Two-event Model Research Articles

Human neutrophil antigen 3 genotype impacts neutrophil-mediated endothelial cell cytotoxicity in a two-event model of TRALI.

Antibodies against human neutrophil antigen (HNA)-3a are associated with severe cases of transfusion-related acute lung injury (TRALI). The HNA-3 system is located on choline transporter-like 2 (CTL-2) protein. CTL-2 is encoded by the gene SLC44A2 and a single-nucleotide polymorphism c.461G>A results in two antigens: HNA-3a and HNA-3b. Three HNA-3 genotypes/ phenotypes exist: HNA-3aa, HNA-3bb, and HNA-3ab. Two different pathways of anti-HNA-3a TRALI have been described: a two-hit neutrophil-dependent pathway and a one-hit neutrophil-independent pathway. However, it is not clear whether HNA-3ab heterozygous patients have a lower risk of anti-HNA-3a-mediated TRALI compared to HNA-3aa homozygous patients. Healthy volunteers were genotyped for HNA-3 by real-time polymerase chain reaction, and phenotyped for HNA-3a by granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT) against two donor sera containing anti-HNA-3a antibodies. The two sera were also used in in vitro models of human pulmonary microvascular endothelial cell (HLMVEC) cytotoxicity to investigate pathways of TRALI development. For both anti-HNA-3a sera, GIFT results matched the genotype, with a lower GIFT ratio for HNA-3ab neutrophils compared to HNA-3aa neutrophils, whereas GAT results showed no difference in agglutination. HLMVEC cytotoxicity was not observed in a one-hit neutrophil-independent model but was observed in a two-hit neutrophil-dependent model. Differences in cytotoxicity were observed between the two anti-HNA-3a sera used. Consistent with reduced HNA-3a antigen density as measured by GIFT, HNA-3ab neutrophils mediated less HLMVEC cytotoxicity than HNA-3aa neutrophils. HNA-3 genotype and HNA-3a antigen expression impacted the severity of anti-HNA-3a-mediated HLMVEC cytotoxicity in a two-hit neutrophil-dependent model of TRALI. Different HNA-3a antibodies might also impact the magnitude of HLMVEC cytotoxicity.

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). 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. 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 β-arrestin-1 with BLT2, protein kinase C (PKC)βI , and PKCδ and served as the first event in a two-event rodent model of ALI. 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.

Antibodies to major histocompatibility complex class II antigens directly prime neutrophils and cause acute lung injury in a two-event in vivo rat model.

Transfusion-related acute lung injury (TRALI) is a significant cause of mortality, especially after transfusions containing antibodies to major histocompatibility complex (MHC) class II antigens. We hypothesize that a first event induces both 1) polymorphonuclear neutrophils (PMNs) to express MHC class II antigens, and 2) activation of the pulmonary endothelium, leading to PMN sequestration, so that the infusion of specific MHC class II antibodies to these antigens causes PMN-mediated acute lung injury (ALI). Rats were treated with saline (NS), endotoxin (lipopolysaccharide [LPS]), or cytokines (interferon-γ [IFNγ], macrophage colony-stimulating factor [MCSF], tumor necrosis factor-α [TNFα]); the PMNs were isolated; and the surface expression of the MHC class II antigen OX6 and priming by OX6 antibodies were measured by flow cytometry or priming assays. A two-event model of ALI was completed with NS, LPS, or IFNγ/MCSF/TNFα (first events) and the infusion of OX6 (second event). Compared with NS incubation, rats treated with either LPS or IFNγ/MCSF/TNFα exhibited OX6 PMN surface expression, OX6 antibodies primed the formyl-methionyl-leucyl phenylalanine (fMLF)-activated respiratory burst, and PMN sequestration was increased. OX6 antibody infusion into LPS-incubated or IFNγ/MCSF/TNFα-incubated rats elicited ALI, the OX6 antibody was present on the PMNs, and PMN depletion abrogated ALI. Proinflammatory first events induce PMN MHC class II surface expression, activation of the pulmonary endothelium, and PMN sequestration such that the infusion of cognate antibodies precipitates TRALI.

α-Enolase Causes Proinflammatory Activation of Pulmonary Microvascular Endothelial Cells and Primes Neutrophils Through Plasmin Activation of Protease-Activated Receptor 2.

