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

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 Fe-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.