Differential regulation of biosynthesis of cell surface and secreted TNF-alpha in LPS-stimulated murine macrophages

Activated macrophages synthesize a 26-kDa cell surface form and a 17-kDa secreted form of tumor necrosis factor alpha (TNF-alpha). 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-alpha 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-alpha than for secreted TNF-alpha (1 mu g/mL). Furthermore, concentrations of actinomycin D that inhibit the synthesis of new mRNA and the production of secreted TNF-alpha did not block the expression of surface TNF-alpha on LPS-stimulated cells. Cycloheximide inhibited the production of both forms of TNF-alpha at similar concentrations. The effects, on the expression of the surface form of TNF-alpha, of various pharmacological agents known to inhibit the production of secreted TNF-alpha 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-alpha at concentrations that inhibited secreted TNF-alpha. The data show that there are differences in the production of surface and secreted TNF-alpha 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.