Nitric oxide (NO) is an important effector molecule on antimicrobial and antitumor effects of macrophages. (1 -> 3)-beta-D-Glucan (beta-glucan) is well known to show various immunopharmacological effects such as antimicrobial effect and antitumor effect by activating various points of host defense mechanisms. This paper deals with NO synthetic activity of peritoneal macrophage (PM) induced by beta-glucan administration in mice. The activity was determined by measuring NO concentration in PM culture by Griess reagent after 24 or 48 h in vitro culture. Administration (i.p. or i.v.) of a branched soluble (1 -> 3)-beta-D-glucan, grifolan (GRN), from Grifola frondosa enhanced NO synthesis of PM dose and time dependently. The activity was abrogated by the addition of N(G)-monomethyl-L-arginine (L-NMMA) in vitro. The most significant activity was observed at 3-7 d after the administration of GRN (250 mu g/mouse). PM from all strains of ICR, C3H/HeN, C3H/HeJ, BALB/c, BALB/c nu/nu, C57BL, and AKR mice showed significant activity by GRN administration. Among beta-glucans tested, SSG and OL-2, highly branched soluble glucans, and a particulate beta-glucan, zymosan, showed similar activity. Addition of GRN directly to in vitro RAW 264.7 or proteose peptone induced peritoneal macrophage (PP-PEC) culture could not enhance NO synthesis. However, NO synthesis of PP-PEC was enhanced in vitro by addition of GRN in the presence of interferon gamma (IFN gamma). Gene expression of IFN gamma mRNA in the liver and PEC were enhanced in GRN administered mice assessed by reverse transcriptase assisted PCR (RT-PCR) method. These facts strongly suggested that beta-glucan has capacity to enhance NO synthesis of PM in vivo through IFN gamma mediated mechanism.

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We have already demonstrated that various activities including NO (nitric oxide) synthesis in vivo and in vitro significantly differ between triple helical (SPG) and single helical (alkaline-treated SPG, SPG-OH) beta-glucans. It was previously suggested that the single helical conformer of beta-glucan (SPG-OH) was dominant in cytokine production and subsequent NO synthesis in vitro. In this study, we analyzed production of inducible nitric oxide synthase (iNOS) induced by beta-glucans in vitro and in vivo. The iNOS production was enhanced in proteose peptone-induced peritoneal macrophages (PMs) cultured with SPG-OH in the presence of IFN-gamma for 24 h, and SPG-OH-induced PMs. Moreover, SPG-OH was effective for iNOS production not only in isolated macrophages but also in tissue macrophages, whereas SPG was less effective. These findings suggest that a single helical conformer is essential for iNOS production, and that NO synthesis by beta-glucans is closely related to iNOS production.

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