Fig 1.
Experimental design and study arms.
Fig 2.
Optimization of human erythrocyte transfusion into rats.
(A) Free hemoglobin present in rat plasma (expressed as an equivalent number of lysed erythrocytes) collected before transfusion (0) or 0.5, 5, 20, 60, 120 or 360 min after 15 (n = 3), 30 (n = 3) or 45% (n = 3) transfusion of human erythrocytes was measured by spectrophotometry. One group of sham animals (n = 3) was analyzed as well. (B) Absolute cell count of surviving human erythrocytes from 15 (n = 3), 30 (n = 3) or 45% (n = 3) transfusion of human erythrocytes were detected using FITC-conjugated anti-human CD235a (glycophorin A) monoclonal antibody at 0.5, 5, 20, 60, 120 and 360 min after transfusion as measured by flow cytometry. Clearance kinetics were standardized to injected erythrocytes at baseline (0 min). Data are means and standard error of the mean.
Fig 3.
Blood was collected from rats before addition of LPS or transfusion (0 min, pre-bleed time point (tp)), (n = 64). Five minutes and 4 hour later, blood was again collected from sham rats (5 min (n = 5) and 4 hours (n = 8)), rats transfused with 15% (5 min (n = 3) and 4 hours (n = 6)), 30% (5 min (n = 3) and 4 hours (n = 9)) or 45% (5 min (n = 3) and 4 hours (n = 6)) human erythrocyte transfusion (xt), LPS only (5 min (n = 4) and 4 hours (n = 10)) or LPS+30% (5 min (n = 3) and 4 hours (n = 13)) transfusion. (A) White blood cells (WBCs). (B) Neutrophils. (C) Monocytes. (D) Lymphocytes. Data are means and standard error of the mean. Statistical analysis was performed using an ANOVA followed by Student t-test. * denotes P ≤ 0.02, ** denotes P ≤ 0.005, respectively, compared to the pre-transfusion control at the corresponding timepoint. # denotes P < 0.05, ## denotes P < 0.01 and ### denotes P < 0.001, respectively, compared to 30% xt at the corresponding timepoint.
Fig 4.
LPS+30% transfusion results in acute lung injury.
Representative histology (H&E stain) of rat lungs. (A) Sham control. (B) 15% transfusion of human erythrocytes. (C) 30% transfusion. (D) 45% transfusion. (E) LPS only. (F) LPS+30% transfusion. Animals receiving transfusion in the absence of LPS and LPS alone demonstrated normal lung architecture as seen in sham treated animals whereas animals receiving LPS+30% transfusion showed severe neutrophil infiltration and thickening of the alveolar walls. Bar represents 100 μm. Tissues were observed with a microscope (BX50, Olympus) at a magnification of 20X at room temperature. Images were acquired with a digital camera (DP70, Olympus).
Fig 5.
LPS+30% transfusion increases neutrophil-mediated lung injury.
Blinded grading of H&E sections for neutrophil infiltration and cell wall thickening from sham (n = 20) animals and animals receiving 15 (n = 15), 30 (n = 30), 45% (n = 15) human erythrocyte transfusion (xt), LPS alone (n = 15) and LPS+30% transfusion (n = 20). Tissues were scored on a scale of 0–4: 0 = normal lungs, 1 = minor lung involvement, 2 = moderate lung involvement, 3 = serious lung involvement, 4 = severe lung involvement. Data are means and standard error of the mean. Statistical analysis was performed using a Kruskal-Wallis test, followed by Mann-Whitney test. * denotes P = 0.05 compared to all other experimental groups.
Fig 6.
Thirty percent erythrocyte transfusion with LPS treatment increases lung weight.
(A) Gross lung weights measured for sham animals (n = 6), rats receiving 15 (n = 3), 30 (n = 6) or 45% (n = 3) human erythrocyte transfusion (xt), LPS only (n = 7) or LPS+30% (n = 11) transfusion. (B) Wet and dry weights measured for sham animals (n = 8), rats transfused with 15 (n = 2), 30 (n = 3) or 45% (n = 3) human erythrocyte transfusion (xt), LPS only (n = 3) or LPS+30% (n = 3) transfusion and expressed as dry to wet ratio. Data are means and standard error of the mean. Statistical analysis was performed using an ANOVA followed by Student t-test. * denotes P < 0.01 compared to the sham. # denotes P < 0.05 compared to LPS only.
Fig 7.
LPS alone and LPS+30% transfusion induces complement activation.
Plasma was isolated from animals prior to receiving LPS alone (n = 3), 15% (n = 3), 30% (n = 3), 45% (n = 3) erythrocyte transfusion (xt) alone or LPS+30% erythrocyte transfusion (n = 3) and then at 5, 60, 120, 180 and 240 minutes after transfusion. C5a was then measured in each sample by ELISA and absorbance was read at 450 nm. Two replicates for each animal were measured for every time point. Data are means and standard error of the mean. Statistical analysis was performed using an ANOVA followed by Student t-test. * denotes P < 0.001 compared to the sham at the corresponding timepoint. # denotes P < 0.05 compared to LPS only at the corresponding timepoint.
Fig 8.
LPS+30% transfusion increases the level of free DNA in circulation.
Plasma was isolated from animals prior to receiving LPS alone (n = 3), 15% (n = 3), 30% (n = 3), 45% (n = 3) erythrocyte transfusion (xt) alone or LPS+30% erythrocyte transfusion (n = 3) and then at 5, 60, 120, 180 and 240 minutes after transfusion. Plasma samples were incubated with PicoGreen. Fluorescence was read at an excitation wavelength of 485 nm and an emission wavelength of 520nm in a microplate reader. All free DNA measurements for each animal were done in triplicate. Data are means and standard error of the mean. Statistical analysis was performed using an ANOVA followed by Student t-test. * denotes P < 0.001 compared to all other experimental groups at the corresponding timepoint.