Myelodysplastic syndrome (MDS) is an incurable hematological malignancy in which clonal hematopoietic stem cells proliferate and expand within bone marrow, leading to cytopenia, dysplasia in one or more of the myeloid lineages, ineffective hematopoiesis, and increased risk of development of acute myeloid leukemia (AML). Clinical studies and experimental mouse models indicate that the bone marrow microenvironment and immune system play important roles in pathogenesis of MDS [
Macrophages are innate immune cells that are positioned throughout the body tissues, where they ingest and degrade abnormal cells, debris, and foreign material and orchestrate inflammatory processes. When monocytes migrate from the circulation and extravasate through the endothelium, they differentiate into macrophages. Monocytes and macrophages are professional phagocytic cells. The various macrophage subsets play either a protective or a pathogenic role in antimicrobial defense, allergy and asthma, autoimmunity, antitumor immune responses, tumorigenesis, metabolism and obesity, atherosclerosis, fibrosis, and wound healing [
The role of macrophages in the pathophysiology of human malignancies has received increasing interest. In solid tumors, 5%–40% of tumor mass consists of tumor-associated macrophages (TAMs). The TAMs are now known to be important for development and progression of malignant diseases, owing to suppression of antitumor immunity. Furthermore, infiltration by TAMs is related to poor outcome in most human malignancies [
In this article, we have investigated the role of monocyte-induced macrophages in the pathogenesis of MDS.
Twenty-four patients diagnosed with MDS were enrolled in this study, as per the criteria of World Health Organization (WHO) (2008). The study was carried out at the Hematology Department of General Hospital, Tianjin Medical University, Tianjin, China, from September 2014 to December 2015. Basic characteristics of the patients are described in Table
Clinical characteristics of MDS patients.
Case | Sex/age | Diagnosis | Cytogenetics | IPSS |
---|---|---|---|---|
1 | Female/61 | 5q− | Good | Int-1 |
2 | Female/79 | RAEB-2 | Good | Int-2 |
3 | Female/68 | RAS | Good | Int-1 |
4 | Female/49 | RAEB-2 | Good | Int-1 |
5 | Male/25 | RCMD | Good | Int-1 |
6 | Female/62 | RAEB-2 | Good | Int-2 |
7 | Male/57 | RAEB-2 | Good | Int-2 |
8 | Male/42 | RAS | Good | Low |
9 | Female/70 | RAEB-2 | Good | Int-2 |
10 | Male/30 | RAEB-2 | Int | High |
11 | Female/29 | RAEB-2 | Poor | High |
12 | Male/58 | RAEB-1 | Poor | Int-2 |
13 | Male/58 | RAEB-2 | Good | Int-2 |
14 | Female/69 | RAEB-2 | Good | Int-2 |
15 | Male/76 | RAEB-2 | Good | Int-2 |
16 | Male/59 | RAEB-2 | Good | Int-2 |
17 | Male/61 | RAEB-2 | Good | Int-2 |
18 | Female/60 | RAEB-2 | Good | Int-2 |
19 | Male/49 | RA | Good | Low |
20 | Male/16 | RCMD | Int | Int-1 |
21 | Male/50 | RCMD | Good | Int-1 |
22 | Female/41 | RCMD | Int | Int-1 |
23 | Female/64 | RCMD | Good | Low |
24 | Male/69 | RCMD | Int | Int-1 |
Peripheral blood mononuclear cells (PBMCs) were separated from fresh heparinized blood samples (5 mL). The PBMCs were seeded at 3–5 million cells/mL in sterile RPMI 1640 (Invitrogen, Carlsbad, CA, USA) and cultured for 7 days with the addition of granulocyte macrophage colony-stimulating factor (GM-CSF) (Huabei Pharmacy, Shijiazhuang, China). The macrophages became attached to the bottom of the culture dishes during the course of the culture. On day 7, the cells were observed under the microscope and collected for counting.
Normal PBMCs were labeled with 0.5
The fluorescent microspheres (80
The level of inducible nitric oxide synthase (iNOS) in the supernatant of macrophage cultures was measured by human ELISA kit (Elabscience Biotechnology, Wuhan, China). In addition, the expression of iNOS was stimulated by lipopolysaccharide (LPS) and IFN-
The effector proteins of macrophages were measured in peripheral blood samples from patients with MDS and normal controls. The cells were stained with CD14, CD68, and CD206 or signal regulatory protein alpha (SIRP
All analyses were performed using SPSS 18.0 software (SPSS Science). Data were presented as mean ± SE. Student’s
The number of monocytes was (659.2 ± 38.6) × 106/L in MDS patients, while that in the controls was (294.0 ± 17.4) × 106/L. The quantity of monocytes in MDS patients was higher than that in the controls (
Quantity of monocytes increased in MDS patients (
The PBMCs from MDS group showed impaired capacity to induce macrophages. The macrophages were observed under the microscope and collected for subsequent experiments. The induced macrophages (CD14+CD68+) in the MDS group and normal controls were 10.06% ± 2.04% and 75.29% ± 5.94%, respectively (
Ability of monocytes to induce macrophages was lower in MDS patients. (a) Monocyte-induced macrophages (CD14+) derived from peripheral blood of patients with MDS and normal controls were measured by flow cytometry. (b) Quantity of CD14+CD68+ cells decreased in MDS patients (
The monocyte-differentiated macrophages in the MDS group showed lower phagocytic capacity than those from the normal controls by fluorescent microspheres.
