Hemorrhagic shock continues to be a major cause of death following traumatic injury (1). In this patient population, the transfusion of blood and blood products is lifesaving and one of the most commonly implemented therapies in modern medicine (2). Treatment with blood products, especially in large volume, has long been associated with adverse clinical outcomes including multisystem organ failure (MOF), immunosuppression, increased postoperative infections, and death (3–6). Other studies have identified a host of molecular and biochemical changes that occur during packed red blood cell (pRBC) storage, collectively known as the red blood cell storage lesion, as the etiology of massive transfusion related morbidity (6–8). Although these changes are documented in the literature, there is little understanding as to how agents in donor blood interact with the transfusion recipient to cause harm.
Microparticles (MPs) are submicron vesicles that are bound by lipid membranes derived from their cell of origin (9). Initially presumed to be rudimentary debris from apoptotic cells, these particles have now been shown to be capable of intercellular signaling and have been implicated in many disease processes (10–13). We have previously shown that pRBC units demonstrate increased MP concentrations during storage and that these MPs are associated with increased lung inflammation. The mechanism of action of pRBC-derived microparticles on transfusion recipients is unknown (14).
In the present study, we hypothesized that MPs generated by stored pRBC activate endothelial cells and promote leukocyte migration into the lung.
Male C57BL/6 mice weighing 21 g to 30 g were purchased from Jackson Laboratories (Bar Harbor, Maine), fed standard laboratory diet and water ad libitum, and acclimated for 1 week in a climate-controlled room with a 12-h light–dark cycle. All experiments were approved by the Institutional Animal Care and Use Committee of the University of Cincinnati.
Murine blood banking
Mice were anesthetized with 0.1 mg/g body weight intraperitoneal pentobarbital and whole blood was collected by open cardiac puncture. Citrate phosphate double dextrose (CP2D) anticoagulant was then added in a ratio of 1:7. Serial centrifugation was used to remove platelet-rich plasma and the leukocyte-rich buffy coat. The red blood cell pellet was resuspended in standard erythrocyte storage medium (additive solution-3 [AS3]) at a ratio of 2:9. These packed red blood cell units were stored at 4°C for 14 days with gentle agitation and protected from light. Previous studies have demonstrated that 14 days of storage in murine red blood cells is approximately equivalent to 42 days of storage in human red blood cells, which is the current Food and Drug Administration (FDA) limit of storage for packed red blood cells in the United States (15). At the end of the storage period, the cellular portion of the pRBC was removed by centrifugation at 300 × g for 10 min. Cellular debris and platelets were removed by centrifugation at 10,000 × g for 10 min. MPs were washed with phosphate buffered saline and isolated using ultracentrifugation at 20,000 × g for 30 min (16, 17).
In control experiments, isolated MPs were stained with antibodies (BD Biosciences, San Jose, Calif) for Ter119 (a murine erythrocyte surface marker), CD45 (leukocyte marker), and CD41 (platelet marker) and analyzed with flow cytometry as previously described (18). This analysis indicated that the microparticles isolated from pRBC units were predominantly erythrocyte in origin, with less than 5% of leukocyte or platelet origin.
In vitro model
Murine endothelial cells were obtained from ATCC microbiology (hemangioendothelioma cells, CRL-2586) (Manassas, Va). Cells were grown to confluence in Dulbecco Modified Eagles Medium supplemented with 10% fetal bovine serum. Confluent layers of endothelial cells were treated at 37°C with MPs derived from 1 mL of murine pRBCs suspended in 1 mL of media. Cells treated with media alone and media with 20 ng/mL tumor necrosis factor-α (TNF-α) served as negative and positive controls respectively. Cell culture supernatants taken at 30 min, and 6, 12, and 24 h were evaluated for interleukin-6 (IL-6) concentration using a pre-adsorbed sandwich ELISA kit from eBioscience (Santa Clara, Calif). Immunofluorescence staining for endothelial-leukocyte adhesion molecule-1 (ELAM-1, E-selectin) and intercellular adhesion molecule-1 (ICAM-1) was performed on confluent layers of endothelial cells grown on glass cover slips after 30 min, 6, 12, and 24 h of treatment.
