The Functional Size of the Primary Complement Lesion in Resealed Erythrocyte Membrane Ghosts

Abstract

We compared the susceptibility to complement (C) lysis of resealed sheep erythrocyte membrane ghosts (M) with that of intact erythrocytes (E). E were lysed hypotonically in the presence of radiolabeled marker molecules that were entrapped in the M during resealing. C-induced lysis was measured by the amounts of ³H-sucrose and hemoglobin released from antibody (A)-treated M (MA) and E (EA), respectively. The degree of lysis produced by different concentrations of guinea pig serum was identical for both systems. Treatment with C8 and C9 resulted in identical levels of lysis of MAC1-7 and EAC1-7; in addition, similar binding of radiolabeled C8 by MAC1-7 and EAC1-7 was obtained. These results demonstrate that the reactivity of M with A and C is similar to that of intact E. To determine the effective pore size of the primary C lesion, radiolabeled molecules of different size were entrapped in the M, and their release by A and C as a function of time or C concentration was investigated. An excess of guinea pig, human, or rabbit serum produced complete release of ³H-sucrose and ³H-inulin at the end of 60 min of incubation at 37°C, but the release of ¹²⁵I-ribonuclease (RNase) proceeded at a slower rate. The rates of release were directly related to the size of the marker molecules and the concentrations of C used. With ¹²⁵I-ovalbumin and larger molecules (hemoglobin, ¹²⁵I-bovine serum albumin, ¹²⁵I-urease, and ¹²⁵I-thyroglobulin) the release was of low level and similar for all molecules. The release in controls with heat-inactivated (56°C, 30 min) guinea pig and human serum or C6-deficient rabbit serum was less than 10% for all markers after 60 min of incubation at 37°C. The patterns of release of marker molecules from MAC1-7 reacted with C8 and C9 were similar to those of EA treated with serum as source of C. However, the differences in rate of release of the various markers were more pronounced. Incubation of MAC1-7 alone resulted in a 10 to 12% release of all markers after 60 min at 37°C, and incorporation of C8 produced an additional 10% increase in release. Because RNase and smaller molecules can readily diffuse across C-treated M, we conclude that the size of the primary C lesion has a minimal diameter of 40 Å. It is also possible that resealing of larger primary C lesions occurs rapidly and thus markedly reduces the egress of large molecules from the lysing M.