Recent in vitro studies indicate that tumor necrosis factor (TNF) production in human monocytic THP-1 cells is suppressed by action of arachidonic acid metabolite prostaglandin-E2 (PGE2). PGE2 stimulation of human monocytic cell line THP-1 demonstrates that PGE2 not only regulates TNF activity at production levels, but does so through the release of two soluble TNF receptors (BP-55, BP-75) as well. PGE2 can thus exert a regulatory effect on TNF biologic activity by interfering with its ability to reach cell membrane receptors. THP-1 cells were activated with PGE2 for either 2- or 6-hr time periods, and the supernatants subsequently tested by ELISA to quantitate the levels of soluble receptor released. In addition, we examined mechanisms of receptor shedding by investigating the rate of membrane internalization and the role of serine proteases. PGE2-stimulated THP-1 cells showed soluble 55- and 75-kDa TNF receptor release levels which exceeded that of spontaneous release at both 2- and 6-hr activation periods. The numbers of both membrane TNF receptors were significantly upregulated as well in PGE2-activated cells, whereas the levels of 55- and 75-kDa TNF receptor mRNA levels remained unchanged. Thus, PGE2 induces TNF receptor release primarily at posttranscriptional levels. Inhibition of serine proteases with Pefabloc, a phenylmethylsulfonyl fluoride analog, resulted in the inhibition of both spontaneous and PGE2-stimulated release. Treatment of THP-1 cells with N-ethylmaleimide and low-temperature incubation, both known to block membrane internalization, also blocked spontaneous and PGE2-induced release. Internalization and cleavage by protease are therefore critical factors in PGE2-induced release of soluble TNF receptor shedding.