Page 74 - Haematologica April 2020

L.A. Hampton O’Neil et al.
erythroblasts and macrophages were determined based on the maximum separation of object perimeters. Multiple erythroblasts could be assigned to a single macrophage. This program can be found at: doi: 10.5281/zenodo.3237585 (Stephen Cross. (2018, March 14). SJCross/RelateCells v1.2.2 (Version v1.2.2). Zenodo.).
Antibodies, cell culture, lentiviral transduction, real-time quantitative polymerase chain reaction, western blotting, flow cytometry, cytospins and statistics
Details of all these methods can be found in the Online Supplementary Methods.
Results
Ephrin-B2 is expressed by M2-like macrophages and bone marrow macrophages
As reported previously by Heideveld et al. macrophages treated with dexamethasone (+Dex) phenotypically resemble the resident macrophages found in BM and fetal liver,31 exhibiting a high level of CD163 and CD169, simi- lar to BM macrophages (Online Supplementary Figure S1). Furthermore, these cultured macrophages are known to be able to help the erythroblasts expand,32,33 form ery- throblastic islands, and phagocytose nuclei.31 However, whereas up to 70% of BM isolated CD14+CD16+ macrophages are VCAM1+, +Dex macrophages are VCAM1–. It is known that BM stromal cells, including macrophages, express ephrinB2.23 Figure 1A and B con- firms that all three macrophage types, +Dex in vitro cul- tured, VCAM1+ and VCAM1– BM macrophages express ephrin-B2, the most potent receptor for EPHB4 and EPHB6 receptors (see below). There was no discernible difference between the expression levels of ephrin-B2 on sorted VCAM1+ and VCAM1– macrophages, but the in vitro cul- ture +Dex macrophages express higher levels (Figure 1C).
EPHB4, EPHB6 and EPHA4 are expressed in expanding and differentiating erythroblasts
To determine which EPH receptors are expressed in ery- throblasts at different stages of development, real-time quantitative polymerase chain reaction (RT-PCR) was per- formed on three independent in vitro cultured differentia- tion courses. In these cultures, CD34+ cells are isolated and expanded for eight days, called D0 to D7. During this time, the cells express CD34 and CD36, markers of the BFU-E and CFU-E. The cells are then differentiated for eight days: T0 to T168. These stages of differentiation can be separated by their morphology using cytospins (Online Supplementary Figure S2A and B). EPHB4, EPHB6 and EPHA4 mRNAs were expressed in expanding day 6 and differentiating T0, representing the proerythroblast/basophilic stage in our culture system. RNA for numerous EPH receptors (EPHA1, EPHA2, EPHA3, EPHA6, EPHA7, EPHA8, EPHB2 and EPHB3) was not detected during erythropoiesis (Figure 1D ). Western blots confirmed EPHB4, EPHB6 and EPHA4 protein expression (Figure 1E). EPHA4 expression diminished but was still retained at the reticulocyte stage. Unlike in mice,9 EPHB1 was not reproducibly detected at either the RNA or protein level (being detected by RT-PCR only once in 4 separate erythroid differentiation courses). Interestingly, the ligand ephrin-B2 is also present in the late stage of expansion in erythroblasts.
Erythroblast surface expression of EPH receptors is dynamic during terminal differentiation
To assess the timeframe in which the EPH receptors are expressed on the surface of erythroblasts during differen- tiation, a surface binding assay was performed. Ephrin-B2 was chosen as the ligand used in this experiment as it binds all EPHB receptors.34 Ephrin-B2 was clustered with a fluorescently-conjugated IgG antibody and added to the live cells. Figure 1F demonstrates that the cells bind ephrin-B2-Fc during the final part of the expansion phase (D5 and D6) when cells are CD34low/CD36high proerythrob- lasts (Figure 2A). As cells commence terminal differentia- tion (T0 hours), there is a steady reduction of ligand bind- ing (no statistically significant binding after T0), and by T72 hours all binding is lost. At T72 hours, the majority of erythroblasts present in culture are beyond the basophilic stage (Online Supplementary Figure S2A), confirming that EPHB receptors are expressed during the early phases of terminal differentiation.
Integrins are crucial in cell-cell contact and adhesion with macrophages through the formation of focal adhe- sion points.6,7,9,35 Therefore, we tested whether the appear- ance of integrins on the erythroblast surface coincided with EPHB4 receptor surface expression. To detect inte- grins, a surface binding experiment was conducted using VCAM1-Fc where the integrins were pre-activated with manganese to ensure VCAM1-Fc construct binding. Without pre-activation, no binding was found (Online Supplementary Figure S3), but when all the integrins were activated, VCAM1-Fc bound throughout erythroblast dif- ferentiation until approximately T144 hours when 50% of the cells were reticulocytes (Figure 1F). Manganese treat- ment was observed to increase cell death and clustering. Therefore flow cytometry analysis was performed only on live single cells during the surface assay by gating on unclustered propidium iodide negative cells.
Baseline activation of integrins occurs during the height of EPHB4 and EPHB6 expression
We next wanted to establish the level of integrin activa- tion during the stages at which EPHB4/B6 become more pronounced at the surface of erythroblasts. To do this, we used an antibody that specifically recognizes the active form of integrin β1, which is present in both the VLA-4 (integrin α4β1) and VLA-5 (integrin α5β1) complexes. Manganese was used to activate integrins beforehand as a positive control. We detected a marked increase of integrin activation in a small percentage of the cells between days 4 and 5 on erythroblasts in the absence of any treatment, representing an increase in 10-30% of cells at day 5 and 90% at day 6 (Figure 2B). This increase represents 50% of the mean fluorescent intensity (MFI) observed with the manganese treatment, which activates all the integrins (Figure 2C). The CD36high/CD34low/- populations displayed this rise in baseline integrin activation (Figure 2A).
To establish the effects of EPH receptor stimulation on integrin activation, the amount of active form of integrin β1 was monitored in the presence of clustered ephrin-B2. Surprisingly, EPH receptor engagement had no significant effect on the level of activation of integrin β1 (Figure 2B).
Peptide inhibition of EPHB4 activation impacts on macrophage-erythroblast interactions
The EPHB4 receptor inhibitor (TNYL-RAW peptide) competes selectively with ephrin-B2 binding to EPHB4
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