To further define the conditions for forming spectrin-hemoglobin cross-linking in human erythrocyte membranes and to examine its possible effects on membrane function, we incubated normal human erythrocytes for up to 3 h in concentrations of H202, varying from 45 to 180 ;.M, in an azide phosphate buffer, pH 7.4. The chemical changes observed indicated that methemoglobin formation occurred early and at a low concentration (45 MM). Morphologic changes characterized by increased echinocyte formation occurred in a dose-dependent fashion. In addition, decreased cell deformability commensurate with increased membrane rigidity was found. Finally, an increase in cell recognition as determined by monocyte phagocytosis and adherence in vitro, as well as decreased phosphatidylcholine accessibility to bee venom phospholipase A2, was found in H202-treated erythrocytes compared with controls. Both of these latter changes were closely correlated with the extent of spectrin-hemoglobin cross-linking.In addition to these protein-mediated interactions, lipid peroxidation also occurred after H202 exposure, as shown by generation of fluorescent amino propene derivatives. The addition of the antioxidant, butylated hydroxytoluene, decreased the fluorescent derivatives, but did not prevent the effects on membrane function. This suggests that lipid peroxidation, though present, was not necessary for the membrane changes found. In contrast, spectrin-hemoglobin aggregation and the alterations in membrane function were completely prevented by prior exposure of the erythrocytes to carbon monoxide.

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Glomerular injury in passive Heymann nephritis (PHN) in rats is mediated by the C5b-9 membrane attack complex (MAC) and is associated with morphologic changes in glomerular visceral epithelial cells (GEC). We determined if the nephritogenic antibody of PHN (gamma 1 sheep anti-Fx1A IgG) directs insertion of the MAC into GEC plasma membranes with consequent cytotoxicity. Antibody-sensitized GEC were exposed to various sera serving as sources of complement. Loss of cell viability was determined by trypan blue uptake and/or by release of cellular lactate dehydrogenase (LDH). Incubation of antibody-sensitized primary and passaged GEC in fresh human serum (FHS) resulted in sigmoidal relationships between cytotoxicity and complement dose (r = 0.97 and 0.94, respectively) such that cytolysis approached 100% with FHS (10% vol/vol). Cytotoxicity was not evident if C8-deficient (C8D) plasma was substituted for FHS, but was restored in a dose-dependent manner by reconstitution with purified rat C8. Sublytic injury was demonstrated by wide separation between simultaneous release curves of cell-incorporated biscarboxyethyl carboxyfluorescein (BCECF; mol wt approximately equal to 520) and LDH at limiting doses of complement (at 2% FHS, BCECF release was 51.1 +/- 0.6% of maximum vs. 3.2 +/- 1.3% for LDH; N = 3) and by blebbing of the plasma membrane on electron microscopy. Thus, the pathogenic antibody of PHN produces complement-mediated sublytic as well as lytic cytotoxicity of GEC.