A Study on the Cytokine Messenger RNA Expression in Bronchial Mucosal Biopsies from Patients with Asthma

为了确定IL-5是否在哮喘患者的支气管粘膜局部产生，并确定产生该细胞因子的确切细胞类型，我们采用非放射性原位杂交和免疫组织化学检查支气管活检。用地高辛- dutp标记IL-5 cDNA探针，并与冷冻组织切片杂交。杂交信号通过免疫组织化学技术可视化。来源于成人T细胞白血病患者的产生il -5的T细胞克隆被用作阳性对照。在8例哮喘患者活检标本中，有6例观察到IL-5 mRNA特异性杂交信号。在未发现潜在疾病的对照组中未检测到杂交信号。使用一组针对淋巴细胞亚群的单克隆抗体对连续切片进行免疫组化染色，强烈表明表达IL-5 mRNA的细胞是T细胞。这些结果提示，T细胞与嗜酸性粒细胞之间通过IL-5的细胞间相互作用可能在哮喘气道炎症中起重要作用。当螨虫抗原刺激时，从螨虫敏感哮喘患者获得的外周血单核细胞（PBMC）释放嗜酸性趋化因子（ECF），这是一种蛋白质。为了鉴定这一因子，我们从受螨抗原刺激和未受螨抗原刺激的PBMC中构建cDNA文库，然后进行减法和鉴别筛选。我们获得了13个因螨抗原而表达增加的基因。其中，9个是线粒体基因，2个是h卫星DNA III， 1个是因子XIII， 1个是铁蛋白重亚基。本实验未获得可能与嗜酸性粒细胞趋化活性相关的基因。然而，由于对两个cDNA文库的聚合酶链反应，IL-5基因只从螨抗原刺激的cDNA文库中扩增出来。

To determine whether IL-5 is locally produced in the bronchial mucosa of asthmatics, and to ascertain the precise cell type producing this cytokine, we examined bronchial biopsies by non-radioactive in situ hybridization and immunohistochemistry. IL-5 cDNA probes were labeled with digoxigenin-dUTP and hybridized to frozen tissue sections. Hybridization signals were visualized by an immunohistochemistry technique. An IL-5-producing T cell clone derived from a patient with adult T cell leukemia was used as a positive control. Specific hybridization signals for IL-5 mRNA were observed in six of the eight biopsy specimens from asthmatics. No hybridization signal was detected in the control subject in whom no underlying disease was found. Immunohistochemistry staining of serial sections using a panel of monoclonal antibodies against lymphocyte subsets strongly suggested that cells expressing mRNA for IL-5 were T cells. These results suggested that a cell-cell inteaction between T cells and eosinohils through IL-5 may play an important role in the airway inflammation in asthma.When stimulated with mite antigens, peripheral blood mononuclear cells (PBMC) obtained from mite-sensitive asthmatics, release eosinophil chemotactic factor(s)(ECF), a type of protein. To characterlize this factor, cDNA libraries were formulated from both PBMC stimulated with mite antigens and those not stimulated with mite antigens, then underwent subtraction and differential screening. We obtained 13 genes whose expression had increased due to mite antigens. Of these, nine were mitochondrial genes, two, h-satellite DNA III,one, factor XIII,and one, ferritin heavy subunit. Gene(s) which might be related to eosinophil chemotactic activity were not obtained in the present experiments. However, as a result of polymerase chain reaction on both cDNA libraries, IL-5 genes were amplified only from the cDNA library which had been stimulated with mite antigens.