Differential toll-like receptor activation in lung ischemia reperfusion injury.
OBJECTIVE
The requirement for toll-like receptors (TLRs) in lung ischemia reperfusion injury (LIRI) has been demonstrated but not fully characterized. Previously, we reported that TLR-4 is required by alveolar macrophages but not pulmonary endothelial or epithelial cells for development of LIRI. Additionally, we demonstrated differential patterns of mitogen-activated protein kinase (MAPK) activation and cytokine release in these cell types during LIRI. Here, we sought to determine whether these differences in activation responses are related to cell-specific TLR activation requirements.


METHODS
Primary cultures of alveolar macrophages, pulmonary endothelial, and immortalized epithelial cells were pretreated with TLR-2 or TLR-4 short interference RNA (ribonucleic acid) before hypoxia and reoxygenation. Cell lysates and media were analyzed for receptor knockdown, MAPK activation, and cytokine production. Rats were pretreated with TLR-2 or TLR-4 short interference RNA before lung ischemia reperfusion and changes in lung vascular permeability were assessed.


RESULTS
Knockdown of TLR-2 in alveolar macrophages did not affect MAPK phosphorylation or cytokine secretion. Conversely, TLR-2 knockdown in pulmonary endothelial and epithelial cells demonstrated significant reductions in extracellular signal-regulated kinase 1/2 activation and cytokine secretion. The lung permeability index in LIRI was decreased by TLR-4 but not TLR-2.


CONCLUSIONS
Differential TLR signaling and MAPK activation in response to LIRI seem to be cell specific. Short interference RNA provides an outstanding tool for examination of the underlying mechanism.
Department of Surgery, University of Washington School of Medicine, Seattle, Wash.
Patrick
P
Phelan
Department of Surgery, University of Washington School of Medicine, Seattle, Wash.
Heather E
HE
Merry
Department of Surgery, University of Washington School of Medicine, Seattle, Wash; Center for Lung Biology, University of Washington, Seattle, Wash. Electronic address: bhwang@u.washington.edu.
Billanna
B
Hwang
Department of Surgery, University of Washington School of Medicine, Seattle, Wash; Center for Lung Biology, University of Washington, Seattle, Wash.
Michael S
MS
Mulligan
D056809:Alveolar Epithelial Cells / Q000378:metabolism / Q000473:pathology; D000818:Animals; D002199:Capillary Permeability; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000378:metabolism / Q000473:pathology; D004789:Enzyme Activation; D008168:Lung / Q000098:blood supply* / Q000378:metabolism* / Q000473:pathology; D055370:Lung Injury / Q000235:genetics / Q000378:metabolism* / Q000473:pathology / Q000517:prevention & control; D016676:Macrophages, Alveolar / Q000378:metabolism / Q000473:pathology; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D018384:Oxidative Stress; D034622:RNA Interference; D020318:Rats, Long-Evans; D015427:Reperfusion Injury / Q000235:genetics / Q000378:metabolism* / Q000473:pathology / Q000517:prevention & control; D015398:Signal Transduction; D051195:Toll-Like Receptor 2 / Q000235:genetics / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D016207:Cytokines; C495343:Tlr2 protein, rat; C495347:Tlr4 protein, rat; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; C535149:Mapk1 protein, rat; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3
Ischemia/reperfusion (lung); TLR-2; TLR-4; proinflammatory mediators; reoxygenation