Proinflammatory activation of vascular endothelium leading to increased surface expression of adhesion molecules and neutrophil (PMN) sequestration and subsequent activation is paramount in the development of acute lung injury and organ injury in injured patients. We hypothesize that α-enolase, which accumulates in injured patients, primes PMNs and causes proinflammatory activation of endothelial cells leading to PMN-mediated cytotoxicity. Proteomic analyses of field plasma samples from injured versus healthy patients were used for protein identification. Human pulmonary microvascular endothelial cells (HMVECs) were incubated with α-enolase or thrombin, and intercellular adhesion molecule-1 surface expression was measured by flow cytometry. A two-event in vitro model of PMN cytotoxicity HMVECs activated with α-enolase, thrombin, or buffer was used as targets for lysophosphatidylcholine-primed or buffer-treated PMNs. The PMN priming activity of α-enolase was completed, and lysates from both PMNs and HMVECs were immunoblotted for protease-activated receptor 1 (PAR-1) and PAR-2 and coprecipitation of α-enolase with PAR-2 and plasminogen/plasmin. α-Enolase increased 10.8-fold in injured patients (P < 0.05). Thrombin and α-enolase significantly increased intercellular adhesion molecule-1 surface expression on HMVECs, which was inhibited by antiproteases, induced PMN adherence, and served as the first event in the two-event model of PMN cytotoxicity. α-Enolase coprecipitated with PAR-2 and plasminogen/plasmin on HMVECs and PMNs and induced PMN priming, which was inhibited by tranexamic acid, and enzymatic activity was not required. α-Enolase increases after injury and may activate pulmonary endothelial cells and prime PMNs through plasmin activity and PAR-2 activation. Such proinflammatory endothelial activation may predispose to PMN-mediated organ injury.

Two-event Transfusion-related Acute Lung Injury Mouse Model

Transfusion-related acute lung injury (TRALI) is defined as acute lung injury that occurs within 6 hours of a blood product transfusion. TRALI continues to be a leading cause of transfusion-related mortality and we have developed a mouse model of TRALI to better understand the mechanisms by which injury occurs and to test therapeutic approaches. Our model is a two-event model based on immune priming and the challenge of BALB/c wild-type mice with cognate MHC Class I monoclonal antibody (MHC I mAb). Immune priming with LPS mimics the primed state of recipients (first event) that is important for the development of TRALI. Donor HLA antibodies are frequently implicated in TRALI reactions, and cognate MHC Class I antibody (second event) produces acute lung injury in primed animals. Here, we describe a detailed protocol with high reproducibility within animals.

The co-operative interaction of puroindolines in wheat grain texture may involve the hydrophobic domain

The puroindoline genes are causatively associated with wheat grain hardness, a commercially significant property. The proteins puroindoline (PIN) A and B are both required in their wild-type (WT) to impart soft grain texture, and absence of/mutations in either/both PIN(s) results in hard wheat. However, there is no biochemical clarity yet that explains this interdependence. This work critically analyses the roles of the tryptophan-rich domain (TRD), the little-known hydrophobic domain (HD), and certain other residues, in the physical associations of PINs. Site-directed mutagenesis-PCR was used to delete the TRD or HD and introduce an Arg39Gly substitution in PINA. The PINB-D1c mutant (Leu60Pro) was also investigated. The yeast two-hybrid system was used to assess the protein–protein interactions (PPI) of proteins. The TRD deletion or Arg39Gly substitution in PINA did not adversely affect its PPI, while deletion of HD resulted in a significant reduction. No effect on PPI was observed for Leu60Pro PINB. The results of this expression system strongly suggest that the HD is essential (but not sufficient) in higher-order associations of PINs. We propose a two-event model that explains the co-operative action of the PINs and why mutations outside the TRD may alter grain texture.

