Cooperative and redundant signaling of leukotriene B4 and leukotriene D4 in human monocytes.

BACKGROUND: Leukotriene B(4) (LTB(4)) and cysteinyl leukotrienes (cysLTs) are important immune mediators, often found concomitantly at sites of inflammation. Although some of the leukotriene-mediated actions are distinctive (e.g., bronchial constriction for cysLTs), many activities such as leukocyte recruitment to tissues and amplification of inflammatory responses are shared by both classes of leukotrienes. OBJECTIVE: We used human monocytes to characterize leukotriene-specific signaling, gene expression signatures, and functions and to identify interactions between LTB(4)- and cysLTs-induced pathways. METHODS: Responsiveness to leukotrienes was assessed using oligonucleotide microarrays, real-time PCR, calcium mobilization, kinase activation, and chemotaxis assays. RESULTS: Human monocytes were found to express mRNA for high- and low-affinity LTB(4) receptors, BLT(1) and BLT(2), but signal predominantly through BLT(1) in response to LTB(4) stimulation as shown using selective agonists, inhibitors, and gene knock down experiments. LTB(4) acting through BLT(1) coupled to G-protein α inhibitory subunit activated calcium signaling, p44/42 mitogen-activated protein kinase, gene expression, and chemotaxis. Twenty-seven genes, including immediate early genes (IEG), transcription factors, cytokines, and membrane receptors were significantly up-regulated by LTB(4). LTB(4) and LTD(4) had similar effects on signaling, gene expression, and chemotaxis indicating redundant cell activation pathways but costimulation with both lipid mediators was additive for many monocyte functions. CONCLUSION: Leukotriene B(4) and LTD(4) display both redundant and cooperative effects on intracellular signaling, gene expression, and chemotaxis in human monocytes. These findings suggest that therapies targeting either leukotriene alone may be less effective than approaches directed at both.

p11 expression in human bronchial epithelial cells is increased by nitric oxide in a cGMP-dependent pathway involving protein kinase G activation.

The effect of nitric oxide on p11 expression was studied in an immortalized human bronchial epithelial cell line (BEAS-2B cells). Three nitric oxide donors were used: spermine NONOate (SP), (+/-)-S-nitroso-N-acetylpenicillamine (SNAP), and S-nitrosoglutathione (SNOG). All three nitric oxide donors had similar effects resulting in dose-dependent and time-dependent accumulation of p11 protein and an increase of steady-state p11 mRNA. Studies using a reporter gene containing the region from -1499 to +89 of the p11 promoter demonstrated an increase in transcriptional activity after stimulation with NO donors for 4 h. These effects were abolished at the promoter and protein level using protein kinase G inhibitors (KT5823 and R(p)-8-pCPT-cGMPS). Incubation of transfected cells with a cell permeable cGMP analogue (8-Br-cGMP) resulted in a dose-related increase of promoter activity. An electrophoretic mobility shift assay of nuclear proteins extracted from BEAS-2B cells identified an AP-1 site located at -82 to -77 of the p11 promoter region as an NO- and cGMP- dependent response element. These data were confirmed using a c-jun dominant negative mutant vector and a c-jun expression plasmid. Therefore, we conclude that nitric oxide-induced p11 expression in human bronchial epithelial cells is mediated at least in part through increased binding of activator protein one to the p11 promoter.