Effect of Dendritic Cells Allergized by K-ras Mutant Peptide on Expressions of CCL19， CCL22, and Fascin-1_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

JIANG Liangxian ¹ , TAN Guang ¹ , WANG Zhongyu ¹ , DOU Chunpeng ¹ , SHAN Lujuan ²

1.The Second Department of General Surgery, The First Affiliated Hospital of Dalian Medical University, Dalian 116011, Liaoning Province, China;;
2.Central Laboratory, The First Affiliated Hospital of Dalian Medical University, Dalian 116011, Liaoning Province, China;

Corresponding author：

Keywords：

Dendritic cell; Chemokine; Cytoskeletal protein; K-ras mutant peptide

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Objective To explore the effect of dendritic cells (DCs) allergized by K-ras mutant peptide on expressions of chemokines CCL19, CCL22, and cytoskeletal protein fascin-1.
Methods DCs were derived from peripheral blood in the presence of granuloceyte/macrophage-colony stimulating factor, interleukin (IL) -4 in vitro. The DCs were collected on day 7 after culture, and were divided into non-K-ras mutant peptide group (addition of RPMI 1604 culture solution 50 μg/ml) and K-ras mutant peptide group (addition of K-ras mutant peptide 50 μg/ml). Phenotype was identified by flow cytometry. The morphological structure was observed by scanning and transmission electron microscopies, respectively. The expressions of IL-12, CCL19, and CCL22 were tested continuously by enzyme-linked immunosorbent assay (ELISA). The expression of cytoskeletal protein fascin-1 was determined by Western blot.
Results ①The expressions of CD1a, CD80, and CD86 after loading K-ras mutant peptide were higher than that before loading K-ras mutant peptide (P lt;0.01). ②The DCs with petal-like and branch-like profections after loading were observed under scanning electron microscopy; The DCs with irregular shapes, branch-like or burr-like were showed under transmission electron microscopy. ③The expressions of IL-12, CCL19, and CCL22 in the Kras mutant peptide group were higher than those in the non-K-ras mutant peptide group at different times (6, 12, 24, and 48 h) after loading Kras mutant peptide (P lt;0.01). ④The expression of fascin-1 in the K-ras mutant peptide group was also higher than that in the non-K-ras mutant peptide group (P lt;0.01).
Conclusion K-ras mutant peptide can promote DC to mature and improve the expression of chemokines and cytoskeletal protein which will strengthen DC migration.

Citation： JIANG Liangxian ,TAN Guang,WANG Zhongyu,DOU Chunpeng,SHAN Lujuan. Effect of Dendritic Cells Allergized by K-ras Mutant Peptide on Expressions of CCL19， CCL22, and Fascin-1. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2010, 17(9): 917-921. doi: Copy

