Secreted tumor necrosis factor-alpha by human myeloid cells: a valuable parameter for evaluation of endotoxin contamination in vitro.
Myeloid cells respond to external stimuli such as gram negative bacterial lipopolysaccharides (LPS) or endotoxin by producing a large array of intermediates and proteins, including the pro-inflammatory cytokines such as, tumor necrosis factor-alpha (TNF-alpha), free radicals, nitric oxide, and other intermediates. The aim of this research was to investigate the response of human myeloid cells to endotoxin, and correlate secreted TNF-alpha concentration with endotoxin exposure. For this purpose, human myeloid cell lines such as, U937, HL-60 (undifferentiated and differentiated) and the human peripheral blood Mononuclear (hpMN) and Polymorphonuclear (hpPMN) cells were treated by bacterial endotoxin in a dose and time dependent manner. Then the cells were isolated from supernatants. The nitro-blue tetrazolium (NBT) reduction potential of isolated cells was quantitatively assessed. The secreted TNF-alpha was assessed by the ELISA and TNF-alpha dependent L929 cells cytotoxicity methods. The hpPMN, hpMN, and differentiated U937 cells showed a higher ability to reduce NBT in response to endotoxin. The amount of produced TNF-alpha by hpPMN and hpMN cells were higher than that of other cells (Up to 300 pg/ml at 15 min and 6000 pg/ml at 24 hrs in response to 10 ng/ml of endotoxin, r = 0.974). The results of this study indicated that, human myeloid cells specially the hpPMN and hpMN cells provide a useful in vitro system for evaluating the endotoxin contamination in culture medium. In response to endotoxin, these cells synthesize and secrete TNF-alpha in a dose and time dependent manner.
Abdolkhaleg
Deezagi
D
National Institute of Genetic Engineering and Biotechnology, Tehran, Iran. deezagi@nigeb.ac.ir
Shokooh
Dorchehzadeh
D
D001681:Biological Assay / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D005609:Free Radicals / Q000276:immunology / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D008070:Lipopolysaccharides / Q000032:analysis* / Q000494:pharmacology*; D022423:Myeloid Cells / Q000276:immunology* / Q000378:metabolism; D009569:Nitric Oxide / Q000276:immunology / Q000378:metabolism; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology* / Q000378:metabolism; D020298:U937 Cells
D005609:Free Radicals; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide