Abstract
Repeated weekly injections of rat erythrocytes produced autoimmune hemolytic anemia (AIHA) in C57BL/6 mice after 5–6 weeks. Using the double in vivo biotinylation (DIB) technique, recently developed in our laboratory, turnover of erythrocyte cohorts of different age groups during AIHA was monitored. Results indicate a significant decline in the proportion of reticulocytes, young and intermediate age groups of erythrocytes, but a significant increase in the proportion of old erythrocytes in blood circulation. Binding of the autoantibody was relatively higher to the young erythrocytes and higher levels of intracellular reactive oxygen species (ROS) were also seen in these cells. Erythropoietic activity in the bone marrows and the spleen of AIHA induced mice was examined by monitoring the relative proportion of erythroid cells at various stages of differentiation in these organs. Cells at different stages of differentiation were enumerated flow cytometrically by double staining with anti-Ter119 and anti-transferrin receptor (CD71) monoclonal antibodies. Erythroid cells in bone marrow declined significantly in AIHA induced mice, erythroblast C being most affected (50% decline). Erythroblast C also recorded high intracellular ROS level along with increased levels of membrane-bound autoantibody. No such decline was observed in spleen. A model of AIHA has been proposed indicating that binding of autoantibodies may not be a sufficient condition for destruction of erythroid cells in bone marrow and in blood circulation. Last stage of erythropoietic differentiation in bone marrow and early stages of erythrocytes in blood circulation are specifically susceptible to removal in AIHA.
Highlights
Autoimmune hemolytic anemia (AIHA) is one of the earliest recognized autoimmune diseases in humans [1], characterized by the production of self-reactive autoantibodies against erythrocytes that can lead to a rapid and profound decline of erythrocyte count and hemoglobinPLOS ONE | DOI:10.1371/journal.pone.0166878 November 21, 2016Stages of Erythroid Differentiation Susceptible to Loss in Auto-Immune Hemolytic Anemia
Our results indicate that while autoantibodies bind to cells in all stages of erythropoiesis in bone marrow and spleen and circulating mature erythrocytes, decline in relative proportion was confined only to the late stages of erythroid differentiation in bone marrow and younger erythrocytes in blood circulation suggesting that these erythroid populations may be preferentially lost in autoimmune hemolytic anemia (AIHA)
The Playfair and Clarke model [20] of experimentally induced AIHA in mice employs one such mechanism, where the immunological unresponsiveness to self erythrocytes is broken by repeated exposure to rat erythrocyte antigens with similar/shared epitopes that activate the otherwise inactive autoreactive B- and T-lymphocytes [22,23]
Summary
Autoimmune hemolytic anemia (AIHA) is one of the earliest recognized autoimmune diseases in humans [1], characterized by the production of self-reactive autoantibodies against erythrocytes that can lead to a rapid and profound decline of erythrocyte count and hemoglobin. Aim of the present study was to look at the complete life cycle of erythroid cells including different stages of differentiation in bone marrow and spleen as well as erythrocytes of different age groups in blood circulation in order to identify stages that are preferentially destroyed in autoimmune hemolytic anemia. In order to accomplish this objective, we studied changes that occur in mice with AIHA in (a) relative proportions of cells in different stages of erythroid differentiation in bone marrow and spleen, as well as the relative proportions of reticulocytes and erythrocytes of different age groups in blood circulation, and (b) the binding of autoantibodies, and the generation of reactive oxygen species (ROS) in cells in different stages of erythroid life cycle in bone marrow, spleen and blood. Our results indicate that while autoantibodies bind to cells in all stages of erythropoiesis in bone marrow and spleen and circulating mature erythrocytes, decline in relative proportion was confined only to the late stages of erythroid differentiation in bone marrow and younger erythrocytes in blood circulation suggesting that these erythroid populations may be preferentially lost in AIHA
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