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Intracellular antioxidant activity is necessary to modulate the macrophage response to endotoxin
Eileen M Bulger1, Iris Garcia, Ronald V Maier
1University of Washington, Department of Surgery, Seattle 98104, USA.
Abstract:
The tissue-fixed macrophage (Mphi) is a key cell in the coordination of the excessive systemic immunoinflammatory response underlying the adult respiratory distress syndrome (ARDS). Macrophage-generated reactive oxygen intermediates (ROIs) are involved in both tissue destruction via lipid peroxidation and in the activation of these inflammatory cells. It is unclear whether oxidant-induced activation involves an extracellular effect and membrane destabilization or occurs through intracellular alteration of the redox state and direct involvement as second messengers. In this study, we compare the differential effects of known intracellular vs. extracellular antioxidants on the Mphi response to endotoxin. Rabbit alveolar Mphi were obtained by bronchoalveolar lavage and exposed to either the extracellular antioxidants [vitamin C (VC) (10-1000 microM), Trolox (100-1000 microM, superoxide dismutase (SOD) (10-500 microM))] or the intracellular antioxidants [N-acetylcysteine (NAC) (0.1-10 mM) or butylated hydroxyanisole (BHA) (10-200 microM)] for 1 h. Cells were subsequently stimulated with lipopolysaccharide at 10 ng/mL. After 18 h, supernatants were analyzed for tumor necrosis factor (TNF) and F2 isoprostane (F2ISP) production and cellular monolayers for procoagulant activity (PCA). A dose response inhibition of both TNF and PCA production was demonstrated after both NAC and BHA pretreatment but not with VC, Trolox, or SOD. In addition, northern blots revealed inhibition of TNF mRNA production by both NAC and BHA. F2ISP, a marker of membrane lipid peroxidation, was inhibited by BHA and Trolox but not NAC, VC, or SOD. In conclusion, antioxidants that are incorporated intracellularly are expected to be beneficial in the treatment of excessive inflammatory responses through the interruption of redox dependent signal transduction pathways and subsequent modulation of the Mphi proinflammatory response.
Insights
Intracellular antioxidants like N-acetylcysteine and BHA effectively reduced inflammatory responses in macrophages, suggesting their potential for treating conditions like ARDS by targeting intracellular pathways.
Area of Science:
- Immunology
- Cell Biology
- Pharmacology
Background:
- Macrophages are central to the inflammatory response in ARDS.
- Macrophage-derived reactive oxygen intermediates contribute to tissue damage and inflammation.
- The precise mechanism of oxidant-induced macrophage activation remains unclear.
Purpose of the Study:
- To investigate the differential effects of intracellular versus extracellular antioxidants on macrophage activation.
- To determine the impact of antioxidants on inflammatory mediator production and cellular activity.
Main Methods:
- Rabbit alveolar macrophages were isolated and pretreated with various antioxidants (intracellular: NAC, BHA; extracellular: VC, Trolox, SOD).
- Cells were stimulated with lipopolysaccharide (LPS).
- Analysis included TNF and F2ISP production, procoagulant activity (PCA), and TNF mRNA levels.
Main Results:
- Intracellular antioxidants N-acetylcysteine (NAC) and butylated hydroxyanisole (BHA) significantly inhibited TNF and PCA production.
- NAC and BHA also reduced TNF mRNA expression.
- Extracellular antioxidants (VC, Trolox, SOD) did not inhibit TNF or PCA.
- F2ISP, a marker of lipid peroxidation, was inhibited by BHA and Trolox.
Conclusions:
- Intracellularly acting antioxidants are effective in modulating macrophage-mediated inflammatory responses.
- Targeting intracellular redox-dependent signaling pathways with antioxidants may offer a therapeutic strategy for ARDS and other inflammatory conditions.