Neutrophil heat shock protein expression and activation correlate with increased apoptosis following transmigration through the endothelial barrier

Polymorphonuclear leukocytes (PMN) undergo endothelial transmigration upon activation or in response to a chemoattractant. Such cells are stressed and have an increased capacity to incite tissue injury. Little is known about the effect of transmigration on PMN stress gene responses, PMN activation, and ultimately programmed cell death (apoptosis). Human endothelial cells (ECV-304) were plated onto transwell membranes to form an endothelial monolayer and PMN transendothelial migration through this endothelial barrier was examined. Chemotaxis was induced by formyl-methionyl-leucyl-phenylalanine (fMLP). Flow cytometry was used to determine PMN receptor expression (CD11b, CD14, CD16, CD18, CD54), phagocytosis, and apoptosis. Heat shock protein (Hsp) expression was evaluated by Western blotting. fMLP-induced PMN transendothelial migration resulted in increased adhesion receptor expression and phagocytosis. Migrated PMN also had an increased rate of apoptosis as evaluated by uptake of propidium iodide and decreased FcgammaR III (CD16) expression. Increased PMN apoptosis coincided with induction of Hsp72 following transmigration. Thus, naive PMN that migrate through endothelium in response to a chemoattractant undergo activation as represented by increased phagocytosis and expression of adhesion receptors.