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Inflammatory Immune Responses by Water-insoluble -glucans

¹ Department of Oral and Molecular Microbiology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita-Osaka 565-0871, Japan;
² Laboratory of Virology and Vaccinology, National Institute of Biomedical Innovation, 7-6-8 Saito-Asagi, Ibaraki-Osaka 567-0085, Japan;
³ Department of Functional Bioscience, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan; and
⁴ Department of Life Science, Nihon University Advanced Research Institute for the Sciences and Humanities, Nihon University Kaikan Daini Bekkan, 12-5, Goban-cho, Chiyoda-ku, Tokyo 102-8251, Japan

View larger version (18K): [in this window] [in a new window]   Figure 1. Cell proliferation and cytokine production by mouse immune cells following stimulation with water-insoluble -glucans. (A) Mouse splenocytes (4 x 105 cells in 200 µL of complete RPMI 1640 medium) were stimulated for 4 days with the indicated concentrations of the test materials indicated in the panel, after which cell proliferation was determined by 3H-thymidine incorporation. (B,C,D) Mouse peritoneal exudate macrophages (5 x 105 cells/mL of complete RPMI 1640 medium) were stimulated for 24 hrs with increasing concentrations of the test materials, after which the production of TNF- (B), IL-6 (C), and IL-1β (D) was assessed with the use of cytokine ELISA kits. Results represent the means ± SE from 9 wells (3 experiments performed in triplicate). *p < 0.01, compared with sucrose-stimulated cells; **p < 0.01, compared with dextran T-2000-stimulated cells; ***p < 0.01, compared with water-soluble -glucan from S. sobrinus-stimulated cells; ****p < 0.01, compared with muramyl dipeptide-stimulated cells. *****p < 0.01, compared with S. sobrinus cell wall extracts-stimulated cells. MDP (); muramyl dipeptide, CW (); S. sobrinus cell wall extracts containing peptidoglycan and lipoteichoic acid, WIG (); water-insoluble -glucans produced by S. sobrinus GTFs, WSG (); water-soluble -glucan produced by S. sobrinus GTFs, Dextran (); dextran T-2000; •, LPS; , sucrose. (E) Effects of NaOH-treated water-insoluble -glucans on cytokine production by peritoneal exudate macrophages. Water-insoluble -glucans (3, 10, 30, or 100 µg) were dissolved in 1 N of NaOH, and diluted 1000-fold with complete RPMI 1640 medium, after which 1 mL of each solution (3, 10, 30, or 100 µg/mL of water-insoluble -glucans) was added to 5 x 105 cells of peritoneal exudate macrophages. TNF- and IL-6 production was assessed with the use of cytokine ELISA kits. Results represent the means ± SE from 9 wells (3 experiments performed in triplicate).

View larger version (9K): [in this window] [in a new window]   Figure 2. Roles of LPS, peptidoglycan, β-glucan, and water-soluble -glucans in the activation of peritoneal exudate macrophages by water-insoluble -glucans. (A) 10 pg/mL of LPS, 2.5 ng/mL of type III peptidoglycan, 25 pg/mL of β-1,3-glucan, 30 µg/mL of water-insoluble -glucans with 10 pg/mL of LPS, 30 µg/mL of water-insoluble -glucans with 2.5 ng/mL of type III peptidoglycan, 30 µg/mL of water-insoluble -glucans with 25 pg/mL of β-1,3-glucan, or 30 and 100 µg/mL of water-insoluble -glucans was added to 5 x 105 cells of peritoneal exudates macrophages for 24 hrs. WIG, water-insoluble -glucans; PGN, peptidoglycan. (B) J774.1 cells (105 cells/mL of complete RPMI 1640 medium) or peritoneal exudate macrophages (5 x 105 cells/mL of complete RPMI 1640 medium) were stimulated with water-insoluble -glucans (WIG; ), water-soluble -glucans from S. sobrinus (WSG; ), dextran T-2000 (Dextran; ), or sucrose () at the indicated concentrations for 24 hrs, after which the production of TNF- was assessed by means of a cytokine ELISA kit. Results represent the means ± SE from 9 wells (3 experiments performed in triplicate). *p < 0.01, compared with cells stimulated with WIG; **p < 0.01, compared with sucrose-stimulated cells. ND; not detectable (less than 78 pg/mL).

