Functional analysis of nitric oxide and nitric oxide synthase expression in allergic rhinitis.

一氧化氮合酶（NOS）异构体在变应性鼻炎患者中的表达：(1)应用免疫细胞化学和RT-PCR方法检测了正常人和常年性变应性鼻炎（AR）患者鼻上皮细胞中NOS异构体的表达差异。AR患者诱导型NOS （iNOS）表达水平明显升高。内皮细胞NOS （eNOS）表达水平在两组间无明显差异。我们还通过使用一种新的荧光指示剂DAF-2 DA，对活细胞中直接NO的产生进行了可视化和量化。结果表明，AR患者上皮细胞产生大量NO。用NOS抑制剂（L-NAME或EIT）预孵育可不同程度地减少NO的产生。(2)体外促炎细胞因子刺激是否会影响不同NOS亚型的表达水平。细胞因子处理（tnf - α、ifn - γ或il -1 β等）显著增加了刷鼻细胞中iNOS的表达，而地塞米松显著抑制了iNOS的表达。我们推测转录因子核因子κ B （NF-kB）的激活可能与转录调控机制密切相关。(3)我们检测了内源性一氧化氮对上皮纤毛细胞纤毛活性的影响。10 ng/ml浓度的tne - α和ifn - γ均以时间依赖性方式降低睫状搏动频率（CBF），同时增加iNOS免疫反应性。我们推测，当细胞内iNOS表达强烈增强时，NO对培养纤毛细胞的CBF有不同的调节作用。变应性鼻炎和慢性鼻窦炎促炎因子和趋化因子的表达：(1)采用RT-PCR半定量分析GM-CSF、il -1 β、IL-6、IL-8、RANTES和eotaxin编码mrna在鼻和鼻窦黏膜中的表达。分析显示，过敏患者GM-CSF、IL-8、RANTES和eotaxin表达显著升高。鼻窦炎患者GM-CSF和IL-8表达明显升高。(2)采用人鼻窦炎粘膜原代外植体培养模型，研究细胞因子的体外表达与NF-kB活性的关系。我们发现NF-kB亚基p50的激活通过启动转录途径负责GM-CSF， IL-6和IL-8的表达。进一步的研究需要评估各种iNOS抑制剂对鼻腔过敏反应的治疗效果，并研究其他分子方法，如反义寡核苷酸的使用。少

Expression of nitric oxide synthase (NOS) isoforms in patients with allergic rhintis :(1) We found the difference in NOS isoform expression in human nasal epithelial cells between normal subjects and patients with perennial allergic rhinitis (AR) by using immunocytochemistry and RT-PCR.AR patients showed significant increases in the levels of inducible NOS (iNOS) expression. However, the levels of endothelial NOS (eNOS) expression were identical between the groups. We also performed visualization and quantification of direct NO production in living cells by using a novel fluorescent indicator, DAF-2 DA.The results indicated that epithelial cells in AR patients produced larger amount of NO.Preincubation with NOS inhibitors (L-NAME or EIT) resulted in decrease in NO production to various degrees.(2) We examined whether in vitro stimulation with proinflammatory cytokines may influence the levels of different NOS isoform expression. The cytokine treatment (TNF-alpha, IFN-gamma, or IL-1beta … More ) significantly augmented iNOS expression in the nasal brushing cells, and dexamethazone significantly suppressed it. We presumed that the activation of transcription factor, nuclear factor-kappa B (NF-kB), might be intimately involved in the transcriptional regulatory mechanisms.(3) We examined the function of endogenously generated NO in the ciliary activity of the epithelial ciliated cells. Both TNE-alpha and IFN-gamma at a concentration of 10 ng/ml decreased ciliary beat frequency (CBF) in a time dependent manner with concomitant increase in iNOS immunoreactivity. We presume that NO modulates CBF in cultured ciliated cells differently under conditions when iNOS expression is strongly augmented inside the cells.Expression of proinflammalory and chemoattractive cytokines in allergic rhintis and chronic sinusitis :(1) We semiquantitatively analyzed the expression of mRNAs encoding GM-CSF, IL-1beta, IL-6, IL-8, RANTES, and eotaxin in nasal and paranasal sinus mucosa by RT-PCR.The analysis revealed that a significant increase in GM-CSF, IL-8, RANTES, and eotaxin expression in allergic patients. A significant increase in GM-CSF and IL-8 expression was observed in sinusitis patients.(2) We employed a primary explant-outgrowth culture model of human paranasal sinus mucosa in order to relate in vitro expression of the cytokines with the NF-kB activity. We found that the activation of NF-kB subunit p50 cultured cells was responsible for the expression of GM-CSF, IL-6, and IL-8 through the initiation of the transcriprional pathway.Further studies need to assesst therapeutic effects of various iNOS inhibitors on allergic responses in the nose as well as to examine other molecular approaches, such as the use of antisense oligonucleotides. Less