Li Y M, Arkins S, McCusker R H, Donovan S M, Liu Q, Jayaraman S, Dantzer R, Kelley K W
Department of Animal Sciences, University of Illinois, Urbana-Champaign 61801, USA.
J Immunol. 1996 Jan 1;156(1):64-72.
Primary (thymus) and secondary (spleen) murine lymphoid tissues express a 25-kDa protein that binds IGF-I. To determine the cellular source of this insulin-like growth factor binding protein (IGFBP), 11 murine or human cell lines representing T, B, and myeloid cells at various stages of differentiation were characterized by IGF-I affinity cross-linkage and Western ligand blotting. Mature myeloid cells, but not T or B cells, secrete a 25-kDa protein that is capable of binding IGF-I. CSF-1-derived bone marrow macrophages also synthesize this 25-kDa IGFBP. Thymic macrophages, which were estimated to secrete 2 ng of binding protein/10(6) cells-h, were used in conjunction with [125I] IGF-I affinity cross-linking to develop a protein binding immunomobility-shift assay to identify which IGFBP is produced by these cells. An anti-IGFBP-4Ab, but not an anti-IGFBP-2 Ab or normal rabbit serum, shifted the [125I] IGF-IGFBP complex to a higher m.w. position, indicating that the single 25-kDa IGFBP is IGFBP-4. Northern blotting confirmed that transcripts for IGFBP-4 as well as IGF-I are expressed in thymic macrophages. A putative 278-bp IGFBP-4 cDNA fragment (residues 341-618) of rat) that contains two unique cysteine residues found only in IGFBP-4 was cloned and sequenced from thymic macrophages. These clones differed from the rat sequence at only six residues (97% homology), and the deduced amino acid sequence from the murine cDNA was identical with that of the rat sequence. Subsequent studies revealed that IGF-I stimulates DNA synthesis in thymic macrophages. However, two different IGF-I analogues differing in the amino-terminus that bind equally well to the IGF-I receptor but poorly to IGFBPs are as effective as IGF-I at 100-fold lower concentrations. These data demonstrate that murine macrophages are a source of a single 25-kDa secreted protein that binds IGF-, that the molecular identity of this protein is IGFBP-4, and that this binding protein may antagonize the extracellular effects of IGF-I.
小鼠的主要（胸腺）和次要（脾脏）淋巴组织表达一种能结合胰岛素样生长因子I（IGF-I）的25kDa蛋白。为确定这种胰岛素样生长因子结合蛋白（IGFBP）的细胞来源，通过IGF-I亲和交联和Western配体印迹法对代表不同分化阶段的T、B和髓样细胞的11种小鼠或人类细胞系进行了表征。成熟的髓样细胞而非T或B细胞分泌一种能结合IGF-I的25kDa蛋白。集落刺激因子-1（CSF-1）来源的骨髓巨噬细胞也合成这种25kDa的IGFBP。胸腺巨噬细胞估计每10⁶个细胞·小时分泌2ng结合蛋白，将其与[¹²⁵I]IGF-I亲和交联结合使用，开发一种蛋白结合免疫迁移率变动分析方法，以鉴定这些细胞产生的是哪种IGFBP。抗IGFBP-4抗体而非抗IGFBP-2抗体或正常兔血清能将[¹²⁵I]IGF-IGFBP复合物迁移至更高分子量位置，表明单一的25kDa IGFBP是IGFBP-4。Northern印迹法证实IGFBP-4以及IGF-I的转录本在胸腺巨噬细胞中表达。从胸腺巨噬细胞中克隆并测序了一个推定的278bp IGFBP-4 cDNA片段（大鼠的341-618位残基），该片段包含仅在IGFBP-4中发现的两个独特半胱氨酸残基。这些克隆与大鼠序列仅在六个残基处不同（同源性97%），从小鼠cDNA推导的氨基酸序列与大鼠序列相同。后续研究表明IGF-I刺激胸腺巨噬细胞中的DNA合成。然而，两种在氨基末端不同但与IGF-I受体结合能力相同但与IGFBPs结合能力较差的不同IGF-I类似物，在浓度低100倍时与IGF-I一样有效。这些数据表明小鼠巨噬细胞是一种单一的25kDa分泌蛋白的来源，该蛋白能结合IGF-，这种蛋白的分子身份是IGFBP-4，并且这种结合蛋白可能拮抗IGF-I的细胞外效应。
