Treatment of erythrocytes with the thiolspecific oxidant azodicarboxylic acid bis(dimethylamide) (diamide) enhances their phagocytosis by adherent monocytes. Phagocytosis of diamide-treated erythrocytes required that the cells were opsonized with whole serum, since complement inactivation abolished phagocytosis. Opsonization with whole serum containing 20-100 times the physiological concentration of naturally occurring anti-band-3 antibodies enhanced phagocytosis of diamide-treated erythrocytes. High inputs of antiband-3 also restored phagocytosis oferythrocytes that had been incubated with complement-inactivated serum. Elevated concentrations of anti-spectrin antibodies were ineffective in whole and complement-inactivated serum.
Immunoglobulin G (IgG) of healthy human blood donors and IgG from pooled sera (Sandoglobulin) contain natural (auto)antibodies to band 3 protein, the major integral membrane protein of human red blood cells. Affinity-purified and 125I-iodinated anti-band 3 antibodies bound specifically to band 3 protein on immunoblots from membrane proteins in the presence of unlabeled, absorbed IgG. Purified (auto)antibodies also bound nonspecifically to band 4.2 and weakly to band 5 and 6, when assayed with second antibody and 125I-iodinated protein A. The antibodies were directed to regions of band 3 protein that were cryptic and in part exoplasmic but with a low accessibility to surface modifications. The antigenic sites were located within the 65K, but not the 38K-dalton chymotryptic fragment of band 3 protein. Antigenic band 3 protein was equally present in membranes of young and senescent red cells. Hence, if these antibodies were involved in recognizing a few exoplasmic sites of band 3 protein on senescent red cells, antigen exposure would require alterations in band 3 accessibility (conformation, topology) rather than an enzymatic generation of antigenic sites.
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