PURIFIED SERUM ANTIBODIES DIRECTED AGAINST Gb₃ INDUCE APOPTOSIS IN VERO AND HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS (HUVEC). † 2150

Introduction: The mechanism of vascular endothelial injury in Hemolytic Uremic Syndrome (HUS) has been speculated to be direct toxin-mediated damage. We propose an alternate hypothesis, in which a natural auto-antibody idiotypic network plays a central role in the damage of vascular endothelium.

Purpose: Our objective was to determine if serum anti-Gb₃ (α-Gb₃) antibodies induce damage to HUVEC and VERO cells.

Methods: α-Gb₃ antibodies were purified from serum by affinity chromatography. α-Gb₃ and anti-anti-Gb₃(α-α-Gb₃) titers were measured by EIA. Fractions with highα-Gb₃ titer were incubated overnight with monolayers of VERO and HUVEC cells stimulated or not stimulated with TNF-α. Cell damage was evaluated by light microscopy, by TdT-mediated dUTP nick end labeling and by detection of DNA fragmentation pattern in agarose gels.

Results: Affinity purified α-Gb₃ antibodies induce cell death in VERO and HUVEC monolayers with changes that are consistent with apoptosis.

Conclusions: Affinity purified α-Gb₃ antibodies induce cell death in VERO and HUVEC monolayers via apoptosis. We propose that in HUS, damage to vascular endothelium is indirectly caused by toxin. In this scenario we hypothesize that toxin binds to α-α-Gb₃ antibodies (which resemble Gb₃) leaving α-Gb₃ antibodies free in circulation; these α-Gb₃ antibodies bind vascular endothelium in which Gb₃ receptors have been upregulated by cytokines such as TNF-α, resulting in cell death by apoptosis. This work was funded in part by NIH-PO1 HD-13021.