PMN Priming Research Articles

G-CSF Infusion for Stem Cell Mobilization Transiently Increases Serum Cell-Free DNA and Protease Concentrations.

G-CSF for stem cell mobilization increases circulating levels of myeloid cells at different stages of maturation. Polymorphonuclear cells (PMNs) are also mobilized in high numbers. It was previously reported that G-CSF primes PMNs toward the release of neutrophils extracellular traps (NETs). Since NETs are often involved in thrombotic events, we hypothesized that high G-CSF blood concentrations could enhance PMN priming toward NET formation in healthy hematopoietic stem cell donors, predisposing them to thrombotic events. However, we found that G-CSF does not prime PMNs toward NETs formation, but increases the serum concentration of cell-free DNA, proteases like neutrophils elastase and myeloperoxidase, and reactive oxygen species. This could possibly create an environment disposed to induce thrombotic events in the presence of additional predisposing factors.

Primed PMNs in healthy mouse and human circulation are first responders during acute inflammation

AbstractPolymorphonuclear neutrophils (PMNs) are the most abundant circulating leukocytes, and the first cells recruited to sites of tissue inflammation. Using a fixation method to preserve native CD marker expression prior to immunophenotyping, we identified a distinct population of “primed for recruitment” PMNs in healthy mouse and human blood that has high expression of adhesion and activation markers compared with the bulk resting-state PMNs. In response to acute tissue inflammation, primed PMNs (pPMNs) were rapidly depleted from the circulation and recruited to the tissue. One hour after acute peritoneal insult, pPMNs became the dominant PMN population in bone marrow (BM) and blood, returning to baseline levels with resolution of inflammation. PMN priming was induced by the granulopoietic factors granulocyte-macrophage–colony-stimulating factor (GM-CSF) and granulocyte–colony-stimulating factor (G-CSF). High levels of pPMNs were observed in neutropenic mice and in pediatric neutropenic patients who were resistant to infection, highlighting an important role of this population in innate immune function.

Blood clotting and traumatic injury with shock mediates complement-dependent neutrophil priming for extracellular ROS, ROS-dependent organ injury and coagulopathy.

Polymorphonuclear (PMN) leucocytes participate in acute inflammatory pathologies such as acute respiratory distress syndrome (ARDS) following traumatic injury and shock, which also activates the coagulation system systemically. Trauma can prime the PMN nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex for an enhanced respiratory burst, but the relative role of various priming agents in this process remains incompletely understood. We therefore set out to identify mediators of PMN priming during coagulation and trauma-shock and determine whether PMN reactive oxygen species (ROS) generated in this manner could influence organ injury and coagulation. Initial experiments demonstrated that PMN are primed for predominantly extracellular ROS production by products of coagulation, which was abrogated by CD88/C5a receptor(C5aR) inhibition. The importance of this was highlighted further by demonstrating that known PMN priming agents result in fractionally different amounts of extracellular versus intracellular ROS release depending on the agent used. Plasma from trauma patients in haemodynamic shock (n=10) also primed PMN for extracellular ROS in a C5a-dependent manner, which correlated with both complement alternative pathway activation and thrombin generation. Furthermore, PMN primed by preincubation with products of blood coagulation directly caused loss of endothelial barrier function in vitro that was abrogated by C5aR blockade or NADPH oxidase inhibition. Finally, we show in a murine model of trauma-shock that p47phox knock-out (KO) mice with PMN incapable of generating ROS were protected from inflammatory end-organ injury and activated protein C-mediated coagulopathy. In summary, we demonstrate that trauma-shock and coagulation primes PMN for predominantly extracellular ROS production in a C5a-dependent manner that contributes to endothelial barrier loss and organ injury, and potentially enhances traumatic coagulopathy.

Endotoxemia induces lung-brain coupling and multi-organ injury following cerebral ischemia-reperfusion

