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Defective translation of tumor necrosis factor mRNA in lipopolysaccharide-tolerant macrophages
A Marchant1, C Gueydan, L Houzet
1Service d'Immunologie, Hôpital Erasme, Université Libre de Bruxelles, Belgium.
Abstract:
Macrophage activation by lipopolysaccharide (LPS) results in the translational activation of tumor necrosis factor (TNF) mRNA. The initial phase of macrophage activation is followed by a refractory state called LPS tolerance characterized by an impaired TNF production in response to a secondary LPS challenge. LPS-tolerant macrophages contain high amounts of TNF mRNA, suggesting a translational regulation of TNF biosynthesis. The induction of LPS tolerance was studied in RAW 264.7 macrophages stably transfected with a chloramphenicol acetyl-transferase (CAT) reporter gene construct driven by a constitutive cytomegalovirus promoter and containing the 3' untranslated region of the murine TNF gene. We found that primary stimulation of transfected cells by LPS (1 ng/ml, 12 hr) resulted in a marked suppression (80%) of CAT accumulation in response to a secondary LPS challenge (1 microgram/ml, 6 hr). In contrast, the accumulation of CAT mRNA was not influenced by LPS tolerance. Using the same CAT reporter, we observed that the serine/threonine phosphatases 1 and 2A inhibitor okadaic acid induced TNF mRNA translation and that this activation was not inhibited by LPS-tolerance. In conclusion, these data indicate that deficient production of TNF in LPS-tolerant macrophages in response to a second LPS challenge is characterized by a defective translation of TNF mRNA. However, this hyporesponsiveness to LPS is specific, since translation of TNF mRNA induced by okadaic acid is not inhibited in LPS-tolerant macrophages.
Insights
Lipopolysaccharide (LPS) tolerance impairs tumor necrosis factor (TNF) production by hindering TNF mRNA translation. This defect is specific, as okadaic acid-induced TNF translation remains unaffected in tolerant macrophages.
Area of Science:
- Immunology
- Molecular Biology
- Cellular Biology
Background:
- Macrophage activation by lipopolysaccharide (LPS) leads to tumor necrosis factor (TNF) mRNA translation.
- LPS tolerance is a refractory state where macrophages show impaired TNF production upon secondary LPS challenge.
- High TNF mRNA levels in tolerant macrophages suggest post-transcriptional regulation of TNF biosynthesis.
Purpose of the Study:
- To investigate the translational regulation of TNF biosynthesis during LPS tolerance in macrophages.
- To elucidate the molecular mechanisms underlying impaired TNF production in LPS-tolerant macrophages.
- To determine the specificity of translational defects in LPS tolerance.
Main Methods:
- Utilized RAW 264.7 macrophages stably transfected with a chloramphenicol acetyl-transferase (CAT) reporter gene construct containing the 3' untranslated region of murine TNF.
- Induced LPS tolerance through primary LPS stimulation followed by a secondary LPS challenge.
- Administered okadaic acid, an inhibitor of protein phosphatases 1 and 2A, to assess TNF mRNA translation.
Main Results:
- Primary LPS stimulation induced LPS tolerance, significantly suppressing CAT accumulation (80%) upon secondary LPS challenge.
- LPS tolerance did not affect CAT mRNA accumulation, indicating translational control.
- Okadaic acid induced TNF mRNA translation, and this process was not inhibited in LPS-tolerant macrophages.
Conclusions:
- Deficient TNF production in LPS-tolerant macrophages is due to defective TNF mRNA translation.
- The hyporesponsiveness to LPS in tolerant macrophages is specific, as TNF mRNA translation induced by okadaic acid is not inhibited.
- These findings highlight a critical role for translational regulation in the development and characteristics of LPS tolerance.