Relationship between solubility of grifolan, a fungal 1,3-beta-D-glucan, and production of tumor necrosis factor by macrophages in vitro

Grifolan, GRN, is a fungal antitumor fl-glucan isolated from Grifola frondo sa. Various studies suggested that the underlying mechanism of the antitumo r activity of GRN is strongly related to immune modulation. In the previous publication (Adachi et al., 1994; Okazaki et al., 1995), we have shown tha t GRN activates macrophages to produce tumor necrosis factor (TNF) in vitro . In this study, the structural unit essential to produce TNF was examined by chemical modifications of GRN. GRN suspended in distilled water was trea ted at 150 degreesC for up to 3 h. Addition of the resulting turbid solutio n to the RAW 264.7 macrophage-like cell line produced TNF, and the relative activity was diminished in relation to the heat treatment period. The frac tions with a heating period longer than 15 min did not show any activity. A fter centrifugation of the resulting solution, significant activity was sho wn by precipitate fractions, suggesting that the insoluble form of GRN is i mportant for TNF production. Interestingly, the precipitate fraction obtain ed from 9 min of treatment also had significant activity. In addition, admi xing the soluble fraction with the particles significantly inhibited the TN F production. In contrast to these observations, the high-molecular-mass su bfraction of the soluble fraction prepared by ultrafiltration produced sign ificant amounts of TNF. Similar phenomena were shown with sodium hydroxide treatment and dimethylsulfoxide treatment. These facts strongly suggested t hat insoluble as well as a high molecular mass soluble form of GRN are requ ired for TNF production by macrophages.