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Phorbol myristate acetate-induced NADPH oxidase activity in human neutrophils: only half the story has been told
H Lundqvist1, P Follin, L Khalfan
1Department of Medical Microbiology and Immunology, University of Göteborg, Sweden.
Abstract:
The paradigm for activation of the neutrophil NADPH oxidase with the protein kinase C activator phorbol myristate acetate (PMA) states that the oxidase assembles the plasma membrane and that the metabolites generated are released extracellularly. This paradigm is challenged by the results presented. Most of the PMA-induced oxidase activity measured as chemiluminescence and dichlorofluorescein fluorescence was insensitive to scavengers of superoxide anion and hydrogen peroxide. This indicates that oxidase activity also takes place in a cellular compartment that the scavengers cannot reach. From the results obtained with granule-deficient HL-60 cells and cord blood neutrophils, we suggest that the scavenger-insensitive part of the NADPH oxidase activity in normal neutrophils resides in an intracellular compartment identical to or originating from granules. Our results also indicate that specific and azurophil granules have to be in very close contact to allow the generated oxygen metabolites to reach and react with myeloperoxidase.
Insights
Neutrophil NADPH oxidase activation by phorbol myristate acetate (PMA) occurs intracellularly, not just at the plasma membrane. This scavenger-insensitive activity suggests a role for neutrophil granules in reactive oxygen species generation.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- The established model posits neutrophil NADPH oxidase activation occurs at the plasma membrane, with extracellular release of reactive oxygen species (ROS).
- Phorbol myristate acetate (PMA) is a common activator of this enzyme complex.
Purpose of the Study:
- To challenge the existing paradigm of neutrophil NADPH oxidase activation and extracellular ROS release.
- To investigate the intracellular localization and compartmentalization of oxidase activity.
Main Methods:
- Measurement of neutrophil NADPH oxidase activity using chemiluminescence and dichlorofluorescein fluorescence.
- Assessment of ROS scavenger sensitivity to determine localization.
- Utilizing granule-deficient HL-60 cells and primary human neutrophils.
Main Results:
- A significant portion of PMA-induced NADPH oxidase activity was insensitive to extracellular ROS scavengers.
- This scavenger-insensitive activity suggests intracellular compartmentalization of the oxidase.
- Results from HL-60 cells and cord blood neutrophils indicate this intracellular activity originates from or resides within neutrophil granules.
- Close contact between specific and azurophil granules appears necessary for ROS to reach myeloperoxidase.
Conclusions:
- Neutrophil NADPH oxidase activation is not exclusively a plasma membrane event; significant intracellular activity occurs.
- Neutrophil granules play a crucial role in housing or facilitating intracellular NADPH oxidase activity.
- The spatial relationship between specific and azurophil granules is critical for myeloperoxidase activation by generated ROS.