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 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.
Caswell
J L
JL
Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Canada. jcaswell@lsd.uoguelph.ca
Middleton
D M
DM
Gordon
J R
JR
D000818:Animals; D001665:Binding Sites; D002417:Cattle / Q000097:blood*; D002630:Chemotactic Factors / Q000096:biosynthesis* / Q000235:genetics; D002634:Chemotaxis, Leukocyte; D016209:Interleukin-8 / Q000096:biosynthesis* / Q000235:genetics*; D009504:Neutrophils / Q000187:drug effects*; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis; D012756:Sheep; D012882:Skin Tests; D013917:Thrombin / Q000378:metabolism
D002630:Chemotactic Factors; D016209:Interleukin-8; D011994:Recombinant Proteins; D013917:Thrombin