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

BACKGROUND: 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). STUDY DESIGN AND METHODS: Ten units of RBCs were collected; 50% (by weight) were leukoreduced (LR-RBCs) and the remainder was unmodified and stored in additive solution-5 (AS-5). An additional 10 units of RBCs were collected, leukoreduced, and stored in AS-3. HMVECs were incubated with [10%-40%]FINAL of the supernatants on Day (D)1 to D42 of storage, lipid extracts, and purified lipids. Endothelial surface expression of intercellular adhesion molecule-1 (ICAM-1), interleukin (IL)-8 release, and PMN adhesion to HMVECs were measured. HMVEC signaling via the BLT2 receptor was evaluated. Supernatants and lipids were also employed as the first event in a two-event model of ALI. RESULTS: The supernatants [10%-40%]FINAL from D21 LR-RBCs and D42 RBCs and LR-RBCs and the lipids from D42 stored in AS-5 induced increased ICAM-1 surface expression on endothelium, IL-8 release, and PMN adhesion. In addition, the supernatants [20%-40%]FINAL from D21 and D42 RBCs in AS-5 also increased endothelial surface expression of ICAM-1. D42 supernatants and lipids also caused coprecipitation of ß-arrestin-1 with BLT2, protein kinase C (PKC)ßI , and PKCδ and served as the first event in a two-event rodent model of ALI. CONCLUSION: Lipids that accumulate during RBC storage activate endothelium and predispose to ALI, which may explain some of the adverse events associated with the transfusion of critically injured patients.

A two-event in vitro model of acute chest syndrome: the role of secretory phospholipase A2 and neutrophils.

BACKGROUND: Acute chest syndrome (ACS) in sickle cell disease is associated with elevation of secretory phospholipase A(2) (sPLA(2) ). We hypothesize that sPLA(2) cleaves membrane lipids from sickled red blood cells (RBCs) causing PMN-mediated endothelial cell injury (ECI) as the second event in a two-event model. METHODS: Whole blood was collected from children when in steady state or daily during admissions for vaso-occlusive pain (VOC) or ACS. The plasma and RBCs were separated, sPLA(2) levels were measured, and the RBCs were incubated with sPLA(2) . Plasma and lipids, extracted from the plasma or the supernatant of sPLA(2) -treated RBCs, were assayed for PMN priming activity and used as the second event in a model of PMN-mediated ECI. Phosphatidylserine (PS) surface expression on RBCs was quantified by flow cytometry. RESULTS: Increased sPLA(2) -IIa levels were associated with ACS. SPLA(2) -liberated lipids from VOC and the plasma, plasma lipids and sPLA(2) -liberated lipids from ACS primed PMNs and caused PMN-mediated ECI (P < 0.01). RBCs from VOC had increased in PS surface expression versus steady state. CONCLUSIONS: ACS plasma and lipids and sPLA(2) -released lipids from RBCs during VOC or ACS induce PMN-mediated ECI. VOC elicited increases in PS surface expression providing a membrane substrate for sPLA(2) lysis of sickle RBCs.

Lysophosphatidylcholines activate G2A inducing G(αi)₋1-/G(αq/)11- Ca²(+) flux, G(ßγ)-Hck activation and clathrin/ß-arrestin-1/GRK6 recruitment in PMNs.

Lyso-PCs (lysophosphatidylcholines) are a mixture of lipids that accumulate during storage of cellular blood components, have been implicated in TRALI (transfusion-related acute lung injury) and directly affect the physiology of neutrophils [PMNs (polymorphonuclear leucocytes)]. Because the G2A receptor, expressed on PMNs, has been reported to recognize lyso-PCs, we hypothesize that lyso-PC activation of G2A causes the increases in cytosolic Ca²(+) via release of G(α) and G(ßγ) subunits, kinase activation, and the recruitment of clathrin, ß-arrestin-1 and GRK6 (G-protein receptor kinase 6) to G2A for signal transduction. PMNs were isolated by standard techniques, primed with lyso-PCs for 5-180 s, and lysed for Western blot analysis, immunoprecipitation or subcellular fractionation, or fixed and smeared on to slides for digital microscopy. The results demonstrated that lyso-PCs cause rapid activation of the G2A receptor through S-phosphorylation and internalization resulting in G(αi)₋1 and G(αq/)11 release leading to increases in cytosolic Ca²(+), which was inhibited by an antibody to G2A or intracellular neutralization of these subunits. Lyso-PCs also caused the release of the G(ßγ) subunit which demonstrated a physical interaction (FRET+) with activated Hck (haemopoietic cell kinase; Tyr4¹¹). Moreover, G2A recruited clathrin, ß-arrestin-1 and GRK6: clathrin is important for signal transduction, GRK6 for receptor de-sensitization, and ß-arrestin-1 both propagates and terminates signals. We conclude that lyso-PC activation of G2A caused release of G(αi)₋1, G(αq/)11 and G(ßγ), resulting in cytosolic Ca²(+) flux, Hck activation, and recruitment of clathrin, ß-arrestin-1 and GRK6.

