A two-insult in vitro model of PMN-mediated pulmonary endothelial damage: requirements for adherence and chemokine release

Lysophosphatidylcholines(lyso-PCs), generated during blood storage, are etiologic in atwo-insult, sepsis-based model of transfusion-related acute lung injury(TRALI). Individually, endotoxin (LPS) and lyso-PCs prime but do notactivate neutrophils (PMNs). We hypothesized that priming of PMNsalters their reactivity such that a second priming agent causes PMNactivation and endothelial cell damage. PMNs were primed or not withLPS and then treated with lyso-PCs, and oxidase activation and elastaserelease were measured. For coculture experiments, activation of humanpulmonary microvascular endothelial cells (HMVECs) was assessed byICAM-1 expression and chemokine release. HMVECs were stimulated ornot with LPS, PMNs were added, cells were incubated with lyso-PCs, andthe number of viable HMVECs was counted. Lyso-PCs activated LPS-primed PMNs. HMVEC activation resulted in increased ICAM-1 andrelease of ENA-78, GRO, and IL-8. PMN-mediated HMVEC damage wasdependent on LPS activation of HMVECs, chemokine release, PMNadhesion, and lyso-PC activation of the oxidase. In conclusion, sequential exposure of PMNs to priming agents activates themicrobicidal arsenal, and PMN-mediated HMVEC damage was the resultof two insults: HMVEC activation and PMN oxidase assembly.