Post-ischemic neurodegeneration remains the principal cause of mortality following cardiac resuscitation. Recent studies have implicated gastrointestinal ischemia in the sepsis-like response associated with the post-cardiac arrest syndrome (PCAS). However, the extent to which the resulting low-grade endotoxemia present in up to 86% of resuscitated patients affects cerebral ischemia-reperfusion injury has not been investigated. Here we report that a single injection of low-dose lipopolysaccharide (50μg/kg, IP) delivered after global cerebral ischemia (GCI) induces blood-brain barrier permeability, microglial activation, cortical injury, and functional decline in vivo, compared to ischemia alone. And while GCI was sufficient to induce neutrophil (PMN) activation and recruitment to the post-ischemic CNS, minimal endotoxemia exhibited synergistic effects on markers of systemic inflammation including PMN priming, lung damage, and PMN burden within the lung and other non-ischemic organs including the kidney and liver. Our findings predict that acute interventions geared towards blocking the effects of serologically occult endotoxemia in survivors of cardiac arrest will limit delayed neurodegeneration, multi-organ dysfunction and potentially other features of PCAS. This work also introduces lung-brain coupling as a novel therapeutic target with broad effects on innate immune priming and post-ischemic neurodegeneration following cardiac arrest and related cerebrovascular conditions.

A Common Genetic Variant in TLR1 Enhances Human Neutrophil Priming and Impacts Length of Intensive Care Stay in Pediatric Sepsis.

Polymorphonuclear leukocytes (PMN) achieve an intermediate or primed state of activation following stimulation with certain agonists. Primed PMN have enhanced responsiveness to subsequent stimuli, which can be beneficial in eliminating microbes but may cause host tissue damage in certain disease contexts, including sepsis. As PMN priming by TLR4 agonists is well described, we hypothesized that ligation of TLR2/1 or TLR2/6 would prime PMN. Surprisingly, PMN from only a subset of donors were primed in response to the TLR2/1 agonist, Pam3CSK4, although PMN from all donors were primed by the TLR2/6 agonist, FSL-1. Priming responses included generation of intracellular and extracellular reactive oxygen species, MAPK phosphorylation, integrin activation, secondary granule exocytosis, and cytokine secretion. Genotyping studies revealed that PMN responsiveness to Pam3CSK4 was enhanced by a common single-nucleotide polymorphism (SNP) in TLR1 (rs5743618). Notably, PMN from donors with the SNP had higher surface levels of TLR1 and were demonstrated to have enhanced association of TLR1 with the endoplasmic reticulum chaperone gp96. We analyzed TLR1 genotypes in a pediatric sepsis database and found that patients with sepsis or septic shock who had a positive blood culture and were homozygous for the SNP associated with neutrophil priming had prolonged pediatric intensive care unit length of stay. We conclude that this TLR1 SNP leads to excessive PMN priming in response to cell stimulation. Based on our finding that septic children with this SNP had longer pediatric intensive care unit stays, we speculate that this SNP results in hyperinflammation in diseases such as sepsis.

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.

A Comparison of Different Methods of RBC Leukoreduction and Their Ability to Minimize Neutrophil Priming Activity during Storage

Background:Pre-storage leukoreduction (LR) of whole blood (WB) or red blood cells (RBC) reduces white blood cell (WBC) and platelet contamination and decreases the amount of biologic response modifiers (BRMs) implicated in the development of transfusion-related acute lung injury (TRALI). There are two primary methods of LR used world-wide, however there has not yet been a side-by-side comparison of the buffy coat method to the in-line filtration, in terms of the ability to minimize the neutrophil (PMN) priming activity in the supernatant. We hypothesize that LR by buffy coat is equal to LR by filtration in minimizing PMN priming activity.Method:Five units of WB were drawn from healthy volunteer donors. Plasma was removed by centrifugation and the RBC units were equally divided by weight; 50% were LR by buffy coat removal and 50% were LR by in-line filtration (Haemonetics RC2D) into bags containing AS-3 additive solution (110 ml) and stored at 2-6oC per AABB criteria. Sterile couplers were used to obtain samples weekly during storage. The supernantant was isolated by centrifugation (5,000g for 7 min, followed by 12,000g for 6 min) and stored at -80oC. PMNs were isolated from heparinized WB from healthy volunteer donors by standard techniques. PMNs were incubated with plasma samples for 5 min at 37oC then activated with 1uM formyl-MLF (fMLF) and the maximal rate of O2- was measured. Priming activity is the augmentation of the maximal rate of superoxide production in response to fMLF. Supernatants were immunoblotted for peroxiredoxin-6, an intracellular RBC enzyme.Results:LR by filtration decreased WBC amount by >3 logs and platelets by >2 logs whereas LR by buffy coat reduced both WBC and platelets by only a single log. The supernatants from both buffy coat and filtration LR showed no difference in PMN priming vs. albumin or buffer-treated controls on day (D) 1 and demonstrated an increase in O2- production on D1 vs. D42 of storage (Table 1). However, the supernatants from buffy coat LR RBCs demonstrated significantly greater PMN priming activity as compared to supernatants from filter LR RBCs (2.4-fold). Samples from D14 and D28 also showed a statistically significant greater increase in O2- production in buffy coat LR samples compared to filtered samples. Additionally, there was increased immunoreactivity for peroxiredoxin-6 in buffy coat supernatants vs. filtered RBC supernatants on identical days of storage (Fig.1).Discussion:Filter LR is more effective in reducing WBC and platelet contamination, and significantly decreases the accumulation of PMN priming activity and peroxiredoxin-6 immunoreactivity in the RBC supernatant. LR via filtration appears to produce RBC units with less cellular contamination, decreased BRMs and less release of intracellular enzymes. Further testing is required to determine if these LR RBC units are clinically safer than RBCs which undergo buffy coat LR. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A sphingosine-1 phosphate agonist (FTY720) limits trauma/hemorrhagic shock-induced multiple organ dysfunction syndrome.

