 min at 4°C, and the protein concentration was determined using the BCA Protein Assay (Perbio Science). The reaction mixture contained 50 mM MES (pH 7.0), 1 mM uridine 5-diphospho-N-acetylglucosamine (GlcNAc; Sigma-Aldrich), 0.1 M GlcNAc, 1 mM p-nitrophenyl 2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl)-{alpha}-D-galactopyranoside (Gal-GalNAc-pnp; Toronto Research Chemicals), 0.1 µCi of uridine diphospho-N-acetyl-D-[U14C]-glucosamine (Amersham) and 26 µl of cell lysate, in a total reaction volume of 50 µl. The mixtures were incubated for 6 h at 37°C then diluted to 1 ml with water and processed by C18 Sep-Pak (Waters) column chromatography. After washing with water, the product was eluted using 70% acetonitrile and every wash and eluate fraction was counted in a scintillation counter. Transferase assays were performed in triplicate.

Cell migration analysis

Immature or mature DC (105 cells), treated or not with BGN, were transferred in the upper chamber of Transwell 24-well plates (pores 8 µm; Costar). To assess the migration, the lower well was filled with 500 µl of AIM-V medium, with or without rhuCCL21 (250 ng/ml–1; R&D Systems). Cells were incubated 5 h at 37°C, then the upper chamber was then removed and cells on the underside of the filter were flushed into the lower chamber. Numbers of viable migrating cells present in the lower chamber were counted using a CASY 1 particle counting system (Schaerfe System).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Differentiation and maturation of monocyte-derived DC

CD14+ cells isolated from blood buffy coats were differentiated into immature DC in the presence of GM-CSF and IL-4 and the DC matured by the addition of TNF-{alpha} plus PGE2 or LPS. The quality of the cells was assessed after selection, differentiation, and maturation by immunofluorescent flow cytometry with Abs directed to phenotypic markers. All the DC used in this study expressed MHC class II, CD40, CD80, CD86, and upon maturation showed increased expression of the of costimulatory molecules (CD80 and CD86). CD83 and the chemokine receptor CCR7 were detected on the cells only after maturation (data not shown). Six representative cultures of mature DC from 6 individual donors were assessed for their functional efficacy. All the cultures of DC were found to efficiently prime autologous T cell-specific responses to a flu peptide used as control (data not shown). In the present study, data obtained from 44 different donors are presented.

sLex expression by monocytes and DC

Due to the requirement of sLex for the initial step of extravasation of circulating immune cells, we investigated sLex expression on monocytes, immature DC, and mature DC. sLex was detected by immunofluorescent flow cytometry analysis using four anti-sLex Abs: CSLEX1 (8 donors), FH6 (14 donors), HECA-452 (5 donors), and CH0-131 (5 donors), the latter being an Ab that specifically reacts with sLex carried on core 2 O-glycans (23). In agreement with published data (29, 30), all Abs positively stained monocytes (Fig. 2). Immature DC were also positive, although the staining intensity was decreased with CSLEX1, CHO-131, and HECA-452 but increased with the FH6 Ab (Fig. 2B). These differences may reflect distinct but subtle differences in the specificities for the Ag, especially regarding the extension of the polylactosaminic chains (23, 30, 31). Strikingly, maturation induced by TNF-{alpha} plus PGE2 consistently and significantly reduced sLex staining by all Abs to a level that was barely detectable (Fig. 2). LPS treatment also reduced the expression of sLex, although to a lesser extent with greater variability among donors (data not shown).


Figure 2
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  		FIGURE 2. sLex is expressed in monocytes and immature DC but not in mature DC. A, Immunostaining of monocytes, immature (untreated), and mature (treated with TNF-{alpha} and PGE2) DC from a representative donor, using four anti-sLex Abs: CSLEX1, FH6, CHO-131, and HECA-452. The specific staining is shown in gray, the thin line is the isotype control. B, Statistical analysis of the variation of mean fluorescence intensity of sLex labeling. Various numbers of patients (n) were analyzed by flow cytometry and the intensity of the staining obtained with CSLEX1, FH6, CHO-131, or HECA-452 related to the isotype control is reported in theses graphs. Means, as indicated by a bar in the graphs, were statistically tested using a Student t test, and p values are indicated on the top.

