Naturally occurring anti-band-3 antibodies and complement together mediate phagocytosis of oxidatively stressed human erythrocytes.

Treatment of erythrocytes with the thiol-specific 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 anti-band-3 also restored phagocytosis of erythrocytes that had been incubated with complement-inactivated serum. Elevated concentrations of anti-spectrin antibodies were ineffective in whole and complement-inactivated serum. Specific recognition of diamide-treated erythrocytes by anti-band-3 antibodies may be due to generation of anti-band-3 reactive protein oligomers on intact diamide-treated erythrocytes. Generation of such oligomers was dose-dependent with respect to diamide. Bound anti-band-3 alone was not sufficient to mediate phagocytosis. It resulted in deposition of complement component C3b on the cells through activation of the alternative complement pathway in amounts exceeding that of bound antibodies by two orders of magnitude. Thus, anti-band-3 and complement together mediate phagocytosis of oxidatively stressed erythrocytes, which stimulate senescent erythrocytes with respect to bound antibody and complement.

Diamide enhances phagocytosis of human red cells in a complement- and anti band 3 antibody-dependent process.

Human red cells treated with 20-200 microM diamide were opsonized by serum and phagocytized by adherent monocytes. Phagocytosis was dependent on complement. It was enhanced by supplementing whole serum and was restored by supplementing complement-inactivated serum with naturally occurring antibodies which bind to the major integral protein of the human red cell, band 3 protein. Diamide induced oligomers of anti band 3 reactive oligomers, and enhanced anti band 3 binding to red cells. Complement C3 binding paralleled that of anti band 3 and exceeded it by two orders of magnitude. Thus, naturally occurring antibodies have functional properties which were not abolished by other serum Ig and may be involved together with complement in the clearance of red cells subjected to oxidant stress.

Naturally occurring autoantibodies to exoplasmic and cryptic regions of band 3 protein, the major integral membrane protein of human red blood cells.

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.