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

一氧化氮合酶(NOS)亚型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，我们发现常年性变应性鼻炎(AR)患者鼻上皮细胞中一氧化氮合酶(NOS)亚型的表达存在差异，AR患者鼻黏膜中诱导型一氧化氮合酶(INOS)的表达水平明显升高。然而，内皮型一氧化氮合酶(ENOS)的表达水平在两组之间是相同的。我们还利用一种新的荧光指示剂DAF-2-DA对活细胞中直接产生的NO进行了可视化和定量。结果表明，AR患者的上皮细胞产生了更多的NO，预先加入一氧化氮合酶抑制剂(L-NAME或EIT)可不同程度地减少NO的产生。(2)我们观察了促炎细胞因子体外刺激是否会影响不同亚型一氧化氮合酶的表达水平。细胞因子治疗(肿瘤坏死因子-α、干扰素-γ或白介素1-β…此外，地塞米松显著抑制iNOS在鼻刷状细胞中的表达。我们推测转录因子-核因子-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NO在纤毛上皮细胞纤毛活动中的作用。TNE-α和干扰素-γ在浓度为10 ng/ml时均以时间依赖的方式降低纤毛搏动频率(CBF)，并伴随iNOS免疫反应增强。变应性鼻炎和慢性鼻窦炎中促炎症和趋化细胞因子的表达：(1)我们用RT-PCR方法半定量分析了GM-CSF、IL-1β、IL-6、IL-8、RANTES和嗜酸性粒细胞趋化因子在变应性鼻炎和慢性鼻窦炎患者鼻和鼻窦黏膜中的表达。鼻窦炎患者鼻窦组织中GM-CSF和IL-8的表达明显增加。(2)采用原代培养的人副鼻窦黏膜组织块生长模型，探讨鼻窦炎患者鼻窦黏膜中细胞因子表达与核因子-kB活性的关系。我们发现，核因子-kB亚单位p50培养细胞的激活是通过转录途径启动GM-CSF、IL-6和IL-8的表达。进一步的研究需要评估各种iNOS抑制剂对鼻部过敏反应的治疗效果，以及其他分子方法，如使用反义寡核苷酸。较少

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