Identification of Stages of Erythroid Differentiation in Bone Marrow and Erythrocyte Subpopulations in Blood Circulation that Are Preferentially Lost in Autoimmune Hemolytic Anemia in Mouse

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.

A Double In Vivo Biotinylation Technique to Assess Erythrocyte Turnover in Blood Circulation

“…The results show that a significant anemia was demonstrable in the immunized mice only after 5-6 weekly administrations of rat erythrocytes [20], when the blood erythrocyte count suffered a 10% decline and hemoglobin a staggering 15% decline (Figure 3, panels G and H, respectively). The anti-mouse erythrocyte autoantibody level also showed a sharp increase at the same time point [20], indicating an autoimmune response. from the four models of murine anemia could be clustered into two distinct categories, with completely contrasting characters.…”

“…The AF-SWCNT treatment resulted in reticulocytosis, showing > twofold increase in the percentage of blood reticulocytes from 2.23 to 5.32% ( Figure 8A) [19]. Autoimmune anemia on the other hand resulted in severe reticulocytopenia, showing a 16-18% decline in reticulocyte proportion as seen in Figure 8, panel B [20]. were stained ex vivo with anti-mouse CD71-PE to determine the proportion of circulating reticulocytes through flow cytometry.…”

“…Generation of anti-mouse erythrocyte autoantibody was estimated by flow cytometric analysis of erythrocytes stained with anti-mouse IgG/IgM-fluorescein isothiocyanate (FITC) polyclonal antibodies [71,72]. The results show that a significant anemia was demonstrable in the immunized mice only after 5-6 weekly administrations of rat erythrocytes [20], when the blood erythrocyte count suffered a 10% decline and hemoglobin a staggering 15% decline (Figure 3, panels G and H, respectively). The anti-mouse erythrocyte autoantibody level also showed a sharp increase at the same time point [20], indicating an autoimmune response.…”

“…There is a significant increase (twofold) in the proportion of older erythrocyte population (biotin high , Figure 7, panels A and C) along with a concomitant decline in the proportion of young erythrocytes (Figure 7, panels B and D). Thus relatively younger erythrocytes in the blood seem to be preferentially eliminated in conditions of AIHA- [20] and CNT-induced anemia [19] either by macrophage-mediated erythrophagocytosis in the reticuloendothelial system of the spleen or by complement-mediated lysis.…”

“…However in the case of anemia, where the homeostasis between erythrocyte destruction and erythropoiesis is disturbed, the erythrocyte turnover pattern may completely alter. To understand the fate of erythrocyte turnover in conditions of anemia, we studied four mouse models of experimental anemia, namely anemia induced by (a) toxic heavy metal cadmium (Cd) ions [17], (b) herbicide Paraquat (PQ) [18], (c) carbon nanotubes (CNTs) [19], and (d) autoantibody in autoimmune hemolytic anemia (AIHA) [20].…”