1. Introduction
1.2 DC-SIGN protein
DC-SIGN is one of several C-type lectin receptors on DCs that functions through calcium-dependent carbohydrate-binding [12]. It is expressed on myeloid DCs dermal DCs and monocyte-derived DCs, DC-SIGN interacts with ICAM-2 [13] and ICAM-3 on endothelial and naive T cells, respectively [14]. On
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DC-SIGN, there are a carbohydrate recognition domain (CRD), a neck region composed of seven and a half repeats, each containing 23 amino acids, and a transmembrane region followed by a cytoplasmic tail containing recycling and internalization motifs [9, 15, 16]. DC-SIGN recognizes mannose, N-acetylglucosamine, and fucose on many pathogens and non-sialylated Lewis (Lewa/b) structures, and can form tetramers in a Ca2+-dependent manner [15].
[15]
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1.3 DC-SIGN pathwayDCs can produce many first-line defensive cytokines, including IL-1, IL-6, IL-12, and TNF-α. Cytokines produced by DCs can shape the adaptive immune response so that it is pathogen-specific. DCs can also produce anti-inflammatory cytokines, such as IL-10 to limit or terminate inflammatory responses. IL-10 is critical for proper immunosuppression, as it is required for the induction of endotoxin tolerance [17].
Mannosylated lipoarabinomannan (ManLAM) induces the DC-SIGN down-stream transcription factor Ras via a signalosome complex consisting of the scaffolding proteins LSP1, KSR1, and CNK. Activated Ras induces a change in Raf-1 conformation, and prolongs NF-kB activity [10, 15, 18]. DC-SIGN signaling leads to the activation of NF-kB, and results in an increase in IL-10 promoter activity.
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[15]
1.4 Interaction between cancers and DC
Cancer cells influence the dendritic cell-driven production of cytokines. When dendritic cells are activated by antigens, they secrete a variety of cytokines that activate T cells, which in turn induce a host of immunoreactions. However, some reports have shown that tumors can escape from the immune response by suppressing DC activity. This includes the inhibition of DC maturation, the blockage of their migration, and the impairment of their antigen presenting ability. Glycosylation changes associated with cancer include the under-expression and/or over-expression of
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naturally occurring glycans [19]. Regulated glycosylation of specific acceptor substrates can affect immune function by creating or masking ligands of endogenous lectins [20]. It is still not clear how cancer cells influence immunoreactions through their interaction with dendritic cells.
1.5 Nasopharyngeal carcinoma
Nasopharyngeal carcinoma (NPC) is a disease in which malignant cells form in the tissues of the nasopharynx. As one of the most common cancers among Chinese and Asian ancestry, it poses one of the serious health problems in southern China where an annual incidence of more than 20 cases per 100,000 is reported.
Men are twice as likely to develop NPC as women. The rate of incidence generally increases from ages 20 to around 50 [21]. EBV has been implicated in a number of human alignancies, such as Burkitt’s and Hodgkin’s lymphoma, nasopharyngeal carcinoma (NPC), and gastric carcinoma. Epstein-Barr virus (EBV) is a well-documented etiologic agent for the development of NPC. EBV is known to infect the vast majority of adults worldwide (~95%),
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usually with lifelong persistence. However, only a small fraction of EBV-infected individuals develop NPC in their lifetime. NPC risk is lower in second- and third-generation Chinese immigrants to the United States than in first-generation immigrants, but the rates are still higher than those of other ethnic groups [22, 23]. The latent membrane protein (LMP) 1 oncogene is encoded by the normal B cell-associated LMP1 strain of EBV (B-LMP1) [24]. LMP-1 induces B lymphocyte transformation, activates NF-kB, and up-regulates B-cell surface markers such as CD40, LFA3, and ICAM-1 [25].
Our study demonstrated that DC-SIGN-recognized ligands were expressed on NPC cells. NPC cells induced immunosuppressive IL-10 secretion from DCs. We further showed that NPC-induced IL-10 secretion was inhibited by DC-SIGN antibody, siRNA, and high concentration of D-mannose under co-cultured conditions. In conclusion, NPC cells escape from the surveillance of the immune system by expressing DC-SIGN ligands, and stimulating immunosuppressive cytokine secretion from DC through DC-SIGN activation.
