Taipei Medical University Institutional Repository:Item 987654321/44539

Tam metin

(1)臺北醫學大學醫學檢驗技生物技術所碩士論文 Master Thesis, School of Medical Laboratory Science and Biotechnology, Taipei Medical University. 鼻咽癌細胞透過 C 型凝集素受器影響樹突狀細胞的功能 Nasopharyngeal carcinoma cells (NPCs) interfere with dendritic cell (DC) function through the C-type Lectin Receptor, DC-SIGN. 研究生: 李丞釩 (Cheng-Fan Lee). 指導教授:. 陳建和 (Chien-Ho Chen, Ph. D). 共同指導教授: 林詠峯 (Yung-Feng Lin, Ph. D). 中華民國 100 年 7 月 1.

(2) 1.

(3) 1.

(4) 1.

(5) 謝誌碩士班就這樣倉倉促促的結束了，在這頁之後的研究雖然不是什麼偉大的研究，但卻是我在這幾年來的努力結晶，也漸漸的發現在粗淺的背後蘊藏是一步步的錯誤堆疊而成的。這一路跌跌撞撞的走，中間也發生很多事情，但是至少這段路中有很多一起奮鬥的夥伴，這本論文雖然晚了一陣子卻夾帶著許多滿滿的回憶。雖然跟陳建和老師相處時間並不長，但他給了我一樣很珍貴的東西，也是這個實驗獨特的，”希望”。陳老師除了研究上的指導外，對於我這個歪斜的學生而言，老師提供了一個尊重學生的環境，能讓我能一步一步的學習，也漸漸對原本失去的熱忱漸漸找回。另外我的共指老師林詠峯老師也給我很大的幫助，常常跟老師討論研究，腦力激當，也漸漸學會對研究的批判精神，而鄭朝文老師也給了我在思考邏輯上的訓練。另外比較特別的是黃嘯谷老師，與黃老師的討論也讓我了解對於一種對於研究上的熱忱與追求，也讓我對於研究這條路有了些展望。在我的求學過程中遇過太多人的幫助，雖然無法在這短短的篇幅中能一一贅數，但每個名字對我都是重要的一段，一開始認識沉穩及博學的林晏任，認份有夢想的王盈純和巧遇的國小同學婷妤，到天兵的林育昌，高調的游培鈞，共患難的蔡易達，以及有著共同夢想的吳浩宇，做完實驗後就像一群小學生一樣忙著打冰蜥蜴，一起去嘶吼，聊著夢想，以及躲在樓梯間打龍的那個暑假，都是滿滿的回憶，同伴很多回憶也很多，老陳家嘴巴永遠維持十點十分的娃娃，從大學時期的學伴到研究所，很直率的小白，實驗室的高輝學長，阿亮學長，以及一起耍笨的育禎，超可愛的謝醫師和我的秘密同伴寄生蟲所的家梅，要當爸爸的阿倫，很有抱負的育汝，大辣辣的小可，你們都是我路途過程中的回憶。而最重要的也特別的是醫技所的朋友，跟經紀人很像兼室友的阿賢，唱雙簧的菁鈴，佩璇，以及很努力，很認真，也真的很漂亮的黎宜寧，也是我研究所生活中，給了我最多回憶的人，也給了我很大的包容，也給我研究上很大的幫助，真的謝謝妳，你給的是我這輩子最棒的回憶。另外也感謝熱力四射，卻又很容易緊張得宴如，很搞笑且會笑到快斷氣的方瑜，很開心他們陪伴我得過程，以及種種的點點滴滴。以及 B 型大正妹楊寶琳，她讓我了解其實 B 型的人其實真的很不錯，以及我分不太清楚的大包和辛苦的苓佑學姐，以及令我又愛又恨的蕙瑩老大，讓我了解當我失業我可以去當按摩師傅，而且你真的很特別可能是水瓶座的關係。另外和我同時期”退伍”的同伴，很認真的偉廷，很有想法的琳雅，很賢慧的佩慈，這些同伴經歷了真的很多事情，簡單的幾句我想也無法形容這段時間的喜怒哀樂。但是只有我們最知道吧。最後再篇幅有限的空間裡，我想把最後的這一段獻給我實驗室的各位，大學部的蘿蔔頭，認真又負責的會長昌佑，可愛又有上進心的雅羽，聰明又努力的懿玟，自認聰明又愛記仇天蠍座的慧姐，都讓阿和的實驗室充滿著歡笑。當初對我很有幫助的助理子芸，以及我想這是我一輩子敬佩的仕翰學長， Qoo，我從你們身上學到好多好多，每每想到都讓我好感動。另外最感謝的是豪. 2.