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14.	Lee SH, Lee SJ, Jung YS, et al. Blocking of p53Snail binding, promoted by oncogenic KRas, recovers p53 expression and function [J]. Neoplasia, 2009; 11(1): 2231.
15.	杨波，何杨，孙冬林，等. Kras(12Val)树突状细胞疫苗诱导抗胰腺癌免疫反应 [J]. 苏州大学学报（医学版）, 2007； 27（5）： 691700.
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17.	Biragyn A, Ruffini PA, Coscia M, et al. Chemokine receptormediated delivery directs selftumor antigen efficiently into the class Ⅱ processing pathway in vitro and induces protective immunity in vivo [J]. Blood, 2004; 104(7): 19611969.
18.	Bertho N, Adamski H, Toujas L, et al. Efficient migration of dendritic cells toward lymph node chemokines and induction of T(H)1 responses require maturation stimulus and apoptotic cell interaction [J]. Blood, 2005; 106(5): 17341741.
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20.	Brivio F, Lissoni P, Rovelli F, et al. Effects of IL2 preoperative immunotherapy on surgeryinduced changes in angiogenic regulation and its prevention of VEGF increase and IL12 decline [J]. Hepatogastroenterology, 2002; 49(44): 385387.
21.	Pietil TE, Veckman V, Lehtonen A, et al. Multiple NFkappaB and IFN regulatory factor family transcription factors regulate CCL19 gene expression in human monocytederived dendritic cells [J]. J Immunol, 2007; 178(1): 253261.
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26.	Gobert M, Treilleux I, BendrissVermare N, et al. Regulatory T cells recruited through CCL22/CCR4 are selectively activated in lymphoid infiltrates surrounding primary breast tumors and lead to an adverse clinical outcome [J]. Cancer Res, 2009; 69(5): 20002009.
27.	Mailloux AW, Young MR. NKdependent increases in CCL22 secretion selectively recruits regulatory T cells to the tumor microenvironment [J]. J Immunol, 2009; 182(5): 27532765.
28.	AlAlwan MM, Rowden G, Lee TD, et al. Fascin is involved in the antigen presentation activity of mature dendritic cells [J]. J Immunol, 2001; 166(1): 338345.
29.	Geyeregger R, Zeyda M, Bauer W, et al. Liver X receptors regulate dendritic cell phenotype and function through blocked induction of the actinbundling protein fascin [J]. Blood, 2007; 109(10): 42884295.
30.	Zanet J, Payre F, Plaza S. Fascin for cell migration in Drosophila [J]. Fly (Austin), 2009; 3(4): 281282.
31.	Hashimoto Y, Loftis DW, Adams JC. Fascin1 promoter activity is regulated by CREB and the aryl hydrocarbon receptor in human carcinoma cells [J]. PLoS One, 2009; 4(4): e5130.

