Role of IL-5 and Receptor System in the Regulation of the Immune System and Inflammatory Response.

(1)我们报道了IL-5和依赖基质细胞的Ly-l^+早期B细胞系在长期骨髓培养系统中的建立（Tominaga et al.）。生长因子1:135,1989)。在这项研究中，我们获得了另外8个不同的细胞系，这些细胞系显示了IgH基因J区片段的种系构型。表面标记为CD45R^+、Ly-l^+、Lyb-2^+、sIgM^-、Thy-l^-、Mac-l^-。其中J8和J10分别用5-氮杂胞苷处理后，与基质细胞ST2和IL-5共培养，分别成为sIgM^+ B细胞和Mac-l^+巨噬细胞样细胞。其他早期淋巴系与ST2和IL-5共培养超过一年后，它们显示出IgH基因J区片段的异质DNA重排谱。Northern blot分析显示，这些细胞系表达Cu-mRNA和5-mRNA。与正常的前b细胞一致。有趣的是，它们组成性地表达c-fms-mRNA。J13细胞与ST2和GM-CSF共培养，取代ST2和IL-5，获得Mac-1表达，保留Ly-1表达。形态学上为巨噬细胞，非特异性酯酶阳性，并表现出对乳胶珠的吞噬。的加入抑制了转化。IL-5。这些结果支持了髓细胞和Ly-1 + B细胞分化途径之间密切关系的证据，并表明我们的il -5依赖克隆是从前B细胞向B细胞和巨噬细胞分化的多电位中间产物。(2) IL-5参与了B细胞和嗜红细胞的体外生长和分化。为了设想IL-5在体内参与这些细胞发育的可能性，我们培育了携带带有金属硫蛋白启动子的小鼠IL-5基因的转基因小鼠。IL-5转基因小鼠血清中IL-5水平升高，血清IgM和IgA水平升高。外周血和脾脏中可见大量嗜酸性粒细胞，肌肉和肝脏中可见嗜酸性粒细胞浸润。转基因小鼠腹腔注射含镉生理盐水，5天后脾脏出现明显的Ly-l^+ B细胞群。在这些细胞上检测到IL-5受体（il - 5r）。在这些转基因小鼠中另一个有趣的发现是IgM类多反应性抗dna抗体的增加。因此，提示IL-5基因的异常表达可能诱导Ly-l^+ B细胞和嗜酸性粒细胞的聚集。此外，该IL-5转基因小鼠可以作为嗜酸性粒细胞模型小鼠，我们可以利用该小鼠来确定IL-5在Ly-l^+ B细胞和嗜酸性粒细胞分化中的作用。(3)我们通过筛选小鼠il -5依赖性早期B细胞系的文库，分离出了编码小鼠IL-5R的CDNA克隆。在COS7细胞中表达CDNA文库，并利用抗il -5受体单克隆抗体进行筛选。推导出的氨基酸序列分析表明，该受体是一个由415个氨基酸组成的糖蛋白（Mr为45,284），包括一个n端疏水区（17个氨基酸）、一个糖基化的胞外结构域（322个氨基酸）、一个跨膜片段（22个氨基酸）和一个细胞质段（54个氨基酸）。转染CDNA的COS7细胞表达了一个60 kd的蛋白，该蛋白以单一类亲和力（K_D = 2-10 nM）结合IL-5。转染小鼠IL-5R CDNA的FDC-PL细胞表达了低亲和力（K_D=6 nM）和高亲和力（K_D=30 pill）的IL-5结合位点，并获得了对IL-5的增殖反应性，而亲代FDC-PL细胞没有检测到任何IL-5结合。Northern blot分析显示，在显示小鼠IL-5结合位点的细胞系中检测到两种mrna （5.0 kb和5.8 kb）。对IL-5受体氨基酸序列的同源性研究表明，IL-5受体包含一个最近发现的细胞因子受体家族的共同基序。(4)小鼠高亲和IL-5R由至少两条膜多肽链组成；a链为p60， B链为P130/ pl40。我们发现B链在依赖il -3的早期B细胞系中组成性表达。为了评估IL-3R的一个成分是否为高亲和力IL-5R的B链，我们检测了抗il -3受体单抗（anti-Aic-2 mAb）对Y16 DNA合成的影响。在Y16细胞裂解液中免疫沉淀p13O/p14O的Anti-Aic-2 mAb部分抑制IL-5和il -3诱导的Y16细胞增殖，而与IL-5R a链不发生反应。因为据报道抗aic -2 mAb与。重组AIC2A和AIC2B，我们检测AIC2A或AIC2B是否参与高亲和力IL-5R的形成。小鼠IL-5R受体a链在L细胞中与AIC2B表达，而不与AIC2A表达，导致高亲和力的IL-5结合位点。单独表达AIC2B不表现出与IL-5的结合。这些转染物的细胞裂解物与放射性标记的IL-5交联形成a链和B链的复合物。这些结果清楚地表明，小鼠IL-5受体的B链是与a链一起形成高亲和力IL-5结合位点不可或缺的，AIC2B很可能是小鼠IL-5受体的B链。发现B链与GM-CSF受体B链相同。少

(1) We reported the establishment of IL-5 and stromal cell-dependent Ly-l^+ early B cell line in long-term bone marrow culture system (Tominaga et al. Growth Factors 1 : 135, 1989). In this study, we obtained another 8 different cell lines that showed germ line configuration of the J region segments of the IgH genes. Their surface markers were CD45R^+, Ly-l^+, Lyb-2^+, sIgM^-, Thy-l^-, and Mac-l^-. When we treated two of them (J8 and J10) with 5-azacytidine followed by coculture with stromal cells (ST2) and IL-5, they became sIgM^+ B cells and Mac-l^+ macrophage-like cells, respectively. After other early lymphold lines were maintained by coculture with ST2 and IL-5 for more than a year, they showed a heterogeneous DNA rearrangement profile of the J region segment of the IgH gene. Northern blot analysis revealed that these cell lines expressed Cu-mRNA, and 5-mRNA. consistent with normal pre-B cells. Intriguingly, they expressed c-fms-mRNA constitutively. When J13 cells were cocultured … More with ST2 and, GM-CSF ih place of ST2 and IL-5, they acquired Mac-1 expression and retained Ly-1 expression. They were morphologically macrophages, nonspeclfic-esterase positive, and showed phagocytosis of latex beads. The conversion was inhibited by addition of. IL-5. These results support evidence for a close relationship between the myeloid and Ly-1^+ B cell pathways of differentiation, and indicate that our IL-5-dependent clones are multipotential intermediates in differentiation from pre-B cells to B cells and macrophages.