Samina Y. Khan

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.

Transfusion-related acute lung injury (TRALI): Current concepts and misconceptions

Transfusion-related acute lung injury (TRALI) is the most common cause of serious morbidity and mortality due to hemotherapy. Although the pathogenesis has been related to the infusion of donor antibodies into the recipient, antibody negative TRALI has been reported. Changes in transfusion practices, especially the use of male-only plasma, have decreased the number of antibody-mediated cases and deaths; however, TRALI still occurs. The neutrophil appears to be the effector cell in TRALI and the pathophysiology is centered on neutrophil-mediated endothelial cell cytotoxicity resulting in capillary leak and ALI. This review will detail the pathophysiology of TRALI including recent pre-clinical data, provide insight into newer areas of research, and critically assess current practices to decrease it prevalence and to make transfusion safer.

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

Introductionu2002 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.

Lysophosphatidylcholines activate G2A inducing Gαi-1-/Gαq/11-Ca2+ flux, Gβγ-Hck activation and clathrin/β-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(βγ) subunits, kinase activation, and the recruitment of clathrin, β-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(βγ) subunit which demonstrated a physical interaction (FRET+) with activated Hck (haemopoietic cell kinase; Tyr⁴¹¹). Moreover, G2A recruited clathrin, β-arrestin-1 and GRK6: clathrin is important for signal transduction, GRK6 for receptor de-sensitization, and β-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(βγ), resulting in cytosolic Ca²(+) flux, Hck activation, and recruitment of clathrin, β-arrestin-1 and GRK6.

Oncogenic and Angiogenic Growth Factors Accumulate during Routine Storage of Apheresis Platelet Concentrates

Purpose: Platelet concentrates are important for support of patients with malignancies requiring myelotoxic chemotherapy. During storage, 10% to 15% of platelets may become activated resulting in the release of α-granules, which contain growth factors. We hypothesize that, during storage, growth factors accumulate in the plasma, specifically platelet-derived growth factor, vascular endothelial growth factor (VEGF), transforming growth factor-β, and fibroblast growth factor-2, which may adversely affect cancer patients. Experimental Design: The concentrations of growth factors were measured by ELISA from the plasma of apheresis platelets serially throughout storage (days 1, 3, 5, and 7) and compared with concentrations in fresh plasma from healthy blood donors. Washing was evaluated as a method of growth factor removal, and an in vitro model of platelet transfusion in a patient receiving Bevacizumab (Avastin) using immunoprecipitation was employed to determine if Bevacizumab would be bound by the VEGF in apheresis platelets. Results: VEGF, platelet-derived growth factor, and transforming growth factor-β were increased on day 1 versus fresh plasma and throughout storage reaching a relative maximum at outdate (P < 0.01, day 5 or 7). Fibroblast growth factor-2 concentrations were significantly increased on day 7 alone versus day 1 or to fresh plasma (P < 0.01). Washing removed 41 ± 11% to 56 ± 2% of the growth factors. Bevacizumab effectively bound the VEGF from apheresis platelets, with significant amounts of VEGF remaining in the supernatant. Conclusions: Significant amounts of growth factors are present in apheresis platelets due to the isolation procedures, and these concentrations increase over storage, which may be partially removed by washing. In addition, apheresis platelet transfusion could affect cancer treatment by binding monoclonal antibodies directed against growth factors of tumor origin.

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

Background and Objectivesu2002 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.

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

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

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.

Tumor necrosis factor-α 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.