BIOLOGY & BIOCHEMISTRY OF MEMBRANE INTERLEUKIN 1

生物学

基本信息

批准号：
3454165
负责人：
EVELYN A. KURT-JONES
金额：
$ 12.33万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-04-01 至 1993-03-31
项目状态：
已结题

项目摘要

Interleukin 1 (IL-1) is an important mediator in immune and inflammatory responses. IL-1 activity is expressed by both secreted and membrane associated molecules. Secreted IL-1 has been extensively characterized. However, the biochemical nature and the function of membrane IL-1 are largely unknown. This proposal addresses basic questions about the biology and biochemistry of membrane IL-1 using human endothelial cells (HEC) as the prototypical membrane IL-1 producing cells. 1) The conditions for induction of membrane IL-1: membrane IL- 1 expression will be examined on human umbilical vein endothelial cells incubated with a panel of immune and inflammatory stimulants in vitro. The treated cells will be either fixed with paraformaldehyde or fractionated by hypotonic lysis and ultracentrifugation to isolate membranes. IL-1 activity will be assayed using the D10.G4.1 IL-1 indicator line. 2) The relationship of membrane and secreted IL-1 will be examined by biochemical, immunochemical, and molecular approaches. The possible precursor-product relationship of membrane and secreted IL-1 will be examined by kinetic and pulse-labeling studies of IL-1 secretion and membrane expression. The antigenic and biochemical relationship of membrane and secreted IL-1 will be examine by anti-IL-1 inhibition of membrane IL-1 function and immunoprecipitation of metabolic or surface labeled cells. Transfection of BW5147 or Cos 7 cells with cDNA encoding known IL-1 alpha or IL-1 beta proteins or endothelial cell-derived IL-1 proteins will be used to establish if membrane and secreted IL-1 are encoded by the same genes and whether expression of membrane versus secreted IL-1 is controlled by transcriptional, post-transcriptional, or post-translational events. 3) The association of IL-1 with the cell membrane will be examined by attempting to extract IL-1 activity from membranes with chaotropic ions, detergents, and proteolytic or lipolytic enzymes. These studies will indicate if membrane IL-1 is an integral membrane protein or associated with an integral membrane protein or membrane lipid. 4) The function of membrane IL-1 will be assessed by comparing membrane and secreted IL-1 as i) co-factors in mitogen induced T cell activation (a prototypical immune activity), ii) stimulants of fibroblast proliferation, and iii) inducers of endothelial cell- leukocyte adhesion (examples of inflammatory responses). The specificity of these responses will be determined by blocking studies with anti-IL-1 antibodies.

白细胞介素1（IL-1）是免疫和免疫系统中的重要介质。炎症反应。 IL-1 活性由两者表达分泌的和膜相关的分子。分泌的IL-1具有被广泛表征。然而，生化性质膜IL-1的功能很大程度上未知。这提案解决了有关生物学的基本问题使用人内皮细胞膜 IL-1 的生物化学 (HEC) 作为原型膜 IL-1 产生细胞。 1) 膜IL-1诱导条件：膜IL- 1 在人脐静脉内皮细胞上检测表达细胞与一组免疫和炎症细胞一起孵育体外兴奋剂。处理过的细胞将被固定多聚甲醛或通过低渗裂解进行分级和超速离心分离膜。 IL-1 活性将使用 D10.G4.1 IL-1 指示线进行测定。 2) 膜与分泌的IL-1的关系为通过生化、免疫化学和分子检测接近。可能的前体-产物关系膜和分泌的 IL-1 将通过动力学和 IL-1 分泌和膜表达的脉冲标记研究。膜和生物化学的关系分泌的IL-1将通过膜的抗IL-1抑制来检查 IL-1 功能和代谢或表面免疫沉淀标记的细胞。用 cDNA 转染 BW5147 或 Cos 7 细胞编码已知的 IL-1 α 或 IL-1 β 蛋白或内皮细胞细胞来源的 IL-1 蛋白将用于确定膜是否和分泌型 IL-1 是否由相同基因编码膜与分泌性 IL-1 的表达受以下因素控制转录、转录后或翻译后事件。 3) IL-1与细胞膜的结合将是通过尝试从膜中提取 IL-1 活性进行检查与离液离子、去污剂和蛋白水解或脂解剂酶。这些研究将表明膜 IL-1 是否是一种整合膜蛋白或与整合膜蛋白相关膜蛋白或膜脂。 4) 通过比较膜IL-1的功能来评估膜和分泌的 IL-1 作为有丝分裂原诱导 T 中的辅助因子 i) 细胞激活（一种典型的免疫活性），ii) 刺激物成纤维细胞增殖，以及 iii) 内皮细胞诱导剂- 白细胞粘附（炎症反应的例子）。这这些反应的特异性将通过阻断来确定抗 IL-1 抗体的研究。