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 188.8.131.52 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.
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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