Kawamoto Hiroko, Takeno Sachio, Yajin Koji
Department of Otolaryngology Head and Neck Surgery, Hiroshima Prefectural Hospital, Hiroshima.
Nihon Jibiinkoka Gakkai Kaiho. 2002 Feb;105(2):158-65. doi: 10.3950/jibiinkoka.105.158.
Interest in the physiological, pathological and therapeutic implications of nitric oxide (NO) have grown exponentially, with human nasal cavity and paranasal sinuses considered a dominant source of NO, indicating that this molecule possesses the diversity of biological effects in the regulation of airway clearance and nonspecific cellular immunity. We previously observed differences in NO synthase (NOS) isoform constitutively expressed in nasal epithelial cells (NECs) from allergic and normal subjects.
We extended the previous work to determine whether in vitro stimulation with proinflammatory cytokines influences levels of different NOS isoform expression.
Nasal epithelial cells were sampled from the inferior turbinate in a group of 16 healthy normal controls and 11 patients with perennial allergic rhinitis against house dust (HD) mite antigen. 1 x 10(5) cells were incubated in conditioned medium for 24 hours. Human recombinant interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), or both (cytomix) were added to a final concentration of 10 ng/ml. Cells were then fixed with 4% paraformaldehyde and processed for fluorescence immunocytochemistry. Immunoreactivity for 2 NOS isoforms, inducible NOS (iNOS) and endothelial NOS (eNOS), was studied by laser scanning confocal microscopy and fluorescence intensity was assessed quantitatively.
We observed constitutive eNOS expression in epithelial cells of all subjects. Different treatments with cytokines did not affect eNOS expression. Cytokine treatment, however, significantly augmented iNOS expression in the control group. The average increase induced with IFN-gamma, TNF-alpha, and cytomix was 1.8, 2.33, and 2.31-fold. Nasal epithelial cells in the HD group showed elevated steady-state iNOS expression even in untreated. Cytokine treatment did not affect the degree of iNOS expression in this group.
These results confirm our previous findings that nasal epithelial cells in patients with allergic rhinitis produce higher levels of NO through the concomitant expression of different NOS isoforms. We also demonstrated that nasal epithelial cells have the potential to express iNOS protein spontaneously or upon stimulation with inflammatory cytokines, such as IFN-gamma and TNF-alpha. Because the high level of exhaled NO is considered a potential marker of allergic airway inflammation, preserving the iNOS gene from its unregulated induction may be important for maintenance of nasal homeostasis and may offer a tool for therapeutic intervention.
一氧化氮（NO）在生理、病理及治疗方面的意义呈指数级增长，人类鼻腔及鼻窦被认为是NO的主要来源，这表明该分子在调节气道清除及非特异性细胞免疫方面具有多种生物学效应。我们之前观察到变应性和正常受试者的鼻上皮细胞（NECs）中组成性表达的一氧化氮合酶（NOS）同工型存在差异。
我们扩展了之前的研究工作，以确定促炎细胞因子的体外刺激是否会影响不同NOS同工型的表达水平。
从16名健康正常对照者和11名对屋尘螨抗原患有常年性变应性鼻炎的患者的下鼻甲采集鼻上皮细胞。将1×10⁵个细胞在条件培养基中孵育24小时。加入人重组干扰素-γ（IFN-γ）、肿瘤坏死因子-α（TNF-α）或两者（细胞因子混合物），终浓度为10 ng/ml。然后用4%多聚甲醛固定细胞，并进行荧光免疫细胞化学处理。通过激光扫描共聚焦显微镜研究两种NOS同工型，即诱导型NOS（iNOS）和内皮型NOS（eNOS）的免疫反应性，并定量评估荧光强度。
我们观察到所有受试者的上皮细胞中均有组成性eNOS表达。细胞因子的不同处理并未影响eNOS表达。然而，细胞因子处理显著增加了对照组中iNOS的表达。IFN-γ、TNF-α和细胞因子混合物诱导的平均增加倍数分别为1.8、2.33和2.31倍。屋尘螨组的鼻上皮细胞即使在未处理时也显示出稳态iNOS表达升高。细胞因子处理并未影响该组中iNOS的表达程度。
这些结果证实了我们之前的发现，即变应性鼻炎患者的鼻上皮细胞通过不同NOS同工型的伴随表达产生更高水平的NO。我们还证明鼻上皮细胞有自发表达iNOS蛋白或在受到炎症细胞因子如IFN-γ和TNF-α刺激时表达iNOS蛋白的潜力。由于呼出NO水平升高被认为是变应性气道炎症的一个潜在标志物，防止iNOS基因的失控诱导对于维持鼻腔内环境稳定可能很重要，并且可能为治疗干预提供一种手段。