In vivo model
In vivo experiments were conducted with healthy male C57BL/6 mice weighing 21 g to 30 g after induction of isoflurane anesthesia. MPs isolated from 1 mL of pRBCs suspended in 200 μL of lactated Ringers were transfused via penile vein injection to simulate the MP burden resulting from a massive transfusion, commonly defined as 10 units of pRBCs in 24 h. Control mice treated with an equivalent volume of lactated Ringers served as controls. Mice were sacrificed at 1, 4, 8, and 24 h and whole lungs were harvested and fixed immediately in neutral buffered formalin and embedded in paraffin. Thin cuts of pulmonary alveolar sections were then stained with monoclonal antibody to murine Ly-6g (eBioscience, San Diego, Calif) to identify pulmonary interstitial immune cells that express murine myeloid antigen Gr-1, a marker for granulocytes. Quantification of leukocytes per high power field (HPF) was performed on sets of four mice per experimental group by an observer blinded to the experimental groups.
Analysis of adhesion molecule expression was performed via immunofluorescent staining. After the exposure settings for each channel were normalized, a total of eight random captures of each slide were performed using imaging software ZEN 2012 version 18.104.22.168 on Axio Imager M2 microscope (Carl Zeiss AG, Jena, Germany). Images were taken at ×10 magnification to maximize the number of cells per capture. The sub-channel specific to the fluorophores representing each adhesion molecule was analyzed using the image analysis package ImageJ version 1.49 v (Wayne Rasband, National Institutes of Health, Bethesda, Md).
In vitro experiments were conducted in triplicate. Animal experiments were performed with four mice in each experimental group and four mice in the control group. Results are reported as means and standard deviations where applicable. Two-tailed Students t tests were performed and P values less than 0.05 were deemed significant.
In initial experiments, we first sought to determine the effect of MPs isolated from stored pRBC units on endothelial cell activation. Treatment of confluent endothelial cells with pRBC-derived MPs resulted in an increased surface expression of ELAM-1 compared with control samples (Fig. 1, A and B). Within 6 h of treatment with MPs, ELAM-1 expression was significantly increased (6.91 vs. 4.07 relative fluorescent intensity [RFI], P < 0.01; Fig. 1C). This was similar to the increase observed in TNF-α stimulated cells compared with controls (7.16 vs. 4.07 RFI, P < 0.01; Fig. 1C). Twelve hours after treatment with MPs, ELAM-1 expression was significantly greater than both TNF-α treatment, and negative controls (10.49 vs. 6.47 and 4.51 RFI respectively, P < 0.01). This difference remained, but to a lesser degree, at 24 h after treatment (5.60 vs. 3.13 and 3.58 RFI, P < 0.01).
Treatment with MPs also increased endothelial cell expression of ICAM-1 (Fig. 2). Twelve hours after treatment with MPs, endothelial cell ICAM-1 expression was significantly increased above that of controls (4.79 vs. 3.84 RFI, P < 0.01). TNF-α induced ICAM-1 expression to a much greater extent than both media and MPs at both 12 and 24 h (Fig. 2).
Endothelial cell expression of IL-6 was also increased by MPs over the course of 24 h. IL-6 levels in cell culture supernatants were significantly increased within 12 h of treatment with MPs compared with controls (1.24 vs. 0.73 ng/mL, P < 0.05). However, this increase was modest compared with the level of IL-6 induced by treatment with TNF- α (8.1 vs. 1.2 ng/mL, P < 0.01, Fig. 3). Neither media nor MPs showed any detectable IL-6 levels prior to exposure to cultured cells (data not shown).
To determine if the observations we made in vitro were operant in vivo, we next transfused mice with MPs and examined the pulmonary vascular endothelium for expression of ELAM-1 and ICAM-1. As shown in Figure 4, transfusion with MPs increased expression of ELAM-1 and ICAM-1 compared with control mice within 1 h of transfusion (18.56 vs. 7.08 RFI, P < 0.01; and 23.66 vs. 6.87 RFI, P < 0.01, respectively). MP-induced ICAM-1 expression returned to control levels within 24 h, whereas MP-induced increases in ELAM-1 expression remained significantly increased compared with controls at all time points (Fig. 4).
Finally, we examined lung sections for leukocyte infiltration after transfusion of MPs. Within 1 h of transfusion with MPs, the density of sequestered leukocytes in pulmonary tissue was increased compared with controls (69.25 vs. 29.25 cells/HPF, P < 0.01) and remained significantly elevated for 24 h (Fig. 5).
