探討台灣金線連免疫調節蛋白對小鼠腹腔巨噬細胞之活化作用

Abstract

台灣金線連 (Anoectochilus Formosanus) 為台灣特有種，為蘭科 (Orchidaceae)金線連屬 (Anoectochilus) 植物，本實驗室自台灣金線連萃取純化出分子量約14 kDa之蛋白，經初步活性測試具有免疫功效，可活化細胞株RAW264.7產生TNF-α 及IL-1β等細胞激素 (cytokine)，並為胸腺非依賴性第一型抗原，不需要 T 淋巴細胞即能直接活化 B 淋巴細胞，增加表面CD69與MHC classl Π表現量，因而命名此蛋白為台灣金線連免疫調節蛋白 (immunomodulatory protein from A. formosanus, IPAF)。然而，IPAF活化巨噬細胞之機制及其活化作用尚未明瞭，因此，本實驗目的為探討IPAF對小鼠腹腔巨噬細胞的免疫活化作用與相關的活化途徑。經實驗結果發現，IPAF可增加小鼠巨噬細胞吞噬活性 (phagocytic activity)，提高細胞激素TNF-α與IL-1β分泌量，增加表面CD86與MHC class Π表現，同時也提高TNF-α、IL-1β、IL-6等細胞激素之mRNA表現。另外，IPAF可相對增加較多的M1型CCL3、CCL4等趨化素 (chemokine)與IL-12細胞激素等mRNA表現量，因此IPAF可促使巨噬細胞分化成 M1 型態，並可推測IPAF有助於 Th1 免疫反應。在訊息傳導途徑方面，分別使用TLR2-/-與TLR4-/-基因剔除小鼠，發現IPAF仍可活化TLR2-/-小鼠腹腔巨噬細胞，而於TLR4-/-小鼠之TNF-α分泌量則顯著下降，因此得知IPAF活化小鼠腹腔巨噬細胞之途徑與TLR4相關。進一步得知IPAF可增加小鼠表面TLR4受體及mRNA基因表現，以及增加TLR-signaling下游相關因子TIRAP、MyD88、TRAF6與NF-κB等mRNA表現，並使用EMSA試驗證明核內NF-κB有增加表現情形。綜合實驗結果可知IPAF能經由TLR4相關途徑活化小鼠巨噬細胞，提升宿主免疫反應。

Anoectochilus formosanus HAYATA is known as a highly valuable traditional Chinese medicine of is an edible medical herb and is originated in Taiwan with regional significance. Our preliminary study has isolated a 14 kDa protein from the whole plant of A. formosanus, which was capable to activate RAW 264.7 macrophages to secrete TNF-α and IL-1β, and resembled type-1 thymus-independent antigen which activated B lymphocytes without assistance of T lymphocytes. As well as up-regulated the expression of surface marker CD69 and MHC class П of B lymphocyte, and this protein was designated as immunomodulatory protein from A. formosanus, IPAF. However, the activation mechanism of IPAF on primary macrophage remained unclear. The objective of this study was aimed to evaluate the immunoregulatory effect of IPAF on murine peritoneal macrophages and to investigate the activation of macrophages by TLR-signaling pathways. Our results demonstrated that IPAF could activate phagocytic activity, TNF-α and IL-1β secretion, expression of surface marker CD86 and MHC class П of macrophages, and increase the mRNA expression of TNF-α, IL-1β and IL-6 of the cells. In addition, IPAF promoted the mRNA expression of IL-12 and M1 type chemokines genes, CCL3 and CCL4 in the cells. The results revealed that IPAF was capable to promote Th1 immune response by inducing M1 polarization of macrophages. We used TLR2 and TLR4 knockout mouse to investingate the signaling pathway, and found that IPAF was capable to activate the macrophages to secrete TNF-α from TLR2 but not in TLR4-deficient mice, suggesting that IPAF activated macrophages through TLR4-signaling pathway. Furthermore, we observed that IPAF could enhance the expression of surface marker TLR2 and TLR4, and increase mRNA expression of the TLR-signaling downstream molecules TIPAP, MyD88, TRAF6 and NF-κB. Moreover, the EMSA result showed a IPAF-induced activation of the transcription factor NF-κB. Taking together, this study clearly demonstrated that IPAF, which activated macrophages through TLR4, was an immune activator and could be helpful to strengthen the host immunity.