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Activation of the respiratory burst in macrophages. Phosphorylation specifically associated with Fc receptor-mediated
J P Brozna1, N F Hauff, W A Phillips
1Department of Pathology, Denver Veterans Administration Medical Center, CO 80220.
Abstract:
Inflammatory macrophages elicited from the peritoneal cavity of mice injected with endotoxin can avidly ingest E opsonized with IgG antibody (EIgG) or with IgM antibody and C (EIgMC). However, only ingestion of EIgG is associated with activation of the respiratory burst and release of superoxide anion. We compared the endogenous phosphorylation of proteins from macrophages stimulated by interaction with EIgG or EIgMC on the premise that proteins phosphorylated after stimulation by EIgG but not EIgMC could play a role in activating the enzyme (oxidase) responsible for the respiratory burst. Proteins were separated by one-dimensional and two-dimensional electrophoresis in polyacrylamide gels. We found that proteins with approximate Mr of 20 kDa, 23 kDa, 46 kDa, 48 kDa (three proteins), 67 kDa, and 130 kDa were more heavily phosphorylated after EIgG stimulation than after EIgMC stimulation. Exposure to PMA, which activates the respiratory burst oxidase, induced phosphorylation of the 23-kDa, 48-kDa group, and 130-kDa proteins that were phosphorylated after stimulation by EIgG. Activity of protein kinase C was found to be significantly increased in the particulate fraction of macrophages stimulated by EIgG but not in the particulate fraction of EIgMC-stimulated cells. These data are compatible with the hypotheses that phosphorylation of specific cellular proteins, especially with a Mr of approximately 48 kDa, is involved in activation of the respiratory burst oxidase, and that function of protein kinase C also plays a part in this activation process.
Insights
Macrophages ingest antibody-coated targets, but only IgG-coated targets trigger the respiratory burst. Protein phosphorylation, particularly of 48 kDa proteins and protein kinase C activity, is linked to this respiratory burst activation.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Inflammatory macrophages play a key role in host defense.
- Macrophages ingest antibody-coated targets, but the signaling pathways differ.
- The respiratory burst, crucial for killing ingested pathogens, is activated differently depending on the antibody type.
Purpose of the Study:
- To investigate the role of protein phosphorylation in activating the respiratory burst.
- To compare protein phosphorylation patterns after stimulation with IgG-opsonized (EIgG) versus IgM and complement-opsonized (EIgMC) targets.
- To identify specific proteins involved in respiratory burst activation.
Main Methods:
- Macrophages were stimulated with EIgG and EIgMC targets.
- Protein phosphorylation was analyzed using one-dimensional and two-dimensional gel electrophoresis.
- Protein kinase C activity was measured in macrophage fractions.
- Phagocytosis assays were performed.
Main Results:
- EIgG stimulation led to increased phosphorylation of specific proteins (20, 23, 46, 48, 67, 130 kDa) compared to EIgMC.
- Phosphorylation of 23, 48, and 130 kDa proteins mirrored the effects of PMA, a known activator of the respiratory burst oxidase.
- Protein kinase C activity was significantly increased in EIgG-stimulated macrophages.
- Only EIgG ingestion triggered the respiratory burst and superoxide anion release.
Conclusions:
- Specific protein phosphorylation events, particularly of ~48 kDa proteins, are critical for activating the respiratory burst oxidase.
- Protein kinase C activation is involved in the EIgG-mediated respiratory burst.
- Differential protein phosphorylation underlies the distinct signaling pathways triggered by EIgG and EIgMC.