PLT-induced TRALI may be the result of two events: 1) the clinical condition of the patient and 2) the infusion of lipids in stored PLTs.

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.

A mixture of lysophosphatidylcholines (lyso-PCs) are generated during blood storage and are etiologic in models of acute lung injury. We hypothesize that lyso-PCs stimulate polymorphonuclear neutrophils (PMNs) through Ca(2)(+)-dependent signaling. The lyso-PC mix (0.45-14.5 micro M) and the individual lyso-PCs primed formyl-Met-Leu-Phe (fMLP) activation of the oxidase (1.8- to 15.7-fold and 1.7- to 14.8-fold; P<0.05). Labeled lyso-PCs demonstrated a membrane association with PMNs and caused rapid increases in cytosolic Ca(2)(+). Receptor desensitization studies implicated a common receptor or a family of receptors for the observed lyso-PC-mediated changes in PMN priming, and cytosolic Ca(2)(+) functions were pertussis toxin-sensitive. Lyso-PCs caused rapid serine phosphorylation of a 68-kD protein but did not activate mitogen-activated protein kinases or cause changes in tyrosine phosphorylation. With respect to alterations in PMN function, lyso-PCs caused PMN adherence, increased expression of CD11b and the fMLP receptor, reduced chemotaxis, provoked changes in morphology, elicited degranulation, and augmented fMLP-induced azurophilic degranulation (P<0.05). Cytosolic Ca(2)(+) chelation inhibited lyso-PC-mediated priming of the oxidase, CD11b surface expression, changes in PMN morphology, and serine phosphorylation of the 68-kD protein. In conclusion, lyso-PCs affect multiple PMN functions in a Ca(2)(+)-dependent manner that involves the activation of a pertussis toxin-sensitive G-protein.

Many receptor-linked agents that prime or activate the NADPH oxidase in polymorphonuclear neutrophils (PMNs) elicit changes in cytosolic Ca2+ concentration and activate mitogen-activated protein (MAP) kinases. To investigate the role of Ca2+ in the activation of p38 and p42/44 MAP kinases, we examined the effects of the Ca2+-selective ionophore ionomycin on priming and activation of the PMN oxidase. Ionomycin caused a rapid rise in cytosolic Ca2+ that was due to both a release of cytosolic Ca2+ stores and Ca2+ influx. Ionomycin also activated (2 μM) and primed (20–200 nM) the PMN oxidase. Dual phosphorylation of p38 MAP kinase and phosphorylation of its substrate activating transcription factor-2 were detected at ionomycin concentrations that prime or activate the PMN oxidase, while dual phosphorylation of p42/44 MAP kinase and phosphorylation of its substrate Elk-1 were elicited at 0.2–2 μM. SB-203580, a p38 MAP kinase antagonist, inhibited ionomycin-induced activation of the oxidase (68 ± 8%, P < 0.05) and tyrosine phosphorylation of 105- and 72-kDa proteins; conversely, PD-98059, an inhibitor of MAP/extracellular signal-related kinase 1, had no effect. Treatment of PMNs with thapsigargin resulted in priming of the oxidase and activation of p38 MAP kinase. Chelation of cytosolic but not extracellular Ca2+ completely inhibited ionomycin activation of p38 MAP kinase, whereas chelation of extracellular Ca2+ abrogated activation of p42/44 MAP kinase. These results demonstrate the importance of changes in cytosolic Ca2+ for MAP kinase activation in PMNs.