Basic and preclinical experiments of IL 8 and MCAF

IL - 8和MCAF的基础和临床前实验我们于1987-1989年在美国国家癌症研究所鉴定、纯化和分子克隆了新型趋化因子IL - 8和MCAF，它们属于新兴的趋化细胞因子家族，CHEMOKINE。在这项国际合作研究中，进行了以下项目。1) IL - 8/MCAF的结构分析及活性位点的确定-----人IL - 8在大肠杆菌中大规模表达并纯化至均匀性。用核磁共振和x射线晶体学分析了IL - 8的结构。该模型表明，IL - 8通过氢键以二聚体的形式存在，两个对称相关的反平行a-螺旋，长24A，间隔14A，位于反平行的六股b-片平台上。根据突变研究和结构分析，推测IL - 8的活性位点在33号His周围区域以及n端区域。2) IL - 8和M - 8受体的结构，更多的CAF----- IL - 8受体在人营养粒细胞上的分子量估计为60000，Kd为lnM。MCAF受体在人单核细胞上的分子量估计为40000，Kd为25nM。虽然IL 8的cDNA已被其他研究小组克隆，但我们已经克隆了几个新的cDNA，它们属于趋化细胞因子受体家族。3) IL - 8产生调控的分子分析-----通过对人IL - 8的cDNA克隆，揭示了IL - 1、TNF、内毒素、外毒素、病毒蛋白、重金属和超氧化物生成物质刺激后，各种类型的细胞都能诱导IL - 8的产生。我们还发现糖皮质激素、维生素D3、脂氧化酶抑制剂和FK506可以抑制IL 8的产生。我们已经确定了人IL - 8基因的增强子由AP-1+NFKB组成。IL - 8产生抑制剂似乎抑制结合这些区域的核因子的激活。4) IL - 8/MCAF的病理生理作用的建立和IL - 8/MCAF可能的临床应用-----我们已经揭示了IL - 8/MCAF在各种人类炎症性疾病的产生，包括溃疡性结肠炎、牛皮癣、肾小球肾炎、尿路感染和关节炎。利用兔IL - 8单克隆中和抗体，建立了IL - 8在兔急性炎症模型（如皮炎、关节炎和肺再灌注）中募集中性粒细胞的重要作用。IL 8/MCAF cDNA转染肿瘤细胞后，这些细胞因子对小鼠的抗肿瘤作用显著。MCAF进一步证明小鼠巨噬细胞具有肿瘤杀伤作用，并具有抗感染活性。这些细胞因子可能的造血活性在体内和体外都没有得到证实。少

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