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Hypoxia enhances lysosomal TNF-alpha degradation in mouse peritoneal macrophages
Nitza Lahat1, Michal A Rahat, Amalia Kinarty
1Immunology Research Unit, Carmel Medical Center, Haifa, Israel.
Abstract:
Infection, simulated by lipopolysaccharide (LPS), is a potent stimulator of tumor necrosis factor-alpha (TNF-alpha) production, and hypoxia often synergizes with LPS to induce higher levels of the secreted cytokine. However, we show that in primary mouse peritoneal macrophages and in three mouse peritoneal macrophage cell lines (RAW 264.7, J774A.1, and PMJ-2R), hypoxia (O(2) < 0.3%) reduces the secretion of LPS-induced TNF-alpha (P < 0.01). In RAW 264.7 cells this reduction was not regulated transcriptionally as TNF-alpha mRNA levels remained unchanged. Rather, hypoxia and LPS reduced the intracellular levels of TNF-alpha by twofold (P < 0.01) by enhancing its degradation in the lysosomes and inhibiting its secretion via secretory lysosomes, as shown by confocal microscopy and verified by the use of the lysosome inhibitor Bafilomycin A1. In addition, although hypoxia did not change the accumulation of the soluble receptor TNF-RII, it increased its binding to the secreted TNF-alpha by twofold (P < 0.05). We suggest that these two posttranslational regulatory checkpoints coexist in hypoxia and may partially explain the reduced secretion and diminished biological activity of TNF-alpha in hypoxic peritoneal macrophages.
Insights
Hypoxia reduces lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-alpha) secretion in macrophages. This occurs post-translationally via enhanced degradation and inhibited secretion, not transcriptional changes.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Lipopolysaccharide (LPS) stimulates tumor necrosis factor-alpha (TNF-alpha) production.
- Hypoxia often synergizes with LPS to increase secreted TNF-alpha.
Purpose of the Study:
- To investigate the effect of hypoxia on LPS-induced TNF-alpha secretion in macrophages.
- To elucidate the regulatory mechanisms behind hypoxia's influence on TNF-alpha.
Main Methods:
- Primary mouse peritoneal macrophages and cell lines (RAW 264.7, J774A.1, PMJ-2R) were used.
- Hypoxia conditions (O(2) < 0.3%) were applied.
- TNF-alpha mRNA levels, intracellular TNF-alpha, secretion, lysosomal degradation, and TNF-RII binding were analyzed.
- Confocal microscopy and Bafilomycin A1 (lysosome inhibitor) were employed.
Main Results:
- Hypoxia significantly reduced LPS-induced TNF-alpha secretion (P < 0.01).
- TNF-alpha mRNA levels remained unchanged, indicating post-transcriptional regulation.
- Intracellular TNF-alpha decreased due to enhanced lysosomal degradation and inhibited secretion.
- Hypoxia increased TNF-RII binding to secreted TNF-alpha (P < 0.05).
Conclusions:
- Hypoxia exerts post-translational control over TNF-alpha secretion in macrophages.
- Enhanced degradation and inhibited secretion via lysosomes, along with increased TNF-RII binding, contribute to reduced TNF-alpha activity under hypoxia.
- These mechanisms may explain diminished TNF-alpha biological activity in hypoxic conditions.
