Plasminogen activator inhibitor type 1 (PAI-1) inhibited microglial phagocytosis in a vitronectin-dependent manner. (A) BV-2 microglial cells were treated with 100 ng/ml of human wild-type PAI-1, the Q123K variant, or the R346A variant for 1 hour, and then incubated for 3 hours with zymosan particles. (A) Microglial phagocytosis of fluorescent zymosan particles was assessed (lower panel) as described above. (Upper panel) Representative images are shown. Results are given as mean ± SD (n = 3). *P < 0.05, NS = not significantly different from untreated control. Scale bar = 20 μm. (B) BV-2 microglial cells were treated with human wild-type PAI-1 or the Q123K mutant (100 ng/ml) in the presence or absence of vitronectin (1 μg/ml) for 1 hour, and then incubated with zymosan particles for 3 hours, followed by phagocytosis assay. Results are given as mean ± SD (n = 3). *P < 0.05, NS = not significant. (C) BV-2 microglial cells were incubated with zymosan particles in the presence or absence of Toll-like receptor (TLR)2 antibody (TLR2 Ab; 2 μg/ml), integrin (ITG)B3 antibody (ITGB3 Ab; 2 μg/ml), normal rabbit serum (2 μg/ml; negative control), or PAI-1 (100 ng/m; positive control). Microglial phagocytosis assay was performed. Results are given as mean ± SD (n = 3). *P < 0.05, **P < 0.01, compared with normal serum.