 
PSGL-1 is the main O-glycoprotein carrying sLex in monocytes and immature DC

As shown in Fig. 3A, monocytes express the O-glycoproteins CD43, CD44, and PSGL-1, all of which could carry sLex on their O-glycan moiety. To detect sLex carried on glycoproteins we used the HECA-452 mAb (32, 33) which we found to be the most sensitive on Western blots. Surprisingly, HECA-452 stained only one 150 kDa band that coincided with the apparent molecular mass of CD43 and PSGL-1. However, we failed to detect sLex on immunoprecipitated CD43 from monocytes, indicating that if present, the Ag was expressed at levels lower than detectable by this assay (data not shown).


Figure 3
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  		FIGURE 3. PSGL-1 is the only glycoprotein carrying sLex in monocytes and immature DC. A, Total cell lysates of monocytes were submitted to an SDS-PAGE electrophoresis, transferred, and probed with Abs specific for O-glycoproteins (CD43, CD44, MUC1, and PSGL-1) and sLex (HECA-452). B, Total cell lysates from monocytes, immature (untreated), and mature (treated with TNF-{alpha} and PGE2) DC were run on SDS-PAGE and blotted with Abs to PSGL-1 and sLex. A fainter band over 250 kDa in the PSGL-1 blot corresponds to some remaining unreduced homodimers. C, PSGL-1 was immunoprecipitated from 250 µg of protein from cell lysates, ran on SD-PAGE and blotted with anti-sLex Ab (HECA-452). *, Partially denatured anti-PSGL-1 Ab or protein G used for immunoprecipitation and detected by the secondary Ab.

 
Although PSGL-1 expression was detected in monocytes, immature DC, and mature DC, distinct gel migration patterns were observed in each cell type (Fig. 3B). Mature DC expressed the fastest migrating form of PSGL-1, with an apparent molecular mass of 130 kDa. The differences in migration pattern are suggestive of changes in posttranslational modifications. Interestingly, the O-glycoprotein detected by HECA-452 showed a similar migration pattern in monocytes and immature DC, but no HECA-452 Ag was detected in mature DC (Fig. 3B). To confirm that the HECA-452 Ag was indeed carried on PSGL-1, PSGL-1 was immunoprecipitated from monocytes, immature DC, and mature DC and blotted with HECA-452 (Fig. 3C). Positive staining was seen on PSGL-1 immunoprecipitated from monocytes and immature DC but in agreement with flow cytometry, no HECA-452 staining was seen on PSGL-1 precipitated from mature DC. Taken together, these results indicate that the main, if not the only, glycoprotein carrying sLex in monocytes and immature DC is PSGL-1. Moreover, although PSGL-1 is expressed in mature DC, it does not carry the sLex glycan.

Transcription of glycosyltransferases is regulated upon differentiation and maturation of DC

To elucidate the mechanisms involved in the changes of sLex expression, the expression of various glycosyltransferases was determined by real-time qRT-PCR, in monocytes, immature DC, and mature DC focusing on two groups of enzymes. As sLex has been shown to be carried on O-linked glycans in PSGL-1 (11), the first group (C2GnT1, C2GnT2, C2GnT3, ST3Gal I, and ST6GalNAc II) consisted of glycosyltransferases acting on core 1 O-glycan structures; the second group (FucT-IV, FucT-VII, ST3Gal III, and ST3Gal IV) is involved in the sLex tetrasaccharide synthesis on both N- and O-linked glycans. Fig. 4A shows the level of mRNA relative to beta-actin mRNA detected for each enzyme in each cell type, while Fig. 4B shows the relative expression of transcript of each enzyme related to its expression in monocytes. Very little variation in mRNA expression was observed between the enzymes involved in sLex synthesis. In contrast, changes associated with differentiation and maturation events were consistently observed in the glycosyltransferases acting on core 1 O-glycans.


