Glucocorticoids are potent immunosuppressive drugs, but their mechanism is poorly understood. Nuclear factor kappa B (NF-kappa B), a regulator of immune system and inflammation genes, may be a target for glucocorticoid-mediated immunosuppression. The activation of NF-kappa B involves the targeted degradation of its cytoplasmic inhibitor, I kappa B alpha, and the translocation of NF-kappa B to the nucleus. Here it is shown that the synthetic glucocorticoid dexamethasone induces the transcription of the I kappa B alpha gene, which results in an increased rate of I kappa B alpha protein synthesis. Stimulation by tumor necrosis factor causes the release of NF-kappa B from I kappa B alpha. However, in the presence of dexamethasone this newly released NF-kappa B quickly reassociates with newly synthesized I kappa B alpha, thus markedly reducing the amount of NF-kappa B that translocates to the nucleus. This decrease in nuclear NF-kappa B is predicted to markedly decrease cytokine secretion and thus effectively block the activation of the immune system.

Monocyte adherence results in the rapid transcriptional activation and mRNA stabilization of numerous mediators of inflammation and tissue repair. While the enhancer and promoter elements associated with transcriptional activation have been studied, mechanisms linking adhesion, mRNA stabilization, and translation are unknown. GRO␣ and interleukin-1␤ (IL-1␤) mRNAs are highly labile in nonadhered monocytes but stabilize rapidly after adherence. GRO␣ and IL-1␤ transcripts both contain A؉U-rich elements (AREs) in the 3 untranslated region (UTR) which have been directly associated with rapid mRNA turnover. To determine if the GRO␣ ARE region was recognized by factors associated with mRNA degradation, we carried out mobility gel shift analyses using a series of RNA probes encompassing the entire GRO␣ transcript. Stable complexes were formed only with the proximal 3 UTR which contained the ARE region. The two slower-moving complexes were rapidly depleted following monocyte adherence but not direct integrin engagement. Deadherence reactivated the two largest ARE-binding complexes and destabilized IL-1␤ transcripts. Antibody supershift studies demonstrated that both of these ARE RNA-binding complexes contained AUF1. The formation of these complexes and the accelerated mRNA turnover are phosphorylation-dependent events, as both are induced in adherent monocytes by the tyrosine kinase inhibitor genistein and the p38 MAP kinase inhibitor of IL-1␤ translation, SK&F 86002. These results demonstrate that cell adhesion and deadhesion rapidly and reversibly modify both cytokine mRNA stability and the RNA-binding complexes associated with AUF1.

We identified IB␣/MAD-3 as an immediate-early gene in human monocytes that is expressed in response to a variety of signals, including adhesion, lipopolysaccharide, and phorbol myristate acetate. Within 5 min of monocyte adhesion, the level of the IB␣ protein is markedly diminished but is rapidly replaced in a cycloheximide-sensitive manner within 20 min. Accompanying the rapid turnover of the IB␣ protein is simultaneous translocation of NF-B-related transcription factors to nuclei of adhered monocytes. The demonstration that NF-B can regulate IB␣/MAD-3 gene transcription in other cell types suggested that the rapid increase in steady-state IB␣/MAD-3 mRNA levels we observed within 30 min of monocyte adherence would result from NF-B-dependent transcriptional stimulation of the IB␣/MAD-3 gene. Nuclear run-on analyses indicated that, instead, while several immediate-early cytokine genes, such as the interleukin 1␤ (IL-1␤) gene, were transcriptionally activated during monocyte adhesion, the rate of IB␣/MAD-3 gene transcription remained constant. The adherence-dependent increase in IB␣/MAD-3 mRNA levels was also not a consequence of mRNA stabilization events. Interestingly, while increases in both IL-1␤ and IB␣/MAD-3 mRNA levels were detected in nuclei of adherent monocytes, cytoplasmic levels of IL-1␤ mRNA increased during adherence whereas those of IB␣/MAD-3 mRNA did not. Taken together, our data suggest that two interactive mechanisms regulate monocytic IB␣/MAD-3 mRNA levels. We propose that adherent monocytes regulate nuclear processing (or decay) of IB␣/MAD-3 mRNA, thereby increasing mRNA levels without stimulating IB␣/MAD-3 gene transcription. Moreover, since inhibition of protein synthesis leads to accumulation of IB␣/MAD-3 mRNA without stimulating IB␣/MAD-3 gene transcription, we suggest that low cytoplasmic levels of IB␣/MAD-3 mRNA are maintained by a translation-dependent degradation mechanism.Transcription factor NF-B plays a critical role in the regulation of many immediate-early response genes associated with the host response to infection and tissue damage (reviewed in references 25 and 42). Rel/NF-B activity is modulated, in part, by a growing family of regulatory molecules, including IB␣, IB␥, the C-terminal domains of precursors for the p50 and p52 components of NF-B (p105 and p100, respectively), and Bcl-3 (for a recent review, see reference 8). The IB inhibitors function by sequestering Rel family members in the cytoplasm, effectively interfering with their transcription activation potential (reviewed in references 8 and 27). Recently, several groups have reported that cytokine, phorbol myristate acetate, or lipopolysaccharide (LPS) stimulation of a variety of malignant cell lines results in rapid degradation of the IB␣ protein (9,11,17,45,47,60). In each case, loss of IB␣ was associated with simultaneous translocation of p50/65 to the nucleus. This indicates that dissociation of NF-B from IB␣, a prerequisite for B-dependent transcriptional activation, requires proteolytic processing of the IB␣ inh...

