行政院國家科學委員會專題研究計畫 成果報告
Toll-like 接受體-9 及其受器在多發性骨髓瘤的生物效應及
可能的臨床應用
計畫類別： 個別型計畫 計畫編號： NSC93-2314-B-002-087- 執行期間： 93 年 08 月 01 日至 94 年 07 月 31 日 執行單位： 國立臺灣大學醫學院內科 計畫主持人： 黃聖懿 共同主持人： 許世明，姚明，田蕙芬 報告類型： 精簡報告 處理方式： 本計畫可公開查詢中 華 民 國 94 年 11 月 14 日
9 3 年 度 行 政 院 國 家 科 學 委 員 會 專 題 研 究 計 畫 報 告 書
一、基本資料 計畫編號：NSC93--2314-B-002-087 計畫種類 一般型研究計畫，A類(研究主持費及執行計畫所需經費) 主持人 單位:台大醫院內科部 姓名:黃聖懿: 職稱:主治醫師 協同主持 單位:台大醫院病理部 姓名: 許世明 職稱:教授 指導教授 單位:台大醫院內科部 姓名:田蕙芬 職稱:教授 計畫 中文 Toll-like 接受體-9 及其受器在多發性骨髓瘤的生物效應及可能的臨 床應用名稱 英文 Biological and clinical implication of toll-like receptor 9 (TLR-9) and its ligand in multiple myeloma
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計畫成果(限四頁以內)
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I. 簡介（Introduction）與背景說明
Multiple myeloma (MM) is a clonal plasma cell neoplasm characterized by proliferation of abnormal plasma cells in bone marrow (BM) that secrete a monoclonal paraprotein (M-protein) in serum and/or urine, and by osteolytic bone destructions. So far, it is an incurable disease and its pathogenesis is largely unknown. The median survival time of MM patients is only three to four years, and then the patients will die on either the disease or its complications. Bacterial infection, usually occurred in the late stage of MM, is the leading reason for mortality. There have been several independent prognostic factors for MM, and one of it is the extent of angiogenesis in BM [1]. Myeloma cells have been reported to be with pro-angiogenic activity [2], but the involved molecules and regulatory mechanisms were still unclear.
The mammalian toll-like receptors (TLRs), originally described for their homology with the Drosophila toll [3], are a family of transmembrane receptors containing extracellular leucine-rich regions, which recognize various microbial components engaging a signaling cascade that results in the response versus such microbes. Exposure to microbial products and cytokines regulate TLRs expression with considerable species-related differences [4]. In human and other mammalians, of the 10 TLRs described to date, different ligands of 9 of the 10 TLRs were identified. Members of the TLR family are differentially expressed on hematopoietic and nonhematopoietic cells [5]. In general, mononuclear phagocytes and dendritic
cells express the widest TLR repertoire [5]. TLRs respond to bacteria and bacterial products by transmitting a ligand induced trans-membrane signal that induces the expression of cytokines such as TNFα, IL-1, IL-6, and IL-12 that are important in the host response to infection [6]. Unmethylated CpG motifs (non-methylated C-G dinucleotides flanked by two 5’purines and three 3’pyrimidines), characteristic of bacterial DNA, are detected by TLR9. Many recent studies have focused on CpG motif and its role as stimulators of immune cells [7] and several studies have suggested a role for TLRs in the stimulatory effects induced by microbial products on human B lymphocytes [8], however, the expression of TLRs in B cells has not been systematically investigated until very recently, a distinct TLR mRNA expression profile, which includes particularly high levels of TLR9 and TLR10, was found on normal and malignant B cells, including MM [9]. Engagement of the B cell receptor or the costimulatory molecule CD40, augmented the TLR9 transcript in resting B cells. Augmented TLR9 mRNA expression was associated with increased responsiveness to its agonist, CpG motif, enhancing proliferation and chemokine production of the immune cells. The regulated expression of selected TLRs in B cells may play an important role in linking innate to adaptive immune responses [8,9]. Therefore, the TLR family of receptors thus appears to provide a critical interface between higher organisms and microorganisms, whereby microorganisms, or distinct components thereof, are recognized, signal cellular activation, and induce a host response against these organisms.
TLR-dependent signaling pathways lead to the activation of the IκB/NF-κB pathway, inducing the expression of genes that participate in innate immune responses, including many inflammatory cytokines and antimicrobial peptides [10]. Very interestingly, an angiogenic switch in macrophages involving synergy between TLR9 and adenosine A2Areceptors (A2AR) were found [6], which hinted that TLR9
might play some role in angiogenesis with coexistence of other molecules. In the absence of adenosine or A2AR agonists, unmethylated CpG motif (TLR9 agonist)
strongly up-regulate TNFαexpression, with no effect on vascular endothelial growth factor (VEGF). On the contrary, in the presence of adenosine or A2AR agonists,
TLR9 agonist strongly up-regulate VEGF expression, while simultaneously down-regulating TNFα[6]. For TLR9 was possibly expressed by myeloma cells (MCs) [9], whether the angiogenic switch involving synergy between TLR9 and A2AR also
occurred in MCs and resulted in the pro-angiogenic activity of MCs is warranted further studies.
By a new xenotransplant animal model we have established for human multiple myeloma [11], we would like to study on the biological effects of TLR9 and other co-receptors, like A2AR, in MM.
