The transcription factor NF-B plays an important role in the regulated expression of cytokines in human monocytes. A p100 subunit of NF-B has IB-like properties by sequestering the p65 transactivating subunit in the cytosol of cells. In transient transfection assays we demonstrated that p100 has an inhibitory effect on the NF-B-dependent IL-6 promoter activity. In view of this finding, we studied the regulation of the p100 subunit in human monocytes in response to LPS, the inflammatory cytokines IL-1␤ and TNF-␣ and lymphokines. The results demonstrate that LPS, IL-1␤, and TNF-␣ induce p100 expression at mRNA and protein level while IFN-␥, IL-3 and IL-4/IL-10 have no effect. The induction of p100 expression was shown to be mediated by a two-fold increase in the p100 transcription rate and a two-fold increase in p100 mRNA stability. Furthermore the p100 mediated upregulation was dependent on a tyrosine kinase dependent pathway rather than the protein kinase C pathway. NF-B is a complex of either p50 homodimers or a p50/p65 heterodimer. The latter is known to strongly autoregulate p100 transcription. We therefore examined the composition of NF-B induced by LPS vs the different lymphokines. LPSinduced NF-B showed a distinct p65 supershift whereas the composition of NF-B induced by different lymphokines did not show a change in p65. We conclude that the p100 subunit of the transcription factor NF-B is induced by different inflammatory mediators while lymphokines fail to induce p100 expression which may be caused by the induction of NF-B predominantly consisting of p50 homodimers.

Human T cells were studied with regard to the regulation of interleukin- 4 (IL-4) and IL-3 gene expression. IL-4 and IL-3 mRNA were undetectable in unstimulated T cells. On activation with the lectin concanavalin A (Con A), both IL-4 and IL-3 mRNA were expressed. Accumulation of IL-4 mRNA peaked after 6 to 12 hours, whereas IL-3 mRNA levels peaked after 3 to 6 hours of stimulation with Con A. Nuclear run-on assays showed a low constitutive transcription for both genes. The transcription rates were increased by Con A resulting in a peak for IL-4 after 1 hour (30% increase) and for IL-3 after 3 hours (40% increase) of Con A treatment. mRNA stability studies demonstrated that on activation with Con A both messages decayed with a half-life of approximately 90 minutes. No IL-4 or IL-3 mRNA expression was induced by the protein kinase C activator phorbol myristate acetate (PMA). However, PMA augmented the Con A- induced IL-4 and IL-3 mRNA accumulation. This was shown to be mediated at posttranscriptional level by a large increase in the stability of both messages (t 1/2 > 3 hours). The transcription rate of both genes was also enhanced by Con A+PMA and reached peak levels for IL-4 after 1 hour (90% increase) and for IL-3 after 3 hours (70% increase) of stimulation. Furthermore, it appeared that the induction of IL-4 mRNA was dependent on protein synthesis because cycloheximide (CHX) blocked the Con A- and Con A+PMA-induced expression of IL-4 mRNA. In contrast, CHX inhibited, but failed to completely block, the Con A- and Con A+PMA- induced IL-3 mRNA expression, whereas the expression of both genes was completely blocked by cyclosporine A. With regard to the secretion of IL-4 protein it was shown that it closely follows the accumulation of IL-4 mRNA. Taken together, the data show that expression of the IL-4 and IL-3 genes in human T cells is controlled by different activation pathways that affect the gene regulation at transcriptional and posttranscriptional levels.

Human recombinant interleukin-4 (IL-4) was studied for its effects on the expression of granulocyte-colony stimulating factor (G-CSF) mRNA in human adherent monocytes in the absence and presence of endotoxin and interleukin 1 (IL-1). IL-4 (15 ng/ml) did not induce G-CSF transcripts in monocytes but suppressed the endotoxin-induced G-CSF expression when added simultaneously. Sequential treatment of monocytes with IL-4 followed by endotoxin suppressed G-CSF mRNA induction totally. This effect was independent of the presence of fetal bovine serum but dependent of the IL-4 dose. Comparable results were obtained with IL-1. IL-1 (50 U/ml) induced G-CSF expression in human adherent monocytes which could be counteracted by IL-4 pretreatment. In addition, it was shown that the induction of G-CSF mRNA by the calcium-ionophore A23187 or by c-AMP elevating agents could be blocked by IL-4. These suppressive effects of IL-4 were not related to changes in the half-life of G-CSF mRNA and were independent of protein synthesis. Finally it was demonstrated that IL-4 had comparable effects on the G-CSF secretion of endotoxin and IL-1 stimulated human monocytes by using a murine bone marrow assay. These results indicate that IL-4 down-regulates the expression of G-CSF gene and secretion of proteins in human activated monocytes.

Interleukin-4 (IL-4) modulates the survival, proliferation, and differentiation of a variety of hematopoietic cells. The effects are mediated through a single class of high-affinity receptors for IL-4. To understand the biologic effects of IL-4 on human T cells, we studied the regulation of IL-4 receptor (IL-4R) gene expression. We showed that IL-4R mRNA accumulation in human T cells is enhanced fourfold after activation of different secondary signaling pathways by concanavalin A (Con A), phorbol myristate acetate (PMA), the calcium ionophore A23187, and combinations of these factors. This could be ascribed to an increase in the IL-4R transcription rate and to stabilization of IL-4R mRNA resulting in a half-life of 80 to 90 minutes (v 35 to 40 minutes in resting T cells). IL-4 did enhance the IL-4R mRNA accumulation by a factor 10, which was caused by an increase in the IL-4R transcription rate and prolonging the half-life of IL-4R transcripts to 140 to 160 minutes. Finally, it was shown that A23187 induced IL-4R mRNA expression is a protein synthesis-dependent process. In contrast, Con A- , PMA-, Con A + PMA-, and Con A + A23187-induced expression of IL-4R mRNA is protein-synthesis independent. Cyclosporine A inhibited the A23187- and Con A + A23187-induced IL-4R mRNA accumulation, whereas Con A-, PMA-, and Con A + PMA-induced IL-4R mRNA expression was not affected by this drug. These data indicate that expression of IL-4 receptors on human T cells can be modulated by different intracellular signaling pathways at both transcriptional and posttranscriptional levels.

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