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Defective translation of tumor necrosis factor mRNA in lipopolysaccharide-tolerant macrophages.

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.