Evaluation of erythroblast macrophage protein related to erythroblastic islands in patients with hematopoietic stem cell transplantation
© Mao et al.; licensee BioMed Central Ltd. 2013
Received: 27 August 2012
Accepted: 27 February 2013
Published: 8 April 2013
Hematopoietic evaluation of the patients after Hematopoietic stem cell transplantation (HSCT) is very important. Erythroblast macrophage protein (Emp) is a key protein with function in normal differentiation of erythroid cells and macrophages. Emp expression correlates with erythroblastic island formation, a process widely believed to be associated with hematopoiesis in bone marrow. We aimed to investigate the hematopoietic function of bone marrow from 46 HSCT patients and 16 inpatients with severe anemia applied to the treatment of EPO by measuring Emp expression level.
Emp mRNA and protein expression levels in mononuclear cells of bone marrow and peripheral blood samples were detected by RT-PCR and Western blotting method respectively.
While hematopoiesis occurs in bone marrow, Emp expression level was elevated and more erythroblastic islands were found , and Emp is upregulated in bone marrow in response to erythropoietin (EPO) treatment.
Emp expression correlates with erythroblastic island formation and has an important function for bone marrow hematopoiesis. Emp could be a potential biomarker for hematopoietic evaluation of HSCT patients.
KeywordsErythroblast macrophage protein Cancer Hematopoietic stem cell transplantation Hematopoiesis Erythroblastic island
The erythroblastic island is a distinct anatomic unit which consists of a central macrophage surrounded by erythroid cells. These islands are present at various stages of differentiation in the fetal liver and the adult bone marrow, and have a key role in erythroid cell proliferation and differentiation [1–4]. Gregory and Eaves found that the erythroid cells differentiated through morphologically defined stages of erythroid progenitors, proerythroblasts, basophilic erythroblasts, polychromatophilic erythroblasts, and orthochromatophilic erythroblasts [5, 6]. The orthochromatophilic erythroblasts then underwent enucleation by extruding their nucleus and became reticulocytes, further expelling all organelles and detaching from their microenvironment to form mature circulating erythrocytes. Over the course of erythroid differentiation, erythroblasts displayed a gradual decrease in cell size and an increase in hemoglobin concentration . The major changes of erythroid cells occurred in erythroblastic island, which correlate with the hematopoiesis of bone marrow. Despite recent advances, the mechanism of erythroblast maturation remains poorly understood. Previous reports showed that within erythroblastic islands, both erythroblast-macrophage and erythroblast-erythroblast maturation occurred by extensive interactions [1, 2, 8–12]. Interestingly, only a few molecules have been definitively shown to enhance the formation of erythroblastic islands. The adhesive interaction involved α4β1 integrin and vascular cell adhesion molecule-1 (VCAM-1) were first identified in erythroblasts and macrophages, respectively . Recently, the role of ICAM-4 in erythroblastic island formation has been clearly documented by gene targeting in-vivo studies [14, 15]. Emp was initially identified as a mediator of erythroblast-macrophage interactions during erythroid differentiation [1, 2, 8, 9]. More recently, it was thought that Emp had an important role in erythroblastic island formation [8, 16, 17]. Using a transplant combination from Emp null fetal liver cells to lethally irradiated wild-type sibling mice, Soniet al. found that loss of Emp function in erythroid cells resulted in impaired proliferation and terminal differentiation. These findings suggest that Emp protein might be a potential molecular marker for hematopoietic evaluation.
Hematopoietic stem cell transplantation (HSCT) is the transplantation of stem cells derived from the bone marrow or blood . Stem cell transplantation is a medical procedure used in the fields of hematology and oncology. Increase in the number of erythroblastic islands in bone marrow always suggests a successful HSCT. However, no clinical correlation between Emp levels and hematopoiesis has been established. In this study, we evaluated hematopoietic function of bone marrow in HSCT patients through the change of Emp expression before and after HSCT. Based on our data, we suggest that Emp is a potential biomarker for hematopoietic evaluation of HSCT patients.
