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Immuno-electron microscopy reveals that the excitotoxin quinolinate is associated with the plasma membrane in human
V Sung1, C N Venkateshan, L Williamson
1Department of Biology, Georgetown University, Washington DC 20057, USA.
Abstract:
Quinolinate (QUIN), a tryptophan-derived excitotoxin, was localized ultrastructurally in human peripheral blood monocytes/macrophages (MO) by immuno-electron microscopy. A combined carbodiimide/glutaraldehyde/paraformaldehyde-based fixation procedure was developed for optimal retention of QUIN in the cell as well as minimal loss of ultrastructure; a silver-enhanced colloidal gold detection system was used for electron-microscopic analysis. Gold particles representing QUIN immunoreactivity were associated with the inner side of the plasma membrane in normal MO. The number of gold particles increased significantly when QUIN levels were elevated by treatment with its precursor kynurenine, but location of the gold particles remained essentially the same under this condition. Treatment with interferon-gamma increased the number of Golgi bodies, vacuoles and pseudopodia, reflecting the activated state of the cell. Significantly increased numbers of gold particles representing QUIN were detectable in approximately the same location as in the case of kynurenine treatment. Combined treatment with kynurenine and interferon-gamma maximally increased the number of gold particles at the periphery of the cell. The pseudopodia were intensely stained with gold particles, while they were not detectable in the inner part of the cytoplasm or in any other organelle even under this activated condition. The significance of the specific location of QUIN revealed in the present study and its relation to the release and subsequent actions of QUIN are discussed.
Insights
Quinolinate (QUIN), an excitotoxin derived from tryptophan, is found on the outer membrane of human monocytes/macrophages. Its presence increases with kynurenine and interferon-gamma treatments, particularly in cell extensions.
Area of Science:
- Immunology
- Neuroscience
- Cell Biology
Background:
- Quinolinate (QUIN) is a tryptophan-derived excitotoxin implicated in neurodegenerative processes.
- Its precise localization within immune cells, particularly monocytes/macrophages (MO), has not been clearly defined.
Purpose of the Study:
- To ultrastructurally localize quinolinate (QUIN) in human peripheral blood monocytes/macrophages (MO) using immuno-electron microscopy.
- To investigate the effect of cellular activation and precursor availability on QUIN localization.
Main Methods:
- Development of a novel fixation technique (carbodiimide/glutaraldehyde/paraformaldehyde) for optimal QUIN and ultrastructure preservation.
- Immuno-electron microscopy utilizing a silver-enhanced colloidal gold detection system.
- Treatment of MO with kynurenine (QUIN precursor) and/or interferon-gamma to modulate QUIN levels and cellular activation.
Main Results:
- QUIN immunoreactivity was primarily localized to the inner side of the plasma membrane in normal MO.
- Kynurenine treatment significantly increased QUIN levels without altering its location.
- Interferon-gamma treatment activated MO, increasing QUIN detection at the cell periphery and within pseudopodia, especially when combined with kynurenine.
Conclusions:
- QUIN is specifically localized at the periphery of monocytes/macrophages, particularly within pseudopodia, suggesting a role in cell-mediated release and action.
- Cellular activation and precursor availability modulate the amount of QUIN detectable at the cell surface.
- This localization provides insights into the potential mechanisms of QUIN's extracellular effects.
