Concurrent presence of agonistic and antagonistic anti-CD95 autoantibodies in intravenous Ig preparations

Abstract

Background Although there have been several reports suggesting the presence of physiologic anti-CD95 (Fas, APO-1) autoantibodies in human intravenous Ig (IVIg) preparations, it is still unclear whether and under which conditions these autoantibodies block or stimulate the CD95 receptor. Objective We examined the effects of IVIg on CD95-mediated apoptosis in CD95-sensitive human blood neutrophils in vitro. Methods The presence of anti-CD95 antibodies was determined by competition assays with flow cytometry. Cell death and apoptosis were assessed by ethidium bromide uptake test and annexin V staining, respectively. Results Pretreatment of neutrophils with IVIg prevented binding of FITC-conjugated anti-CD95 mAb to the cell surface, suggesting that IVIg contains CD95 autoantibodies. By using low concentrations of IVIg (1 to 10 mg/mL), we observed a dose-dependent inhibition of anti-CD95 mAb (CH11)–mediated neutrophil apoptosis. Higher concentrations of IVIg (20 to 50 mg/mL), however, induced neutrophil death and apoptosis in a dose-dependent manner. This effect was partially blocked by soluble CD95 receptors (recombinant Fc-Fas) but not by an anti-CD95 blocking mAb, which was shown to recognize the CH11 epitope of CD95. Conclusion Both agonistic and antagonistic anti-CD95 antibodies are present in IVIg, and the effect on CD95 is dose-dependent. Our findings have potential implications for IVIg treatment, which is intended to target the CD95 receptor.