Page 36 - Haematologica3

E. Heideveld et al.
pressor gene in mice lead to embryonic death as erythrob- lasts fail to enucleate.11 These data show that in vivo, macrophages are important in regulating erythropoiesis in adults and during development.
Previously, we found that blood-derived monocytes induced to differentiate using stem cell factor (SCF), ery- thropoietin (EPO) and glucocorticoids enhance in vitro ery- thropoiesis by supporting HSPC survival.12 These macrophages display a tissue-resident profile expressing CD14 (lipopolysaccharide [LPS]-receptor), CD16 (FcγRIII), scavenger receptor CD163, CD169, CD206 (mannose receptor), CXCR4 and minimal expression of dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN).12 We hypothesized that these cultured monocyte-derived macrophages may have a sim- ilar role as mouse CD169+ macrophages in both hematopoiesis and erythropoiesis. This would provide an easy-to-use in vitro human model system to mimic ery- throblastic islands allowing for the study of functional interactions between macrophages and erythroid cells, which is currently limited to harvesting BM or involves genetic modification.13 A better understanding of the mechanism(s) through which human macrophages inter- act and regulate erythroblast maturation and enucleation is important in order to understand the pathology of ery- thropoietic disorders, such as erythrocytosis in poly- cythemia vera or erythrophagocytosis in several types of hemolytic anemia, as well as to improve in vitro erythroid differentiation protocols for erythrocyte production.14,15
In mice BM, erythroblasts are bound to macrophages via interactions between integrin-α4β1 on erythroblasts and VCAM1 on macrophages, and blocking these molecules disrupts erythroblastic islands.16 Chow et al. described human BM macrophages as also expressing VCAM1. However, Ulyanova et al. have shown that Vcam-/- mice do not display an erythroid phenotype during homeostasis or phenylhydrazide-induced stress.17 During terminal differ- entiation erythroblasts enucleate, resulting in reticulocytes and pyrenocytes. The latter are also still encapsulated by plasma membrane. In mice, clearance of pyrenocytes occurs via TAM-receptors on the central macrophages that recognize and bind phosphatidylserine (PS) exposed on pyrenocytes resulting in phagocytosis in a protein S- dependent manner.18,19 The TAM-receptor family of tyro- sine kinases (TYRO3, AXL, and MERTK) play an impor- tant role in the phagocytic ability of macrophages as triple knock-out mice fail to clear apoptotic cells in multiple tis- sues. These mice develop normally, but eventually devel- op autoimmunity, such as systemic lupus erythematosus (SLE).20 This is in line with studies showing that SLE has been associated with failure of macrophages to phagocy- tose apoptotic cells and pyrenocytes in both humans and mice.21-24 In addition, anemia is found in about 50% of SLE patients; Toda et al. showed that embryos suffer from severe anemia caused by failure of macrophages to phago- cytose pyrenocytes.25 These data indicate that macrophages are essential during all stages of erythro- poiesis, including enucleation, and display inherent fea- tures that are indispensable to the functionality of these macrophages.
Herein, we show that peripheral blood monocytes can be differentiated to erythropoiesis-supporting macrophages that interact with erythroid cells, phagocy- tose pyrenocytes and phenotypically resemble human CD169+ BM and FL macrophages.
Methods
Human materials
Human blood, BM and FL mononuclear cells were purified by density separation, following manufacturer’s protocol. Regarding blood, informed consent was given in accordance with the Declaration of Helsinki, the Dutch National and Sanquin Internal Ethic Boards, and by the Bristol Research Ethics Committee (REC; 12/SW/0199). Following informed consent, adult BM aspi- rates were obtained from the sternum of patients undergoing car- diac surgery, and approved by the Medical Ethical Review Board of the AMC (MEC:04/042#04.17.370). Fetal tissues (week 15-22) were obtained from elective abortions contingent on informed consent and approval by the Medical Ethical Commission of the Erasmus University Medical Center Rotterdam (MEC-2006-202).
Cell culture
CD14 and CD34 MicroBeads (Miltenyi Biotec, Gladbach, Germany) were used for cell isolation from peripheral blood. CD14+ monocytes were cultured at 1.5-3x106 cells/well (CASY® Model TTC, Schärfe System GmbH, Reutlingen, Germany) in a 12-well plate as described.12 Cells were treated with 1-20μM mifepristone (Sigma-Aldrich, Munich, Germany) directly after isolation or 4-24 hours after three days of culture. CD34+ cells were differentiated towards erythroblasts,12 with the addition of 1ng/ml IL-3 (R&D systems, Abingdon, UK) at the start of culture. Media was replenished every two days. After 8-10 days, cells were differentiated towards reticulocytes by removing dexam- ethasone, increasing EPO (10U/ml, ProSpec; East Brunswick, NJ, USA) and adding heparin (5U/ml, LEO Pharma B.V., Breda, The Netherlands), 5% pooled AB+ plasma and holotransferrin (700μg/ml, Sanquin, Amsterdam, The Netherlands). Every other day, half the media was replenished. For co-culture experiments, CD14+ cells were differentiated with (GC-macrophages) or with- out dexamethasone for three days and co-cultured with ery- throblasts (day 8-10 of culture; ratio 1:1.5) or more differentiated erythroid cells (day 6 of differentiation; ratio 1:4) for 24 hours.
Flow cytometry
Cells were washed in phosphate-buffered saline (PBS) and resuspended in 1% bovine serum albumin (BSA)/PBS. Cells were incubated with primary antibodies for 30min at 4C, measured on LSRII or LSRFortessa (both BD Biosciences, Oxford, UK) and ana- lyzed using FlowJo software (FlowJo v10; Tree Star, Inc., Ashland, OR, USA) (antibodies listed in Online Supplementary Methods).
Mass spectrometry
See Online Supplementary Methods.
ImageStreamX and IncuCyte
GC-macrophages or unstimulated cells were incubated with 100μg/ml fluorescein isothiocyanate (FITC)-labeled zymosan (S. cerevisiae; MP Biomedicals, Solon, OH, USA) for 40min at 37C. Zymosan was removed and cells were fixed in 4% paraformalde- hyde (PFA) for 20min at 4C. Cells were transferred to 1% BSA/PBS and stained with human leukocyte antigen-antigen D- related R-phycoerythrin (HLA-DR PE; BD Biosciences). Furthermore, erythroid cells at day seven of differentiation were stained with Deep Red Anthraquinone 5 (DRAQ5; Abcam, Cambridge, UK). Imaging was performed on the ImageStreamX (Amnis Corporation, Seattle, WA, USA) and images were ana- lyzed using IDEAS Application v6.1 software (Amnis Corporation). For IncuCyte experiments see Online Supplementary Methods.
396
haematologica | 2018; 103(3)