Influence of different erythrocyte storage times on the macrophage response in haemorrhagic shock mice.

OBJECTIVE: To investigate the characteristics of the macrophage response to transfusion of erythrocytes kept at different storage times in the mouse model of haemorrhagic shock.
METHODS: Erythrocytes were isolated from mice and stored for 7, 21 or 35 days and samples injected intravenously into haemorrhagic shock mice. Changes in macrophages, inflammatory cytokines and T cell differentiation were assessed using flow cytometry or enzyme-linked immunosorbent assay (ELISA). In a second experiment, haemorrhagic shock mice were injected with 21D-erythrocytes and the expression of nuclear factor erythroid 2 p45-related factor 2 (Nrf2), arginine -1 (Arg-1) and inducible nitrous oxide (iNOS) determined.
RESULTS: The proportion of M1-polarized macrophages was greatest in the 21D group while M2 macrophages tended to increase with the erythrocyte storage time. Levels of inflammatory cytokines and T helper 1 (Th1) cells increased in proportion to erythrocytes storage time. Most regulatory T cells (Treg) were found at 21D. Arg-1 expression was significantly increased in a group that received an heme oxygenase 1 (HO-1) agonist and significantly decreased in a group that received an HO-1 inhibitor but there were no differences in the expression of iNOS or Nrf2.
CONCLUSION: 21D storage time may be an important time point for erythrocyte storage and immunity response and Arg-1 may have a role in the macrophage response to erythrocyte infusion.

Introduction

Macrophages, the most plastic cells of the hematopoietic system are found in most every organ of the body.[1] Activation or polarization of macrophages plays a critical role in many cell activities, including homeostasis, immune response disease pathogenesis and erythropoiesis.[2-4] Based on different converging signals from the cellular environment and inflammatory stimuli, activation of macrophages can be broadly described in terms of M1 or M2 phenotypes.[5] Generally, M1 polarization depends on the signalling interferon (IFN)-γ and lipopolysaccharide (LPS) while M2 polarization depends on signalling of interleukin-4 (IL-4), immune complexes, glucocorticoids combined with or without transforming growth factor beta (TGF-β).[5] M1 macrophages produce pro-inflammatory and cytotoxic cytokines (e.g., inducible nitric oxide synthase (iNOS), IL-12, major histocompatibility complex (MHC) class II, IL-8 and CCL2).[6] By contrast, M2 macrophages produce more anti-inflammatory cytokines and substances involved in repairing function (e.g., arginase/ornithine, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and transforming growth factor-beta (TGF-β).[6]

Studies suggest that prolonged erythrocyte storage before transfusion increases morbidity and mortality, in some hospital patients.[7] Although the exact mechanism for this remains unknown, it has been suggested that the acute delivery of large amounts of iron to the monocyte/macrophage system could produce adverse effects.[7] Indeed, macrophages are intimately involved in the regulation of iron homeostasis because they scavenge old and damaged erythrocytes to release iron contained within the heme moiety.[3] In turn, heme modulates macrophage function and differentiation as well as macrophage inflammatory response.[3] Heme oxygenase 1 (HO-1), one of the isoforms of heme oxygenase, the enzyme involved in the degradation of heme, is a nuclear factor erythroid 2 p45-related factor 2 (Nrf2)-regulated gene.[8,9] Normally present in low levels, HO-1 is upregulated by several oxidative stress stimuli, including its substrate heme, heavy metals, UV irradiation, reactive oxygen species (ROS), modified lipids, and inflammatory cytokines.[9] Therefore, for the successful management of haemorrhagic shock, it is important to investigate the role of the Nrf2/HO-1 axis on the macrophage response to a protracted erythrocyte transfusion. Accordingly, we investigated the characteristics of the macrophage response in haemorrhagic shock mice injected with erythrocytes at different storage times.

Materials and methods

Animals

40 male C57 mice aged 8∼10 weeks were purchased from Model Animal Research Centre of Nanjing University, China. The specific pathogen free (SPF) mice were housed at constant room temperature with a 12:12-h light-dark cycle with free access to food and water. All animal procedures were performed in accordance with the National Institutes of Health guidelines for the Care and Use of Laboratory Animals and the study was authorized by the Animal Research Ethics Committee of The Shanghai Gongli Hospital, the Second Military Medical University.

