Study on the assessment of industrial respiratory allergens using the analysis of cytokine

为建立工业化学品致敏小鼠呼吸道过敏性的评价方法，本文对化学品致敏的适宜方法和呼吸道过敏指标进行了研究。首先，采用甲苯二异氰酸酯（TDI）鼻内吸入致敏小鼠，并以TDI吸入激发，同时采用皮肤吸入致敏小鼠。采用双抗体夹心ELISA法检测支气管肺泡灌洗液（BALF）及脾细胞PHA培养上清中细胞因子的分泌模式，并检测血清总IgE抗体。在连续4天两次吸入TDI致敏的小鼠中显示总IgE抗体显著升高，暴露后休息6天，最后一次致敏暴露后7天。同时，致敏组PHA刺激脾细胞产生IL-4和GM-CSF的水平显著升高。而IFN-γ的产生水平在攻击后24和48 h与对照组相比升高。表皮途径致敏的小鼠也显示出类似的细胞因子分泌模式。BALF中细胞因子分泌模式的评估受到对照组细胞因子水平显著升高的干扰。这些结果表明，通过鼻内或表皮途径致敏以及在首次致敏暴露后约20天通过鼻内途径激发是有用的。用ELISA夹心法检测PHA刺激的脾细胞IL-4、GM-CSF和IFN-γ的分泌模式，可作为评价化学物质致呼吸道变态反应的指标。

To develop the method for assessment of respiratory sensitivity of industrial chemicals in mice, we have examined the proper methods to induce sensitization with chemicals and the indices of respiratory allergy. Firstly, mice were sensitized with inhaled toluene diisocyanate (TDI) by intranasal route for several schedules of exposure and challenged with inhaled TDI.In addition to this method, mice were sensitized by epicutaneous route and challenged by inhalation. The analysis of cytokine secretion patterns in bronchoalveolar lavage fluid (BALF) and the supernatants in which splenocytes were cultured with PHA, and the measurement of serum total IgE antibody were carried out using sandwich ELISA.Consequently, the significant elevation of total IgE antibody was shown in mice which were sensitized with inhaled TDI in the twice of 4 consecutive days-exposure which has 6 days of rest and 7 days after last sensitization exposure. At the same time, the significant elevation of IL-4 and GM-CSF production levels in PHA-stimulated splenocytes were shown in the sensitized groups. While IFN-gamma production level elevated with the control groups 24 and 48 h after challenge. The similar patterns of cytokine secretion were also shown in mice sensitized by the epicutaneous route. Assessment of cytokine secretion patterns in BALF was disturbed by the significant elevation of cytokine levels in the control groups. These results indicated that the sensitization by intranasal or epicutaneous route and the challenge by intranasal route about 20 days after the first sensitization exposure was useful. The analysis of cytokine, IL-4, GM-CSF, and IFN-gamma, secretion patterns in PHA-stimulated splenocytes using sandwich ELISA was also suggested to be available for the indices of assessment of chemicals causing respiratory allergy.