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In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration
Published on: January 6, 2014
Involvement of phospholipase A2 in Pseudomonas aeruginosa-mediated PMN transepithelial migration
Bryan P Hurley1, Natecia L Williams, Beth A McCormick
1Mucosal Immunology Laboratories, Massachusetts General Hospital, 3503 Charlestown, MA 02129, USA. Bphurley@partners.org
Abstract:
Inflammation resulting from bacterial infection of the respiratory mucosal surface during pneumonia and cystic fibrosis contributes to pathology. A major consequence of the inflammatory response is recruitment of polymorphonuclear cells (PMNs) to the infected site. To reach the airway, PMNs must travel through several cellular and extracellular barriers, via the actions of multiple cytokines, chemokines, and adhesion molecules. Using a model of polarized lung epithelial cells (A549 or Calu-3) grown on Transwell filters and human PMNs, we have shown that Pseudomonas aeruginosa induces PMN migration across lung epithelial barriers. The process is mediated by epithelial production of the eicosanoid hepoxilin A(3) (HXA(3)) in response to P. aeruginosa infection. HXA(3) is a PMN chemoattractant metabolized from arachidonic acid (AA). Given that release of AA is believed to be the rate-limiting step in generating eicosanoids, we investigated whether P. aeruginosa infection of lung epithelial cells resulted in an increase in free AA. P. aeruginosa infection of A549 or Calu-3 monolayers resulted in a significant increase in [(3)H]AA released from prelabeled lung epithelial cells. This was partially inhibited by PLA(2) inhibitors ONO-RS-082 and ACA as well as an inhibitor of diacylglycerol lipase. Both PLA(2) inhibitors dramatically reduced P. aeruginosa-induced PMN transmigration, whereas the diacylglycerol lipase inhibitor had no effect. In addition, we observed that P. aeruginosa infection caused an increase in the phosphorylation of cytosolic PLA(2) (cPLA(2)), suggesting a mechanism whereby P. aeruginosa activates cPLA(2) generating free AA that may be converted to HXA(3), which is required for mediating PMN transmigration.
Insights
Pseudomonas aeruginosa infection triggers lung epithelial cells to release arachidonic acid (AA), a key step in producing hepoxilin A3 (HXA3). This process is crucial for the migration of polymorphonuclear cells (PMNs) during respiratory infections.
Area of Science:
- Respiratory Medicine
- Cell Biology
- Immunology
Background:
- Bacterial infections like pneumonia and cystic fibrosis cause inflammation in the respiratory tract.
- Inflammation leads to the recruitment of polymorphonuclear cells (PMNs) to the infection site.
- PMN migration across lung epithelial barriers is essential for clearing infections but also contributes to pathology.
Purpose of the Study:
- To investigate the role of Pseudomonas aeruginosa infection in mediating PMN transmigration across lung epithelial cells.
- To identify the specific molecular mechanisms, particularly eicosanoid production, involved in P. aeruginosa-induced PMN migration.
- To determine if increased free arachidonic acid (AA) release is a prerequisite for PMN transmigration.
Main Methods:
- Utilized a model of polarized lung epithelial cells (A549 or Calu-3) and human PMNs.
- Measured the release of radiolabeled arachidonic acid ([(3)H]AA) from infected lung epithelial cells.
- Assessed the effect of phospholipase A2 (PLA(2)) and diacylglycerol lipase inhibitors on PMN transmigration and AA release.
- Investigated the phosphorylation status of cytosolic PLA(2) (cPLA(2)) following P. aeruginosa infection.
Main Results:
- P. aeruginosa infection significantly increased the release of free AA from lung epithelial cells.
- PLA(2) inhibitors (ONO-RS-082 and ACA) partially blocked AA release and dramatically reduced P. aeruginosa-induced PMN transmigration.
- Inhibition of diacylglycerol lipase did not affect PMN transmigration.
- P. aeruginosa infection led to increased phosphorylation of cPLA(2), indicating its activation.
Conclusions:
- P. aeruginosa infection activates cytosolic PLA(2) (cPLA(2)) in lung epithelial cells, leading to increased free arachidonic acid (AA) release.
- The released AA is likely converted to hepoxilin A3 (HXA3), a chemoattractant essential for mediating P. aeruginosa-induced PMN transmigration.
- Targeting PLA(2) activity represents a potential therapeutic strategy to reduce PMN infiltration in respiratory bacterial infections.
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