(6) 哥，一起熬夜做實驗，一起努力奮鬥到天明，今天能做出這個實驗，真的謝謝豪哥大力的幫助，如果沒有他我想我會又要多待一陣子，在研究的生涯中也許我將寫下一個句點，是一個故事的結束，也是另一個故事的開始，當我踏進北醫後我的故事開始了，而現在也到了尾聲，雖然這個故事不夠精采卻句句深刻，雖然這個故事不夠刺激但卻平淡幸福，學到的很多但體認到的更多，這篇謝誌也只是代表我在這段時間證明我來過也證明我走過，這將事一段記憶的碎片，卻也歷歷在目，將來的路還很長，至少這段路我遇到很棒的人，謝謝你們。. 謹致於台北醫學大學台北醫學大學醫學檢驗技生物技術所一百年七月. 3.

(7) Contents. Page. 中文摘要………….……………………………………………7 Abstract………….……………..……….……………………..8 Abbreviation………………..……….…..……………………10 1. Introduction……………………………..……..…………..11 1.1 Dendritic cells………………………...…………………………11 1.2 DC-SIGN protein……….……………………………………....13 1.3 DC-SIGN pathway……………………………………………...15 1.4 Interaction between cancers and DC……………………………16 1.5 Nasopharyngeal carcinoma……………………………………..17. 2. Material and Methods………………………………….....19 2.1 Chemicals and reagents…………………………………………...19 2.2 Buffers and medium………………………………………………20 2.3 Antibodies………………..……………………………………….21 2.4 Cell culture………………………………………………………..22 2.3.1 Preparation of immature MDDCs………………………….....22 2.3.2 Preparation of Nasopharyngeal carcinoma cells...………..…..22 2.5 Enzyme-linked immunosorbent assay (ELISA)..…………….. …23 2.5.1 Detection of cytokine production……………………………..23 2.5.2 Cytokine production in blockage of DC-SIGN under co-cultured conditions………………………………..……………………23 2.5.3 Treatment with monosaccharides under co-cultured conditions……………..…………….……….……………..…24. 4.

(8) 2.6 Flow cytometry……………………………………………………24 2.6.1 Expression of DC-SIGN ligands on NPC cell membranes...…24 2.6.2 Identification of maturation markers of MDDCs..…….……...25 2.7 Immunofluorescence staining………………….…………………..25 2.8 RNA interference…………………………………………………..26 2.9 MTT assay…………………………………………………………26 2.10 Western blotting…………………………………………………..27 2.11 Statistics…………………………………………………………..27. 3. Results……………………………………………………...28 3.1 The cytokine production and maturation of MDDCs co-cultured with NPC cells…………………………………………………........…..28 3.2 NPC cells expressed DC-SIGN-recognized ligands……………….29 3.3 The membrane components on NPC cells affected DCs….……….29 3.4 DC-SIGN functions under co-culture conditions……………….....30 3.5 NPC cells affect DCs through other means than cell-cell contact...30. 4. Discussion………………………………………………….32 5. References……………………………………………..…...37 6. Figures.….……..………………………………….……..…40 Fig. 1…………………………………………………..…………….40 Fig. 2………...………………………………………..……………..42 Fig. 3 ..…….………………………………...……………..………..43 Fig. 4…………………………………………...………..…………..45 Fig. 5 ……………………..……………………………..…………..46 Fig. 6…………………………………………………….…………..48. 5.

(9) Fig. 7 …………………………………………………..……..……..49 Fig. 8………………………………………………..……………….52 Fig. 9...………………………………………………………………53. 6.