In the present study, we examined the role of RBC-derived MPs on endothelial cell activation. Using both in vitro and in vivo model systems, we found that MPs derived from stored pRBC units induce endothelial cell expression of the leukocyte adhesion molecules, ELAM-1 and ICAM-1, and the cytokine, IL-6. The time course over which ELAM-1 is expressed on the plasma membrane of confluent microvascular endothelial cells is consistent with de novo synthesis. The lack of immunofluorescent staining observed on unstimulated cells is consistent with a quiescent endothelium. Likewise, stimulation with TNF-α confirms reactivity of the in vitro model to typical pro-inflammatory stimuli.
Patients suffering from traumatic injury continue to require lifesaving packed red blood cell transfusion, sometimes in large volumes, during resuscitation (19). During storage, pRBCs develop a series of biochemical and physical changes known as the red blood cell storage lesion (7). Although several previous studies have suggested that transfusion of older units may result in harm (20, 21), clinical data has suggested that the storage age of pRBCs does not influence clinical outcome (22–24). One challenge in interpreting this literature is that the definition of “old” pRBCs varies between studies, ranging from 14 days (25) to 35 days (26) of storage. Critically ill, trauma, and complex surgical patients are the most vulnerable to adverse effects from the transfusion of older pRBC units (25–27). Due to the potential harm resulting from the transfusion of stored pRBCs, the development of strategies to mitigate the storage lesion, including novel storage solutions (28) and cryopreservation (29), remains an area of intense study. Our findings are important because they indicate that microparticles present in stored packed red blood cells lead to endothelial cell activation in the transfusion recipient.
Endothelial cell activation is a prerequisite for leukocyte binding and transmigration, and is an integral step in the pathogenesis of lung injury. Activation of the endothelium involves expression of leukocyte adhesion molecules as well as development of a pro-inflammatory cytokine gradient. We validated our in vitro findings using a murine model in which MPs were intravenously transfused. In these experiments, we found that pulmonary endothelial activation results from the transfusion of pRBC-derived MPs, with associated sequestration of leukocytes in pulmonary tissues. Because leukocyte adhesion to the endothelium requires both selectins (ELAM-1) and integrins (ICAM-1), and we found that transfusion of MPs increased the expression of both surface proteins in vitro and in vivo, it is possible that MPs induce pulmonary inflammation via direct activation of the endothelium.
Several important points regarding potential harm from microparticles must be considered. The current studies utilized microparticles isolated from stored pRBC units. Although no clinical situation involves the transfusion of microparticles alone, previous studies from our laboratory indicate that microparticles isolated from stored red blood cell units exert similar biological effects as those present in stored pRBCs (14). In addition, previous work from our laboratory indicates that the microparticle concentration present in stored human pRBC units equals or exceeds the number of microparticles found in stored murine units (30). Additionally, previous studies have shown that higher MP concentrations increase their biological effects in a dose-response fashion (14). Thus, we suspect that potential harm from microparticles from stored pRBC units may become clinically relevant in patients who receive multiple units of pRBCs, especially in the setting of massive transfusion.
A second point addresses the clinical relevance of microparticles from stored pRBC units. Several studies have demonstrated an association between the age of transfused pRBCs and adverse clinical outcomes, such as deep vein thrombosis (20), transfusion-related acute lung injury (31), morbidity (26), and mortality (20, 26, 32). An important goal of the current project has been to determine whether pRBC-derived microparticles could be responsible, at least in part, for these negative outcomes. Based on data from our and other laboratories, we have found that MPs are not present in fresh pRBCs, but accumulate gradually over the duration of the storage period (14, 33). Further research is necessary to determine the point during storage at which pRBC microparticles become potentially harmful, as well as potential methods of microparticle removal or neutralization.
One potential limitation of our experimental design is the focus on murine models. In the present study, we intentionally focused on murine cells for two reasons. First, we wished to remain consistent between our in vitro and in vivo models. Our primary endpoints of increased adhesion molecules and proinflammatory cytokines can vary between species, both in concentration and in time course, so we elected to remove this potentially confounding variable in our experimental design. Second, previous studies have suggested that the development of the RBC storage lesion, including microparticle formation, is significantly affected by poorly understood characteristics of the pRBC donor (34). Thus, use of a murine model minimizes human donor variability and maximizes our ability to understand the potential impact of microparticles on the inflammatory response. Another limitation relates to the injection of microparticle into naive animals. Previous experiments from our laboratory have demonstrated that microparticle treatment in a hemorrhagic shock model can result in lung injury (14). In the present study, we sought to examine the effects of RBC-derived microparticles with minimized confounding factors, including hemorrhage and neutrophil activation. By removing these factors, we were able to investigate the interaction between microparticles and endothelial cells in a focused environment.
