Marguerite R. Kelher

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.

Plasma and lipids from stored platelets cause acute lung injury in an animal model

BACKGROUND : Transfusion of PLT concentrates may cause TRALI, a life‐threatening reaction that has been linked to the infusion of anti‐WBC immunoglobulins or older, stored PLTs that contain bioactive lipids. We hypothesize that lipids generated during storage of PLTs cause TRALI in a two‐event animal model.

Plasma from stored packed red blood cells and MHC class I antibodies causes acute lung injury in a 2-event in vivo rat model

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion death. We hypothesize that TRALI requires 2 events: (1) the clinical condition of the patient and (2) the infusion of antibodies against MHC class I antigens or the plasma from stored blood. A 2-event rat model was developed with saline (NS) or endotoxin (LPS) as the first event and the infusion of plasma from packed red blood cells (PRBCs) or antibodies (OX18 and OX27) against MHC class I antigens as the second event. ALI was determined by Evans blue dye leak from the plasma to the bronchoalveolar lavage fluid (BALF), protein and CINC-1 concentrations in the BALF, and the lung histology. NS-treated rats did not evidence ALI with any second events, and LPS did not cause ALI. LPS-treated animals demonstrated ALI in response to plasma from stored PRBCs, both prestorage leukoreduced and unmodified, and to OX18 and OX27, all in a concentration-dependent fashion. ALI was neutrophil (PMN) dependent, and OX18/OX27 localized to the PMN surface in vivo and primed the oxidase of rat PMNs. We conclude that TRALI is the result of 2 events with the second events consisting of the plasma from stored blood and antibodies that prime PMNs.

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.

Physiological levels of interleukin-18 stimulate multiple neutrophil functions through p38 MAP kinase activation

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<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.

Platelet-Activating Factor-Induced Clathrin-Mediated Endocytosis Requires β-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 β-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 μM 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, β-arrestin-1 or dynamin-2 were neutralized by intracellular introduction of specific Abs. PAFR ligation caused 1) coprecipitation of the PAFR and clathrin with β-arrestin-1, 2) fluorescent resonance energy transfer-positive interactions among the PAFR, β-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 β-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 β-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, β-arrestin-1 recruitment of a p38 MAPK signalosome, and specific actin bundle formation at the PAFR for transduction before endosomal scission.

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

Transfusion-related acute lung injury (TRALI) remains a significant cause of transfusion-related mortality with red cell transfusion. We hypothesize that prestorage filtration may reduce proinflammatory activity in the red blood cell (RBC) supernatant and prevent TRALI. Filters were manufactured for both small volumes and RBC units. Plasma containing antibodies to human lymphocyte antigen (HLA)-A2 or human neutrophil antigen (HNA)-3a was filtered, and immunoglobulins and specific HNA-3a and HLA-2a neutrophil (PMN) priming activity were measured. Antibodies to OX27 were added to plasma, and filtration was evaluated in a 2-event animal model of TRALI. RBC units from 31 donors known to have antibodies against HLA antigens and from 16 antibody-negative controls were filtered. Furthermore, 4 RBC units were drawn and underwent standard leukoreduction. Immunoglobulins, HLA antibodies, PMN priming activity, and the ability to induce TRALI in an animal model were measured. Small-volume filtration of plasma removed >96% of IgG, antibodies to HLA-A2 and HNA-3a, and their respective priming activity, as well as mitigating antibody-mediated in vivo TRALI. In RBC units, experimental filtration removed antibodies to HLA antigens and inhibited the accumulation of lipid priming activity and lipid-mediated TRALI. We conclude that filtration removes proinflammatory activity and the ability to induce TRALI from RBCs and may represent a TRALI mitigation step.

The role of endothelial activation in the pathogenesis of transfusion-related acute lung injury

ALI = acute lung injury; ARDS = acute respiratory distress syndrome; EBS = Earl’s buffered saline; FP = fresh plasma; ICAM-1 = intracellular adhesion molecule-1; NBF = National Blood Foundation; PMN(s) = polymorphonuclear leukocyte(s).From the Bonfils Blood Center and the Departments of Pediatrics and Surgery, University of Colorado School of Medicine, Denver, Colorado.

Proteomic Analyses of Human Plasma: Venus versus Mars

BACKGROUND: Plasma is vital for the resuscitation of injured patients and to restore necessary procoagulants, especially Factors (F)II, FV, FVII, FX, and FXIII; however, female plasma has been implicated in the majority of transfusion‐related acute lung injury (TRALI) cases and male‐only plasma transfusion regimens have significantly decreased the incidence of TRALI. Little is known about the human plasma proteome, and no comparisons have been made between male and female plasma; therefore, we hypothesize that there are significant differences between plasma from male and female donors.