David J. Elzi

Plasma and lipids from stored packed red blood cells cause acute lung injury in an animal model.

Transfusion-related acute lung injury (TRALI) is a serious complication of hemotherapy. During blood storage, lipids are generated and released into the plasma. In this study, the role of these lipids in TRALI was investigated using an isolated, perfused rat lung model. Rats were pretreated with endotoxin (LPS) or saline in vivo and the lungs were isolated, ventilated, and perfused with saline, or (a) 5% (vol/ vol) fresh human plasma, (b) plasma from stored blood from the day of isolation (D.0) or from the day of outdate (D.42), (c) lipid extracts from D.42 plasma, or (d) purified lysophosphatidylcholines. Lungs from saline or LPS-pretreated rats perfused with fresh (D.0) plasma showed no pulmonary damage as compared with saline perfused controls. LPS pretreatment/D.42 plasma perfusion caused acute lung injury (ALI) manifested by dramatic changes in both pulmonary artery pressure and edema. Incubation of LPS pre-tx rats with mibefradil, a Ca2+ channel blocker, or WEB 2170, a platelet-activating factor (PAF) receptor antagonist, inhibited ALI caused by D.42 plasma. Lung histology showed neutrophil sequestration without ALI with LPS pretreatment/saline or D.0 plasma perfusion, but ALI with LPS pretreatment/D.42 plasma perfusion, and inhibition of D.42 plasma induced ALI with WEB 2170 or mibefradil. A significant increase in leukotriene E4 was present in LPS-pretreated/D.42 plasma-perfused lungs that was inhibited by WEB 2170. Lastly, significant pulmonary edema was produced when lipid extracts of D.42 plasma or lysophosphatidylcholines were perfused into LPS-pretreated lungs. Lipids caused ALI without vasoconstriction, except at the highest dose employed. In conclusion, both plasma and lipids from stored blood produced pulmonary damage in a model of acute lung injury. TRALI, like the adult respiratory distress syndrome, may be the result of two insults: one derived from stored blood and the other from the clinical condition of the patient.

Identification of lipids that accumulate during the routine storage of prestorage leukoreduced red blood cells and cause acute lung injury

BACKGROUND: Lipids accumulate during the storage of red blood cells (RBCs), prime neutrophils (PMNs), and have been implicated in transfusion‐related acute lung injury (TRALI). These lipids are composed of two classes: nonpolar lipids and lysophosphatidylcholines based on their retention time on separation by high‐pressure liquid chromatography. Prestorage leukoreduction significantly decreases white blood cell and platelet contamination of RBCs; therefore, it is hypothesized that prestorage leukoreduction changes the classes of lipids that accumulate during storage, and these lipids prime PMNs and induce acute lung injury (ALI) as the second event in a two‐event in vivo model.

Lysophosphatidylcholines prime the NADPH oxidase and stimulate multiple neutrophil functions through changes in cytosolic calcium.

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 Ca2+‐dependent signaling. The lyso‐PC mix (0.45–14.5 μM) 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 Ca2+. Receptor desensitization studies implicated a common receptor or a family of receptors for the observed lyso‐PC‐mediated changes in PMN priming, and cytosolic Ca2+ 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 Ca2+ 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 Ca2+‐dependent manner that involves the activation of a pertussis toxin‐sensitive G‐protein.

Platelet-Activating Factor-Mediated Endosome Formation Causes Membrane Translocation of p67phox and p40phox That Requires Recruitment and Activation of p38 MAPK, Rab5a, and Phosphatidylinositol 3-Kinase in Human Neutrophils

Neutrophils (polymorphonuclear leukocytes, PMNs) are vital to innate immunity and receive proinflammatory signals that activate G protein-coupled receptors (GPCRs). Because GPCRs transduce signals through clathrin-mediated endocytosis (CME), we hypothesized that platelet-activating factor (PAF), an effective chemoattractant that primes the PMN oxidase, would signal through CME, specifically via dynamin-2 activation and endosomal formation resulting in membrane translocation of cytosolic phagocyte oxidase (phox) proteins. PMNs were incubated with buffer or 2 μM PAF for 1–3 min, and in some cases activated with PMA, and O2− was measured, whole-cell lysates and subcellular fractions were prepared, or the PMNs were fixed onto slides for digital or electron microscopy. PAF caused activation of dynamin-2, resulting in endosomal formation that required PI3K and contained early endosomal Ag-1 (EEA-1) and Rab5a. The apoptosis signal-regulating kinase-1/MAPK kinase-3/p38 MAPK signalosome assembled on Rab5a and phosphorylated EEA-1 and Rab GDP dissociation inhibitor, with the latter causing Rab5a activation. Electron microscopy demonstrated that PAF caused two distinct sites for activation of p38 MAPK. EEA-1 provided a scaffold for recruitment of the p40phox-p67phox complex and PI3K-dependent Akt1 phosphorylation of these two phox proteins. PAF induced membrane translocation of p40phox-p67phox localizing to gp91phox, which was PI3K-, but not p47phox-, dependent. In conclusion, PAF transduces signals through CME, and such GPCR signaling may allow for pharmacological manipulation of these cells to decrease PMN-mediated acute organ injury.

