Mita S, Harada N, Naomi S, Hitoshi Y, Sakamoto K, Akagi M, Tominaga A, Takatsu K
Department of Biology, Kumamoto University Medical School, Japan.
J Exp Med. 1988 Sep 1;168(3):863-78. doi: 10.1084/jem.168.3.863.
T cell-replacing factor (TRF)/IL-5 is a glycosylated polypeptide that acts as a key factor for B cell growth and differentiation. Since IL-5 action is probably mediated by specific cell surface receptor(s), we have characterized the binding of IL-5 to cells using biosynthetically [35S]methionine-labeled IL-5 and 125I-IL-5 that had been prepared using Bolton-Hunter reagent. The radiolabeled IL-5 binds specifically to BCL1-B20 (in vitro line) (a murine chronic B cell leukemic cell line previously shown to differentiate into IgM-secreting cells in response to IL-5) within 10 min at 37 degrees C. There are two classes of binding sites with high affinity (Kd = 66 pM) and low affinity (Kd = 12 nM) for IL-5 and an average number of binding sites for high affinity and for low affinity were 400 and 7,500 per cell, respectively. The specificity of binding of radiolabeled IL-5 has been confirmed by demonstrating that only unlabeled IL-5 and anti-IL-5 mAb but not by IL-1, IL-2, IL-3, IFN-gamma, and GM-CSF inhibit radiolabeled IL-5 binding to BCL1-B20 cells. Treatment of surface-bound radiolabeled IL-5 with bivalent crosslinkers identified a membrane polypeptide of Mr 46,500 to which IL-5 is crosslinked. A variety of cell types have been surveyed for the capacity to bind specifically radiolabeled IL-5 with high affinity. BCL1 cells MOPC104E (murine myeloma cell line) expressed IL-5-R, whereas BAL. 17 and L10 A (B cell lymphoma) did not. T cell line, mastocytoma cell line, or macrophage tumor cell line did not display detectable levels of IL-5-R. were hardly detectable on normal resting B cells but were expressed on LPS-activated B cells, fitting the function of IL-5 that acts on activated B cells for their differentiation into Ig-secreting cells. Intriguingly, early B cell lines (J-87 and T-88) that grow in the presence of IL-5 expressed significant but low numbers of high-affinity binding sites for IL-5. The biological effects of IL-5 were mediated by high-affinity binding sites. The identification and characterization of IL-5-R should provide new insight into the apparent diverse biological activities of IL-5.
T细胞替代因子（TRF）/白细胞介素-5（IL-5）是一种糖基化多肽，是B细胞生长和分化的关键因子。由于IL-5的作用可能由特定的细胞表面受体介导，我们使用生物合成的[35S]甲硫氨酸标记的IL-5和用博尔顿-亨特试剂制备的125I-IL-5来表征IL-5与细胞的结合。放射性标记的IL-5在37℃下10分钟内特异性结合到BCL1-B20（体外细胞系）（一种小鼠慢性B细胞白血病细胞系，先前已证明其可响应IL-5分化为分泌IgM的细胞）。对于IL-5存在两类结合位点，高亲和力（Kd = 66 pM）和低亲和力（Kd = 12 nM），高亲和力和低亲和力结合位点的平均数量分别为每个细胞400个和7500个。通过证明只有未标记的IL-5和抗IL-5单克隆抗体能抑制放射性标记的IL-5与BCL1-B20细胞的结合，而IL-1、IL-2、IL-3、干扰素-γ和粒细胞-巨噬细胞集落刺激因子不能抑制，从而证实了放射性标记的IL-5结合的特异性。用二价交联剂处理表面结合的放射性标记的IL-5，鉴定出一种Mr为46,500的膜多肽，IL-5与之交联。已对多种细胞类型进行了检测，以确定其特异性结合高亲和力放射性标记的IL-5的能力。BCL1细胞MOPC104E（小鼠骨髓瘤细胞系）表达IL-5受体，而BAL.17和L10 A（B细胞淋巴瘤）不表达。T细胞系、肥大细胞瘤细胞系或巨噬细胞瘤细胞系未显示可检测水平的IL-5受体。在正常静止B细胞上几乎检测不到，但在LPS激活的B细胞上表达，这符合IL-5作用于激活的B细胞使其分化为分泌Ig的细胞的功能。有趣的是，在IL-5存在下生长的早期B细胞系（J-87和T-88）表达了大量但数量较少的IL-5高亲和力结合位点。IL-5的生物学效应由高亲和力结合位点介导。IL-5受体的鉴定和表征应为深入了解IL-5明显多样的生物学活性提供新的见解。