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2. Materials and methods
2.1 Chemicals and Reagents：
Acrylamide buffer was from Bioreagent. Albumin, bovine serum
(BSA), ammonium peroxodisulfate (APS),
ethylenediaminetetraacetic acid (EDTA), lipopolysaccharides, sodium pyruvate, thiazolyl blue tetra-zolium bromide (MTT), triton X-100, trypsin-EDTA were from Sigma. Protein assay reagent was from Bio-Rad. D(+)Fucose, protease inhibitor cocktail Set I were from Calbiochem. DC-SIGN siRNA, stealth RNAiTM siRNA, and primers for GAPDH, IL-10 and IL-12β were from Invitrogen.
Dimethyl sulfoxide (DMSO) was from Scharlau. Fetal bovine serum (FBS) was from Biowest. Galactose and mannose were from Bio basic inc. Glycerol, sodium dodecyl sulfate (SDS), trizma base (Tris), Tween 20 and N,N,N`,N`-Tetramethylethylenediamine (TEMED) were from Amersco. Glycine was from Riedel-deHaen.
Isopropanol was from Fluka. Methanol and 2-mercaptoethanol were from Merk. RhDC-SIGN/Fc Chimera, Recombinant Human GM-CSF and IL-4 were from R&D systems. Prestained poretin
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marker was from Fermentas. PureFection was from Biosciences.
PVDF membrane ws from Life science.
2.2 Buffers and Media:
Buffers used were as follows:
SDS-PAGE Running buffer (25 mM Tris, 192 mM glycine, 0.1%
SDS),
5X SDS-PAGE Sample buffer (10% SDS, 10 mM
beta-mercapto-ethanol, 20 % Glycerol, 0.2 M Tris-HCl, pH 6.8, and 0.05% Bromophenolblue)
Western transfer buffer (25 mM Tris, 192 mM glycine, 10%
methanol)
Western blocking buffer (1% BSA in PBS)
Phosphate-buffer saline (PBS; 137 mM NaCl, 10 mM Phosphate and 2.7 mM KCl, pH 7.4)
Tris-buffered saline tween-20 (TBST; 50 mM Tris.HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20)
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RIPA buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium
deoxycholate, 0.1% SDS, 50mM Tris, pH 7.5, 1mM PMSF and 10 g/ml Leupeptin) was from Genestar
Cell lysis buffer (1X protease inhibitor cocktail in RIPA buffer)
Media used were as follows:
Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM) and RPMI Medium 1640 were purchased from Gibco
2.3 Antibodies
Anti-CD11c-FITC, anti-HLA-DR-FITC, anti-CD-86-PE, anti-DC-SIGN-PE, and anti-CD14-PE were purchased for flow cytometry from eBioscience (USA). Anti-Raf-1, anti-phosphorylation-Raf-1 at serine 338, and anti- phosphorylation-p65 were purchased from Cell Signaling (USA).
Anti-p65 was purchased from Millipore (USA). In this study, Anti-
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DC-SIGN used to block the DC-SIGN receptor was from R&D system.(USA). Alkaline phosphatase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were from Bioscience. Human IL-10 and IL-12 antibodies were from PeproTech. Mouse IgG (H+L) preadsorbed secondary antibody was form GeneTex.
2.4 Cell culture
2.4.1 Preparation of immature MDDCs
Primary monocytes (CD14+) were separated from the peripheral blood mononuclear cells of healthy donors by magnetic beads conjugated with anti-CD14 (MACS, Germany). Primary cells were maintained in RPMI 1640 medium containing 10% FBS (BIOLOGICAL INDUSTRIES, Israel). Monocytes were cultured in a medium containing IL-4 (10 ng/ml) and granulocyte monocyte colony–stimulating factor (50 ng/ml) for seven days to induce differentiation into immature dendritic cells (MDDC).
2.4.2 Preparation of NPC cells
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The NPC cell line tw01 with and without Epstein-Barr virus was derived from the primary nasopharyngeal tumors of Taiwanese patients with de novo NPC (NPC cell lines were provided by Tsai SC, National Taiwan University, Taiwan). All cell lines were maintained in DMEM containing 10% FBS.
2.5 Enzyme-linked immunosorbent assay (ELISA)
2.5.1 Detection of cytokine production
Cells were cultured in 24-well plates at a density of 5×104 cells per-well. They were stimulated with 5x105 NPC cells, pre-treated for one hour with 1 μg/ml DC-SIGN blockage antibody, and the supernatant was collected. The concentrations of IL-10 and IL-12 were detected by a commercially available Enzyme linked immunosobent assay (R＆D system, USA).
2.5.2 Cytokine production in blockage of DC-SIGN under co-cultured conditions
MDDCs (5x104) were pre-treated with 1μg/ml blockage
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DC-SIGN antibody for one hour, and co-cultured with 5x105 NPCs.
