Author: Antoine Roux, Ethan Hughes, Mia Foster
Research Article
Hemo and Immunoregulatory Cytokine Production by Erythroblast Antigen+ Glycophorin A+ Bone Marrow Cells
Ethan Hughes1*, Antoine Roux2 and Mia Foster2
1Department of Biochemistry, Imperial College London, London, UK
2Department of Immunology, Aix-Marseille University, Marseille, France
Published: 14 April2015
Abstract
Background: The bone marrow microenvironment orchestrates hematopoiesis through a complex interplay of cell-cell interactions and soluble factors, including cytokines. While stromal cells and leukocytes are recognized cytokine sources, the contribution of developing hematopoietic cells themselves, particularly erythroid progenitors, is less understood. Erythropoiesis occurs in specialized niches (erythroblastic islands) involving close contact between erythroblasts and macrophages, suggesting potential for bidirectional signaling. Glycophorin A (GPA) is a definitive erythroid lineage marker, while other antigens recognized by historical ‘Erythroblast Antigen’ (EBA) antibodies (often targeting GPA itself or associated markers like Band 3 or Transferrin Receptor/CD71) mark specific developmental stages.
Objective: This study aimed to determine whether human bone marrow cells expressing erythroid markers (defined operationally here as co-expressing GPA and an EBA marker recognized by a specific anti-erythroblast antibody, potentially reflecting CD71 high expression) are capable of producing key hematopoietic and immunoregulatory cytokines, both constitutively and upon stimulation.
Methods: Human bone marrow mononuclear cells (BMMCs) were obtained from healthy donors. Cells co-expressing GPA (CD235a) and an ‘Erythroblast Antigen’ (EBA, defined by reactivity with a specific monoclonal antibody known to bind erythroblasts, e.g., clone 10F7 MN recognizing Band 3 or similar, or alternatively defined as CD71high/GPA+) were isolated using fluorescence-activated cell sorting (FACS). GPA-negative cells served as controls. Sorted populations were cultured in vitro under basal conditions or stimulated with lipopolysaccharide (LPS), phytohemagglutinin (PHA), or interleukin-1β (IL-1β). Cytokine secretion into culture supernatants was measured using Enzyme-Linked Immunosorbent Assays (ELISA) and multiplex bead array assays for a panel of cytokines (e.g., IL-1β, IL-6, TNF-α, G-CSF, GM-CSF, IL-10, TGF-β1). Cytokine mRNA expression was assessed by quantitative real-time PCR (RT-qPCR).
Results: Highly purified populations of EBA+/GPA+ erythroblasts were successfully isolated from human bone marrow (>95% purity). Under basal culture conditions, these cells secreted detectable, albeit low, levels of certain cytokines, notably TGF-β1 and IL-10. Upon stimulation, particularly with LPS or IL-1β, EBA+/GPA+ cells significantly upregulated the production and secretion of several pro-inflammatory and hematopoietic cytokines, including IL-6, G-CSF, and GM-CSF, as confirmed by both protein secretion assays and increased mRNA expression. Production of TNF-α and IL-1β was minimal or absent. The immunoregulatory cytokines IL-10 and TGF-β1 were also produced, with IL-10 secretion potentially increasing after stimulation. In contrast, GPA-negative bone marrow cells exhibited a different cytokine profile, with higher basal and stimulated production of pro-inflammatory cytokines like TNF-α.
Conclusion: This study demonstrates that human bone marrow erythroid progenitor cells, specifically the EBA+/GPA+ population, are not merely passive recipients of regulatory signals but are themselves capable of producing a distinct repertoire of biologically active cytokines. Their ability to secrete factors like IL-6, G-CSF, GM-CSF, IL-10, and TGF-β1, especially upon stimulation, suggests that erythroblasts may actively participate in regulating their own development (autocrine/paracrine loops), influencing other hematopoietic lineages, and modulating the immune microenvironment within the bone marrow niche.
Keywords: Erythropoiesis; Erythroblast; Glycophorin A (GPA); Erythroblast Antigen (EBA); Cytokines
