Nitric oxide (NO) possesses antiinflammatory effects, which may be exerted via its ability to inhibit the transcription factor, NF-B. A commonly proposed mode of action for inhibition of NF-B by NO involves interference with NF-B binding to DNA. Because activation of inhibitory B kinase (IKK), the prerequisite enzyme complex necessary to induce NF-B, is subject to redox regulation, we assessed whether IKK could present a more proximal target for NO to inhibit NF-B activation. We demonstrate here that S-nitrosothiols (SNO) caused a dose-dependent inhibition of the enzymatic activity of IKK, in lung epithelial cells and in Jurkat T cells, which was associated with S-nitrosylation of the IKK complex. Using biotin derivatization of SNO, we revealed that IKK␤, the catalytic subunit required for NF-B activation, was a direct target for S-nitrosylation. A mutant version of IKK␤ containing a Cys-179-toAla mutation was refractory to inhibition by SNO or to increases in S-nitrosylation, in contrast to wild-type IKK␤, demonstrating that Cys-179 is the main target for attack by SNO. Importantly, inhibition of NO synthase activity in Jurkat T cells resulted in activation of IKK, in association with its denitrosylation. Moreover, NO synthase inhibition enhanced the ability of tumor necrosis factor ␣ to activate IKK, illustrating the importance of endogenous NO in regulating the extent of NF-B activation by cytokines. Collectively, our findings demonstrate that IKK␤ is an important target for the redox regulation of NF-B by endogenous or exogenous NO, providing an additional mechanism for its antiinflammatory properties.

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The effect of formoterol, alone and in combination with budesonide, upon tumour necrosis factor-a stimulated (10 ng?mL -1 ) human bronchial epithelial cells was investigated.Addition of formoterol (¢10 -10 M) reduced granulocyte macrophage-colony stimulating factor (GM-CSF) levels, as assessed by enzyme-linked immunosorbent assay, by 40 -50% and increased interleukin (IL)-8 levels by y50%. The effects of formoterol were long lasting (23 h). Budesonide (10 -8 M) reduced the amounts of both cytokines (GM-CSF and IL-8) by 40%. Simultaneous addition of formoterol and budesonide reduced GM-CSF levels y75%, while IL-8 levels were decreased y40%, similar to the reduction obtained with budesonide alone. The glucocorticoid receptor (GR) antagonist RU486 did not influence the effect of formoterol, suggesting no involvement of the GR. Formoterol rapidly induced an elevation in intracellular cyclic adenosine monophosphate, which was reduced in the presence of propranolol. In addition, the alterations in cytokine secretion induced by formoterol could be fully blocked by propranolol, demonstrating that these effects are b 2 -receptor mediated.In conclusion, the combination of budesonide and formoterol reduces the secretion of granulocyte macrophage-colony stimulating factor to basal levels and counteracts the capacity of formoterol alone to induce interleukin-8 production, modulations which may facilitate improved asthma control.