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.
Highlights
Lipopolysaccharide(LPS,endotoxin) is a potent acterized by its ability to cause the lysis of tumor cells ( l ), is stimulator of tumor necrosisfactor a (TNFa) synthesis amultifunctional cytokine (2), capable of influencing the and secretion inmouse macrophage tumor cel(lsGolen- growth, differentiation, and function of a broad range of cells bock, D
The a full-length cDNA has revealed that TNFais synthesized as effect of PMA has been investigated at each stage of a proprotein (26 kDa) with an N-terminal extension of 76 sLTdioPNoenSFs aaonflbooiTtnoegNineFrcneareessumaissl.RtesTNTirAneNa.FrtCamampoeinRndct,NootAmfraiMstnaysonnintetoahndetM,dsiiaatscniod6anncfyeuollflflsuPewrMxtihptAehrre,s-asinemqitunheoencareco,iudbgurhet sieitdnmudeoasypl(fa9us,nm1ci0tci)o.rnTehttioicsualpnurcmehs,oerqluitkheenecaeTNtiysFpniaocpatrlecclseuiagrvsnoeadrl but it prolongs the half-life of Tumor necrosis factor a (TNFa) mRNA about 3- polypeptide in the plasma membrane(11-13)
Later events include the stimulation of cytokine synthesis (35), the production of TNFa (36, 37)
Summary
RAW 264.7and Mono Mac 6 Cells Differ in Their Ability to Secrete TNFa in Response to LPS-The ability of the two cell lines, mouse RAW264.7 andhuman Mono Mac 6, to synthesize and secrete TNFa in response to LPSwas examined. To exclude the possibility that the low levels of TNFa secretion in response to LPSalone might have resulted from the production of an inhibitor that blocked the TNFa-monoclonal antibody interaction or the cytotoxic activity of TNFa, equal volumes of culture supernatantsfrom LPS-treated cells and PMA/LPS-treated cells were mixed and assayed for the presence of TNFa by radioimmunoassay. The desensitization observed with Mono Mac 6 cells after a long-term preincubation with PMA with respect to TNFa secretion (Fig. 2 B ) was investigated at the level of TNFa mRNA expression.
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