促發炎細胞激素對軟骨細胞SW-1353的影響

The effects of pro-inflammatory cytokines on chondrocyte SW-1353 cells

陳威宇(Wei-Yu Chen)

指導教授 ： 陳孟琦

中山醫學大學/醫學院/免疫學研究所/碩士(2010年)

摘要

摘要

前言︰IL-1beta在退化性關節炎患者的軟骨組織，扮演發炎重要的角色，IL-1beta所誘發的趨化因子 (IL-8, MCP-1, IP-10) 分別能吸引並活化嗜中性白血球、單核球細胞及毒殺性T淋巴細胞，涵蓋先天及後天免疫，影響病程的進展，這些基因可能由不同的訊號傳遞路徑所調控。IL-1beta能提高細胞鈣離子的活化，鈣離子的濃度能影響訊息傳遞路徑及基因表現，也可能影響細胞的存活。IFN-gamma為活化的毒殺性T淋巴細胞所分泌，能增加抗原呈獻等後天免疫反應，但它對先天免疫反應的影響則未知。
研究目的︰(1) 利用訊號傳遞路徑抑制劑，我們將釐清IL-1beta所誘發基因表現的相關調控機制；(2) 分析鈣離子對IL-1beta所誘發的趨化因子基因表現及相關訊息傳遞路徑的影響；(3) 分析IFN-beta對IL-1beta所誘發的趨化因子基因表現及相關訊息傳遞路徑的影響。
實驗方法︰SW-1353細胞加入不同濃度的細胞激素IL-1beta或IFN-gamma，利用HO33342 (Hoechst 33342) 和PI (propidium iodide) 染劑以正立螢光顯微鏡觀察細胞存活；以RT-PCR分析基因表現；利用Fura-2AM (Fura-2 acetoxymethyl ester) 測定鈣離子濃度的變化；用ELISA來測量趨化激素的分泌；以Western blot來分析ERK、p38以及CaMK-II磷酸化程度；以核質蛋白分離以及EMSA探討轉錄因子入核情況以及與DNA probe結合的能力。
結果︰IL-1beta可誘發鈣離子的驅動，但是濃度過高則會導致細胞壞死。IL-1beta所誘發的IL-8可被ERK路徑抑制劑PD98059所抑制；IL-1beta所誘發的IP-10則可被p38及NF-kappaB路徑抑制劑SB203580及MG132所抑制。Ionomycin能增加細胞質鈣離子濃度，也改變IL-1beta對激活酶磷酸化的影響，它造成IL-1beta所誘發的ERK、CaMK-II的磷酸化更增強，相反的p38磷酸化則減弱；在轉錄因子方面，它造成IL-1beta所誘發C/EBP與DNA probe的結合更增強，而NF-kappaB入核則減少；在趨化激素表現方面，Ionomycin一方面增強IL-1beta所誘發的IL-8，另一方面卻抑制IL-1beta所誘發的IP-10基因表現以及分泌量。IFN-gamma 減少IL-1beta所誘發的IL-8，相反的它會增加IL-1beta所誘發IP-10。
結論︰IL-1beta濃度過高則會導致細胞壞死，可能和鈣離子調控失衡有關。IL-1beta所誘發的IL-8, IP-10由不同的激活酶及轉錄因子所調控，而鈣離子濃度上升時會增加細胞壞死及透過改變訊息傳遞路徑增強先天免疫反應相關基因的表現。相反的IFN-gamma則能將IL-1beta所引發的發炎基因表現轉向後天免疫反應，但其如何影響訊息傳遞路徑則有待進一步探討。

關鍵字

關節炎 ； 鈣離子 ； 趨化激素 ； 干擾素-gamma ； 白細胞介素-1beta

並列摘要

Abstract

Background: Pro-inflammatory cytokine IL-1beta plays a central role in the pathogenesis of osteoarthritis. Chemokines IL-8, MCP-1, IP-10 induced by this cytokine are repectively capable of attracting and activating neutrophils, monocytes and T cells which are involved in innate or adaptive immunity. The regulation of signal transduction for the induction of these genes is not completely clear. IL-1beta can elicit elevated intracellular [Ca2+], which may affect signal transduction pathways and downstream gene exparession. IFN-gamma, secreted by activated T cells, can increase MHC expression and antigen presentation and thus an important cytokine in adaptive immunity.
Aims of study: (1) To clarify the signal transduction pathways involved in IL-1-beta-induced chemokine genes using pathway-specific inhibitors; (2) To investigate the effects of elevated intracellular [Ca2+] on IL-1-beta-induced activation of signal transduction pathways and chemokine gene expression. (3) To study the effects of IFN-gamma on IL-1beta-induced gene expression.
Methods: SW-1353 cells were treated with different concentrations of cytokines IL-1beta or IFN-gamma.. Cell viability was analyzed by staining the cells with HO33342 (Hoechst 33342) or PI (propidium iodide), and observed with fluroscent microscopy. Gene expression was analyzed by RT-PCR. Fura-2AM staining was used for cytosolic [Ca2+] measurement. Chemokines release was measured by ELISA. Phosphrylation of ERK, p38 and CaMK-II was analyzed by Westen blot. Translocation of transcriptional factors into the nucleus and their ability to bind with the DNA probes was analyzed by EMSA using nuclear protein extracts.
Results: IL-1beta elicited elevated intracellular [Ca2+]. At concentration above 100 U/ml, the cytokine caused cell necrosis. IL-1beta-induced IL-8 was suppressed by ERK pathway inhibitor PD98069. While IL-1beta-induced IP-10 was suppressed by the p38 inhibitor (SB203580) and NF-kappaB inhibitor (MG132). Ionomycin, which increased intracellular [Ca2+], enhanced phosphorylation of ERK and CaMK-II, but suppressed that of p38 indcued by IL-1beta. Ionomycin also enhanced IL-1beta-induced C/EBP binding, but it suppressed IL-1beta-induced nuclear translocation of NF-kappaB. This drug enhanced IL-8, but suppressed IP-10 expression and protein secretion-induced by IL-1beta. IFN-gamma, however, enhanced IP-10, but suppressed IL-8 expression and protein secretion-induced by IL-1beta.
Conclusions: Cell necrosis induced by high concentration of IL-1beta is correlated with loss of calcium homeostasis caused by this cytokine. IL-1beta-induced IL-8 and IP-10 are differentially regulated by distinct signal transduction pathways. Elevated intracellular [Ca2+] increases IL-1beta-induced cell necrosis and shifts these pathways, thus favors the expression of innate immunity-related genes. In contrast, IFN-gamma alters the IL-1beta-induced gene expression pattern, which favors the progression of adaptive immunity. The mechanisms involved in the suppressive effect on IL-1beta-induced gene expression remains to be elucidated.

並列關鍵字

arthritis ； calcium ion ； chemokines ； interferon-gamma ； interleukin-1beta

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