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

Neutrophils (PMNs) are a vital part of host defense and are the principal leukocyte in innate immunity. Interleukin (IL)-18 is a proinflammatory cytokine with roles in both innate and adaptive immunity. We hypothesize that PMNs contain preformed IL-18, which is released in response to specific inflammatory stimuli. Isolated PMNs were stimulated with a battery of chemoattractants (5 min to 24 h), and IL-18 release was measured. PMNs were also separated into subcellular fractions and immunoblotted with antibodies against IL-18 or were fixed and probed with antibodies to IL-18 as well as to the contents of granules, intracellular organelles, and filamentous actin (F-actin), incubated with fluorescent secondary antibodies, and examined by digital microscopy. Quiescent PMNs contained IL-18 in the cytoplasm, associated with F-actin, as determined by positive fluorescence resonance energy transfer (FRET+). In turn, TNF-alpha stimulation disrupted the association of IL-18 with F-actin, induced a FRET+ interaction of IL-18 with lipid rafts, and elicited IL-18 release. Manipulation of F-actin status confirmed the relationship between IL-18 and F-actin in resting PMNs. Consequently, incubation with monomeric IL-18 binding protein inhibited TNF-alpha-mediated priming of the PMN oxidase. We conclude that human PMNs contain IL-18 associated with F-actin in the cytoplasm and TNF-alpha stimulation causes dissociation of IL-18 from F-actin, association with lipid rafts, and extracellular release. Extracellular IL-18 participates in TNF-alpha priming of the PMN oxidase as demonstrated by inhibition with the IL-18 binding protein.

Amantadine inhibits platelet-activating factor induced clathrin-mediated endocytosis in human neutrophils.

Receptor signaling is integral for adhesion, emigration, phagocytosis, and reactive oxygen species production in polymorphonuclear neutrophils (PMNs). Priming is an important part of PMN emigration, but it can also lead to PMN-mediated organ injury in the host. Platelet-activating factor (PAF) primes PMNs through activation of a specific G protein-coupled receptor. We hypothesize that PAF priming of PMNs requires clathrin-mediated endocytosis (CME) of the PAF receptor (PAFr), and, therefore, amantadine, known to inhibit CME, significantly antagonizes PAF signaling. PMNs were isolated by standard techniques to >98% purity and tested for viability. Amantadine (1 mM) significantly inhibited the PAF-mediated changes in the cellular distribution of clathrin and the physical colocalization [fluorescence resonance energy transfer positive (FRET+)] of early endosome antigen-1 and Rab5a, known components of CME and similar to hypertonic saline, a known inhibitor of CME. Furthermore, amantadine had no effect on the PAF-induced cytosolic calcium flux; however, phosphorylation of p38 MAPK was significantly decreased. Amantadine inhibited PAF-mediated changes in PMN physiology, including priming of the NADPH oxidase and shape change with lesser inhibition of increases in CD11b surface expression and elastase release. Furthermore, rimantadine, an amantadine analog, was a more potent inhibitor of PAF priming of the N-formyl-methionyl-leucyl-phenylalanine-activated oxidase. PAF priming of PMNs requires clathrin-mediated endocytosis that is inhibited when PMNs are pretreated with either amantadine or rimantadine. Thus, amantadine and rimantadine have the potential to ameliorate PMN-mediated tissue damage in humans.

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.

Transfusion-related acute lung injury.

Transfusion-related acute lung injury (TRALI) is a life-threatening adverse event of transfusion, which has an increasing incidence in the United States and is the leading cause of transfusion-related death. TRALI and acute lung injury (ALI) share a common clinical definition except that TRALI is temporally- and mechanistically-related to transfusion of blood or blood components. A number of different models have been proposed to explain the pathogenesis. The first is an antibody-mediated event whereby transfusion of anti-HLA, class I or class II, or anti-granulocyte antibodies into patients whose leukocytes express the cognate antigens. The antibody:antigen interaction causes complement-mediated pulmonary sequestration and activation of neutrophils (PMNs) resulting in TRALI. The second is a two-event model: the first event is the clinical condition of the patient resulting in pulmonary endothelial activation and PMN sequestration, and the second event is the transfusion of a biologic response modifier (including anti-granulocyte antibodies, lipids, and CD40 ligand) that activates these adherent PMNs resulting in endothelial damage, capillary leak, and TRALI. These hypotheses are discussed with respect to animal models and human studies that provide the experimental and clinical relevance. The definition of TRALI, patient predisposition, treatment, prevention and reporting guidelines are also examined.

