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

Abstract

Background Although transfusion is a life-saving intervention, it may be associated with significant morbidity in injured patients. We hypothesize that stored red blood cells induce pro-inflammatory activation of human pulmonary microvascular endothelium (HMVECs) resulting in neutrophil (PMN) adhesion and predisposition to acute lung injury (ALI). Methods Ten units of red blood cells (RBCs) were collected and 50% (by weight) were leukoreduced (LR-RBCs) and the remainder was unmodified and stored in additive solution-5 (AS-5). Another 10 units of RBCs were collected, leukoreduced, and stored in AS-3. HMVECs were incubated with [10–40%]FINAL of the supernatants day (D)1-D42 of storage, lipid extracts and purified lipids. Endothelial surface expression of ICAM-1, IL-8 release, and PMN adhesion to HMVECs were measured. HMVEC signaling via the BLT2 receptor was evaluated. Supernatants and lipids were also employed as the first event in a two-event model of ALI. Results The supernatants [10–40%]FINAL from D21 LR-RBCs and D42 RBCs and LR-RBCs and the lipids from D42 stored in AS-5 induced increased ICAM-1 surface expression on endothelium, IL-8 release, and PMN adhesion. In addition, the supernatants [20–40%]FINAL from D21 and D42 RBCs in AS-5 also increased endothelial surface expression of ICAM-1. D42 supernatants and lipids also caused co-precipitation of β-arrestin-1 with BLT2, PKCβI, and PKCδand served as the first event in a two-event rodent model of ALI. In conclusion, lipids that accumulate during RBC storage activate endothelium and predispose to ALI, which may explain some of the adverse events associated with the transfusion of critically injured patients.

Contrasting effects of stored allogeneic red blood cells and their supernatants on permeability and inflammatory responses in human pulmonary endothelial cells

“…In agreement with earlier studies, we also found that RBC supernatants possess barrier-disruptive properties (38,46,50,52,56,67). Surprisingly, supernatants from fresh RBCs or old RBCs had same degree of deleterious effects on ECs with that of old RBCs, suggesting that these harmful substances are present in RBC from the beginning, opposing the existing theory that they accumulate during storage.…”

“…Many in vitro and in vivo studies suggested possible mechanisms of RBC transfusion-associated pathologies (40,42,68). It was shown that accumulation of bioactive lipids detected in RBC with longer storage duration (49,52) can cause neutrophil activation through a G protein-coupled receptor on the neutrophil (18) and through the leukotriene B4 receptor BLT2 and activation of protein kinase C on pulmonary endothelium (50). Other studies demonstrated the increased production of microparticles from RBC and platelets during storage, which triggered coagulation through tissue factor signaling and induced the secretion of proinflammatory cytokines (11,19).…”

Succinate Activation of SUCNR1 Predisposes Severely Injured Patients to Neutrophil-mediated ARDS

“…Histological changes were visualized using the 2-event rat model on H&E stained lung sections (40X) read by a blinded observer. 20,36 PMN sequestration was measured following the first event in flushed, fixed lungs as described. 20,36 Measurement of BALF chemoattractant-1 (CINC-1), Lung Myeloperoxidase (MPO), Liver and Kidney Injury BALF chemokine-induced neutrophil CINC-1 and MPO activity were quantified as described.…”

Outcomes Associated With Early RBC Transfusion in Pediatric Severe Sepsis: A Propensity-Adjusted Multicenter Cohort Study

“…We had hypothesized that early RBC transfusion would be independently associated with fewer organ failure-free days. In preclinical studies, RBC transfusion can contribute to many of the pathophysiologic processes that result in organ dysfunction, including lung injury, renal injury, inflammation, and immune suppression (24)(25)(26)(27)(28)(29)(30)(31). The extent to which RBC transfusion contributes to organ dysfunction in critically ill children remains unclear.…”