We have previously demonstrated that Il-1 and TNF could rapidly, but transiently, induce gene expression of Il-1 beta in human polymorphonuclear leukocytes (PMN) at both the protein and mRNA level. Additionally, we demonstrated a cooperative effect of Il-1 and TNF on the kinetics of induction of Il-1 beta mRNA and protein. In order to better understand the molecular basis of Il-1 beta induction, we have further investigated the regulation of Il-1 and TNF-induced gene expression in the PMN. Using nuclear run-on transcription analysis, we found that within 1 h Il-1, TNF, and TNF plus Il-1 induced the transcription of the Il-1 beta gene by 33-, 61-, and 99-fold, respectively. By 2 h, the levels of transcription had been reduced to approximately 50% of peak levels for TNF- and TNF plus Il-1-treated PMN, and to near noninduced levels in Il-1-treated PMN. We also found that these cytokines induced stable mRNA, i.e., Il-1 beta mRNA t1/2 for Il-1-, TNF-, and TNF plus Il-1-induced PMN were 57, 94, and 86 min, respectively. By 2 h, when steady state levels of Il-1 beta mRNA were found to decrease, Il-1 beta mRNA t1/2 had fallen to approximately 18 min for all cytokine treatments. To determine if protein synthesis was required for induction of Il-1 beta gene expression, we treated PMN simultaneously with cytokines and cycloheximide, and found that cycloheximide enhanced the accumulation of Il-1-induced Il-1 beta mRNA, but abrogated the accumulation of Il-1 beta mRNA, by TNF- or TNF plus Il-1-treated PMN. This abrogation of Il-1 beta mRNA accumulation was not caused by inhibition of induction of Il-1 beta transcription because TNF induction of transcription of Il-1 beta was not affected by simultaneous treatment with cycloheximide. Thus, we report that Il-1 and TNF regulate IL-1 beta gene expression via both transcriptional and post-transcriptional mechanisms in vitro.
Cycloheximide Gene Expression Regulation