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Human peripheral blood mononuclear lymphocytes produce interferon gamma (IFN-gamma) in response to stimulation by mitogens. Previous studies on the kinetics of IFN-gamma mRNA production upon mitogen induction, showed that steady-state levels of mRNA increased to a maximum at 12-24 h post-induction after which they declined to levels not detectable by the assay used. We show here that in mitogen induced peripheral blood lymphocytes, inhibition of protein synthesis using three different inhibitors (cycloheximide, puromycin, pactamycin) resulted in an increase in the steady-state levels of IFN-gamma mRNA. The levels of mRNA in cells treated with inhibitor 16 h post-induction were up to 3-fold higher than in untreated cells. Superinduction was possible up to 40 h post-induction after which the steady-state levels of mRNA had declined to limits below detection; IFN-gamma mRNA was not superinduced by cycloheximide in the presence of actinomycin D.

The effect of inhibition of protein synthesis on interferon gamma (IFN-gamma) mRNA induction has been examined in human peripheral blood leukocytes and growing T lymphoblasts. Inhibition of protein synthesis by cycloheximide or puromycin when lymphocytes were stimulated with mitogen alone had little effect on the steady-state levels of IFN-gamma mRNA induced. Activation of the IFN-gamma gene appears to occur independently of synthesis of protein factors. Synergistic induction of IFN-gamma by mitogen plus the phorbol ester mezerein was at least in part accounted for by increased levels of IFN-gamma mRNA in both fresh lymphocytes and growing lymphoblasts. Inhibition of protein synthesis abolished the synergistic effect on mRNA leading to levels similar to those observed in cells treated with mitogen alone. Synergistic induction is dependent upon synthesis of protein factors either within the cells or produced as soluble mediators; these factors are not simply lymphokines since addition of exogenous factors to the cultures did not reverse the block on synergy by the inhibition. These data suggest that protein factors though they may be important in exerting qualitative control on the level of production of IFN-gamma have no role in the initial activation of the gene.

Fresh human peripheral blood mononuclear lymphocytes and lymphoblasts that had been grown for a period in T-cell growth-factor containing medium were stimulated with staphylococcal enterotoxin A plus mezerein to produce interferon-gamma (IFN-gamma). Growing lymphoblasts produced peak levels of IFN-gamma much earlier after induction than fresh lymphocytes. Quantitation of the steady-state levels of IFN-gamma mRNA showed these to differ markedly between the two cell types over a period of time post-induction. In fresh lymphocytes the steady-state levels of IFN-gamma mRNA increased to a peak level over a period of 4 days while in growing lymphoblasts the peak level occurred after 8 hours. These differences in IFN-gamma mRNA production were shown to be not the result of gross alteration of RNA metabolism following blast transformation.