Trauma/hemorrhagic shock (T/HS) is one of the major consequences of battlefield injury as well as civilian trauma. FTY720 (sphingosine-1-phosphate agonist) has the capability to decrease the activity of the innate and adaptive immune systems and, at the same time, maintain endothelial cell barrier function and vascular homeostasis during stress. For this reason, we hypothesize that FTY720, as part of resuscitation therapy, would limit T/HS-induced multiple organ dysfunction syndrome in a rodent T/HS model. Rats subjected to trauma/sham shock (T/SS) or T/HS (30 mm Hg × 90 min) were administered FTY720 (1 mg/kg) post-T/HS during volume resuscitation. Lung injury (permeability to Evans blue dye), polymorphonuclear leukocyte (PMN) priming (respiratory burst activity), and red blood cell (RBC) rigidity were measured. In addition, lymph duct-cannulated rats were used to quantify the effect of FTY720 on gut injury (permeability and morphology) and the biologic activity of T/HS versus T/SS lymph on PMN-RBC and RBC deformability. Trauma/hemorrhagic shock-induced increased lung permeability, PMN priming, and RBC rigidity were all abrogated by FTY720. The systemic protective effect of FTY720 was only partially at the gut level, because FTY720 did not prevent T/HS-induced gut injury (morphology or permeability); however, it did abrogate T/HS lymph-induced increased respiratory burst and RBC rigidity. FTY720 limited T/HS-induced multiple organ dysfunction syndrome (lung injury, red cell injury, and neutrophil priming) as well as T/HS lymph bioactivity, although it did not limit gut injury.

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

AbstractTransfusion-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.

Endotoxin Priming of Neutrophils Requires Endocytosis and NADPH Oxidase-dependent Endosomal Reactive Oxygen Species

NADPH oxidase 2 (Nox2)-generated reactive oxygen species (ROS) are critical for neutrophil (polymorphonuclear leukocyte (PMN)) microbicidal function. Nox2 also plays a role in intracellular signaling, but the site of oxidase assembly is unknown. It has been proposed to occur on secondary granules. We previously demonstrated that intracellular NADPH oxidase-derived ROS production is required for endotoxin priming. We hypothesized that endotoxin drives Nox2 assembly on endosomes. Endotoxin induced ROS generation within an endosomal compartment as quantified by flow cytometry (dihydrorhodamine 123 and Oxyburst Green). Inhibition of endocytosis by the dynamin-II inhibitor Dynasore blocked endocytosis of dextran, intracellular generation of ROS, and priming of PMN by endotoxin. Confocal microscopy demonstrated a ROS-containing endosomal compartment that co-labeled with gp91(phox), p40(phox), p67(phox), and Rab5, but not with the secondary granule marker CD66b. To further characterize this compartment, PMNs were fractionated by nitrogen cavitation and differential centrifugation, followed by free flow electrophoresis. Specific subfractions made superoxide in the presence of NADPH by cell-free assay (cytochrome c). Subfraction content of membrane and cytosolic subunits of Nox2 correlated with ROS production. Following priming, there was a shift in the light membrane subfractions where ROS production was highest. CD66b was not mobilized from the secondary granule compartment. These data demonstrate a novel, nonphagosomal intracellular site for Nox2 assembly. This compartment is endocytic in origin and is required for PMN priming by endotoxin.

