Caswell J L, Middleton D M, Gordon J R
Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Canada.
Vet Immunol Immunopathol. 1999 Mar 1;67(4):327-40. doi: 10.1016/s0165-2427(99)00007-0.
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 microg 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.
白细胞介素-8是细胞因子趋化因子家族的一员，在许多非啮齿类动物中是一种有效的中性粒细胞趋化因子。在本研究中，重组牛白细胞介素-8（rbIL-8）在细菌中作为谷胱甘肽-S-转移酶融合蛋白表达。融合蛋白通过谷胱甘肽-琼脂糖亲和层析纯化，重组rbIL-8通过凝血酶切割洗脱。纯化的rbIL-8分子约为8 kDa，经蛋白质印迹分析确认为 authentic IL-8。重组牛IL-8在低至1.0 ng/ml的剂量下即可诱导中性粒细胞产生特异性剂量依赖性的体外趋化作用，并且这种活性可被用抗绵羊IL-8单克隆抗体预处理rbIL-8所抑制。通过显微镜分析和流式细胞术测定，暴露于rbIL-8的中性粒细胞形成伪足并变长。向正常小牛皮肤注射3.3 ng至3.3 μg的rbIL-8可诱导中性粒细胞的剂量依赖性募集，但不会募集嗜酸性粒细胞。在注射rbIL-8后15至60分钟，注射部位中性粒细胞的血管内边缘化明显，注射后1至18小时血管外中性粒细胞数量稳步增加。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在体外和体内作为中性粒细胞而非嗜酸性粒细胞的趋化因子发挥作用。
