The Role of Glutathione Reductase in Neutrophil Respiratory Burst and ERK Signaling

Abstract

Abstract Respiratory burst plays a crucial role in the killing of bacteria by phagocytes. During the respiratory burst, NADPH oxidase complexes are assembled on the membrane to produce superoxide, which is then converted to H2O2 for pathogen killing. Glutathione (GSH) is crucial for the detoxification of H2O2 that has diffused into the cytosol. GSH is generated via both de novo synthesis and regeneration from glutathione disulfide. The rate-limiting step in GSH synthesis is catalyzed by γ-glutamate cysteine ligase, composed of a catalytic subunit (Gclc) and a regulatory subunit (Gclm), whereas GSH regeneration is catalyzed by glutathione reductase (Gsr). Mice with a mutation in the Gsr gene exhibit a profound defect in immune defense against bacteria, which is associated with compromised respiratory burst activity. To address the mechanisms underlying the compromised respiratory burst, we purified neutrophils from wildtype (WT) and Gsr−/− and Gclm−/− mice, and assessed their respiratory burst in response to phorbol ester (PMA). Compared to WT neutrophils, Gsr−/− neutrophils exhibited a dramatic decrease in respiratory burst activity. A similar trend was observed in neutrophils derived from Gclm−/− mice, which have 10–40% of normal tissue GSH levels. However, no significant defect in immune defense against E. coli was found in Gclm−/− mice. Cellular fractionation indicated that the NADPH oxidase subunits underwent membrane translocation in response to PMA in both WT and Gsr−/− neutrophils. Interestingly, Gsr−/− neutrophils also displayed a dramatic enhancement in activation of the ERK MAP kinase pathway. Our results indicate that Gsr plays an important role in the regulation of both the respiratory burst and cell signaling in neutrophils.