Blood Neutrophil Immune Functions are Altered in PFAPA Syndrome

Background: PFAPA syndrome is a non-mendelian autoinflammatory disease of unknown etiology characterized by Periodic Fever, Aphthous stomatitis, Pharyngitis, and cervical Adenitis. In typical cases, attacks begin before the age of five and occur every 2-8 weeks with striking periodicity. We have previously profiled the blood cells and serum cytokine levels in a cohort study of patients with PFAPA (Brown et al, BMC Pediatric 2010:10, 65) and the purpose of the present study was to investigate neutrophil function during the febrile and afebrile state in the same cohort. Methods: Neutrophils (PMN) were isolated from blood collected from twelve PFAPA patients during febrile and afebrile phase (median age of 3.5 yrs). PMN apoptosis was assessed by annexin V staining, production of reactive oxygen species (ROS) by luminol/isoluminol-amplified chemiluminescence, MPO quantity by ELISA, and degranulation/priming as surface expression of CD11b and L-selectin by flow cytometry. Results: We found a significantly increased intracellular (ic) production of ROS in response to phorbol myristate acetate in PMN during the febrile phase, as compared to cells from the afebrile phase and controls. No differences were found in MPO quantity between the samples, indicating that the increase in icROS-production was due to increased NADPH-oxidase activation. We also found decreased spontaneous apoptosis in PMN during the febrile phase of PFAPA, while apoptosis was instead increased during the afebrile phase. Increased expression of CD11b indicated that the PMN were slightly primed during the febrile phase, supported by increased oxidative response to galectin-3, a priming-dependent process. Conclusion: Alterations in PMN function, e.g., icROS-production, priming and spontaneous apoptosis, may give mechanistic clues to the so-far undefined etiology of the PFAPA syndrome.

Experimental Antibody Filtration Inhibits Antibody-Mediated Neutrophil Priming and TRALI in An In Vivo model

Abstract 41Transfusion-related acute lung injury (TRALI) is a leading cause of transfusion-related death with a majority of the reported cases secondary to the infusion of antibodies (Abs) contained within the plasma/blood component. An experimental filter that removes IgG was developed. We hypothesize that filtration of plasma with antibodies to leukocyte antigens will decrease both antibody-mediated priming of PMNs and antibody-mediated TRALI in a two-event in vivo model. Methods: Human plasma was drawn from healthy volunteers and IgG concentrations were measured before and after filtration. Plasma was obtained from two multiparous female donors: one with antibodies to HLA-A2 and to DR7 and the other with antibodies against HNA-3a. These plasma samples were filtered (F-Plas) or left as an unmodified control (Plas) and the anti-leukocyte antibodies were measured in a blinded fashion in referral labs using flow cytometry and Luminex™ beads or standard granulocyte antibody detection assays. These plasma samples were then used to prime the fMLP-activated respiratory burst, measured as the SOD-inhibitable reduction of cytochrome c (nmol O2−/min), of PMNs from HNA-3a+ donors or donors homozygous donors for HLA-A2, respectively. For the two-event in vivo modeling rats were incubated with 2 μg/ml endotoxin (LPS, S. enteritides) or saline (NS) for 2 hours (first event) and then were transfused with heat-treated human plasma that contained 25 μg/ml of an antibody against the MHC class I antigen OX27 that was either filtered (or left unmodified) prior to infusion (second event) followed by Evans Blue dye (EBD). ALI was measured as %EBD leak from the plasma into the bronchoalveolar lavage fluid. Statistical differences were measured via paired (PMN priming) or independent (in vivo TRALI) ANOVA, and data are reported as the mean ± the standard error of the mean. *=p<.05 vs. all groups (Table). Results: Plasma filtration removed 98±2.1% of IgG from normal plasma and both the antibodies to HNA-3a and HLA-A2 such that they were no longer detected (HLA-A2: 94 Luminex™ units (LU) pre-filtration and 0 LU units post-filtration and DR7: 30 LU pre-filtration and 0 LU post-filtration). In addition, filtration also inhibited the priming activity of the plasma containing antibodies to HNA-3a and HLA-A2 on HNA-3A+ PMNs and HLA-A2+ PMNs, respectively. Moreover, the plasma spiked with antibodies to OX27 caused ALI in LPS-treated rats, but not NS treated animals, which was inhibited by filtration. We conclude that this experimental filter removes IgG and detectable amounts of specific antibodies to HLA and HNA ligands as well as obviating the priming activity of these antibodies in PMNs which express the cognate antigens. Filtration of plasma spiked with specific antibodies to MHC ligands also abrogated the antibody-induced TRALI in a two-event, in vivo model. Such a filtration step could mitigate antibody-mediated TRALI.Abs/Tx'sfMLP (O2− nmol/min)Plas+ fMLPF-plas+ fMLPNS/plas (% EBD)NS/F-PlasLPS/PlasLPS/F-PlasHNA-3a0.7 ± .31.1 ± .2*0.6 ± .2HLA-A21.6 ± .63.4 ± .4*1.5 ± .4OX270.09 ± .020.13 ± .030.4 ± .14*0.13 ± .03 Disclosures:Silliman:Pall Corporation: Honoraria. Sowemimo-Coker:Pall Medical Corporation: Employment.

