Alternative pathway-mediated rebinding of immune complexes to human red blood cells

Antigen-antibody complexes (Ag-Ab) prepared from 125I-bovine serum albumin (BSA) and guinea-pig anti-BSA were (i) incubated at 37 degrees for 4 min with undiluted normal human serum and autologous red blood cells (RBC) together, or (ii) incubated first at 37 degrees for 30 min with serum diluted optimally for binding and then with RBC. Reactions were stopped by dilution and cooling, and RBC bearing antigen-antibody-complement complexes (Ag-Ab-C) were washed and resuspended in either untreated normal human serum (undiluted or diluted), serum treated with zymosan (SZYM), ethylenediamine tetracetic acid (SEDTA) or ethyleneglycol tetracetic acid-Mg++ (SEGTA), serum heated at 56 degrees for 30 or 120 min (S delta 30 or S delta 120), or buffer alone. The mixtures were placed at 37 degrees and the percentage of Ag-Ab-C dissociated from RBC after progressively increasing times determined. (i) Ag:Ab:C bound to RBC with undiluted serum dissociated more rapidly following resuspension in SZYM, SEDTA, or S delta 30 than following resuspension in untreated serum. Rate of dissociation in SEGTA paralleled that in untreated serum. (ii) Ag-Ab-C bound to RBC with diluted serum dissociated following resuspension in SZYM, SEDTA, or S delta 30, but rebound and dissociated a second time following resuspension in untreated serum or SEGTA. Initial dissociation occurred in less than 1 min in undiluted serum, took place at 0 degrees as well as 37 degrees, and was diminished but detectable in greater than 8- and greater than 64-fold-diluted serum, respectively. Rebinding required 37 degrees, factors B and D and C3, and was maximal at 4-8 min. Subsequent dissociation had similar complement requirements to initial dissociation, but occurred only at 37 degrees and was 90% complete at 15 min. No dissociation of Ag-Ab-C bound to RBC in (i) or in (ii) occurred following resuspension in S delta 120 or in buffer. These findings suggest that after initial binding, release of experimental immune complexes from RBC in whole serum involves concurrent dissociation and alternative pathway-dependent rebinding.