Erythrocyte-Derived Microparticles Activate Pulmonary Endothelial Cells in a Murine Model of Transfusion

Abstract

Erythrocyte-derived microparticles (MPs) are sub-micrometer, biologically active vesicles shed by red blood cells as part of the biochemical changes that occur during storage. We hypothesized that MPs from stored red blood cells would activate endothelial cells. MPs from aged murine packed red blood cells (pRBCs) were isolated and used to treat confluent layers of cultured endothelial cells. Endothelial expression of leukocyte adhesion molecules, endothelial-leukocyte adhesion molecule-1 (ELAM-1) and intercellular adhesion molecule-1(ICAM-1), and inflammatory mediator, interleukin-6 (IL-6), was evaluated at 0.5, 6, 12, and 24 h of treatment. Healthy C57BL/6 mice were transfused with a MP suspension and lung sections were analyzed for adhesion molecules and sequestered interstitial leukocytes. Increased levels of ELAM-1 and ICAM-1 were found on cultured endothelial cells 6 h after MP stimulation (6.91 vs. 4.07 relative fluorescent intensity [RFI], P < 0.01, and 5.85 vs. 3.55 RFI, P = 0.01, respectively). IL-6 in cell culture supernatants was increased after 12 h of MP stimulation compared with controls (1.24 vs. 0.73 ng/mL, P = 0.03). In vivo experiments demonstrated that MP injection increased ELAM-1 and ICAM-1 expression at 1 h (18.56 vs. 7.08 RFI, P < 0.01, and 23.66 vs. 6.87 RFI, P < 0.01, respectively) and caused increased density of pulmonary interstitial leukocytes by 4 h of treatment (69.25 vs. 29.25 cells/high powered field, P < 0.01). This series of experiments supports our hypothesis that erythrocyte-derived MPs are able to activate pulmonary endothelium, leading to the pulmonary sequestration of leukocytes following the transfusion of stored pRBCs.

Figure 1

Immunofluorescence imaging of murine endothelial cells in confluent culture after 6 hour incubation with (A) Dulbecco’s Modified Eagles Media vs (B) DMEM with microparticles derived from 1 ml of murine packed red blood cells. Endothelial cells were labelled with fluorescent antibodies to ELAM-1 (red). Nuclei were counterstained with DAPI (blue) and actin filaments with FITC-phalloidin (green). (C) Quantification of red fluorescence intensity after 30 min, 6, 12 and 24 hours of incubation with plain media, microparticles, or TNF-α. * p<0.05 compared to control, n=4 at each time point.

Figure 2

Immunofluorescence imaging of murine endothelial cells in confluent culture after 6 hour incubation with (A) Dulbecco’s Modified Eagles Media vs (B) DMEM with microparticles derived from 1 ml of murine packed red blood cells. Endothelial cells were labelled with a fluorescent antibody to ICAM-1 (red) Nuclei were counterstained with DAPI (blue) and actin filaments with FITC-phalloidin (green). (C) Quantification of red fluorescence intensity after 30 min, 6, 12 and 24 hours of incubation with plain media, microparticles and TNF-α. * p<0.05 compared to control, n=4 at each time point.

Figure 3

IL-6 concentration in endothelial cell culture supernatant after 30 minutes or 6, 12 and 24 hours of stimulation with plain media, and media with red blood cell derived microparticles, or TNF-α. * p<0.05 compared to control, n=4 at each time point in each group.

Figure 4

In vivo expression of ICAM-1 by murine pulmonary endothelium as determined by immunofluorescence 4 hours after injection of mice with (A) 200 μL lactated Ringer’s solution or (B) red cell derived microparticles. Sections were stained with red fluorescence labelled ICAM-1 and nuclei were counterstained with DAPI (blue). Quantification of (C) ICAM-1 and (D) ELAM-1 staining intensity at 1, 4, 8, and 24 hours after injection of red cell derived microparticles as compared to vehicle control. * p<0.05 compared to control, n=4 in each group.

Figure 5

Immunohistochemical staining of thin lung sections 4 hours after injection of mice with (A) 200 μL lactated Ringer’s solution or (B) red blood cell derived microparticles. Sections were stained with Ly-6G/GR1 antibody to demonstrate leukocytes. (C) Quantitation of pulmonary leukocytes at 1, 4, 8, and 24 hours after microparticle injections as compared to vehicle controls. * p<0.05 compared to control, n=4 in each group.