No abstract

Cystic fibrosis (CF) is an inherited disorder associated with severe inflammation and repeated bacterial infection and colonization in the lung. Airway epithelium is involved in defence against bacteria, but this system may be defective in CF. Pro‐inflammatory cytokines can stimulate the expression of inducible nitric oxide synthase (iNOS), an enzyme generating nitric oxide, which functions as an important mediator in host defence mechanisms. To understand better the poor resistance to infections in the CF lung, the expression of the iNOS gene was investigated in explanted lungs from patients with cystic fibrosis (n‐13), bronchiectasis (n‐3), emphysema (n‐14), and in normal lungs (n‐8). In addition, bronchial epithelial cell lines were examined to study iNOS gene expression in vitro. Strong immunoreactivity for iNOS was seen in inflammatory cells and bronchial epithelium in all the diseased lungs, except for bronchial epithelium in CF. Quantitative analysis showed a significant reduction in the area of epithelium immunostained in CF [CF 6·8±1·6 (%±SEM); emphysema 18·2±2·8; normal 9·6±0·8, P<0·01], regardless of steroid treatment. These results were supported by in situ hybridization of iNOS mRNA, which showed a pattern of gene expression in CF, emphysema, and normal lung which paralleled that of protein immunoreactivity. Stimulation with cytokines (IL‐1β, TNF‐α, and IFN‐γ) increased the expression of iNOS mRNA detected by reverse transcriptase‐polymerase chain reaction (RT‐PCR) in cultures of normal (16HBE14o−), but not CF (CFBE41o−, with ΔF508 CFTR mutation) epithelial cells. Expression of iNOS in inflammatory cells suggests that the gene is normal in CF. Absence of iNOS from bronchial epithelium may be due to low expression of the gene resulting from abnormalities in the signalling system that normally causes induction, such as cytokine receptors, second messengers or transcription factors. The resulting deficiency of the nitric oxide defence system may be relevant to the susceptibility of CF patients to pulmonary bacterial colonization. © 1998 John Wiley & Sons, Ltd.

The production of inducible nitric oxide synthase (iNOS) within vascular smooth muscle (VSM) cells following exposure to proinflammatory cytokines is a major cause of the vasorelaxation and hypotension of septic shock. We have defined the cytokine-responsive element of the murine iNOS promoter, transfected into a VSM cell line, and the role of the NF-kappa B/Rel family of proteins in iNOS gene activation in these cells. The combination of interleukin-1, interferon-gamma, and tumor necrosis factor-alpha stimulates promoter activity by a factor of 8.1-fold; single cytokines show little activity, while pairs of cytokines produce an intermediate effect. Using a series of promoter deletion mutants, we have defined the cytokine-responsive element from position -890 to -1002; this region contains an NF-kappa B-binding site as well as a number of interferon response elements. Nuclear proteins from cytokine-stimulated VSM cells which bind to an oligonucleotide containing this kappa B site are composed of p65 together with an unidentified protein of 50 kDa, which is not a known Rel family member. A promoter mutant with a 2-base pair change within this kappa B site, which abolishes NF-kappa B binding, has an activity of only approximately 34% (S.E. +/- 1.5) of the wild-type promoter. In addition, protein binding to this site is abolished by a specific inhibitor of NF-kappa B activation, which also abrogates iNOS activity. Residual inducibility in such mutant promoters is attributable to the presence of an independently functioning downstream kappa B site (-85 to -75). The mechanism by which NF-kappa B activates the iNOS promoter in VSM cells in response to cytokines appears to be markedly different to that operative in macrophages in response to lipopolysaccharide.