(10) 中文摘要. 鼻咽癌細胞是一種盛行於東南亞的惡性上皮細胞癌。目前已知在癌症的形成過程中，對免疫系統的躲避作用是一個重要的關鍵，然而鼻咽癌細胞是透過何種方式來躲避免疫系統監控目前尚未清楚。在人類免疫系統裡擔任重要抗原呈現角色的樹突狀細胞上有一種 C-type lectin 受體，樹突狀細胞特異性細胞間黏附分子-3-辨認非整合素 (DC-SIGN)，它會辨認一些特殊的醣類結構並與其結合，然後誘發抑制型的免疫反應。研究指出癌細胞會表現許多不正常醣化蛋白，而這些醣化蛋白與免疫抑制有關。於是我們先用流式細胞儀以及螢光染色法偵測到鼻咽癌細胞上確實有 DC-SIGN 可辨認的配體（ligand）;接下來再以細胞共同培養的方式，並用酵素免疫吸附法(ELISA）去檢測，也發現鼻咽癌細胞會刺激樹突狀細胞分泌免疫抑制的細胞激素，如介白素 10 （IL-10）;我們更進一步利用 DC-SIGN 抗體和 siRNA 去拮抗 DC-SIGN 的作用，發現 IL-10 的量明顯減少;用高濃度醣類作競爭抑制實驗，也發現在高濃度的右旋甘露醣 (D-mannose) 可以抑制樹突狀細胞分泌 IL-10。以上結果顯示，鼻咽癌細胞上表現 DC-SIGN 可辨認的醣基 ligand，並透過樹突狀細胞上的 DC-SIGN 刺激其分泌免疫抑制性的 IL-10，進而躲避免疫系統的監控。. 7.

(11) Abstract Nasopharyngeal carcinoma (NPC) is a malignant epithelial cell-derived tumor that is predominately found among populations in Southeast Asia, including Taiwan. The interaction that exists between tumors and the immune system is important in tumor progression. Because the mechanisms underlying NPC’s ability to escape the surveillance of the immune system are still unclear, our study sought to investigate the interaction between one type of antigen presenting cells, known as Dendritic cells (DCs), and NPC. DCs express a C-type lectin receptor, DC-pecific ICAM-3-Grabbing Nonintegrin (DC-SIGN) that can induce suppressive immune responses by recognizing carbohydrate structure. Recent studies indicated that cancer cells express many abnormal glycosylated proteins, and these glycoproteins are involved in the immune suppressive responses. Therefore we used flow cytometry and immunostaining, and found that DC-SIGN-recognized ligands were expressed on NPC cells. Using enzyme-linked immunosorbent assay (ELISA), we demonstrated that NPC cells induced immunosuppressive interleukin (IL)-10 secretion from DC. We. 8.

(12) further showed that IL-10 secretion was inhibited by DC-SIGN antibody and siRNA; high concentration of D-mannose also decreased IL-10 secretion under co-cultured conditions. In conclusion, NPC cells express DC-SIGN ligands, and stimulate immunosuppressive IL-10 secretion from DC through DC-SIGN activation, resulting in the escape from surveillance of the immune system.. 9.

(13) Abbreviation. DC: Dendritic cell IL-: interleukinNPC: nasopharyngeal carcinoma MDDC: monocyte-derived dendritic cell DC-SIGN: Dendritic cell-specific intracellular adhesion molecules (ICAM)-3 grabbing non-integrin LPS: lipopolysaccharide CLR: C-type lectin receptor TLR: Toll-like receptor. 10.

(14) 1. Introduction. 1.1 Dendritic cells Dendritic cells (DCs) are potential antigen-presenting cells (APCs), which recognize pathogen and mediate adaptive immune response [1, 2]. There are different DC subsets resident in circulating blood, lymph node and some tissues including myeloid DC, plasmacytoid DC and langerhans cell. Generally speaking myeloid DC and plasmacytoid DC are considered that they could polarize naïve T cells toward Th1 or Th2. The polarization results of Th1/Th2 play an important role on the way to specify immune response, especially in the anti-tumor immune response. DCs recognize pathogen-associated molecular patterns (PAMPs) by specific pattern-recognition receptors (PRRs). DCs are specialized APCs that recognize, acquire, process, and present antigens to naive resting T cells for the induction of an Ag- specific immune response [3-5]. There are two receptor families on DC involved in pathogen recognition.. Toll-like. receptors. 11. (TLRs). recognize.

(15) pathogen-associated molecular patterns, while C-type lectin receptors (CLRs) recognize glycans of antigen [6]. TLRs signal via conserved. pathways. to. induce. the. activation. of. several. transcription factors, of which nuclear factor-kB (NF-kB) appears pivotal for DC maturation as well as cytokine production [7]. Immature monocyte- derived dendritic cells (MoDCs) strongly express DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a kind of CLRs [8, 9]. DC-SIGN binding to ICAM-3 is calcium dependent, and DC-SIGN carbohydrate recognition domain (CRD) binds two Ca2+ ions, one essential for the tertiary structure and the other for coordinating ligand binding [10]. DC functions were inhibited by cancer cells, and make cancer cells to escape from immunoserveillance. The major mechanisms of making inactive DCs include inhibiting their maturation, blocking their migration, and impairing their ability of antigen presenting [11].. 12.

