Secreted tumor necrosis factor-α by human myeloid cells: a valuable parameter for evaluation of endotoxin contamination in vitro

Abstract

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-α (TNF-α), 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-α 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-α was assessed by the ELISA and TNF-α 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-α 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-α in a dose and time dependent manner.

Keywords::

• Endotoxin Measurement
• Human Myeloid Cell Lines
• Human Peripheral Blood Leukocytes
• Tumor Necrosis Factor-α

Acknowledgment

This work was supported by research funds from National Institute of Genetic Engineering and Biotechnology (R.P. grant number 162), Tehran-Iran.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.