Figure 4
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  		FIGURE 4. Expression of glycosyltransferases involved in O-glycosylation is regulated during DC differentiation and maturation. For each donor, 1–2 million cells were snap frozen at days 0 and 8 of the culture. Total mRNA extracted from frozen cells was used as a template for retrotranscription before quantitative PCR using the pairs of primers listed in Table I. A first group of enzymes (bold font) is active on O-glycan core structures, while the second group (normal font) is involved in sLex synthesis. A, Transcript expression of various glycosyltransferases relative to the level of expression of the mRNA of beta-actin, in monocytes (four donors), immature (untreated) and mature (treated with TNF-{alpha} and PGE2) DC (three donors). Little interindividual variability was observed, especially in monocytes. B, The level of expression of each glycosyltransferase in immature and mature DC relative to the level detected in monocytes. These histograms represent the means of the three donors presented in A, and are representative of three independent experiments performed on a total of nine donors. Bars, SD. Statistical significance was tested using the Student t test; **, p ≤ 0.001; *, p ≤ 0.05; n.s., not significant. C, Histogram represents the ST3Gal I/C2GnT1 transcript expression ratio in monocytes ({square}), immature DC (Figure 4), and mature DC ({blacksquare}). Exact values calculated from the level of expression relative to beta-actin (A) are indicated on the top of the bars.

 
Three GlcNAc transferases (C2GnT1, C2GnT2, C2GnT3) have the ability to initiate the synthesis of core 2 by adding GlcNAc to core 1 (Fig. 1). ST3Gal I can compete for the core 1 acceptor substrate, adding sialic acid to give a product that can only be modified by subsequent addition of another sialic acid, thus preventing conversion to core 2 or core 1 extension. We observed that the three C2GnTs enzymes were expressed in monocytes at relatively low levels while C2GnT1 and C2GnT2 mRNAs were significantly up-regulated by 10- to 15-fold in immature DC compared with monocytes (Fig. 4, A and B). However, upon maturation, levels of C2GnTs were similar to those observed in monocytes. Importantly, although ST3Gal I mRNA was expressed at similar levels in monocytes and immature DC, levels were significantly up-regulated 5-fold when DC were matured (Fig. 4, A and B). ST6GalNAc II tended to show a pattern of expression similar to ST3Gal I, although, due to a broader individual variability (Fig. 4A, right panel) statistical significance was only reached by increasing the number of donors (data not shown).

ST3Gal I and C2GnT1 have been shown to have locational overlap in the Golgi, and the relative mRNA expression of these enzymes defines the type of core structures expressed on O-glycoproteins (34, 35). Fig. 4C shows the ratio of ST3Gal I and C2GnT1 mRNA expression. Although ST3Gal I mRNA is always expressed at a higher level than C2GnT1 mRNA, the ratio is decreased 8-fold in immature DC compared with monocytes but increased by a 100-fold in mature DC compared with immature cells. These data strongly suggest that mature DC are more likely to express less core 2-based O-glycans than immature DC.

O-glycoprofiling of monocytes, immature, and mature DC

All the above data imply differences in the O-glycan profile of monocytes, immature DC, and mature DC. To definitively determine the glycan structures carried by these cells, the carbohydrates were analyzed by MS. MALDI-TOF analysis of the O-glycans derived from the monocytes showed major signals for both core 1 and core 2 glycans, the majority of which were sialylated (Fig. 5, top panel). Both the core 1 and core 2 families remained abundant in the immature DC (Fig. 5, middle panel) but in agreement with the glycosyltransferase mRNA expression data, matured DC showed a dramatic reduction in core 2 structures (Fig. 5, bottom panel) and a prevalence of sialylated core 1.


Figure 5
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  		FIGURE 5. O-glycoprofile of monocytes, immature, and mature DC. MALDI-TOF mass spectra of O-glycans from monocytes (top panel), immature DC (middle panel), and DC matured for 48 h with TNF-{alpha} plus PGE2 (bottom panel). O-glycans were released by reductive elimination of the peptide/glycopeptide mixture obtained by Sep-Pak purification of tryptic/PNGase F digests of detergent extracts of cells. They were permethylated and subjected to Sep-Pak clean-up before MS analysis. The sequences given in the annotations were deduced from compositional information and MS/MS data. Squares indicate the m/z 1518 and 1879 corresponding to O-linked sLex.

