探討白細胞介素IL-32於EB病毒感染B細胞中之調控機制及生理功能

Abstract

EB病毒為人類致癌皰疹病毒，能不朽化初代B細胞為不斷增生的淋巴母細胞株 (lymphoblastoid cell line，LCL)。越來越多的研究證實許多EB病毒的基因產物在使細胞轉型的過程中可誘發細胞激素的產生，而這些細胞激素為細胞增生所必需。於本實驗室先前的cDNA微陣列分析結果中，我們發現白細胞介素32 (interleukin 32，IL-32) 的表現量在EB病毒感染B細胞後有增加的現象。IL-32近年來被認為是一促炎細胞激素，因此本研究的主要目的為探討IL-32在EB病毒不朽化的LCL細胞中，其表現的位置、調控機制和生物活性為何。 首先以RT-Q-PCR證實IL-32在九株本實驗室建立的LCL細胞中皆有表現。以RT-PCR的方式偵測LCL中IL-32表現的亞型 (isoform)，結果顯示IL-32的六種剪接變異體中，IL-32β為主要表現的亞型。IL-32的蛋白質在與同一捐贈者得來的LCL與初代B細胞相比也具有高量表現。以免疫螢光染色法觀察細胞內的IL-32，顯示主要表現於LCL的細胞質中。進一步發現EB病毒的潛伏膜蛋白LMP1和溶裂極早期蛋白Rta於表皮細胞中可誘發IL-32的mRNA和蛋白質表現，而LMP1在表皮細胞和B細胞皆可誘發IL-32。以shRNA技術將LCL細胞中內生性的LMP1降解，會抑制IL-32的表現，顯示LMP1在LCL細胞中具正向調控IL-32之功能。分子機制上，LMP1透過其C端活化區域CTAR1及CTAR2以調控IL-32基因表現。在螢光酵素報導基因分析結果中則顯示活化IL-32需透過其啟動子上-8至+2的NF-κB結合位和-30至-23的CRE (cAMP response element) 位點。染色質免疫沉澱法則指出在LMP1表現的Akata細胞中，NF-κB的p65會與IL-32啟動子結合。此外，IL-32啟動子上+272至+281的Sp1結合位對於Rta活化IL-32的螢光報導質體是重要的。於表皮細胞同時表現LMP1和Rta時，IL-32的表現具有加成作用，暗示LMP1和Rta透過不同的訊息路徑誘發IL-32。 在LMP1表現的Akata細胞中以shRNA技術降解IL-32，結果發現TNFα的mRNA表現下降。另外在降解內生性IL-32的LCL細胞中，以phorbol myristate acetate (PMA) 誘發發炎細胞激素產生，結果發現IL-8、IL-1β及IL-10的mRNA表現無法被誘發。基於這些結果我們假設於LCL細胞中LMP1誘發的IL-32能增強細胞激素的產生，並能參與體外EB病毒引起的發炎反應。生理上，將IL-32降解不會影響LCL細胞的增生或凋亡。

Epstein-Barr virus (EBV) is a human oncogenic herpes virus, which has the potential to immortalize primary B cells into unlimitedly proliferating lymphoblastoid cell line (LCL). The growing body of evidence indicated that several EBV products induce cytokines during immortalization process and these cytokines are requisite for cell proliferation. In previous cDNA microarray results, we found that interleukin 32 (IL-32), a recently discovered proinflammatory cytokine, is upregulated after EBV infection. So, the main purpose of this thesis is to examine the expression pattern, regulatory mechanism and biological activities of IL-32 in EBV-immortalized LCLs. The expression of IL-32 was confirmed by RT-Q-PCR in 9 LCLs, which were established in our lab. Among the six isoforms of IL-32, IL-32β was the major isoform detected in LCLs by RT-PCR. The up-regulation of IL-32 proteins was demonstrated in LCLs, compared to their corresponding primary B cells. The location of these intracellular IL-32 was mainly in the cytoplasm of LCLs using immunofluorescence assay. Furthermore, the EBV latent membrane protein 1 (LMP1) and immediate-early protein Rta were responsible to induce IL-32 expression at both mRNA and protein levels in epithelial cells, while LMP1 was found to induce IL-32 in both epithelial and B cells. Short hairpin RNA (shRNA)-mediated depletion of endogenous LMP1 in LCLs suppressed the IL-32 expression, suggesting that LMP1 is the key factor to induce IL-32 in LCLs. Molecularly, we demonstrated that the COOH-terminal activating region (CTAR) 1 and CTAR2 of LMP1 was required to induce IL-32. The NF-κB site located at the -8 to +2 and the CRE (cAMP response element) site at the -30 to -23 of IL-32 promoter are required to activate IL-32 in the reporter assays. Furthermore, the results from ChIP assay indicated that NF-κB subunit p65 is recruited to IL-32 promoter region in LMP1-expressing Akata cells. In addition, the Sp1 site at the +272 to +281 of IL-32 promoter was essential for Rta-induced IL-32 expression. LMP1 and Rta induced IL-32 additively while co-expressed in epithelial cells, suggesting the distinct pathways to induce IL-32. In LMP1-overexpressed Akata cells, shRNA-mediated silence of endogenous IL-32 reduced TNFα mRNA expression. The mRNA expression of phorbol myristate acetate (PMA)-induced inflammatory cytokines, including IL-8, IL-10 and IL-1β were decreased in IL-32-depleted LCLs, as compared with the control cells. So, we assumed that LMP1-induced IL-32 production enhance the cytokine production in EBV-immortalized LCLs, which may contribute to the involvement of EBV-induced inflammation in vivo. Biologically, knockdown IL-32 did not influence the proliferation abilities and apoptosis of LCLs.