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研究生:童建學
研究生(外文):TUNG, CHIEN-HSUEH
論文名稱:氯奎寧透過NFAT訊息抑制紅斑狼瘡病患的CD4+ T細胞表面CD154的表現之機轉
論文名稱(外文):The mechanistic role of Hydroxychloroquine on NFAT signaling in CD154 expression on CD4+ T lymphocyte of systemic lupus erythematosus
指導教授:吳淑芬吳淑芬引用關係
指導教授(外文):WU, SHU-FEN
口試委員:吳淑芬陳永恩戴建國江明格張聰賢王狀銘
口試委員(外文):WU, SHU-FEN
口試日期:2017-11-08
學位類別:博士
校院名稱:國立中正大學
系所名稱:生命科學系分子生物研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2017
畢業學年度:106
語文別:中文
論文頁數:73
中文關鍵詞:紅斑性狼瘡氯奎寧CD154
外文關鍵詞:systemic lupus erythematosushydroxychloroquineCD154
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氯奎寧(hydroxychloroquine, HCQ)是一種經常使用在治療全身性紅斑狼瘡的藥物,但是它對T淋巴細胞的作用機轉卻仍不清楚,以前的研究發現,全身性紅斑狼瘡(SLE)的病人的T淋巴細胞,其細胞膜的CD154過度表達與紅斑狼瘡疾病嚴重程度有正相關。在我們的研究中發現,用氯奎寧預先處理紅斑狼瘡病患血中T淋巴細胞,再用ionomycin或IL-15刺激,發現氯奎寧可以去抑制因ionomycin刺激所造成的T淋巴細胞表面CD154的表現,而這個抑制作用隨氯奎寧濃度增加,效果更明顯,此外我們的研究中還發現這個抑制現象是藉由抑制細胞中鈣離子的釋放,進一步減少訊息路徑下游的NFATc2的細胞核位移及NFATc1的生成,而達成減少CD154的合成。然而氯奎寧無法去抑制IL-15刺激造成的T淋巴細胞表面CD154的表現及STAT5訊息路徑,由結果可以發現使用氯奎寧處理T淋巴細胞,能夠抑制T淋巴細胞的鈣離子訊息路徑,進而抑制CD154的表現及後續免疫反應。這發現為氯奎寧治療SLE的機轉提供了進一步的了解。
Background: Over-expression of membranous CD154 in T lymphocytes has been previously found in systemic lupus erythematosus (SLE). As hydroxychloroquine (HCQ) has been frequently used in the treatment of lupus, we sought to identify the effects of HCQ on CD154 and a possibly regulatory mechanism.
Methods: CD4+ T cells were isolated from the blood of lupus patients. After stimulated with ionomycin or IL-15 and pre-treated with various concentrations of HCQ, expression of membranous CD154, NFAT and STAT5 signaling were assessed.
Results: HCQ pre-treatment had significant dose-dependent suppressive effects on membranous CD154 expression in ionomycin-activated T cells from lupus patients. Furthermore, HCQ inhibited intracellular sustained calcium storage release, and attenuated the nuclear translocation of NFATc2 and the expression of NFATc1. However, the expression of CD154 through IL-15 mediated STAT5 signaling was not inhibited by HCQ treatment.
Conclusions: HCQ inhibited NFAT signaling in activated T cells and blocked the expression of membranous CD154, but not STAT5 signaling. These findings provide one of the mechanistic insights into SLE in HCQ treatment.