Isolation of Erythrocytes

Whole blood was taken from six healthy C57 mice. From each mouse, 0.1 ml blood was taken by tail vein blood collection and placed in anticoagulant tubes. The leukocyte buffy-coat was obtained using Ficoll discontinuous density gradient centrifugation and was diluted using Ringer buffer followed by 3000 rpm centrifugation for 10 min at 4°C. The pellet was washed twice using Ringer buffer (5 mM KCl, 125 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 32 mM, HEPES, 25 mM Tris 10 mM glucose, pH 7.4). Erythrocytes were re-suspended in Ringer buffer to obtain a 2% haematocrit and stored at 4°C for 7 days (7D), 21 days (21D) or 35 days (35D).

Animal model

An established mouse model of trauma-haemorrhagic shock was used.[10,11] Erythrocytes (0.5 ml) with different storage times were injected into haemorrhagic shock mice via tail vein intravenous injection. Each group (7D, 21D and 35D) included six mice. The macrophage polarization, levels of inflammatory cytokines, and T cell differentiation were assessed after 72 h. The mice were then anaesthetized by intraperitoneal injection with 10% chloral hydrate (300 mg/kg) and sacrificed by cervical dislocation.

Subsequently, in a separate experiment, 18 haemorrhagic shock mice were injected intravenously with 21D-erythrocytes. The mice were separated into three groups (six mice in each group) : controls (haemorrhagic shock mice without any treatment); HO-1 activity induced by tert butyl hydroquinone (TBHQ, 16.7 mg/kg, intraperitoneal injection tid for 3 days before establishing the model); HO-1 inhibited by zinc protoporphyrin (ZnPP, one intraperitoneal injection [5 mg/kg] before establishing the model).

Macrophage isolation

The mice were sacrificed after 72 h following treatment. Lymph nodes were collected and enzymatically digested using collagenase. Macrophages were isolated from the digested lymphatic samples by 1.5 h adherence culturing on tissue culture plastic. The non-adherent cells were rinsed away with phosphate-buffered saline (PBS) in triplicate and the remaining macrophages were collected and purified by using CD11b+ microbeads (Miltenyi Biotec, Leiden, The Netherlands). Cells were harvested when the purity of CD11b+ F4/80+ cells was >96%. The F4/80 antigen is widely-used as a monocyte-macrophage marker in mice. The isolated macrophages were subjected to flow cytometry analyses.

Flow cytometry

For cell surface examination, macrophages were incubated in 1% bovine serum albumen (BSA)-PBS buffer at room temperature for 20 min. Thereafter, cells were incubated in the M1 antibody marker CD68 or the M2 antibody marker CD200R (eBioscience, San Diego, CA, USA) in a dark room at 4°C.

For intracellular staining, cells were stimulated with 1 μg/ml brefeldin A (Sigma, USA) for 6 h at 37°C under 5% CO2/95% air atmosphere, and stained for 20 min at room temperature in 0.3% saponin/1% BSA-PBS buffer. After rinsing, cells were incubated for 1 h at 4°C with the following antibodies: iNOS (eBioscience lnc.); arginase 1 (Arg-1) (eBioscience lnc.); Nrf2 (eBioscience lnc.). Expression of iNOS and Arg-1 are key markers associated with M1 and M2 phenotypes, respectively.[6]

For measurement of T helper 1 (Th1) and regulatory T cells (Treg) cells, anti-mouse CD4 FITC (Abcam) was added and cells were incubated for 30 min at 4°C. Thereafter, cells were incubated in anti-mouse Foxp3 PE (Abcam) and incubated for 30 min at 4°C.

Subsequently, all cells were subjected to flow cytometry detection (Becton Dickinson Biosciences, San Jose, CA, USA) and analysed using CellQuest software.

