Production and functional characterization of recombinant bovine interleukin-8 as a specific neutrophil activator and chemoattractant
Production and functional characterization of recombinant bovine interleukin-8 as a specific neutrophil activator and chemoattractant
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DOI：
10.1016/s0165-2427(99)00007-0
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发表时间：
1999-03-01
影响因子：
1.8
通讯作者：
Gordon, JR
中科院分区：
文献类型：
--
作者：
Caswell, JL;Middleton, DM;Gordon, JR
白细胞介素-8是细胞因子趋化因子家族的一员，在许多非啮齿类物种中是一种有效的中性粒细胞化学引诱物。在这项研究中，重组牛白细胞介素-8（rbIL-8）在细菌中表达的谷胱甘肽-S-转移酶融合蛋白。融合蛋白经谷胱甘肽-琼脂糖亲和层析纯化，凝血酶裂解洗脱重组rbIL-8。纯化的rbIL-8分子约为8 kDa，并通过Western分析证实为真实的IL-8。重组牛IL-8在低至1.0 ng/ml的剂量下诱导特定的剂量依赖性中性粒细胞的体外趋化性，并且这种活性通过用羊IL-8的单克隆抗体预处理rbIL-8而被抑制。通过显微镜分析和流式细胞术测定，暴露于rbIL-8的中性粒细胞产生伪足并变得细长。将3.3 ng至3.3 μ g rbIL-8注射到正常小牛的皮肤中可诱导嗜中性粒细胞的剂量依赖性募集，但不诱导嗜酸性粒细胞的募集。注射rbIL-8后15 ~ 60 min，中性粒细胞在血管内聚集明显，注射后1 ~ 18 h，中性粒细胞在血管外稳定增加。在注射后18和30小时，在这些病变中检测到具有凋亡形态特征的中性粒细胞，这与真皮中性粒细胞数量的减少相关。这些结果明确证实，牛IL-8在体外和体内起中性粒细胞，而不是嗜酸性粒细胞，趋化剂的作用。(C)1999 Elsevier Science B. V.保留所有权利。
Interleukin-8, a member of the chemokine family of cytokines, is a potent neutrophil chemoattractant in many non-rodent species. In this study, recombinant bovine interleukin-8 (rbIL-8) was expressed in bacteria as a glutathione-S-transferase fusion protein. The fusion protein was purified by glutathione-Sepharose affinity chromatography and recombinant rbIL-8 was eluted by cleaving with thrombin. The purified rbIL-8 molecule was approximately 8 kDa and was confirmed as authentic IL-8 by Western analysis. Recombinant bovine IL-8 induced specific dose-dependent in vitro chemotaxis of neutrophils at doses as low as 1.0 ng/ml, and this activity was inhibited by pre-treatment of rbIL-8 with a monoclonal antibody to ovine IL-8. Neutrophils exposed to rbIL-8 developed pseudopodia and became elongated as determined by microscopic analysis and flow cytometry. Injection of 3.3 ng to 3.3 mu g of rbIL-8 into the skin of a normal calf induced dose-dependent recruitment of neutrophils but not eosinophils. Intravascular margination of neutrophils was obvious at the injection sites from 15 to 60 min after administration of rbIL-8, and extravascular neutrophil numbers increased steadily from 1 to 18 h after injection. Neutrophils with morphologic features of apoptosis were detected in these lesions at 18 and 30 h after injection, and this correlated with reduction in the number of dermal neutrophils, These results confirm unequivocally that bovine IL-8 functions as a neutrophil, but not an eosinophil, chemoattractant in vitro and in vivo. (C) 1999 Elsevier Science B.V. All rights reserved.