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.

Feeding cadmium chloride (50 or 1000 ppm CdCl2 in drinking water, ad libitum) to C57BL/6 mice resulted in a significant and sustained fall in blood erythrocyte count and hemoglobin levels that started 4 and 3 weeks after the start of 50 and 1000 ppm cadmium doses respectively. A transient yet significant reticulocytosis occurred during the first 4 weeks of cadmium treatment. Using the recently developed double in vivo biotinylation (DIB) technique, turnover of erythrocyte cohorts of different age groups was simultaneously monitored in control and cadmium treated mice. A significant accumulation of younger erythrocytes and a concomitant decline in the relative proportions of older erythrocytes in circulation was observed in both 50 and 1000 ppm cadmium groups indicating that older erythrocytes were preferentially eliminated in cadmium induced anemia. A significant increase in the erythropoietin levels in plasma was seen in mice exposed to 1000 ppm cadmium. Levels of inflammatory cytokines (IL1A, IL6, TNFα, IFNγ) were however not significantly altered in cadmium treated mice. A significant increase in cellular levels of reactive oxygen species (ROS) was observed in older erythrocytes in circulation but not in younger erythrocytes. Erythropoietic activity in the bone marrows and spleens of cadmium treated mice was examined by monitoring the relative proportion of cells belonging to the erythroid line of differentiation in these organs. Erythroid cells in bone marrow declined markedly (about 30%) in mice in the 1000 ppm cadmium group but the decline was not significant in the 50 ppm cadmium group. Cells representing various stages of erythroid differentiation in bone marrow and spleen were enumerated flow cytometrically by double staining with anti-Ter119 and anti-transferrin receptor (CD71) monoclonal antibodies. Decline of erythroid cells was essentially confined to pro-erythroblast and erythroblast-A, along with a concurrent increase in the splenic erythroid population indicating a stress response. In short cadmium exposure causes preferential clearance of older erythrocytes from circulation along with a depressed erythropoietic activity at higher doses.

We have developed a new double in vivo biotinylation (DIB) technique that may be used for assessing turnover patterns of erythrocytes in circulation. This technique involves two successive in vivo biotinylation steps, interspersed by a period of 5-30 days, which would enable us to tag with biotin a population of erythrocytes entering blood circulation over a defined period of time, between the two biotinylation steps. As such we can track the age-related changes in a lifetime of the circulating erythrocytes, or we can simultaneously study two defined age cohorts of aged as well as young erythrocytes in circulation. We have extensively used this technique to look at erythrocyte loss in mouse models of anemia induced by (a) heavy metal cadmium (Cd), (b) herbicide Paraquat (PQ), (c) carbon nanotubes (CNTs), and (d) autoantibody in autoimmune hemolytic anemia (AIHA). We have found that the pattern of erythrocyte removal is distinctly different in different models of murine anemia. In certain types of anemia (CNT and AIHA), younger erythrocytes in blood circulation are preferentially removed, whereas in other cases (Cd and PQ), old erythrocytes are specifically eliminated.

C57BL/6 mice were given repeated weekly injections of rat erythrocytes to induce autoimmune hemolytic anemia (AIHA), characterized by decreased erythrocyte count and hemoglobin content, presence of membrane bound autoantibodies on circulating erythrocytes and a decline in the bone marrow erythropoietic activity. Erythroid cells in the bone marrow and spleen of AIHA mice were examined for the presence of membrane bound autoantibodies using a fluorochrome conjugated anti-mouse IgG antibody. Apoptotic activity was also assessed flow cytometrically using Annexin V and 7AAD. Results showed that there is significant binding of autoantibodies to both spleen and bone marrow, but the apoptosis is evident only in bone marrow, that too in later stages of differentiation.