THE RELEASE OF SOLUBLE P55 TNF RECEPTOR FROM U937 CELLS STUDIED BY A NEW P55 IMMUNOASSAY

A highly specific and sensitive immunoassay for soluble p55 tumor necr osis factor receptor (TNFR) has been established. The immunoassay was based on a newly developed monoclonal antibody (IV4E) recognizing a no n-TNF-binding site of the p55 TNFR. The IV4E antibody immunoprecipitat ed a 55 kDa TNF binding protein from HL-60 cells. No binding of IV4E t o the p75 TNFR could be detected. Bound TNFR to IV4E was detected with digoxigenin (DIG) labeled TNF. This assay could detect down to 300 pg /ml of soluble p55, which represents an 8-10-fold increase in sensitiv ity compared to earlier developed immunoassays. The assay was specific for soluble p55 TNFR present in serum and cell culture supernatants, since addition of excess unlabeled TNF together with DIG labeled TNF i nhibited the signal. TNF concentrations up to 10 ng/ml in the TNFR sam ple did not affect the assay, indicating that TNFRs can be measured in samples containing TNF. The new immunoassay was used to study the mec hanisms underlying the release of soluble p55 TNFR from U937 cells sti mulated with TPA. The TPA induced release of soluble p55 TNFR from U93 7 cells occurred in two phases. First, a rapid increase of soluble p55 was observed after the addition of TPA. Later, the release of p55 occ urred at a slower rate, and this release was inhibited by known inhibi tors of protein synthesis and intracellular transport. Addition of TPA increased the p55 mRNA expression in U937 cells. The results suggest that TPA induces both release and new synthesis of p55 in U937 cells.