Neutrophilic superoxide production can assess pharmacological and pharmacogenetic ß-adrenoreceptor effects.

Asthma can be controlled well in most patients by inhaled ß-adrenoreceptor (ß2 AR) agonists and steroids. Poor response to ß2 AR agonists is difficult to predict, especially in young children and by lung function testing, which may be affected by multiple influences. As an alternative approach, we analyzed ex vivo neutrophilic superoxide inhibition in response to ß2 AR stimulation. In 60 healthy volunteers, this assay was unaffected by sex, age, smoking, atopy or asthma status. Furthermore, we assessed effects of genetic variants in ß2 AR by sequencing the ADRB2 gene in our cohort and relating genotypes to ß2 AR-mediated neutrophilic superoxide inhibition. Gly16Arg genotypes correlated with minor decrease in overall adrenoresponse in this small study population. Taken together, ex vivo testing of the ß2 AR response in human neutrophils represents a robust tool with good signal-to-noise ratio at physiological ß2 AR agonist concentrations, and this assay may be useful to complement future pharmacogenetic studies in asthma.

The MAPK kinase kinase TAK1 plays a central role in coupling the interleukin-1 receptor to both transcriptional and RNA-targeted mechanisms of gene regulation.

Mechanisms of fulminant gene induction during an inflammatory response were investigated using expression of the chemoattractant cytokine interleukin-8 (IL-8) as a model. Recently we found that coordinate activation of NF-kappaB and c-Jun N-terminal protein kinase (JNK) is required for strong IL-8 transcription, whereas the p38 MAP kinase (MAPK) pathway stabilizes the IL-8 mRNA. It is unclear how these pathways are coupled to the receptor for IL-1, an important physiological inducer of IL-8. Expression of the MAP kinase kinase kinase (MAPKKK) TAK1 together with its coactivator TAB1 in HeLa cells activated all three pathways and was sufficient to induce IL-8 formation, NF-kappaB + JNK2-mediated transcription from a minimal IL-8 promoter, and p38 MAPK-mediated stabilization of a reporter mRNA containing IL-8-derived regulatory mRNA sequences. Expression of a kinase-inactive mutant of TAK1 largely blocked IL-1-induced transcription and mRNA stabilization, as well as formation of endogenous IL-8. Truncated TAB1, lacking the TAK1 binding domain, or a TAK1-derived peptide containing a TAK1 autoinhibitory domain were also efficient in inhibition. These data indicate that the previously described three-pathway model of IL-8 induction is operative in response to a physiological stimulus, IL-1, and that the MAPKKK TAK1 couples the IL-1 receptor to both transcriptional and RNA-targeted mechanisms mediated by the three pathways.

The NF-kappa b repressing factor is involved in basal repression and interleukin (IL)-1-induced activation of IL-8 transcription by binding to a conserved NF-kappa b-flanking sequence element.

Interleukin (IL)-8, a prototypic chemokine, is rapidly induced by the pro-inflammatory cytokine IL-1 but is barely detectable in noninduced cells. Although there is clear evidence that the transcription factor NF-kappaB plays a central role in inducible IL-8 transcription, very little is known about the cis-elements and trans-acting factors involved in silencing of the IL-8 promoter. By sequence comparison with the interferon-beta promoter, we found a negative regulatory element (NRE) in the IL-8 promoter overlapping partially with the NF-kappaB response element. Here we show that an NF-kappaB-repressing factor (NRF) binds to the IL-8 promoter NF-kappaB-NRE. Reduction of cellular NRF by expressing NRF antisense RNA results in spontaneous IL-8 gene expression. In contrast, IL-1-induced IL-8 secretion is strongly impaired by expressing NRF antisense RNA. Mutation of the NRE site results in loss of NRF binding and increased basal IL-8 transcription. On the other hand IL-1-induced IL-8 transcription is decreased by mutating the NRE. These data provide evidence for a dual role of the NRF in IL-8 transcription. Although in the absence of stimulation it is involved in transcriptional silencing, in IL-1-induced cells it is required for full induction of the IL-8 promoter.