In this study, we found a novel mechanism for azurophil granule translocation in human neutrophils, wherein LPC induces azurophil granule translocation through G2A/Rho/ROCK/F-actin polymerization, complementing the previously identified LPC-induced i increase/p38 MAPK activation pathway ( Fig. It is to be noted that while fMLP in the presence of CB increases mean fluorescence intensity (MFI) by a shift of the whole distribution to the right, LPC increases MFI with making the distribution more bimodal by increasing the proportion of cells with the highest values ( Fig. These results indicate that LPC induces CD63 translocation via F-actin polymerization in human neutrophils. LPC increased F-actin polymerization as shown by increased staining of FITC-conjugated phalloidin, a marker of F-actin ( Fig. This result suggests that LPC enhances CD63 surface expression by induction of F-actin polymerization in human neutrophils. Intriguingly, however, CB pretreatment blocked LPC-induced CD63 surface expression ( Fig. However, in the presence of CB (2.5 μg/ml, i.e., 5.2 μM) that by itself did not affect CD63 surface expression, fMLP markedly increased surface expression of CD63 ( Fig. Keywords: Azurophil granule, Lysophosphatidylcholine, Neutrophil, TranslocationĬontrarily, fMLP (1 μM) alone expectedly could not induce surface expression of CD63 ( Fig.
Theses results suggest a novel regulatory mechanism for azurophil granule translocation where LPC induces translocation of azurophil granules via Rho/ROCK/F-actin polymerization pathway. NSC23766, a Rac inhibitor, however, did not affect LPC-induced CD63 surface expression.
LPC-induced CD63 surface expression was inhibited by both a Rho specific inhibitor, Tat-C3 exoenzyme, and a Rho kinase (ROCK) inhibitor, Y27632 which also inhibited LPC-induced F-actin polymerization. Interestingly, cytochalasin B, an inhibitor of action polymerization, blocked LPC-induced CD63 surface expression. Treatment of neutrophil with LPC significantly increased CD63 (an azurophil granule marker) surface expression. However, the precise mechanism of LPC-induced azurophil granule translocation was not fully understood. Previously, we reported that lysophosphatidylcholine (LPC), an endogenous lipid, enhances bactericidal activity of human neutrophils via increasing translocation of azurophil granules. Translocation of azurophil granules is pivotal for bactericidal activity of neutrophils, the first-line defense cells against pathogens.
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