Concentrations of immunoreactive human tumor necrosis factor alpha produced by human mononuclear cells in vitro

• Published in: Journal of leukocyte biology 1988-03, Vol.43 (3), p.216-223
• Main Authors: Meer, Jos W.M., Endres, Stefan, Lonnemann, Gerhard, Cannon, Joseph G., Ikejima, Takashi, Okusawa, Seijiro, Gelfand, Jeffrey A., Dinarello, Charles A.
• Format: Article
• Language: English
• Subjects: Analysis of the immune response. Humoral and cellular immunity; Animals; Biological and medical sciences; Female; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; Immunobiology; In Vitro Techniques; Interleukin-1 - blood; interleukin‐1; Leukocytes, Mononuclear - analysis; Leukocytes, Mononuclear - metabolism; Lipopolysaccharides - pharmacology; lymphocyte; Mice; Miscellaneous; monocyte; Rabbits; Radioimmunoassay; Regulatory factors and their cellular receptors; tumor necrosis factor; Tumor Necrosis Factor-alpha - biosynthesis; Tumor Necrosis Factor-alpha - blood; Tumor Necrosis Factor-alpha - immunology
• ISSN: 0741-5400; 1938-3673
• DOI: 10.1002/jlb.43.3.216

The concentrations of tumor necrosis factor (TNF) produced by human peripheral blood mononuclear cells (MNC) were measured using a radioimmunoassay (RIA) for human TNF. This was developed using a rabbit antiserum against human recombinant TNF (Hu rTNF), and Hu rTNF labeled with Na1251 by a modification of the chloramine T method. This RIA does not detect human lymphotoxin, interieukin‐1 alpha or beta, interleukin 2, interleukin 6, interferon alpha or gamma, granulocyte‐macrophage‐colony stimulating factor, and C5a des arg. A good correlation (r = 0.89) was found between the RIA and the cytolytic bioassay for TNF. The sensitivity of the RIA is between 3 and 78 pg/ml (median 11 pg/ml). The mean concentration of TNF in 24‐h culture supernatants of human MNC exposed to different concentrations of lipopolysaccharide (LPS) was found to increase in dose‐dependent fashion and then level off between 50 and 100 ng/ml. The concentrations of IL‐1 beta and alpha detected by specific RIAs in these supernatants were between 0.2 and 19 ng/ml and 0.04 and 1 ng/ml, respectively. The amount ot TNF produced by human MNC in vitro was determined in a cohort of 50 normal volunteers. Without exogenous stimuli, TNF concentrations were almost always below the detection limit; with 0.5 ng/ml LPS, the median concentration of TNF was 2 ng/ml, and with PHA the median was 3.8 ng/ml. In cultures performed in the presence of indomethacin significantly (p< 0.005) more TNF was produced. Using this RIA, we could detect TNF in the circulation of mice injected with Hu rTNF. When plasma samples of patients with febrile illnesses were added directly to the RIA, TNF was not detectable, with the exception of patients with malaria. These studies demonstrate the range and sensitivity of LPS‐induced and mitogen‐induced production of immunoreactive TNF by human MNC in vitro without interference of similar cytokines in bioassays.