Human Monocytes - CD14, CD16 - Ziegler-Heitbrock

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The use of human monocytoid lines as indicators of endotoxin

Abstract

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.

Authors: Eperon, S., Jungi, T.W.
Journal: J. Immunol. Meth., 194: 121-129
Year: 1996
PubMed: Find in PubMed