The pro‐inflammatory effects of platelet contamination in plasma and mitigation strategies for avoidance

Plasma and platelet concentrates are disproportionately implicated in transfusion-related acute lung injury (TRALI). Platelet-derived pro-inflammatory mediators, including soluble CD40 ligand (sCD40L), accumulate during storage. We hypothesized that platelet contamination induces sCD40L generation that causes neutrophil [polymorphonuclear leucocyte (PMN)] priming and PMN-mediated cytotoxicity. Plasma was untreated, centrifuged (12,500 g) or separated from leucoreduced whole blood (WBLR) prior to freezing. Platelet counts and sCD40L concentrations were measured 1-5 days post-thaw. The plasma was assayed for PMN priming activity and was used in a two-event in vitro model of PMN-mediated human pulmonary microvascular endothelial cell (HMVEC) cytotoxicity. Untreated plasma contained 42±4·2×10(3)/μl platelets, which generated sCD40L accumulation (1·6-eight-fold vs. controls). Priming activity and HMVEC cytotoxicity were directly proportional to sCD40L concentration. WBLR and centrifugation reduced platelet and sCD40L contamination, abrogating the pro-inflammatory potential. Platelet contamination causes sCD40L accumulation in stored plasma that may contribute to TRALI. Platelet reduction is potentially the first TRALI mitigation effort in plasma manufacturing.

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.

Thrombin Activation of Protease-Activated Receptor-2 on Neutrophils Primes the Respiratory Burst.

AbstractAbstract 1119Thrombin is a multifunctional serine protease involved in hemostasis, fibrinolysis, and pro-inflammatory activation of innate immunity through stimulation of protease-activated receptors (PARs). Thrombin is considered the prototypical protease to activate PARs through cleavage of the N-terminal exodomain of the receptor, thereby unmasking a tethered ligand which activates it. Neutrophils (PMNs) are reported to express PAR-2; however, little is known about the changes in PMN physiology following protease activation of PAR-2. In addition, proteomic analyses (two dimensional gel electrophoresis and mass spectroscopy (MALDI-TOF)) of injured patients (blunt trauma) who developed acute lung injury (ALI) and multiple organ failure (MOF) have identified a number of serine proteases which accumulate post-injury. Moreover, these identical proteases were also found via proteomics in stored, but not fresh, packed red blood cells, which are used to resuscitate these injured patients. We hypothesize that thrombin primes PMNs through activation of PAR-2. Methods: PMNs were isolated from whole blood drawn from healthy human donors using dextran sedimentation, ficoll-hypaque gradient centrifugation and hypotonic lysis of red blood cells. The proteins from whole cell lysates were separated by SDS-polyacrylamide electrophoresis, transferred to nitrocellulose and immunoblotted with a monoclonal antibody to PAR-2. The PMNs were also incubated for 3–30 minutes at 37°C with thrombin [1-10 U/ml] followed by fMLP [1 μM] activation of the NADPH oxidase. Oxidase activity was measured by the superoxide dismutase-inhibitable reduction of cytochrome c at 550 nm. Results: PMNs express PAR-2 as detected by western blotting of whole cell lysates. Thrombin [1 U/ml] for 30 min, significantly primed the fMLP-activated respiratory burst: buffer control: 1.6 +/− 0.5 vs. 1 U/ml thrombin: 2.2 +/− 0.3* (*p<.05, n=5). Higher concentrations of thrombin or shorter incubation times did not induce PMN priming. Furthermore, pre-incubation with leupeptin [50 μM] and 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) [250 μM], two effective protease inhibitors, abrogated the thrombin-induced PMN priming by 100±18%. We conclude that thrombin primes the PMN respiratory burst, which is dependent upon its protease activity. Because PMNs are the effector cell in ALI/MOF and priming agents have been linked to the development of clinical ALI and MOF, proteases that accumulate in the patient plasma post-injury or are transfused with stored PRBCs may be etiologic in post-injury ALI/MOF. Disclosures:No relevant conflicts of interest to declare.

