Page 81 - Haematologica April 2020

EPHB4 drives macrophage-erythroblasts interactions
act in concert as a coincidence detector, to ensure that the macrophage associates and binds to the differentiating progenitor cell at the appropriate time (Figure 6).
Furthermore, contact inhibition of locomotion is thought to be controlled by a ratio of EPHB and EPHA receptors.16 Interestingly, we observed that erythroblasts have a high level of EPHB and EPHA receptors during early stages of differentiation but lose EPHB receptors by the final stages of differentiation, while EPHA is retained (albeit at lower levels). We hypothesize that EPH recep- tors may contribute throughout the whole process of dif- ferentiation; in the early stages of differentiation, EPHB receptor is present in higher amounts than EPHA leading to recognition between the cells, supported by the EPHB4 inhibition disrupting this interaction. As the cells differen- tiate, the amount of EPHA receptor becomes dominant over EPHB, tantalisingly suggesting that, once the ery- throblast has enucleated to form a reticulocyte, EPHA4 may be involved in the separation of the cells; a hypothe- sis that will need to be explored in future experiments.
It is notable that, in humans, EPH receptor activation did not affect integrin activation at day 5, which contradicts the findings of a recent report,9 which showed that, in mice, EPHB1 receptor engagement caused a rise in CD29 and its activation at the surface of erythroblasts after agrin treatment. We cannot exclude the possibility that agrin still has a role in the integrin β1 activation observed in this study, separate from its additional role in activating the EPHB receptors at the surface. However, our results indi- cate that these two events are likely independent and that the difference between the previous study and ours is due to differences in species.
Our observation here that VCAM1– macrophages are capable of interacting with erythroblasts confirms work by Wei and Frenette41 and Falchi et al.33 All three types of macrophages tested in this study were capable of binding erythroblasts to some degree. This change in the level of binding could be related to the subtle changes which exist between these cells, be it the presence of VCAM1, differ- ent levels of ephrin-B2 at their surface or other unknown differences. Indeed, it is known that varying levels of ephrin-B2 can influence the function of EPHB6.42 As the BM macrophages express lower amounts of ephrin-B2 expression than cultured macrophages, this may explain the former’s interaction sensitivity to EPHB6 silencing. Taken together, the experiments have shown that EPH receptors are more important to the recognition between erythroblasts and macrophages than VCAM1. This mirrors the results found by Wei and Frenette who reported that EMP is more important than VCAM1 for the further stages of the interaction.41 However, we do not fully understand the hierarchy between EPH receptors and the integrins. Our experiments do, however, demonstrate that both EPH and integrins together are essential for the interaction and recognition of the macrophages to the erythroblasts.
The evidence presented here also suggests a VCAM1– independent interaction for integrins in erythroblastic island formation, or perhaps associations are more promiscuous than anticipated. In addition, although a large number of mature macrophages in the human BM are VCAM1+, indicating a preference for this phenotype in island formation, the ability for VCAM1– macrophages to form interactions with erythroblasts indicates a flexibility in terms of the type of macrophages which can participate in erythroblastic island formation, which may be impor- tant during stress erythropoiesis. The role of VCAM1 in helping erythroblasts expand40 would explain this prefer- ence.
In addition to driving interactions, the EPH receptors may play additional supportive roles within the erythrob- lastic island. It was previously reported that overexpres- sion of EPHB4 increases HSC numbers;20 therefore, EPHB4 might have a proliferative role within the niche. Finally, the observation that erythroblasts express both ephrin-B2 and EPHB4, raises the potential for homophilic cell inter- action that could lead to higher proliferation, and also explains the ability of erythroblasts to proliferate in vitro in the absence of macrophages.
In summary, it has previously been difficult to deter- mine the specific contribution certain receptors play in human macrophage-erythroblast interactions during ery- thropoiesis. This study has successfully employed live imaging of human macrophages and erythroblasts to probe the role of a potential receptor interaction between the cells over time. This has shown, for the first time, a dependence on EPHB interactions for the macrophage and erythroblast interaction, and we believe this imaging assay can help further delineate the importance of other receptors in the interaction between erythroblasts and macrophages in future experiments.
Acknowledgments
We would like to thank Dr. Emile van den Akker for discussion on development of an antibody panel for macrophages; Dr. Andrew Herman and Lorena Sueiro for the Biomedical Sciences Flow Cytometry Facility for FACS sorting; the Wolfson Bioimaging facility for access to the Incucyte, widefield and all their help; Abi Gartner for his help with analysis. We thank the Elizabeth Blackwell Institute Wellcome Trust ISSF Award for providing funding for Stephen Cross.
Funding
This work was funded by NHS Blood and Transplant R&D grants (WP15-05); a Wellcome Trust PhD studentship (LHO- N; 105385/Z/14/Z). This work is also funded by the National Institute for Health Research (NIHR) Blood and Transfusion Research Unit (NIHR BTRU) in Red Cell Products (NIHR- BTRU-2015-10032). The views expressed are those of the authors and not necessarily those of NIHR or the Department of Health and Social Care.
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