Mammalian erythropoiesis occurs within erythroblastic islands (EBIs), niches where maturing erythroblasts interact closely with a central macrophage. While it is generally accepted that EBI macrophages play an important role in erythropoiesis, thorough investigation of the mechanisms by which they support erythropoiesis is limited largely by inability to identify and isolate the specific macrophage sub-population that constitute the EBI. Early studies utilized immunohistochemistry or immunofluorescence to study EBI morphology and structure, while more recent efforts have used flow cytometry for high-throughput quantitative characterization of EBIs and their central macrophages. However, these approaches based on the expectation that EBI macrophages are a homogeneous population (F4/80+/CD169+/VCAM-1+ for example) provide an incomplete picture and potentially overlook critical information about the nature and biology of the islands and their central macrophages. Here, we present a novel method for analysis of EBI macrophages from hematopoietic tissues of mice and rats using multispectral imaging flow cytometry (IFC), which combines the high-throughput advantage of flow cytometry with the morphological and fluorescence features derived from microscopy. This method provides both quantitative analysis of EBIs, as well as structural and morphological details of the central macrophages and associated cells. Importantly, the images, combined with quantitative software features, can be used to evaluate co-expression of phenotypic markers which is crucial since some antigens used to identify macrophages (e.g., F4/80 and CD11b) can be expressed on non-erythroid cells associated with the islands instead of, or in addition to the central macrophage itself. We have used this method to analyze native EBIs from different hematopoietic tissues and evaluated the expression of several markers that have been previously reported to be expressed on EBI macrophages. We found that VCAM-1, F4/80, and CD169 are expressed heterogeneously by the central macrophages within the EBIs, while CD11b, although abundantly expressed by cells within the islands, is not expressed on the EBI macrophages. Moreover, differences in the phenotype of EBIs in rats compared to mice point to potential functional differences between these species. These data demonstrate the usefulness of IFC in analysis and characterization of EBIs and more importantly in exploring the heterogeneity and plasticity of EBI macrophages.

Terminal erythroid differentiation occurs in the bone marrow, within specialized niches termed erythroblastic islands. These functional units consist of a macrophage surrounded by differentiating erythroblasts and have been described more than five decades ago but their function in the pathophysiology of erythropoiesis has remained unclear until recently. Here we propose that the central macrophage in the erythroblastic island contributes to the pathophysiology of anemia of inflammation. After introducing erythropoiesis and the interactions between the erythroblasts and the central macrophage within the erythroblastic islands, we will discuss the immunophenotypic characterization of this specific subpopulation of macrophages. We will then integrate these concepts into the currently known pathophysiological drivers of anemia of inflammation and address the role of the central macrophage in this disorder. Finally, as a means of furthering our understanding of the various concepts, we will discuss the differences between murine and rat models with regards to developmental and stress erythropoiesis in an attempt to define a model system representative of human pathophysiology.

Purpose of review The erythroid progenitors BFU-E (burst-forming unit-erythroid) and CFU-E (colony-forming unit-erythroid) have a critical role in erythropoiesis. They represent a heterogeneous and poorly characterized population of cells with modifiable self-renewal, proliferation and differentiation capabilities. This review focuses on the current state of erythroid progenitor biology with regard to immunophenotypic identification and regulatory programs; in addition, we will discuss the therapeutic implications of using these erythroid progenitors as pharmacologic targets. Recent findings Erythroid progenitors are classically defined by the appearance of morphologically defined colonies in semisolid cultures. However, these prior systems preclude a more thorough understanding of the composite nature of progenitor populations. Recent studies employing novel flow cytometric and cell-based assays have helped to redefine hematopoiesis, and suggest that erythroid progenitors may arise from different levels of the hematopoietic tree. Moreover, the identification of cell surface marker patterns in human BFU-E and CFU-E enhance our ability to perform downstream functional and molecular analyses at the population and single cell level. Advances in these techniques have already revealed novel subpopulations with increased self-renewing capacity, roles for erythroid progenitors in globin gene expression, and insights into pharmacologic mechanisms of glucocorticoids and pomalidomide. Summary Immunophenotypic and molecular characterization resolves the diversity of erythroid progenitors and may ultimately lead to the ability to target these progenitors to ameliorate diseases of dyserythropoiesis.