台灣金線連免疫調節蛋白IPAF活化免疫細胞之機制

Abstract

摘要 本研究探討台灣金線連免疫調節蛋白(immunomodulatory protein from A. formosanus Hayata, IPAF)之生化特性及免疫調節活性，並進一步瞭解IPAF在免疫細胞中可能之訊息傳導路徑。台灣金線連經30%硫酸銨沈澱可得分子量為14 kDa之IPAF蛋白，名為台灣金線連免疫調節蛋白，等電點約為5.4。IPAF能活化RAW 264.7巨噬細胞產生一氧化氮(nitric oxide, NO)，並能刺激TNF-α及IL-1β的產生。IPAF亦可活化淋巴細胞，提升細胞激素IFN-γ的分泌量，以及刺激小鼠脾臟細胞的增生，但不引起IL-4的產生。經由流式細胞儀分析得知小鼠脾臟細胞主要之增生細胞群為B細胞，並於去除T細胞之環境下仍能受IPAF刺激進行增生反應，故認為IPAF屬於胸腺非依賴性抗原(thymus-independent antigen)。在訊息傳導路徑方面，C57BL/10ScN小鼠試驗發現IPAF刺激TLR4-/-腹腔巨噬細胞所產生之TNF-α分泌量降為TLR4+/+腹腔巨噬細胞分泌量之7.35 %，故推測IPAF於巨噬細胞中之訊息傳導路徑與TLR4高度相關；另一方面，IPAF在去除T細胞之環境下仍能使TLR4-/-之B細胞增生，故認為IPAF在B細胞中之訊息傳導路徑與TLR4無明顯相關，應有另一路徑傳遞其增生訊息。綜合實驗結果可知IPAF能活化B細胞，並能經由TLR4活化巨噬細胞，可提升宿主免疫反應，極具研究開發價值。

Abstract IPAF (immunomodulatory protein from Anoectochilus formosanus Hayata) was purified from Anoectochilus formosanus Hayata by 30% saturation of ammonium sulfate precipitation. SDS-PAGE analysis showed that IPAF has a molecular weight of 14 kDa and isoelectric focusing electrophoresis revealed the isoelectric point of IPAF was 5.4. Culturing RAW 264.7 macrophages with IPAF in vitro showed that IPAF could activate the cells and then increase nitric oxide, TNF-α and IL-1β production. Moreover, IPAF also stimulated the proliferation of murine splenocytes and increased their IFN-γ secretion, but did not result in IL-4 production. However, the cell depletion test demonstrated that IPAF increased the B cell proliferation without T cells existing. These results suggested that IPAF was a thymus-independent antigen. Peritoneal cells from C57BL/10ScN mice, which have a null mutation in TLR4 (TLR4-/-), were hyporesponsive in TNF-α secretion to both LPS and IPAF, suggesting that LPS and IPAF may share a same signaling pathway involving TLR4. On the other hand, IPAF could increase TLR4-/- B cells proliferation. Therefore, we propose that IPAF might be not involved in TLR4 signaling pathway and have another signaling pathway in B cell proliferation. Taking together, this study clearly demonstrated that IPAF, which activated B cells and also activated macrophages through TLR4, is an immune stimulus and could be helpful to strength the immunity of its host.