Polymorphonuclear Cell Transmigration Induced by Pseudomonas aeruginosa Requires the Eicosanoid Hepoxilin A3

Abstract

Lung inflammation resulting from bacterial infection of the respiratory mucosal surface in diseases such as cystic fibrosis and pneumonia contributes significantly to the pathology. A major consequence of the inflammatory response is the recruitment and accumulation of polymorphonuclear cells (PMNs) at the infection site. It is currently unclear what bacterial factors trigger this response and exactly how PMNs are directed across the epithelial barrier to the airway lumen. An in vitro model consisting of human PMNs and alveolar epithelial cells (A549) grown on inverted Transwell filters was used to determine whether bacteria are capable of inducing PMN migration across these epithelial barriers. A variety of lung pathogenic bacteria, including Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa are indeed capable of inducing PMN migration across A549 monolayers. This phenomenon is not mediated by LPS, but requires live bacteria infecting the apical surface. Bacterial interaction with the apical surface of A549 monolayers results in activation of epithelial responses, including the phosphorylation of ERK1/2 and secretion of the PMN chemokine IL-8. However, secretion of IL-8 in response to bacterial infection is neither necessary nor sufficient to mediate PMN transepithelial migration. Instead, PMN transepithelial migration is mediated by the eicosanoid hepoxilin A3, which is a PMN chemoattractant secreted by A549 cells in response to bacterial infection in a protein kinase C-dependent manner. These data suggest that bacterial-induced hepoxilin A3 secretion may represent a previously unrecognized inflammatory mechanism occurring within the lung epithelium during bacterial infections.

<em>In vitro</em> Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

“…This occurs via a pathogen-specific induction of an epithelial cell-derived neutrophil chemotactic gradient 3,23 . For example, pathogenic P. aeruginosa interacting with the apical surface of lung epithelial cells causes a substantial number of neutrophils to migrate across the epithelial layer 18,22,25,26,33,34 . This clinically relevant assay system can be manipulated in numerous ways to unveil key pathogen and host molecular contributors when paired with appropriate controls.…”

“…Each of these pathogenic organisms is capable of driving neutrophils across polarized intestinal epithelial monolayers grown on inverted collagen coated permeable transwell filters. This experimental system has been adapted with modifications to explore lung epithelial barriers and pathogen induced neutrophil trans-epithelial migration 18,26 . Several airway epithelial cell lines apart from H292 have been used for these studies including commonly cited A549, BEAS-2B, and Calu-3 lung epithelial cell lines 22,26 .…”

“…Note: All transwells should be subjected to the same handling and washing regimen, whether or not they have been infected with bacteria, to ensure that all transwells being assayed have been manipulated in the same manner. A 60 min infection with either PAO1 or MC1000 does not reduce viability or alter the barrier properties of the H292 monolayer as assessed by a lactate dehydrogenase based toxicology assay and the HRP flux assay, respectively 18,22 . 10.…”

“…Note: We suggest waiting a minimum of 8 days post seeding of transwells to ensure that the epithelial monolayers have fully formed and exhibit a functional barrier. One can test the H292 epithelial barrier by determining whether the monolayer is capable of restricting trans-epithelial flux of the protein horse radish peroxidase (HRP) following addition to the basolateral surface 18 .…”

Alveolar Macrophages from Septic Mice Promote Polymorphonuclear Leukocyte Transendothelial Migration via an Endothelial Cell Src Kinase/NADPH Oxidase Pathway

“…While not addressed in the present study, others have assessed PMN migration across 549 cells (human pulmonary type II-like epithelial cells). Stimulation of A549 cell monolayers with either TNF-␣ or Pseudomonas aeruginosa enhanced PMN transepithelial migration (64,65). While these cells produced IL-8, this chemokine was not the major mediator of PMN migration.…”