目錄 I
中文摘要 1
英文摘要 2-3
緒論 3
一、 全身性紅斑性狼瘡(systemic lupus erythematosus,SLE) 4
二、 全身性紅斑性狼瘡與T淋巴球CD154的關係 7
三、 CD154(CD40 ligand) 10
四、 鈣離子訊息路徑 11
五、 活化的T細胞核因子 (nuclear factor of activated T cell, NFAT) 11
六、 IL-15訊息路徑 15
七、 可溶性CD154, soluble CD154 17
八、 氯奎寧 hydroxychloroquine 17
研究目的 19
材料與方法 21
實驗材料 21
一、 紅斑狼瘡病患 21
二、 試劑氯奎寧 21
三、 人體血液T淋巴細胞(Blood T lymphocytes)之分離 21
四、 人體血液T淋巴細胞(Blood T lymphocytes)之培養 22
五、 Jurkat 細胞株之培養 22
六、 人體血液T淋巴細胞及Jurkat 細胞株之活化 23
七、 細胞之表面抗原分析 23
八、 細胞內部的細胞激素染色法 (Intracellular Cytokine Staining) 23
九、 西方墨點法免疫印跡 (Western blotting) 24
十、 鈣離子訊息路徑分析 24
十一、 細胞生存力測定法 24
實驗方法 26
一、 實驗用人類周邊血單核細胞之分離及培養 26
二、 實驗用T淋巴細胞之分離及培養 26
三、 實驗用Jurkat細胞之培養 27
四、 周邊血單核細胞(PBMC),CD4+ T細胞和Jurkat細胞的刺激及氯奎寧hydroxychloroquine處理 28
五、 利用流式細胞儀來探討hydroxychloroquine對CD154的影響 29
六、 細胞表面的細胞標記染色法(cell marker staining) 29
七、 細胞內部的細胞激素染色法 (intracellular cytokine staining) 30
八、 細胞生存力測定法 30
九、 細胞核和細胞質蛋白萃取和免疫蛋白質分析西方墨點法 31
十、 免疫細胞化學染色及螢光顯微鏡 32
十一、 探討氯奎寧對細胞內鈣離子路徑的影響 33
十二、 統計分析 33
實驗結果 35
一、 紅斑狼瘡病人T細胞刺激後表達的細胞膜CD154表現量與臨床上年齡,臨床表現,自身抗體或抗風濕藥的相關性 35
二、 使用各種時間或濃度的離子黴素去刺激,來自狼瘡患者的T細胞比健康對照組表達更高量的CD154 36
三、 氯奎寧治療可以減少CD4+ T細胞在刺激後細胞表面CD154的表達 37
四、 氯奎寧治療對在刺激後的CD4+ T淋巴細胞細胞表面CD154表達的抑制效應,跟劑量有關係 37
五、 氯奎寧治療對CD154的效果具有特異性 38
六、 氯奎寧治療的效果不是由於氯奎寧的非特異性細胞毒性 39
七、 氯奎寧治療可以通過抑制內質網的Ca2+流出而來抑制CD154表達 40
八、 氯奎寧治療可以降低CD4+ T細胞的NFATc2核轉位 41
九、 氯奎寧治療可以降低CD4+ T細胞的NFATc1表達 43
十、 氯奎寧治療無法抑制IL-15誘導的CD154表達 44
十一、 氯奎寧治療無法抑制IL-15誘導的STAT5訊息路徑 44
結果討論 45
表一、 SLE患者的基本資料 52
圖一、刺激後的細胞膜CD154在SLE患者中過度表達,且與SLE疾病嚴重程度相關 ….… 53
圖二、使用各種時間或濃度的離子黴素去刺激,來自狼瘡患者的純化CD4+ T細胞比健康對照組表達更高量的CD154 54
圖三、氯奎寧可以下降SLE患者純化的周邊血液單核細胞刺激後增加的細胞表面CD154 55
圖四、刺激後SLE患者的純化的周邊血液單核細胞上調CD154,而氯奎寧則下調CD154,效果具有劑量依賴性 56
圖五、氯奎寧減少CD4+ T細胞的CD154表達,效果具有劑量依賴性 57
圖六、氯奎寧治療無法抑制CD4的表達 58
圖七、氯奎寧具有有限的細胞毒性效果 59
圖八、氯奎寧治療可以抑制離子黴素刺激後的細胞內鈣離子從內質網流出,效果具有劑量依賴性 60
圖九、氯奎寧治療可以減少NFATc2的核轉位 61
圖十、氯奎寧治療可以減少NFATc1的表達 62
圖十一、氯奎寧治療不能抑制通過IL-15誘導表達的CD154 63
圖十二、氯奎寧治療不能抑制通過IL-15誘導的STAT5訊息路徑 64
圖十三、氯奎寧治療抑制CD154的可能模式 65
參考文獻 66


1.Lederman S, Yellin MJ, Inghirami G, Lee JJ, Knowles DM, Chess L. Molecular interactions mediating T-B lymphocyte collaboration in human lymphoid follicles. Roles of T cell-B-cell-activating molecule (5c8 antigen) and CD40 in contact-dependent help. J Immunol. 1992;149:3817-26.
