Basic and preclinical experiments of IL 8 and MCAF

IL 8和MCAF的基础和临床前实验

• 批准号: 03044066
• 负责人: MATSUSHIMA Kouji
• 金额: $ 6.4万
• 依托单位: Cancer Research Institute, Kanazawa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03044066/
• 关键词: Leukocyte Chemotactic factors; cytokine; interleukin 8; MCAF; structural analysis; receptors; gene expression; pathophysialogical roles; インタ-ロイキン8; McAF; 構造と機能; 前臨床実験

BASIC AND PRECLINICAL EXPERIMENTS OF IL 8 AND MCAFNovel chemotactic cytokines, IL 8 and MCAF were identified, purified, and molecularly cloned by us at National Cancer Institute, USA in 1987-1989, belong a family of emerging chemotactic cytokine family, CHEMOKINE. In this international collabolative research, the following projects were performed.1)Structural analysis of IL 8/MCAF and determining their active site(s)-----Human IL 8 was expressed in E coli and purified to homogeneity in a large scale. The structure of IL 8 was analyzed by both NMR and X-ray crystallography. The proposed model shows that IL 8 exists as dimer through hydrogen bonding and two symmetry-related anti-parallel a-helices, 24A long and separated by 14A, lie on top of a six-stranded anti-parallel b-sheet platform. The active site of IL 8 was presumed to be the region surrounding His at 33 as well as N-terminal region based on the mutation studies and structural analyses.2)Structure of the receptors for IL 8 and M … More CAF-----The MW of the receptors for IL 8 on human nutrophils was estimated to be 60,000 with Kd of lnM. The MW of the receptors for MCAF on human monocytes was estimated to be 40,000 with Kd of 25nM. Although the cDNA for IL 8 was cloned by other groups, we have cloned several novel cDNA which belong to the family of chemotactic cytokine receptors.3)Molecular analysis of the regulation of the production of IL 8-----After cDNA cloning of human IL 8, we revealed the induction of the production of IL 8 by various types of cells after stimulating with IL 1, TNF, endotoxin, exotoxin, viral proteins, heavy metals, and superoxide generating substances. We also showed the suppression of the production of IL 8 by glucocorticoids, vitamin D3, lipooxygenase inhibitors, and FK506. We have determined the enhancer of the human IL 8 gene to be consisted of AP-1+NFKB. IL 8 production suppressive agents seem to inhibit the activation of nuclear factors which bind to these regions.4)Establishment of pathophysiological roles of IL 8/MCAF and examining possible clinical application of IL 8/MCAF-----We have revealed the production of IL 8/MCAF in various human inflammatory diseases including ulcerative colitis, psoriasis, glomerulonephritis, urinary tract infection, and arthritis. Essential involvement of IL 8 in recruiting neutrophils in acute inflammation models in rabbits, such as dermatitis, arthritis, and lung reperfusion was established using monoclonal neutralizing antibody against rabbit IL 8. IL 8/MCAF cDNA transfection into tumor cells showed significant anti-tumor effect of these cytokines in mice. MCAF is further shown to prime murine macrophages to be tumor cytocidal and also have anti-infectious activity in mice. Possible hematopoietic activity of these cytokines was not proved either in vitro or in vivo. Less

1987年至1989年，美国在美国国家癌症研究所（National Cancer Institute）在美国国家癌症研究所（National Cancer Institute）鉴定，纯化和分子克隆了IL 8和MCAFNOVEL趋化细胞因子，IL 8和MCAF的基本和临床前实验。在这项国际合作研究中，进行了以下项目。1）IL 8/MCAF的结构分析，并确定其主动地点----人类IL 8在e Coli中表达，并大规模纯化为同质性。通过NMR和X射线晶体学分析了IL 8的结构。提出的模型表明，IL 8通过氢键作为二聚体存在，并且两个与对称相关的抗平行A螺旋长24A，并由14a隔开，位于六链的抗平行B-steet平台的顶部。根据突变研究和结构分析，IL 8的活性位点被预计为围绕33的区域以及N末端区域。2）IL 8和M的受体结构IL 8和M…更多的Caf -----更多的IL受体MW在人类营养区域的受体MW估计为60,000，估计为60,000。人类单核细胞上MCAF的受体MW估计为40,000，KD为25nm。尽管IL 8的cDNA由其他组克隆，但我们克隆了几个属于趋化性细胞因子受体家族的新型cDNA。蛋白质，重金属和超氧化物质。我们还显示了糖皮质激素，维生素D3，脂氧酶抑制剂和FK506抑制IL 8的产生。我们已经确定人IL 8基因的增强子是AP-1+NFKB一致的。 IL 8产量抑制剂似乎抑制了与这些区域结合的核因素的激活。4）建立IL 8/MCAF的病理生理作用，并检查了IL 8/MCAF的可能临床应用 - 我们已经揭示了IL 8/MCAF在各种人类炎症性疾病中的产生，包括各种人类炎症性疾病，包括溃疡性疾病，包括溃疡性炎症，脉络膜炎，脉络膜炎，幼发学，尿布素，尿布素，尿布素，尿布素，尿布素，尿布素。使用单克隆性中和对兔8。IL8/MCAF cDNA转染的抗体，建立了IL 8的急性炎症模型中嗜中性粒细胞的基本参与。 MCAF进一步证明了鼠巨噬细胞为肿瘤巨噬细胞，并且在小鼠中也具有抗感染活性。这些细胞因子的可能造血活性在体外或体内均未证明。较少的

项目成果

Mahe,Y.: "Hepatitis B virus X protein transactivates human interleukin 8 gene throughhacting on muclear factor KB and CCAAT/enhancer-binding protein-like cise-lementls" J.Biol,Chem.266. 13759-13763 (1991)

Mahe,Y.：“乙型肝炎病毒 X 蛋白通过作用于核因子 KB 和 CCAAT/增强子结合蛋白样顺式元件反式激活人白细胞介素 8 基因”J.Biol,Chem.266。

Smyth, M.J., etal.: "Interleukin 8 gene expression and production in human peripheral blood lymphocyte subset" J.Immunol.146. 3815-3823 (1991)

Smyth, M.J. 等人：“人外周血淋巴细胞亚群中白细胞介素 8 基因的表达和产生”J.Immunol.146。

Oppenheim,J.J., Zachariae,C.O.C., Mukaida,N., Matsushima,K.: "Properties of the novel proinflammatory supergene″intercrine″cytokine family." Annu.Rev.Immunol., 31 (1991)

Oppenheim, J.J.、Zachariae,C.O.C.、Mukaida,N.、Matsushima,K.：“新型促炎超基因“间分泌”细胞因子家族的特性。”Annu.Rev.Immunol.，31（1991）

Oppenheim, J.J. etal.: Annu.Rev.Immunol.Properties of the novel proinflammatory supergene"intercrine"cytokine family, 617-648 (1991)

奥本海姆，J.J.

Baldwin,E.T.: "Crystal structure of interleukin 8" Proc.Natl.Acad.Sci.88. 502-506 (1991)

Baldwin,E.T.：“白细胞介素 8 的晶体结构”Proc.Natl.Acad.Sci.88。