endotoxininduced tumor necrosis factor alpha synthesis in murine embryo fibroblasts murine embryo fibroblasts mef were found to secrete tumor necrosis factor tnf in response to stimulation with endotoxin endotoxininduced tnf production by mef was inhibited by cycloheximide however reversal of the effect of this inhibitor on protein synthesis results in tnf being secreted in amounts equivalent to those produced by endotoxininduced mef not treated with cycloheximide actinomycin d treatment of mef blocked the production of endotoxininduced tnf maximal production of tnf required mef gene transcription during the first 6 h of incubation with endotoxin to determine whether endotoxininduced tnf alpha tnfalpha andor tnf beta were produced by mef cdna was synthesized from the total rna isolated from endotoxininduced mef and amplified by the polymerase chain reaction in the presence of oligonucleotide primers specific for each cytokine on the basis of the polymerase chain reaction analysis it was determined that tnfalpha mrna levels were increased in endotoxininduced mef thus production of tnfalpha by fibroblasts in response to the endotoxin component of bacterial cell walls is likely to contribute to the expression of tnfmediated effects occurring in fibroblastrich tissues infected with gramnegative bacteria