A novel microfluidic assay reveals a key role for protein kinase C δ in regulating human neutrophil-endothelium interaction

A key step in neutrophil-mediated tissue damage is the migration of activated neutrophils across the vascular endothelium. Previously, we identified protein kinase C delta as a critical regulator of neutrophil migration in sepsis but did not identify specific steps in migration. In this study, we used our novel biomimetic microfluidic assay to delineate systematically the mechanism by which protein kinase C delta regulates individual steps in human neutrophil-endothelial interaction during inflammation. The biomimetic microfluidic assay includes a network of vascular channels, produced from in vivo images connected to a tissue compartment through a porous barrier. HUVECs cultured in vascular channels formed a complete lumen under physiologic shear flow. HUVECs were pretreated with TNF-alpha +/- a protein kinase C delta inhibitor, and the tissue compartment was filled with a chemoattractant (fMLP or IL-8). Under physiologic shear flow, the role of protein kinase C delta on spatial and temporal neutrophil adherence/migration was quantified. Protein kinase C delta inhibition significantly reduced neutrophil adhesion in response to fMLP and IL-8 only under low shear rate and near bifurcations. Protein kinase C delta inhibition also decreased adherence to nonactivated HUVECs in response to fMLP or IL-8. Protein kinase C delta inhibition reduced neutrophil migration into the tissue compartment in response to fMLP and to a lesser degree, to IL-8. Antibody-coated microparticles demonstrated that protein kinase C delta inhibition down-regulated E-selectin and ICAM-1 but not VCAM-1 expression. With the use of a physiologically relevant in vitro model system, we demonstrate that protein kinase C delta plays an important role in the regulation of neutrophil adherence/migration during inflammation and identifies key steps regulated by protein kinase C delta in neutrophil-endothelial interactions.