Physiologically aged red blood cells undergo erythrophagocytosis in vivo but not in vitro

Abstract

Background: The lifespan of red blood cells is terminated when macrophages remove senescent red blood cells by erythrophagocytosis. This puts macrophages at the center of systemic iron recycling in addition to their functions in tissue remodeling and innate immunity. Thus far, erythrophagocytosis has been studied by evaluating phagocytosis of erythrocytes that were damaged to mimic senescence. These studies have demonstrated that acquisition of some specific individual senescence markers can trigger erythrophagocytosis by macrophages, but we hypothesized that the mechanism of erythrophagocytosis of such damaged erythrocytes might differ from erythrophagocytosis of physiologically aged erythrocytes.

Design and methods: To test this hypothesis we generated an erythrocyte population highly enriched in senescent erythrocytes by a hypertransfusion procedure in mice. Various erythrocyte-aging signals were analyzed and erythrophagocytosis was evaluated in vivo and in vitro.

Results: The large cohort of senescent erythrocytes from hypertransfused mice carried numerous aging signals identical to those of senescent erythrocytes from control mice. Phagocytosis of fluorescently-labeled erythrocytes from hypertransfused mice injected into untreated mice was much higher than phagocytosis of labeled erythrocytes from control mice. However, neither erythrocytes from hypertransfused mice, nor those from control mice were phagocytosed in vitro by primary macrophage cultures, even though these cultures were able to phagocytose oxidatively damaged erythrocytes.

Conclusions: The large senescent erythrocyte population found in hypertransfused mice mimics physiologically aged erythrocytes. For effective erythrophagocytosis of these senescent erythrocytes, macrophages depend on some features of the intact phagocytosing tissue for support.

Figure 1.

Enrichment of sRBCs by hypertransfusion. (A) A schematic presentation of the ht protocol. Five mice received blood from age- and sex-matched mice by peritoneal injection. Two weeks later, blood from two mice was injected into one of the transfused mice and a day later blood from two more mice was injected into the same mouse. Four weeks later, the hypertransfused (ht) mouse was used for further analyses. (B) Blood cells of a non-hypertransfused control mouse were biotinylated and after 6 weeks the blood was labeled with streptavidin-fluorescein isothiocyanate and analyzed by FACS (C) Blood-cells were biotinylated 2 days after the first transfusion in all five mice of the hypertransfusion group. Blood cells of mice that were not hypertransfused were biotinylated at the same time and served as a control. The percentage of biotinylated sRBC was determined at several time-points on a single mouse pair (C) and 6 weeks after the initiation of hyper-transfusions (ht mice: n=4, control mice: n=5, P<0.001) (D).

Figure 2.

Markers of aging on sRBC of ht-mice are identical to aging markers on control mice. Six weeks after biotinylation, RBC were stained for biotin by streptavidin and for phosphatidylserine (PS) by phycoerythrin-annexin V (A), for CD47 by anti-CD47 antibody (C) or analyzed for esterase activity (D). Using flow cytometry, RBC were gated into non-biotinylated and biotinylated populations. The expression of each marker was determined separately in each population. (A) Externalized PS was detected with annexin V and was higher on sRBC than on young RBC in both ht- and control-mice (n=4). Results are normalized to non-biotinylated (young) RBC, (taken as 100%). (B) Serum PS measured with the two-step FACS procedure was nearly 5-fold higher in the serum of ht-mice than in the serum of control-mice (n=5). (C) Surface CD47 was detected with anti-CD47 antibodies and was significantly lower in sRBC than in young RBC of both the ht- and the control-mice (n=3). Results are normalized to non-biotinylated (young) RBC (taken as 100%). (D) Esterase activity was measured by incubation of RBC with calcein-AM (2 μM) followed by incubation for 1 h with 100 μmol/L deferiprone (L1). Esterase activity was calculated based on the calcein fluorescence of L1-treated RBC. Senescent RBC showed 40–50% lower esterase activity compared to young RBC from the same blood sample. The results of one representative experiment are represented by the histogram and the mean of three independent experiments is shown in the insert. In (A) (C) and (D) -insert, young and sRBC were compared within each group (blood from control or ht mice) with a two-tailed paired t-test (*P<0.05, **P<0.01). Biotinylated sRBC were compared between ht- and control-mice and were found not to be significantly different.

Figure 3.

Reactive oxygen species (ROS) in sRBC from control and ht-mice. RBC were stained for biotin by streptavidin and for ROS by either (A) dichlorofluorescein (DCF) (n=5), or (B) rhodamine 123 (n=2). For DCF, young and sRBC were compared within each group (blood from ht- or control-mice) by the two-tailed paired t-test *P<0.05, **P<0.01. Biotinylated sRBC were compared between ht-and control-mice and were found not to be significantly different.

Figure 4.

In vivo phagocytosis of sRBC. RBC of normal and ht-mice were stained with PKH26 and injected intraperitoneally into mice of the same age and sex. After 4 days, mice were perfused with heparin-PBS to remove non-phagocytosed RBC. The spleens were then removed, washed and phagocytosed RBC visualized with a fluorescence microscope (A and B). Quantification of PKH26 staining of spleen sections indicated that erythrophagocytosis was higher following injection of RBC from ht-mice than after injection of RBC from control-mice (C). The phagocytosed RBC were localized mainly in the peripheral red pulp area closest to the white pulp. (D) Bright field, white pulp (WP), red pulp (RP) (F) PKH26 fluorescence, and (E) merged, arrows pointing to peripheral red pulp.

Figure 5.

In vitro phagocytosis of damaged and senescent RBC. (A) Untreated RBC and RBC treated with calcium and the calcium ionophore A23187 were compared with respect to externalized PS/RBC by labeling with fluorescent annexin V. The upper panel shows fluorescence (FL2-H) versus forward light scatter (FSC-H) dot plots. A cursor line was set based on annex-in V-unlabeled RBC. The lower panel shows the FL2-H distribution histograms of the indicated populations. One representative experiment out of three is shown. (B) Bone marrow (BM)-derived macrophages and (C) spleen-derived macrophages were incubated for 90 or 30 min, respectively, with RBC from ht- or control-mice or with RBC treated with calcium-ionophore, BHP or opsonized with rabbit anti mouse IgG. Non-phagocytosed RBC were lysed with a hypotonic buffer. Cell cultures were stained with benzidine and the percentage of macrophages that phagocytosed one or more RBC was determined (200–700 cells were counted).