The use of human monocytoid lines as indicators of endotoxin.
We wish to develop an in vitro test system for pyrogenic substances. A major source of pyrogen activity is endotoxin. Here we describe a highly sensitive endotoxin-monitoring system based on cytokine measurement in human cell lines of myelomonocytoid origin. The following measures were taken to develop an endotoxin monitoring system of high sensitivity. (i) Mono Mac 6 (MM6) and THP-1 cells, which both represent advanced stages of myelomonocytic development, were better suited as endotoxin indicators than the more immature U-937 line. (ii) In order to enhance cell surface expression of CD14, a major lipopolysaccharide (LPS) receptor, cells were pretreated for 2 days with calcitriol. (iii) The use of fetal calf serum (FCS) without detectable endotoxin traces was essential for maintaining a high LPS sensitivity. (iv) Selected subclones of either THP-1 or MM6 were significantly more sensitive LPS indicators than bulk cultures from which the clones originated. (v) Based on stimulation indices, a commercial tumor necrosis factor-alpha (TNF-alpha) immunoassay proved to be a more sensitive LPS indicator than other cytokine assays or the expression of procoagulant activity/tissue factor. Thus we were able to eliminate the disadvantage of previous cell line-based systems (i.e., low sensitivity) without loss of reproducibility which is seen when using fresh blood, monocytes or monocyte-derived macrophages. High endotoxin sensitivity is a prerequisite for a test system specifically indicating pyrogen activity, because it permits the testing of substances at higher dilutions, thereby minimizing nonspecific interference.
Eperon
S
S
Institute of Veterinary Virology, University of Berne, Switzerland.
Jungi
T W
TW
D002117:Calcitriol / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D003029:Coagulants / Q000032:analysis / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004731:Endotoxins / Q000032:analysis*; D006801:Humans; D008033:Limulus Test; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects; D009000:Monocytes / Q000166:cytology* / Q000187:drug effects*; D010641:Phenotype; D011749:Pyrogens / Q000032:analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis
D003029:Coagulants; D004731:Endotoxins; D008070:Lipopolysaccharides; D011749:Pyrogens; D014409:Tumor Necrosis Factor-alpha; D002117:Calcitriol