Antibodies to the HLA-A2 antigen prime neutrophils and serve as the second event in anin vitromodel of transfusion-related acute lung injury

Transfusion-related acute lung injury (TRALI) is the most common cause of transfusion-related mortality and has been linked to the infusion of donor antibodies directed against recipient HLA class I antigens. We hypothesize that antibodies against HLA class I antigens bind to the antigens on the neutrophil (PMN) surface and induce priming and PMN cytotoxicity as the second event in a two-event in vitro model of PMN-mediated cytotoxicity. Isolated PMNs from HLA-A2 homozygotes, heterozygotes and null donors were incubated with a monoclonal antibody to HLA-A2 and a human polyclonal IgG to HLA-A2 and priming of the oxidase was measured. The monoclonal antibodies and PMNs from these three groups were then used in a two-event model of PMN cytotoxicity. The antibodies to HLA-A2 both primed PMNs from HLA-A2 homozygotes but not from heterozygotes or nulls. Antibodies to HLA-A2 also served as the second event in a two-event model to induce PMN cytotoxicity of HLA-A2 homozygous PMNs. Antibodies to HLA class I antigens may directly prime/activate PMNs through the ligation of the antigen on the cell surface, and the antigen density appears to be important for these changes in PMN physiology.

Human platelets pathogen reduced with riboflavin and ultraviolet light do not cause acute lung injury in a two‐event SCID mouse model

Pathogen reduction technologies (PRTs) can induce platelet (PLT) lesions that reduce PLT survival and recovery from circulation and may be associated with acute lung injury (ALI). Human PLTs (hPLTs) in plasma with or without single or multiple Mirasol PRT treatments were assessed in vitro by aggregation and percentage of P-selectin expression. In vivo studies included PLT recovery in SCID mice and assessment of ALI in a two-event mouse model in which the sensitizing event was lipopolysaccharide injection and the second event was infusion of Mirasol-treated hPLTs. A single-dose Mirasol treatment (5 J/cm(2) ) did not induce any change in aggregation in response to adenosine 5'-diphosphate (ADP) while a five-times-repeat Mirasol treatment (5×) increased aggregation response to low concentration of ADP. Mirasol PLTs (1×-5×) had increased percentage of P-selectin-positive PLTs after treatment and decreased aggregation with TRAP as the agonist. In vivo recovery in SCID mice was reduced extensively with Mirasol treatments (1× and 5×). In the two-event model of ALI, only the 5× Mirasol PLTs accumulated in the lung and this was not accompanied by changes in lung histology or increases in MIP-2 levels in bronchoalveolar lavage fluid. Mirasol PRT treatment induced PLT activation and reduced in vivo recovery in a SCID mouse model of transfusion. In our two-event mouse model of ALI, the 5× Mirasol hPLTs accumulated in the lung, but did not cause signs of ALI. The 1× Mirasol treatment did not lead to PLT lung accumulation or ALI in this model.

Lipids: Free Fatty Acids, Eicosanoids, and Lysophospholipids and the Pro-Inflammatory Effects of Transfusion