(16) [10]. 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. 13.

(17) 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]. 14.

(18) 1.3 DC-SIGN pathway DCs 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. Mannosylated. the. induction. of. lipoarabinomannan. endotoxin (ManLAM). tolerance induces. [17]. 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.. 15.

(19) [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. 16.

(20) 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%),. 17.

(21) 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.. 18.

(22) 2. Materials and methods. 2.1 Chemicals and Reagents： Acrylamide buffer was from Bioreagent. Albumin, bovine serum (BSA),. ammonium. ethylenediaminetetraacetic. peroxodisulfate acid. (EDTA),. (APS),. 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. 19.

(23) 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). 20.

(24) 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. anti-phosphorylation-Raf-1. at. serine. (USA). 338,. Anti-Raf-1, and. anti-. phosphorylation-p65 were purchased from Cell Signaling (USA). Anti-p65 was purchased from Millipore (USA). In this study, Anti-. 21.

(25) 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. 22.

(26) 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. 23.

(27) 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 DCs at 5x104 per well were seeded on a 24 well-plate containing 500 μl of RPMI medium over-night. DCs were pre-treated with 20 mM mannose, fucose, and galactose for one hour. Then, the DCs and 5x105 NPC cells were co-cultured and incubated for 24 hours. After incubation, the supernatant was 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. 24.

(28) 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.. 25.

(29) 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. 26.

(30) (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.. 27.

(31) 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.. 28.

(32) 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. 29.

(33) 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 cells and DCs in the presence of DC-SIGN siRNA, and detected IL-10 production. The NPC-induced IL-10 production of DCs was decreased. (Fig.. 6).. It. was. known. that. DC-SIGN. binds. carbohydrates on antigens [15]. We then used high doses of monosaccharides to compete with ligands on NPC on binding DC-SIGN. The data showed that 20 mM mannose inhibited IL-10 production of DCs in co-cultured condition, while fucose and galactose did not (Fig. 7). DC-SIGN on DCs recognizes mannose-containing ligands on NPC, and induces IL-10 production in DCs.. 3.5 NPC cells affect DCs through other means than cell-cell. 30.

(34) contacts We 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.. 31.

(35) 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. 32.

(36) 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.. 33.

(37) 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. 34.

(38) [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. 35.

(39) independent. In the future we would also like to determine the correlations of EBV-derived proteins in NPC and immune responses.. 36.

(40) 5. Reference. 1.. Dominguez PM, Ardavin C. Differentiation and function of mouse monocyte-derived dendritic cells in steady state and inflammation. Immunol. 2.. Rev 2010,234:90-104. Shortman K, Naik SH. Steady-state and inflammatory dendritic-cell. 3.. development. Nat Rev Immunol 2007,7:19-30. Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, et al.. 4.. Immunobiology of dendritic cells. Annu Rev Immunol 2000,18:767-811. Lanzavecchia A, Sallusto F. Regulation of T cell immunity by dendritic cells.. 5.. Cell 2001,106:263-266. 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.. 6.. 7. 8.. 9.. Brain Res Bull 2003,59:421-427. 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. 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. 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. 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. 10.. 2000,100:575-585. den Dunnen J, Gringhuis SI, Geijtenbeek TB. Dusting the sugar fingerprint:. 11.. C-type lectin signaling in adaptive immunity. Immunol Lett 2010,128:12-16. Vicari AP, Caux C, Trinchieri G. Tumour escape from immune surveillance. 12.. through dendritic cell inactivation. Semin Cancer Biol 2002,12:33-42. 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.. 37.

