探討CCL3及CCL4於EB病毒感染B細胞中之調控機制及生理功能

EB病毒為一廣泛流行於人類間的疱疹病毒，目前已發現許多與EB病毒相關的疾病，包含鼻咽癌、何杰金氏淋巴瘤等。這些疾病都具有淋巴球浸潤以及病變細胞分泌多種細胞素的特徵，EB病毒似乎可藉由引發類似發炎反應的現象改變週遭微環境，進而幫助病毒感染細胞的存活。在以細胞素蛋白質微陣列分析後，發現許多細胞素在EB病毒感染B細胞後，表現量皆會上升，其中CCL3及CCL4兩個具有引發發炎反應及聚集白血球能力的細胞素，在病毒感染B細胞後表現量也有上升的現象。進一步以即時同步偵測定量聚合酶連鎖反應 (RT-Q-PCR)以及酶聯免疫吸附試驗 (ELISA)檢測，發現EB病毒感染B細胞3天時，CCL3及CCL4 mRNA表現量會達高峰，接著會有些微的下降，但仍會持續表現直至細胞被轉形為淋巴母細胞株 (LCL)；蛋白質部份則可觀察到兩種細胞素在感染第3天表現量達到高峰後，會維持相同的表現量直到細胞被轉形，因此推測EB病毒感染B細胞後，可藉由某種機制誘發CCL3及CCL4表現。當轉染不同EB病毒基因至鼻咽癌及巴氏淋巴瘤細胞株後，發現LMP1具有誘發CCL3及CCL4表現的能力。利用短干擾RNA (shRNA)於淋巴母細胞中降解LMP1表現時，可發現CCL3及CCL4表現量隨著LMP1表現量的下降也有下降的情形，更進一步證實EB病毒的確是透過LMP1誘發並維持細胞中CCL3及CCL4的表現。將LMP1刪除突變體轉導至B細胞後，實驗結果顯示LMP1兩大功能性區域-CTAR1及CTAR2，皆參與在LMP1誘發CCL3及CCL4的機制中，且這樣的調控從基因轉錄階段即開始。進一步以報導基因分析法 (reporter assay)檢測，可發現LMP1具有活化CCL4啟動子活性的能力。但於電泳位移分析 (EMSA)實驗結果顯示，轉錄因子與CCL4啟動子的結合，並不受LMP1影響，LMP1可能是藉由磷酸化轉錄因子活化其功能，進而啟動CCL4基因表現。於生理功能分析實驗中，當利用抗體阻擋CCL4的功能後，以流氏細胞儀及西方點墨法分析，皆可觀察到淋巴母細胞出現細胞凋亡的現象，顯示CCL4作用於細胞上，可提供細胞存活之訊息，抑制細胞凋亡。我們的實驗結果顯示，EB病毒感染B細胞後，會經由LMP1誘發CCL3及CCL4表現。透過活化CCL4啟動子活性誘發CCL4表現，分泌至細胞外的CCL4會以autocrine或paracrine的方式作用於細胞上，提供EB病毒感染B細胞存活的訊息，進而幫助EB病毒感染B細胞之存活。

關鍵字

CCL3 ； CCL4 ； EB病毒； LMP1

並列摘要

Many Epstein-Barr virus (EBV)-associated malignancies, such as Hodgkin's lymphoma, nasopharyngeal carcinoma (NPC), are characterized by intensive lymphocytes infiltration. EBV-induced chemokine secretion contributes to the recruitment of the lymphocytes. As analyzed by antibody array, we found that CCL3 and CCL4 secretion were induced in B cells after EBV infection. CCL3 and CCL4 harbored the ability to recruit leukocyte and induce inflammation. These factors may contribute to the lymphocyte infiltration of EBV-associated neoplasms, so we are interesting in investigating the role of CCL3 and CCL4 in EBV-associated diseases. When investigated by using RT-Q-PCR, transcripts of both chemokines reached the peak on day 3 post-infection, and then maintained lower expression levels until the cells transformed to lymphoblastoid cell lines (LCL). Both CCL3 and CCL4 proteins could be detected by ELISA, and stably expressed until day 28 post-infection. We revealed that EBV latent membrane protein-1 (LMP1) harbored the ability to induce CCL3 and CCL4 expression in NPC cell lines and B cells. Knockdown of LMP1 expression in LCLs, CCL3 and CCL4 levels decrease at the same time, suggesting that LMP1 can induce and maintain CCL3 and CCL4 expression in EBV-infected B cells. Deletion of LMP1 major functional domains, CTAR1 and CTAR2, abolished LMP1-mediated induction of CCL3 and CCL4, suggesting that both CTAR domains are important for inducing CCL3 and CCL4 expression. As analyzed by reporter assay, it shows that LMP1 can turn on CCL4 promoter activity. However, in the study of EMSA, it seems that LMP1 dosen’t influence on the binding of transcription factors on CCL4 promoter. We suggest that LMP1 may activate CCL4 promoter through influence on the activity of transcription factors. We also found that CCL4 is important for LCL survival. When using CCL4 neutralization antibody, it will induced LCL apoptosis. In our study, we revealed that EB virus induces CCL3 and CCL4 expression through LMP1. CCL4 harbor the ability to provided B cell survival signal and may contribute to the pathogenesis of EBV-associated lymphoproliferative disease .

並列關鍵字

CCL3 ； CCL4 ； Epstein-Barr virus ； LMP1

參考文獻

Aggarwal, B. B., Shishodia, S., Sandur, S. K., Pandey, M. K., and Sethi, G. (2006). Inflammation and cancer: how hot is the link? Biochem Pharmacol 72, 1605-1621.

Ai, M. D., Li, L. L., Zhao, X. R., Wu, Y., Gong, J. P., and Cao, Y. (2005). Regulation of survivin and CDK4 by Epstein-Barr virus encoded latent membrane protein 1 in nasopharyngeal carcinoma cell lines. Cell Res 15, 777-784.

Anderson, P. (2008). Post-transcriptional control of cytokine production. Nat Immunol 9, 353-359.

Atwater, J. A., Wisdom, R., and Verma, I. M. (1990). Regulated mRNA stability. Annu Rev Genet 24, 519-541.

Babcock, G. J., Decker, L. L., Volk, M., and Thorley-Lawson, D. A. (1998). EBV persistence in memory B cells in vivo. Immunity 9, 395-404.

國際替代計量

探討CCL3及CCL4於EB病毒感染B細胞中之調控機制及生理功能

全文下載

主題瀏覽