N-乙醯幾丁寡糖之免疫刺激機轉

Abstract

在動物實驗中，人們觀察到N-乙醯幾丁寡糖(NACOS)具有免疫刺激、抗腫瘤、抗微生物等活性，然而對於導致這現象的機制並不是相當清楚。在不同聚合度的NACOS中，我們發現只有N-乙醯幾丁六醣(NACOS-6)與N-乙醯幾丁五醣(NACOS-5)具有刺激小鼠腹腔巨噬細胞、RAW264.7小鼠巨噬細胞株以及THP-1人類單核球細胞株產生TNF-a的活性。添加NF-kB與MAPK (JNK, p38, ERK)路徑之專一性抑制劑，可阻斷NACOS引起之巨噬細胞活化。推斷NACOS藉由與一專一性受體之結合，導致MAPK與NF-kB路徑之活化，產生細胞激素。添加核內體(endosome)酸化抑制劑─氯奎寧，並無法阻斷NACOS-6所引起的巨噬細胞活化。推測NACOS-6刺激巨噬細胞活化不需要經由胞吞作用(endocytosis)。添加其他醣類對受體進行競爭性抑制之結果，發現N-乙醯葡萄糖胺與甘露聚醣(mannan)可對NACOS-6之受體產生競爭性抑制，而甘露糖(mannose)不行，推測甘露糖受體(Mannose receptor)不是NACOS之受體，且藉由其他實驗排除了DC-SIGN與TLR-4等受體。NACOS-6可誘導小鼠樹突細胞成熟，並且提升CD86、CD80等表面分子標記之表現。以NACOS-6作為佐劑，以卵清蛋白(ovalbumin, OVA)為抗原對小鼠進行免疫之模式中，發現NACOS-6可有效刺激對OVA具專一性IgG2a與IFN-r等Th1反應之產生，並且可以有效防護黑色素瘤MO5之侵襲。

N-acetyl-chitooligosaccharides (NACOS) have been shown to exhibit immunopotentiating, antitumor, and antimicrobial activities in vivo; however, the mechanisms behind those observations have not been fully understood. We found that among NACOS of different degrees of polymerization (n = 1-6), penta-N-acetylchitopentaose (NACOS-5) and hexa-N-acetylchitohexaose (NACOS-6) were able to stimulate mouse peritoneal macrophages, macrophage-like RAW264.7 cells, and human THP-1 monocytic cells to produce TNF- α and IL-1 α. NACOS stimulation induced the phosphorylation of MAP kinases (JNK, p38 and ERK) and nuclear translocation of NF-κB in RAW264.7 cells, and TNF-α production was blocked by inhibitors of JNK, p38, ERK, and NF-κB activation. Added chloroquine to block the endocytosis-dependent pathway did not inhibit the macrophage activation by NACOS. Macrophage activation by NACOS was competitively blocked by the addition of N-acetyl-D-glucosamine, suggesting that NACOS stimulates macrophages through the binding of a specific receptor. The polysaccharide mannan could also block macrophage activation by NACOS; however mannose did not show any inhibitory effect. NACOS were able to induce the maturation of murine bone-marrow-derived dendritic cells in vitro. These findings indicate that NACOS may stimulate macrophages and dendritic cells through the binding of a specific receptor, leading to the activation of NF-κB and MAP kinases, and the production of proinflammatory cytokines. Using NACOS-6 as an adjuvant, immunization with OVA in C57BL/6 mice resulted in production of OVA specific IgG2a and IFN-γ and the protection from MO5 melanoma challenge.