Protein Kinase C-Delta (PKCδ) Tyrosine Phosphorylation is a Critical Regulator of Neutrophil-Endothelial Cell Interaction in Inflammation.

BACKGROUND:Neutrophil dysfunction plays an important role in inflammation-induced tissue injury. Previously, we identified protein kinase C-δ as a critical controller of neutrophil activation and trafficking but how is regulated in inflammation has not been delineated. duanyu1531δ activity is regulated by tyrosine phosphorylation on multiple sites. Tyrosine155 is a key regulator of apoptosis and gene expression, but its role in proinflammatory signaling is not known. METHODS:In-vitro studies - superoxide anion (O2) and neutrophil extracellular traps (NETs) were measured in bone marrow neutrophils (BMN) isolated from wild type (WT) and knock-in mice tyrosine 155 → phenylalanine). Our novel 3D biomimetic microfluidic assay (bMFA) was used to delineate regulation of individual steps in neutrophil adhesion and migration using WT and duanyu1531δY155F BMN and mouse lung microvascular endothelial cells (MLMVEC). In-vivo studies - WT and duanyu1531δY155F knock-in mice underwent sham or cecal ligation and puncture surgery and the lungs harvested 24 h post-surgery. RESULTS:In vitro - duanyu1531δY155F BMN had significantly reduced O2 and NETs release compared with WT. WT BMN, but not duanyu1531δY155F BMN, demonstrated significant adhesion and migration across tumor necrosis factor-activated MLMVEC in bMFA. duanyu1531δ inhibition significantly reduced WT BMN adhesion and migration under low shear and near bifurcations, but had no effect on duanyu1531δY155F BMN. In vivo - mutation of duanyu1531δ tyrosine 155 significantly decreased neutrophil migration into the lungs of septic tyrosine 155 is a key phosphorylation site controlling proinflammatory signaling and neutrophil-endothelial cell interactions. These studies provide mechanistic insights into duanyu1531δ regulation during inflammation.

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