 
Analysis of the spectrum for sLex-containing core 2 O-glycans showed these to be present on monocytes at m/z 1518 and 1879. Although minor, these signals are well above the background noise. Moreover, when they were selected for collisional activation in MALDI-TOF/TOF experiments, they yielded abundant fragment ions confirming the sequences shown in Fig. 5 (data not shown). However, in immature, as well as mature DC, these sLex components were no longer visible above background. In addition, no fragment ions corresponding to sLex sequences were observed when m/z 1518 and 1879 were subjected to collisional activation. Moreover, sLex was found on N-linked glycans isolated from monocytes but was also lost from immature and mature DC (data not shown). These observations are in apparent conflict with the flow cytometry and Western blot data, which show expression of sLex by immature DC. This may be a reflection of the difference in sensitivity of MS and the other techniques; however, MS analysis also showed that N-glycans and O-glycans were carrying longer polylactosaminic chains in immature DC than in monocytes (data not shown). Thus, it is possible that sLex in immature DC is synthesized at the end of polylactosaminic chains on core 2-based glycans, rather than directly on the core structure, making them difficult to identify above background in the high mass region of the MALDI spectrum.

The kinetics of regulation of glycosyltransferases mRNA during maturation is similar to that observed for CCR7 and MMP-9

Once activated, mature DC migrate to the lymph nodes where they interact with T lymphocytes (1). To investigate the functional relevance of the O-glycosylation changes upon maturation, we studied the time course of C2GnT1, ST3Gal I, and ST6GalNAc II mRNA expression after maturation stimuli. The down-regulation of C2GnT1 mRNA was found to be an early event, starting during the first 2 h after the addition of the maturation stimuli (Fig. 6A). Interestingly, TNF-{alpha} plus PGE2 treatment induced a quicker and more marked effect on down-regulation of C2GnT1 expression than LPS, the C2GnT1 mRNA being decreased by >10-fold after only 2 h of exposure to TNF-{alpha} plus PGE2. By 48 h, both maturation stimuli resulted in a 100-fold down-regulation of C2GnT1. In accordance with the mRNA regulation, GlcNAc-transferase activity was significantly found decreased by 57% at 24 h and 81% at 48 h of TNF-{alpha} plus PGE2 treatment (Fig. 6B).


Figure 6
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  		FIGURE 6. Time-course analysis of the regulation of glycosyltransferases during DC maturation. A, At day 6 of culture, cells were treated with TNF-{alpha} plus PGE2 or LPS for 48 h or left untreated. For each culture condition, 1–2 million cells were snap frozen at times 0, 2, 4, 8, 24, and 48 h. Expression of the transcript of C2GnT1, ST3Gal I, ST6GalNAc II, CCR7, and MMP-9 was then analyzed by qRT-PCR. Graphs show the time course of expression relative to the level of transcript measured at time 0 h. Values are the mean of three different donors. B, At times 0, 2, 8, 24, and 48 h after stimulation with TNF-{alpha} plus PGE2, 5–10 million cells were collected and lysed to measure the enzymatic activity of GlcNAc transferase. Enzymatic activity was determined by measuring the transfer of 14C-labeled GlcNAc to the Galbeta1–3GalNAc-PnP acceptor (see Materials and Methods), so the measured C2GnT activity was specific for core 2 synthesis. The percentage of transfer was then standardized to the total amount of protein in the cell lysate and related to the percentage counted at time 0 h. The figure represents the mean of four different donors from two independent experiments. Statistical significance was tested using the paired Student t test; *, p ≤ 0.05. C, effect of PGE2 on the regulation of transcript of glycosyltransferases. For each culture condition, 1–2 million cells were collected and snap frozen at times 0, 4, and 48 h. Expression of the transcripts of C2GnT1, ST3Gal I, ST6GalNAc II, CCR7, and MMP-9 was then analyzed by qRT-PCR. The results are expressed as fold change compared with the level of expression detected in cells before treatment (time = 0 h). Histograms are the mean of two different donors. Bars, SD.

 
Both maturation stimuli also induced an early up-regulation of ST3Gal I, and TNF-{alpha} plus PGE2 induced up-regulation of ST6GalNAc II, which was particularly pronounced at the early time points (Fig. 6A). However, LPS treatment resulted in a reduction in ST6GalNAc II expression (Fig. 6A).

