Natural killer T (NKT) cells recognize CD1d loaded with glycosphingolipid (GSL) antigens. After being stimulated by GSL antigens, NKT cells immediately secrete cytokines.
2-4)The variety of cytokines release from NKT cells can promote immunoresponses against tumors and microbial infections, and regulate autoimmune responses, 5,6) although a puzzling aspect of these responses is that both Th1 and Th2 cytokines can be secreted in response to the stimulation.The fungal glycolipids are exogenous antigens for NKT cells 7) ; therefore, we focused on acidic glycosphingolipids (AGLs) of edible mushrooms (Hypsizigus marmoreus and Pleurotus eryngii) 8,9) and reported that AGLs induced interferon-g (IFN-g) and interleukin-4 (IL-4) release from murine T cells in a CD11c-positive cell-dependent manner.
10)Invariant natural killer T (iNKT) cells, that are more than 80% of murine NKT cells, express an invariant T cell receptor Va14 chain in combination with certain Vb chains. In mice, CD1d-dependent NKT cells are either CD4CD8-double negative or CD4-positive. The significance of CD4 expression on NKT cells is ambiguous because there is no evidence of any interaction between CD4 and MHC class II molecules on antigen-presenting cells (APCs), although CD4 can directly interacts with CD1d and enhances NKT cell activation.11) Additionally, NKT cells mainly leave the thymus when NK1.1 is negative. 6) In this study, we reported that edible mushroom AGLs activated CD4-positive NK1.1-negative iNKT hybridoma cells that express Va14-Ja18/Vb8.2 12) in a CD1d-dependent manner and intravenous AGL injection elevated the cytokine level.
MATERIALS AND METHODS
MaterialsPurified AGLs of H. marmoreus and P. eryngii were structurally elucidated according to previous reports, as described briefly.10,13) Saponified and dialyzed extracts were fractionated on a DEAE-Sephadex A-25 column (acetate form) (Amersham Biosciences AB, Uppsala, Sweden), and further purification was performed using a silica gel-6RS-8060 Iatrobeads column (Iatron Laboratories Inc., Tokyo, Japan). The composition of fatty acids and sugars, identification of phosphate bonds, confirmation of inositolphosphate content, determination of linkages among sugars and between sugar and inositol in purified AGLs, and AGL purity was confirmed by treatment with the appropriate reagents and using assays including columns, thin layer chromatography, a microwave oven, gas chromatography, gas-liquid chromatography/mass spectrometry, matrix-assisted laser desorption ionization-time of flight/mass spectrometry and 1 H-NMR. a-Galactosylceramide (a-GalCer) (KRN7000: ALX-306-027) and isoglobotrihexosylceramide (iGb3) (ALX-306-028) were purchased from ALEXIS Biochemicals (Lausanne, Switzerland).Mice and Protocols for in Vivo Experiments AGLs were dissolved in 100% polysorbate-20, diluted with sterilized phosphate buffered saline (PBS) to 0.5% polysorbate-20, and then kept at 4°C until used. Stock solutions were further diluted to 200 and 100 mg/ml AGL or 5 mg/ml a-GalCer in PBS containing 0.025% polyso...