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

Abstract

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

Experimental prestorage filtration removes antibodies and decreases lipids in RBC supernatants mitigating TRALI in vivo

“…[21][22][23] These antibodies, lipids, or sCD40L activate neutrophils (PMNs), allowing for PMN-mediated endothelial damage, capillary leak, and acute lung injury (ALI). [6][7][8]13,24 We hypothesize that a prestorage filter that removes antibodies, leukocytes, and platelets and decreases lipid bioactivity would significantly decrease the presence of donor HLA antibodies and the accumulation of biologically active lipids and sCD40L and would abrogate TRALI in a 2-event animal model.…”

Immunomodulatory Actions of Lysophosphatidylcholine

“…LPC (C16:0, 15 µM) induces de-ramification of murine microglia, and nonselective cation current, and calciumactivated potassium current, with the subsequent hyperpolarization (Schilling et al, 2004) NEUTROPHILS LPC (1 µM) alone slightly increases superoxide anion generation, and when co-treated with PMA, synergistically enhances PMA-induced superoxide anion generation (Englberger et al, 1987). Subsequent study showed that LPCs having fatty acids with more than 10 carbons enhance superoxide responses of stimulated human neutrophils (Ginsburg et al, 1989;Wyman et al, 2002). Lipids generated from stored blood were found to prime neutrophils, and identified to be LPCs (Silliman et al, 1994;Silliman et al, 1996).…”

“…One of characteristics of LPC actions is that LPC per se is a weak stimulator or even inactive, however, when LPC is combined with other stimulators, synergistic interactions often occurs (Englberger et al, 1987;Ginsburg et al, 1989;Asaoka et al, 1991;Asaoka et al, 1992;Sakai et al, 1994;Wyman et al, 2002). The most prominent example is seen in the priming of respiratory burst of neutrophils.…”

Transfusion Medicine

“…28,29 These compounds effectively prime the PMN oxidative burst and can activate primed adherent PMNs both in vitro. [28][29][30][31] In addition an in vitro In response to an infection in the tissues, inflammatory signals (arrows) diffuse to the vasculature and activate the vascular endothelium causing release of chemokines (4-pointed stars), which attract PMNs to the endothelial surface. Attraction is followed by selectin-mediated PMN rolling and β 2 -integrin:ICAM-1 mediated firm adhesion of PMNs to endothelial cells (ECs).…”