CCR7 and MMP-9 are expressed by mature DC and both proteins have been shown to be required for DC migration from peripheral tissues to the draining lymph nodes (16, 19). We therefore compared the kinetics of mRNA expression of CCR7 and MMP-9 with that of the three glycosyltransferases. As shown in Fig. 6A, MMP-9 transcript expression significantly increased 2 h after the addition of the maturation stimuli, particularly when the cells were matured with TNF plus PGE2. As previously reported (36), expression of the CCR7 transcript was found to be dramatically up-regulated during maturation, notably more rapidly and to a higher level when cells were treated with TNF-{alpha} plus PGE2 rather than LPS (Fig. 6A). Significantly, the kinetics of up-regulation of CCR7 mRNA correlated with the kinetics of down-regulation of the C2GnT1 transcript. This observation, together with the up-regulation of ST6GalNAc II at the early time points, suggest that changes in glycosyltransferase expression may be related to migration.

Effect of PGE2 on glycosyltransferase expression

PGE2 is synthesized by cyclo-oxygenase as a by-product of the arachidonic acid pathway (37) and has been shown to be necessary for the migration of human DC (36, 38, 39, 40). Because the mRNA of glycosyltransferases seemed to be coregulated with transcripts of proteins involved in migration (CCR7 and MMP-9), we investigated the effect of PGE2 on the regulation of glycosyltransferase mRNA expression. DC were treated for 48 h with either TNF-{alpha}, LPS, PGE2, or a combination of these molecules (TNF-{alpha} plus PGE2 and LPS plus PGE2). PGE2 treatment induced a significant and early overexpression of CCR7 (Fig. 6C) but the stimulatory effect was reduced with time. TNF-{alpha} or LPS synergized with PGE2 in increasing CCR7 mRNA levels at 48 h. An early effect of PGE2 on transcription of MMP-9 was also seen. These observations are in accordance with previous reports indicating that PGE2 is a strong modulator of the expression of factors known to be involved in DC migration, particularly early after DC maturation (36, 38, 39, 40).

Interestingly, PGE2 alone also induced an early decrease of C2GnT1 expression, and an increase of both ST3Gal I and ST6GalNAc II sialyltransferases (Fig. 6C) at 4 h. However, by 48 h C2GnT1 expression was only slightly reduced and ST6GalNAc II had returned to levels expressed by untreated cells. The expression of ST3Gal I mRNA remained elevated.

A specific O-linked glycan pattern may be required for mature DC to migrate

To investigate the effect of the O-glycan profile on the migratory ability of DC, immature and mature DC were treated for 48 h with BGN and assayed for their ability to migrate toward CCL21, a ligand for CCR7. BGN is a competitive inhibitor of O-glycosyltransferases that largely prevents transfer on core 1 structure, thereby preventing the addition of sialic acid, conversion to core 2 or core 1 extension. BGN treatment had no effect on the expression of CD14, MHC class II, CD80, CD86, and CD83, nor induced any loss of viability (data not shown). Immature DC showed very little migration due to lack of CCR7 expression. However, despite the fact that a slight increase in CCR7 expression was seen (Fig. 7A), all three cultures of mature DC tested showed a decreased in migratory ability after treatment with BGN, which ranged from 12.5 to 57% reduction depending on the donor. Fig. 7B shows that BGN significantly decreased CCR7-induced migration by an average of 18%.


