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Ganoderma sinense is a Chinese unique medicinal fungus that has been used in folk medicine for thousands of years. Polysaccharides are considered to be biologically active ingredients due to their immune-modulating functions. Previously we found that GSP-2, a new polysaccharide isolated from Ganoderma sinense, exerts an immunomodulatory effect in human peripheral blood mononuclear cells but the underlying mechanism is unclear. The present study aimed to investigate how GSP-2 triggers immunologic responses and the implicated signaling pathways. GSP-2 effects were investigated both in a macrophagic cell line, RAW264.7, and in primary macrophages. Moreover, the molecular basis of GSP-2 recognition by immune cells, and the consequent activation of signaling cascades, were explored by employing recombinant human HEK293-TLR-Blue clones, individually overexpressing various Toll-like receptors. GSP-2 dose-dependently induced the overexpression of Toll-like receptor 4 (TLR4) but did not affect the expression of other TLRs. Moreover, GSP-2 induced TNFα secretion in primary macrophages from wild-type, but not TLR4-knockout mice. In addition, GSP-2 upregulated TLR4 protein expression and activated the MAPK pathway in RAW246.7 macrophages. Finally, GSP-2 induced the production of the cytokines TNFα, IL1β, and IL6. Our data demonstrated that GSP-2 was specifically recognized by TLR4, promoting cytokine secretion and immune modulation in macrophages. [ABSTRACT FROM AUTHOR]

Soluble lactoferrin (LTF) is a versatile molecule that not only regulates the iron homeostasis, but also harbors direct microbicidal and immunomodulating abilities in mammalian body fluids. In contrast, little is known about the function of membrane-bound LTF (mbLTF), although its expression on human polymorphonuclear leukocytes (huPMNs) has been reported for decades. Given that LTF/anti-LTF antibodies represent a potential diagnostic/prognostic biomarker and a therapeutic target in patients with immune disorders, we wished, in the present study, to generate a novel human LTF- (huLTF-) specific mAb suitable for detailed analyses on the expression and function of mbLTF as well as for deciphering the underlying mechanisms. By using the traditional hybridoma cell fusion technology, we obtained a murine IgG1 (kappa) mAb, M-860, against huLTF. M-860 recognizes a conformational epitope of huLTF as it binds to natural, but not denatured, huLTF in ELISA. Moreover, M-860 detects mbLTF by FACS and captures endogenous huLTF in total cell lysates of huPMNs. Functionally, M-860 induces the activation of huPMNs partially through TLR4 but independently of phagocytosis. M-860 is thus a powerful tool to analyze the expression and function of human mbLTF, which will further our understanding of the roles of LTF in health and disease. [ABSTRACT FROM AUTHOR]