ELISA assay

Samples of blood (0.1 ml) were taken from the haemorrhagic shock mice injected intravenously with erythrocytes at different storage times (six in each group) and analysed for inflammatory cytokines using the enzyme-linked immunosorbent assay (ELISA) kit purchased from R&D System lnc. (Minneapolis, MN, USA). The cytokines that were investigated were as follows: tumour necrosis factor alpha [TNF-α]; IL-6; iNOS; IL-18; IL-1β; CCL22; TGF-β; IL-10. All procedures were conducted according to the manufacturer’s protocols.

Statistical analyses

All assays were repeated at least three times and data were expressed as mean ± standard deviation (SD). Data were analysed using Graph Prism 7.0. Comparisons among groups were analysed using one-way analysis of variance (ANOVA) followed by post hoc Tukey test. A P-value <0.05 was considered to indicate statistical significance.

Results

The effect of different erythrocyte storage times on macrophage polarization

Figure 1 shows the variation of macrophage polarization in haemorrhagic shock mice after injection of erythrocytes at different storage times. The proportion of M1-polarized macrophages was greatest in the 21D group followed by 7D and 35D groups. The proportion of M2-polarized macrophages tended to be in proportion with erythrocyte storage times and was greatest in the 35D group followed by 7D and 21D groups. Thus, the ratio of M2/M1 increased significantly from 7D (8.3% vs. 9.5%) to 35D (2.2% vs. 15.7%) (P<0.05). These findings suggest that extended storage times for erythrocytes may promote macrophage polarization from M1 to M2.

Figure 1.

Flow cytometric analysis of macrophages taken from the blood of haemorrhagic shock mice injected with erythrocytes stored at different times (7, 21 and 35 days [7D, 21D and 35D]). The macrophages were incubated with the M1 antibody CD68 or the M2 antibody CD200R and fluorescein isothiocyanate (FITC) was measured at 530 nm. Y axis is the percentage of CD68 or CD200R. The figures show the variation of macrophage polarization in haemorrhagic shock mice after injection of erythrocytes at different storage times.

(1a) The proportion of M1-polarized macrophages was greatest in the 21D group followed by 7D and 35D groups. The proportion of M2-polarized macrophages was greatest in the 35D group followed by 7D and 21D groups.

(1b) Counts for M1 (CD68) (dashed line) and M2 (CD200R) (solid line).

The effect of different erythrocyte storage times on the secretion of inflammation cytokines

Results of the ELISA assay on blood from haemorrhagic shock mice injected with erythrocytes showed that the expression levels of the inflammatory cytokines (i.e., TNF-α, IL-6, iNOS, IL-18, IL-1β, CCL22, TGF-β, and IL-10) were in proportion to the erythrocyte storage times (Figure 2a-h).

Figure 2

a–h. Expression of inflammatory cytokines in the blood of haemorrhagic shock mice injected with erythrocytes stored at different times (7, 21 and 35 days) as measured by enzyme-linked immunosorbent assay (ELISA). The following cytokines were investigated: tumour necrosis factor alpha [TNF-α]; interleukin (IL)-6); inducible nitric oxide synthase (iNOS); IL-18, IL-1β, CCL22, transforming growth factor-beta (TGF-β); IL-10. *P<0.05 vs 7D; **P<0.01 vs. 7D; ##P<0.01 vs 21D.

The effect of different erythrocyte storage times on T lymphocyte differentiation

Flow cytometric analysis showed that the percentage of Th1 cells increased in proportion to erythrocyte storage time (Figure 3b). However, for Treg cells the highest proportion was seen in the 21D group, followed by the 35D group and finally the 7D group (Figure 3b).

Figure 3.

Flow cytometric analysis of T helper (Th) cells and regulatory T cells (Treg) taken from the blood of haemorrhagic shock mice injected with erythrocytes stored at different times (7, 21 and 35 days [7D, 21D and 35D]).

The effect of different erythrocyte storage times on the expression of Arg-1, NOS2 and Nrf2 in macrophages

Data from a second experiment where haemorrhagic shock mice had been injected intravenously with 21D-erythrocytes, showed that by comparison with the control group, the expression of Arg-1 was significantly increased in the group that received the HO-1 agonist and significantly decreased in the group that received the HO-1 inhibitor, ZnPP (Figure 4a). However, there were no significant differences between groups in the expression of inducible nitric oxide synthase (NOS2) or Nrf2 (Figure 4b and c).