Abstract SCI-48Stored packed red blood cells (PRBCs) have been implicated in increased morbidity and mortality in critically ill patients, patients requiring cardiothoracic surgery, and injured patients following blunt and/or penetrating trauma. Lipids are generated during the routine storage of cellular blood components and have been implicated in transfusion-related acute lung injury (TRALI) and postinjury multiple organ failure (MOF). These lipids are comprised of two classes, as denoted by their retention times on normal-phase HPLC: 1) nonpolar lipids, including arachidonic acid, 5-hydroxy-eicosotetranoic acid (HETE), 12-HETE, 15-HETE, and 5-oxo-eicosotetranoic acid (5-oxo-ETE); and 2) a mixture of lysophosphatidylcholines (lyso-PCs), including stearoyl-, oleoyl-, or palmitoyl-lyso-PCs as well as C16 or C18 lyso-platelet activating factor. Importantly, all of these lipids were identified by quadrupole LC/MS/MS. The former are derived from red blood cells, because they are generated during the routine storage of prestorage leuko- and platelet-reduced PRBCs, while the latter are platelet-derived, and are generated during storage of platelet concentrates (PC) and unmodified PRBCs but not leukocyte-reduced PRBCs (LR-PRBCs). Generation of the nonpolar lipids requires an active phospholipase, such as peroxiredoxin-6, which accumulates during PRBC and LR-PRBC storage and appears to be active and T-phosphorylated. The nonpolar lipids do not accumulate during PC storage. These lipids are released into the plasma fraction of the stored component and have been shown to: 1) prime the polymorphonuclear leukocytes (PMN) oxidase; 2) activate primed PMNs; and 3) serve as the second event in a two-event model of TRALI in both rodents and sheep. This lipid priming of PMNs occurs through activation of specific receptors, for example G2A, for lyso-PCs, and causes activation of G-protein-linked cellular receptor, G-protein release, and stimulation of kinase cascades. This results in translocation of the cytosolic oxidase components and a change from a resting PMN phenotype to a hyperactive, adherent phenotype. Conversely, stored PRBCs have also been implicated in MOF. However, acute lung injury does not occur until 72 hours postinjury, implicating PRBCs as a first event(s). The nonpolar lipids that accumulate during routine storage activate primary human pulmonary microvascular endothelial cells (HMVECs) and primary human liver sinusoidal endothelial cells (LSECs), as quantified by increased surface expression of intercellular adhesion molecule-1 (ICAM-1) and the synthesis and release of chemokines, for example IL-8. This proinflammatory activation results in PMN adherence, and such HMVEC activation occurs through activation of the BLT2 receptor and activation of a PKC-dependent kinase cascade. Stored PRBCs (day 42) may serve as the first event in a two-event rodent model of acute lung injury. We conclude that lipids generated during the routine storage of cellular blood components have proinflammatory effects in vitro and in vivo, which may explain some of the adverse events of transfusion, including TRALI and postinjury MOF, and that inhibition and avoidance of these compounds may make transfusions safer. Further research is already under way in several prospective trials, including: 1) Age of Blood Components transfused in the PICU (ABC-PICU); 2) red cell storage duration and outcomes in cardiac surgery at the Cleveland Clinic; 3) Red Cell Storage duration Study (RECESS); and 4) the Age of Blood Evaluation trial (ABLE). Disclosures:Silliman:Pall Corporation: Honoraria, Research Funding.

Host Platelets and, in Part, Neutrophils Mediate Lung Accumulation of Transfused UVB-Irradiated Human Platelets in a Mouse Model of Acute Lung Injury

We previously reported that ultraviolet light B (UVB)-treated human platelets (hPLTs) can cause acute lung injury (ALI) in a two-event SCID mouse model in which the predisposing event was Lipopolysaccharide (LPS) injection and the second event was infusion of UVB-treated hPLTs. To delineate contributions of host mouse platelets (mPLTs) and neutrophils in the pathogenesis of ALI in this mouse model, we depleted mPLTs or neutrophils and measured hPLT accumulation in the lung. We also assessed lung injury by protein content in bronchoalveolar lavage fluid (BALF). LPS injection followed by infusion of UVB-treated hPLTs resulted in sequestration of both mPLTs and hPLTs in the lungs of SCID mice, although the numbers of neutrophils in the lung were not significantly different from the control group. Depletion of mouse neutrophils caused only a mild reduction in UVB-hPLTs accumulation in the lungs and a mild reduction in protein content in BALF. In comparison, depletion of mPLTs almost completely abolished hPLTs accumulation in the lung and significantly reduced protein content in BALF. UVB-treated hPLTs bound to host mPLTs, but did not bind to neutrophils in the lung. Aspirin treatment of hPLTs in vitro abolished hPLT accumulation in the lung and protected mice from lung injury. Our data indicate that host mPLTs accumulated in the lungs in response to an inflammatory challenge and subsequently mediated the attachment of transfused UVB-hPLTs. Neutrophils also recruited a small percentage of platelets to the lung. These findings may help develop therapeutic strategies for ALI which could potentially result from transfusion of UV illuminated platelets.

