Production and functional characterization of recombinant bovine interleukin-8 as a specific neutrophil activator and chemoattractant

Interleukin-8, a member of the chemokine family of cytokines, is a potent n eutrophil chemoattractant in many non-rodent species. In this study, recomb inant bovine interleukin-8 (rbIL-8) was expressed in bacteria as a glutathi one-S-transferase fusion protein. The fusion protein was purified by glutat hione-Sepharose affinity chromatography and recombinant rbIL-8 was eluted b y cleaving with thrombin. The purified rbIL-8 molecule was approximately 8 kDa and was confirmed as authentic IL-8 by Western analysis. Recombinant bo vine IL-8 induced specific dose-dependent in vitro chemotaxis of neutrophil s at doses as low as 1.0 ng/ml, and this activity was inhibited by pre-trea tment of rbIL-8 with a monoclonal antibody to ovine IL-8. Neutrophils expos ed to rbIL-8 developed pseudopodia and became elongated as determined by mi croscopic analysis and flow cytometry. Injection of 3.3 ng to 3.3 mu g of r bIL-8 into the skin of a normal calf induced dose-dependent recruitment of neutrophils but not eosinophils. Intravascular margination of neutrophils w as 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 we re detected in these lesions at 18 and 30 h after injection, and this corre lated with reduction in the number of dermal neutrophils, These results con firm unequivocally that bovine IL-8 functions as a neutrophil, but not an e osinophil, chemoattractant in vitro and in vivo. (C) 1999 Elsevier Science B.V. All rights reserved.