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Heat shock proteins and macrophage resistance to the toxic effects of nitric oxide
M R Hirvonen1, B Brüne, E G Lapetina
1Department of Toxicology, National Public Health Institute, Kuopio, Finland.
Abstract:
Nitric oxide (NO) functions as a pathophysiological mediator in mammalian tissues. Activated macrophages produce NO as a non-specific immune response directed against invading bacteria or micro-organisms. The same macrophages that initiate the production of NO also can be toxically affected by NO. Incubation of RAW 264.7 macrophages with lipopolysaccharide (LPS) and/or interferon-gamma (INF-gamma) induced the formation of NO by the activation of a cytokine-inducible NO synthase (NOS). The viability of these macrophages was inversely correlated with the formation of nitrite, a final NO-oxidation product measurable in the incubation medium. The addition of an NOS inhibitor, NG-monomethyl-L-arginine, diminished NO formation and preserved cell viability in a dose- and time-dependent fashion. Treatment of macrophages with ten cycles of non-lethal doses of LPS and INF-gamma, each followed by subculturing of the surviving cells, resulted in cell resistance to the NO toxic insult induced by LPS and INF-gamma. These resistant macrophages showed a 2-fold increase in the expression of the constitutive heat shock protein (HSC 70) which is known to be involved in protecting cells against the action of various metabolic insults. Our results establish a link between cell resistance to the toxic effects of NO, and the expression of heat shock proteins in RAW 264.7 macrophages.
Insights
Macrophages exposed to nitric oxide (NO) can develop resistance. This resistance is linked to increased expression of heat shock proteins (HSCs), offering protection against NO toxicity.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Nitric oxide (NO) acts as a mediator in mammalian tissues, produced by macrophages during immune responses.
- Macrophages producing NO can also be negatively affected by it, impacting their viability.
Purpose of the Study:
- To investigate the development of resistance in macrophages to nitric oxide (NO) toxicity.
- To explore the relationship between NO resistance and heat shock protein expression.
Main Methods:
- RAW 264.7 macrophages were stimulated with lipopolysaccharide (LPS) and interferon-gamma (INF-gamma) to induce NO production.
- Nitrite levels (NO oxidation product) and cell viability were measured.
- An NO synthase (NOS) inhibitor, NG-monomethyl-L-arginine, was used to assess NO's role in toxicity.
- Macrophages were subjected to repeated cycles of non-lethal stimuli to induce resistance.
Main Results:
- Macrophage viability decreased with increasing nitrite formation, indicating NO toxicity.
- NOS inhibition preserved cell viability and reduced NO formation.
- Repeated exposure to sub-lethal stimuli induced resistance to NO's toxic effects.
- Resistant macrophages exhibited a two-fold increase in constitutive heat shock protein 70 (HSC 70) expression.
Conclusions:
- Macrophage resistance to NO toxicity can be acquired through repeated exposure to immune stimuli.
- Increased expression of heat shock proteins, specifically HSC 70, is associated with this acquired NO resistance.
- HSC 70 may play a protective role against the metabolic insults caused by NO in macrophages.