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Processing and secretion of tumor necrosis factor alpha in endotoxin-treated Mono Mac 6 cells are dependent on
A Pradines-Figueres1, C R Raetz
1Merck Research Laboratories, Department of Biochemistry, Rahway, New Jersey 07065.
Abstract:
Lipopolysaccharide (LPS, endotoxin) is a potent stimulator of tumor necrosis factor alpha (TNF alpha) synthesis and secretion in mouse macrophage tumor cells (Golenbock, D. T., Hampton, R. Y., Qureshi, N., Takayama, K., and Raetz, C. H. R. (1991) J. Biol. Chem. 266, 19490-19498). In contrast, addition of LPS (10 ng/ml) to human monomyelocytic (Mono Mac 6) cells induces very little production of TNF alpha, as judged by immunoassay of the growth medium. When 30 ng/ml 4-beta-phorbol-12-myristate 13-acetate (PMA) is added together with LPS, large amounts of TNF alpha are secreted. PMA alone is inactive. Maximal TNF alpha levels in the medium are achieved at 1 ng/ml of LPS. Protein kinase C inhibitors, such as H7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine), staurosporine, and sphingosine, reduce TNF alpha secretion stimulated by PMA. The effect of PMA has been investigated at each stage of TNF alpha biogenesis. Treatment of Mono Mac 6 cells with LPS alone results in rapid, transient, and full expression of TNF alpha mRNA. Concomitant addition of PMA does not increase TNF alpha mRNA synthesis any further, but it prolongs the half-life of TNF alpha mRNA about 3-fold. However, mRNA stabilization does not account for the striking effect of PMA on TNF alpha secretion. Analysis of TNF alpha synthesis and secretion by immunoprecipitation indicates that LPS alone is fully effective in stimulating the formation of the intracellular 26-kDa TNF alpha precursor. LPS alone is not sufficient to allow processing of the precursor and secretion of mature 17-kDa TNF alpha. The rate of TNF alpha secretion observed immediately after the addition of PMA to LPS-pretreated cells is similar to the maximum rate from LPS/PMA-treated cells, but without the lag observed in cells after being exposed to LPS and PMA simultaneously. In summary, PMA is required for the completion of TNF alpha precursor processing and secretion in LPS-treated human Mono Mac 6 cells, whereas murine RAW cells are able to complete the terminal steps of TNF alpha processing in the absence of PMA.
Insights
Lipopolysaccharide (LPS) stimulates tumor necrosis factor alpha (TNF alpha) mRNA in human cells, but requires 4-beta-phorbol-12-myristate 13-acetate (PMA) for TNF alpha precursor processing and secretion.
Area of Science:
- Immunology
- Cell Biology
Background:
- Lipopolysaccharide (LPS) potently stimulates tumor necrosis factor alpha (TNF alpha) synthesis in mouse cells.
- Human monomyelocytic cells (Mono Mac 6) produce minimal TNF alpha in response to LPS alone.
Purpose of the Study:
- To investigate the role of 4-beta-phorbol-12-myristate 13-acetate (PMA) in TNF alpha production in human Mono Mac 6 cells stimulated with LPS.
- To elucidate the stages of TNF alpha biogenesis affected by PMA.
Main Methods:
- Stimulation of Mono Mac 6 cells with LPS and/or PMA.
- Measurement of TNF alpha levels in cell culture medium via immunoassay.
- Analysis of TNF alpha mRNA expression and half-life.
- Immunoprecipitation to assess TNF alpha precursor synthesis and processing.
Main Results:
- LPS alone induced TNF alpha mRNA but not significant TNF alpha secretion.
- PMA, in combination with LPS, dramatically increased TNF alpha secretion.
- PMA prolonged TNF alpha mRNA half-life but did not increase its synthesis.
- LPS stimulated precursor formation, while PMA was essential for precursor processing and secretion of mature TNF alpha.
- Protein kinase C inhibitors reduced PMA-stimulated TNF alpha secretion.
Conclusions:
- PMA is crucial for the post-transcriptional processing and secretion of TNF alpha in LPS-stimulated human Mono Mac 6 cells.
- Human cells require an additional factor (PMA) for TNF alpha secretion, unlike murine RAW cells.