Iijima H, Duguet A, Eum S Y, Hamid Q, Eidelman D H
Meakins-Christie Laboratories, Respiratory Division, Department of Medicine, McGill University Health Centre, Montréal, Québec, Canada.
Am J Respir Crit Care Med. 2001 Apr;163(5):1233-40. doi: 10.1164/ajrccm.163.5.2003145.
To explore the possible role of eosinophils in NO-mediated tissue injury, we studied a murine model of allergic asthma. Male A/J mice were sensitized and challenged intranasally with ovalbumin (OVA). Following challenge, the number of eosinophils in bronchoalveolar lavage fluid (BALF) increased from 0.4% of total cells at baseline (0.02 x 10(4) cells/ml) to 60.2% at 48 h after the challenge (9.34 x 10(4) cells/ml). The rise in eosinophil count was accompanied by a 40.3% increase in total NO(2-) plus NO(3-) (NO(x)) in BALF. This in turn was accompanied by expression of inducible NO synthase (NOS II) in airway epithelial and inflammatory cells, as well as by evidence of staining for 3-nitrotyrosine (3NT) in peribronchial inflammatory cells and at the epithelial surface. Both NO(x) production and 3NT were significantly reduced by pretreatment of the challenged mice with the highly specific NOS II inhibitor N-3-aminomethyl-benzyl-acetamidine-dihydrochloride (1400W), as well as by the nonselective NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME). L-NAME and 1400W also reduced the number of BALF eosinophils (37.2% and 61.5%, respectively, as compared with the control value), suggesting that NO production by NOS II contributes to eosinophil recruitment. To further examine the role of eosinophils, we pretreated additional mice with an anti-interleukin (IL)-5 antibody, which reduced BALF eosinophilia following OVA challenge by 90.1%. In concert with the decrease in eosinophils, the anti-IL-5 antibody reduced NO(x) in BALF almost to the baseline value, and decreased the number of 3NT-positive cells in the peribronchial region by 74.4%. Western blot analysis of protein extracted from whole lung confirmed the reduction in tyrosine nitration by anti-IL-5 antibody. These findings indicate that NO and eosinophilic inflammation are closely coupled, and suggest that eosinophils are an important source of tyrosine nitration.
为了探究嗜酸性粒细胞在一氧化氮（NO）介导的组织损伤中可能发挥的作用，我们研究了一种过敏性哮喘的小鼠模型。雄性A/J小鼠经卵清蛋白（OVA）鼻内致敏和激发。激发后，支气管肺泡灌洗液（BALF）中嗜酸性粒细胞的数量从基线时占总细胞数的0.4%（0.02×10⁴个细胞/ml）增加到激发后48小时的60.2%（9.34×10⁴个细胞/ml）。嗜酸性粒细胞计数的增加伴随着BALF中总NO₂⁻加NO₃⁻（NOₓ）增加40.3%。这反过来又伴随着气道上皮和炎症细胞中诱导型一氧化氮合酶（NOS II）的表达，以及支气管周围炎症细胞和上皮表面3-硝基酪氨酸（3NT）染色的证据。用高度特异性的NOS II抑制剂N-3-氨基甲基-苄基-乙脒二盐酸盐（1400W）以及非选择性NOS抑制剂Nⁿ-硝基-L-精氨酸甲酯（L-NAME）对激发的小鼠进行预处理，均可显著降低NOₓ的产生和3NT的水平。L-NAME和1400W还减少了BALF中嗜酸性粒细胞的数量（分别比对照值减少37.2%和61.5%），表明NOS II产生的NO有助于嗜酸性粒细胞的募集。为了进一步研究嗜酸性粒细胞的作用，我们用抗白细胞介素（IL）-5抗体对另外一些小鼠进行预处理，该抗体使OVA激发后BALF中的嗜酸性粒细胞增多减少了90.1%。与嗜酸性粒细胞的减少相一致，抗IL-5抗体使BALF中的NOₓ几乎降至基线值，并使支气管周围区域3NT阳性细胞的数量减少了74.4%。对全肺提取的蛋白质进行的蛋白质印迹分析证实了抗IL-5抗体可减少酪氨酸硝化作用。这些发现表明NO与嗜酸性粒细胞炎症密切相关，并提示嗜酸性粒细胞是酪氨酸硝化作用的重要来源。