Figure 7
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  		FIGURE 7. Effect of BGN treatment on the chemotaxis of DC. Immature DC and DC treated with TNF-{alpha} plus PGE2 from three donors were treated or not with BGN to artificially alter any subsequent modification of the core 1 structure. A, Level of expression of CCR7 (chemokine receptor for CCL21) assessed by immunofluorescent flow cytometry. This is representative of six donors. B, Cells were submitted to a chemotaxis assay using CCL21 (250 ng/ml–1) as chemoattractant. For each donor, the percentage of migrating cells was counted in three wells for each condition. Histograms represent the means for three donors. Bars, SD. Statistical significance was tested using the Student t test. n.s., Not significant.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
A necessary requirement of the immune response is the trafficking of immune cells to the appropriate site to perform their function. In this study, we show that monocytes and in vitro-generated immature DC express sLex which, at the level of the sensitivity of the methods used here, is exclusively expressed on PSGL-1, the high-affinity counterreceptor for P-selectin (41). In contrast, although mature DC express PSGL-1, they do not express sLex. The expression of sialyl- and fucosyltransferases involved in sLex biosynthesis remained constant during the differentiation and maturation of DC indicating that the regulation of these enzymes was not responsible for the regulation of sLex expression. However, the differentiation of monocytes into immature DC induced an up-regulation of C2GnT1 and C2GnT2, and the subsequent maturation of DC dramatically decreased both C2GnTs mRNA expression and the corresponding enzymatic activity. Concomitantly, maturation increased ST3Gal I and ST6GalNAc II mRNA expression. The data suggest that while monocytes and immature DC can express glycoproteins carrying core 2-based glycans on glycoproteins, mature DC carry predominantly core 1-based structures. That this is indeed the case was confirmed by direct analysis of glycan structures on monocytes, immature and mature DC. Because sLex is carried on core 2 in PSGL-1 (11), we conclude that the control of the core 1/core 2 ratio is driving the expression of sLex in these cells. In agreement with this, expression of C2GnT1 has been previously shown to control P-selectin ligand expression in T cells (28, 42) and sLex expression in pre-B lymphocytic cells (43).

Immature DC showed the lowest ST3Gal I to C2GnT1 ratio (Fig. 4), suggestive of an increase in the expression of core 2-based O-glycans. This, together with the extended polylactosamic O-linked chains observed by MS, may explain the shift in molecular mass of PSGL-1 observed in immature DC. Similar mass shift of PSGL-1 has previously been shown to be a consequence of overexpressing C2GnT1 in a cell line expressing PSGL-1 and endogenous C2GnT1 (44). These authors also reported that the higher isoform resulted in slower rolling on P-selectin, indicative of a better tethering and extravasation (44). Based on these observations and our data, one could predict that immature DC will have a better interaction with P-selectin than monocytes.

Monocytes are established precursors for dendritic cells and macrophages (45), although whether the differentiation into DC in the presence of GM-CSF and IL-4 as established in vitro has an in vivo counterpart is unclear. However, this culture system is an important tool for studying human DC biology where it is not possible to carry out in vivo experiments. Here, we show the presence of sLex on PSGL-1 expressed by immature DC. This would allow these cells to extravasate into the tissues. In accordance with this finding, in the murine system interaction of immature DC with P and E selectin is required for their entry into inflamed tissues (7).

The absence of sLex on mature DC, despite the expression of PSGL-1, avoids any possibility of interaction with E- or P-selectin. Although the possible re-emergence of mature DC into the blood has been documented (46), such mature DC would be unable to migrate back to peripheral tissues. Furthermore, because these cells lack the expression of L-selectin (CD62L) (47) (data not shown), they cannot enter lymph nodes via blood circulation (48, 49). This finding is of particular importance in the context of immunotherapy using monocyte-derived DC, because i.v. injection of mature DC lacking both L-selectin and sLex adhesion molecules would appear be the least efficient way to address DC to the lymph nodes (49).

Maturation of DC induced by pathogens or cytokines in vivo results in the migration of DC to the secondary lymphoid organs such as the lymph nodes. This migration is known to be dependent on the expression of chemokine receptors (3). As observed in vivo, when monocyte-derived DC are induced to mature in vitro they turn on expression of the chemokine receptor, CCR7, and other factors involved in migration such as MMP-9 and CD44 (Fig. 6) (18, 19). Concomitant with the turning on of CCR7 and MMP-9, we show a down-regulation of C2GnT1 and an up-regulation of ST3Gal 1 and ST6GalNAc II. The regulation of the mRNA encoding C2GnT1, ST3Gal I, and ST6GalNAc II occurred early in the maturation process (Fig. 6A). Importantly, the time course of loss of C2GnT1 expression induced by TNF-{alpha} plus PGE2 mirrors that of the increase in CCR7 mRNA. However, maturing DC do still show 43% of their C2GnT enzyme activity 24 h after stimulation, indicating that the loss of activity is delayed, probably reflecting C2GnT protein half-life. However, ST6GalNAc II and ST3Gal I mRNA are rapidly up-regulated after stimulation with TNF-{alpha} plus PGE2 (Fig. 6A). Overexpression of competing enzymes is an efficient way to impair some pre-existing yet decreasing enzymatic activity. Thus, the fast induction of ST6GalNAc II and ST3Gal I mRNA expression will effectively prevent the core 2 synthesis by C2GnTs on newly synthesized glycoproteins in maturing DC.

