Bilirubin impairs bactericidal activity of neutrophils through an antioxidant mechanism in vitro

Background. Liver failure accompanied by hyperbilirubinemia after major hep atic resection is profoundly associated with septic complications. Although the immune dysfunction in cholestasis has been intensively investigated, t he contribution of increased serum bilirubin to the impaired resistance to bacterial infection remains to be elucidated. Because bilirubin possesses a n antioxidant activity, we hypothesized that bilirubin may scavenge reactiv e oxygen species (ROS) produced by neutrophils and consequently impair neut rophil bacterial killing. To address this, we evaluated the effects of bili rubin on the bactericidal activity of ROS or of neutrophils in vitro. Materials and methods. The antioxidant activity of bilirubin was determined using an ROS-sensitive fluorophore, dichlorofluorescin diacetate (DCFH-DA) . Bilirubin concentration in the buffer solution was monitored spectorophot ometrically after incubation with ROS. The effect of bilirubin on killing o f Escherichia coli by ROS or by isolated human neutrophils was determined b y counting the viable E. coli after incubation on nutrient agar. Results. The bilirubin concentration in the buffer solution was decreased b y the addition of hydrogen peroxide, especially in the presence of peroxida se or ferrous iron. DCFH-DA oxidation by ROS or activated neutrophils was i nhibited by bilirubin in a dose-dependent manner. The bactericidal activity of ROS or of isolated neutrophils was significantly attenuated by bilirubi n, Conclusions. Bilirubin impairs bactericidal activity of neutrophils through scavenging ROS. Increased levels of serum bilirubin may well be responsibl e for the impaired bacterial clearance in patients with hyperbilirubinemia. (C) 2001 Academic Press.