Yang Mhan Pyo, Lee Keum Jong, Yun So Mi, Kim Ju Hyang, Ko In Kyung, Jeung Eui Bae
Department of Veterinary Medicine, College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungbuk National University, Cheongju, 361-763, Chungbuk, South Korea.
Vet Immunol Immunopathol. 2002 May;86(1-2):43-53. doi: 10.1016/s0165-2427(02)00010-7.
Feline peripheral blood mononuclear cells (PBMC)-derived chemotactic factor induced by egg white derivatives (EWD) treatment was analyzed at the protein and messenger ribonucleic acid (mRNA) level. EWD itself was not active chemotactic for feline peripheral blood polymorphonuclear cells (PMN). But chemotaxis of PMN was enhanced by either culture supernatant from PBMC treated with EWD or human recombinant (hr) interleukin (IL)-8. Both hr IL-8 and the culture supernatant from PBMC treated with EWD yielded a distinct band, molecular weight of 6-8kDa, in sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 15% loading gel. Therefore, to identify this chemotactic factor, culture supernatant from PBMC treated with EWD was partially purified by anion exchange chromatography on diethylaminoethyl (DEAE)-Sepharose CL-6B and concentrated by ultrafiltration. Only the fraction, which was eluted with 0.3M NaCl, showed a high concentration of total protein and also enhanced the chemotactic activity of PMN. This activity was thereafter designated as eluate. The chemotactic activity of eluate was inhibited by anti-hr IL-8 polyclonal antibody (pAb). A single protein band with 6-8kDa was shown in both the eluate and hr IL-8 when analyzed by SDS-PAGE and Western blotting using anti-hr IL-8 pAb, suggesting that the chemotactic factor for feline PMN is IL-8, 6-8kDa, produced by PBMC treated with EWD. The physicochemical characteristics of eluate were stable in heated (60-100 degrees C), acid (pH 3.0), and alkaline (pH 9.0) conditions. The eluate under these conditions also showed a distinct band in molecular weight of 6-8kDa in SDS-PAGE and Western blotting and was very active in chemotactic activity of PMN.IL-8 mRNA gene expression on feline PBMC was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) assay using a series of oligonucleotides, each 22 mer, derived from feline IL-8. Feline IL-8 mRNA showed low level in 3-h incubation without EWD, but it was increased in a dose-dependent manner by addition of EWD. Following EWD (10 microg/ml) treatment, IL-8 mRNA expression was rapidly increased up to 6h and decreased by 12h although it was not expressed in freshly prepared PBMC. This study strongly suggested that immunoenhancing effect of EWD on chemotactic response of PMN is mediated by feline IL-8, 6-8kDa, produced by PBMC stimulated with EWD. In addition, the expression of feline IL-8 mRNA on PBMC is increased when stimulated with EWD.
在蛋白质和信使核糖核酸（mRNA）水平上分析了蛋清衍生物（EWD）处理诱导的猫外周血单个核细胞（PBMC）来源的趋化因子。EWD本身对猫外周血多形核细胞（PMN）没有趋化活性。但是，用EWD处理的PBMC培养上清液或人重组（hr）白细胞介素（IL）-8均可增强PMN的趋化性。在含15%浓缩胶的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）中，hr IL-8和用EWD处理的PBMC培养上清液均产生了一条明显的条带，分子量为6-8 kDa。因此，为了鉴定这种趋化因子，用EWD处理的PBMC培养上清液通过二乙氨基乙基（DEAE）-琼脂糖CL-6B阴离子交换色谱进行部分纯化，并通过超滤浓缩。只有用0.3M NaCl洗脱的部分显示出高浓度的总蛋白，并且还增强了PMN的趋化活性。此后将该活性指定为洗脱液。洗脱液的趋化活性被抗hr IL-8多克隆抗体（pAb）抑制。当通过SDS-PAGE和使用抗hr IL-8 pAb的蛋白质印迹分析时，洗脱液和hr IL-8中均显示出一条6-8 kDa的单一蛋白条带，表明猫PMN的趋化因子是由用EWD处理的PBMC产生的6-8 kDa的IL-8。洗脱液的理化特性在加热（60-100摄氏度）、酸性（pH 3.0）和碱性（pH 9.0）条件下是稳定的。在这些条件下的洗脱液在SDS-PAGE和蛋白质印迹中也显示出一条分子量为6-8 kDa的明显条带，并且在PMN的趋化活性方面非常活跃。使用一系列源自猫IL-8的22聚体寡核苷酸，通过逆转录-聚合酶链反应（RT-PCR）分析猫PBMC上的IL-8 mRNA基因表达。在没有EWD的3小时孵育中，猫IL-8 mRNA水平较低，但通过添加EWD以剂量依赖性方式增加。在EWD（10微克/毫升）处理后，IL-8 mRNA表达在6小时内迅速增加，在12小时时下降，尽管在新鲜制备的PBMC中未表达。这项研究强烈表明，EWD对PMN趋化反应的免疫增强作用是由用EWD刺激的PBMC产生的6-8 kDa的猫IL-8介导的。此外，当用EWD刺激时，猫PBMC上的IL-8 mRNA表达增加。