2.Armitage RJ, Fanslow WC, Strockbine L, Sato TA, Clifford KN, Macduff BM, Anderson DM, Gimpel SD, Davis-Smith T, Maliszewski CR et al. Molecular and biological characterization of a murine ligand for CD40. Nature. 1992; 357:80-2.
3.Cron RQ. CD154 transcriptional regulation in primary human CD4 T cells. Immunol Res. 2003;27:185-202.
4.Grammer AC, Slota R, Fischer R, Gur H, Girschick H, Yarboro C, Illei GG, Lipsky PE. Abnormal germinal center reactions in systemic lupus erythematosus demonstrated by blockade of CD154-CD40 interactions. J Clin Invest. 2003;112:1506-20.
5.Huang W, Sinha J, Newman J, Reddy B, Budhai L, Furie R, Vaishnaw A, Davidson A. The effect of anti-CD40 ligand antibody on B cells in human systemic lupus erythematosus. Arthritis Rheum. 2002;46:1554-62.
6.Xu H, Liu J, Cui X, Zuo Y, Zhang Z, Li Y, Tao R, Li Y, Pang J. Increased frequency of circulating follicular helper T cells in lupus patients is associated with autoantibody production in a CD40L-dependent manner. Cell Immunol. 2015;295:46-51.
7.Crow MK, Kirou KA. Regulation of CD40 ligand expression in systemic lupus erythematosus. Curr Opin Rheumatol. 2001;13:361-9.
8.Grewal IS, Flavell RA. The role of CD40 ligand in costimulation and T-cell activation. Immunol Rev. 1996;153:85-106.
9.Howard LM, Miller SD. Immunotherapy targeting the CD40/CD154 costimulatory pathway for treatment of autoimmune disease. Autoimmunity. 2004;37:411-8.
10.Kyttaris VC, Zhang Z, Kampagianni O, Tsokos GC. Calcium signaling in systemic lupus erythematosus T cells: a treatment target. Arthritis Rheum. 2011;63:2058-66.
11.Alaaeddine N, Hassan GS, Yacoub D, Mourad W. CD154: an immunoinflammatory mediator in systemic lupus erythematosus and rheumatoid arthritis. Clin Dev Immunol. 2012:490148.
12.Vakkalanka RK, Woo C, Kirou KA, Koshy M, Berger D, Crow MK. Elevated levels and functional capacity of soluble CD40 ligand in systemic lupus erythematosus sera. Arthritis Rheum. 1999;42:871-81.
13.Goules A, Tzioufas AG, Manousakis MN, Kirou KA, Crow MK, Routsias JG. Elevated levels of soluble CD40 ligand (sCD40L) in serum of patients with systemic autoimmune diseases. J Autoimmun. 2006;26:165-71.
14.Mehta J, Genin A, Brunner M, Scalzi LV, Mishra N, Beukelman T, Cron RQ. Prolonged expression of CD154 on CD4 T cells from pediatric lupus patients correlates with increased CD154 transcription, increased nuclear factor of activated T cell activity, and glomerulonephritis. Arthritis Rheum. 2010;62:2499-509.
15.Desai-Mehta A, Lu L, Ramsey-Goldman R, Datta SK. Hyperexpression of CD40 ligand by B and T cells in human lupus and its role in pathogenic autoantibody production. J Clin Invest. 1996;97:2063-73.
16.Santos-Argumedo L, Alvarez-Maya I, Romero-Ramirez H, Flores-Romo L. Enforced and prolonged CD40 ligand expression triggers autoantibody production in vivo. Eur J Immunol. 2001;31:3484-92.
17.Koshy M, Berger D, Crow MK. Increased expression of CD40 ligand on systemic lupus erythematosus lymphocytes. J Clin Invest. 1996;98:826-37.
18.Vavassori S, Covey LR. Post-transcriptional regulation in lymphocytes: the case of CD154. RNA Biol. 2009;6:259-65.
19.Kyttaris VC, Wang Y, Juang YT, Weinstein A, Tsokos GC. Increased levels of NF-ATc2 differentially regulate CD154 and IL-2 genes in T cells from patients with systemic lupus erythematosus. J Immunol. 2007;178:1960-6.