Figure 4.

Flow cytometric analysis of macrophages taken from the blood of haemorrhagic shock mice injected with erythrocytes stored for 21 days. The mice were separated into three groups (six in each group): controls (haemorrhagic shock mice without any treatment); HO-1 activity induced by tert butyl hydroquinone (TBHQ, 16.7 mg/kg, intraperitoneal injection tid for 3 days before establishing the model); HO-1 inhibited by zinc protoporphyrin (ZnPP, 5 mg/kg, intraperitoneal injection at once before establishing the model). By comparison with the control group, arginase 1 (Arg-1) expression was significantly increased in the group that received the HO-1 agonist and significantly decreased in the group that received the HO-1 inhibitor, ZnPP. However, there were no significant differences between groups in inducible nitric oxide synthase (NOS2) or nuclear factor erythroid 2 p45-related factor 2 (Nrf2) expression.

Discussion

Using the mouse model of haemorrhagic shock, we investigated the consequences of transfusions of erythrocytes kept at different storage times on macrophage and inflammatory responses. The results showed that while M2 macrophages tended to increase in proportion with erythrocyte storage time, the highest proportions of M1 macrophages was in the 21D group. However, inflammatory cytokines increased in proportion with the erythrocyte storage time as did the percentage of Th1 cells but Treg cells were greatest in number in the 21D group.

According to stimuli inducing their polarization and cytokine profile, macrophages have been classified as pro-inflammatory M1 type (killer cells) and anti-inflammatory M2 type (repair type cells).[5] A switch from M1 to M2 macrophages usually occurs during inflammation resolution when anti-inflammatory or reparative function is required.[12,13]

In this study, the proportion of M1 and M2 macrophages in the 7D day group were similar, but in the 21D group, M1 become polarization to M2 stage. In the 35D group, M2 macrophages were increased and M1 macrophages were markedly decreased, indicating that long time storage of erythrocytes may promote the polarization of macrophage from M1 to M2.

Inflammation is an important part of the macrophage response and, as shown by this study, the inflammatory cytokines (i.e., TNF-α, IL-6, iNOS, IL-18, IL-1β, CCL22, TGF-β, and IL-10) were significantly increased in proportion with erythrocyte storage time. These findings suggest that a long storage time may impair the M1/M2 macrophage balance and increase inflammation.

The Th1/Treg cell balance plays a critical role in cell immune response and inflammatory control.[14] Specifically, Treg cells can control “collateral damage” resulting from protective immunity induced by infection and inhibit sterile inflammation.[15] Th1 cells are essential for the immunity response against intracellular pathogens and are generated from naive precursors depending on multiple signalling activation.[16] Treg cells can control the response of different types of Th cells via expression of specific Th cell-associated transcription factor.[17,18] In this current study, Th1 cells were shown to be significantly increased in proportion to increased blood storage time while the Treg cells achieved the highest percentage at 21D. These findings suggest that 21day storage time might be an important time point for macrophage regulation and immunity response.

Previous studies have found that macrophages respond to inflammation via the Nrf2 signalling pathway, which also play a critical role in ROS regulation.[9,19,20] In this study, data from a second experiment where haemorrhagic shock mice had been injected intravenously with 21D-erythrocytes, showed that the induction or inhibition of HO-1 had no effect on the expression of Nrf2 or on the MI marker, iNOS. However, Arg-1 expression, a marker for M2 macrophages, was significantly increased after treatment with an HO-1 agonist and decreased after treatment with an HO-1 inhibitor.

The study had some limitations. For example, the molecular mechanisms involved in the effects of erythrocyte storage times on macrophage response were not invstigated. Furthermore, more in vivo and in vitro studies are required to confirm these results.

In conclusion, these data suggest that macrophage polarization is involved in the response to transfusion of erythrocytes at different storage times in the mouse model of haemorrhagic shock. It appears that the 21D storage time might be an important time point for erythrocytes storage and immunity response. In addition, although Nrf2/HO-1 axis did not appear to be involved in the response to 21D erythrocyte transfusion, Arg-1 may have a role in the macrophage response.