Mirasol Pathogen Reduction Technology®treatment does not affect acute lung injury in a two-event in vivo model caused by stored blood components

Mirasol Pathogen Reduction Technology (PRT) treatment uses riboflavin and UV light to inactivate pathogens in blood components. Neutrophil [polymorphonuclear cells (PMN)] priming activity accumulates during routine storage of cellular blood components, and this activity has been implicated in transfusion-related acute lung injury (TRALI). We hypothesize that PRT-treatment of blood components affects the priming activity generated during storage of packed RBCs (PRBCs) or platelet concentrates (PCs), which can elicit ALI in vivo. Plasma, PRBCs and PCs were isolated from healthy donor's whole blood or by apheresis. Half of a collected unit was treated with PRT treatment and the remainder was left as an unmodified control. Supernatant was collected during storage of PCs and PRBCs and assayed for PMN priming activity and used as the second event in a two-event in vivo model of TRALI. PRT treatment did not induce priming activity in plasma or affect the priming activity generated during storage of PCs or PRBCs as compared with the unmodified controls. The supernatants from stored, but not fresh, PCs and PRBCs did cause ALI as the second event in a two-event animal model of TRALI, which was unaffected by PRT treatment. We conclude that the PRT treatment does not induce priming activity in plasma nor does it affect the priming activity generated during storage of PCs or PRBCs or their ability to cause ALI as the second event in a two-event in vivo model of TRALI. Moreover, the amount of priming activity in TRIMA-isolated PCs was significantly less than SPECTRA-isolated PCs.

Systemic Not Just Mesenteric Lymph Causes Neutrophil Priming After Hemorrhagic Shock

Inflammatory mediators in postshock mesenteric lymph have been causally linked to systemic polymorphonuclear cells (PMNs) priming resulting in acute lung injury (ALI) and multiple organ failure. Earlier human and animal studies demonstrated ALI after lower limb ischemia/reperfusion (I/R) injury. As hemorrhagic shock (HS) is in essence a systemic I/R insult, we postulated that systemic lymph after HS would exhibit PMN priming and this was studied in vitro. Lymph was collected at intervals from the hind limb of dogs subjected to sham or HS and crystalloid resuscitation. Human PMNs isolated from heparinized blood of normal volunteers were incubated with buffer, sham lymph, or lymph after 120 minutes of shock or resuscitation. PMN priming was indexed by CD11b expression (mean fluorescence intensity), superoxide anion (O2(-)) generation (nanomoles/mg protein), and elastase release (%) after the addition of fMLP (1 micromol). PMNs with buffer served as control. PMN priming after exposure to either shock or postshock resuscitation lymph was noted by increased expression of CD11b, superoxide generation, and elastase release after exposure to fMLP. No priming effect was noted with sham lymph. Maximal bioactivity of shock or postresuscitation shock lymph was noted at 2 hours postresuscitation. Exposure with systemic lymph after HS resulted in PMN priming. These results question the unique properties attributed to post-HS lymph from the splanchnic bed in causing PMN priming and ALI after shock. The causal agent(s) for these effects are unclear.

ABERRANT REGULATION OF POLYMORPHONUCLEAR PHAGOCYTE RESPONSIVENESS IN MULTITRAUMA PATIENTS

A systemic inflammatory response often follows severe trauma. Priming (preactivation) of polymorphonuclear phagocytes (PMNs) is an essential first step in the processes that lead to damage caused by the systemic activation of innate immune response. Until recently, priming could only accurately be measured by functional assays, which require isolation of cells, thereby potentially inducing artificial activation. The aim of this study was to identify primed PMNs in response to trauma by using a whole blood analysis with a broad detection range. Twenty-two trauma patients were analyzed for PMN priming with novel developed antibodies recognizing priming epitopes by flow cytometric analysis. Expression of priming epitopes on PMNs was analyzed with respect to time, injury, and disease severity. Expression of priming epitopes in the circulation was compared with expression profiles of PMNs obtained from lung fluid. Fourteen healthy volunteers served as controls. Expression of priming epitopes on peripheral blood PMNs of injured patients was similar, as found in healthy controls, whereas highly primed cells were found in the lung fluid of injured patients (increase of >50 times as compared with peripheral blood cells). In fact, the responsiveness of PMNs toward the bacteria-derived stimulus N-formyl-methionyl-leucyl-phenylalanine was markedly decreased in trauma patients. Lack of expression of priming epitopes and the unresponsiveness to N-formyl-methionyl-leucyl-phenylalanine demonstrates the presence of partially refractory cells in the circulation of trauma patients. An increased expression of epitopes found on pulmonary PMNs suggests that optimal (pre)activation of these cells only occurs in the tissues.