20.Vassilopoulos D, Kovacs B, Tsokos GC. TCR/CD3 complex-mediated signal transduction pathway in T cells and T cell lines from patients with systemic lupus erythematosus. J Immunol. 1995;155:2269-81.
21.Lowe RM, Genin A, Orgun N, Cron RQ. IL-15 prolongs CD154 expression on human CD4 T cells via STAT5 binding to the CD154 transcriptional promoter. Genes Immun. 2014;15:137-44.
22.Baranda L, de la Fuente H, Layseca-Espinosa E, Portales-Perez D, Nino-Moreno P, Valencia-Pacheco G, Abud-Mendoza C, Alcocer-Varela J, Gonzalez-Amaro R. IL-15 and IL-15R in leucocytes from patients with systemic lupus erythematosus. Rheumatology (Oxford). 2005;44:1507-13.
23.Aringer M, Stummvoll GH, Steiner G, Koller M, Steiner CW, Hofler E, Hiesberger H, Smolen JS, Graninger WB. Serum interleukin-15 is elevated in systemic lupus erythematosus. Rheumatology (Oxford). 2001;40:876-81.
24.Bijl M, Horst G, Limburg PC, Kallenberg CG. Expression of costimulatory molecules on peripheral blood lymphocytes of patients with systemic lupus erythematosus. Ann Rheum Dis. 2001;60:523-6.
25.Harigai M, Hara M, Fukasawa C, Nakazawa S, Kawaguchi Y, Kamatani N, Kashiwazaki S. Responsiveness of peripheral blood B cells to recombinant CD40 ligand in patients with systemic lupus erythematosus. Lupus. 1999;8:227-33.
26.Yellin MJ, Thienel U. T cells in the pathogenesis of systemic lupus erythematosus: potential roles of CD154-CD40 interactions and costimulatory molecules. Curr Rheumatol Rep. 2000;2:24-31.
27.Wang X, Huang W, Schiffer LE, Mihara M, Akkerman A, Hiromatsu K, Davidson A. Effects of anti-CD154 treatment on B cells in murine systemic lupus erythematosus. Arthritis Rheum. 2003;48:495-506.
28.Yellin MJ, D'Agati V, Parkinson G, Han AS, Szema A, Baum D, Estes D, Szabolcs M, Chess L. Immunohistologic analysis of renal CD40 and CD40L expression in lupus nephritis and other glomerulonephritides. Arthritis Rheum. 1997;40:124-34.
29.Fox R. Anti-malarial drugs: possible mechanisms of action in autoimmune disease and prospects for drug development. Lupus. 1996;5:S4-10.
30.Sailler L, Puissant B, Meliani P, Castex JO, Saivin S, Adoue D, Fournie B, Arlet P, Montastruc JL, Lapeyre-Mestre M et al. Blood concentrations of hydroxychloroquine and its desethyl derivative correlate negatively with the percentage of CD45RO+ cells among CD4+ lymphocytes in hydroxychloroquine-treated lupus patients. Ann N Y Acad Sci. 2007;1108:41-50.
31.Willis R, Seif AM, McGwin G, Jr., Martinez-Martinez LA, Gonzalez EB, Dang N, Papalardo E, Liu J, Vila LM, Reveille JD et al. Effect of hydroxychloroquine treatment on pro-inflammatory cytokines and disease activity in SLE patients: data from LUMINA (LXXV), a multiethnic US cohort. Lupus. 2012;21:830-5.
32.de Sanctis JB, Garmendia JV, Chaurio R, Zabaleta M, Rivas L. Total and biologically active CD154 in patients with SLE. Autoimmunity. 2009;42:263-5.
33.Wallace DJ, Gudsoorkar VS, Weisman MH, Venuturupalli SR. New insights into mechanisms of therapeutic effects of antimalarial agents in SLE. Nat Rev Rheumatol. 2012;8:522-33.
34.Lombard-Platlet S, Bertolino P, Deng H, Gerlier D, Rabourdin-Combe C. Inhibition by chloroquine of the class II major histocompatibility complex-restricted presentation of endogenous antigens varies according to the cellular origin of the antigen-presenting cells, the nature of the T-cell epitope, and the responding T cell. Immunology. 1993;80:566-73.
