環狀核苷酸磷酸二酯酶4與cAMP訊息傳導在小鼠巨噬細胞中對於CCL3生成之調控 ;Regulation of CCL3 production by phosphodiesterase 4 and cAMP signaling in mouse macrophages

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題名:	環狀核苷酸磷酸二酯酶4與cAMP訊息傳導在小鼠巨噬細胞中對於CCL3生成之調控;Regulation of CCL3 production by phosphodiesterase 4 and cAMP signaling in mouse macrophages
作者:	賴秋蓉;Lai,Ciou-rong
貢獻者:	生命科學系
關鍵詞:	CC趨化激素配體3;環磷酸腺苷;環狀核苷酸磷酸二酯酶4;巨噬細胞;T細胞;趨化作用;cAMP;CCL3;Chemotaxis;Macrophage;PDE4;T cell
日期:	2012-10-22
上傳時間:	2012-11-12 14:27:25 (UTC+8)
出版者:	國立中央大學
摘要:	CCL3又稱為MIP-1α，是CC趨化激素家族的一員。它可由巨噬細胞、淋巴細胞、嗜中性白血球與樹突細胞生成及釋放，並藉由其趨化與促發炎特性來調控發炎反應。然而，目前對CCL3生成與作用的調控機制尚不甚明瞭。文獻報導已顯示，在巨噬細胞內增加cAMP濃度可抑制LPS對CCL3的釋放。已知PDE4在許多發炎細胞中藉著水解cAMP來調控cAMP的濃度，而抑制PDE4活性有減緩發炎反應的功效。因此本研究的主要目的是探討PDE4是否藉由影響cAMP的訊息傳導路徑來參與調控CCL3的生成。實驗結果顯示，LPS處理RAW 264.7與小鼠腹腔巨噬細胞可刺激CCL3的釋放，且隨著時間與LPS濃度增加而上升。PDE4抑制劑rolipram則可有效地抑制此LPS刺激所產生之CCL3 mRNA的表現與蛋白質的釋放，其IC50約為0.1 μM。再者，單獨處理dibutyryl-cAMP或PKA活化劑6-Bnz-cAMP也會抑制CCL3的釋放，而Epac活化劑8-pCPT-2’-O-Me-cAMP則無影響，同時，PKA抑制劑Rp-8-CPT-cAMPS會部分回復rolipram對CCL3的抑制作用。由此我們推論， PDE4抑制劑對CCL3釋放的抑制是藉由增加cAMP進而活化PKA所致。本研究以LPS刺激PDE4基因剔除巨噬細胞，進一步證實，剔除PDE4B而非PDE4A或PDE4D可顯著降低CCL3的釋放，而此下降程度與野生型巨噬細胞處理rolipram時的抑制作用相當。此結果證明rolipram對CCL3的抑制是由於抑制了PDE4B的活性所致。此外，CCL3對小鼠脾臟T細胞的趨化作用也會被rolipram所抑制。綜合本研究的結果證明，在巨噬細胞內PDE4B可調控cAMP的訊息傳導進而影響LPS/TLR4/CCL3的免疫反應。CCL3, also known as macrophage inflammatory protein 1 alpha (MIP-1α), is a member of the C-C chemokine family. It is produced by macrophages, lymphocytes, neutrophils, and dendritic cells, and exerts chemotactic and proinflammatory effects. The mechanism underlying its production and action, however, is largely unknown. It has been shown that increasing intracellular cAMP concentration in macrophage suppresses the LPS-induced CCL3 secretion. Type 4 cAMP-specific phosphodisterases (PDE4s) are known to regulate cAMP levels in most inflammatory cells by hydrolyzing cAMP and, thereby inhibition of PDE4 activity can increase cAMP concentrations in these cells. In this study, we aimed to determine whether PDE4 is involved in regulation of the CCL3 production via activation of cAMP signal pathways. By stimulation of Raw264.7 and mouse peritoneal macrophages with LPS, we found that the CCL3 release was increased in a time- and dose-dependent manner within 8 h. The PDE4 inhibitor rolipram effectively suppressed the CCL3 mRNA expression and protein release with the IC50 of approximately 0.1 μM. Moreover, the LPS-induced CCL3 release was also dose-dependently inhibited by the PKA activator 6-Bnz-cAMP, but not by the Epac activator 8-pCPT-2’-O-Me-cAMP, suggesting that the effect of PDE4 inhibition on the CCL3 release was caused by increasing cAMP and activation of PKA. In addition, the attenuated CCL3 release caused by rolipram was partially reversed by the PKA inhibitor Rp-8-CPT-cAMPS. Using PDE4-deficient macrophages, we further found that LPS-stimulated PDE4B-/-, but not PDE4A-/- or PDE4D-/-, macrophages produced a significant decrease in the CCL3 release, and this reduction was similar to that observed in the wild-type macrophages inhibition by rolipram. This demonstrated that the inhibitory effect of rolipram on the CCL3 release was mediated by the inhibitor of the PDE4B isoform. Moreover, the chemotaxis of the splenic T cells induced by CCL3 was also inhibited by rolipram. Taken together, these findings demonstrated that PDE4B and the cAMP/PKA signaling play an essential role in the LPS/TLR4/CCL3 signal pathway in macrophages.
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