Extracellular DNA Traps Are Associated with Pathogenesis of TRALI in Humans and Mice

Abstract 37Transfusion-related acute lung injury (TRALI) is a serious complication of blood transfusion occurring within 6 hours of transfusion. It is characterized by hypoxemia, respiratory distress and pulmonary infiltrates (Kleinman et al. Transfusion 2004) and is the leading cause of transfusion-induced death. The biological processes contributing to TRALI are still poorly understood. All blood products can cause TRALI, and to date no specific treatment is available. A “two-event model” has been proposed as the trigger (Silliman et al Blood 2007). The first event may include surgery, trauma or infection; the second involves the transfusion of anti-neutrophil antibodies or bioactive lipids within the blood product. Together these events induce neutrophil activation and sequestration in the pulmonary capillaries (Looney et al J Clin Invest 2006) resulting in endothelial damage and capillary leakage (Shaz et al Blood 2011). In response to pathogens or under stress, activated neutrophils have the ability to release neutrophil extracellular traps (NETs) (Brinkmann Science 2004; Fuchs et al J Cell Biol 2007) that are composed of DNA fibers decorated with histones and antimicrobial proteins (Urban PLoS Pathog 2009) originally contained in the neutrophil granules. Although protective against infection, these NETs are injurious to tissue (Xu et al Nat Med 2009, Xu et al J Immunol 2011), activate platelets (Fuchs et al PNAS 2010) and can contribute to the pathology of inflammatory diseases (Kessenbrock et al Nat Med 2009, Garcia-Romo Sci Transl Med 2011). The purpose of this study was to determine whether NETs are formed in patients during TRALI and whether they contribute to TRALI in a mouse model.Here we show that NETs biomarkers are present in TRALI patients' blood and that the antibody responsible for the most severe TRALI cases and that is directed against human neutrophil antigen (HNA)-3a (Davoren et al Transfusion 2003; Greinacher et al Nat Med 2010) stimulates NETs generation from primed human neutrophils when compared to a control antibody. This effect was inhibited when FcgRIIa-binding sites were blocked prior to antibody incubation. Moreover, anti-HNA-3a F(ab')2 fragments had no effect on NETs generation. These results allowed us to conclude that FcgRIIa activation was required in anti-HNA-3a antibody-mediated NETosis.We next used an in vivo model of antibody-induced TRALI in mice (Looney et al J Clin Invest 2006; Looney et al. J Clin Invest 2009; Hidalgo et al Nat Med 2009). In this two-event model, BALB/c mice are injected i.p. with a low-dose of LPS and 24 hours later are infused with an anti-MHC class I monoclonal antibody (anti-H-2Kd). Two hours later, we measured arterial blood oxygen saturation to document lung function and the lung injury markers were quantified. As observed in blood from TRALI patients, we detected a significant increase in the concentration of circulating NETs biomarkers in mice with TRALI compared to mice that were challenged only with LPS. Multiphoton analysis of fixed lung tissue showed strong staining for citrullinated histone H3 (a phenomenon already described to correlate with NETs formation (Wang et al J Cell Biol 2009; Li et al J Exp Med 2010)) collocalizing with DNA streaks in the alveoli of mice with TRALI. Moreover, we found NETs in abundance in the TRALI-affected alveoli of mice by transmission electron microscopy. We demonstrate that intranasal DNaseI pretreatment of the mice prior to antibody infusion prevented NETs accumulation in the alveoli and also improved the lung function of the mice experiencing TRALI by increasing their arterial blood oxygenation. These results support the hypothesis that NETs are indeed formed in the inflamed lungs during TRALI and contribute to the disease process and thus, could be targeted to prevent or treat TRALI. Disclosures:No relevant conflicts of interest to declare.

A two‐event In vitro model of acute chest syndrome: The role of secretory phospholipase A2 and neutrophils

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. 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. 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. 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.

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

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. RBC units were divided: 50% was leukoreduced (LR-RBCs), stored, and sampled on Day 1 and at the end of storage, Day 42. Priming activity was evaluated on isolated PMNs, and the purified lipids from Day 1 or Day 42 were used as the second event in the in vivo model. The plasma and lipids from RBCs and LR-RBCs primed PMNs, and the LR-RBC activity decreased with longer storage. Unlike RBCs, nonpolar lipids comprised the PMN-priming activity from stored LR-RBCs. Mass spectroscopy identified these lipids as arachidonic acid and 5-, 12-, and 15-hydroxyeicsotetranoic acid. At concentrations from Day 42, but not Day 1, three of four of these lipids individually, and the mixture, primed PMNs. The mixture also caused ALI as the second event in a two-event model of TRALI. We conclude that the nonpolar lipids that accumulate during LR-RBC storage may represent the agents responsible for antibody-negative TRALI.

