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The role of glutathione reductase in maintaining human granulocyte function and sensitivity to exogenous H2O2
Abstract:
Human granulocytes (polymorphonuclear leukocytes, PMN) produce H2O2 and other reactive oxygen species while undergoing phagocytosis. To examine the role of the glutathione cycle in metabolizing H2O2, we incubated PMN with 1,3-bis (2-chloroethyl) nitrosourea (BCNU). Incubation of PMN with BCNU results in a dose-dependent inhibition of PMN glutathione reductase (GRED), with 50% inhibition occurring at approximately 2 micrograms/mL BCNU. PMN hexose monophosphate shunt activity stimulated with an exogenous H2O2-generating system was inhibited only when the GRED activity was reduced to less than 30% of control. BCNU-treated cells contained lower levels of reduced sulfhydryls and reduced glutathione, which decreased even more in the presence of an exogenous H2O2-generating system. The effect of BCNU and exogenous H2O2 on various aspects of phagocytosis were examined. Exposure of BCNU-treated PMN to an H2O2-generating system resulted in an inhibition of chemotactic peptide-induced shape changes and degranulation. The ability of BCNU-treated cells to produce O2- was diminished only when the PMN were incubated with an H2O2-generating system in the presence of cyanide. Ingestion of opsonized bacteria by BCNU-treated PMN was unaffected by incubation in an H2O2-generating system even in the presence of cyanide. We conclude that PMN GRED is inhibited by BCNU, the ability of PMN to metabolize H2O2 is affected only when GRED is reduced more than 70%, this inhibition affects the glutathione content of these cells, and some, but not all of the phagocytic functions of GRED-inhibited PMN are inhibited after exposure to an H2O2-generating system.
Insights
BCNU inhibits glutathione reductase (GRED) in human granulocytes (PMN). This impacts their ability to metabolize hydrogen peroxide (H2O2), affecting phagocytosis functions when GRED activity is significantly reduced.
Area of Science:
- Biochemistry
- Immunology
- Cell Biology
Background:
- Human granulocytes (polymorphonuclear leukocytes, PMN) generate reactive oxygen species (ROS) like hydrogen peroxide (H2O2) during phagocytosis.
- The glutathione cycle is crucial for metabolizing H2O2 within PMN.
- Understanding this cycle's role is vital for comprehending PMN function and oxidative stress response.
Purpose of the Study:
- To investigate the impact of 1,3-bis (2-chloroethyl) nitrosourea (BCNU) on PMN glutathione reductase (GRED) activity.
- To determine how BCNU-induced GRED inhibition affects H2O2 metabolism and PMN phagocytic functions.
Main Methods:
- Incubation of PMN with varying doses of BCNU to assess GRED inhibition.
- Measurement of PMN hexose monophosphate shunt activity under H2O2 stimulation.
- Analysis of reduced sulfhydryl and glutathione levels in treated PMN.
- Evaluation of phagocytic functions (shape change, degranulation, superoxide production, bacterial ingestion) in BCNU-treated PMN exposed to H2O2.
Main Results:
- BCNU caused dose-dependent inhibition of PMN GRED, with 50% inhibition at ~2 µg/mL.
- Hexose monophosphate shunt activity was impaired only when GRED activity fell below 30%.
- BCNU-treated PMN showed reduced glutathione levels, further depleted by exogenous H2O2.
- Chemotactic peptide-induced shape changes and degranulation were inhibited in BCNU-treated PMN exposed to H2O2.
- Superoxide production was diminished only under cyanide presence with H2O2.
- Bacterial ingestion remained unaffected even with H2O2 and cyanide.
Conclusions:
- BCNU effectively inhibits PMN GRED.
- PMN H2O2 metabolism is compromised when GRED is inhibited by over 70%.
- This inhibition impacts cellular glutathione levels.
- Certain phagocytic functions of GRED-inhibited PMN are impaired following H2O2 exposure, but not all.