BIOACTIVITY OF POSTSHOCK MESENTERIC LYMPH DEPENDS ON THE DEPTH AND DURATION OF HEMORRHAGIC SHOCK

Mesenteric hypoperfusion due to circulatory shock is a key event in the pathogenesis of subsequent distant organ injury. Postshock mesenteric lymph (PSML) has been shown to contain proinflammatory mediators elaborated from the ischemic gut. We hypothesize that the relative bioactivity of PSML depends on the depth and duration of circulatory shock. To first determine the timing of PSML bioactivity, we subjected rats to hemorrhagic shock (30 mm Hg x 45 min) and then resuscitation with 50 vol% of shed blood and normal saline (4x shed blood) over 2 h. Mesenteric lymph was collected hourly up to 6 h after shock. Superoxide release was measured from human neutrophils (polymorphonuclear neutrophils [PMNs]) incubated with lymph fractions collected from each of the hourly time points. Rats were then subjected to four different shock variations: (1) 30 mm Hg x 45 min, (2) 30 mm Hg x 15 min, (3) 45 mm Hg x 45 min, and (4) 45 mm Hg x 15 min, and were resuscitated. PSML flow depends on depth of shock, but not duration of shock or resuscitation volume. Maximal PSML bioactivity, as measured by PMN priming for the respiratory burst, occurred during the third postshock hour, which correlated with peak lymph flow rate. PSML bioactivity was greatest with 30 mm Hg x 45 min, followed by 30 mm Hg x 15 min, 45 mm Hg x 45 min, and 45 mm Hg x 15 min. Hemorrhagic shock provokes the release of bioactive agents in PSML that is dependent on both depth and duration of shock.

NEUTRALIZATION OF KC AND MIP-2; DIVERGENT EFFECTS ON ACTIVATION PATHWAYS IN MOUSE PMN CELL LINE -CONSISTENT WITH MOUSE BLOOD PMN

Acute lung injury (ALI) is associated with the targeting and sequestration of PMN in the lung. Chemokines, produced by resident and non-resident pulmonary cells in response to inflammatory stimuli, activate pathways that regulate PMN chemotaxis, apoptosis and cytolytic activity. We have shown that systemic antibody blockade of murine PMN chemokines keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2) and local pulmonary silencing with KC or MIP-2 siRNA differentially effect lung tissue injury and inflammation in a mouse model of hemorrhage (Hem) induced PMN priming for ALI following septic challenge. To further investigate the divergent roles KC and MIP-2 play while sharing affinity for a common CXCR2-like receptor, we examined phosphorrylation patterns for proteins associated with PMN activation. Previously we reported differences in the %-phospho-AKT, ERK1/2 and p38, 12-14H-post systemic antibody neutralization of KC or MIP-2 following Hem. This data suggested that KC and MIP-2 function through different downstream signaling pathways. In order to further test our hypothesis of divergent activation pathways, an inducible, terminally differentiated PMN cell line (MPRO) was used. MPRO cells, 3 × 104 cells/well in 96 well plates, were stimulated with 50 nM platelet activating factor and treated with either 100 ng/ml anti-KC or 25 ng/ml anti-MIP-2 or IgG. Phospho- vs. total protein was assessed for pathway activation proteins AKT, ERK1/2 and p38 by ELISA method (SuperArray CASE Kit). Phosphorylation profiles for antibody vs. IgG treated MPRO cells for AKT, ERK1/2 and p38 and were consistent with those obtained from mouse blood PMN. The %-phospho-p38 and AKT decreased below IgG levels in the anti-MIP-2 treated cells. While %-phospho-ERK1/2 was decreased below IgG, there was no difference between antibody treatments. These findings support our hypothesis that murine PMN chemokines, KC and MIP-2, differentially stimulate activation pathways that regulate PMN activity. (NIH HL73525)