Transfusion-Related Acute Lung Injury in a Rat Model of Trauma-Hemorrhage

Major trauma often causes hemorrhage and predisposes to transfusion-related acute lung injury (TRALI). TRALI is a leading cause of transfusion-related deaths; however, its pathophysiology is uncertain. In the existing two-event models of TRALI, infection (lipopolysaccharide injection) is followed by the infusion of aged blood products. Our objective was to develop a trauma-relevant two-event model of TRALI by examining the effect of aged packed red blood cells (PRBC) on lung injury in rats with trauma-hemorrhage. Male Lewis rats were used. Rat PRBC were prepared similar to human PRBC. Recipients were implanted with femoral arterial and venous catheters (isoflurane anesthesia) and then subjected to 30% controlled arterial hemorrhage after 16-hour recovery. After a 60-minute shock period, rats were resuscitated with crystalloid and PRBC (0-35 days old; 3:1 ratio) and followed for up to 6 hours. Lung edema was evaluated by Evans blue dye (EBD), protein, and cytokine-induced neutrophil chemoattractant-1 (CINC-1) accumulation in bronchoalveolar lavage fluid, and arterial blood gases were measured (iSTAT). CINC-1 levels increased over time in our PRBC stored for over 21 days. Transfusion survival was reduced, and Evans blue dye, protein, and CINC-1 accumulation in bronchoalveolar lavage fluid were increased in rats transfused with 28-day-old and 35-day-old PRBC compared with the 0-day group. Arterial PO2 and O2 saturation were decreased in rats transfused with 28-day-old and 35-day-old PRBC. However, pH and PCO2 were not different between groups. These results suggest that transfusion of 28-day-old and 35-day-old PRBC reliably promotes lung edema in a rat model of catheter surgery and hemorrhage. We propose that this model can be used as a trauma-relevant two-event model of TRALI.

A novel in vivo ovine model of transfusion‐related acute lung injury (TRALI)

Even with the introduction of specific risk-reduction strategies, transfusion-related acute lung injury (TRALI) continues to be a leading cause of transfusion-related morbidity and mortality. Existing small animal models have not yet investigated TRALI resulting from the infusion of heat-treated supernatant from whole blood platelet concentrates. In this study, our objective was the development of a novel in vivo two-event model of TRALI in sheep. Lipopolysaccharide (LPS; 15 μg/kg) as a first event, modelled clinical infection. Transfusion (estimated at 10% of total blood volume) of heat-treated pooled supernatant from date-of-expire human whole blood platelet concentrates (d5-PLT-S/N) was used as a second event. TRALI was defined by both hypoxaemia that developed either during the transfusion or within two hours of its completion and post-mortem histological evidence of pulmonary oedema. LPS infusion did not cause lung injury itself, but did result in decreased circulating levels of lymphocytes and neutrophils with evidence of the latter becoming sequestered in the lungs. Sheep that received LPS (first event) followed by d5-PLT-S/N (second event) displayed decreased pulmonary compliance, decreased end tidal CO(2) and increased arterial partial pressure of CO(2) relative to control sheep, and 80% of these sheep developed TRALI. This novel ovine two-event TRALI model presents a new tool for the investigation of TRALI pathogenesis. It represents the first description of an in vivo large animal model of TRALI and the first description of TRALI caused by transfusion with heat-treated pooled supernatant from human whole blood platelet concentrates.

α-Enolase From Injured Patients and Stored Packed Red Blood Cells Activates Pulmonary Endothelium and Serves as the First Event In a Two-Event Model of Neutrophil Cytotoxicity

