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.