Structural organization of the neutrophil NADPH oxidase: phosphorylation and translocation during priming and activation.

The reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is part of the microbicidal arsenal used by human polymorphonuclear neutrophils (PMNs) to eradicate invading pathogens. The production of a superoxide anion (O2-) into the phagolysosome is the precursor for the generation of more potent products, such as hydrogen peroxide and hypochlorite. However, this production of O2- is dependent on translocation of the oxidase subunits, including gp91phox, p22phox, p47phox, p67phox, p40phox, and Rac2 from the cytosol or specific granules to the plasma membrane. In response to an external stimuli, PMNs change from a resting, nonadhesive state to a primed, adherent phenotype, which allows for margination from the vasculature into the tissue and chemotaxis to the site of infection upon activation. Depending on the stimuli, primed PMNs display altered structural organization of the NADPH oxidase, in that there is phosphorylation of the oxidase subunits and/or translocation from the cytosol to the plasma or granular membrane, but there is not the complete assembly required for O2- generation. Activation of PMNs is the complete assembly of the membrane-linked and cytosolic NADPH oxidase components on a PMN membrane, the plasma or granular membrane. This review will discuss the individual components associated with the NADPH oxidase complex and the function of each of these units in each physiologic stage of the PMN: rested, primed, and activated.

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 to 10 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 ω-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.

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 muM PAF for 1-3 min, and in some cases activated with PMA, and O(2)(-) 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 p40(phox)-p67(phox) complex and PI3K-dependent Akt1 phosphorylation of these two phox proteins. PAF induced membrane translocation of p40(phox)-p67(phox) localizing to gp91(phox), which was PI3K-, but not p47(phox)-, 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.

Platelet-activating factor-induced clathrin-mediated endocytosis requires beta-arrestin-1 recruitment and activation of the p38 MAPK signalosome at the plasma membrane for actin bundle formation.

Clathrin-mediated endocytosis (CME) is a common pathway used by G protein-linked receptors to transduce extracellular signals. We hypothesize that platelet-activating factor (PAF) receptor (PAFR) ligation requires CME and causes engagement of beta-arrestin-1 and recruitment of a p38 MAPK signalosome that elicits distinct actin rearrangement at the receptor before endosomal scission. Polymorphonuclear neutrophils were stimulated with buffer or 2 microM PAF (1 min), and whole cell lysates or subcellular fractions were immunoprecipitated or slides prepared for colocalization and fluorescent resonance energy transfer analysis. In select experiments, beta-arrestin-1 or dynamin-2 were neutralized by intracellular introduction of specific Abs. PAFR ligation caused 1) coprecipitation of the PAFR and clathrin with beta-arrestin-1, 2) fluorescent resonance energy transfer-positive interactions among the PAFR, beta-arrestin-1, and clathrin, 3) recruitment and activation of the apoptosis signal-regulating kinase-1/MAPK kinase-3/p38 MAPK (ASK1/MKK3/p38 MAPK) signalosome, 4) cell polarization, and 5) distinct actin bundle formation at the PAFR. Neutralization of beta-arrestin-1 inhibited all of these cellular events, including PAFR internalization; conversely, dynamin-2 inhibition only affected receptor internalization. Selective p38 MAPK inhibition globally abrogated actin rearrangement; however, inhibition of MAPK-activated protein kinase-2 and its downstream kinase leukocyte-specific protein-1 inhibited only actin bundle formation and PAFR internalization. In addition, ASK1/MKK3/p38 MAPK signalosome assembly appears to occur in a novel manner such that the ASK1/p38 MAPK heterodimer is recruited to a beta-arrestin-1 bound MKK3. In polymorphonuclear neutrophils, leukocyte-specific protein-1 may play a role similar to fascin for actin bundle formation. We conclude that PAF signaling requires CME, beta-arrestin-1 recruitment of a p38 MAPK signalosome, and specific actin bundle formation at the PAFR for transduction before endosomal scission.