secreted TNF-a in LPS-stimulated murine macrophages

Activated macrophages synthesize a 26-kDa cell surface form and a 17-kDa secreted form of tumor necrosis factor a (TNF-a). This study was designed to investigate possible differences between the biosynthesis ofthese two forms by murine peritoneal exudate cells (PEC) and a murine macrophage cell line (RAW 264.7) stimulated with lipopolysaccharide (LPS). Both PEC and RAW 264.7 cells produced surface and secreted TNF-a in response to LPS in a dose-dependent manner. However, much lower concentrations of LPS (100 ng/mL) were needed for optimal expression of surface TNF-a than for secreted TNF-a (1 j.tg/mL). Furthermore, concentrations of actinomycin D that inhibit the synthesis ofnew mRNA and the production , ofsecreted TNF-a did not block the expression of surface TNF-a on LPS-stimulated cells. Cycloheximide inhibited the production of both forms of TNF-a at similar concentrations. The effects, on the expression of the surface form of TNF-a, of various pharmacological agents known to inhibit the production of secreted TNF-a were studied. It was found that: (1) dexamethasone, a glucocorticoid agonist and (2) Eli and ETYA, inhibitors oflipoxygenase, had no effect on cell surface TNF-a at concentrations that inhibited secreted TNF-a. The data show that there are differences in the production of surface and secreted TNF-a and indicate that the regulation of synthesis of this protein is more complex than that suggested by a mere precursor/product relationship between the two forms. J. Leukoc. Riot. 62: 249-257; 1997.