Leukotriene b4 and its metabolites prime the neutrophil oxidase and induce proinflammatory activation of human pulmonary microvascular endothelial cells.

Leukotrienes are proinflammatory lipid mediators, derived from arachidonic acid via 5-lipoxygenase (5-LO). Leukotriene B4 (LTB4) is an effective polymorphonuclear neutrophil (PMN) chemoattractant, as well as being a major product of PMN priming. Leukotriene B4 is rapidly metabolized into products that are thought to be inactive, and little is known about the effects of LTB4 on the pulmonary endothelium. We hypothesize that LTB4 and its metabolites are effective PMN priming agents and cause proinflammatory activation of pulmonary endothelial cells. Isolated PMNs were primed (5 min, 37°C) with serial concentrations 10−11 to 10−5 M of LTB4 and its metabolites: 6-trans-LTB4, 20-OH-LTB4, and 20-COOH-LTB4, and then activated with fMLP. Primary human pulmonary microvascular endothelial cells (HMVECs) were incubated with these lipids (6 h, 37°C, 5% CO2), and intercellular adhesion molecule 1 was measured by flow cytometry. Polymorphonuclear neutrophil adhesion was measured by myeloperoxidase assays, and to ensure that these reactions were specific to the LTB4 receptors, BLT1 and BLT2 were antagonized with CP105,696 (BLT1) or silenced with siRNA (BLT1 and BLT2). Leukotriene B4 and its metabolites primed PMNs over a wide range of concentrations, depending on the specific metabolite. In addition, at high concentrations these lipids also caused increases in the surface expression of intercellular adhesion molecule 1 on HMVECs and induced HMVEC-mediated adhesion of PMNs. Silencing of BLT2 abrogated HMVEC activation, and blockade of BLT1 inhibited the observed PMN priming activity. We conclude that LTB4 and its &ohgr;-oxidation and nonenzymatic metabolites prime PMNs over a range of concentrations and activate HMVECs. These data have expanded the repertoire of causative agents in acute lung injury and postinjury multiple organ failure.

LysoPCs induce Hck- and PKCδ-mediated activation of PKCγ causing p47phox phosphorylation and membrane translocation in neutrophils.

Lysophosphatidylcholines (lysoPCs) are effective polymorphonuclear neutrophil (PMN) priming agents implicated in transfusion‐related acute lung injury (TRALI). LysoPCs cause ligation of the G2A receptor, cytosolic Ca2+ flux, and activation of Hck. We hypothesize that lysoPCs induce Hck‐dependent activation of protein kinase C (PKC), resulting in phosphorylation and membrane translocation of 47 kDa phagocyte oxidase protein (p47phox). PMNs, human or murine, were primed with lysoPCs and were smeared onto slides and examined by digital microscopy or separated into subcellular fractions or whole‐cell lysates. Proteins were immunoprecipitated or separated by polyacrylamide gel electrophoresis and immunoblotted for proteins of interest. Wild‐type (WT) and PKCγ knockout (KO) mice were used in a 2‐event model of TRALI. LysoPCs induced Hck coprecipitation with PKCδ and PKCγ and the PKCδ:PKCγ complex also had a fluorescence resonance energy transfer (FRET)+ interaction with lipid rafts and Wiskott‐Aldrich syndrome protein family verprolin‐homologous protein 2 (WAVE2). PKCγ then coprecipitated with p47phox. Immunoblotting, immunoprecipitation (IP), specific inhibitors, intracellular depletion of PKC isoforms, and PMNs from PKCγ KO mice demonstrated that Hck elicited activation/Tyr phosphorylation (Tyr311 and Tyr525) of PKCδ, which became Thr phosphorylated (Thr507). Activated PKCδ then caused activation of PKCγ, both by Tyr phosphorylation (Τyr514) and Ser phosphorylation, which induced phosphorylation and membrane translocation of p47phox. In PKCγ KO PMNs, lysoPCs induced Hck translocation but did not evidence a FRET+ interaction between PKCδ and PKCγ nor prime PMNs. In WT mice, lysoPCs served as the second event in a 2‐event in vivo model of TRALI but did not induce TRALI in PKCγ KO mice. We conclude that lysoPCs prime PMNs through Hck‐dependent activation of PKCδ, which stimulates PKCγ, resulting in translocation of phosphorylated p47phox.

Supernatants and lipids from stored red blood cells activate pulmonary microvascular endothelium through the BLT2 receptor and protein kinase C activation.

Although transfusion is a lifesaving intervention, it may be associated with significant morbidity in injured patients. We hypothesize that stored red blood cells (RBCs) induce proinflammatory activation of human pulmonary microvascular endothelial cells (HMVECs) resulting in neutrophil (PMN) adhesion and predisposition to acute lung injury (ALI).