Inhibition of marker influx into complement-treated resealed erythrocyte ghosts by anti-C5.

Abstract

Resealed human erythrocyte ghosts were used to study the nature of membrane damage induced by complement (C). Resealed ghosts containing a small water soluble dye, carboxyfluorescein (CF), and one of a series of rhodamine-modified globular proteins were treated with C, and marker release was followed both by fluorometry of the supernatant and fluorescence microscopy. Release of markers was size dependent: four proteins of 35,000 m.w. or less were released to the same extent as CF, but eight proteins of 40,000 m.w. or more were not released from ghosts even though CF was released from the same ghosts. Low concentrations of detergents, such as sodium deoxycholate, were lytic to red cells, caused release of CF, and demonstrated protein marker sieving similar to that of complement. Marker influx into C-treated ghosts was assessed by preparing ghosts containing CF and anti-Ars hapten and allowing proteins modified with Ars and rhodamine to diffuse into ghosts and become trapped by the anti-Ars antibody. Proteins of 35,000 m.w. or less penetrated into C-treated ghosts and proteins of 40,000 m.w. or more did not. Thus a similar sieving size cut-off was observed for marker influx though the C channel compared to that observed for marker efflux. Antibodies against purified C components were tested for their ability to block marker influx into C-treated ghosts. After washing, such ghosts were incubated with various antisera, washed again, and then incubated with the 18,000 and 35,000 m.w. markers to determine their penetration through the C pore. Antibodies against red cell antigens or C3, C8, C9 did not prevent marker penetration. However, anti-C5 blocked entry of both markers greater than 90% at dilutions to 1/1000. These anti-C5 preparations did not block detergent pores in these ghosts. These results suggest that determinants on membrane-bound C5b are close to the functional C lesion.