Studies on identification of environmental substances resulting in human respiratory diseases.

在本研究中，我们将鉴定导致人类呼吸系统疾病的环境物质集中在3个方面：(a)环境物质进入巨噬细胞，(b)巨噬细胞产生细胞因子，(c)细胞因子诱导产生超氧化物。由于很难常规获得人肺泡巨噬细胞，我们用人外周血单核细胞代替。同样，我们用细菌细胞壁骨架代替环境物质。结果表明：(1)嗜中性粒细胞和单核细胞均能与其他细胞分离，纯度均在90%以上。(2)单核细胞吞噬荧光标记的细胞壁骨架呈剂量依赖性。(3)吞噬细胞壁骨架激活的单核细胞产生的细胞因子呈剂量依赖性增加，但细胞因子的峰值时间和量在不同细胞因子之间存在差异。(4)不同细胞因子产生超氧化物的能力也存在差异；GM-CSF刺激超氧化物产生的效果最为显著，G-CSF、tnf - α、IL-6、IL-2和il -1 β的效果仅次于GM-CSF，而il -1 α、il -1 β和ifn - α几乎不受刺激。超氧化物产生与成纤维细胞增殖关系的研究尚未开展，但我们已经建立了体外方法来测量巨噬细胞中环境物质的掺入，巨噬细胞分泌细胞因子以及细胞因子对超氧化物产生的刺激。在不久的将来，我们希望在分化后建立新的白血病细胞系，这些细胞系具有更高的细胞因子和超氧化物的产生能力。这些细胞系可能会使我们的方法更加普遍。

In this study, we concentrated identification of environmental substances resulting in human respiratory diseases on 3 matters : (a) incorporation of environmental substances into macrophages, (b) production of cytokines in the macrophages, and (c) production of superoxide induced by cytokines. Since it was difficult to obtain human alveolar macrophages routinely, we substituted human peripheral blood monocytes. Similarly, we used bacterial cell wall skeletones in stead of environmental substances. Results are as follows : (1) Both neutrophiles and monocytes were able to be separated from other cells in purity of more than 90%. (2) Monocytes phagocytized the fluorescence-labeled cell wall skeletones dose-dependently. (3) Production of cytokines in the monocytes activated by phagocytosis of the cell wall skeletones increased dose-dependently, whereas there was difference in its peak time and amount between cytokines. (4) There was difference also in potency of superoxide production among cytokines; GM-CSF stimulated superoxide production most prominently, G-CSF, TNF-alpha, IL-6, IL-2 and IL-1beta did prominently next to GM-CSF, and any of IL-1alpha, IL-1beta and IFN-alpha scarcely stimulated.The study on relationship between superoxide production and proliferation of fibroblast has not been carried out, we, however, have established in vitro methods to measure incorporation of environmental substances into macrophages, secretion of cytokines from macrophages and stimulation of superoxide production by cytokines.In the near future, we would like to establish new leukemic cell lines with higher potency of the cytokine and superoxide production after their differentiation. These cell lines may be expected to get our method more general.