The superantigen toxic shock syndrome toxin (TSST)-1 can induce tumor necrosis factor (TNF)-alpha expression in T cells and monocytes, through different signaling pathways. We have stimulated peripheral blood mononuclear cells with TSST-1 and found that the major cell producers of TNF-alpha as detected by cytofluorimetry and immunocytochemistry were CD4(+) T lymphocytes. The expression of TNF-alpha by CD4(+) T cells can be inhibited by either, wortmannin (WN) or LY 294002, two phosphatidylinositol 3-kinase (PI 3-K) inhibitors. The inhibitory effect is not transcriptional as WN does not change the mRNA steady state of TNF-alpha at any of the concentrations tested and LY 294002 when preincubated with mononuclear cells at its median inhibitory concentration (IC(50) = 1. 4 microM) significantly inhibited the expression of TNF-alpha but not its mRNA. Immunoprecipitation of pulse-labeled intracellular TNF-alpha showed a specific decrease in the synthesis of this cytokine on cells treated with PI 3-K inhibitors. The p38 mitogen-activated protein kinase (MAPK) is involved in control of TNF-alpha translation in human macrophages. In T cells, we have found that the p38 MAPK inhibitor SB 203580 significantly decreased the secretion of TNF-alpha but not its mRNA. In addition, the combined use of WN and SB 203580 had an additive inhibitory effect on secretion of TNF-alpha. Therefore, both PI 3-K and p38 MAPK signaling pathways control TNF-alpha production in T cells.

Summary Tumour necrosis factor‐α (TNF‐α) is a proinflammatory cytokine produced by several cell types, including T cells upon antigen stimulation. Its production is crucial for the development of an early defence against many pathogens, but its beneficial effects are dependent on the strength and duration of its expression. In this paper we present evidence indicating that serum increases translational efficiency of TNF‐α in human peripheral blood mononuclear cells stimulated with superantigen. The increase in translation of TNF‐α due to serum could be inhibited by the phosphatidylinositol (PI) 3‐K inhibitors, wortmannin and LY294002, suggesting that PI 3‐K is involved in the translational control of TNF‐α by serum. Similarly to primary T cells, stimulation of Jurkat T cells with superantigen led to TNF‐α secretion and this was up‐regulated by serum. Transfection of Jurkat cells with a constitutively active form of PI 3‐Kα increased the production of TNF‐α in cells stimulated with superantigen. Additionally, we used the specific inhibitors targeting ERK kinase and p38 mitogen‐activated protein kinase (MAPK), potentially downstream of PI 3‐kinase, PD98059 and SB203580. Differently from with PI 3‐K inhibitors, the accumulation of TNF‐α mRNA was inhibited by PD98059 or SB203580. These results suggest that, in T cells, activation of PI 3‐K is an important step in controlling TNF‐α protein synthesis in response to growth factors.

Tumor necrosis factor α (TNF-α), a proinflammatory cytokine, is produced abundantly by monocytes and macrophages. We have compared LPS-stimulated TNF-α production and regulation in freshly isolated human monocytes and macrophages differentiated in vitro. A significant increase in LPS-induced TNF-α protein secretion was observed in macrophages over freshly isolated monocytes without comparable differences in TNF-α mRNA induction. Polysome gradient analysis showed polysome-mRNA distribution did not change, whereas TNF-α mRNA stability increased in macrophages. Tristetraprolin mRNA expression was constitutive and decreased with differentiation-linked kinetics. Blockable LPS-inducible MAP kinase activity (p38, ERK) affected TNF-α biosynthesis differentially at the transcriptional and post-transcriptional level throughout the culture period. We suggest that the increase in TNF-α secretion in macrophages relates to changes in post-transcriptional processing, which is regulated indirectly by the expression of RNA-binding proteins. Changes in gene expression throughout monocytic differentiation equip the cell to act as a more potent producer of this proinflammatory cytokine.