Anti-Fx1A produces complement-dependent cytotoxicity of glomerular epithelial cells

Abstract

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 (γ1 sheep anti-Fx1A IgG) direct 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 ~ 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.