Normal rabbit serum was used as a control. Abolishment of PAI 1 activity using anti PAI selleck kinase inhibitor 1 antibody significantly inhibited the effect of LPSIFN stimulated ACM on microglial migration. PAI 1 neutralization also attenuated the effect of unstimulated ACM, indicating the presence of a low Inhibitors,Modulators,Libraries concentration of PAI 1 in the control ACM. These results further support that PAI 1 plays an important role in neu roinflammation by promoting microglial migration. Plasminogen activator inhibitor type 1 inhibited microglial phagocytosis of zymosan particles The effect of PAI 1 protein on the phagocytic activity of microglia was next investigated using zymosan par ticles as a prey. Zymosan particles are components of yeast cell wall, and served as a model for the phago cytosis of invading microbes.
Inhibitors,Modulators,Libraries The recombinant mouse PAI 1 protein inhibited the engulfment of zymosan particles in both BV 2 microglial cells and primary microglia cultures. PAI 1 inhibited the microglial phagocytic activity Inhibitors,Modulators,Libraries in a dose dependent manner, as 1000 ngml of PAI 1 treatment produced greater inhibition than 100 ngml. BSA did not inhibit the phagocytic activity of microglia. To identify the role of LRP1 in the PAI 1 inhibition of microglial phagocytosis, primary microglial cultures were treated with PAI 1 in the presence of RAP pep tide. The addition of RAP did not affect the PAI 1 inhibition of microglial phagocytic activity, indicating that LRP1 is not involved Inhibitors,Modulators,Libraries in the PAI 1 re duction of microglial phagocytosis. TLR2, TLR6 and glucan receptor dectin 1 have been previously impli cated in the recognition and phagocytosis of zymosan particles in either a cooperative or independent man ner.
The mRNA and protein levels of TLR2 and TLR6 were markedly decreased after 6 hours of PAI 1 treatment, but there was no significant difference in dectin 1 mRNA or TLR9 protein levels. Consistent with TLR2 mRNAprotein reduction, PAI Inhibitors,Modulators,Libraries 1 inhibited TLR2 mediated microglial activation as determined by NO production after stimulation with the TLR2 agon ist LTA in primary microglia cultures. To further define the inhibitory mechanism of PAI 1 in microglial phagocytosis, we used wild type human PAI 1 protein, and the R346A and Q123K mutants of this protein. The wild type protein and the R346A mutant inhibited the engulf ment of zymosan particles, whereas the Q123K mu tant did not have an inhibitory effect.
The addition of recombinant vitronectin protein to PAI 1 treated microglial cells rescued the phagocytic activity. We speculate add to your list that PAI 1 may inhibit the engulfment of zymosan particles by interfering with vitronectinITGB3 interaction. Vitronectin is a multi functional molecule that binds to PAI 1, ITGB3, and bacteria. To verify our hypothesis, the anti TLR2 or anti ITGB3 antibodies were applied to BV 2 micro glial cells together with zymosan particles.