| 摘要: | 趨化激素IFNγ-induced protein 10（IP-10）又稱為CXCL10，可由多種免疫及非免疫細胞所產生，它與許多發炎和自體免疫疾病有關。文獻指出，不同的促發炎介質包括LPS會刺激巨噬細胞釋放IP-10，然而目前對IP-10產生的調控機制還不太清楚。已知在多數發炎細胞中活化cAMP的訊息傳導可有效抑制細胞激素的產生，而分解cAMP的磷酸二酯?PDE4在發炎細胞中為調控cAMP濃度的主要酵素。因此，本研究主要的目的是想探討在巨噬細胞中PDE4是否參與調控IP-10的產生以及它的作用機制。利用LPS處理Raw 264.7 和小鼠腹腔巨噬細胞，我們發現IP-10的釋放會隨著時間和濃度的增加而上升，而PDE4抑制劑rolipram會有效的抑制IP-10的釋放，其IC50分別約為0.2 μM 和 0.02 μM。同時，Rolipram也會抑制IP-10 mRNA的表現。再者， 以PKA活化劑6-Bnz-cAMP處理巨噬細胞，其LPS刺激IP-10的釋放也會隨著藥劑濃度的增加而下降，然而Epac活化劑8-pCPT-2’-O-Me-cAMP只在高濃度時才有部分抑制作用。此外，PKA inhibitor Rp-8-CPT-cAMPS也會部份回復rolipram對IP-10的抑制作用。由這些結果顯示，rolipram對IP-10的抑制主要是藉由活化cAMP/PKA的路徑所致。我們進一步利用LPS處理PDE4基因剔除小鼠之腹腔巨噬細胞，發現PDE4B-/-細胞釋放IP-10會顯著下降，且其下降程度與rolipram處理PDE4B+/+巨噬細胞相當，同時rolipram不再進一步抑制PDE4B-/-巨噬細胞釋放IP-10。PDE4A-/-與PDE4D-/-細胞其IP-10的反應與PDE4A+/+與PDE4D+/+細胞一致。這些結果證明rolipram對IP-10的抑制作用是由於抑制PDE4B活性所致。另外，利用小鼠脾臟T細胞進行趨化反應，結果顯示IP-10誘導T細胞的移動也會被rolipram抑制。?合以上結果得知，在小鼠巨噬細胞中PDE4B對LPS刺激IP-10的產生與釋放是不可或缺的，且剔除或抑制PDE4B活性會活化cAMP/PKA訊息傳導進而抑制IP-10的反應。IFNγ-induced protein 10 (IP-10), also known as CXCL10, is a chemokine that can be produced by a variety of immune and non-immune cells during an inflammatory condition, and is associated with several inflammatory disorders. Accumulating evidence indicates that IP-10 is secreted by macrophages in response to various pro-inflammatory mediators, including lipopolysaccharide（LPS）. The regulatory mechanism underlying the production of IP-10, however, remains unclear. Activation of cAMP signaling is known to have negatively modulatory effects on cytokine production in most inflammatory cells. Type 4 phosphodiesterases（PDE4）, the enzymes responsible for cAMP degradation, play a key role in regulation of cAMP concentration in inflammatory cells. Thus, in this study, we aimed to determine whether and how PDE4 is involved in regulation of IP-10 production in macrophages in response to LPS. By stimulation of Raw 264.7 and mouse peritoneal macrophages with LPS, we observed that the IP-10 release increased in a time- and dose-dependent manner, and the PDE4 inhibitor rolipram effectively suppressed the IP-10 release with the IC50 of approximately 0.2 μM and 0.02 μM, respectively. The inhibition of IP-10 by rolipram was also obtained at the transcriptional level. Additionally, the LPS-induced IP-10 release was does-dependently inhibited by the PKA activator 6-Bnz-cAMP, but less effectively by the Epac activator 8-pCPT-2’-O-Me-cAMP. Moreover, the rolipram-inhibited IP-10 release was partially reversed by the PKA inhibitor Rp-8-CPT-cAMPS. These results indicated that the effect of rolipram on the IP-10 response was primarily mediated by the PKA activation. Using PDE4-deficient peritoneal macrophages treated with LPS, we found that the release of IP-10 in PDE4B-/- macrophages, but not PDE4A-/- or PDE4D-/- macrophages, was significantly decreased, to the level similar to that in the PDE4B+/+ macrophages treated with rolipram. Moreover, rolipram did not further decrease the IP-10 release in PDE4B-/- macrophages. These results demonstrated that the effect of rolipram on the IP-10 release was mediated by inhibition of PDE4B activity. Using mouse splenic T cells in a chemotactic reaction, we found that the IP-10-induced migration of T cells was also attenuated by rolipram. Taken together, these findings demonstrate that PDE4B is essential in the LPS-stimulated IP-10 production in mouse macrophages, and by activation of cAMP/PKA signaling ablation or inhibition of PDE4B can downregulate the IP-10 response. |