1. Santegoets SJ, van den Eertwegh AJ, van de Loosdrecht AA, et al. Human dendritic cell line models for DC differentiation and clinical DC vaccination studies [J]. J Leukoc Biol, 2008; 84(6): 13641373.
2. Kim HS, Choo YS, Koo T, et al. Enhancement of antitumor immunity of dendritic cells pulsed with heattreated tumor lysate in murine pancreatic cancer [J]. Immunol Lett, 2006; 103(2): 142148.
3. Xia Y, Dai J, Lu PR, et al. Distinct effect of CD40 and TNFsignaling on the chemokine/chemokine receptor expression and function of the human monocytederived dendritic cells [J]. Cell Mole Immunol, 2008; 5(2)： 4656.
4. Ishimoto H, Yanagihara K, Araki N, et al. Singlecell observation of phagocytosis by human blood dendritic cells [J]. Jpn J Infect Dis, 2008; 61(4): 294297.
5. Koido S, Hara E, Homma S, et al. Cancer vaccine by fusions of dendritic and cancer cells [J].Clin Dev Immunol, 2009; 2009: ID657369. doi: 10.1155/2009/657369.
6. Huang CF, Monie A, Weng WH, et al. DNA vaccines for cervical cancer [J]. Am J Transl Res, 2010; 2(1): 7587.
7. 薛刚，曹永宽，王培红，等. 肿瘤细胞裂解物致敏的树突状细胞对结肠癌LoVo细胞的杀伤作用 [J]. 中国普外基础与临床杂志， 2009； 16(1)： 4851.
8. 谭广，任金帅，王忠裕，等. TNFα增强树突状细胞诱导的抗胰腺癌免疫应答的实验研究 [J]. 中国普外基础与临床杂志， 2006； 13(6)： 689692.
9. Lee DH. Dendritic cellsbased vaccine and immune monitoring for hepatocellular carcinoma [J]. Korean J Physiol Pharmacol, 2010; 14(1): 1114.
10. Srivastava PK. Therapeutic cancer vaccines [J]. Curr Opin Immunol, 2006; 18(2): 201205.
11. Kikuchi T. Genetically modified dendritic cells for therapeutic immunity [J]. Tohoku J Exp Med, 2006; 208(1): 18.
12. Recktenwald CV, Kellner R, Lichtenfels R, et al. Altered detoxification status and increased resistance to oxidative stress by Kras transformation [J]. Cancer Res, 2008; 68(24): 1008610093.
13. Shen YM, Yang XC, Yang C, et al. Enhanced therapeutic effects for human pancreatic cancer by application Kras and IGFIR antisense oligodeoxynucleotides [J]. World J Gastroenterol, 2008; 14(33): 51765185.
14. Lee SH, Lee SJ, Jung YS, et al. Blocking of p53Snail binding, promoted by oncogenic KRas, recovers p53 expression and function [J]. Neoplasia, 2009; 11(1): 2231.
15. 杨波，何杨，孙冬林，等. Kras(12Val)树突状细胞疫苗诱导抗胰腺癌免疫反应 [J]. 苏州大学学报（医学版）, 2007； 27（5）： 691700.
16. Wang YX, Gao L, Ji ZZ. Inhibitary effects of antisense oligonucleotide specific to Kras point mutation on the target gene expression in human pancreatic carcinoma cells [J]. Chin Med J (Engl), 2007; 120(16): 14481450.
17. Biragyn A, Ruffini PA, Coscia M, et al. Chemokine receptormediated delivery directs selftumor antigen efficiently into the class Ⅱ processing pathway in vitro and induces protective immunity in vivo [J]. Blood, 2004; 104(7): 19611969.
18. Bertho N, Adamski H, Toujas L, et al. Efficient migration of dendritic cells toward lymph node chemokines and induction of T(H)1 responses require maturation stimulus and apoptotic cell interaction [J]. Blood, 2005; 106(5): 17341741.
19. Trinchieri G. Interleukin12 and the regulation of innate resistance and adaptive immunity [J]. Nat Rev Immunol, 2003; 3(2): 133146.
20. Brivio F, Lissoni P, Rovelli F, et al. Effects of IL2 preoperative immunotherapy on surgeryinduced changes in angiogenic regulation and its prevention of VEGF increase and IL12 decline [J]. Hepatogastroenterology, 2002; 49(44): 385387.
21. Pietil TE, Veckman V, Lehtonen A, et al. Multiple NFkappaB and IFN regulatory factor family transcription factors regulate CCL19 gene expression in human monocytederived dendritic cells [J]. J Immunol, 2007; 178(1): 253261.
22. Nakano H, Lin KL, Yanagita M, et al. Bloodderived inflammatory dendritic cells in lymph nodes stimulate acute TH1 immune responses [J]. Nat Immunol, 2009; 10(4): 394402.
23. Britschgi MR, Link A, Lissandrin TK, et al. Dynamic modulation of CCR7 expression and function on naive T lymphocytes in vivo [J]. J Immunol, 2008; 181(11): 76817688.
24. 李立, 张升宁, 冉江华, 等. 受体来源未成熟树突状细胞诱导大鼠肝移植免疫低反应性的实验研究 [J]. 中国普外基础与临床杂志, 2009; 16(1): 3238.
25. Yamashita U, Kuroda E. Regulation of macrophagederived chemokine (MDC, CCL22) production [J]. Crit Rev Immunol, 2002; 22(2): 105114.
26. Gobert M, Treilleux I, BendrissVermare N, et al. Regulatory T cells recruited through CCL22/CCR4 are selectively activated in lymphoid infiltrates surrounding primary breast tumors and lead to an adverse clinical outcome [J]. Cancer Res, 2009; 69(5): 20002009.
27. Mailloux AW, Young MR. NKdependent increases in CCL22 secretion selectively recruits regulatory T cells to the tumor microenvironment [J]. J Immunol, 2009; 182(5): 27532765.
28. AlAlwan MM, Rowden G, Lee TD, et al. Fascin is involved in the antigen presentation activity of mature dendritic cells [J]. J Immunol, 2001; 166(1): 338345.
29. Geyeregger R, Zeyda M, Bauer W, et al. Liver X receptors regulate dendritic cell phenotype and function through blocked induction of the actinbundling protein fascin [J]. Blood, 2007; 109(10): 42884295.
30. Zanet J, Payre F, Plaza S. Fascin for cell migration in Drosophila [J]. Fly (Austin), 2009; 3(4): 281282.
31. Hashimoto Y, Loftis DW, Adams JC. Fascin1 promoter activity is regulated by CREB and the aryl hydrocarbon receptor in human carcinoma cells [J]. PLoS One, 2009; 4(4): e5130.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Effect of Dendritic Cells Allergized by K-ras Mutant Peptide on Expressions of CCL19， CCL22, and Fascin-1

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