Fig. 5. Glycine enhances azurophil granule-phagosome fusion via p38 mitogen-activated protein kinase (MAPK).
(A) Effects of strychnine, diphenylene iodonium (DPI) and SB203580 on glycine-induced enhancement of fluid-phase pinocytosis. Uptake of Lucifer yellow in neutrophils was measured as fluid-phase pinocytosis. Neutrophils were exposed to E. coli for 20 min. Unphagocytosed E. coli was washed away and neutrophils were further incubated with glycine (500 μM) for 15 min in the presence of 0.5 mg/ml Lucifer yellow. Strychnine (1 μM), DPI (1 μM) or SB203580 (10 μM) was added during 15 min of incubation. Neutrophils were lysed with ice-cold distilled water for 1 h and fluorescence was measured. (B) Effects of strychnine, DPI, and SB203580 on zymosan-CD63 fusion. Neutrophils were allowed to ingest Texas Red-conjugated zymosan for 20 min. Unphagocytosed zymosan particles were washed away and neutrophils were further incubated with glycine in the presence of strychnine, DPI, or SB203580. Neutrophils were fixed and stained with anti-CD63 antibody. Stacks of 12–16 confocal images were collected with an LSM 510 laser-scanning confocal microscope (Carl Zeiss, Jena, Germany) and analyzed by LSM Image Examiner software (Carl Zeiss). The fusion of CD63 and zymosan particles was analyzed by measuring the area of co-localization between CD63 and zymosan particles, which was divided by total intracellular zymosan area in confocal images of neutrophils at each slice of 0.52 μm in thickness. (C) Effects of protease inhibitor cocktail and SB203580 on glycine-induced enhancement of bactericidal activity. Neutrophils were pretreated with a protease inhibitor cocktail or SB203580 for 1 h before E. coli exposure. (A–C) The average of three experiments is shown (mean ± SE). ***p < 0.001 by Student’s t-test.
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