Patient characteristics of HSCT
Patient characteristics, HSCT type, underlying disease, and immunosuppressive regimens used before HSCT
Chronic myelogenous leukemia
Acute myelogenous leukemia
Severe aplastic anemia
Immunosuppressive conditioning regimen
Busulfan and melphalan
Busulfan and cyclophosphamide
Total body irradiation and melphalan
Total body irradiation and cyclophosphamide
Peripheral blood and bone marrow were taken from HSCT recipients at the point of 30, 90, and 180 days, respectively. Before HSCT, all patient samples of peripheral blood and bone marrow were acted as the second control group, and that 10 donors constituted the first control group. Samples were processed for peripheral blood and bone marrow smears, and isolation of mononuclear cells.
Isolation of mononuclear cells in peripheral blood and bone marrow
Heparinized (10 IU/mL) peripheral blood and bone marrow was layered on a Ficoll-Hypaque discontinuous gradient system (Sigma) and centrifuged at 1200 × g for 30 min. The mononuclear cells at the interface of plasma and Ficoll-Hypaque were collected and re-suspended in serum-free dulbecco’s modified eagle medium (DMEM). Then the quantity (A) of mononuclear cells were detected by the blood cell analyzer (Beckman Coulter), and their smears were examined by Wright/Giemsa staining (Maker Biotechnology Co., China) for the erythroblastic cell ratio (B%) of mononuclear cells. The erythroblastic cell of number was calculated using the equation: erythroblastic cells (× 109/L) = A(× 109/L) × B%
The morphology of peripheral blood and bone marrow cells was examined by Wright/Giemsa staining. In addition to using blood cell analyzer, cells were directly smeared onto glass plate in an appropriate cell concentration (10 μL blood or bone marrow) and stained with Wright/Giemsa solution. The morphology of blood cells was observed under microsope and at least 200 blood cells with nucleus were counted to determine the ratio of erythroblastic cell in peripheral blood, or the hyperplastic degree in bone marrow.
Mononuclear cells in bone marrow and peripheral blood samples were adjusted to the same quantity (1 × 106 cells in PBS). Total RNA was extracted from the cells and single strand cDNA synthesis was performed by using Blood RNA extraction kit (QIAGEN, Hilden, Germany) and moloney murine leukemia virus-reverse transcription kit (SuperSciptII, Life Technologies, Inc.) according to the manufacturer’s directions. PCR primers used for Emp and β-actin mRNA detection are as follows: Emp sense 5’-ACCCGACCCTCAAGGTGCCC-3’, antisense 5’-GTGGCCG TCTCACGCCTCTC-3’; β-actin5’-TTCCTGGGCATGGAGTCCT-3’, antisense 5’-TGATCTTCATTGTGCTGGGTG-3’. The gene expression levels were normalized by β-actin.
Cells were lysed with lysis buffer (20 mMTris–HCl pH 7.8, 1 mM EDTA, 50 mM sodium chloride, and 0.5% NP-40), and protein concentration was determined by BCA assay kit (Pierce, USA). Western blot analysis was carried out as directions of manufacturer (Cell signaling Biotech). Antibodies against human Emp (Abcam plc. USA. ab65239) and β-actin (Cell signaling Biotech, USA) were used. Results were visualized with horseradish peroxidase-conjugated secondary antibodies (Sigma, USA; 1:1000) and enhanced chemiluminescence.
The patient characteristics before EPO treatment
46.3 ± 2.3
Mean value in 12 inpatients with severe anemia
Low or extremely low
4.7 ± 1.2 × 109/L
15.4 ± 1.3%
The Emp strip was invisible
Statistical analysis between groups was performed using Student t-test. Results were considered statistically significant if P values were <0.05.