AbstractAbstract 3355A significant number of injured patients with intermediate injury severity scores (15<ISS<30) develop multiple organ failure (MOF), which clinically begins with acute lung injury (ALI). Transfusion of >6 units of stored PRBCs (≥28 days) is associated with the development of ALI/MOF on day 3 post injury (Am J Surg 178:502-4, 1999). The pro-inflammatory mediators, e.g. cytokines, responsible for MOF/ALI in the injured have remained elusive; therefore, we hypothesize that “moonlighting” proteins, which have defined intracellular function when released in the circulation, activate innate immunity and are etiologic in the development of ALI/MOF post-injury. Methods:Proteomics on the field blood (plasma) of 3 patients with blunt trauma who later developed ALI/MOF and the plasma from 3 units of packed red blood cells (PRBCs) on day 1 and day 42 were completed using 2-dimensional gel electrophoresis/mass spectroscopy (MALDI/TOF) with computer analyses of the resultant peptides. The proteins from whole cell lysates from Human pulmonary microvascular endothelial cells (HMVECs) were separted by SDS-PAGE, transferred to nitrocellulse and immunoblotted with antibodies to protease activated receptor-1 (PAR-1) and PAR-2. HMVECs were also incubated for 6 hours and 1) ICAM-1 was measured by flow cytometry, 2) isolated neutrophils (PMNs) were added allowed to settle and in selected wells PMN adherence to these activated HMVECs was measured by myeloperoxidase content in the lysate, or 3) after the PMNs settled, lysophosphatidylcholines (lyso-PCs) [4.5μM], lipids from stored platelets implicated in TRALI, were added and the number of viable HMVECs/mm2 were counted by microscopy. Results:HMVECs display immunoreactivity for both PAR-1 and PAR-2. Of the 243 proteins identified in the injured patients and the stored vs. fresh PRBCs, α-enolase increased by 10.8-fold and 4.4-fold respectively (p<.05 & p<.005). Both thrombin and α-enolase induced ICAM-1 expression in HMVECs (Table 1) which was inhibited (60±8%) by pre-treatment with the anti-protease leupeptin. α-Enolase also induced significant PMN adhesion vs. media control: Media: 3.1±1.5; α-enolase (50 μg/ml): 14.4±4.7; LPS: 35.5±0.7*. The α-enolase-activated HMVECs vs. buffer-treated lyso-PCs induced significant PMN-mediated cytotoxicity (Table 2). We conclude that α-enolase from the injured and stored but not fresh PRBCs causes pro-inflammatory activation of HMVECs resulting in PMN adherence and PMN cytotoxicity in a two-event in vitro model through activation of PARs receptors. Moonlighting proteins like the glycolytic lyase α-enolase may have unexpected pro-inflammatory activity, which predispose the injured patient to increased morbidity and mortality. Disclosures:No relevant conflicts of interest to declare.

Platelet depletion and aspirin treatment protect mice in a two-event model of transfusion-related acute lung injury

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-associated mortality in the US. Previously, we established an immune-mediated TRALI mouse model, wherein mice with cognate antigen were challenged with MHC class I mAb. In this study, when mice housed in a rodent, specific pathogen-free barrier room were challenged with MHC I mAb, there was significant protection from TRALI compared with nonbarrier mice. Priming mice with LPS restored lung injury with mAb challenge. Using TLR4-deficient bone marrow chimeras, the priming phenotype was restricted to animals with WT hematopoietic cells, and depletion of either neutrophils or platelets was protective. Both neutrophils and platelets were sequestered in the lungs of mice with TRALI, and retention of platelets was neutrophil dependent. Interestingly, treatment with aspirin prevented lung injury and mortality, but blocking the P selectin or CD11b/CD18 pathways did not. These data suggest a 2-step mechanism of TRALI: priming of hematopoietic cells, followed by vascular deposition of activated neutrophils and platelets that then mediate the severe lung injury. Furthermore, our data offer an explanation for the increased incidence of TRALI in patients with immune priming conditions, and we suggest what we believe to be a novel therapeutic approach.

Lesión pulmonar aguda producida por transfusión

The term Transfusion-Related Acute Lung Injury (TRALI) was coined in 1985. It is a relatively rare, life-threatening clinical syndrome characterized by acute respiratory failure and non-cardiogenic pulmonary edema during or following a blood transfusion. Although its true incidence is unknown, a rate 1 out of every 5000 transfusions has been quoted. TRALI has been the most common cause of transfusion-related fatalities during three years in the USA. Two different etiologies have been proposed. The first is a single antibody-mediated event involving the transfusion of anti-HLA or antigranulocyte antibodies into patients whose leukocytes express the cognate antigens. The second is a two-event model: the first event is related to the clinical condition of the patient (sepsis, trauma, etc.) resulting in pulmonary endothelial activation and neutrophil sequestration, and the second event is the transfusion of a biologic response modifier that activates these adherent polymorphonuclear leukocytes resulting in endothelial damage and capillary leak. The patient management is support as needed based on the severity of the clinical picture and strategies to prevent TRALI are focused on: donor-exclusion policies, product management strategies and avoidance of unnecessary transfusions.

