Differential regulation of biosynthesis of cell surface and secreted TNF‐α in LPS‐stimulated murine macrophages

Activated macrophages synthesize a 26‐kDa cell surface form and a 17‐kDa secreted form of tumor necrosis factor α (TNF‐α). This study was designed to investigate possible differences between the biosynthesis of these 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‐α 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‐α than for secreted TNF‐α (1 μg/mL). Furthermore, concentrations of actinomycin D that inhibit the synthesis of new mRNA and the production of secreted TNF‐α did not block the expression of surface TNF‐α on LPS‐stimulated cells. Cycloheximide inhibited the production of both forms of TNF‐α at similar concentrations. The effects, on the expression of the surface form of TNF‐α, of various pharmacological agents known to inhibit the production of secreted TNF‐α were studied. It was found that: (1) dexamethasone, a glucocorticoid agonist and (2) ETI and ETYA, inhibitors of lipoxygenase, had no effect on cell surface TNF‐α at concentrations that inhibited secreted TNF‐α. The data show that there are differences in the production of surface and secreted TNF‐α 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. Biol. 62: 249–257; 1997.