Isolation, characterization, and immunoprecipitation studies of immune complexes from membranes of beta-thalassemic erythrocytes

Abstract

beta-Thalassemia, a hemoglobinopathy that results in the precipitation of denatured alpha-globin chains on the membrane, is characterized by erythrocytes with significantly reduced lifespans. We have demonstrated previously that hemoglobin denaturation on the membrane can promote clustering of integral membrane proteins, and that this clustering in turn leads to autologous antibody binding, complement fixation, and rapid removal of the cell by macrophages. To evaluate whether this pathway also occurs in beta-thalassemic cells, we have isolated and characterized the immune complexes from the membranes of these cells. We observe that autologous IgG-containing complexes obtained by either immunoprecipitation or simple centrifugation of nondenaturing detergent extracts of beta-thalassemic cell membranes contain globin, band 3, IgG, and complement as major components. Absorption spectra of these complexes demonstrate that the globin is, indeed, mainly in the form of hemichromes. Immunoblotting studies further show that much of the band 3 protein in the aggregates is covalently cross-linked to a dimeric or tetrameric form, consistent with the preference of the autologous IgG for clustered band 3. Although the insoluble aggregates constitute only approximately 1.6% of the total membrane protein, they still contain 27% of the total IgG and 35% of the total complement C3 on the thalassemic cell surface. Because cell surface IgG and complement component C3 are thought to trigger removal of erythrocytes from circulation, the hemichrome-induced clustering of band 3 may contribute to the beta-thalassemic cell's shortened lifespan.

Growth of Plasmodium falciparum induces stage‐dependent haemichrome formation, oxidative aggregation of band 3, membrane deposition of complement and antibodies, and phagocytosis of parasitized erythrocytes

“…Data presented here suggest that parasite development induces, in sequence, deposition of haemichromes and oxidative aggregation of band 3; deposition of autologous IgG and complement; and final recognition by phagocytes. This sequence is similar to that observed in normally senescent RBCs (Lutz, 1990), artificially modified RBCs (Lutz et al , 1987; Beppu et al , 1990; Turrini et al , 1991) or pathologic RBCs, most notably sickle (Kannan et al , 1988) and thalassaemic RBCs (Yuan et al , 1992; Mannu et al , 1995; Cappellini et al , 1999).…”

Prevalence and clinical significances of red cell alloimmunization and red cell bound immunoglobulin G in polytransfused patients with thalassemias

“…In the present study, there was an association between increased transfusion requirements with the formation of RBC autoantibody. It is possible that deposition of opsonizing autologous immunoglobulins and possibly also complement C3 fragments [33], increases phagocytic removal of the thalassemia erythrocytes resulting in increased requirement for transfusions. In agreement with our study, Dhawan et al [8] found that the development of RBC autoantibody was significantly higher in patients receiving more transfusions.…”

From the Cradle to the Grave: The Role of Macrophages in Erythropoiesis and Erythrophagocytosis

“…Besides inadequate ROS detoxification free heme and iron may as well give rise to enhanced oxidative stress since partially oxygenated hemoglobin is susceptible to redox reactions ( 118 , 131 ). In all these diseases, high levels of anti-band 3 antibodies and C3b are found bound to RBCs ( 132 , 133 ). Increased RBC phagocytosis is observed which can be ameliorated in many patients by a splenectomy ( 124 ).…”