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Enhanced oxidative mechanisms in immunologically activated versus elicited polymorphonuclear neutrophils:
C J Morrison1, R A Isenberg, D A Stevens
1Department of Medicine, Santa Clara Valley Medical Center, San Jose, CA 95128.
Abstract:
Peritoneal polymorphonuclear neutrophils (PMN) from mice were tested for their ability to kill the yeast form of Blastomyces dermatitidis (Bd) in vitro and for their fungicidal mechanisms. PMN elicited from immune mice by the intraperitoneal injection of non-viable Bd (referred to as immunologically activated PMN or ActPMN) showed significantly enhanced fungicidal activity in comparison with PMN elicited with thioglycollate medium (ThioPMN) [means = 44.7% (SD 12.8%) and 16.4% (SD 9.2%) killed; n = 14; p less than 0.001]. Production of superoxide anion (O2-) by ActPMN after stimulation with phorbol myristate acetate was enhanced in comparison with production by ThioPMN. Superoxide dismutase, which removes O2-, inhibited ActPMN killing by 75% (p less than 0.001) when added to cultures immediately before challenge with Bd (optimal concentration: 6000 U/ml). Sodium azide, which inhibits myeloperoxidase and scavenges singlet oxygen (1O2), and catalase, which breaks down hydrogen peroxide (H2O2), inhibited ActPMN killing by 64% (p less than 0.001) and 52% (p less than 0.001), with optimal concentrations of 1 mM and 10,000 U/ml, respectively. Two agents that both scavenge 1O2 and antagonise hypochlorous acid (HOCl-), histidine and tryptophan, were also powerful inhibitors of ActPMN killing. Quenchers of hydroxyl radical (.OH), dimethylsulfoxide and sodium benzoate, had less effect, and required higher concentrations. These data suggest that the enhanced killing of Bd by ActPMN involves one or more oxidative mechanisms, and that there is a prominent role for O2-, either directly or as a precursor of other active oxygen species, a probable role for H2O2, and possible roles for 1O2, HOCl-, and .OH.
Insights
Immunologically activated neutrophils (ActPMN) show enhanced killing of Blastomyces dermatitidis (Bd) compared to thioglycollate-elicited PMN. Oxidative mechanisms, particularly superoxide anion, are crucial for this enhanced fungicidal activity against Bd.
Area of Science:
- Immunology
- Microbiology
- Cell Biology
Background:
- Blastomyces dermatitidis (Bd) is a fungal pathogen causing blastomycosis.
- Polymorphonuclear neutrophils (PMN) are key immune cells involved in combating fungal infections.
- Understanding the mechanisms of PMN-mediated fungal killing is crucial for developing effective treatments.
Purpose of the Study:
- To investigate the fungicidal activity of immunologically activated PMN (ActPMN) against the yeast form of Blastomyces dermatitidis (Bd) in vitro.
- To elucidate the specific oxidative mechanisms employed by ActPMN during Bd killing.
Main Methods:
- Peritoneal PMN were elicited from mice using either non-viable Bd (ActPMN) or thioglycollate medium (ThioPMN).
- Fungicidal activity was assessed by incubating PMN with Bd yeast cells.
- The role of reactive oxygen species (ROS) was investigated using specific inhibitors and scavengers, including superoxide dismutase, sodium azide, catalase, histidine, tryptophan, dimethyl sulfoxide, and sodium benzoate.
Main Results:
- ActPMN exhibited significantly enhanced fungicidal activity against Bd compared to ThioPMN (44.7% vs. 16.4% killing).
- ActPMN showed increased production of superoxide anion (O2-) upon stimulation.
- Inhibition of O2- by superoxide dismutase reduced ActPMN killing by 75%.
- Inhibition of myeloperoxidase and singlet oxygen (1O2) by sodium azide, and hydrogen peroxide (H2O2) by catalase, also significantly reduced Bd killing.
- Agents scavenging 1O2 and hypochlorous acid (HOCl-) were potent inhibitors, while hydroxyl radical (.OH) quenchers had a lesser effect.
Conclusions:
- Enhanced killing of Bd by ActPMN involves multiple oxidative mechanisms.
- Superoxide anion (O2-) plays a prominent role, either directly or as a precursor to other ROS.
- Hydrogen peroxide (H2O2) likely contributes to fungicidal activity.
- Singlet oxygen (1O2), hypochlorous acid (HOCl-), and hydroxyl radical (.OH) may also be involved in the ActPMN-mediated killing of Bd.