(41) 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. 14.. DC-SIGN. J Exp Med 2004,200:979-990. Stambach NS, Taylor ME. Characterization of carbohydrate recognition by. 15.. langerin, a C-type lectin of Langerhans cells. Glycobiology 2003,13:401-410. 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. 16.. immunity. Cell Signal 2010,22:1397-1405. Kwon DS, Gregorio G, Bitton N, Hendrickson WA, Littman DR. DC-SIGN-mediated internalization of HIV is required for trans-enhancement. 17. 18.. 19.. 20.. of T cell infection. Immunity 2002,16:135-144. Grutz G. New insights into the molecular. mechanism. of. interleukin-10-mediated immunosuppression. J Leukoc Biol 2005,77:3-15. Gringhuis SI, den Dunnen J, Litjens M, van der Vlist M, Geijtenbeek TB. Carbohydrate-specific signaling through the DC-SIGN signalosome tailors immunity to Mycobacterium tuberculosis, HIV-1 and Helicobacter pylori. Nat Immunol 2009,10:1081-1088. Aarnoudse CA, Garcia Vallejo JJ, Saeland E, van Kooyk Y. Recognition of tumor glycans by antigen-presenting cells. Curr Opin Immunol 2006,18:105-111. Royle L, Mattu TS, Hart E, Langridge JI, Merry AH, Murphy N, et al. An analytical and structural database provides a strategy for sequencing O-glycans from microgram quantities of glycoproteins. Anal Biochem. 21.. 2002,304:70-90. Cho WC. Nasopharyngeal carcinoma: molecular biomarker discovery and. 22.. progress. Mol Cancer 2007,6:1. Chen CJ, You SL, Lin LH, Hsu WL, Yang YW. Cancer epidemiology and. 23.. control in Taiwan: a brief review. Jpn J Clin Oncol 2002,32 Suppl:S66-81. Sun LM, Epplein M, Li CI, Vaughan TL, Weiss NS. Trends in the incidence rates of nasopharyngeal carcinoma among Chinese Americans living in Los Angeles County and the San Francisco metropolitan area, 1992-2002. Am J. 24.. Epidemiol 2005,162:1174-1178. Miller G, Niederman JC, Stitt DA. Infectious mononucleosis: appearance of neutralizing antibody to Epstein-Barr virus measured by inhibition of. 25.. formation of lymphoblastoid cell lines. J Infect Dis 1972,125:403-406. Demachi-Okamura A, Ito Y, Akatsuka Y, Tsujimura K, Morishima Y, Takahashi T, et al. Epstein-Barr virus (EBV) latent membrane. 38.

(42) protein-1-specific cytotoxic T lymphocytes targeting EBV-carrying natural 26.. 27.. 28.. killer cell malignancies. Eur J Immunol 2006,36:593-602. Hsu SC, Chen CH, Tsai SH, Kawasaki H, Hung CH, Chu YT, et al. Functional interaction of common allergens and a C-type lectin receptor, dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN), on human dendritic cells. J Biol Chem 2010,285:7903-7910. Gringhuis SI, den Dunnen J, Litjens M, van Het Hof B, van Kooyk Y, Geijtenbeek TB. C-type lectin DC-SIGN modulates Toll-like receptor signaling via Raf-1 kinase-dependent acetylation of transcription factor NF-kappaB. Immunity 2007,26:605-616. Geijtenbeek TB, Van Vliet SJ, Koppel EA, Sanchez-Hernandez M, Vandenbroucke-Grauls CM, Appelmelk B, et al. Mycobacteria target. 29.. DC-SIGN to suppress dendritic cell function. J Exp Med 2003,197:7-17. Kuo P-L, Hung J-Y, Huang S-K, Chou S-H, Cheng D-E, Jong Y-J, et al. Lung Cancer-Derived Galectin-1 Mediates Dendritic Cell Anergy through Inhibitor of DNA Binding 3/IL-10 Signaling Pathway. The Journal of Immunology. 30.. 2011,186:1521-1530. Raetz CRH, Whitfield C. LIPOPOLYSACCHARIDE ENDOTOXINS. Annual. 31.. Review of Biochemistry 2002,71:635-700. Fiorentino DF, Bond MW, Mosmann TR. Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones. J. 32.. Exp Med 1989,170:2081-2095. Heidt S, Segundo DS, Chadha R, Wood KJ. The impact of Th17 cells on transplant rejection and the induction of tolerance. Curr Opin Organ. 33.. Transplant 2010,15:456-461. Chang J, Kunkel SL, Chang CH. Negative regulation of MyD88-dependent signaling by IL-10 in dendritic cells. Proc Natl Acad Sci U S A 2009,106:18327-18332.. 39.

(43) 6. Figures Figure 1 (a). (b). 40.

(44) 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.. 41.

(45) 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.. 42.

(46) Figure 3 (a). 43.

(47) (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.. 44.

(48) 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.. 45.

(49) Figure 5. (a). 46.

(50) (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.. 47.

(51) Figure 6. Figure 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.. 48.

(52) Figure 7 (a). 49.

(53) (b). 50.

(54) (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.. 51.

(55) Figure 8. Figure 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.. 52.

(56) Figure 9. Figure 9. Ongoing experiments. 53.

(57)