Analysis of morphology
Characteristics of morphology before or after HSCT
The first control
5.7 ± 1.3
1.1 ± 0.4
2.7 ± 1.5
1.4 ± 0.5
21.4 ± 4.5
12.8 ± 2.5
48 ± 4.3
The second control
2.1 ± 0.7
6 ± 1.2
1.7 ± 0.6
10.3 ± 1.7
Low or extremely low
3.2 ± 1.4a
5.3 ± 1.4a
16.7 ± 1.6a
30 days after HSCT
2.5 ± 1.0a
13.6 ± 1.6a
1.5 ± 0.4
17.8 ± 1.9a
43.3 ± 4.1a,b
6.6 ± 1.7a
23.5 ± 1.2a
90 days after HSCT
3.2 ± 1.2a
15.4 ± 1.8a
2.2 ± 0.7
19.7 ± 1.4a
46.2 ± 3.6a,b
10.4 ± 1.5a
31.2 ± 1.8a
180 days after HSCT
3.0 ± 1.1a
14.2 ± 1.5a
2.1 ± 0.8
18.5 ± 1.6a
44.7 ± 4.2a,b
8.8 ± 1.2a
33.4 ± 1.1a
The Emp expression
Analysis of EPO treatment
HSCT is a potentially curative therapy for a variety of hematological disorders and cancers of the blood or bone marrow, such as multiple myeloma or leukemia. Hematopoietic evaluation of the patients after HSCT is very important.
In humans, the functional unit for definitive erythropoiesis is the erythroblastic island, a multicellular structure composed of a central macrophage surrounded by developing erythroblasts. Erythroblast-macrophage interactions play a central role in the terminal maturation of erythroblasts [1, 2, 9]. Reconstitution and increase of erythroblastic islands in bone marrow was usually used as a marker for successful HSCT. Erythroblast-macrophage protein (Emp) was initially identified as a mediator of erythroblast-macrophage interactions during erythroid differentiation. The Emp protein [1, 2, 8, 16, 18] plays an important role in normal maturation process of erythroblastic cells in erythroblastic islands in bone marrow. More recent studies have shown that targeted disruption of Emp leads to abnormal erythropoiesis. Unfortunately, the involvement of Emp in HSCT remains uncertain. In this report, we showed that Emp protein levels were correlated with erythroblastic island formation in bone marrow.
Emp was expressed in a variety of hematopoietic (and indeed many non-hematopoietic) cells, including erythroblasts and macrophages . However, Emp expression increased in erythroblasts when hematopoiesis took place in bone marrow (Table 3). Interestingly in this study, Emp mRNA was detected in all the groups, whereas the Emp protein was only detectable in bone marrow mononuclear cells in HSCT group (Figure 2C). These results suggested that Emp might be a potential marker for bone marrow hematopoietic evaluation.
EPO, a kind of hormone preventing erythroid progenitors’ apoptosis , had been widely used in clinics [22–24]. Erythroblastic islands were barely found in bone marrow smears before EPO therapy and were dramatically increased after EPO treatment (Table 2, Figure 3A). Meanwhile, Emp protein level was significantly increased in mononuclear cells from bone marrow in patients receiving EPO treatment (Figure 3B). The data indicate that Emp protein expression could have a close correlation with hematopoietic island formation in bone marrow.
Taken together, our findings provide that Emp protein expression in bone marrow has a close relation with hematopoietic island formation, which might be a potential marker for hematopoietic evaluation in clinic. Thus, our findings suggest that erythobalst island formation is likely an essential feature of erythropoiesis after HSCT. Future studies will be required to assess if Emp levels directly correlate with functional erthropoiesis in this model.
The Emp expression correlates with erythroblastic island formation and has an important function for bone marrow hematopoiesis. Based on our data, we suggest that the Emp is a potential biomarker for hematopoietic evaluation of HSCT patients.
We thank Dr. ManKong for his technical assistance and Dr. Jianxin Yang for helpful discussion. This work was supported by Health Department Fund Project in Hubei Province (grant number CGX2008-2).
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