The supernatants of the co-cultures were harvested and analyzed for IL-10 and IL-12.
2.5.3 Treatment with monosaccharides under co-cultured conditions collected and tested for IL-10 production.
2.6 Flow cytometry
2.6.1 Detection of DC-SIGN ligands on NPC cell membranes To detect the DC-SIGN ligand expression on NPC cell membranes, 2x105 NPCs were collected and washed with PBS containing 0.1% FBS. They were subsequently incubated with 10
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μg/ml DC-SIGN recombinant protein (R&D system, USA) for one hour at room temperature. After incubation, we detected the DC-SIGN protein on NPCs with anti-DC-SIGN antibodies. Stained cells were analyzed with FACSCalibur (BD Biosciences, USA) to determine the number of double positive cells. Data were processed with CellQuest software (BD Biosciences, Franklin Lakes, NJ).
2.6.2 Identification of maturation markers of MDDCs
MDDCs at 2x105 per well were incubated with fluorescencence-labeled primary antibodies at 4°C for 20 minutes, and then washed twice with PBS containing 1% BSA. The stained cells were analyzed with FACSCalibur to determine the number of CDC11C double positive cells. Data were processed with CellQuest software.
2.7 Immunofluorescent staining
NPCs at 8x104 per well were washed twice with 1x PBS and fixed with 4% paraformaldehyde for one hour at room temperature.
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Following incubation, the cells were washed twice with 1x PBS and blocked by PBS containing 1% BSA. The cells were incubated with 10 μg/ml DC-SIGN recombinant protein for one hour at room temperature and incubated with primary anti-DC-SIGN antibody overnight. The following day, we detected the DC-SIGN protein on NPC cell membranes by immunofluorescent analysis.
2.8 RNA interference
CD14+ monocyte was treated with 10 ng/ml DC-SIGN siRNA and control siRNA at day 1, and supplied with 10ng/ml siRNA at day 3. Transfected MDDCs at 2x104 per well were seeded on a 24-well plate and co-cultured with 2x105 NPC for 24 hours. The co-cultured supernatant was harvested and analyzed for IL-10 and IL-12.
2.9 MTT assay
DCs at 5x104 per well were seeded on a 24-well plate containing 500 μl of RPMI medium overnight. Treatment with mannose, fucose, or galactose were performed in different doses
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(0 mM, 10 mM, 20 mM, 40 mM) for 24 hours. The cells were then collected for MTT assay. The cells were washed with 1x PBS, and then incubated in 400 μl of medium and 100 μl of MTT (5mg/ml) at 37℃ for one hour. After incubation, we removed the medium and added 80 μl of DMSO. We added 50 μl of the resultant solution to each well in a 96-well plate, and detected the absorbance at 540 nm.
2.10 Western blotting
MDDCs were treated with NPC membranes, and proteins were harvested by lysis buffer. SDS PAGEs were performed with 20 μg of samples, and signaling protein expressions were detected by western blotting using specific antibodies.
2.11 Statistics
All results are expressed as means ± standard deviations.
Data were compared using the Student’s t-test. Significant differences with p < 0.05 were applied.
28 3. Results
In this project, the integrated hypothesis was that nasopharyngeal carcinoma cells interacted with the DC-SIGN receptor of dendritic cells to affect dendritic cell maturation and cytokine production.
3.1 The cytokine production and maturation of MDDCs co-cultured with NPC cells
We co-cultured the monocyte-derived dendritic cells and nasopharyngeal carcinoma cells for 24 hours and collected the supernatant. Under co-cultured conditions, the IL-10 production was increased, and IL-12 was decreased (Fig. 1a, b). These data indicated that NPC cells affected DC cytokine production. On the other hand, we also detected dendritic cell maturation marker expression under co-cultured conditions. The antigen presenting marker, HLA-DR, and the DC-SIGN protein were also decreased under co-cultured conditions (Fig. 2a, b). The NPCs interfered with the expression of DC markers and cytokine production.
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3.2 NPC cells expressed DC-SIGN-recognized ligands
NPC cells were incubated with 10 μg/ml DC-SIGN recombinant protein. The presence of DC-SIGN on NPC cell membrane was detected under certain conditions. DC-SIGN binding on NPC was detected only in the presence of calcium by flow cytometry (Fig. 3a). Consistent results were also seen in immunofluorescence staining (Fig. 3b). These data showed that DC-SIGN ligands are expressed on the NPC cell membrane, and NPC cells interact with DC-SIGN on DCs.
