Establishment of an immunoassay system for monocyte chemotacticand activating factor (MCAF) and its clinical application

本项目的研究成果总结如下：1.我们通过将MCAF cDNA转染小鼠骨髓瘤细胞系，建立了MCAF产生细胞系，并从该细胞系的培养上清液中纯化MCAF。我们观察到预先给予纯化的重组MCAF可以预防正常小鼠或粒细胞减少小鼠由铜绿假单胞菌或伤寒沙门氏菌引起的致死性感染.我们建立了一种特异性和敏感性的抗人MCAF的ELASA，使用这种ELASA，我们观察到在给予免疫调节剂OK-432后，恶性胸膜炎患者的MCAF水平增加。此外，大多数人胶质母细胞瘤细胞系在IL-1或TNF α刺激下可产生MCAF，提示MCAF在肿瘤免疫中的潜在作用.我们还研究了MCAF相关分子，白细胞介素-8（IL-8）在急性炎症中的作用，通过给予针对IL-8的中和抗体。这些实验证明IL-8在脂多糖诱导的皮炎和肺再灌注损伤中的嗜中性粒细胞浸润和嗜中性粒细胞依赖性组织损伤中起重要作用.用ELISA法测定尿IL-8水平，发现尿路感染患者及狼疮性肾炎、急性肾小球肾炎、伊加肾病急性加重期等多种类型肾小球肾炎患者尿IL-8水平升高，提示尿IL-8水平测定可用于多种泌尿系疾病的诊断和监测。我们从分子生物学的角度探讨了IL-8基因激活的机制，并证实NF_<-k>B位点在我们检测的每一个细胞系中都参与了IL-8基因的转录。

The results of the project is summarized as follows.1. We established MCAF-producing cell line by transfecting MCAF cDNA into a mouse myeloma cell line and purified MCAF from the culture supernatants of this cell line. We observed that the preadministration of purified recombinant MCAF could prevent either normal or granulocytopenic mice from lethal infection caused by pseudomonas aeruginosa o r Salmonella typhymurium.2. We established a specific and sensitive ELASA against human MCAF.Using this ELASA, we observed that the pleural fluid levels of MCAF increased in patients with malignant pleurisy upon the administration of an immunomodulator, OK-432. In addition, most human glioblastoma cell lines could produce MCAF in response to IL-1 or TNFalpha, suggesting a potential role of MCAF in tumor immunity.3. We also examined the role of MCAF-related molecule, interleukin-8 (IL-8) in the acute inflammation by administering a neutralizing antibody against IL-8. these experiments demonstrated that IL-8 plays an essential role in neutrophil infiltration and neutrophi-dependent tissue injuries in lipopolysaccharide-induced dermatitis and lung reperfusion injury.4. The determination of urinary IL-8 lavels using ELISA revealed that urinary IL-8 levels increased in the urines of patients with urinary tract infections and those with several types of glomerulonephritis such as lupus nephritis, acute glomerulonephritis, and IgA nephropathy with acute exacerbations.These results imply that the determination of urinary IL-8 level can be used for diagnosing and monitoring several diseases in urinary tracts.In addition, we explored the molecular biological aspects of IL-8 gene activation and established that NF_<-k>B site was essentially involved in the transcription of IL-8 gene in every cell line that we examined.