PGE2 has been shown to be essential for CCR7-mediated migration of mature DC (36, 38, 39, 40). Remarkably, PGE2 on his own is sufficient to induce a rapid up-regulation of ST6GalNAc II and ST3Gal I (Fig. 6) and down-regulation of C2GnT1. This provides further evidence that the regulation of these glycosyltransferases could be related to migration of mature DC. Moreover, a chemotaxis-enhancing function has recently been demonstrated for PSGL-1 that is independent of sLex and is inhibited by core 2 glycans on PSGL-1 (17). Chemotaxis of DC to CCL21 was assessed in vitro after inhibiting O-glycan elongation with BGN. The results showed an effect of BGN treatment on the motility of mature DC, albeit variable among donors. The O-glycans expressed by mature DC were mostly sialylated core 1 structures and treatment of these cells with BGN would inhibit the addition of sialic acid to these O-glycans. Therefore, we suggest that the sialylation of the core 1 O-glycans is required to allow mature DC to migrate with maximum efficiency, possibly through a mechanism that involves CCL21 binding to PSGL-1.

A switch from core 2 to sialylated core 1 O-glycans is also well-documented in the change to malignancy in breast epithelial cells, and has been shown to be controlled by the relative expression of GlcNAc- and sialyltransferases, which compete for the core 1 structure as acceptor substrate (Fig. 1) (34, 35, 50, 51). It should be noted that the most common route of metastasis in breast cancer is via the lymphatic system (52) and overexpression of ST3Gal I is associated with grade, and therefore lymph node involvement (53). Given the data shown here, it could be hypothesized that the expression of a core 1 O-glycan pattern is required for cells to migrate to, and/or settle in, the lymph nodes.

In this article, we clearly demonstrate that regulation of glycosyltransferases controls the expression of O-glycan core structures in monocytes, and in vitro-generated DC, and that the ratio of core 1/core 2 O-glycans is associated with the level of expression of sLex on PSGL-1. Upon maturation, there is a rapid regulation of glycosyltransferases involved in O-linked glycosylation. This results in a change in the profile of O-glycans expressed by PSGL-1. Such changes would also affect other proteins involved in the migration to lymph nodes, such as CD44 and MMP-9. The independent effect of PGE2 on this regulation, and the influence of O-glycosylation on the migration of mature DC in vitro, also suggest that the changes of O-glycosylation upon DC maturation may be required for the efficient chemokine-dependent migration of mature DC to the lymph nodes.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Cancer Research U.K., the Biotechnology and Biological Sciences Research Council, and the Wellcome Trust. A.D. is a Biotechnology and Biological Sciences Research Council Professorial Fellow. Back

2 Address correspondence and reprint requests to Dr. Joy M. Burchell, Breast Cancer Biology Group, Third Floor, Thomas Guy House, Guy’s Hospital, London, SE1 9RT, U.K. E-mail address: joy.burchell{at}kcl.ac.uk Back

3 Abbreviations used in this paper: DC, dendritic cell; sLex, sialyl-Lewisx; PSGL-1, P-selectin glycoprotein ligand-1; MMP, matrix metalloprotease; rhu, recombinant human; BGN, 1-benzyl-2-acetamido-2-deoxy-{alpha}-D-galactopyranoside; qRT-PCR, quantitative RT-PCR; Ct, cycle threshold; MS, mass spectrometry; GlcNAc, N-acetylglucosamine. Back

Received for publication May 1, 2007. Accepted for publication August 13, 2007.


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 Introduction
 Materials and Methods
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Z. S Kawar, T. K Johnson, S. Natunen, J. B Lowe, and R. D Cummings
PSGL-1 from the murine leukocytic cell line WEHI-3 is enriched for core 2-based O-glycans with sialyl Lewis x antigen
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