View larger version (39K): [in this window] [in a new window]   Figure 3. Effect of macrophage activation stimulated with water-insoluble -glucans on suppression of TLR2, TLR4, NOD1, and NOD2 mRNA expression. (A) The primers used in the RT-PCR assay and sequence of the target mRNA for TLR2, TLR4, NOD1, and NOD 2. (B) J774.1 cells were transfected with siRNA of TLR2, TLR4, NOD1, NOD2, or buffer only with the use of TransIT-TKO transfection reagent. Total RNA was extracted 24 hrs after transfection, and RT-PCR was performed. (C) J774.1 or transfected J774.1 cells (105 cells/mL) were stimulated with 1 mg/mL of water-insoluble -glucan for 24 hrs, and the TNF- in the culture supernatants was measured by a TNF- ELISA kit.

View larger version (37K): [in this window] [in a new window]   Figure 4. Cytokine production in human monocytes induced by water-insoluble -glucans and chemotaxis and H2O2 production in human polymorphonuclear cells, following stimulation with water-insoluble -glucans. (A,B) Human monocytes (5 x 105 cells/mL of complete RPMI 1640 medium) were stimulated for 48 hrs with increasing concentrations of water-insoluble -glucans (WIG; ), water-soluble -glucans from S. sobrinus (WSG; ), S. sobrinus cell wall extracts including peptidoglycan and lipoteichoic acid (CW; ), dextran T-2000 (Dextran; ), muramyl dipeptide (MDP; ), LPS (•), or sucrose (), after which the production of TNF- (A) and IL-8 (B) was assessed with the use of cytokine ELISA kits. Results represent means ± SE from 12 wells (4 experiments performed in triplicate). *p < 0.01, compared with sucrose-stimulated cells; **p < 0.01, compared with dextran T-2000-stimulated cells; ***p < 0.01, compared with water-soluble -glucan from S. sobrinus-stimulated cells; ****p < 0.01, compared with muramyl dipeptide-stimulated cells. *****p < 0.01, compared with S. sobrinus cell wall extract-stimulated cells. (C) Polymorphonuclear cells (5 x 106 cells/mL of complete RPMI 1640 medium) were labeled with BCECF-AM, and chemotaxis assays were performed with the indicated concentrations of water-insoluble -glucans (WIG) and water-soluble -glucans (WSG). Chemotaxis of polymorphonuclear cells was measured following a 24-hour treatment with the supernatant from monocytes stimulated with the indicated concentrations of sucrose (light-gray columns), water-insoluble -glucans (black columns), or water-soluble -glucans from S. sobrinus (dark-gray columns). (D) Polymorphonuclear cells (5 x 106 cells/mL of complete RPMI 1640 medium) were pre-treated with C5a, sucrose, water-insoluble -glucans (WIG), or water-soluble -glucans from S. sobrinus (WSG) for 1 hr at 37°C. Following pre-treatment, phorbol 12-myristate 13-acetate and DCFH-DA were added to the cell suspensions. After a one-hour incubation at 37°C, the increase in total fluorescence was determined. Results represent the means ± SE from 15 wells (5 experiments performed in triplicate). #p < 0.01, compared with polymorphonuclear cells stimulated with the supernatant from PBS-stimulated human monocytes; ##p < 0.01, compared with polymorphonuclear cells stimulated with PBS.

Journal of Dental Research, Vol. 86, No. 3, 242-248 (2007)
DOI: 10.1177/154405910708600309


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