II.關鍵材料及方法 (Subjects and Methods)
I. Immunostaining of TLR-9 on MM plasma cells
我們首先嘗試用免疫化學染色法來檢視是否病人的漿細胞和骨髓瘤細胞株的 漿細胞上有無 TLR-9 的表現。由於 TLR-9 主要存在細胞質中，因此要做染色 前必須在細胞表面上打洞，染色用的單株抗體才得以進入細胞質中。此種免疫 化學染色法的作法可見我們先前的文獻發表[12]。我們收集到 10 例新診斷為 MM 的病案，取其骨髓檢體加以 Ficoll-Paque 溶液分離取得 mononuclear cells (MNCs) [11]一部分 MNCs 則利用磁珠吸附的原理(magnetic cell sorting)來純化
其中的 myeloma cells (MCs)。一部分純化的 MCs 作成短期細胞培養，並做染 色 TLR-9 用。
II Real-time PCR of expression of TLR-9 gene from clinical samples of MM patients 另一部分之MCs則直接抽取RNA用real-time PCR的方式半定量其TLR-9的基因表 現[9]。TLR9基因表現的狀況便會和病人臨床表現作比較，看臨床上是否有何特 徵。 III.重要之結果 (Results) 1. 我們確定TLR-9幾乎表現在我們所有骨髓瘤病人和骨髓瘤細胞株的漿細胞細 胞質中(除了細胞株RPMI-8226以外)(如圖一所示)。 2. real-time PCR的方式半定量這些檢體其TLR-9的基因表現則互有差異。 3. TLR-9的基因表現的多少與臨床上的特徵或是預後暫無關聯性。 VI.討論 (Discussion)與成果之貢獻 1. 我們確定TLR-9可以表現在癌化的漿細胞上。 2. TLR-9的生物意義仍需更多的檢體和用其ligand刺激才能得知。 圖一 漿細胞型態，病人檢體，劉氏染色 漿細胞，病人檢體，免疫化學染色 (negative control)
漿細胞，病人檢體，免疫化學染色 (positive for TLR-9 staining)
漿細胞型態，細胞株IM-9，劉氏染色
漿細胞，細胞株IM-9，免疫化學染色 (negative control)
漿細胞，細胞株IM-9，免疫化學染色 (positive for TLR-9 staining)
參考文獻 (References)
1. Anderson KC. Advances in the biology of multiple myeloma: therapeutic applications. Seminars in Oncology, 1999. 26:10~22.
2. Giuliani N, Colla S, Lazzaretti M, Sala R, Roti G, Mancini C, Bonomini S, Lunghi P, Hojden M, Genestreti G, Svaldi M, Coser P, Fattori PP, Sammarelli G, Gazzola GC, Bataille R, Almici C, Caramatti C, Mangoni L, Rizzoli V: Pro-angiogenetic properties of human myeloma cells: production of angiopoietin-1 and its potential relationship with myeloma-induced angiogenesis. Blood 2003, 102:638-645. 3. Medzhitov, R., Preston-Hurlburt, P. & Janeway, C.A. Jr. (1997) A human
homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature, 388, 394-397.
4. Rehli, M. (2002) Of mice and men: species variations of Toll-like receptor expression. Trends in Immunology, 23, 375-378.
5. Zarember, K.A. & Godowski, P.J. (2002) Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. Journal of Immunology, 168, 554-561.
6. Pinhal-Enfield, G., Ramanathan, M., Hasko, G., Vogel, S.N., Salman, A.L., Boons, G..J. & Leibovich, S.J. (2003) An angiogenic switch in macrophages involving synergy between toll-like receptors 2, 4, 7, and 9 and adenosine A2AReceptors.
American Journal of Pathology, 163, 711-721.
7. Leadbetter, E.A., Rifkin, I.R., Hohlbaum, A.M., Beaudette, B.C., Shlomchik, M.J. & Marshak-Rothstein, A. (2002) Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors. Nature, 416, 603-607.
8. Hartmann, G.. & Krieg, A.M. (2000) Mechanism and function of a newly
identified CpG DNA motif in human primary B cells. Journal of Immunology, 164, 944-953.
9. Emer, B., Bosisio, D., Golay, J., Polentarutti, N. & Mantovani, A. (2003) The toll-like receptor repertoire of human B lymphocytes: inducible and selective
expression of TLR9 and TLR10 in normal and transformed cells. Blood, 102, 956-963.
10. Akira, S., Takeda, K. & Kaisho, T. (2001) Toll-like receptors: critical proteins linking innate and acquired immunity. Nature Immunology, 2, 675-680.
11. Huang, S.Y., Tien, H.F, Su, F.S. & Hsu, S.M. (2003) Non-irradiated NOD/SCID-human chimeric animal model for primary NOD/SCID-human multiple myeloma- a potential in vivo culture system. American Journal of Pathology. (in press)
12. Huang, S.Y., Tang, J.L., Jou, S.T., Tsay, W., Hu, C.H., Lin, D.T., Lin, K.S., Lin, K.S., Wang, C.H., Chen, Y.C., Shen, M.C. & Tien, H.F. (1999) Minimally
differentiated acute myeloid leukemia in Taiwan: predominantly occurs in children less than 3 years and adults between 51 and 70 years. Leukemia, 13, 1506-1512. 13. Chen, J.W., et al. (1996) Non-radioisotopic differential display method to directly
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