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.

In Huntington disease (HD), polyglutamine expansion causes the disease protein huntingtin to aggregate and accumulate in the nucleus and cytoplasm. The cytoplasmic huntingtin aggregates are found in axonal terminals and electrophysiological studies show that mutant huntingtin affects synaptic neurotransmission. However, the biochemical basis for huntingtin-mediated synaptic dysfunction is unclear. Using electron microscopy on sections of HD mouse brains, we found that axonal terminals containing huntingtin aggregates often had fewer synaptic vesicles than did normal axonal terminals. Subcellular fractionation and electron microscopy revealed that mutant huntingtin is co-localized with huntingtin-associated protein-1 (HAP1) in axonal terminals in the brains of HD transgenic mice. Mutant huntingtin binds more tightly to synaptic vesicles than does normal huntingtin, and it decreases the association of HAP1 with synaptic vesicles in HD mouse brains. Brain slices from HD transgenic mice that had axonal aggregates showed a significant decrease in [(3)H]glutamate release, suggesting that neurotransmitter release from synaptic vesicles was impaired. Taken together, these findings suggest that mutant huntingtin has an abnormal association with synaptic vesicles and this association impairs synaptic function.

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.

At the onset of endospore formation in Bacillus subtilis the DNA-binding protein Spo0A directly activates transcription from promoters of about 40 genes. One of these promoters, Pskf, controls expression of an operon encoding a killing factor that acts on sibling cells. AbrB-mediated repression of Pskf provides one level of security ensuring that this promoter is not activated prematurely. However, Spo0A also appears to activate the promoter directly, since Spo0A is required for Pskf activity in a ⌬abrB strain. Here we investigate the mechanism of Pskf activation. DNase I footprinting was used to determine the locations at which Spo0A bound to the promoter, and mutations in these sites were found to significantly reduce promoter activity. The sequence near the ؊10 region of the promoter was found to be similar to those of extended ؊10 region promoters, which contain a TRTGn motif. Mutational analysis showed that this extended ؊10 region, as well as other base pairs in the ؊10 region, is required for Spo0A-dependent activation of the promoter. We found that a substitution of the consensus base pair for the nonconsensus base pair at position ؊9 of Pskf produced a promoter that was active constitutively in both ⌬abrB and ⌬spo0A ⌬abrB strains. Therefore, the base pair at position ؊9 of Pskf makes its activity dependent on Spo0A binding, and the extended ؊10 region motif of the promoter contributes to its high level of activity.

A. Tumor necrosis factor-␣ causes release of cytosolic interleukin-18 from human neutrophils.

Patients with sepsis and acute lung injury have increased interleukin (IL)-18 levels systemically. We hypothesize that IL-18 stimulates neutrophils (PMNs) at physiologic concentrations. IL-18 primed the oxidase at 15 min (10–100 ng/ml), 30 min (0.1–100 ng/ml), and 60 min (100 ng/ml; P&lt;0.05) and caused translocation of p47phox to the membrane similar to lipopolysaccharides. CD11b surface expression was increased by IL-18 in a time- and concentration-dependent manner. IL-18 caused up-regulation of the formyl-Met-Leu-Phe receptor, changes in PMN size, and elastase release. Investigation of signaling demonstrated IL-18-mediated activation of p38 mitogen-activated protein (MAP) kinase in a concentration (0.1–100 ng/ml)-, time (5–15 min)-, and Ca2+-dependent manner. IL-18 directly increased cytosolic Ca2+ concentration. IL-18 activation of PMNs was blocked by inhibition of p38 MAP kinase activity (SB203580) or by inhibition of p38 MAP kinase activation by chelation of cytosolic Ca2+. We conclude that IL-18, at physiologic concentrations, is an effective PMN priming agent that requires p38 MAP kinase activity.