2. Material and Methods
2.6 Flow cytometry
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.
26
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
27
(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.
29
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
30
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
31
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.
32
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
33
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.
34
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
35
[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
36
independent. In the future we would also like to determine the correlations of EBV-derived proteins in NPC and immune responses.
37
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40 6. Figures Figure 1
(a)
(b)
41
Figure 1. Cytokine expressions changed in MDDC co-cultured with NPC. MDDC were co-cultured with NPC in various conditions (DC alone, with NPC culture medium, DC:NPC in 1:10 ratio, DC:NPC in 1:100 ratio, DC:NPC in 1:10 ratio without cell-cell contacts, and DC:NPC in 1:100 ratio without cell-cell contacts) in several time periods (6, 24, 36 and 72 hours). (a) The IL-10 expression increased in MDDC and NPC co-cultured medium. (b) The IL-12 expression decreased in MDDC and NPC co-cultured medium.
42 Figure 2
(a)
(b)
Figure 2. NPC affected the maturation of MDDC. (a) Detection of the HLA-DR expression levels to compare the maturation and presentation ability of MDDC. HLA-DR represents the maturation maker. (b) Detection of the DC-SIGN expression levels to compare the capturing ability of MDDC.
43 Figure 3
(a)
44
(b)Figure 3. NPC expresses DC-SIGN ligand(s) on the cell surface.
The utilization of DC-SIGN-Fc recombinant protein was to detect the existence of DC-SIGN ligands on the cell surface of NPC. NPC cells were incubated with DC-SIGN recombinant protein, and detected by anti-DC-SIGN antibody. DC-SIGN ligand expression on NPC cell surface was detected by (a) flow cytometry, and (b) immunofluorescence staining with and without Ca+2.
45 Figure 4
(a)
(b)
Figure 4. NPC cells interacted with DCs via DC-SIGN. (a) IL-10 production of DCs treated with DC-SIGN blockage antibody. (b) IL-10 production was induced by NPC in co-cultured condition, and reversed in the addition of 1μg/ml anti-DC-SIGN antibody. n=4.
46 Figure 5.
(a)
47 (b)
Figure 5. IL-10 production of DCs treated with NPC lysates. (a) IL-10 production was induced by NPC lysates, and was reversed in the addition of 1 μg/ml anti-DC-SIGN antibody. n=3. (b) IL-10 production was induced by NPC cell membranes, and reversed in the addition of 1 μg/ml anti-DC-SIGN antibody.
48
Figure 6Figure 6. IL-10 production in DC-SIGN-knocked-down DCs. Under co-cultured conditions, IL-10 production in DCs was induced by NPC, and reversed in DC-SIGN siRNA knock-down.
49 Figure 7
(a)
50
(b)
51 (c)
Figure 7. Effects of monosaccharides on the interaction between NPC cells and DCs. (a) Effects of mannose and fucose on DC viability by MTT assay. (b) Effects of mannose and fucose on IL-10 production of DCs. (c) IL-10 production of DCs in NPC-co-cultured condition, and effects of 20 mM monosaccharides. n=2.
52
Figure 8Figure 8. IL-10 production of DCs treated with NPC condition medium. IL-10 production of DCs was induced by NPC condition medium, but was not reversed in the addition of 1 μg/ml anti-DC-SIGN antibody. n=3.
53
Figure 9Figure 9. Ongoing experiments