Microparticles generated during storage of pRBCs lead to increased endothelial cell expression of adhesion molecules as well as pulmonary leukocyte infiltration. Removal or neutralization of red blood cell derived microparticles may be beneficial to patients receiving a large volume of stored pRBCs.
1. Kauvar DS, Lefering R, Wade CE. Impact of hemorrhage on trauma outcome: an overview of epidemiology, clinical presentations, and therapeutic considerations. J Trauma
3. Hess JR, Thomas MJ. Blood use in war and disaster: lessons from the past century. Transfusion
4. Makley AT, Goodman MD, Belizaire RM, Friend LA, Johannigman JA, Dorlac WN, Lentsch AB, Pritts TA. Damage control resuscitation decreases systemic inflammation after hemorrhage. J Surg Res
5. Ferraris VA, Ferraris SP, Saha SP, Hessel EA 2nd, Haan CK, Royston BD, Bridges CR, Higgins RS, Despotis G, Brown JR, et al. Society of Thoracic Surgeons Blood Conservation Guideline. Perioperative blood transfusion and blood conservation in cardiac surgery: the Society of Thoracic Surgeons and The Society of Cardiovascular Anesthesiologists clinical practice guideline. Ann Thorac Surg
6. Zimrin AB, Hess JR. Current issues relating to the transfusion of stored red blood cells. Vox Sang
7. Hoehn RS, Jernigan PL, Chang AL, Edwards MJ, Pritts TA. Molecular mechanisms of erythrocyte aging. Biol Chem
8. Lagerberg JW, Truijens-de Lange R, de Korte D, Verhoeven AJ. Altered processing of thawed red cells to improve the in vitro quality during postthaw storage at 4 degrees C. Transfusion
9. Mullier F, Bailly N, Chatelain C, Chatelain B, Dogne JM. Pre-analytical issues in the measurement of circulating microparticles: current recommendations and pending questions. J Thromb Haemost
10. Chironi GN, Boulanger CM, Simon A, Dignat-George F, Freyssinet JM, Tedgui A. Endothelial microparticles in diseases. Cell Tissue Res
11. Hargett LA, Bauer NN. On the origin of microparticles: from “platelet dust” to mediators of intercellular communication. Pulm Circ
12. Italiano JE Jr, Mairuhu AT, Flaumenhaft R. Clinical relevance of microparticles from platelets and megakaryocytes. Curr Opin Hematol
13. Simak J, Gelderman MP. Cell membrane microparticles in blood and blood products: potentially pathogenic agents and diagnostic markers. Transfus Med Rev
14. Belizaire RM, Prakash PS, Richter JR, Robinson BR, Edwards MJ, Caldwell CC, Lentsch AB, Pritts TA. Microparticles from stored red blood cells activate neutrophils and cause lung injury after hemorrhage and resuscitation. J Am Coll Surg
15. Makley AT, Goodman MD, Friend LA, Johannigman JA, Dorlac WC, Lentsch AB, Pritts TA. Murine blood banking: characterization and comparisons to human blood. Shock
16. Baron M, Boulanger CM, Staels B, Tailleux A. Cell-derived microparticles in atherosclerosis: biomarkers and targets for pharmacological modulation? J Cell Mol Med
17. Shah MD, Bergeron AL, Dong JF, Lopez JA. Flow cytometric measurement of microparticles: pitfalls and protocol modifications. Platelets
18. Kasten KR, Tschop J, Goetzman HS, England LG, Dattilo JR, Cave CM, Seitz AP, Hildeman DA, Caldwell CC. T-cell activation differentially mediates the host response to sepsis. Shock
19. Holcomb JB, Tilley BC, Baraniuk S, Fox EE, Wade CE, Podbielski JM, del Junco DJ, Brasel KJ, Bulger EM, Callcut RA, et al. Transfusion of plasma, platelets, and red blood cells in a 1:1:1 vs a 1:1:2 ratio and mortality in patients with severe trauma: the PROPPR randomized clinical trial. JAMA
