Journal
INFLAMMATION RESEARCH
Volume 57, Issue 5, Pages 216-229Publisher
SPRINGER BASEL AG
DOI: 10.1007/s00011-007-7031-4
Keywords
neutrophil; transendothelial migration; PKC; LTB4; TNF-alpha
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Objective and Design: The process of neutrophil extravasation is involved in acute and chronic inflammatory diseases; however our understanding of the role of endothelial second messengers in the regulation of leukocyte emigration is still incomplete. Materials and Methods: We investigated this using an in vitro model of neutrophil migration across human endothelial cells. Results: Activation of endothelial protein kinase C (PKC) by either phorbol myristate acetate (PMA) or bryostatin-1 (a potent PKC delta and epsilon activator) completely abolished neutrophil migration mediated by either endothelial TNF-alpha stimulation or LTB4. Pretreatment with Go-6983 (PKC alpha, beta, delta, inhibitor) prior to addition of bryostatin-1 restored LTB4 induced PMN migration, while pretreatment with Go-6976 (PKC alpha and beta inhibitor) did not. PKC delta specific siRNA knockdown of PKC delta expression in endothelial cells also restored LTB4 induced PMN migration. In addition, PMA and bryostatin-1 both increased endothelial adhesion to the substratum that was also reversed using PKC delta siRNA knockdown of PKC delta expression. PMA and bryostatin-1 additionally altered the staining pattern of FAK[pY(397)], paxillin, and vinculin from a dot-like pattern to a dash-like pattern around the cell perimeter. While PMA reduced transendothelial resistance (TER), bryostatin-1 had no effect on TER. Conclusions: These observations show that endothelial PKC delta activation eliminates neutrophil transendothelial migration through a mechanism unrelated to endothelial barrier integrity. These data are consistent with PKC delta mediated increased cell substrate adhesion as a limiting factor for neutrophil transendothelial migration towards a chemoattractant.
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