Fig. 5
Red blood cell (RBC) transfusions activate intestinal leukocytes. a Scatter plots show CD11b expression in macrophages from anemic and anemic-transfused intestine; Mann–Whitney U test, **P < 0.01; photomicrographs (Wright–Giemsa; ×1000) below show “activated” morphology of macrophages from the anemic-transfused intestine. N = 5 mice per group. Scale bar =5 µm. b Fluorescence photomicrographs (magnification ×400) show immunoreactivity for 4-hydroxynonenal (4-HNE) in Ly6C (+) macrophages in anemic-transfused (solid arrows), but not in anemic control (open arrows). 4-HNE was also detected in nearby epithelium. Scale bar = 50 µm; N = 5 mice per group. c Scatter plots show relative nuclear factor-κB (NF-κB) activity (optical density) in NF-κB reporter RAW264.7 macrophages treated with fresh RBCs (control), 7-day RBCs, fresh RBCs resuspended in phosphate-buffered saline (PBS), 7-day RBCs in PBS, plasma supernatant from fresh and 7-day RBCs, fresh RBCs suspended in plasma from 7-day blood, and 7-day RBCs mixed with fresh plasma; N = 4 different donor mice, each with two technical replicates; Kruskal–Wallis H test, *P < 0.05, **P < 0.01, †P < 0.001 vs. control. d Scatter plots show oxyhemoglobin (oxyHb), methemoglobin (metHb), and heme concentrations in supernatants from 7-day RBCs. N = 6 donor mice. e Scatter bar diagrams show NF-κB activity in reporter macrophages treated with 7-day RBCs vs. 7-day RBCs + haptoglobin (10 μg mL−1). N = 8 donor mice. Hemin was used as a positive control. †P < 0.001 vs. 7-day RBCs, Mann–Whitney U test. f Scatter plots show severity of bowel injury in anemic-transfused pups pre-treated with haptoglobin. N = 5 anemic control, 4 haptoglobin-treated anemic control, and 8 each in anemic-transfused and haptoglobin-treated anemic-transfused; Kruskal–Wallis H test, *P < 0.05, **P < 0.01 vs. anemic control, #P < 0.05 vs. haptoglobin-treated anemic, and ζP < 0.05 vs. anemic-transfused. g Kaplan–Meier curves summarize survival data from above experiment. One haptoglobin-treated mouse died 2 h after the procedure and was excluded; Mantel–Cox log-rank test, *P < 0.05. h Top: Representative photographs show intestinal injury in anemic-transfused mice, but not in haptoglobin-treated anemic-transfused mice. Bottom: Representative photomicrographs (hematoxylin–eosin; magnification ×250) of ileum (left) and colon (right) from these groups. N = 8 mice per group. Scale bars = 100 µm. All scatter plots summarize the data as means ± standard error of the mean
