Ishida H, Yang G, Harada N, Hastings R L, Castle B E, Kastelein R, Miyajima A, Howard M
DNAX Research Institute of Molecular and Cellular Biology, Incorporated, Palo Alto, California 94304.
Cell Immunol. 1991 Aug;136(1):142-54. doi: 10.1016/0008-8749(91)90389-s.
Anti-receptor antibodies have previously been used in two cytokine systems (IL-1 and TNF alpha) to identify the existence of different cytokine receptors on different cell types. In this study, we have similarly used two approaches to evaluate whether IL-4 receptors on different cell types are identical, or whether more than one species of IL-4 receptor exists. The first approach involved production of monoclonal antibodies specific for the IL-4 receptor expressed by the murine mast cell line, MC/9. Six anti-IL-4 receptor monoclonal antibodies were produced against the purified soluble extracellular domain of the recombinant IL-4 receptor derived from MC/9 cells. These antibodies were capable of binding to and specifically immunoprecipitating the soluble extracellular domain of the recombinant mast cell IL-4 receptor. Following biotinylation of the antibodies and addition of phycoerythrin-streptavidin, their binding to cell associated IL-4 receptors on MC/9 mast cells could be readily visualized by immunofluorescence. Using this approach, the anti-mast cell IL-4R antibodies were found to specifically bind IL-4 receptors expressed on a variety of other murine cell types, including T cells, B cells, macrophages, fibroblasts, and L cells. The antibodies did not bind to two human cell lines known to bind human but not murine IL-4. The intensity of staining was directly related to the number of IL-4 binding sites identified previously by receptor-ligand equilibrium binding analyses. As a second approach to evaluating potential receptor heterogeneity, we constructed S1 nuclease protection assay probes for two separate regions of the mast cell IL-4 receptor, one located in the extracellular domain and one in the intracellular domain. Subsequent S1 analyses showed that both regions are expressed by the following types of cells: T cells, B cells, macrophages, myeloid cells, L cells, and stromal cells. The two approaches used in this study therefore indicate that the same or highly similar IL-4 receptor species is expressed by a wide variety of hemopoietic and nonhemopoietic cells. Since the anti-IL-4 receptor antibodies produced in this study did not block binding of IL-4 to its receptor, we cannot exclude the possible existence of a second type of IL-4R coexpressed on the cells tested in this study, or expressed uniquely by other cell types that were not investigated.
抗受体抗体先前已用于两种细胞因子系统（白细胞介素-1和肿瘤坏死因子α），以确定不同细胞类型上不同细胞因子受体的存在。在本研究中，我们同样采用了两种方法来评估不同细胞类型上的白细胞介素-4受体是否相同，或者是否存在不止一种类型的白细胞介素-4受体。第一种方法涉及制备针对小鼠肥大细胞系MC/9表达的白细胞介素-4受体的单克隆抗体。针对源自MC/9细胞的重组白细胞介素-4受体的纯化可溶性细胞外结构域制备了六种抗白细胞介素-4受体单克隆抗体。这些抗体能够结合并特异性免疫沉淀重组肥大细胞白细胞介素-4受体的可溶性细胞外结构域。在抗体进行生物素化并添加藻红蛋白-链霉亲和素后，它们与MC/9肥大细胞上细胞相关白细胞介素-4受体的结合可通过免疫荧光轻松观察到。使用这种方法，发现抗肥大细胞白细胞介素-4R抗体特异性结合多种其他小鼠细胞类型上表达的白细胞介素-4受体，包括T细胞、B细胞、巨噬细胞、成纤维细胞和L细胞。这些抗体不与已知能结合人而非小鼠白细胞介素-4的两种人细胞系结合。染色强度与先前通过受体-配体平衡结合分析确定的白细胞介素-4结合位点数量直接相关。作为评估潜在受体异质性的第二种方法，我们构建了针对肥大细胞白细胞介素-4受体两个不同区域的S1核酸酶保护分析探针，一个位于细胞外结构域，一个位于细胞内结构域。随后的S1分析表明，这两个区域在以下细胞类型中均有表达：T细胞、B细胞、巨噬细胞、髓细胞、L细胞和基质细胞。因此，本研究中使用的两种方法表明，多种造血和非造血细胞表达相同或高度相似的白细胞介素-4受体类型。由于本研究中产生的抗白细胞介素-4受体抗体并未阻断白细胞介素-4与其受体的结合，我们不能排除在本研究测试的细胞上共表达的第二种类型白细胞介素-4受体的可能存在，或者未研究的其他细胞类型独特表达的白细胞介素-4受体的可能存在。
