Inducement of Cytokine Release by GFPBW2, a Novel Polysaccharide from Fruit Bodies of Grifola frondosa, through Dectin-1 in Macrophages

Polysaccharides, especially beta-glucans isolated from various species of mushrooms, are considered as biological response modifiers (BRMs) to be widely used in the treatment of cancer, especially due to their immunostimulatory activity. We herein characterized the structure of a novel water-soluble homogeneous polysaccharide (GFPBW2) from the fruit bodies of mushroom Grifola frondosa and investigated its immunomodulatory activity in vitro. GFPBW2 was purified from the alkali-extracted fractions by stepwise elution with a molecular weight of 26.2 kDa. On the basis of infrared and NMR spectroscopy, methylation and monosaccharide composition analysis, partial acid hydrolysis, and Smith degradation, its structure was elucidated to possess a backbone consisting of beta-D-1,3- and beta-D-1,4-linked glucopyranosyl residues, with branches attached to O-6 of beta-D-1,3-linked glucopyranosyl residues. Functionally, it is an effective inducer of tumor necrosis factor-a (TNF-alpha) and interleukin-6 (IL-6) secretion in murine resident peritoneal macrophages. Using quartz crystal microbalance (QCM) analysis, we found that GFPBW2 could bind dendritic cell-associated C-type lectin-1 (Dectin-1) with an affinity constant (K-d) value of 1.08 X 10(-7) M, while it could activate Syk and enhance TNF-alpha production in RAW264.7 cells overexpressing wild type but not mutant Dectin-1. Furthermore, Syk, NF-kB signaling, and cytokine release in resident peritoneal macrophages induced by GFPBW2 could be significantly inhibited by a specific Dectin-1 blocking reagent, Laminarin. These data suggested that GFPBW2 might be a potential ligand of Dectin-1, and the potential of GFPBW2 to activate macrophage through triggering cytokine secretion might be attributed, at least in part, to the involvement of Dectin-1.