Hydroxyurea Treatment of Children with Sickle Cell Disease Inhibits RBC Membrane Degradation by Secretory Phospholipase a2.

Background: Secretory phospholipase A2 is an enzyme that is elevated in SCD patients with acute chest syndrome (ACS) and vaso-occlusive pain crisis (VOC) and inhibition of its enzymatic activity is in trials for ACS prevention (Styles LA. Blood. 1996; 87:2573). This enzyme cleaves arachidonic acid from the phospholipids of red blood cell (RBC) membranes; and arachidonic acid can be converted to other pro-inflammatory lipid compounds (Murakami M. J Biochem. 2002; 131:285). sPLA2 cleaves phosphatidylserine (PS) expressing lipid membranes. PS is usually in the inner leaflet (unexposed) in normal red blood cells (RBCs) but becomes externalized in sickle RBCs especially during VOC or ACS. There is modest exposure of PS during in the steady state among certain RBC populations (de Jong K. Br J Haematol. 2006; 133:427). PS exposure allows sPLA2 to cleave RBC membranes, producing lipids which prime neutrophils (PMNs) and cause PMN-mediated pulmonary endothelial cell injury as the second event in a two-event model (Ball JB, Blood submitted).Objective: We hypothesize that sickle RBCs treated with hydroxyurea (HU) will be more resistant to digestion by sPLA2, thereby inhibiting the release of bioactive lipids.Design/Methods: Whole blood was collected from children with SCD when healthy or daily during admissions for VOC or ACS. The plasma and RBCs were separated. Plasma and lipids extracted from the plasma were used as priming agents of quiescent PMNs isolated from healthy donors. Additionally, the separated RBCs were treated with exogenous sPLA2, creating sPLA2-liberated lipids, which were also used as priming agents. The plasma sPLA2 levels were measured.Results: There was no difference in the sPLA2 levels of untreated and HU-treated SCD patients in the healthy condition (non-treated SCD: 12.8±3.1 ng/ml, HU: 12.2±4.2 ng/ml, p=0.95) and in VOC (non-treated SCD: 89.2 ± 79.3 ng/ml, HU: 86.5 ± 44.3 ng/ml, p=0.85). The PMN priming of the sPLA2-liberated lipids of untreated SCD patients, both healthy (*, Table 1) and those with VOC (#, Table 1), was elevated compared to the sPLA2-liberated lipids from HU-treated patients. We conclude that hydroxyurea treatment in vivo induces resistance to sPLA2 cleavage of RBC membrane lipids thereby inhibiting priming of PMNs, which has been implicated in PMN-mediated endothelial cell injury (Ball JB, Blood submitted).Table 1: PMN priming activity of plasma lipids and sPLA2-liberated lipids from patients with SCD.HealthyVOC painplasma lipidssPLA2-liberated lipidsplasma lipidssPLA2-liberated lipidsHgb AA2.55 ± 0.62 nmol O2-/min1.94 ± 0.20 nmol O2-/minuntreated SCD2.21 ± 0.17 nmol O2-/min3.19 ± 0.25 * nmol O2-/min2.73 ± 0.24 nmol O2-/min3.99 ± 0.28 # nmol O2-/minHU-treated SCD2.34 ± 0.47 nmol O2-/min2.75 ± 0.49 nmol O2-/min2.23 ± 0.4 nmol O2-/min2.69 ± 0.52 nmol O2-/min* - p<0.01 compared to untreated plasma lipids from healthy untreated SCD patients# - p<0.01 compared to untreated plasma lipids from untreated SCD patients with VOC

α1-Antitrypsin deficiency is a possible first event in the two-event model of transfusion-related acute lung injury: a proposal and case report

α1-Antitrypsin deficiency is a possible first event in the two-event model of transfusion-related acute lung injury: a proposal and case report