環狀核?酸磷酸二酯?4對LPS刺激小鼠巨噬細胞產生IL-1Ra之影響; Effects of Cyclic Nucleotide Phosphodiesterase 4 on LPS-stimulated IL-1Ra Production in Mouse Macrophages

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Title:	環狀核?酸磷酸二酯?4對LPS刺激小鼠巨噬細胞產生IL-1Ra之影響;Effects of Cyclic Nucleotide Phosphodiesterase 4 on LPS-stimulated IL-1Ra Production in Mouse Macrophages
Authors:	楊景行;Jing-xing Yang
Contributors:	生命科學研究所
Keywords:	環狀核苷酸磷酸二酯酶4;白細胞介素-1受體拮抗劑;巨噬細胞;Macrophage;IL-1Ra;PDE4;LPS
Date:	2011-07-27
Issue Date:	2012-01-05 14:28:30 (UTC+8)
Abstract:	Interleukin-1 receptor antagonist (IL-1Ra)是IL-1家族的成員之一，可以與IL-1的受體結合而抑制IL-1受體的訊息傳導。文獻指出，利用LPS刺激免疫細胞TNF-?與IL-1β的產生會被IL-1Ra所抑制，故IL-1Ra被認為是一種抗發炎的細胞激素。此外，IL-1Ra在臨床用於治療許多發炎疾病，包括類風溼性關節炎。在本研究中，我們證實利用LPS刺激Raw 264.7細胞及小鼠腹腔巨噬細胞，IL-1Ra釋放量會隨著LPS刺激的時間及濃度增加而上升。Rolipram是PDE4抑制劑，可使巨噬細胞內cAMP的濃度上升，進而使LPS所誘導的IL-1Ra釋放顯著增加，EC50約為0.3~1 μM；同時，Rolipram也會增加IL-1Ra的轉錄作用。我們發現利用PKA活化劑6-Bnz-cAMP處理巨噬細胞可增加IL-1Ra的釋放，而Epac活化劑8-pCPT-2’-O-Me-cAMP則無此作用。此外，PKA抑制劑Rp-8-CPT-cAMPS也可阻斷Rolipram所增加的IL-1Ra。這些結果指出，Rolipram是經由活化cAMP/PKA訊息傳導來調節IL-1Ra的釋放。我們進一步利用PDE4剔除小鼠的腹腔巨噬細胞來分析PDE4四個亞型(PDE4A、4B、4C及4D)如何調節IL-1Ra的產生。結果顯示，LPS刺激PDE4B-/-巨噬細胞其IL-1Ra釋放量與利用LPS與Rolipram共同處理的野生型細胞相當，表示Rolipram使IL-1Ra釋放上升是由於抑制PDE4B所致。此外，利用anti-IL-1Ra抗體將巨噬細胞釋放的IL-1Ra中和做進一步分析，結果顯示LPS刺激所釋放的IL-1Ra可部分抑制TNF-a的釋放，然而Rolipram所增加的IL-1Ra卻無法抑制TNF-a釋放。綜合以上結果，我們發現抑制或剔除PDE4B可活化cAMP/PKA的訊息傳導，使LPS刺激IL-1Ra的釋放上升，然而此訊息傳導不能直接影響TNF-a的釋放。 Interleukin-1 receptor antagonist (IL-1Ra) is a member of the IL-1 cytokine family that binds to IL-1 receptors and subsequently prevents IL-1 from triggering a signal in the cell. IL-1Ra can be produced by a variety of cell types in response to inflammatory stimuli. Accumulating evidences indicates that IL-1Ra functions as an anti-inflammatory cytokine that can block LPS-induced TNF-a and IL-1β production in inflammatory cells. Moreover, the human recombinant IL-1Ra is currently used as a therapeutic agent for rheumatoid arthritis. In this study, we demonstrated that stimulation of Raw 264.7 cells and mouse peritoneal macrophages with LPS increased IL-1Ra release in a time- and dose-dependent manner. The cAMP-specific phosphodiestrase PDE4 inhibitor Rolipram, a cAMP-elevating agent, significantly enhanced the LPS-stimulated IL-1Ra release with the EC50 of approx. 0.3~1 uM. This induction of IL-1Ra by LPS and Rolipram was also observed at the transcriptional level. Moreover, the increase in the IL-1Ra release was mimicked by the treatment of the cells with the PKA activator 6-Bnz-cAMP, whereas the Epac activator 8-pCPT-2’-O-Me-cAMP did not produce the similar effect. In addition, the Rolipram-enhanced IL-1Ra production was reversed by the PKA inhibitor Rp-8-CPT-cAMPS. These results indicated that the effect of Rolipram was mediated by activation of the cAMP/PKA signal pathway. To further dissect among the four PDE4 subtypes (PDE4A, 4B, 4C, 4D) which one is responsible for the regulation of the IL-1Ra production, PDE4-deficient peritoneal macrophages were employed. The results showed that ablation of PDE4B enhanced LPS-stimulated IL-1Ra release to the level similar to that produced by the WT cells treated with both LPS and Rolipram, indicating the effect of Rolipram was mediated by inhibition of PDE4B. Additionally, using anti-IL-1Ra antibody to deplete IL-1Ra in the culture medium, our results indicated that in the presence of LPS alone the TNF-a release could be suppressed by the release of IL-1Ra in the medium. However, the Rolipram-enhanced IL-1Ra release was not responsible for the inhibition of TNF-a release caused by Rolipram. Taken together, these findings suggested that ablation or inhibition of PDE4B activates cAMP/PKA signaling and leads to upregulation of LPS-stimulated IL-1Ra production, which however does not contribute to its down-regulation of TNF-a release in mouse macrophages.
Appears in Collections:	[Graduate Institute of Life Science] Electronic Thesis & Dissertation

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