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).