3.3 The membrane components on NPC cells affected DCs In NPC co-cultured condition, IL-10 production of DCs was significantly increased (Fig. 4b). We then treated DCs with NPC cell lysates and membranes, respectively, and detected the production of IL-10 in DCs (Fig. 5). The IL-10 production was inhibited under conditions in which DC-SIGN was blocked by the DC-SIGN blockage antibody. Note that the DC-SIGN antibody did not affect IL-10 secretion of DCs in the absence of NPC (Fig. 4a). These data
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suggested that the component(s) on NPC cell membrane interacted with DC-SIGN on DCs, and induced IL-10 production.
3.4 DC-SIGN functions under co-culture conditions
To confirm the role of DC-SIGN in mediating interaction of DCs with NPC, we further tested the effects of DC-SIGN inhibition by RNA interference and sugar competition. We co-cultured NPC mannose-containing ligands on NPC, and induces IL-10 production in DCs.
3.5 NPC cells affect DCs through other means than cell-cell
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contactsWe treated the dendritic cells with 24-hour NPC-conditioned medium. The data showed that NPC condition medium induced the IL-10 production in DCs (Fig. 8). The effect was not affected by the DC-SIGN antibody, suggesting that NPCs may have non-cell contact pathways to induce IL-10 production of DCs.
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4. Discussion
In this study, we found that NPC cells indeed express DC-SIGN ligands, and affect DC maturation and IL-10 production via DC-SIGN signaling. Activation of DC-SIGN signals through Ras/Raf-1 and NF-κB pathway, thus increases NF-κB p65 to bind IL-10 promoter, and induces high IL-10 expression [26, 27]. This is the first report of NPC to influence the immune system via such pathway, in which NPC cells affect DC functions.
Recent studies of colon cancer revealed interactions of
DC-SIGN on DCs and a glycoprotein on the cancer cells. The interaction interfered with DC maturation, and increased IL-10 production of DCs [6, 28]. In our study, we demonstrated that NPC cells inhibit DC maturation as HLA-DR, an antigen presenting marker, was reduced in NPC-cocultured conditions (Fig. 2a).
Immunosuppressive IL-10 was highly produced by DCs during NPC interactions (Fia. 1a). NPC cells can indeed affect dendritic cell functions by expressing antigen(s) recognized by surface receptor DC-SIGN as DC-SIGN proteins were located in the NPC
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cell surface (Fig. 3). In order to confirm whether it is through this receptor to influence the dendritic cells, we used anti-DC-SIGN blockage antibody, and found more than 50% inhibition of IL-10 secretion was achieved (Fig. 4). However, we also found that NPC cell culture medium can cause a similar phenomenon, in which DC-SIGN antibody did not reverse it (Fig 1a, Fig. 2a and Fig. 8), suggesting a DC-SIGN-independent pathway in IL-10 production of DCs. One possibility is galectin-1 which is a lung cancer-derived soluble lectin, and was shown to mediate DC anergy [29]. NPC may produce soluble factors, such as galectin-1, and mediate DC anergy other than the DC-SIGN pathway. It is worth investing the role of galectin-1 in NPC.
NPC cell lysates induced DC IL-10 production, but it was not significantly reduced by the DC-SIGN blockage antibody (Fig. 5a).
It is consistent with the idea that NPC may produce non-DC-SIGN ligands to mediate immunosuppressive responses in DCs. An alternative possibility is that the lysates may contain unknown substances which antagonized the effects the antibody.
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LPS are lipoglycans consisting of a lipid and a polysaccharide.
They are found in the outer membrane of bacteria, and elicit strong immune responses in animals [30]. We used LPS to activate the Toll-like receptor and NF-κB pathway, which induces IL-10 and IL-12 production [15]. Our experiments showed that 10 ng/ml LPS stimulated DCs with highest IL-10 and IL-12 amount at 12 hour, and then they decreased as time increased. When the NPC cells and dendrite-like cells were co-cultured, IL-10 was significantly higher than in the control group at 24 and 48 hour, while the trends of IL-12 were similar to the controls (Fig.1). Without LPS stimulation, DCs produced moderate amount of cytokines; however, the effects of NPC were not obvious (data not shown).
DC-SIGN, a C-type lectin receptor, recognizes various carbohydrate structures, such as mannose, fucose, galactose, and N-acetylglucosamine. A high dose of mannose inhibits the IL-10 production under the co-cultured condition (Fig. 7c). It is similar to the findings in the study of colon cancer [6]. Another study on Man-LAM of Mycobacterium tuberculosis indicated its interaction with DC-SIGN to induce IL-10 production and inhibit DC maturation
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[28]. Of note, Man-LAM activates DC-SIGN signaling pathway through Ras [10, 12]. Very possibly the DC-SIGN ligands on NPC may induce Ras pathway in DCs to affect their functions.