To determine the role of macrophages, the monocyte-differentiated macrophages from patients with MDS and normal controls were evaluated. The phagocytic percentage (PP, the count of macrophages engulfing fluorescent microspheres/total macrophage cell number × 100%) of monocyte-differentiated macrophages (23.69% ± 3.22%) was significantly decreased in the MDS group compared to that in normal controls (42.75% ± 2.13%,
Phagocytosis of monocyte-induced macrophages as demonstrated by fluorescent microspheres. (a) Phagocytic capacity of differentiated macrophages derived from peripheral blood from patients with MDS and normal controls was tested with fluorescent microspheres by flow cytometry. In the picture, the left represents the macrophages not engulfing the fluorescent microspheres; the right represents the macrophages engulfing the fluorescent microspheres. R3 suggests that the macrophages are swallowing a fluorescent microsphere; R4 suggests that the macrophages are swallowing two fluorescent microspheres; R5 suggests that the macrophages are swallowing three fluorescent microspheres; R6 suggests that the macrophages are swallowing four fluorescent microspheres. (b) The PI and PP of monocyte-induced macrophages from MDS and normal controls are shown, respectively (
The ability of macrophages to engulf CFSE-labeled normal PBMCs was decreased in the MDS group compared to the normal controls, as evidenced by immunofluorescence microscopy.
We applied another method to confirm the impaired phagocytosis of macrophages in MDS patients. The CFSE-labeled normal PBMCs were incubated with macrophages from MDS patients or normal controls and then assessed for phagocytosis by immunofluorescence microscopy. The PI of macrophages in the MDS patients (0.24 ± 0.04) was significantly lower than that in the normal controls (0.48 ± 0.06,
Phagocytosis of monocytes-induced macrophage as demonstrated by CFSE. CFSE-labeled normal PBMCs were incubated with monocyte-induced macrophages from either normal controls (a) or patients with MDS (b). These cells were assessed by immunofluorescence microscopy for the presence of fluorescently labeled normal PBMCs within the macrophages (indicated by arrows). (c) Phagocytic capacity of differentiated macrophages from patients with MDS and normal controls was tested by immunofluorescence microscopy. The PI of differentiated macrophages from MDS and normal controls are shown (
The expression of the macrophage mannose receptor (CD206) on macrophages in MDS patients was significantly reduced compared to that in normal controls (9.73% ± 2.59% versus 51.15% ± 10.82%, respectively;
Expression of CD206 (CD206+/CD14+CD68+) on macrophages from peripheral blood from patients with MDS and normal controls was tested by flow cytometry (
The expression of SIRP
Expression of SIRP
The level of iNOS in the supernatant of macrophage cultures from MDS patients was increased compared to that in normal controls (35.87 ± 6.25 pg/mL versus 22.05 ± 3.67 pg/mL, respectively;
Comparison of levels of iNOS in the supernatant of macrophage cultures from either MDS patients or normal controls, as measured by ELISA (
Cancer development is a multistep process involving sequential mutations in oncogenes and tumor suppressor genes of normal cells, resulting in the transformation into a tumor cell [
Traditionally, macrophages have been described as tumoricidal cells. Macrophages have a pleiotropic biological role that includes antigen presentation, target cell cytotoxicity, removal of debris and tissue remodeling, regulation of inflammation, induction of immunity, thrombosis, and various forms of endocytosis. Increasing evidence indicates that macrophages can also adopt a protumor phenotype in both primary tumors and metastases, as they can promote growth, angiogenesis, metastasis, and immunosuppression [
In our study, we observed that although the number of monocytes in majority of the MDS patients was increased, the macrophages derived from MDS monocytes were fewer and exhibited impaired phagocytosis. This suggested that the ability of abnormal monocytes to develop into normal macrophages was inhibited in MDS patients. Moreover, when abnormal MDS clonal cells arise, the macrophages fail to phagocytose them. In addition, macrophages and monocytes could be partly progenies of MDS clone in most cases, which leads to bone marrow protumor microenvironment.
We observed that the levels of CD206 and SIRP
In this study, we found that iNOS expression was upregulated in MDS patients, compared to normal controls. Human carcinomas are associated with upregulation of iNOS, which is otherwise generally not expressed in normal (noncancerous) tissues, with the exception of the kidney, brain, and placenta [
In this study, we explored the role of macrophages in the pathogenesis of MDS by inducing the monocytes to become macrophages. Compared with normal controls, the macrophage phagocytosis activity in MDS patients was abnormal. The expressions of recognized receptors CD206 and SIRP
The author should be contacted for data requests.
The study was approved by the Ethics Committee of the General Hospital, Tianjin Medical University.
Informed written consent was obtained from all patients or their guardians in accordance with the Declaration of Helsinki.
The authors declare that they have no competing interests.
Yu Han performed the research and analyzed the data. Huaquan Wang designed the study, ensured correct analysis of the data, and wrote the manuscript. Zonghong Shao assisted in designing the experiments, oversaw the collection of the data, and contributed to the writing of the manuscript. All authors critically revised and approved the manuscript.
This project is partly supported by the National Natural Science Foundation of China (nos. 81170472, 81400088, and 81570111) and Application Bases and Advanced Technology Research Program of Tianjin (nos. 14JCYBJC27200 and 09JCYBJC11200). These funders have no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.