35.Sperber K, Quraishi H, Kalb TH, Panja A, Stecher V, Mayer L. Selective regulation of cytokine secretion by hydroxychloroquine: inhibition of interleukin 1 alpha (IL-1-alpha) and IL-6 in human monocytes and T cells. J Rheumatol. 1993;20:803-8.
36.Goldman FD, Gilman AL, Hollenback C, Kato RM, Premack BA, Rawlings DJ. Hydroxychloroquine inhibits calcium signals in T cells: a new mechanism to explain its immunomodulatory properties. Blood. 2000;95:3460-6.
37.Pinelli DF, Ford ML. Novel insights into anti-CD40/CD154 immunotherapy in transplant tolerance. Immunotherapy. 2015;7:399-410.
38.Liang MH, Socher SA, Larson MG, Schur PH. Reliability and validity of six systems for the clinical assessment of disease activity in systemic lupus erythematosus. Arthritis Rheum. 1989;32:1107-18.
39.Munster T, Gibbs JP, Shen D, Baethge BA, Botstein GR, Caldwell J, Dietz F, Ettlinger R, Golden HE, Lindsley H et al. Hydroxychloroquine concentration-response relationships in patients with rheumatoid arthritis. Arthritis Rheum. 2002;46:1460-9.
40.Matthies KM, Newman JL, Hodzic A, Wingett DG. Differential regulation of soluble and membrane CD40L proteins in T cells. Cell Immunol. 2006;241:47-58.
41.Roy M, Waldschmidt T, Aruffo A, Ledbetter JA, Noelle RJ. The regulation of the expression of gp39, the CD40 ligand, on normal and cloned CD4+ T cells. J Immunol. 1993;151:2497-510.
42.So JS, Kim GC, Song M, Lee CG, Park E, Kim HJ, Kim YS, Jun CD, Im SH. 6-Methoxyflavone inhibits NFAT translocation into the nucleus and suppresses T cell activation. J Immunol. 2014;193:2772-83.
43.Timmerman LA, Healy JI, Ho SN, Chen L, Goodnow CC, Crabtree GR. Redundant expression but selective utilization of nuclear factor of activated T cells family members. J Immunol. 1997;159:2735-40.
44.Zhou B, Cron RQ, Wu B, Genin A, Wang Z, Liu S, Robson P, Baldwin HS. Regulation of the murine Nfatc1 gene by NFATc2. J Biol Chem. 2002;277:10704-11.
45.Costedoat-Chalumeau N, Amoura Z, Hulot JS, Lechat P, Piette JC. Hydroxychloroquine in systemic lupus erythematosus. Lancet. 2007;369:1257-8.
46.The Canadian Hydroxychloroquine Study Group. A randomized study of the effect of withdrawing hydroxychloroquine sulfate in systemic lupus erythematosus. N Engl J Med. 1991;324:150-4.
47.Meng XW, Feller JM, Ziegler JB, Pittman SM, Ireland CM. Induction of apoptosis in peripheral blood lymphocytes following treatment in vitro with hydroxychloroquine. Arthritis Rheum. 1997;40:927-35.
48.Brandt C, Liman P, Bendfeldt H, Mueller K, Reinke P, Radbruch A, Worm M, Baumgrass R. Whole blood flow cytometric measurement of NFATc1 and IL-2 expression to analyze cyclosporine A-mediated effects in T cells. Cytometry A. 2010;77:607-13.
49.Dominguez-Gutierrez PR, Ceribelli A, Satoh M, Sobel ES, Reeves WH, Chan EK. Reduced levels of CCL2 and CXCL10 in systemic lupus erythematosus patients under treatment with prednisone, mycophenolate mofetil, or hydroxychloroquine, except in a high STAT1 subset. Arthritis Res Ther. 2014;16:R23.
50.Daikh DI, Finck BK, Linsley PS, Hollenbaugh D, Wofsy D. Long-term inhibition of murine lupus by brief simultaneous blockade of the B7/CD28 and CD40/gp39 costimulation pathways. J Immunol. 1997;159:3104-8.
51.Kalled SL, Cutler AH, Datta SK, Thomas DW. Anti-CD40 ligand antibody treatment of SNF1 mice with established nephritis: preservation of kidney function. J Immunol. 1998;160:2158-65.

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