(2) IL-5 has been suggested to be5 lnvolved in in vitro growth and differentiation of B cells and eoginophlls. To envisage the possible engagement of IL-5 in the development of these cells in vivo, transgenic mice carrying the mouse IL-5 gene legated with a metallothionein promoter were generated. The IL-5 transgenic mice exibited elevated levels of IL-5 In the serum and an increase in the levels of serum IgM and IgA. A massive eosinophilia In peripheral blood and spleen and an infiltration of eosinophils In muscle and liver were observed. When cadmium-containing saline was injected i. p. into transgenic mice, a distinctive Ly-l^+ B cell population became apparent in the spleen after 5 days. IL-5 receptors (lL-5R) were detected on those cells by mabs against la-5R. Another interesting finding in these transgenic mice was an increase in polyreactive anti-DNA antibodies of IgM class. It Is suggested, therefore, that aberrant expression of the IL-5 gene may Induce accumulation of Ly-l^+ B cells and eosinophils. Furthermore, this IL-5 transgenic mouse can be a model mouse for eosinophilia and we can determine the role of IL-5 in the differentiation of Ly-l^+ B cells and eosinophils by using this mouse.(3) We have Isolated CDNA clones encoding a murine IL-5R by expression screening of a library prepared from a murine IL-5-dependent early B cell line. A CDNA library was expressed In COS7 cells and screened by panning with the use of anti-IL-5 receptor monocional antibodies. The deduced amino acid sequence analysis demonstrates that the receptor Is a glycoprotein of 415 amino acids (Mr 45, 284), Including an N-terminal hydrophobic region (17 amino acids), a glycosylated extracellular domain (322 amino acids), a single transmembrane segment (22 amino acids) and a cytoplasmic tall (54 amino acids). COS7 cells transfected with the CDNA expressed a 60-kD protein that bound IL-5 with a single class of affinity (K_D = 2-10 nM). FDC-PL cells transfected with the CDNA for murine IL-5R showed the expression of IL-5 binding sites with both low (K_D=6 nM) and high affinity (K_D=30 pill) and acquired responsiveness to IL-5 for proliferation, although parental FDC-PL cells did not show any detectable IL-5 binding. Northern blot analysis showed that two species of mRNAs (5.0 kb and 5.8 kb) were detected In cell lines that display binding sites for murine IL-5. Homology search for the amino acid sequence of the IL-5 receptor reveals that the IL-5 receptor contains a common motif of a cytokine receptor family that is recently identified.(4) The murine high-affinity IL-5R consists of at least two membrane polypeptide chains ; a chain of p6O and B chain of P130/Pl4O. We found that the B chain is constitutively expressed in IL-3-dependent early B cell-line. To evaluate whether a component of IL-3R is the B chain of the high affinity IL-5R, effect of anti-IL-3 receptor mAb (anti-Aic-2 mAb) on DNA synthesis of Y16 was examined. Anti-Aic-2 mAb that immunoprecipitated p13O/p14O in the cell lysates of Y16 cells partially inhibited the IL-5- and IL-3-induced proliferation of Y16 cells, whereas it did not react with the a chain of IL-5R. Since anti-Aic-2 mAb is reported to react with. recombinant AIC2A and AIC2B, we examined whether AIC2A or AIC2B is involved in the formation of the high affinity IL-5R. The expression of the murine IL-5R receptor a chain with AIC2B, but not with AIC2A in L cells resulted in the high affinity IL-5 binding sites. The expression of AIC2B alone did not show any IL-5 binding by itself. Cell lysates of these transfectants were crosslinked to form the complex of a and B chains with radiolabeled IL-5. These results clearly indicate that B chain of the murine IL-5 receptor is an indispensable for the formation of high-affinity IL-5 binding sites together with the a chain and that AIC2B is likely the the B chain of the mouse IL-5 receptor. The B chain was found to be identical to the B chain of GM-CSF receptor. Less