20. Spinella PC, Carroll CL, Staff I, Gross R, Mc Quay J, Keibel L, Wade CE, Holcomb JB. Duration of red blood cell storage is associated with increased incidence of deep vein thrombosis and in hospital mortality in patients with traumatic injuries. Crit Care
21. Weinberg JA, McGwin G Jr, Griffin RL, Huynh VQ, Cherry SA 3rd, Marques MB, Reiff DA, Kerby JD, Rue LWJ3rd. Age of transfused blood: an independent predictor of mortality despite universal leukoreduction. J Trauma
22. Alexander PE, Barty R, Fei Y, Vandvik PO, Pai M, Siemieniuk RA, Heddle NM, Blumberg N, McLeod SL, Liu J, et al. Transfusion of fresher vs older red blood cells in hospitalized patients: a systematic review and meta-analysis. Blood
23. Lacroix J, Hebert PC, Fergusson DA, Tinmouth A, Cook DJ, Marshall JC, Clayton L, McIntyre L, Callum J, Turgeon AF, et al. Age of transfused blood in critically ill adults. N Engl J Med
24. Steiner ME, Ness PM, Assmann SF, Triulzi DJ, Sloan SR, Delaney M, Granger S, Bennett-Guerrero E, Blajchman MA, Scavo V, et al. Effects of red-cell storage duration on patients undergoing cardiac surgery. N Engl J Med
25. Spadaro S, Reverberi R, Fogagnolo A, Ragazzi R, Napoli N, Marangoni E, Bellini T, Volta CA. Transfusion of stored red blood cells in critically ill trauma patients: a retrospective study. Eur Rev Med Pharmacol Sci
26. Goel R, Johnson DJ, Scott AV, Tobian AA, Ness PM, Nagababu E, Frank SM. Red blood cells stored 35 days or more are associated with adverse outcomes in high-risk patients. Transfusion
27. Horvath KA, Acker MA, Chang H, Bagiella E, Smith PK, Iribarne A, Kron IL, Lackner P, Argenziano M, Ascheim DD, et al. Blood transfusion and infection after cardiac surgery. Ann Thorac Surg
28. Cancelas JA, Dumont LJ, Maes LA, Rugg N, Herschel L, Whitley PH, Szczepiokowski ZM, Siegel AH, Hess JR, Zia M. Additive solution-7 reduces the red blood cell cold storage lesion. Transfusion
29. Scott KL, Lecak J, Acker JP. Biopreservation of red blood cells: past, present, and future. Transfus Med Rev
30. Hoehn RS, Jernigan PL, Japtok L, Chang AL, Midura EF, Caldwell CC, Kleuser B, Lentsch AB, Edwards MJ, Gulbins E, et al. Acid Sphingomyelinase inhibition in stored erythrocytes reduces transfusion-associated lung inflammation. Ann Surg
2016; [Epub ahead of print].
31. Peters AL, van Hezel ME, Juffermans NP, Vlaar AP. Pathogenesis of non-antibody mediated transfusion-related acute lung injury from bench to bedside. Blood Rev
32. Weinberg JA, McGwin G Jr, Vandromme MJ, Marques MB, Melton SM, Reiff DA, Kerby JD, Rue LW 3rd. Duration of red cell storage influences mortality after trauma. J Trauma
33. Kriebardis AG, Antonelou MH, Stamoulis KE, Economou-Petersen E, Margaritis LH, Papassideri IS. RBC-derived vesicles during storage: ultrastructure, protein composition, oxidation, and signaling components. Transfusion
34. Tzounakas VL, Georgatzakou HT, Kriebardis AG, Voulgaridou AI, Stamoulis KE, Foudoulaki-Paparizos LE, Antonelou MH, Papassideri IS. Donor variation effect on red blood cell storage lesion: a multivariable, yet consistent, story. Transfusion
Keywords Abbreviations: Diapedesis; endothelial activation; microparticles; red blood cell storage lesion; AS3; additive solution-3; CP2D; citrate phosphate double dextrose; ELAM-1; endothelial-leukocyte adhesion molecule-1; ELISA; enzyme-linked immunosorbent assay; FDA; Food and Drug Administration; HPF; high powered field; ICAM-1; intercellular adhesion molecule-1; IL-6; interleukin-6; MOF; multisystem organ failure; MPs; microparticles; pRBCs; packed red blood cells; RFI; relative fluorescent intensity; TNF-α; tumor necrosis factor-alpha