IL-4 and IL-13 comprise promising targets for therapeutic interventions in asthma and other Th2-associated diseases, but agents targeting either IL-4 or IL-13 alone have shown limited efficacy in human clinical studies. Because these cytokines may involve redundant function, dual targeting holds promise for achieving greater efficacy. We describe a bifunctional therapeutic targeting IL-4 and IL-13, developed by a combination of specific binding domains. IL-4-targeted and IL-13-targeted single chain variable fragments were joined in an optimal configuration, using appropriate linker regions on a novel protein scaffold. The bifunctional IL-4/IL-13 antagonist displayed high affinity for both cytokines. It was a potent and efficient neutralizer of both murine IL-4 and murine IL-13 bioactivity in cytokine-responsive Ba/F3 cells, and exhibited a half-life of approximately 4.7 days in mice. In a murine model of ovalbumin-induced ear swelling, the bifunctional molecule blocked both the IL-4/IL-13-dependent early-phase response and the IL-4-dependent late-phase response. In the ovalbumin-induced lung inflammation model, the bifunctional IL-4/IL-13 antagonist reduced the IL-4-dependent rise in serum IgE titers, and reduced IL-13-dependent airway hyperresponsiveness, lung inflammation, mucin gene expression, and serum chitinase responses. Taken together, these findings demonstrate the effective dual blockade of IL-4 and IL-13 with a single agent, which resulted in the modulation of a more extensive range of endpoints than could be achieved by targeting either cytokine alone.

Monocyte adhesion resulted in rapid tyrosine phosphorylation and subsequent cytokine mRNA induction. The objective of this study was to determine the role of specific tyrosine phosphorylation events, particularly those involving members of the MAP kinase family, in regulating adhesioninduced cytokine expression. Using nuclear run-on analyses, we demonstrated that on adhesion, monocytes rapidly transcriptionally activated numerous cytokine mRNAs, coincident with the activation of the transcription factors NF-B and AP-1. Both an inhibitor of tyrosine phosphorylation, genistein, and the cytoplasmic tyrosine phosphatase PTP1B, were unable to prevent adhesion-mediated transcriptional activation. However, both blocked adhesion-induced ERK and JNK but not p38 kinase activation and at the same time decreased the stability of interleukin-1␤ (IL-1␤) and IL-8 transcripts. In addition, whereas adhesive events occurred in the presence of genistein and PTP1B, monocyte spreading was markedly inhibited. Our results suggest that the majority of protein phosphorylation events are associated with adhesioninduced cytokine expression through transcript stabilization and cytoskeletal organization. A minority of protein phosphorylation events, not sensitive to genistein or PTP1B exposure, may be instrumental in regulating transcription. Thus the spectrum of protein tyrosine kinases required for transcription appear distinct from those involved in maintaining the stability of some cytokine mRNAs and the integrity of the cytoskeleton to which mRNA destined for translation must be associated. J. Leukoc. Biol. 67: 216-225; 2000.