Endotoxin induces leukocyte transmigration and changes in permeability of the airway epithelium via protein-kinase C and extracellular regulated kinase activation

Abstract

Lipopolysaccharide (LPS) endotoxin of Gram-negative bacteria compromises the integrity of the airway epithelial barrier and initiates migration of leukocytes across the epithelium. The goal of the present study was to identify the role of extracellular regulated kinase (ERK1/2) transduction pathways in these processes. The first aim was to determine whether LPS induces ERK1/2 activation and changes in epithelial permeabilityin epithelial cells alone or only in the presence of immune cells. The second aim was to determine whether the changes in the epithelial permeability were diminished by ERK1/2 blockade. The third aim was to investigatethe role of protein kinase C (PKC) activation as an upstream event in activation of ERK1/2. In vitro 20 μg/ml LPS challenge reduced epithelial barrier function, and induced ERK1/2 phosphorylation in primary cultures of bovine tracheal epithelium and in the transformed human airway epithelial cell line, Calu-3. LPS initiated migration of neutrophil-like and monocyte-like transformed HL-60 cell across sheets of Calu-3 cells. The migration rate and the associated changes in the electrical resistance, permeability to albumin, and ERK1/2 phosphorylation were all blocked by calphostin C, the specific blocker of PKC and by PD98059 (2'-amino-3'methoxyflavone), a selective cell-permeable inhibitor of MAP kinase kinase. In rats, in vivo perfusion of the lumen of an isolated segment of trachea with LPS (0.1 mg/ml) initiated migration of neutrophils and increased the permeability to albumin. Again, these effects were markedly inhibited by PD98059 and calphostin C (by > 50%). We conclude that epithelial ERK1/2 is activated by endotoxin via PKC and is an important pathway in regulation of epithelial permeability.

Keywords

Endotoxin leukocyte migration (chemotaxis)bronchial epithelium permeability PKC ERK1/2

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