ABSTRACT
In this study, an immunologically active novel microparticulate mushroom ß-glucan (PRA-1p) was prepared using an alkali-soluble glucan PRA-1 by an emulsification and cross-linking method. PRA-1 was a hyperbranched (1â3),(1â6)-ß-d-glucan with a degree of branching of 0.89, isolated from the sclerotia of Polyporus rhinocerus. PRA-1 had a rod-like conformation, while PRA-1p exhibited a monodisperse and homogeneous spherical conformation with a diameter ranging from 0.3 to 2.0 µm in water. PRA-1p significantly induced nitric oxide and reactive oxygen species production as well as morphological changes of murine macrophages (RAW 264.7 cells) and upregulated their phagocytic activity. Furthermore, PRA-1p treatment markedly enhanced the secretion of cytokines, including cutaneous T cell-attracting chemokine 27, granulocyte-colony-stimulating factor, monocyte chemoattractant protein 1, macrophage inflammatory protein 1α, macrophage inflammatory protein 2, regulated on activation, normal T cell expressed and secreted, soluble tumor necrosis factor receptor 1, and tissue inhibitors of metalloproteinases. Activation of RAW 264.7 cells triggered by PRA-1p was associated with activation of inducible nitric oxide synthase, nuclear factor κB, extracellular signal-regulated kinase, and protein kinase B. This work suggests that novel PRA-1p derived from the mushroom sclerotia of P. rhinocerus has potential application as an immunostimulatory agent.
Subject(s)
Immunologic Factors/chemistry , Immunologic Factors/pharmacology , Plant Extracts/chemistry , Plant Extracts/pharmacology , Polyporus/chemistry , beta-Glucans/chemistry , beta-Glucans/pharmacology , Animals , Chemokine CCL27/genetics , Chemokine CCL27/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Immunologic Factors/isolation & purification , Macrophages/drug effects , Macrophages/immunology , Mice , NF-kappa B/genetics , NF-kappa B/immunology , Nitric Oxide/immunology , Phagocytosis/drug effects , Plant Extracts/isolation & purification , Polyporus/immunology , RAW 264.7 Cells , Reactive Oxygen Species/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , beta-Glucans/isolation & purificationABSTRACT
Treatment of hot water extract of the sclerotium of Polyporus rhinocerus (PRW) with murine macrophages including RAW 264.7 cell line and primary macrophages (PMs) could enhance their functional activities. These include a significant up-regulation of pinocytosis; an increase in the production of reactive oxygen species (ROS) and nitric oxide (NO); an increase in tumor necrosis factor alpha (TNF-alpha) production and inducible nitric oxide synthase (iNOS) expression in both RAW 264.7 cells and PMs. Cell surface receptors for yeast-derived beta-glucan, including Dectin-1, CR3, and TLR2, were determined by flow cytometry, and the expression of Dectin-1+ cells on the cell surface decreased in the responses of PMs to PRW. PRW increased phosphorylation of IkappaBalpha, which could trigger the nuclear factor kappa B (NF-kappaB) signal pathway for macrophage activation in RAW 264.7 cells. Therefore, the immunomodulatory effect of PRW could be mediated by macrophage activation via the NF-kappaB signal pathway.
Subject(s)
Complex Mixtures/pharmacology , Macrophage Activation/drug effects , Macrophages/immunology , NF-kappa B/metabolism , Polyporus/chemistry , Animals , Cell Line , Complex Mixtures/chemistry , Gene Expression Regulation, Enzymologic/drug effects , I-kappa B Proteins/metabolism , I-kappa B Proteins/pharmacology , Lectins, C-Type/metabolism , Macrophage-1 Antigen/metabolism , Macrophages/drug effects , Macrophages/metabolism , Mice , Mycelium/chemistry , Mycelium/immunology , NF-KappaB Inhibitor alpha , NF-kappa B/antagonists & inhibitors , NF-kappa B/genetics , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Phosphorylation , Pinocytosis/drug effects , Polyporus/immunology , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects , Toll-Like Receptor 2/metabolism , Tumor Necrosis Factor-alpha/metabolism , Up-Regulation/drug effects , beta-Glucans/metabolismABSTRACT
In this study, we report that a polysaccharide isolated from a Chinese medicinal herb, Zhu Ling (the sclerotium of Polyporus umbellatus (Per) Fr), induces phenotypic and functional maturation of murine bone-derived dendritic cells (BMDCs). Treatment of BMDCs with Polyporus polysaccharide (PPS) resulted in enhanced cell-surface expression of CD86, as well as enhanced production of both interleukin (IL)-12 p40 and IL-10 in a dose-dependent manner. In addition, treatment of BMDCs with PPS resulted in increased T cell-stimulatory capacity and decreased phagocytic ability. PPS-induced production of IL-12 p40 was inhibited by monoclonal antibodies to Toll-like receptor 4 (TLR4). Flow cytometric analysis showed that fluorescence-labeled PPS (f-PPS) bound specifically to BMDCs. This binding was blocked by both unlabeled PPS and anti-TLR4, but not by anti-TLR2 and anti-CR3 monoclonal antibodies. Taken together, our data show that PPS promotes the activation and maturation of murine BMDCs via TLR4.