IL-10 is a cytokine associated with immune suppression. It was identified as a cytokine synthesis inhibitor, and inhibited antigen presentation [31]. Inhibitory effect of IL-10 on T cells was indirect and mediated mainly through antigen-presenting cells like macrophages and DCs [17]. High concentration of IL-10 promote naïve T cells to differentiate into T regulatory (Treg) cells [32], and Treg cells produce more IL-10 in positive feedback regulation [33].
These mechanisms decrease production of pro-inflammation T helper cell, Th1 and Th2, to inhibit immune responses. It seems likely that cancer cells, including NPC in our study, commonly regulate immune suppression by stimulating IL-10 production in DCs.
Currently we are thriving to identify DC-SIGN-interacting proteins on NPC cell membrane, and characterize their carbohydrate structures. We also hope to determine whether the NPC-induced DC-SIGN signaling pathways are Raf-1 dependent or
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independent. In the future we would also like to determine the correlations of EBV-derived proteins in NPC and immune responses.
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5. Reference1. Dominguez PM, Ardavin C. Differentiation and function of mouse monocyte-derived dendritic cells in steady state and inflammation. Immunol Rev 2010,234:90-104.
2. Shortman K, Naik SH. Steady-state and inflammatory dendritic-cell development. Nat Rev Immunol 2007,7:19-30.
3. Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, et al.
Immunobiology of dendritic cells. Annu Rev Immunol 2000,18:767-811.
4. Lanzavecchia A, Sallusto F. Regulation of T cell immunity by dendritic cells.
Cell 2001,106:263-266.
5. Sreekumaran E, Ramakrishna T, Madhav TR, Anandh D, Prabhu BM, Sulekha S, et al. Loss of dendritic connectivity in CA1, CA2, and CA3 neurons in hippocampus in rat under aluminum toxicity: antidotal effect of pyridoxine.
Brain Res Bull 2003,59:421-427.
6. Nonaka M, Ma BY, Murai R, Nakamura N, Baba M, Kawasaki N, et al.
Glycosylation-dependent interactions of C-type lectin DC-SIGN with colorectal tumor-associated Lewis glycans impair the function and differentiation of monocyte-derived dendritic cells. J Immunol 2008,180:3347-3356.
7. Ouaaz F, Arron J, Zheng Y, Choi Y, Beg AA. Dendritic cell development and survival require distinct NF-kappaB subunits. Immunity 2002,16:257-270.
8. Geijtenbeek TB, Kwon DS, Torensma R, van Vliet SJ, van Duijnhoven GC, Middel J, et al. DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell 2000,100:587-597.
9. Geijtenbeek TB, Torensma R, van Vliet SJ, van Duijnhoven GC, Adema GJ, van Kooyk Y, et al. Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses. Cell 2000,100:575-585.
10. den Dunnen J, Gringhuis SI, Geijtenbeek TB. Dusting the sugar fingerprint:
C-type lectin signaling in adaptive immunity. Immunol Lett 2010,128:12-16.
11. Vicari AP, Caux C, Trinchieri G. Tumour escape from immune surveillance through dendritic cell inactivation. Semin Cancer Biol 2002,12:33-42.
12. den Dunnen J, Gringhuis SI, Geijtenbeek TB. Innate signaling by the C-type lectin DC-SIGN dictates immune responses. Cancer Immunol Immunother 2009,58:1149-1157.
38
13. Bergman MP, Engering A, Smits HH, van Vliet SJ, van Bodegraven AA, Wirth HP, et al. Helicobacter pylori modulates the T helper cell 1/T helper cell 2 balance through phase-variable interaction between lipopolysaccharide and DC-SIGN. J Exp Med 2004,200:979-990.
14. Stambach NS, Taylor ME. Characterization of carbohydrate recognition by langerin, a C-type lectin of Langerhans cells. Glycobiology 2003,13:401-410.
15. Svajger U, Anderluh M, Jeras M, Obermajer N. C-type lectin DC-SIGN: an adhesion, signalling and antigen-uptake molecule that guides dendritic cells in immunity. Cell Signal 2010,22:1397-1405.
16. Kwon DS, Gregorio G, Bitton N, Hendrickson WA, Littman DR.
DC-SIGN-mediated internalization of HIV is required for trans-enhancement
DC-SIGN-mediated internalization of HIV is required for trans-enhancement